?

flpbm^o^qflkp^ka>k^ivpbp
/--6*/-.-
Click anywhere on page. Type desired page number. Then click OK.

Peptide Separation Technology Columns 8

 Suitable for a wide range of peptides, including large, small, acid, basic, hydrophobic and hydrophillic
 Good peak shape and retention in formic acid and TFA for optimal chromatography and detection
 Available in 130Å and 300Å pore sizes
www.waters.com/PST

Protein Separation Technology 20

 Separates proteins of various sizes, hydrophobicities, and isoelectric points
 Available as 3.5 μm packing for HPLC, and 1.7 μm packing for UPLC methods
 Maximizes recovery and minimizes protein carryover
 Quality control trested with protein mixture
www.waters.com/proteins

Amino Acid Analysis 36

 A turnkey total solution for amino acid analysis
 Specifically designed for accurate, robust, and sensitive amino acid analysis
 Proven assured performance in the areas for protein characterization, cell culture monitoring,
and analysis of food and feeds
www.waters.com/AAA

Oligonucleotide Separation Technology 41

 Exceptional column lifetime for reduced cost per analysis
 Scalable column offering for lab scale isolation needs
 Separation efficiencies equivalent to or better than PAGE, CGE, or ion-exchange HPLC methods
 Resolve failure sequences from detritylated full length products
www.waters.com/OST

MassPREP Consumables and Kits 47

 Developed and tested specifically to obtain high quality MS and LC/MS results
 Prepared under strict quality control to ensure optimal performance
 Conveniently packaged for optimal efficiency
www.waters.com/biosep
 BIOSEPARATIONS AND ANALYSES

Click anywhere on page. Type desired page number. Click OK.

Bioseparations and Analysis

Introduction ...........................................................................................2 Amino Acid Analysis

Peptide Isolations and Analyses Amino Acid Analysis ......................................................... 35
Peptide Isolation and Analysis .............................................4 UPLC Amino Acid Analysis Solution ................................. 36
Peptide Separation Technology .............................................8 ½ !BB14@F½!LHMN½!BHC½!M@KXRHR
Atlantis HPLC and UPLC Columns ...................................... 12 Using HPLC ......................................................................... 38
Prep OBD Columns—Ideal for Purification Labs ................ 13 ½ 0HBN4@F½(0,#½-DSGNC ....................................................... 39
BioSuite HPLC Peptide Analysis Columns .......................... 14 Oligonucleotide Separation Technology
BioSuite Cation-Exchange HPLC Columns ........................... 15 Oligonucleotide Purification and Analysis ........................ 40
Delta-Pak HPLC Columns .................................................... 15 Oligonucleotide Separation Technology ............................ 41
Waters Insulin HMWP HPLC Column ................................... 15 Columns for Large DNA/RNA Species ................................ 44
Symmetry HPLC Columns ................................................... 16 Gen-Pak FAX Anion-Exchange Columns ............................. 44
Protein Isolations and Analyses MassPREP OST Standard ..................................................... 45
Protein Isolation and Characterization............................... 18 Oasis μElution Plates ......................................................... 45
Protein Separation Technology .......................................... 20 MS and LC/MS Consumables for Biomolecules
BioSuite HPLC Columns ...................................................... 22 MS and LC/MS Consumables
BioSuite Ion-Exchange HPLC Columns ................................ 23 for Biomolecules ................................................................ 47

Protein-Pak HR Ion-Exchange RapiGest SF and MassPREP Oligonucleotide,

Glass Columns .................................................................... 24 Peptide, and Protein Digest Standards............................... 48

Protein-Pak PW Series Columns ......................................... 24 MassPREP Peptide Standards .............................................. 48

Advanced Purification (AP) Glass Columns ....................... 25 MassPREP Protein Digestion Standards .............................. 49

Advanced Purification (AP) Glass Column MassPREP Phosphopeptide Standards ................................. 49

Accessories and Spare Parts .............................................. 26 MassPREP Digestion Standard Mixtures ............................. 50
Fittings and Connectors for Advanced MassPREP OST Standard ..................................................... 50
Purification (AP) Glass Columns ........................................ 26 RapiGest SF Protein Digestion Surfactant .......................... 51
Accell Plus Ion-Exchange Packings ..................................... 27 MassPREP On-Line Desalting Devices ................................ 52
BioSuite Size-Exclusion Columns (SEC).............................. 28 NanoEase Trap, Capillary, and Nano Columns ................... 53
Protein-Pak and Shodex NanoEase Trapping Columns .............................................. 53
Size-Exclusion Columns (SEC) ............................................ 31
NanoEase Nano and Capillary Columns ............................. 54
Ultrahydrogel HPLC Columns ............................................. 31 nanoACQUITY UltraPerformance LC Columns .................... 55
Symmetry300 C4 HPLC Columns ....................................... 32 nanoACQUITY UPLC 2D Kit ................................................ 56
Protein-Pak Affinity Columns............................................. 32
MassPREP Kits ..................................................................... 57
BioSuite pC18 and pPhenyl Reversed-Phase
MassPREP Glycoanalysis Kit ............................................... 57
Chromatography (RPC) HPLC Columns ................................ 33
MassPREP Phosphopeptide Enrichment Kit .......................... 58
BioSuite Hydrophobic-Interaction
Waters Protein Expression System Kits .............................. 58
Chromatography (HIC) HPLC Columns................................. 34
UPLC Technology for Biomolecules .................................................... 59
Protein HIC and Protein-Pak Phenyl HIC
HPLC Columns ..................................................................... 34

www.waters.com Bioseparations and Analyses 1

BIOSEPARATIONS AND ANALYSES

Innovative Technologies From the Leaders in Separation

Science and Analytical Biochemistry

Advances in the areas of genomics, proteomics, metabanomics, and Waters comprehensive chemistry and consumables family includes:
molecular and system biology continue to revolutionize the diagnosis
½ 0DOSHCD½3DO@Q@SHNM½4DBGMNKNFX½BNKTLMR½ENQ½M@MN ½B@OHKK@QX
and treatment of disease and increase our fundamental understanding of
analytical, and preparative peptide applications
biological processes.
½ 0QNSDHM½3DO@Q@SHNM½4DBGMNKNFX½QDUDQRDC OG@RD½BNKTLMR ½"HN3THSD™ SEC,
As a leading analytical supplier of instrumentation, software, service and
IEX, RP, and HIC columns for analytical and purification applications
support, and chemistry products, Waters is uniquely positioned to provide
researc hers the tools, tec hnologies, and integrated solutions desired ½ !BB14@F™ Ultra chemistry specific for Waters UPLC Amino Acid
to tackle the formidable c hallenges involving various biomolecules. !M@KXRHR½3NKTSHNM ½@R½VDKK½@R½0HBN4@F™½@MC½!BB14@F½ENQ½(0,# A@RDC½
Beginning with a keen understanding of today’s biomolecule-related amino acid analyses
challenges, Waters scientists and engineers continuously seek purposeful ½ /KHFNMTBKDNSHCD½3DO@Q@SHNM½4DB GMNKNFX½BNKTLMR½ENQ½RXMSGDSHB
innovations that help deliver impactful solutions in applications ranging oligonucleotide and DNA/ RNA fragment isolations and analyses
from proteomics and biomarker discovery through the commercialization
of advanced biopharmaceuticals. We continue to develop new, innovative ½ -@RR02%0™ c hemistry consumables and kits for MS and LC/MS
columns and sample preparation consumables that support the HPLC, applications of peptides, proteins, and other biomolecules
UPLC® tec hnology, and LC/MS analyses of peptides, oligonucleotides,
proteins, and amino acids.

2 Bioseparations and Analyses www.waters.com

 BIOSEPARATIONS AND ANALYSES

Peptide and Oligonucleotide Isolations and Analyses Amino Acid Analysis

Synthetic peptides and oligonucleotides can be effectively separated by Waters solutions in this area have evolved from more than 25 years of
various chromatographic techniques including reversed-phase, ion-exchange, experience in amino acid analysis and include both HPLC and UPLC
and size-exclusion chromatography. Waters Peptide Separation Technology A@RDC½NEEDQHMFR½!BB14@F½@MC½0HBN4@F½OQD BNKTLM½CDQHU@SHY@SHNM½
and BioSuite columns are designed to address various peptide analysis and chemistries and columns are designed for traditional HPLC applications.
lab-scale purification needs. Our Oligonucleotide Separation Tec hnology In 2006, we introduced our UPLC Amino Acid Analysis Solution that
reversed-phase and GenPak™ Fax IEX columns address various high-resolu- TRDR½NTQ½MDWS½FDMDQ@SHNM½!BB14@F½5KSQ@½B GDLHRSQX½SN½OQNUHCD½RTODQHNQ½
tion analysis and lab-scale isolation c hallenges involving various DNA component resolution and separation speed compared to results possible
and RNA species. using established HPLC-based methods.

Protein Analysis and Characterization Proteomics, Protein Expression, and Biomarker Discovery
As the major building blocks of life, many biopharmaceutical advances Experiments in proteomics begin with samples that are both very complex
have resulted from the detailed knowledge and characterization of these and limited in volume and concentration. Good sample preparation and
molecules. Consequently, detailed protein c haracterization studies are separation of the various analytes prior to MS analysis can greatly enhance
necessary that incorporate powerful analytical tec hniques, suc h as detection sensitivity and provide more meaningful results. Waters MS and
peptide mapping and mass spectrometry using a variety of HPLC and LC/MS consumables that include NanoEase™ and nanoACQUIT Y® trap,
UPLC column c hemistries that include size exclusion, ion exc hange, capillary, and nano columns, RapiGest™ SF reagent, as well as MassPREP
hydrophobic interaction, and reversed phase. Waters Protein Separation standards and kits help ensure the best results from MS and LC/MS
Technology reversed-phase, BioSuite SEC, IEX, HIC, and RP HPLC columns, instrumentation.
offer outstanding performance when applied to the separation of these
macromolecules.

www.waters.com Bioseparations and Analyses 3

PEPTIDE ISOLATIONS AND ANALYSES

Peptides Isolation and Analysis

T he separation of peptides plays a fundamental role in many areas of shape since the peptide basic groups are shielded from secondary inter-
researc h and development. Some peptides have significant biological actions with the surface of the packing material.
activity and require analysis in a complex matrix. It may be necessary to
TFA, however, reduces the sensitivity of MS detection of peptides. W hile
isolate these molecules as natural products or as synthetic peptides. In
usable signal is usually obtained by using lower concentrations of TFA,
addition, the separation of peptides is a fundamental tool for the analysis
many investigators prefer to use formic acid as the mobile phase modifier.
of protein structure because the small, reproducible fragments are a tool
In this case, the column surface has a larger effect on the separation.
for dissecting the structural details of the much larger protein. T he same
Retention is often muc h less, and many of the peptide peaks will be
principle underlies the use of peptide separations for analyzing proteom-
asymmetrical and tailing. It is common practice to choose columns desig-
ics, protein expression, and biomarker discovery. All of these applications
nated as “no TFA” or “low TFA” for these separations. T he BioSuite PA-A
focus on a family of molecules with similar properties suc h that the
column described below was developed for this purpose. More recently the
separation can be approac hed in the same way. T he principles behind
development of Ethylene-Bridged Hybrid (BEH Technology™) packing materi-
each separation mechanism can be considered in selecting a column for
als provided an improved option. T he ethane bridges reduce the surface
a particular application. T he choice of chromatographic mode will depend
concentration of silanol groups so that there is little secondary interaction
on the type of peptide sample you are separating. T he guidelines below
between peptides and the base particle. Packing materials based on these
will help direct you to the appropriate column selection.
particles can be used for peptide separations in either T FA or formic
Reversed-Phase Peptide Separations acid containing mobile phases without significant loss of retention or
band-broadening. T he BEH Tec hnology particles for the basis of the
Mechanisms
Peptide Separation Technology family of columns described below.
Reversed-phase separations generally give the highest resolution of
With the Peptide Separation Technology columns, the minimal secondary
peptide mixtures, often allowing for effective separation of peptides
interactions create the greatest number of options for adjusting selectivity
differing by a single amino acid. In reversed-phase separations, the
with mobile phase manipulation. T he selectivity of a peptide separation
hydrophobicity of the peptide determines the elution order, with the least
is different for mobile phases containing TFA and formic acid. T he use of
hydrophobic peptides eluting first. T his hydrophobicity is related to the
columns that give good performance with either modifier create an option
size of the peptide, with larger molecules being more hydrophobic
for developing methods in addition to the desirable impact on sensitivity
in general, and also to the hydrophobicity of the side c hains of the
in MS detection. Furthermore, because peak shape and resolution are not
constituent amino acids. Both the retention and the separation selectivity
dependent on the presence of TFA, the concentration of TFA can be adjusted
are also influenced by the three-dimensional structure of the peptide.
to fine tune the selectivity of a peptide separation.
T he packing material interacts with these peptide properties based
foremost on the hydrophobicity of the column surface, reflecting bonded More dramatic changes in selectivity can be induced by changing the pH
phase chain length and density. Other column properties that can strongly of the separation from low pH to pH 7 or even pH 10 (e.g., using XBridge
influence the separation selectivity include the pore size of the packing BEH130 C18 columns).
material and the surface chemistry of the particle of the packing material.
Ion-Exchange Chromatography
Column Selectivity
In ion-exc hange c hromatography, the c harge on the amino acid side
T he retention mechanism of peptide separations is similar on reversed- c hains is the determining factor in the separation. T he benefits of
phase HPLC columns in general. T he selectivity of the separation is ion-exc hange c hromatography are the high-capacity packings and the
significantly affected by the base sorbent, pore size, alkyl ligand ability to easily load ion-exc hange fractions onto a reversed-phase
density, and residual silanol endcapping. T hese small differences in column for desalting or final purification. W hile ion-exc hange separa-
separation selectivity can be a part of developing a peptide separa- tions of peptides are not generally as highly resolving as reversed
tion. If it is difficult to ac hieve a desirable separation on a particular phase, the tec hnique can provide an orthogonal selectivity for difficult
R P-HPLC column, then the use of an alternative R P-HPLC column samples. Peptides most often separate best on strong cation exc hangers.
chemistry may be tested. For example, different separation selectivities T he ion-exc hange column packings described in the section on protein
will be observed using XBridge™ BEH300 C18 compared to Atlantis T3. c hromatography are suitable for peptides.
Mobile-Phase Selectivity Size-Exclusion Chromatography
Peptides are c harged molecules that do not freely interact with the Size-exclusion chromatography is sometimes used for peptide separations.
hydrophobic surface of reversed-phase columns. T he mobile phase T he peptides are separated based on their size in solution. T he resolution
is typically, therefore, acidified to protonate the peptide carboxyl seldom compares favorably to either reversed phase or ion exchange, but
groups, increasing retention. Trifluoroacetic acid is often preferred for it can provide an alternative for some samples. T he composition of the
this purpose because it also forms ion pairs with the basic groups of separation buffer will require careful adjustment for each particular peptide
peptides. T he combination of protonation and ion pairing leads to the sample. Elevated ionic strength and the addition of some organic solvent,
best retention and solubility for peptides. It also gives the best peak typically methanol or propanol, may be necessary for good peak shape. T he

4 Peptide Isolations and Analyses www.waters.com

 PEPTIDE ISOLATIONS AND ANALYSES

size-exclusion packings, particularly those with smaller pore sizes, described BioSuite PA-A column is especially suitable for polar peptides and for
in the section on protein chromatography, are suitable for peptides. use with MS-compatible eluents. T he BioSuite PA-B column has 300Å
pores for the analysis of large and hydrophobic peptides. Both BioSuite
Applications of Peptide Separations Peptide Analysis columns are specifically QC-tested with protein digests
Peptide Mapping to ensure batc h-to-batc h product consistency and column-to-column
performance reproducibility.
Peptide mapping continues to be the preferred technique for the comprehen-
sive characterization of biopharmaceutical products and proteins in general. Many other Waters columns have been used for particular peptide mixtures.
Waters provides an array of products that facilitate the development of It is often observed that most peptides will give reasonable chromatography
information-rich peptide maps. Before applying the separation techniques on many different columns. T here is, however, often a particular column
discussed above, the purified protein sample must be digested. T his step that, for some subtle and unpredictable reason, works better than any other
often proves difficult because of incomplete and irreproducible digestion, for a particular pair of peptides.
non-specific cleavages, precipitation of large core peptides, and slow Isolation of Natural and Synthetic Peptides
digestion. RapiGest SF is a surfactant that unfolds proteins so that the sites
of enzymatic cleavage are freely accessible for digestion. T his improves Purification of large amounts of individual peptides is most often achieved
reproducibility and speed. It also helps to keep all the sample components with reversed-phase chromatography using the same principles and ideas
in solution throughout the digestion process and up to the analysis. T he described above. T he columns are often c hosen to provide economical
properties and use of RapiGest are fully described below in the section on separations on a larger scale so they are packed with larger size particles.
MS and LC/MS consumables. T his section also describes several standard T he Peptide Separation Technology columns described above are available
mixtures of peptides and protein digests that are used to test and confirm in larger particle sizes and larger dimension columns for this purpose. T he
the performance of columns and instruments before committing any minimal secondary interactions mean that these columns can be used for
samples to the peptide mapping system. peptides representing a wide range of sizes and chemical properties. T he
surface chemistry of these packings is consistent and scalable across the
T he primary c hoice for the separation component of peptide mapping range of particle sizes. As necessary, separations may be optimized on
is the Peptide Separation Tec hnology family of columns. T his packing small scale columns and then transferred to columns large enough for the
material is based on BEH Tec hnology particles to minimize second- required sample.
ary interactions and to maximize column lifetime. T hese materials
are available with either 130Å or 300Å pores to adapt to the sizes For some complex mixtures, two step separations may give the highest yield
of peptides being analyzed. Both pore sizes are available in a range and purity. In general, the best first step is cation-exchange chromatography
of column dimensions suited to the sample size. T he same separation followed by a reversed-phase step for final isolation and desalting.
c hemistry is provided with either 3.5 m particles for HPLC analysis or Proteomics, Analysis of Protein Expression, and Biomarker Discovery
with 1.7 m for use with ACQUIT Y UPLC systems. Peptide Separation
For analyses used in proteomics and biomarker discovery, the available
Tec hnology columns are specifically QC-tested with protein digests
samples are extremely limited so the requirements of c hromatographic
to confirm batc h to batc h reproducibility. T his column c hemistry is,
separation must be coupled with extreme sensitivity. T he sample
therefore, suitable for the development of methods that are to be used
c hromatographic tools and principles described above are implemented
routinely for extended periods.
on the smallest scale in the nanoACQUIT Y UPLC® system with nanoAC-
Other Waters columns also have proven suitable for peptide mapping. QUIT Y columns. T he application solutions are described below in the
T he BioSuite Peptide Analysis columns are two premier reversed-phase section on MS and LC/MS consumables.
column c hemistries specifically optimized for peptide mapping. T he

www.waters.com Peptide Isolations and Analyses 5

PEPTIDE ISOLATIONS AND ANALYSES

Preparative Peptide Separations

T he isolation and purification of peptides from both natural and synthetic Scale-up
sources have been the focus of many scientists in research and develop-
ment. T his process has been difficult because of unpredictable separations, Some peptide samples may require an optimum isolation method. Test
short column lifetime, and lack of scalability. runs on an analytical-scale column define conditions to transfer to
a larger column. OBD columns are an integral part of the process of
Samples can be complex so separations must be developed and optimized. transferring a separation because the packed bed is stable and uniform.
T hese experiments are usually performed on pilot or analytical-scale If preferred, large particles are available with the same selectivity as
columns, but inconsistent performance of preparative columns complicates all other particule sizes. Peptide Separation Tec hnology packings are
the development of purification protocols. Peptide Separation Technology available in a wide range of particle sizes and column dimensions, all
columns provide a stable surface c hemistry that can be used for most with the same, consistent c hemistry.
peptides. Scaling the optimized separation requires identical column
chemistry and performance across a range of column dimensions. Peptide Separation of 13 Residue Peptide at Various Sample Loads
Separation Technology column chemistry is consistent across all particle
sizes and column dimensions. Scalable Peptide Purification
T he loss of samples due to early column failure must be minimized. Waters 2.6e-1
developed the patent-pending Optimum Bed Density (OBD™) prepara-
2.4e-1
tive column design. T his format provides the most rugged, efficient, and ACQUITY UPLC BEH 130 C18
2.2e-1 1.7 μm, 2.1 x 100 mm
reproducible preparative columns. W hen this column design is coupled with 200 pmol on column
2.0e-1
the stable surface chemistry of Peptide Separation Technology columns in
a range of particle sizes, the process of peptide isolation and purification 1.8e-1
AU

becomes more efficient, and overall costs are reduced. 1.6e-1

1.4e-1
Utility and Suitability for a 1.2e-1

Wide Range of Peptides 1.0e-1

Peptide Separation Technology columns are suitable for a wide variety of 8.0e-2

30 32 34 36 38 40 42 44 46 48 50 Min.
peptides, including acidic and basic, long and short, hydrophilic and hydro-
phobic, and modified sequences. T here is little need for screening columns 1.5
to match a particular sample, or for maintaining an inventory of different XBridge BEH 130 C18
1.3
column chemistries. Peptide Separation Technology columns are compat- 5 μm, 4.6 x 50 mm
40 ugs on column
ible with alternative mobile phases for flexibility in developing purification 1.1

methods. Good peak shapes and separations can be obtained with both TFA 9.0e-1
AU

and short chain organic acids as mobile phase modifiers, and the columns 7.0e-1

can be used with both acetonitrile and alcohols. Good purification and yield
5.0e-1
can be obtained with bio-compatible solvents so that the isolated peptide
3.0e-1
can be used in bio-assays.
1.0e-1

Versatility for Peptide Isolation and Purification 2 3 4 5 6 7 8 9 10 11 12 Min.

0.70 0.38
5.25e-2
0.65 0.36
0.34
0.60 4.75e-2
0.55 Average
0.32
0.30 Basic XBridge BEH 130 ODB Prep C18
0.28
0.50
0.45
pI .4 0.26 pI 10.3 4.25e-2 5 μm, 19 x 100 mm
17 Residues 0.24 15 Residues
0.40
0.35
1772 da
0.22
0.20 1809 da 3.75e-2
3.4 mg injected
0.18
0.30 0.16
0.25 0.14
0.20
0.12 3.25e-2
0.10
0.15 0.08
0.10 0.06 2.75e-2
AU

0.04
0.05
0.02
0 0
2.25e-2
0.70 0.34
0.65 0.32

0.60
Large 0.30 Hydrophobic 1.75e-2
0.28
0.55 pI 4.7 0.26
pI 7.3
0.50 ~ 40 Residues 0.24 16 Residues 1.25e-2
0.45 ~ 4000 da 0.22
1872 da
0.40 0.20

0.35
0.18
0.16
7.5e-3
0.30 0.14
0.25 Peptide 0.12
0.20 0.10 2.5e-3
0.15 0.08
Preservative 0.06
0.10
0.04
0.05 0.02 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Min.
0 0
15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Min.

Peptide Separation Technology columns are compatible with a broad range of peptides. A single column can Waters Peptide Separation Technology columns, available in different particle size
be used to purify the wide range of sample types generated in a peptide synthesis laboratory. There is no and column configurations, are well-suited for various lab-scale purification needs.
need to switch columns for separating peptides of extreme sizes, isoelectric point, or hydrophobicity.

6 Peptide Isolations and Analyses www.waters.com

 PEPTIDE ISOLATIONS AND ANALYSES

Column Lifetime Cation-Exchange Peptide and

Columns fail both physically and c hemically. One major cause of Polypeptide Separations
short column lifetime in low pH mobile phases is hydrolysis of the For most analytical and preparative peptide separations, cation-exchange
bonded phase that leads to significant changes in peptide retention. BEH chromatography is used mainly when alternative selectivity is required. In
Technology columns incorporates proprietary procedures for bonding and some large-scale purifications, cation exchange can take on a more central
endcapping that yield stable bonded phases. In low pH stability test, BEH role. In these cases, cation exchange is frequently used as the first step in
C18 columns showed very little retention loss. T he physical stability of these the separation, followed by a secondary purification step using reversed-
columns is ensured by the OBD design that was developed to produce the phase methods.
most stable packed beds.
Waters offers Protein-Pak™ SP HR packings for cation-exc hange separa-
Long-Term Stability tions. T hese packings are useful both for analytical and preparative work.
T hey are based on rigid, hydrophilic polymethacrylate particles with large
100 1000Å pores. T he naturally hydrophilic polymer reduces non-specific
Peptide Separation
Technology adsorption, resulting in better recovery of peptide/polypeptide mass and
90
B bioactivity. T hese packings are stable in the pH range of 2-12.

C Protein-Pak SP HR 8 and 15 μm packing material is available in prepacked

80 glass columns.
% Initial Retention

70
UPLC for Complementing
Isolation and Purification
60
UPLC tec hnology provides useful information on the composition of a
peptide sample. UPLC analytical methods suggest appropriate conditions
50 for isolation, and analysis with UPLC identifies the fractions to pool. Purity
0 50 100
Hours in 1% TFA in H2O pH 1.0 at 80 °C of the final product can then be confirmed.

Several columns were tested with repeated injections. Retention was monitored to determine column lifetime.

Reduce Cost of Isolation

Peptide purification is expensive but Peptide Separation Tec hnology
columns can reduce the cost per sample. T he same column can be used for
many sample types and mobile phases. Column life is extended with the
stable bonding chemistry and OBD packing. Consistent stability ensures
that the best methods and column dimensions are used for each sample.

www.waters.com Peptide Isolations and Analyses 7

PEPTIDE ISOLATIONS AND ANALYSES

Peptide Separation Technology

Waters Peptide Separation Tec hnology consists of a unique family of
columns that meet the demanding requirements of peptide separations,
from proteomics, to peptide mapping and isolation. Key product charac-
teristics include:
½ O(½@MC½SDLODQ@STQD½SNKDQ@MS½"%(½4DBGMNKNFX½O@QSHBKDR
½ ^½@MC½^½ONQD½RHYDR½ENQ½RL@KK½@MC½K@QFD½ODOSHCDR
½  ½ ½ ½@MC½½¿L½NEEDQHMFR½ENQ
analytical through preparative
½ /UDQ½½(0,# ½50,# ½@MC
Nano LC column choices

Two Pores Sizes for Flexible Method Development Peptide Separation Technology provides:
½ )LOQNUDC½BGQNL@SNFQ@OGX½NE½ODOSHCDR
A 4 residue 507.3
– Narrow, symmetrical peaks for best resolution
B 25 residue 2447.2 ACQUITY BEH130

C 20 residue 1565.8 – Successful separation of a wide range of peptides: large

1.2e-1
C and small, acidic and basic, hydrophobic and hydrophilic
A B
1.0e-1
– Good peak shape and retention in formic acid and
8.0e-2

trifluoroacetic acid for optimal chromatography and detection

AU

6.0e-2

4.0e-2
– Available in 130Å and 300Å pore sizes for varying
2.0e-2

0.0
sample requirements

6.0e-2
C ½ 7HCD½Q@MFD½NE½O@QSHBKD½RHYDR½@MC½BNKTLM½CHLDMRHNMR½ENQ½BNMRHRSDMS½
B
ACQUITY BEH300
5.0e-2
A separations in applications from from very sensitive proteomic analyses
4.0e-2
to. high mass load purification
AU

3.0e-2

2.0e-2
– Chemically stable bonding for extended column life at low pH
1.0e-2

0.0
and high temperatures
20 25 30 35 40 45 50 55 60 65 70 75 80 Min.

– BEH Technology particles stable to pH 12 for peptide

Comparison of the separation of tryptic digest of phosphorylase b on ACQUITY UPLC BEH130 chromatography at high pH
and BEH300 1.7 μm Peptide Separation Technology columns.
½ 5MHPTD½BNKTLM½BGDLHRSQX½BNLAHMDC½VHSG½SGD½ADMDEHSR½NE½RTA ½ÅL½
particles of UPLC Technology for the ultimate resolution

8 Peptide Isolations and Analyses www.waters.com

 PEPTIDE ISOLATIONS AND ANALYSES

BEH Technology
Peptide Separation Tec hnology columns are based on synthetic particles Higher Resolution Peptide Mapping with UPLC
to ensure the highest quality and the most consistent performance. T his
7.5e-2

second generation of Waters patented BEH Technology is a result of Waters 7.0e-2

6.5e-2 HPLC
6.0e-2
Peak Capacity = 372
commitment to continued investment in c hromatography researc h and 5.5e-2
5.0e-2

development. T he columns not only meet the demanding manufacturing

AU
4.5e-2
4.0e-2
3.5e-2

and performance specifications for the BEH Technology particles, but are 3.0e-2
2.5e-2
2.0e-2

also specifically QC-tested with a peptide map. 1.5e-2

1.0e-2

9.0e-2

8.0e-2

7.0e-2 UPLC
6.0e-2
Peak Capacity = 723

AU
5.0e-2

4.0e-2

3.0e-2

2.0e-2

30 35 40 45 50 55 60 65 70 75 80 85 90 Min.

Resolution of complex peptide digests phosphorylase b as shown here, improves when BEH Technology columns
are used. Peptide Separation Technology columns with 1.7 μm particles produce highest resolution
with the expected increase in sensitivity and decrease in length.

Peptide Separation Technology Columns

Peptide Separation Technology provides a consistent set of chromatographic peptide separation methods used in biotechnology development as well as
tools usable across all research and development applications that require predictable behavior with many different samples encountered in proteom-
analysis and isolation of peptides. Peptide Separation Technology columns ics, protein characterization, and peptide synthesis. T he surface chemistry
are based on the C18 BEH Technology particles and are available from sizes of all particle sizes, in either the 130Å or 300Å pore sizes, gives sharp
1.7 μm to 10 μm. T he range of column dimension spans from i.d. of 75 μm symmetrical peaks over a wide range of peptide properties and application
to 30 mm and lengths from 50 to 250 mm. T here are over 125 standard requirements. Peptide Separation Technology columns are integral elements
columns and others can be custom prepared. T here is no need to compro- of Waters Assured Performance Solutions, combination of instrumentation,
mise in matching the column to the application. T hese columns are specifi- chemistry and software optimized for particular applications.
cally QC-tested with a peptide map ensuring the stability of well-developed

Peptide Separation Technology

applications: From discovery
proteomics to isolation of synthetic
peptides; From capillary LC/MS
to HPLC and UPLC

UPLC Peptide Analysis Solution

nanoACQUITY UPLC Columns

Analytical HPLC Columns

NanoEase Columns

MassPREP
Standards
ACQUITY UPLC Columns
nanoACQUITY
UPLC System

OBD Prep Columns

Waters Prep System

www.waters.com Peptide Isolations and Analyses 9

PEPTIDE ISOLATIONS AND ANALYSES

Peptide Nano and Capillary Columns

Peptide Separation Technology nanoACQUITY UPLC® ACQUITY UPLC VanGuard Pre-Column and Column In-Line Filter Unit
Columns (10,000 psi)
Inner Particle Description Part No.
Description Diameter Length Size Part No. BEH300, C18, 1.7 μm, 2.1 x 5 mm, 3/pk 186004629
nanoACQUITY UPLC BEH130 C18 75 μm 100 mm 1.7 μm 186003542 BEH130, C18, 1.7 μm VanGuard Pre-Column, 2.1 x 5 mm, 3/pk 186003975
nanoACQUITY UPLC BEH130 C18 75 μm 150 mm 1.7 μm 186003543 In-line filter holder and six 0.2 μm
205000343
nanoACQUITY UPLC BEH130 C18 75 μm 200 mm 1.7 μm 186003544 stainless steel replacement filters
nanoACQUITY UPLC BEH130 C18 75 μm 250 mm 1.7 μm 186003545 Five 0.2 μm stainless steel replacement filters
700002775
nanoACQUITY UPLC BEH130 C18 100 μm 100 mm 1.7 μm 186003546 and End Nuts for 205000343
nanoACQUITY UPLC BEH130 C18 150 μm 100 mm 1.7 μm 186003550
nanoACQUITY UPLC BEH300 C18 75 μm 100 mm 1.7 μm 186003810
nanoACQUITY UPLC BEH300 C18 100 μm 100 mm 1.7 μm 186003811 Peptide Separation Technology HPLC Columns
nanoACQUITY UPLC BEH300 C18 150 μm 100 mm 1.7 μm 186003812 Inner Particle
nanoACQUITY UPLC BEH300 C18 75 μm 150 mm 1.7 μm 186003813 Description Diameter Length Size Part No.
nanoACQUITY UPLC BEH300 C18 75 μm 200 mm 1.7 μm 186003814
nanoACQUITY UPLC BEH300 C18 75 μm 250 mm 1.7 μm 186003815 XBridge BEH130 C18 1 mm 50 mm 3.5 μm 186003560
XBridge BEH130 C18 1 mm 100 mm 3.5 μm 186003561
XBridge BEH130 C18 1 mm 150 mm 3.5 μm 186003562
Peptide Analytical Columns XBridge BEH130 C18 2.1 mm 50 mm 3.5 μm 186003563
XBridge BEH130 C18 2.1 mm 100 mm 3.5 μm 186003564
XBridge BEH130 C18 2.1 mm 150 mm 3.5 μm 186003565
Peptide Separation Technology NanoEase™ Columns XBridge BEH130 C18 2.1 mm 250 mm 3.5 μm 186003566
Inner Particle XBridge BEH130 C18 4.6 mm 50 mm 3.5 μm 186003567
Description Diameter Length Size Part No. XBridge BEH130 C18 4.6 mm 100 mm 3.5 μm 186003568
XBridge BEH130 C18 4.6 mm 150 mm 3.5 μm 186003569
XBridge BEH130 C18 75 μm 50 mm 3.5 μm 186003588 XBridge BEH130 C18 4.6 mm 250 mm 3.5 μm 186003570
XBridge BEH130 C18 75 μm 100 mm 3.5 μm 186003589 XBridge BEH130 C18 1 mm 50 mm 5 μm 186003571
XBridge BEH130 C18 75 μm 150 mm 3.5 μm 186003590 XBridge BEH130 C18 1 mm 100 mm 5 μm 186003572
XBridge BEH130 C18 100 μm 50 mm 3.5 μm 186003591 XBridge BEH130 C18 1 mm 150 mm 5 μm 186003573
XBridge BEH130 C18 100 μm 100 mm 3.5 μm 186003592 XBridge BEH130 C18 2.1 mm 50 mm 5 μm 186003574
XBridge BEH130 C18 100 μm 150 mm 3.5 μm 186003593 XBridge BEH130 C18 2.1 mm 100 mm 5 μm 186003575
XBridge BEH130 C18 150 μm 50 mm 3.5 μm 186003594 XBridge BEH130 C18 2.1 mm 150 mm 5 μm 186003576
XBridge BEH130 C18 150 μm 100 mm 3.5 μm 186003595 XBridge BEH130 C18 2.1 mm 250 mm 5 μm 186003577
XBridge BEH130 C18 150 μm 150 mm 3.5 μm 186003596 XBridge BEH130 C18 4.6 mm 50 mm 5 μm 186003578
XBridge BEH130 C18 300 μm 50 mm 3.5 μm 186003597 XBridge BEH130 C18 4.6 mm 100 mm 5 μm 186003579
XBridge BEH130 C18 300 μm 100 mm 3.5 μm 186003598 XBridge BEH130 C18 4.6 mm 150 mm 5 μm 186003580
XBridge BEH130 C18 300 μm 150 mm 3.5 μm 186003599 XBridge BEH130 C18 4.6 mm 250 mm 5 μm 186003581
XBridge BEH300 C18 75 μm 50 mm 3.5 μm 186003632 XBridge BEH300 C18 1 mm 50 mm 3.5 μm 186003604
XBridge BEH300 C18 75 μm 100 mm 3.5 μm 186003633 XBridge BEH300 C18 1 mm 100 mm 3.5 μm 186003605
XBridge BEH300 C18 75 μm 150 mm 3.5 μm 186003634 XBridge BEH300 C18 1 mm 150 mm 3.5 μm 186003606
XBridge BEH300 C18 100 μm 50 mm 3.5 μm 186003635 XBridge BEH300 C18 2.1 mm 50 mm 3.5 μm 186003607
XBridge BEH300 C18 100 μm 100 mm 3.5 μm 186003636 XBridge BEH300 C18 2.1 mm 100 mm 3.5 μm 186003608
XBridge BEH300 C18 100 μm 150 mm 3.5 μm 186003637 XBridge BEH300 C18 2.1 mm 150 mm 3.5 μm 186003609
XBridge BEH300 C18 150 μm 50 mm 3.5 μm 186003638 XBridge BEH300 C18 2.1 mm 250 mm 3.5 μm 186003610
XBridge BEH300 C18 150 μm 100 mm 3.5 μm 186003639 XBridge BEH300 C18 4.6 mm 50 mm 3.5 μm 186003611
XBridge BEH300 C18 150 μm 150 mm 3.5 μm 186003640 XBridge BEH300 C18 4.6 mm 100 mm 3.5 μm 186003612
XBridge BEH300 C18 300 μm 50 mm 3.5 μm 186003641 XBridge BEH300 C18 4.6 mm 150 mm 3.5 μm 186003613
XBridge BEH300 C18 300 μm 100 mm 3.5 μm 186003642 XBridge BEH300 C18 4.6 mm 250 mm 3.5 μm 186003614
XBridge BEH300 C18 300 μm 150 mm 3.5 μm 186003643 XBridge BEH300 C18 1 mm 50 mm 5 μm 186003615
XBridge BEH130 C18 300 um 50 mm 5 μm 186003682 XBridge BEH300 C18 1 mm 100 mm 5 μm 186003616
XBridge BEH300 C18 1 mm 150 mm 5 μm 186003617
Peptide Separation Technology ACQUITY UPLC Columns XBridge BEH300 C18 2.1 mm 50 mm 5 μm 186003618
XBridge BEH300 C18 2.1 mm 100 mm 5 μm 186003619
Inner Particle XBridge BEH300 C18 2.1 mm 150 mm 5 μm 186003620
Description Diameter Length Size Part No. XBridge BEH300 C18 2.1 mm 250 mm 5 μm 186003621
XBridge BEH300 C18 4.6 mm 50 mm 5 μm 186003622
ACQUITY UPLC BEH130 C18 2.1 mm 50 mm 1.7 μm 186003554
XBridge BEH300 C18 4.6 mm 100 mm 5 μm 186003623
ACQUITY UPLC BEH130 C18 2.1 mm 100 mm 1.7 μm 186003555
XBridge BEH300 C18 4.6 mm 150 mm 5 μm 186003624
ACQUITY UPLC BEH130 C18 2.1 mm 150 mm 1.7 μm 186003556
XBridge BEH300 C18 4.6 mm 250 mm 5 μm 186003625
ACQUITY UPLC BEH300 C18 2.1 mm 50 mm 1.7 μm 186003685
ACQUITY UPLC BEH300 C18 2.1 mm 100 mm 1.7 μm 186003686
ACQUITY UPLC BEH300 C18 2.1 mm 150 mm 1.7 μm 186003687

10 Peptide Isolations and Analyses www.waters.com

 PEPTIDE ISOLATIONS AND ANALYSES

Peptide Preparative Columns Peptide Separation Technology OBD Columns

Peptide Separation Technology HPLC Columns BEH130 and BEH300 C18 OBD Prep Columns, 5 μm and 10 μm
Inner Particle Configuration
Description Diameter Length Size Style Part No. Inner μmoles of a Weight of a Single Typical Flow Rate
Diameter Length Single Peptide Peptide (mg) (mL/min)
XBridge BEH130 C18 10 mm 50 mm 5 μm Column 186003582
XBridge BEH130 C18 10 mm 100 mm 5 μm Column 186003583 10 mm 50 mm 0.25-5 0.5-10 4.5-9
XBridge BEH130 C18 10 mm 150 mm 5 μm Column 186003584 10 mm 100 mm 0.25-5 0.5-10 4.5-9
XBridge BEH130 C18 10 mm 250 mm 5 μm Column 186003585 10 mm 150 mm 0.25-5 0.5-10 4.5-9
XBridge BEH130 C18 19 mm 50 mm 5 μm OBD Column 186003586 10 mm 250 mm 0.25-5 0.5-10 4.5-9
XBridge BEH130 C18 19 mm 100 mm 5 μm OBD Column 186003587 19 mm 50 mm 1-18 2.0-36 16-32
XBridge BEH130 C18 19 mm 150 mm 5 μm OBD Column 186003945 19 mm 100 mm 1-18 2.0-36 16-32
XBridge BEH130 C18 4.6 mm 50 mm 10 μm Column 186003648 19 mm 150 mm 1-18 2.0-36 16-32
XBridge BEH130 C18 4.6 mm 100 mm 10 μm Column 186003649 19 mm 250 mm 1-18 2.0-36 16-32
XBridge BEH130 C18 4.6 mm 150 mm 10 μm Column 186003650
XBridge BEH130 C18 4.6 mm 250 mm 10 μm Column 186003651 BEH130 and BEH300 C18 OBD Prep Columns, 10 μm
XBridge BEH130 C18 10 mm 50 mm 10 μm Column 186003652
XBridge BEH130 C18 10 mm 100 mm 10 μm Column 186003653
XBridge BEH130 C18 10 mm 150 mm 10 μm Column 186003654 Inner μmoles of a Weight of a Single Typical Flow Rate
XBridge BEH130 C18 10 mm 250 mm 10 μm Column 186003655 Diameter Length Single Peptide Peptide (mg) (mL/min)
XBridge BEH130 C18 19 mm 50 mm 10 μm OBD Column 186003656 30 50 2.5-25 5-100 40-80
XBridge BEH130 C18 19 mm 150 mm 10 μm OBD Column 186003657 30 100 2.5-25 5-100 40-80
XBridge BEH130 C18 19 mm 250 mm 10 μm OBD Column 186003658 30 150 2.5-25 5-100 40-80
XBridge BEH130 C18 30 mm 50 mm 10 μm OBD Column 186003659 30 250 2.5-25 5-100 40-80
XBridge BEH130 C18 30 mm 100 mm 10 μm OBD Column 186003660
XBridge BEH130 C18 30 mm 150 mm 10 μm OBD Column 186003661
XBridge BEH130 C18 30 mm 250 mm 10 μm OBD Column 186003662
XBridge BEH300 C18 10 mm 50 mm 5 μm Column 186003626
XBridge BEH300 C18 10 mm 100 mm 5 μm Column 186003627 Literature
XBridge BEH300 C18 10 mm 150 mm 5 μm Column 186003628 References
XBridge BEH300 C18 10 mm 250 mm 5 μm Column 186003629
XBridge BEH300 C18 19 mm 50 mm 5 μm OBD Column 186003630
XBridge BEH300 C18 19 mm 100 mm 5 μm OBD Column 186003631
Peptide Separation Technology Brochure,
XBridge BEH300 C18 19 mm 150 mm 5 μm OBD Column 186003946 Literature Reference 720001835EN
XBridge BEH300 C18 4.6 mm 50 mm 10 μm Column 186003663
XBridge BEH300 C18 4.6 mm 100 mm 10 μm Column 186003664 UPLC Peptide Analysis Solution Brochure,
Literature Reference 720001836EN
XBridge BEH300 C18 4.6 mm 150 mm 10 μm Column 186003665
XBridge BEH300 C18 4.6 mm 250 mm 10 μm Column 186003666 Optimum Bed Density (OBD) Preparative
XBridge BEH300 C18 10 mm 50 mm 10 μm Column 186003667 Column Technology Brochure,
Literature Reference 720002336EN
XBridge BEH300 C18 10 mm 100 mm 10 μm Column 186003668
XBridge BEH300 C18 10 mm 150 mm 10 μm Column 186003669
XBridge BEH300 C18 10 mm 250 mm 10 μm Column 186003670
XBridge BEH300 C18 19 mm 50 mm 10 μm OBD Column 186003671
XBridge BEH300 C18 19 mm 150 mm 10 μm OBD Column 186003672
XBridge BEH300 C18 19 mm 250 mm 10 μm OBD Column 186003673
XBridge BEH300 C18 30 mm 50 mm 10 μm OBD Column 186003674 Purification and Isolation Cartridge Holders
XBridge BEH300 C18 30 mm 100 mm 10 μm OBD Column 186003675
XBridge BEH300 C18 30 mm 150 mm 10 μm OBD Column 186003676
XBridge BEH300 C18 30 mm 250 mm 10 μm OBD Column 186003677

Peptide Separation Technology Guard Columns

Inner Particle Configuration
Description Diameter Length Size Style Part No.
Description Qty. Part No.
XBridge BEH130 C18 10 mm 10 mm 5 μm Guard Column 186004469
XBridge BEH130 C18 19 mm 10 mm 5 μm Guard Column 186004468 10 x 10 Cartridge Holder 289000779
XBridge BEH130 C18 10 mm 10 mm 10 μm Guard Column 186004465 19 x 10 Cartridge Holder 186000709
XBridge BEH130 C18 19 mm 10 mm 10 μm Guard Column 186004464
XBridge BEH300 C18 10 mm 10 mm 5 μm Guard Column 186004471 Replacement o-ring 7.8 mm 2/pkg 700001019
XBridge BEH300 C18 19 mm 10 mm 5 μm Guard Column 186004470 Replacement o-ring 10 mm 2/pkg 700001436
XBridge BEH300 C18 10 mm 10 mm 10 μm Guard Column 186004467 Replacement o-ring 19 mm 2/pkg 700001020
XBridge BEH300 C18 19 mm 10 mm 10 μm Guard Column 186004466

www.waters.com Peptide Isolations and Analyses 11

PEPTIDE ISOLATIONS AND ANALYSES

Waters Atlantis UPLC and HPLC Columns

for Peptide Separations
Comparative Peptide Separations on Waters BEH and
Silica-Based C18 Column Offerings
Columns: 2.1 x 100 mm
Sample: 30 pmoles MassPREP phosphorylase digestion standard
Detection: ESI+; Waters Q-Tof Premier™
Run Time: 70 min
Temperature: 40 °C
Solvent A: 0.1% Formic Acid in Water
Solvent A: 0.1% Formic Acid in Acetonitrile
Gradient: Time(min) Flow Rate %A %B Curve
Initial 0.200 95.0 5.0
1.00 0.200 95.0 5.0 6
59.00 0.200 50.0 50.0 6
60.00 0.200 30.0 70.0 6
61.00 0.200 95.0 5.0 6

T he Peptide Separation Tec hnology family of reversed-phase columns

ACQUITY UPLC BEH130 C18
was developed to provide good resolution for both the analysis and
lab-scale isolation of peptides. In addition to these offerings, Waters

%
Atlantis ® family of UPLC and HPLC offerings are useful when alterna- 8

tive column c hemistries that exhibit different peptide separation selec- ACQUITY UPLC BEH300 C18

tivities are required for a particular application. %

T he Atlantis T3 series of analytical and preparative columns, available 7

in 3-10 mm particles, are well suited for HPLC-based peptide analysis ACQUITY UPLC HSS T3 C18

and purification needs. Waters ACQUIT Y® UPLC HSS C 18, HSS Shield
%

RP18, HSS C18 SB and HSS T3 columns, when combined with the UPLC 6

Instrumentation provide peptide component resolution and speed of ACQUITY UPLC HSS C18

analysis similar to that obtained with our BEH130 or BEH300, C 18

%

UPLC columns. 6

ACQUITY UPLC HSS C18 SB

%

7
5 10 15 20 25 30 35 40 45 MIn.

Atlantis T3 HPLC and ACQUITY HSS UPLC Analytical Columns

Inner Atlantis Atlantis ACQUITY HSS ACQUITY HSS ACQUITY HSS

Diameter Length Type T3, 3 μm T3, 5 μm T3, 1.8 μm C18, 1.8 μm C18 SB, 1.8 μm
1.0 mm 50 mm Column 186003713 186003730 186003535 186003529 186004114
1.0 mm 100 mm Column — — 186003536 186003530 186004115
1.0 mm 150 mm Column 186003714 186003731 186003537 186003531 186004116
2.1 mm 10 mm Guard 1860037561 1860037591 — — —
2.1 mm 30 mm Column 186003716 186003733 186003944 186003987 186004117
2.1 mm 50 mm Column 186003717 186003734 186003538 186003532 186004118
2.1 mm 100 mm Column 186003718 186003735 186003539 186003533 186004119
2.1 mm 150 mm Column 186003719 186003736 186003540 186003534 186004120
3.0 mm 50 mm Column 186003721 186003738 — — —
3.0 mm 100 mm Column 186003722 186003739 — — —
3.0 mm 150 mm Column 186003723 186003740 — — —
3.0 mm 250 mm Column — 186003741 — — —
3.9 mm 20 mm Guard 1860037572 1860037602 — — —
4.6 mm 20 mm Guard 1860037582 1860037612 — — —
4.6 mm 30 mm Column 186003725 186003743 — — —
4.6 mm 50 mm Column 186003726 186003744 — — —
4.6 mm 75 mm Column 186003727 186003745 — — —
4.6 mm 100 mm Column 186003728 186003746 — — —
4.6 mm 150 mm Column 186003729 186003747 — — —
4.6 mm 250 mm Column — 186003748 — — —
1 Requires Sentry Guard Holder WAT097958
2 Requires Sentry Guard Holder WAT046910

12 Peptide Isolations and Analyses www.waters.com

 PEPTIDE ISOLATIONS AND ANALYSES

Atlantis Prep Columns for Peptides Purification and Isolation Cartridge Holders

Inner Configuration Part No.

Diameter Length Style Particle Size T3
10 mm 10 mm Guard 5 μm 1860036951
10 mm 50 mm Column 5 μm 186003691
10 mm 100 mm Column 5 μm 186003692
10 mm 150 mm Column 5 μm 186003693
10 mm 250 mm Column 5 μm 186003694
19 mm 10 mm Guard 5 μm 1860036992
Description Qty. Part No.
19 mm 50 mm OBD Column 5 μm 186003696
19 mm 100 mm OBD Column 5 μm 186003697 10 x 10 Cartridge Holder 289000779
19 mm 150 mm OBD Column 5 μm 186003698 19 x 10 Cartridge Holder 186000709
19 mm 250 mm OBD Column 5 μm 186004026
30 mm 50 mm OBD Column 5 μm 186003700 Replacement o-ring 7.8 mm 2/pkg 700001019
30 mm 75 mm OBD Column 5 μm 186003701 Replacement o-ring 10 mm 2/pkg 700001436
30 mm 100 mm OBD Column 5 μm 186003702 Replacement o-ring 19 mm 2/pkg 700001020
30 mm 150 mm OBD Column 5 μm 186003703
50 mm 50 mm OBD Column 5 μm 186004080
50 mm 100 mm OBD Column 5 μm 186004081
50 mm 150 mm OBD Column 5 μm 186004082

10 mm 10 mm Guard 10 μm 1860037061
10 mm 150 mm Column 10 μm 186003704
10 mm 250 mm Column 10 μm 186003705
19 mm 10 mm Guard 10 μm 1860037102
19 mm 50 mm OBD Column 10 μm 186003707
19 mm 150 mm OBD Column 10 μm 186003708
19 mm 250 mm OBD Column 10 μm 186003709
30 mm 150 mm OBD Column 10 μm 186003711
30 mm 250 mm OBD Column 10 μm 186003712
50 mm 50 mm OBD Column 10 μm 186004083
50 mm 100 mm OBD Column 10 μm 186004084
50 mm 150 mm OBD Column 10 μm 186004085
50 mm 250 mm OBD Column 10 μm 186004086
1
Requires 10 x 10 mm Prep Guard Holder 289000779
2
Requires 19 x 10 mm Prep Guard Holder 186000709

Prep OBD Columns—Ideal for Purification Labs

Column lifetime of a preparative column is no longer a random event Long, predictable lifetimes
½ $DUDKNODC½HM½ ½7@SDQR½G@R½RGHOODC½NUDQ½ ½OQDO½BNKTLMR½ ½ #TRSNLDQR½G@UD½CNBTLDMSDC½BNMRHRSDMSKX½@BGHDUHMF½NUDQ½ ½HMIDB-
without a single OBD column bed failure due to voiding. No other tions with their Waters Prep OBD columns, increasing productivity and
column manufacturer has been able to match Waters performance. saving money.
T he critical difference
½ 7@SDQR½OQNOQHDS@QX½/"$½4DBGMNKNFX½DMRTQDR½DWBDKKDMS½RB@K@AHKHSX
and equivalent performance across a range of dimensions.
T he right size to get the job done
½ 7@SDQR½NEEDQR½@M½DWSDMRHUD½RDS½NE½0QDO½/"$½BNKTLMR½HMBKTCHMF An Exploded View of the Elements of an Empty OBD Column
19, 30, and 50 mm i.d.

(Actual Prep OBD column lifetime depends on the quality of injected samples and operating conditions throughout the use of the column.
For maximum column lifetime for peptide separations, injection of soluble, particulate-free peptides is required. Please refer to the
OBD Care and Use Instructions or contact your Waters Representative for more information.)

www.waters.com Peptide Isolations and Analyses 13

PEPTIDE ISOLATIONS AND ANALYSES

BioSuite HPLC Peptide Analysis Columns

½ 4VN½(0,#½BNKTLM½BGDLHRSQHDR½SG@S½OQNUHCD½@KSDQM@SHUD Consistent Results Due to Superior

chemistries for peptide separations
Batch-to-Batch Reproducibility
½ $DRHFMDC½ENQ½L@WHLTL½QDRNKTSHNM½NE½BNLOKDW½CHFDRSR
Waters batch release protocol includes a tryptic map of cytochrome c which
½ !U@HK@AKD½HM½U@QHNTR½BNMEHFTQ@SHNMR½ENQ½,#½NQ½,#-3½@OOKHB@SHNMR½ is used to test for reproducibility to retention times and resolution. The
½ %WBDKKDMS½A@SBG SN A@SBG½QDOQNCTBHAHKHSX½ENQ½BNMRHRSDMS½QDRTKSR three test chromatograms below show the results of the protein digest test
for different batches of PA-B material.
½ 5MHPTDKX½1# SDRSDC½RODBHEHB@KKX½ENQ½ODOSHCD½L@OOHMF

Cytochrome c Tryptic Map Q.C. Test

BioSuite Peptide Analysis Series Eluent A: 0.05% TFA in water

Eluent B: 0.01% TFA in MeCN
BioSuite PA Series consists of two of Waters premier reversed-phase column Flow rate: 1.0 ml/min
Gradient: Time Profile
c hemistries specifically optimized for peptide mapping from simple to (min) %A %B
0.0 100 0
complicated digests. 45 72 28
Injection Volume: 20 μL
Temperature: 35 °C
Detection: UV 220 nm

BioSuite C18 3 μm PA-A Batch 112

BioSuite C18 3 μm PA-A is a100Å, difunctional bonded, low ligand density, Batch 113

silica-based column.
Batch 114

½ 3ODBHEHB@KKX½CDRHFMDC½ENQ½DWBDKKDMS½QDSDMSHNM½NE½ONK@Q½ODOSHCDR½
½ )CD@K½BGNHBD½ENQ½,#-3½@OOKHB@SHNMR½TRHMF½ENQLHB½@BHC½&! ½SG@S½
10 15 20 25 30 35 40 45 50 55 Min.
minimizes ion-suppression
½ %WBDKKDMS½ODQENQL@MBD½ENQ½SQ@CHSHNM@K½(0,#½RDO@Q@SHNMR½TRHMF½KNV½
TFA concentrations (e.g., 0.025% TFA)
Inner BioSuite C18 BioSuite C18
Diameter Length 3 μm PA-A 3.5 μm PA-B

BioSuite C18 3.5 μm PA-B 2.1 mm

2.1 mm
50 mm
100 mm
186002425
186002426
186002433
186002434
BioSuite C18 3.5 μm PA-B is a 300Å, high ligand density, monofunctional, 2.1 mm 150 mm 186002427 186002435
silica-based column. 2.1 mm 250 mm 186002428 186002436
4.6 mm 50 mm 186002429 186002437
½ /TSRS@MCHMF½ODQENQL@MBD½VGDM½RDO@Q@SHMF½BNLOKDW½CHFDRSR½BNMS@HM- 4.6 mm 100 mm 186002430 186002438
ing hydrophilic, hydrophobic, and basic peptides 4.6 mm 150 mm 186002431 186002439
4.6 mm 250 mm 186002432 186002440
½ 3TODQHNQ½OD@J½RG@OD½@MC½B@O@BHSX½ENQ½ODOSHCD½RDO@Q@SHNMR½TRHMF½4&!½
containing eluents (e.g., 0.1% TFA)
½ 'NNC½BGNHBD½ENQ½SGD½RDO@Q@SHNM½NE½K@QFDQ½ODOSHCD½EQ@FLDMSR½FDMDQ-
ated by some endoproteases (e.g., Lys-C)

14 Peptide Isolations and Analyses www.waters.com

 PEPTIDE ISOLATIONS AND ANALYSES

BioSuite Cation-Exchange HPLC Columns

BioSuite SP NP, SP-PEEK, and SP cation-exchange chemistries (CXC) consists Separation of Angiotensins on BioSuite SP-PEEK
of the “strong” sulfopropyl ligand bonded to a pH stable (i.e., pH 2–12), Cation-Exchange HPLC Column
methacrylic ester-based polymeric resin. The availability of different pore
and particle size materials provides chromatographers with the flexibility Column: BioSuite SP-PEEK, 4.6 mm ID x 50 mm, PEEK
Eluent: A: 20 mmol/L sodium acetate buffer, pH 5.0
required to isolate and or characterize peptides based upon minor charge B: 20 mmol/L sodium acetate buffer containing
1.0 mol/L NaCl, pH 5.0 [Val5]-II [Asn1, Val5]-II
differences. Non-porous (NP) and porous IEX columns are also available Linear gradient from eluent
[Sar1, IIe6]-II
[Sar1, T hr8]-II Des-Asp1, [IIe8]-II
A to B for 20 minutes
to meet various separations requirements. Speed and superior chromato- Flow rate: 1.0 mL/min.
[Sar1, Val5, Ala8]-II

[Val5]-II
graphic resolution are possible using the non-porous IEX offerings while Temperature: 25°C
Detection: UV @ 280nm
porous BioSuite offerings are available for applications requiring greater III

peptide binding capacity. In addition, BioSuite SP material is offered in II

PEEK™ hardware as well as in 21.5 mm i.d. stainless steel “lab-scale”

preparative column dimensions.
0 5 10 15 20 25 Min.

Exclusion Limit # Approx Protein

(Daltons) against Column Binding Capacity
Pore Polyethylene Inner Volume Per Pre-Packed
Description Matrix Size Glycol Diameter Length (mL) Column Part No.
BioSuite SP-PEEK 7 μm CXC Polymer 1300Å >4,000,000 4.6 mm 50 mm 0.83 58 mg* 186002182
BioSuite SP 2.5 μm NP CXC Polymer n/a 500 4.6 mm 35 mm 0.58 2.9 mg** 186002183
BioSuite SP 10 μm CXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 132 mg** 186002184
BioSuite SP 13 μm CXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 2,178 mg** 186002185
BioSuite Q-PEEK and
SP-PEEK columns are available * = Data generated with Gamma Globulin ** = Data generated with Hemoglobin
in 4.6 x 50 mm # Note: For best resolution of complex samples, do not exceed 20% of the column’s protein binding capacity

Delta-Pak HPLC Columns

Delta-Pak™ packings, ideal for the separation of peptides, proteins, and pore size materials (100Å and 300Å) with a C18 or C4 bonded phase.
natural products, are based on a highly stable, bonded, endcapped For more information and part numbers, visit waters.com/biosep.
5 μm or 15 μm spherical silica. Delta-Pak is available in two different
Synthetic Peptide Separation on Delta-Pak C18 HPLC Column
Physical Characteristics
Column: Delta-Pak C18 5 μm 300Å
Particle Particle Pore Carbon End-
3.9 x 150 mm
Packing Chemistry Size Shape Size Load capped Part Number: WAT011793
Sample: Synthetic peptide-neurotensin (5 mg/mL)
Injection: 10 μL (50 μg)
Delta-Pak C18 5 μm Spherical 100Å 17% Yes Mobile Phase: Solvent A: H2O with 0.1% TFA
C18 5 μm Spherical 300Å 7% Yes Solvent B: CH3CN with 0.1% TFA
Gradient: 0-2 min, 5% B
C4 5 μm Spherical 100Å 7% Yes Conditions: 2-27 min, 5-9% B
30-31 min, 90-5% B
C4 5 μm Spherical 300Å 3% Yes Flow Rate: 1 mL/min
Detection: 230 nm
C18 15 μm Spherical 100Å 17% Yes
C18 15 μm Spherical 300Å 7% Yes
C4 15 μm Spherical 100Å 7% Yes
C4 15 μm Spherical 300Å 3% Yes

Waters Insulin HMW P HPLC Column

T he Waters Insulin HMW P column is specifically designed for use in the
Description Dimensions Part No.
manufacture and quality control of insulin products. T his column is tested
Waters Insulin HMWP Column 7.8 x 300 mm WAT201549
for performance in the analysis of impurities with molecular masses greater
Protein-Pak 125 Sentry Guard Column, 3.9 x 20 mm
than that of insulin. 2/pkg (requires holder) 186000926
Sentry™ Universal Guard Column Holder WAT046910
Tested to perform in the method published in PharmaEuropa Vol. 8, No 3, pages 359-360, September 1996.

www.waters.com Peptide Isolations and Analyses 15

PEPTIDE ISOLATIONS AND ANALYSES

Symmetry HPLC Columns

Waters Symmetry®, reversed-phase, silica-based particles are synthesized columns with minimal non-desired secondary interactions between
using ultrapure organic reagents, resulting in high purity material with very bound ligand and biomolecules.
low silanol activity. When combined with the high surface coverage of the
There are several factors to assess when developing a reversed-phase
bonded phase, outstanding peptide separations and recoveries are possible.
HPLC method for peptides. Batch-to-batch reproducibility of the column
½ 3TODQHNQ½L@MTE@BSTQHMF½BNMSQNK½ENQ½BNMRHRSDMS is an important consideration when developing a validated and trans-
batch-to-batch and column-to-column results ferable method.Symmetry300 columns have outstanding batch-to-batch
and column-to-column reproducibility.
½^½@MC½^½ONQD½RHYD½NEEDQHMFR½ENQ
small or larger size peptides
Pore Size Effects on Peptide Selectivity: Comparative Results
½ 3XLLDSQX3GHDKC™ column chemistry offers complementary on Symmetry 100Å vs. Symmetry300 Columns
selectivity to Symmetry column offerings
Column: A. Symmetry C18, 5 μm,
½ 3XLLDSQX0QDO™ columns provide direct scale up 100Å, 4.6 x 150 mm
Gradient:
Injection:
0-50 min, 0-30% B
20 μL
while maintaining resolution B. Symmetry300 C18, 5 μm,
300Å, 4.6 x 150 mm
Flow Rate: 0.75 mL/min
Temperature: 35 °C
Sample: Tryptic digests of cytochrome c (bovine) Detection: 214 nm
Symmetry300 Columns: T he First Columns Specifically Engineered Mobile Phase: Solvent A: 0.1% TFA in water
Solvent B: 0.1% TFA in acetonitrile
for the Discovery and Development of New Biopharmaceuticals 10

13
As a scientist, you know that tremendous time and resources are required
9 12
when developing HPLC assays for well-characterized biopharmaceuticals. A
4
56
8
23 11
You also know that column-to-column variability is the “Achilles heel” of 1 7

this demanding process.

In the past you’ve had no choice but to deal with variability by finding
costly and time-consuming ways around this problem. The regulatory guide-
10
lines being adopted worldwide as a result of the International Conference 13
5,6
on Harmonization (ICH) and the U.S. Food and Drug Administration’s B
9 12

specified biological products initiatives are placing increasingly stringent 2

4 8
3 11
demands on this process. Full analytical characterization is becoming 1 7

more important for regulatory filing, making column variability an

unacceptable risk. 0 20 40 Min.

Now, regardless of whether you’re developing HPLC assays for purity,

stability, or identity, you can have confidence in the long-term compli- The key to a successful separation is the selection of a column that gives
ance of your methods, batch-to-batch, column-after-column, year-after-year. the highest chemistry resolution with maximum peak capacity and recovery.
That’s because Waters has developed Symmetry300™ columns, specifically
engineered for the discovery and development of new biopharmaceuticals. Symmetry300 Columns
Symmetry300 columns are a 300Å reversed-phase addition to the Inner Particle
existing Symmetry family of columns. They have been specifically Diameter Length Size C18 C4
designed to provide maximum batch-to-batch and column-to-column 1.0 mm 150 mm 3.5 μm 186000185 186000276
performance consistency and recovery of protein and peptide applica- 2.1 mm 50 mm 3.5 μm 186000187 186000277
tions. Symmetry300 columns are offered in two particle sizes (3.5 m 2.1 mm 100 mm 3.5 μm 186000188 186000278
and 5 m) and in two chemistries (C4 for large peptides and proteins 2.1 mm 150 mm 3.5 μm 186000200 186000279
and C18 for smaller peptides) to address various needs. 4.6 mm 50 mm 3.5 μm 186000201 186000280
4.6 mm 75 mm 3.5 μm 186000189 186000281
High Recoveries of Peptides and Proteins 4.6 mm 100 mm 3.5 μm 186000190 186000282
4.6 mm 150 mm 3.5 μm 186000197 186000283
The heart of the column is high purity-based deactivated silica. Waters 2.1 mm 150 mm 5 μm WAT106172 186000285
dedicated chromatography chemistry manufacturing plant operates 3.9 mm 150 mm 5 μm WAT106154 186000286
under the stringent standards of cGMP supplemented with FDA registra- 4.6 mm 50 mm 5 μm WAT106209 186000287
tion for Class 1 medical devices and ISO 9002. The silica used in the 4.6 mm 150 mm 5 μm WAT106157 186000288
4.6 mm 250 mm 5 μm WAT106151 186000289
manufacture of our Symmetry300 columns is synthesized using ultra- 19 mm 10 mm 5 μm 186001847 —
pure organic reagents that yields high purity particles with very low 19 mm 50 mm 5 μm 186001848 —
silanol activity. These particles when combined with innovative ligand 19 mm 100 mm 5 μm 186001849 —
(i.e., C4 and C18) bonding techniques helps produce reversed-phase
For more complete listings, go to www.waters.com

16 Peptide Isolations and Analyses www.waters.com

X ? F L J L I B @ R I B P Z

Waters Hybrid Particle Technology has helped chromatographers better separate and analyze M O L Q BFKP
amino acids, peptides, and oligonucleotides. Now see how Waters new BEH300 C4 UPLC® and M B M Q FAB P
HPLC columns can improve your protein characterizations. > JFKL>@ FAP

Learn more at www.waters.com/proteins LI FDLKR@ I BL Q FAB P

© 2009 Waters Corporation. Waters, UPLC and The Science of What’s

Possible are trademarks of Waters Corporation.
PROTEIN ISOLATIONS AND ANALYSES

Protein Isolation and Characterization

Selecting the Separation Mode Hydrophobic-Interaction Chromatography
T here are five principals of chromatography used for protein purification; In this mode, proteins are bound to a moderately hydrophobic surface
ion exc hange, size exclusion, reversed phase, affinity, and hydrophobic at high ionic strength. T he protein’s solubility is lowered under these
interaction. conditions. T he protein is then eluted with a gradient of decreasing ionic
strength. Hydrophobic-interaction chromatography often has good resolu-
A protein purification protocol will always include several steps. T he
tion and retains biological activity. Aqueous buffer systems are used in
tec hniques should be used in a sequence that reflects different selectiv-
hydrophobic interaction. Many different factors affect resolution, includ-
ity mec hanisms. T he order of steps should be c hosen to move from high
ing buffer salt concentration and type, buffer pH, surfactant, modifiers,
capacity and high speed to higher resolution.
column temperature, and packing material functionality. Since proteins
Ion-Exchange Chromatography may also interact with one another under conditions of high ionic strength,
better resolution is achieved when the starting material is already partially
In ion-exc hange separations, the distribution and net c harge on the
purified. It is also inconvenient to load large volumes of sample at high
protein’s surface determines the interaction of the protein with the
ionic strength. T his would suggest that hydrophobic interaction would
c harged groups on the packing material’s surface. T he c harges on the
normally be a good second or subsequent step in an isolation scheme, such
protein and the packing material must be opposite for interaction to
as when an ammonium sulfate precipitation occurs prior to the chromato-
occur. T he variables that can be manipulated, suc h as buffer pH, buffer
graphic protocol.
composition, gradient slope, and gradient forming salt, offer a wide
range of options for optimizing a separation. Particle Considerations for Lab-Scale Purifications
Size-Exclusion Chromatography High resolution traditional HPLC packings for protein c hromatography
offer familiar c hemical functionalities bonded onto rigid, spherical
T he apparent size and shape of proteins in solution is the basis for
polymeric or silica supports. T hese packings provide improved c hemi-
separation in size-exclusion c hromatography. T his simple separa-
cal and physical stability, better reproducibility and dramatically faster
tion mec hanism relies on protein size differences, packing material,
separations than traditional soft gels. Columns containing lab-scale
pore size, and particle size as well as the operating conditions, suc h
preparative packings (8, 15, or 40 μm) produce more concentrated
as sample concentration and volume for the successful resolution of
fractions. T he hydrophilic polymer with familiar functional groups result
protein mixtures. Size-exclusion packing materials have evolved from
in high recovery of biological activity.
the large particle polysaccharide soft gels to small, rigid packings that
offer enhanced resolution and significantly faster separations. Pore Size Considerations
Size exclusion has a wide range of applicability in protein isolations. It is Good peak shape and recovery for proteins had been observed on the
often used to assay fractions, as run times are relatively short, and the 100-130Å pore size materials; however, the bias is toward the 300Å
technique relatively straightforward. It can also be used for buffer exchange, packings for protein samples. T he best advice is to test both 130Å and
as the low molecular weight salt molecules are easily separated from the larger pore sizes (>300Å) as a step in the development of an optimized
much larger proteins. protein separation. Purification and analysis media is available in pore
sizes ranging from 130Å to 4000Å.
Reversed-Phase Chromatography
Reversed phase is an alternative hydrophobic-separation technique that Sample Preparation
relies on the interaction between the protein’s non-polar amino acid side Protein samples derived from tissue homogenates, cell lysis, or physiologic
chains with the packing surface and ligands. T his mechanism provides fluids frequently require removal of particulate contaminants prior to LC,
another high resolution dimension for protein chromatography that can MS, or LC/MS analysis. In some situations, use of an on-line protein desalt-
yield good recovery of biological activity with lower molecular weight ing device (e.g., MassPREP on-line desalting cartridge) can effectively be
proteins and peptides. It is an excellent tool for assessing fraction purity used to remove ion-suppressing compounds prior to mass determination by
and for preparing proteins for sequencing or structural analysis. LC/MS techniques. Other situations exist where simple filtration or centrifu-
gation can be effectively used to remove insoluble components prior to
Affinity Chromatography analysis. Glycoproteins offer additional challenges that can be addressed
Affinity is the only chromatographic mode in which proteins are isolated via the use of specialty reagents such as Waters RapiGest SF surfactant
on the basis of their biospecific interaction with an immobilized ligand. that has been shown to significantly enhance protein deglycosylation. In
Proteins can be recovered with retention of bioactivity and high purity. addition, solid-phase extraction techniques, such as those offered in Waters
Modern affinity packings take advantage of rigid base materials that MassPREP Deglycosylation Kit and Plate, can effectively isolate and concen-
combine enhanced bed stability for crude sample processing with high trate glycans, cleaved from gylcoproteins, prior to analysis. Accell™ Plus
throughput for rapid microgram to gram scale affinity purifications. Many ion-exc hanger cartridges are also available for solid-phase extraction of
separations can be achieved in a single step resulting in more cost- proteins from complex matrices or for isolation or sample concentration.
effective separations.

18 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

Strengths of Different Modes of Chromatography

Ion Exchange Hydrophobic Interaction Affinity Reversed Phase Size Exclusion

Concentrates sample Concentrates sample Concentrates sample Concentrates sample Rapid

Column can be cleaned Column can be cleaned Superb selectivity Good resolution for Good recovery of activity
using NaOH using NaOH proteins and peptides
Outstanding resolution Reliable medium to high
Moderate to high resolution Moderate resolution Simple technique resolution technique
High capacity
High capacity High capacity Moderate capacity Can be used to buffer exchange
Good recovery of activity
Good recovery of activity Good recovery of activity Compatible with detergents
Significant purification
Widespread applicability in a single step

Limitations of Different Modes of Chromatography

Ion Exchange Hydrophobic Interaction Affinity Reversed Phase Size Exclusion

Fractions may need to be Fractions may need to be Ligand leakage Limited applicability with Moderate resolution
desalted prior to the next desalted prior to the next high MW proteins
purification step purification step Column cannot be cleaned Non concentrating
using aggressive chemicals Column cannot be cleaned
using aggressive chemicals Moderate capacity
Stationary phases can
be expensive Organic mobile phase
may denature protein

Planning a Protein Isolation Protocol Typical Isolation Protocol

T he design of an overall c hromatography protocol depends on many Taking all these facts into consideration, a typical purification protocol
factors, such as the sample volume, the cleanliness of the starting material, would start with an ion-exchange step, as the resolving power is good where
whether economic affinity supports are available, and what the final use of a dilute feed stream can be concentrated prior to elution and the packing
the purified protein might be. materials can be cleaned using aggressive agents. As the protein is usually
eluted using an increasing salt gradient, the active fractions often contain
For instance, many of the initial feed streams for protein isolations are
high concentrations of salt. It is sometimes necessary to desalt the fractions
relatively dilute protein solutions. Chromatographic modes that concentrate
prior to the next step. T his can be avoided if the next step is hydrophobic
samples (ion exchange, hydrophobic interaction, and affinity) are good start-
interaction, where the protein is loaded onto the column in a concentrated
ing points for these solutions.
salt solution. In some instances, especially where the feedstream comes
For samples such as cell lysates and homogenates or physiologic fluids, from an ammonium sulfate precipitation, hydrophobic interaction may be
there are additional considerations. T hese mixtures contain cell fragments, a good first purification step, followed by ion exchange.
lipids, and nucleic acid fragments, which will bind strongly to a column.
T he eluate from this ion-exc hange and/or hydrophobic-interaction step
Columns becoming contaminated in this way are typically cleaned using
is usually free of lipids and other contaminants that may cause problems
0.1 M - 1.0 M NaOH prior to re-equilibration with the starting buffers.
with an affinity column. It can now be put onto an affinity column
T his would destroy a peptide or protein ligand on an affinity column
should one be available. T he use of an affinity column is possible, if
and would irreversibly damage a silica-based, reversed-phase column.
available, once lipids and other contaminants have been removed by
T herefore, ion exchange or hydrophobic interaction would be appropriate
the ion-exc hange and/or hydrophobic-interaction step. Alternatively,
modes for a first purification step.
reversed-phase c hromatography or a second ion-exc hange step under
In reversed-phase chromatography, the sample is loaded onto the column different conditions or with higher resolving power (smaller particle size
in an aqueous environment and then eluted using an increasing gradient of or longer column) can be used. Both of these approac hes result in high
an organic solvent, such as acetonitrile or propanol. T he major drawback to resolution and sample concentration.
this mode of chromatography is that some proteins will denature (causing
T he final step in the purification is often size exclusion, which will separate
loss of biological activity) in the presence of the organic solvents at the
out dimers and polymers of the protein of interest, and potentially desalt
concentration required to elute the protein. As a general rule, the likelihood
the protein.
of denaturation increases with increasing molecular weight of the protein.

www.waters.com Protein Isolations and Analyses 19

PROTEIN ISOLATIONS AND ANALYSES

Protein Separation Technology

T he analysis and characterization of protein samples requires the detec-
tion of small c hemical differences between large molecules. Most often
these analyses have employed an array of analytical tec hniques, eac h
sensitive to a different property of the protein. Reversed-phase HPLC has
not been fully exploited in these tests because the separation of proteins
often yields relatively broad and asymmetrical peaks with poor recovery
and significant carryover. Waters new reversed-phase, ethylene-bridged
hybrid (BEH Tec hnology) Protein Separation Tec hnology columns are
specifically designed for the high resolution analysis of proteins.
Waters New Family of BEH300 C4 columns for protein separations:
½3DO@Q@SDR½OQNSDHMR½NE½U@QHNTR½RHYDR
hydrophobicities, and isoelectric points
½!U@HK@AKD½@R½½¿L½O@BJHMF½ENQ½(0,#½@MC
1.7 μm packing for UPLC methods
½-@WHLHYDR½QDBNUDQX½@MC½LHMHLHYDR½OQNSDHM½B@QQXNUDQ
½4NKDQ@SDR½DWSQDLD½O(½@MC½SDLODQ@STQD
½1T@KHSX BNMSQNK½SDRSDC½VHSG
protein mixture
½#NTOKDR½CHQDBSKX½SN
ESI-MS for protein
identification

300Å C4 Columns Developed for Protein Chromatography Batch-to-Batch Reproducibility

Column: XBridge™ BEH300 C4, 3.5 μm, Sample: Column: ACQUITY UPLC BEH300 C4, 1.7 μm, Sample:
2.1 x 50 mm Protein mixture prepared in 2.1 mm x 50 mm Protein mixture prepared in
Mobile Phase A: 0.1% TFA in water 0.1% TFA in 5% acetonitrile Eluent A: 0.1% TFA in 100% HPLC grade water 0.1% TFA in 5% acetonitrile
Mobile Phase B: 0.075% TFA in 71.4% acetonitrile 1. Ribonuclease A (0.04 mg/mL) Eluent B: 0.075% TFA in 71.4% acetonitrile 1. Ribonuclease
Flow Rate: 0.2 mL/min 2. Cytochrome c (0.06 mg/mL) Flow Rate: 0.2 mL/min 2. Cytochrome c
Gradient: 28-100% B in 25 min 3. Bovine Serum Albumin (0.20 mg/mL) Gradient: 28-100% B in 25 min 3. BSA
Column Temp.: 40 °C 4. Myoglobin (0.13 mg/mL) Column Temp.: 40 °C 4. β-Lactoglobulin
Injection: 5 μL 5. Enolase (0.22 mg/mL) Detection λ: 220 nm 5. Enolase
Detection λ: 220 nm 6. Phosphorylase b (0.59 mg/mL) 6. Phosphorylase b

4 4
0.15

5 6
2
1 3

2
0.10 Batch A
3
6
AU

0.05
Batch B

0.00 Batch C

6 12 18 min 0 2 4 6 8 10 12 14 16 18 20 22 Min.

BEH300 C4 columns can be used with proteins that have a wide range of properties. This protein mix was This comparison shows the consistent batch-to-batch performance with a protein separation.
chosen to represent a range of isoelectric points, molecular weights, and hydrophobicities.

20 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

Minimal Protein Carryover

Ovalbumin Absence of detectable Ovalbumin carryover

0.05
Sample: 1.3 μg Ovalbumin injected
Columns: ACQUITY UPLC BEH300 C4, 1.7 μm,
2.1 mm x 50 mm
Eluent A: 0.1% TFA in 100% HPLC grade water
Eluent B: 0.075% TFA in 71.4% acetonitrile
AU

Flow Rate: 0.2 mL/min

Gradient: 28-100% B in 25 min
Hold at 100 %B for 2 min then
re-equilibrate column at 28% B for 18 min
Gradient restarted at 45 min and
0.00 at 90 min
Column Temp.: 40 °C
0 10 20 30 40 50 60 70 80 90 100 110 120 130 min Detection λ: 220 nm

1st gradient without 2nd gradient without

sample injection sample injection
Column carryover was tested by running multiple gradients following a single injection. Protein peaks observed during the first gradient are not found in subsequent gradients.

LC/UV Analysis of Reduced Monoclonal Antibody Ordering information for Waters BEH300 C4 offerings for traditional HPLC
and advanced UPLC protein separations are shown below.
Sample: Reduced monoclonal antibody Flow Rate: 0.2 ml/min
(IgG1), 0.5 μg/μl Gradient: 20-50% B in 20 min
Column: ACQUITY UPLC BEH 300 C4, 2.1 x 50 mm, Column Temp: 80 °C
1.7 μm (top), 3.5 μm (bottom) Injection: 5 μl
Mobile Phase A: 0.03%TFA in Water Detection: 215 nm UPLC Columns Particle Size Dimensions Part No.
Mobile Phase A: 0.03%TFA in ACN

ACQUITY UPLC BEH300 C4 1.7 μm 2.1 x 50 mm 186004495

LC HC ACQUITY UPLC BEH300 C4 1.7 μm 2.1 x 100 mm 186004496
ACQUITY UPLC BEH300 C4 1.7 μm 2.1 x 150 mm 186004497
0.3

BEH300 C4,
0.2
1.7 μm, 2.1 x 50 mm ACQUITY UPLC BEH300 C4 1.7 μm VanGuard™ Pre-Column 186004623
AU

0.1

14.40 15.20 16.00 16.80 Min.

Note: ACQUIT Y UPLC BEH300 C4, 1.7 μm columns are designed for
10 12 14 16 18 20 Min.
use with the ACQUITY UPLC system. T he benefits of the small particle
packing in ACQUITY UPLC BEH300 C4, 1.7 μm columns are only realized
0.3 HC
LC with the low system volume and low detector dispersion of an ACQUITY
UPLC system.
AU

0.2
BEH300 C4,
3.5 μm, 2.1 x 50 mm
0.1

nanoACQUITY UPLC
10 12 14 16 18 20 Min.
Columns (10,000 psi) Particle Size Dimensions Part No.
nanoACQUITY UPLC BEH300 C4 1.7 m 75 m x 100 mm 186004639
BEH300 C4 Columns for Protein Characterization with UPLC / MS nanoACQUITY UPLC BEH300 C4 1.7 m 100 m x 100 mm 186004640
nanoACQUITY UPLC BEH300 C4 1.7 m 150 m x 100 mm 186004641
Sample: Lys-C digested (limited) Waters LCT Premier™ XE MS Detection
reduced monoclonal antibody Capillary Voltage: 3200 V
Column: ACQUITY UPLC BEH300 C4, Cone Voltage: 45 V
For use with nanoACQUITY UPLC systems rated to 10,000 psi only.
1.7 m, 2.1 x 50 mm Source Temp.: 150 °C Not for use with nanoACQUITY UPLC systems rated to 5,000 psi.
Eluent A: 0.1% FA in 100% HPLC grade water Desolvation Temp.: 350 °C
Eluent B: 0.1% FA in 100% acetonitrile Desolvation Gas: 000 L/h
Flow Rate: 0.2 mL/min
Gradient: 5% B for 5 min Fd
Then 5-21% B in 0.1 min.
LC
HPLC Columns Particle Size Dimensions Part No.
Then 21-29% B in 30 min.
Column Temp.: 80 °C XBridge BEH300 C4 3.5 μm 2.1 x 50 mm 186004498
XBridge BEH300 C4 3.5 μm 2.1 x 100 mm 186004499
Fc/2
XBridge BEH300 C4 3.5 μm 2.1 x 150 mm 186004500
XBridge BEH300 C4 3.5 μm 2.1 x 250 mm 186004501
XBridge BEH300 C4 3.5 μm 4.6 x 50 mm 186004502
%

XBridge BEH300 C4 3.5 μm 4.6 x 100 mm 186004503

XBridge BEH300 C4 3.5 μm 4.6 x 150 mm 186004504
Fc/2 XBridge BEH300 C4 3.5 μm 4.6 x 250 mm 186004505
Clip 2
Fc/2 Custom BEH300 C4 186004506
Clip 1

9
8 10 12 14 16 18 20 22 24 26 28 30 32 34 Min

T he large fragments obtained through LysC digestion of a monoclonal antibody can be separated on the UPLC
BEH300 C4 column coupled directly to ESI/Tof MS for identification of the individual peptide products.

www.waters.com Protein Isolations and Analyses 21

PROTEIN ISOLATIONS AND ANALYSES

BioSuite HPLC Columns for Protein Separations

½ )NM DWBG@MFD ½RHYD DWBKTRHNM ½GXCQNOGNAHB HMSDQ@BSHNM

and reversed-phase column offerings
½ %WBDKKDMS½QDRNKTSHNM½@MC½QDBNUDQX½NE½OQNSDHMR
and peptides
½ !U@HK@AKD½HM½CHEEDQDMS½O@QSHBKD½@MC½ONQD½RHYDR
½ 3B@K@AKD½EQNL½@M@KXSHB@K½SN½šK@A RB@KD›
preparative applications

Waters BioSuite HPLC columns for protein and peptide separations contain Predictable Ion-Exchange Chromatography on BioSuite Analytical
new high-performance chemistries dedicated to the isolation, analysis, and and “Lab-Scale” Preparative DEAE AXC Columns
characterization of biomolecules. Separation offerings include ion-exchange,
size-exclusion, hydrophobic-interaction, and reversed-phase columns and A) BioSuite DEAE 10 μm, B) BioSuite DEAE 13 μm,
support Waters array of LC and LC/MS systems. 7.5 mm i.d. x 75 mm 21.5 mm i.d. x 150 mm

1
2
2

15 30 Min. 30 60 Min.

Sample: Mixture of Ovalbumin (peak 1) and Trypsin Inhibitor (peak 2)

A: 0.2 mg each in 0.1 mL Eluent A
B: 2 mg each in 1 mL Eluent A
Eluent A: 20 mM Tris-HCl, pH 8.0
Eluent B: 20 mM Tris-HCl, pH 8.0 containing 0.5 M NaCl
Flow Rate: A: 1.0 mL/min
B: 6.0 mL/min
Gradient: A: Linear gradient from 0 to 100%B in 60 min.
B: Linear gradient from 0 to 100%B in 120 min.
Temperature: 25 °C
Detection: 280 nm

Waters Alliance ® Bioseparations System

T he latest information on the BioSuite family of application-driven solutions

that address separations needs in the areas of well characterized biophar-
maceuticals, genomics, proteomics, and well characterized biotherapeutics
are available online at www.waters.com/biosep.

22 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

BioSuite Ion-Exchange HPLC Columns

BioSuite ion-exchange column offerings include strong and weak, cation- available. Speed and superior chromatographic resolution are possible
(CXC) and anion exchangers (AXC) bonded to a pH stable (i.e., pH 2-12), using the NP IEX offerings. Waters porous ion exchangers are available
methacrylic ester-based polymeric resin. T he availability of four separa- for applications requiring greater protein or peptide binding capacity.
tion chemistries provides chromatographers with the flexibility required In addition, selected BioSuite ion-exchange columns are offered in PEEK
to develop methods that separate proteins or peptides based upon minor hardware as well as in 21.5 mm i.d. preparative column sizes.
c harge differences. Non-porous (NP) and porous IEX columns are also

Protein Selectivity Differences on BioSuite CM (Weak Cation- Enhanced Compound Resolution by Ion-Exchange Chromatography
Exchange) vs. SP (Strong Cation-Exchange) Columns on BioSuite SP Non-Porous (NP) vs. Porous CXC Columns

3 4
A) BioSuite CM, A) BioSuite SP, B) BioSuite SP,
10 μm CXC 5 2.5 μm NP CXC 10 μm CXC
1 2

4 1

2 2

B) BioSuite SP, 3
10 μm CXC
5
1 2 4 4

0 10 20 30 40 Min. 3 4 5 6 Min. 20 30 40 Min.

Sample: Trypsinogen (Peak 1), Ribonuclease (Peak 2), Sample: Alpha-Chymotrypsinogen A (Peak 1) and Ribonuclease (Peak 2)
Alpha-Chymotrypsinogen A (Peak 3), Columns: A: BioSuite SP, 2.5 μm NP CXC (4.6 x 35 mm)
Cytochrome c (Peak 4), and Lysozyme (Peak 5) B: BioSuite SP, 10 μm CXC (7.5 x 75 mm)
Columns: A: BioSuite CM, 10 μm CXC (7.5 x 75 mm) Eluent: A: 20 mM MES [2[N-morpholino]ethanesulfonic acid], pH 6.0
B: BioSuite SP, 10 μm CXC (7.5 x 75 mm) Eluent B: 20 mM MES, pH 6.0 containing 0.5 M NaCl
Eluent: A: 20 mM Sodium Phosphate, pH 7.0 Flow Rate: A: 1.5 mL/min
Eluent B: 20 mM Sodium Phosphate, pH 7.0 containing 0.5 M NaCl B: 1.0 mL/min
Flow Rate: 1.0 mL/min Gradient: A: Linear gradient from 0 to 100% B in 10 min.
Gradient: Linear gradient from 0 to 100% B in 60 min. B: Linear gradient from 0 to 100% B in 60 min.
Temperature: 25 °C Temperature: 25 °C
Detection: 280 nm Detection: 280 nm

Exclusion Limit # Approx Protein

(Daltons) against Column Binding Capacity
Pore Polyethylene Inner Volume Per Pre-Packed
Description Matrix Size Glycol Diameter Length (mL) Column Part No.
BioSuite Q-PEEK 10 μm AXC Polymer 4000Å >5,000,000 4.6 mm 50 mm 0.83 58 mg* 186002176
BioSuite SP-PEEK 7 μm CXC Polymer 1300Å >4,000,000 4.6 mm 50 mm 0.83 58 mg** 186002182

BioSuite DEAE 2.5 μm NP AXC Polymer n/a 500 4.6 mm 35 mm 0.58 2.9 mg* 186002179
BioSuite SP 2.5 μm NP CXC Polymer n/a 500 4.6 mm 35 mm 0.58 2.9 mg*** 186002183
BioSuite Q-PEEK and
SP-PEEK columns are available
BioSuite Q 10 μm AXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 331 mg* 186002177
in 4.6 x 50 mm
BioSuite Q 13 μm AXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 5,445 mg* 186002178
BioSuite DEAE 10 μm AXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 99 mg* 186002180
BioSuite DEAE 13 μm AXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 1633 mg* 186002181

BioSuite SP 10 μm CXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 132 mg*** 186002184
BioSuite SP 13 μm CXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 2,178 mg*** 186002185
BioSuite CM 10 μm CXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 149 mg*** 186002186
BioSuite CM 13 μm CXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 2,450 mg*** 186002187

* Data generated with BSA ** Data generated with Gamma Globulin *** Data generated with Hemoglobin
# Note: For best resolution of complex samples, do not exceed 20% of the column’s protein binding capacity

www.waters.com Protein Isolations and Analyses 23

PROTEIN ISOLATIONS AND ANALYSES

Protein-Pak HR Ion-Exchange Glass Columns

T he Protein-Pak HR 8 μm and 15 μm packing materials are available solutions, such as 0.1-1.0 M sodium hydroxide and acetic solutions, such as
prepacked in Waters Advanced Purification (AP) glass columns in a choice of 20% acetic acid, for cleaning purposes.
5 mm i.d. (minicolumn) or 10 mm i.d. by 100 mm in length. T he 5 mm i.d.
Protein-Pak HR ion exc hangers are available with a Q functional group,
column is also available in a 50 mm length. T hese columns are compatible
a strong anion exc hanger; DEAE, a weak anion exc hanger; SP a strong
with any HPLC and FPLC system with the use of an adapter kit.
cation exchanger; and CM, a weak cation exchanger. T he principle differ-
Waters Protein-Pak HR packing materials are based on rigid, hydrophilic, ence between a weak and strong ion exchanger does not lie in the protein
polymethacrylate particles with large 1000Å pores. T he naturally hydro- binding capacity, but in the pH range of operation. Weak ion exchangers
philic polymer reduces non-specific adsorption, resulting in quantitative tend to have a more restricted useful pH range of operation.
recovery of protein mass and bioactivity. T hese packings are compatible
with buffers in the pH range 2-12, and will withstand exposure to caustic

Ion-Exchange Particle Pore Protein Resolution on Protein-Pak DEAE 15HR

Packing Type Size Size Dimensions Part No. Anion-Exchange Column
Protein-Pak Polymeric strong 8 μm 1000Å 5 x 50 mm WAT039575
Q 8HR anion exchanger 5 x 100 mm WAT039630 Column: AP-1 Glass Column,
10 x 100 mm 2
10 x 100 mm WAT035980 Buffer A: 20 mM Tris-HCl pH 8.2
Protein-Pak Polymeric strong 15 μm 1000Å 5 x 50 mm WAT039782 Buffer B: Buffer A with 1M Sodium Chloride 3

Q 15HR anion exchanger 10 x 100 mm WAT037663 Flow Rate: 1.56 mL/min

Gradient: 0 to 25% Buffer B over 38 min
Protein-Pak Polymeric weak 8 μm 1000Å 5 x 50 mm WAT039791 Load: 0.5 mg protein
5
DEAE 8HR anion exchanger 5 x 100 mm WAT039783 Detection: 280 nm
1 4
1. Adenosine
10 x 100 mm WAT035650 2. Carbonic anhydrase
Protein-Pak Polymeric weak 15 μm 1000Å 5 x 50 mm WAT039780 3. Human transferrin
4. Ovalbumin
DEAE 15HR anion exchanger 5 x 100 mm WAT039786 5. Soybean trypsin inhibitor
10 x 100 mm WAT038564 40 Min.

Protein-Pak Polymeric strong 8 μm 1000Å 5 x 50 mm WAT039570

SP 8HR cation exchanger 5 x 100 mm WAT039625
10 x 100 mm WAT035655
Protein-Pak
SP 15HR
Polymeric strong
cation exchanger
15 μm 1000Å 10 x 100 mm WAT038567 Protein-Pak PW Series Columns
Protein-Pak Polymeric weak 8 μm 1000Å 5 x 50 mm WAT039790
Waters also offers a line of 10 μm polymer-based ion-exchangers prepacked
CM 8HR cation exchanger 5 x 100 mm WAT039785
10 x 100 mm WAT035970 in steel or glass columns. The Protein-Pak™ 5PW columns are available as
Protein-Pak Polymeric weak 15 μm 1000Å 5 x 50 mm WAT039787 DEAE and SP ion-exchangers. These columns can be used on HPLC and FPLC
CM 15HR cation exchanger systems in both analytical and preparative configurations.
Note: Additional custom column dimensions are available.
Description Dimensions Part No.
Polymeric Weak Anion-Exchanger 7.5 x 75 mm WAT088044
Approximate Protein Binding Capacity per Prepacked Column* Protein-Pak™ DEAE 5PW Glass Column 8 x 75 mm WAT011783
Protein-Pak™ DEAE 5PW Steel Column 21.5 x 150 mm WAT010640
Protein-Pak HR Packing Polymeric Strong Cation-Exchanger 7.5 x 75 mm WAT088043
Prepacked Column Q** DEAE** SP*** CM*** Protein-Pak™ SP 5PW Glass Column 8 x 75 mm WAT011784
5 x 50 mm 60 mg 40 mg 40 mg 25 mg
5 x 100 mm 130 mg 150 mg 80 mg 45 mg
10 x 100 mm 500 mg 300 mg 300 mg 180 mg

Properties of Protein-Pak HR* Columns

Protein-Pak Protein-Pak Q HR ** Protein-Pak DEAE HR ** Protein-Pak CM HR *** SP HR ****

Type of material Polymer Polymer Polymer Polymer * For best resolution do not exceed 20% of the protein
binding capacity.
Protein binding capacity 60 mg/mL 40 mg/mL 25 mg/mL 40 mg/mL ** Bovine serum albumin in 20mM Tris/Cl pH 8.2
Ion-exchange capacity 200 μeq/mL 250 μeq/mL 175 μeq/mL 225 μeq/mL was used to measure protein binding capacity of
Protein-Pak Q and DEAE HR.
Nominal pK 11.7 9.0 5.7 2.2 *** Cytochrome c in 25mM MES pH 5.0 was used to
measure protein binding capacity of Protein-Pak SP
Typical protein recovery >95% >95% >95% >95% and CM HR.
Typical recovery of biological activity >90% >90% >90% >90% **** Same conditions as CM. Protein binding capacity
of Protein-Pak SP 40 HR is 20 mg/mL.
pH stability 2-12 2-12 2-12 2-12

24 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

Advanced Purification (AP) Glass Columns

Bed Volume Flow Rate Pressure Rating
Dimensions (mL) (mL/min) (psi/MPa) Part No.
5 x 50 mm 0.8-1.2 0-4 1500 psi/10 MPa WAT064-01
5 x 100 mm 1.8-2.2 0-4 1500 psi/10 MPa WAT064-02
10 x 100 mm 5-8 0-4 1500 psi/10 MPa WAT021901
10 x 200 mm 13-16 0-4 1500 psi/10 MPa WAT021902
10 x 300 mm 21-24 0-4 1500 psi/10 MPa WAT021903
10 x 600 mm 45-48 0-4 1500 psi/10 MPa WAT021906
Waters Advanced Purification (AP) series of glass columns are constructed 20 x 100 mm 22-31 4-16 1000 psi/6.8 MPa WAT027501
of biocompatible glass and polymeric materials and can be easily used with 20 x 200 mm 53-62 4-16 1000 psi/6.8 MPa WAT027502
20 x 300 mm 85-94 4-16 1000 psi/6.8 MPa WAT027503
silica, polymer, or soft gel packings. To optimize flow and ensure uniform
20 x 600 mm 179-188 4-16 1000 psi/6.8 MPa WAT027506
sample distribution onto the packed bed, eac h column incorporates a 50 x 100 mm 137-196 16-100 500 psi/3.4 MPa WAT023331
distributor. A replaceable filter protects the packing from large particulate 50 x 200 mm 333-392 16-100 500 psi/3.4 MPa WAT023332
contaminants. Empty AP glass columns are available in a variety of sizes 50 x 300 mm 530-589 16-100 500 psi/3.4 MPa WAT023333
and utilize the same design to ensure predictable methods transfer 50 x 600 mm 1118-1177 16-100 500 psi/3.4 MPa WAT023336
among them. AP glass columns are compatible with both analytical and Additional connectors and fitting are available.
preparative HPLC and FPLC systems.

Advanced Purification (AP) Glass Column Accessories and Spare Parts

Waters Advanced Purification (AP) glass columns feature non-metallic AP-2 Column Accessories and Spare Parts
construction and adjustable bed height with easy-to-use coarse and
fine adjustments. T he AP glass columns are available in a variety Description Dimensions Part No.
of dimensions. Glass Tube 20 x 100 mm WAT019891
20 x 200 mm WAT019892
20 x 300 mm WAT019893
AP Minicolumn Accessories and Spare Parts Plastic Shield 20 x 100 mm WAT027542
20 x 200 mm WAT027543
Description Dimensions Part No. 20 x 300 mm WAT027544
20 x 600 mm WAT027545
Glass Tube 5 x 50 mm WAT038802 O-rings, 5/pkg WAT027528
5 x 100 mm WAT038803 Filters, 2/pkg WAT027530
Column Jacket 5 x 50 mm WAT038804 Replacement Tubing (Tefzel) WAT023344
5 x 100 mm WAT038805 1/8 inch o.d. x 0.040 inch i.d. x 10 feet
Filters, 10/pkg WAT038806 (3.2 mm o.d. x 1.02 mm i.d. x 3 m)
O-rings, 13/pkg WAT038807
(includes 10 inlet/outlet and 3 funnel)
AP-5 Column Accessories and Spare Parts

AP-1 Column Accessories and Spare Parts Description Dimensions Part No.
Glass Tube 50 x 100 mm WAT019876
Description Dimensions Part No. 50 x 200 mm WAT019877
Glass tube 10 x 100 mm WAT021992 50 x 300 mm WAT019878
10 x 200 mm WAT022033 Plastic Shield 50 x 100 mm WAT023370
10 x 300 mm WAT022034 50 x 200 mm WAT023371
10 x 600 mm WAT022035 50 x 300 mm WAT023372
Plastic shield 10 x 100 mm WAT021927 50 x 600 mm WAT023373
10 x 200 mm WAT021945 O-rings, 5/pkg WAT023345
10 x 300 mm WAT021946 Filter, 2/pkg WAT023343
10 x 600 mm WAT021947 Replacement Tubing (Tefzel) WAT023344
O-rings, 5/pkg WAT021907 1/8 inch o.d. x 0.040 inch i.d. x 10 feet)
Filters, 10/pkg WAT021910 (3.2 mm o.d. x 1.02 mm i.d. x 3 m)
Replacement tubing (Tefzel) WAT021950
1/16 inch o.d. x 0.009 inch i.d. x 10 feet Consult your appropriate Care and Use Manual for
(1.6 mm o.d. x 0.23 mm i.d. x 3 m) additional spare parts information at www.waters.com/chemcu

www.waters.com Protein Isolations and Analyses 25

PROTEIN ISOLATIONS AND ANALYSES

Fittings and Connectors for Advanced Purification (AP) Glass Columns

Connection of an AP Minicolumn and an AP-1 Column to 1/8” o.d. Tubing
1
1 3 2

Description Qty. Part No. Description Qty. Part No.

1 Collet and nut assembly (3/8-24) 10/pkg WAT005138 1 Compression screw and ferrule ‘Z’ fitting, plastic 1/pkg WAT082708
2 Ferrule 1/8” tube 10/pkg WAT005136 2 Union ‘Z’ fitting to ‘Z’ fitting, plastic 1/pkg WAT082745
3 Union 3/8-24 x ‘Z’ fitting 5/pkg WAT005137

Connection of an AP-2 and an AP-5 Column to 1/8” o.d. Tubing Connection of Pharmacia Fitting to 1/16” o.d. Tubing

2 1
1 1
3

2 2

Description Qty. Part No. Description Qty. Part No.

1 Collet and nut assembly (3/8-24) 10/pkg WAT005138 1 Compression screw and ferrule ‘Z’ fitting, plastic 1/pkg WAT082708
2 Ferrule 1/8” tube 10/pkg WAT005136 2 Union, plastic 1/pkg WAT021951
3 Union 3/8-24 x 3/8-24 1/pkg WAT082734

Connection of a Protein-Pak™ Steel Column to 1/16” Connection of 1/8” or 1/16” Flanged Type Fitting
and 1/8” o.d. Tubing to 1/8” o.d. Tubing
1 4
3 3
6 2

2
5 1

Description Qty. Part No. Description Qty. Part No.

1 Collet and nut assembly (3/8-24) 10/pkg WAT005138 1 Collet and nut assembly (3/8-24) 10/pkg WAT005138
2 Ferrule 1/8” tube 10/pkg WAT005136 2 Ferrule 1/8” tube 10/pkg WAT005136
3 Union 3/8-24 x ‘Z’ fitting 5/pkg WAT005137 3 Adapter 3/8-24 x 1/4-28 1/pkg WAT082735
4 Compression screw and ferrule ‘Z’ fitting, plastic 1/pkg WAT082708
5 Compression screw ‘Z’ fitting, steel 10/pkg WAT005070
6 Ferrule 1/16” steel 10/pkg WAT005063

26 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

Accell Plus Ion-Exchange Packings

Solid-phase extraction for protein sample preparation Ion-Exchange Sample Preparation
Waters Accell Plus ion-exchange packings are 40 μm, 300Å polymer- with Sep-Pak Cartridges
coated, silica-based materials for both lab- and process-scale chroma-
tography. Accell Plus, available as a QMA strong anion exchanger or To perform ion-exchange sample preparation with Sep-Pak cartridges, use
CM, a weak cation exchanger, is easy to pack and is excellent for the a gradient of pH or ionic strength with Accell Plus CM, Accell Plus QMA or
purification of proteins, enzymes, and immunoglobulins. The rigid NH2 as a sorbent.
silica-based packing material will withstand very high flow rates during ½ #NMCHSHNM½SGD½B@QSQHCFD½VHSG½RHW½SN½SDM½GNKC TO½UNKTLDR
cleaning and re-equilibration cycles. Normal flow rates are used during of deionized water or weak buffer
sample loading and elution to obtain the best possible resolution.
½ ,N@C½SGD½R@LOKD½CHRRNKUDC½HM½@½RNKTSHNM½NE½CDHNMHYDC
Accell Plus bulk material may be packed into our Advanced Purification water or buffer
(AP) glass columns. Prepacked 0.7 mL Sep-Pak® cartridges can be used for
½ %KTSD½TMV@MSDC½VD@JKX½ANTMC½BNLONMDMSR
rapid method screening or solid phase extraction applications.
with a weak buffer
To estimate packed bed volume for a known amount of Accell Plus:
½ %KTSD½SGD½EHQRS½BNLONMDMS½NE½HMSDQDRS½VHSG½@½RSQNMFDQ
Accell Plus used (g) x 2 = packed bed volume (mL). buffer (change the pH or ionic strength)
½ %KTSD½NSGDQ½BNLONMDMSR½NE½HMSDQDRS½VHSG½OQNFQDRRHUDKX
Accell Plus PrepPak Cartridges* stronger buffers

(47 x 300 mm) ½ 7 GDM½XNT½QDBNUDQ½@KK½NE½XNTQ½BNLONMDMSR ½CHRB@QC

the used cartridge in an appropriate manner
Economical, convenient preparative separations in the 500 mg to
10 g range. For a complete listing of Waters products for preparative
chromatography, go to www.waters.com. General Elution Protocol for Ion-Exchange Chromatography on
Sep-Pak Cartridges (NH2, Accell Plus QMA, Accell Plus CM)
Particle Particle Pore
Basic Steps for SPE
Description Size Size Part No.
Accell Plus CM * 40 μm 300Å WAT036545 1 2 3

PrepPak™ 1000 Module WAT089592 Condition/ Load Sample Step Step Step
Equilibrate (Black) Elute 1 Elute 2 Elute 3
* Requires PrepPak 1000 Module

Protein Binding Capacity* of Accell Plus

Accell Plus QMA** Accell Plus CM***

200 mg BSA/g packing 175 mg Cytochrome c/g packing
* For best resolution do not exceed 20% of the protein binding capacity.

** Bovine serum albumin in 20 mM Tris/Cl pH 7.0 was used to measure protein binding capacity 1. Condition and equilibrate the cartridge
of Accell Plus QMA. (not required for normal-phase)
2. Load sample
*** Cytochrome c in 20 mM sodium phosphate pH 6.3 was used to measure protein binding 3. Elute components—increase strength
capacity of Accell Plus CM.
of mobile phase in steps

Accell Plus Sep-Pak Cartridges

Accell Plus Ion-Exchange Bulk Packings
Sep-Pak Plus cartridges packed with Accell Plus ion exchangers provide a
For all preparative isolations based on ionic interactions, particularly rapid, economical means to clean up heavily contaminated samples that
proteins, enzymes, and immunoglobulins. would damage a high resolution column. They can also be used to rapidly
screen chromatographic conditions. These are also available in a variety
of configurations.
Particle Particle Pore
Description Size Size Qty. Part No.
Description Ion-Exchange Type Part No.
Accell Plus QMA 40 μm 300Å 100 g WAT010742
Anion Exchanger 500 g WAT010741 Accell Plus CM Weak Cation Exchanger WAT020550
Accell Plus CM 40 μm 300Å 100 g WAT010740 Accell Plus QMA Strong Anion Exchanger WAT020545
Cation Exchanger 500 g WAT010739

www.waters.com Protein Isolations and Analyses 27

PROTEIN ISOLATIONS AND ANALYSES

BioSuite Size-Exclusion HPLC Columns (SEC)

BioSuite Ultra High Resolution (UHR), High Resolution (HR), and Standard determine separation efficiency. BioSuite 4 μm particle size, UHR (Ultra-
size-Exclusion Column packings use a rigid yet “wettable” silica-based High Resolution) columns provide maximum separation efficiency, followed
media that is stable from pH 2.5-7.5. As indicated in the calibration curve by BioSuite HR (High Resolution) columns and BioSuite Standard SEC
tables, the exclusion limits of BioSuite SEC packings are determined by columns. To maximize column life of analytical (i.e., 4.6 mm or 7.8 mm
the particle and pore size of the silica-based material. Particle size of the i.d.) or preparative (i.e., 21.5 mm i.d.) SEC columns, use of BioSuite Guard
SEC packing media as well as column length are important parameters that columns is recommended.

LC/MS Analysis of BSA Aggregation Using BioSuite 250, 5 μm HR SEC Column

100 100
TIC 66420

%
66443

%
66548

62801
68030

0 mass
60000 80000 100000 120000 140000

100 132835
0

2 4 6 8 10 12 14 Min.

132991
LC/MS: Waters Alliance HPLC with 2996 PDA and ZQ (top)
or Q-Tof Mass Detectors (bottom) 66417*
Sample: BSA (heat aggregated)
%
Column: BioSuite 250, 5 μm, HR SEC (7.8 x 300 mm)
Injection: 10 μL
131011 133095
Mobile Phase: 50 mM Ammonium formate (pH 6.5) 66506

Flow Rate: 1 mL/min 133175

135141
Temp: 25 °C 66555
MS Source: ESI (positive)
65504 67505
MS Capillary (kV): 3.3 64540 67648
MS Cone (V): 30
Mass Range: 400 – 3000 m/z 0 mass
60000 70000 80000 90000 100000 110000 120000 130000 140000

LC/MS Analysis of Protein Standards Using BioSuite 250, 5 μm High Resolution (HR) SEC Column (LC/MS conditions as above)
4
100

3
1. T hyrogobulin 669 KDa
2. BSA Dimer 136 KDa
3. BSA 66 KDa
5
4. ß-Lactoglobulin 36 KDa
% 5. Myoglobin 16 KDa
6. Cytochrome C 12 KDa

2
1

0
2 4 6 8 10 12 14 16 18 20 Min.

BioSuite SEC Reference: SEC- MS Analysis of Aggregates in Protein Mixtures. Application Book Supplement
of LC/GC Europe. Sept. 2003. (Waters Literature Code Number: 720000743EN)

28 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

Protein Calibration Curves for BioSuite Ultra-High Resolution (UHR) SEC Columns

1,000,000 Sample: T hyroglobulin (MW 670,000 Da), Gamma globulin (MW 155,000 Da),
Bovine Serum Albumin (66,330Da), Beta Lactoglobulin (MW18,400 Da),
BioSuite 250, Lysozyme (14,300 Da), Cytochrome c (12,400 Da),Triglycine (189 Da)
4 μm UHR SEC Columns: BioSuite 250, 4 μm UHR SEC (4.6 x 300 mm)
BioSuite 125, 4 μm UHR SEC (4.6 x 300 mm)
Eluent: 0.15 M Sodium Phosphate, pH 6.8
Flow rate: 0.35 mL/min
Temperature: 25 °C
100,000
Detection: 280 nm (220 nm for triglycine)
MWWeight
Molecular

10,000
Globular Protein Branched Linear
Column Mol. Wt. Range Dextrans PEG/PEO
BioSuite 125,
BioSuite 125 5,000-150,000 1,000-30,000 500-15,000
4 μm UHR SEC
BioSuite 250 10,000-500,000 2,000-70,000 1,000-35,000

1,000

Note: Operating flow rates for BioSuite Ultra-High Resolution (UHR) SEC columns (4.6 mm i.d.) are from
0.1 - 0.4 mL/min. Use of an HPLC system (e.g. Waters Alliance HPLC System) capable of operating at these
flows is essential for optimal UHR SEC column performance.

100
6 8 10 12 MIn.

Protein Calibration Curves for BioSuite High Resolution (HR) SEC Columns

Sample: T hyroglobulin (MW 670,000 Da), IgG (MW 156,000 Da),

BioSuite 250, BSA (66,330Da), Ovalbumin (MW 43,000 Da), Peroxidase
10 6 5 μm HR SEC (40,200 Da),Beta Lactoglobulin (MW18,400 Da), Myoglobin
(MW 16,900 Da), Ribonuclease A (MW 13,700 Da),
Cytochrome C (12,400 Da), Glycine tetramer (246 Da)
BioSuite 450, Columns: BioSuite 450, 8 μm HR SEC (7.8 x 300 mm)
8 μm HR SEC BioSuite 250, 5 μm HR SEC (7.8 x 300 mm)
BioSuite 125, 5 μm HR SEC (7.8 x 300 mm)
Eluent: 0.1 M Sodium Phosphate, pH 7.0 containing 0.3M Sodium Chloride
10 5 Flow rate: 1.0 mL/min
Temperature: 25 °C
Detection: 220 nm
Molecular Weight

10 4
Globular Protein Branched Linear
Column Mol. Wt. Range Dextrans PEG/PEO

BioSuite 125, BioSuite 125 5,000-150,000 1,000-30,000 500-15,000

5 μm HR SEC BioSuite 250 10,000-500,000 2,000-70,000 1,000-35,000
10 3 BioSuite 450 20,000-7,000,000 4,000-500,000 2,000-250,000

10 2

6 8 10 12 MIn.

www.waters.com Protein Isolations and Analyses 29

PROTEIN ISOLATIONS AND ANALYSES

Protein Calibration Curves for BioSuite Standard SEC Columns

BioSuite 250,
Sample: T hyroglobulin (MW 670,000 Da), IgG (MW 156,000 Da),
10 μm SEC BSA (66,330Da), Ovalbumin (MW 43,000 Da), Peroxidase
10 6 (40,200 Da),Beta Lactoglobulin (MW18,400 Da), Myoglobin
(MW 16,900 Da), Ribonuclease A (MW 13,700 Da),
Cytochrome C (12,400 Da), Glycine tetramer (246 Da)
Columns: BioSuite 450, 13 μm SEC (7.5 x 300 mm)
BioSuite 250, 10 μm SEC (7.5 x 300 mm)
BioSuite 125, 10 μm SEC (7.5 x 300 mm)
BioSuite 450, Eluent: 0.1M Sodium Phosphate, pH 7.0 containing 0.3M Sodium Chloride
Flow rate: 1.0 mL/min
10 5 13 μm SEC Temperature: 25 °C
Detection: 220 nm
Molecular Weight

Globular Protein Branched Linear

10 4 Column Mol. Wt. Range Dextrans PEG/PEO

BioSuite 125 5,000-100,000 1,000-30,000 500-15,000

BioSuite 250 10,000-500,000 2,000-70,000 1,000-35,000
BioSuite 450 20,000-7,000,000 4,000-500,000 2,000-250,000
BioSuite 125,
10 μm SEC
10 3

20 30 40 Min.

BioSuite 4.6 mm, 7.8 mm, and 7.5 mm i.d. Analytical

or 21.5 mm i.d. Preparative Columns are Available.

Suggested Protein Suggested Volume

Globular Mass Load for Maximum Load for Maximum
Inner Column Protein Mol. Wt. Multicomponent Multicomponent
Description Matrix Diameter Length Volume Range Resolution* Resolution* Part No.
BioSuite 125, 4 μm UHR SEC Silica 4.6 mm 300 mm 4.98 mL 5,000-150,000 Less than 8 mg/mL Less than 40 μL 186002161
BioSuite 250, 4 μm UHR SEC Silica 4.6 mm 300 mm 4.98 mL 10,000 - 500,000 Less than 8 mg/mL Less than 80 μL 186002162
BioSuite UHR Guard SEC Silica 4.6 mm 35 mm 186002163

BioSuite 125, 5 μm HR SEC Silica 7.8 mm 300 mm 14.33 mL 5,000-150,000 Less than 8 mg/mL Less than 200 μL 186002164
BioSuite 250, 5 μm HR SEC Silica 7.8 mm 300 mm 14.33 mL 10,000 - 500,000 Less than 8 mg/mL Less than 200 μL 186002165
BioSuite 450, 8 μm HR SEC Silica 7.8 mm 300 mm 14.33 mL 20,000-7,000,000 Less than 8 mg/mL Less than 200 μL 186002166
BioSuite HR Guard SEC Silica 6 mm 40 mm 186002167

BioSuite 125, 10 μm SEC Silica 7.5 mm 300 mm 13.25 mL 5,000-150,000 Less than 8 mg/mL Less than 200 μL 186002168
BioSuite 125, 13 μm SEC Silica 21.5 mm 300 mm 108.9 mL 5,000-100,000 Less than 8 mg/mL Less than 1.6 mLs 186002169
BioSuite 250, 10 μm SEC Silica 7.5 mm 300 mm 13.25 mL 10,000 - 500,000 Less than 8 mg/mL Less than 200 μL 186002170
BioSuite 250, 13 μm SEC Silica 21.5 mm 300 mm 108.9 mL 10,000 - 500,000 Less than 8 mg/mL Less than 1.6 mLs 186002171
BioSuite 450, 13 μm SEC Silica 7.5 mm 300 mm 13.25 mL 20,000-7,000,000 Less than 8 mg/mL Less than 200 μL 186002172
BioSuite 450, 17 μm SEC Silica 21.5 mm 300 mm 108.9 mL 20,000-7,000,000 Less than 8 mg/mL Less than 1.6 mLs 186002173
BioSuite Guard SEC Silica 7.5 mm 75 mm 186002174
BioSuite Guard SEC Silica 21.5 mm 75 mm 186002175

* Using a BSA protein standard in a 50 mM phosphate buffer containing salt (either 0.1 M NaCl or 0.1 M Na2SO4 ) eluent.
Useful protein mass loads will vary depending upon separation eluent, complexity of sample, and on the type of proteins contained in mixture.
In general, maximum component resolution is obtained by injecting the smallest possible volume of a dilute protein solution.
* Note: Operating flow rates for BioSuite Ultra-High Resolution (UHR) SEC columns (4.6 mm i.d.) are from 0.1-0.4 mL/min. Use of an HPLC system
(e.g. Waters Alliance HPLC System) capable of operating at these flows is essential for optimal UHR SEC column performance.

30 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

Protein-Pak and Shodex Size-Exclusion HPLC Columns

Waters offers two families of packings for size-exclusion chromatogra- Standard Protein Mix on KW-803 Column
phy. Protein-Pak packings are based on a 10 μm diol-bonded silica and
are available in a selection of pore sizes and column configurations. Column: Protein KW-803 8

In addition, Waters offers a series of Shodex™ 7 μm high-resolution, gel Eluent:

Flow Rate:
25 mM Sodium Phosphate pH 6.8
0.72 mL/min
filtration packings. Detection: 280 nm
1. Blue Dextran
2. Ferritin
The Protein-Pak size-exclusion columns can be expected to resolve proteins 3. Aldolase
that differ in molecular weight by a factor of two and to distinguish 4. Bovine Serum Albumin
5. Ovalbumin 6
7

proteins differing by as little as 15% in molecular weight. The degree of 6. Chymotrypsinogen 3

4 5

7. Cytochrome c
resolution is more dependent on the sample mass and volume than the 8. Cytidine 1
2

interaction between the sample and the stationary phase. Ideally, there
should be no interaction between the stationary phase and the sample
molecules. Secondary interactions are most often ionic and can, therefore,
25 Min.
be reduced by increasing the ionic strength of the mobile phase. Typical,
salt concentrations range to 0.2-0.5M NaCl. It may also be useful in This gel filtration separation of protein standards demonstrates the ability to separate proteins in a wide range of
some cases to consider adding 10-20% methanol to eliminate hydro- molecular weights in minutes for high sensitivity analysis or protein isolation up to the milligram scale.

phobic and other hydrogen-bonding interactions.

Protein-Pak Columns and Packings Shodex Size-Exclusion Columns

Steel Column Dimensions MW Range Part No.
Column Dimensions Particle Size MW Range Part No.
Protein-Pak 60 7.8 x 300 mm 1,000-20,000 WAT085250 Protein KW-802.5 8 x 300 mm 7 μm 100-50,000 WAT035943
Protein-Pak 125 7.8 x 300 mm 2,000-80,000 WAT084601 Protein KW-803 8 x 300 mm 7 μm 100-150,000 WAT035946
Protein-Pak 300SW 7.5 x 300 mm 10,000-300,000 WAT080013 Protein KW-804 8 x 300 mm 7 μm 500-600,000 WAT036613
Protein-Pak 125 Sentry Guard Column Protein-Pak 125 Sentry Guard Column
3.9 x 20 mm, 2/pkg (requires holder) 186000926 3.9 x 20 mm, 2/pkg (requires holder) 186000926
Sentry Universal Guard Column Holder WAT046910 Sentry Universal Guard Column Holder WAT046910

Glass Column
Protein-Pak 200SW 8 x 300 mm 500-60,000 WAT011786
Protein-Pak 300SW 8 x 300 mm 10,000-300,000 WAT011787
Inquire for additional offerings including prep.

Ultrahydrogel HPLC Columns

Packed with hydroxylated polymethacrylate-based gel, Waters Ultra hydrogel Columns (7.8 x 300 mm)
Ultra hydrogel™ SEC columns are ideal for the analysis of aqueous-
soluble samples, suc h as oligomers; oligosacc harides; polysacc harides; Column Pore Size Exclusion Limit Part No.
and cationic, anionic, and amphoteric polymers. Measuring 7.8 x 300 mm, Ultrahydrogel 120 120 Å 5x 103 WAT011520
these high-resolution columns offer many advantages over conventional Ultrahydrogel 250 250 Å 8 x 104 WAT011525
aqueous SEC columns, such as: Ultrahydrogel 500 500 Å 4 x 105 WAT011530
Ultrahydrogel 1000 1000 Å 1 x 106 WAT011535
½ !½VHCD½O(½Q@MFD½  Ultrahydrogel 2000 2000 Å 7 x 106 WAT011540
Ultrahydrogel Linear Blend 7 x 106 WAT011545
½ #NLO@SHAHKHSX½VHSG½GHFG½BNMBDMSQ@SHNMR½NE½NQF@MHB½RNKUDMSR
Ultrahydrogel DP* 120 Å 5 x 103 WAT011550
(up to 20% organic, 50% organic if the mobile phase is Ultrahydrogel Guard Column N/A N/A WAT011565
introduced by gradient) Ultrahydrogel Guard Column DP* N/A N/A WAT011570
½ 'QD@SDQ½EKDWHAHKHSX½ENQ½SGD½LNAHKD½OG@RD * DP = Degree of Polymerization, choice of column when working with glucose oligomers.

½ -HMHL@K½MNM RHYD DWBKTRHNM½DEEDBSR

www.waters.com Protein Isolations and Analyses 31

PROTEIN ISOLATIONS AND ANALYSES

Symmetry300 C4 HPLC Columns

Compared to our BEH300 C4 offerings, Symmetry300 C4 particles are ½ ^½ONQD½ENQ½ODOSHCD½@MC½OQNSDHM½@OOKHB@SHNMR
100% silica-based and are synthesized using ultrapure organic reagents
½ &TKKX½DMC B@OODC½SN½LHMHLHYD½TMCDQRHQDC½RDBNMC@QX½HMSDQ@BSHNMR
resulting in high purity material with very low silanol activity for outstand-
ing peptide and protein separations and recoveries. ½ !KSDQM@SHUD½RDO@Q@SHNM½RDKDBSHUHSX½BNLO@QDC½SN
Waters BEH300 C4 hybrid material
½ 1#½SDRSDC½VHSG½ODOSHCD½R@LOKDR½SN½GDKO½DMRTQD½DWBDKKDMS
Protein: Symmetry300 C4 Versus Competitors
batch-to-batch consistency
Mobile Phase A: 0.1% TFA in water Injection Volume: 20 μL
Mobile Phase B: 0.08% TFA in acetonitrile Temperature: 40 °C
Flow Rate: 0.75 mL/min Detection: UV @ 220 nm
Symmetry300 C4 Columns
Gradient: Time Profile Instrument: Alliance 2790, 996 PDA
(min) %A %B Analytes: 1. RNAse
0.00 80 20 2. BSA Inner Particle
15.00 35 65 3. B-Lactoglobulin Description Diameter Length Size Part No.
4. Ovalbumin
2 4
Symmetry300 C4 1.0 mm 150 mm 3.5 μm 186000276
1 Symmetry300 Symmetry300 C4 2.1 mm 50 mm 3.5 μm 186000277
3 C4 column
P = 138.56 Symmetry300 C4 2.1 mm 100 mm 3.5 μm 186000278
Symmetry300 C4 2.1 mm 150 mm 3.5 μm 186000279
Symmetry300 C4 4.6 mm 50 mm 3.5 μm 186000280
Symmetry300 C4 4.6 mm 75 mm 3.5 μm 186000281
1
4 Competitive 300Å
Symmetry300 C4 4.6 mm 100 mm 3.5 μm 186000282
2 3 C18 column Symmetry300 C4 4.6 mm 150 mm 3.5 μm 186000283
P = 90.09 Symmetry300 C4 2.1 mm 150 mm 5 μm 186000285
Symmetry300 C4 3.9 mm 150 mm 5 μm 186000286
Symmetry300 C4 4.6 mm 50 mm 5 μm 186000287
0 2 4 6 8 10 12 14 Min. Symmetry300 C4 4.6 mm 150 mm 5 μm 186000288
Symmetry300 C4 4.6 mm 250 mm 5 μm 186000289

Protein-Pak Affinity Columns

T he Protein-Pak™ Affinity Epoxy-Activated packing consists of 40 μm, 500Å Purification of Carbonic Anhydrase
pore size particles having a hydrophilic bonding layer with a glycidoxypro-
pyl functionality resulting in a seven atom spacer arm. T he epoxy-activated Ligand: Sulfanilamide, 180 μmoles/g
Column: AP-1 Glass Column, 10 x 100 mm
surface can immobilize a wide range of ligands via a covalent linkage with Buffer A: 100 mM Tris sulfate with 200 mM sodium sulfate pH 8.7
Buffer B: 200 mM potassium thiocyanate in 50 mM Tris sulfate pH 6.5
amino, hydroxyl or sulfhydryl groups using simple coupling procedures. Gradient: Initial A B Flow
For method screening or small scale separation, choose the convenience of 1 min 100% 0% 1 mL/min
2 min 100% 0% 2 mL/min
prepacked microcolumns. Larger-scale separations are easily achieved by 7 min 0% 100% 2 mL/min
17 min 100% 0% 2 mL/min
packing bulk material in our Advanced Purification (AP) glass column. Detection: 280 nm
Protein Load: 24 mg of bovine hemolysate yielded 0.6 mg of carbonic anhydrase
To estimate packed bed volume for a known amount of Protein-Pak ™
with a 100% recovery of esterase activity.
1. Carbonic Anhydrase
Affinity Epoxy-Activated packing:
Protein-Pak Affinity Epoxy-Activated used
(g) x 2 = packed bed volume (mL). 1

Particle Particle Pore

Packing Size Size Qty. Part No.
Protein-Pak Affinity 40 μm 500Å 25 g WAT030653
Epoxy-Activated Packing 100 g WAT030654
Protein-Pak Affinity
Epoxy-Activated Microcolumn 40 μm 500Å 10/box WAT035955
30 Min.
(500 mg of material in a 3 cc syringe barrel)
Inquire for additional offerings.

32 Protein Isolations and Analyses www.waters.com

 PROTEIN ISOLATIONS AND ANALYSES

BioSuite pC18 and pPhenyl Reversed-Phase Chromatography (RPC) HPLC Columns

Reversed-phase chromatography (RPC) has become a widely accepted Hydrophobic Proteins are Well Resolved by Reversed-phase
tool for the separation of proteins, peptides, synthetic oligonucle- Chromatography on BioSuite™ pPhenyl RP Column
otides, and other biomolecules. For many applications, Symmetry
and Symmetry300, Atlantis, T3, or XBridge BEH130 and BEH300 BioSuite™ pPhenyl, 1000Å, 10 μm RPC
chemistries can be successfully used for the isolation and analyses of Sample: Ribonuclease (Peak 1), bovine Insulin (Peak 2bovine), Cytochrome-C
these biocompounds. However for some applications, the large pore size (Peak 3), Lysozyme (Peak 4), Transferrin (Peak 5), Bovine serum
albumin (Peak 6), Myoglobin (Peak 7), Ovalbumin (Peak 8)
and high chemical stability of BioSuite pC18 and pPhenyl resin-based Column: BioSuite™ pPhenyl, 1000Å, 10 μm RPC (4.6 x 75 mm)
Eluent: A: HPLC grade water with 0.05% TFA
packings may be preferred. BioSuite RPC column offerings include a Eluent B: Acetonitrile with 0.05% TFA 1 4 5 6
2
C18 (pC18) and a phenyl (pPhenyl) chemistry bonded to a pH stable, Flow rate: 1.0 mL/min
7
8

Gradient: Linear gradient from 5 to 80% B in 60 min. 3

methacrylic ester-based polymeric resin. The 500Å pore size of the Temperature: 25 °C
Detection: 220 nm
pC18 base matrix accommodates proteins up to 2,500,000 Daltons
while the 1,000Å pore size of the pPhenyl base matrix accommodates
proteins up to 5,000,000 Daltons.
The BioSuite pC18, 2.5 μm, NP column contains a non-porous chemistry
that yields superior chromatographic resolution in less time compared
to chromatography performed on the porous, pC 18, 500Å, 7 μm RPC 15 30 Min.

selection. Waters porous, pC18, 500Å, 7 μm RPC column is available for

applications requiring greater binding capacity. The pC18 and pPhenyl
RPC chemistries are available in 21.5 x 150 mm columns for “lab- LC/MS Analysis of a Reduced Monoclonal IgG1
scale” isolations while a 2.0 x 75 mm column is well suited for narrow- Antibody on a BioSuite pPhenyl RPC Column
bore HPLC and LC/MS applications.
Sample: Reduced monoclonal IgG1 antibody Peak No. Identity
Column: BioSuite pPhenyl, 1000 A, 10 μm RPC (2.0 x 75 mm) 1 HC Clip1 (with glycan variants)
Eluent A: Water with 0.1% Formic Acid 2 LC (1-2 S-S Intact)
Reversed-phase Chromatography at Elevated pH on BioSuite™ pC18 Eluent B: Acetonitrile with 0.1% Formic Acid 3 LC
Flow Rate: 0.2 ml/min 4 LC+1 Da
RP Column Possible on Polymer Based Material Gradient: 10% B for 6 min (waste), then 20-35% B in 30 min 5 HC Clip 2 (w/o glycan variants)
Temperature: 80°C 6 HC Clip 3 (w/o glycan variants)
Detection: Waters LCT Premier™ ESI-TOFMS 7 Antibody Half mer
8 HC (with glycan variants)
BioSuite™ pC18, 500Å, 7 μm RPC 9 HC Fragment (w/o glycan variants)
Sample: Met-enkephalin (Peak 1), bradykinin (Peak 2), leu-enkephalin (Peak 3), 10 HC Fragment-18 Da
Neurotensin (Peak 4), bombesin (Peak 5), Angiotensin l (Peak 6), (w/o glycan variants)
Somatostatin (Peak 7), Insulin (Peak 8)
LC HC: Heavy Chain, LC: Light Chain
Column: BioSuite pC18, 500Å, 7 μm RPC (4.6 x 150 mm) HC TIC: Total Ion Chromatogram
Eluent: A: 0.2 M NH4OH, pH 10.8 3 8
Eluent B: 0.2 M NH4OH, pH 10.8 (20%) / Acetonitrile (80%)
Flow rate: 1.0 mL/min TIC of BioSuite pPhenyl,
Gradient: Linear gradient from 0 to 80% B in 50 min. Reduced IgG1 1000 A, 10 μm RPC
3
Temperature: 25 °C 1 6 antibody
Detection: 220 nm
7
5
4

9
7
10
8

6
2 4
1 5

0 15 30 Min. 10 12 14 16 18 20 22 24 26 28 30 Min.

Note: The BioSuite pPhenyl, 1000Å RPC columns have a higher ligand Description Matrix Inner Diameter Length Part No.
density compared to the BioSuite Phenyl, 1000Å HIC columns and BioSuite pC18, 2.5 μm NP RPC Polymer 4.6 mm 35 mm 186002152
are not recommended for hydrophobic-interaction separations. BioSuite pC18, 500, 7 μm RPC Polymer 2.0 mm 150 mm 186002153
BioSuite pC18, 500, 7 μm RPC Polymer 4.6 mm 150 mm 186002154
BioSuite pC18, 500, 13 μm RPC Polymer 21.5 mm 150 mm 186002155

BioSuite pPhenyl, 1000, 10 μm RPC Polymer 2.0 mm 75 mm 186002156

BioSuite pPhenyl, 1000, 10 μm RPC Polymer 4.6 mm 75 mm 186002157
BioSuite pPhenyl, 1000, 13 μm RPC Polymer 21.5 mm 150 mm 186002158

www.waters.com Protein Isolations and Analyses 33

PROTEIN ISOLATIONS AND ANALYSES

BioSuite Hydrophobic-Interaction Chromatography (HIC) HPLC Columns

The separation of proteins and peptides based upon hydrophobic charac- Hydrophobic-Interaction Chromatography on BioSuite™ Phenyl HIC
teristics is a powerful chromatographic technique. However, some proteins Column is an Excellent Alternative to Reversed-phase Methods.
denature at elevated organic solvent concentrations making reversed-phase
chromatography (RPC) difficult. BioSuite Phenyl HIC columns provide a BioSuite Phenyl, 4
Sample: Cytochrome c (Peak 1),
Myoglobin (Peak 2), Ribonuclease
viable separation alternative to RPC. HIC is characterized by the adsorption 10 μm HIC (Peak 3), Lysozyme (Peak 4),
of compounds to a weakly hydrophobic surface at high salt concentrations, Alpha-chymotrypsin (Peak 5), and
Alpha-Chymotrypsinogen A (Peak 6)
followed by elution with a decreasing salt gradient. HIC combines the
Column: BioSuite Phenyl, 10 μm HIC
non-denaturing characteristics of salt precipitation with the precision of (7.5 x 75 mm)
2 5 Eluent: A: 0.1 M Sodium Phosphate,
HPLC to yield excellent separation of biologically active material. pH 7.0 containing
BioSuite Phenyl, 1000Å, 10 μm HIC column media consists of a phenyl 1 6
2 M Ammonium Sulfate
6 Eluent B: 0.1 M Sodium Phosphate, pH 7.0
3
group bonded to a methacrylic ester-based polymeric resin. The large Flow Rate: 1.0 mL/min
Gradient: Linear gradient from
1000Å pore size accommodates proteins up to 5,000,000 Daltons. A 0 to 100% B in 60 min.
21.5 mm i.d. x 150 mm column is also available for “lab-scale” isolations. Temperature: 25 °C
Detection: 280 nm

Note: T he BioSuite Phenyl, 1000Å HIC columns have a lower ligand

density compared to the BioSuite pPhenyl, 1000Å RPC columns and are 0 30 60 Min.
not recommended for reversed-phase separations.

Inner
Description Matrix Diameter Length Part No.
BioSuite Phenyl 10 μm HIC Polymer 7.5 mm 75 mm 186002159
BioSuite Phenyl 13 μm HIC Polymer 21.5 mm 150 mm 186002160

Protein HIC and Protein-Pak Phenyl HIC HPLC Columns

Waters Protein HIC and Protein-Pak Phenyl hydrophobic-interaction columns Protein Purification by Hydrophobic-Interaction Chromatography
are packed with rigid, 10 μm polymethacrylate packing materials with
500Å pore size to ensure rapid protein diffusion. A low density bonding Sample: Wheat Germ Extract
Column: Protein HIC PH-814, 8 x 75 mm
with phenyl groups results in a hydrophobic surface that allows for protein Buffer A: 1.7 M Ammonium sulfate in Buffer B
Buffer B : 0.1M Sodium phosphate, pH 7.0
purification with a high recovery of both mass and biological activity. Gradient: 0-100% Buffer B in 15 minutes linear
Flow Rate: 1.1 mL/min
Detection: 280 nm

HIC Packing Material Capacities

Protein HIC PH-814 40 mg/mL * Lectin

Protein-Pak Phenyl 5PW 25 mg/mL *
* Ovalbumin in 1.8 M ammonium sulfate, 0.1 M sodium phosphate pH.

Hydrophobic-Interaction Columns

Description Dimensions Part No.

Protein HIC PH-814 Steel Column 8 x 75 mm WAT035520
Protein-Pak Phenyl-5PW Glass Column 8 x 75 mm WAT011785
30 Min
Inquire for additional offerings.
Unique selectivity of hydrophobic interaction packings provides resolution
and recovery of biologically active proteins with little denaturation.

34 Protein Isolations and Analyses www.waters.com

 AMINO ACID ANALYSIS

Amino Acid Analysis

Overview Food and feed samples for assessing nutritional content are much more
complex. Hydrolysis requires large volumes of 6N HCl to ensure both
Amino Acid Analysis (AAA) is required for many applications. Amino acids dispersal of the sample and a sufficient excess of acid. T he sulfur-contain-
are the constituents of proteins and are intermediates in many metabolic ing amino acids are particularly important in these assays because they
pathways. Bound amino acids are measured following hydrolysis of the are growth-limiting. Performic acid oxidation is preferred for accurate
protein to determine the concentration of protein, to identify a protein, and determination of methionine and cysteine in these complex samples.
to detect structural variants. Amino acid composition is a critical component T he most complete profile is obtained by analyzing the sample with and
of the nutritional value of foods and feeds. Free amino acids are measured without oxidation.
without hydrolysis using the same analytical tools used to monitor cell
culture and fermentation processes. Similar assays are applied in the food In cell culture and fermentations, free amino acids are supplied in the media
industry to recognize the origin of the natural products based on the free as nutrients. A larger group of amino acids, including especially glutamine
amino acids released by extraction. AAA is also used to assess the status and tryptophan must be included in the measurement for assessing the
of metabolic pathways in clinical researc h laboratories. Eac h of these health of the cell culture and supporting a feeding schedule.
applications requires specific sample-handling and some modification
to the method.
Analysis of Amino Acids
After hydrolysis or other sample preparation, the amino acids are ready
Sample Preparation for analysis. T his is a c hallenging analytical problem because the set of
Samples for AAA can be divided into two groups. W here the protein in the compounds covers a wide range of c hemical properties but with pairs of
sample is the ultimate object of the analysis, the sample must be hydro- components that are very similar. T hey also lack common chemical features
lyzed to release the amino acids. In contrast, where the amino acids are that can be used for convenient detection. Several suitable solutions have
measured as metabolic indicators, no hydrolysis is required. However, the been developed to address these concerns.
amino acids may need to be separated from other sample constituents that Amino acids were first separated by cation-exc hange c hromatography.
could bind amino acids or otherwise interfere with the analysis. Decades of development and continual improvements have produced
Purified proteins and peptides are hydrolyzed with acid at high temperature, methods that give complete resolution in less than 60 minutes. Ion-
commonly 6N HCl at 100-105 °C for 24 hours. Higher temperatures for exc hange methods are not compatible with direct detection. An added
shorter times are sometimes used for specific purposes but with reduced post-column reaction system uses a colorimetric reagent like ninhydrin or
recovery and increased side reactions. Vapor-phase hydrolysis is useful for a fluorescent reactant like o-pthalaldehyde.
sensitive analysis of small amounts of pure proteins. T he differences between the amino acids are more suitable for reversed-
T here are artifacts of hydrolysis. T he amine, asparagine, and glutamine, phase c hromatography. Because most of the compounds are polar,
are converted to the corresponding acids. Tryptophan is completely lost in reversed-phase separations incorporate an ion-pairing reagent like a
hydrolysis, and significant amounts of the sulfur-containing amino acids perfluorinated acid. T hese separations may be coupled to an electro-
are also destroyed. Some amino acids, e.g., threonine, serine, and tyrosine spray mass spectrometer for detection.
may be oxidized while the bonds between hydrophobic residues are hydro- A more general approach to reversed-phase AAA is pre-column deriva-
lyzed slowly, leading to underestimates in both cases. Tryptophan may be tization. T he derivatized amino acids retain better on the reversed-phase
preserved with alkaline hydrolysis or with the use of methanesulfonic acid. column and can be more easily separated. Most common derivatization
T he sulfur amino acids may be quantitatively oxidized with performic acid reagents react with the amines. Some reagents react only with primary
to the very stable methionine sulfone and cysteic acid. Oxidation degrades amines, but the generally useful ones also react with secondary amines so
other amino acids, including tyrosine, serine, and threonine, so reduction that proline is detected. In addition to improving chromatography, derivati-
and alkylation of cysteine residues and carefully excluding oxygen from zation can make the amino acids readily detectable. T he detection may be
the hydrolysis to preserve methionine is more useful. To correct for gradual UV absorbance or fluorescence. Several reagents have been used, and two
destruction or release, data from three hydrolysis times, for example, 24, of the most thoroughly developed and best understood are described below.
48, and 72 hours, are extrapolated to constant values.
Both approaches to AAA yield satisfactory results. Ion exchange with post-
column detection is the oldest and has the larger knowledge base about
unusual amino acids. Reversed-phase methods with pre-column derivatiza-
tion are faster, more sensitive, and do not require specialized equipment.

www.waters.com Amino Acid Analysis 35

AMINO ACID ANALYSIS

UPLC® Amino Acid Analysis Solution

T he UPLC Amino Acid Analysis Solution

Over the years, Waters has provided three distinct products utiliz- T he UPLC Amino Acid Analysis Solution leverages Waters experience
HMF½OQD BNKTLM½CDQHU@SHY@SHNM½@MC½QDUDQRDC OG@RD½(0,#½!TSN4@F™, in separation science, derivatization c hemistries, and information
0HBN4@F ½@MC½!BB14@F½4NC@X ½7@SDQR½BNMSHMTDR½SN½KD@C½SGD½V@X½AX½ management. It is a total application solution optimized for accurate,
bringing together its most ground-breaking and popular tec hnolo- reliable, and reproducible analysis of amino acids. Based on Waters
gies: ACQUIT Y UPLC @MC½!BB14@F Ultra, the newest addition to the !BB14@F½5KSQ@½B GDLHRSQX ½BNLAHMDC½VHSG½NTQ½@V@QC½VHMMHMF½!#15)4 9½
!BB14@F½E@LHKX½NE½OQNCTBSR½ UPLC separation tec hnology, users can feel confident with assured
performance in the areas of protein c haracterization, cell culture
T he UPLC Amino Acid Analysis Solution is holistically designed and
monitoring, and nutritional analysis of foods and feeds.
optimized specifically for amino acid analyses. Derivatized amino acids
are separated using the ACQUIT Y UPLC System, enhancing resolution T he UPLC Amino Acid Analysis Solution consists of:
to ensure accurate and precise qualitative and quantitative results. Just
½ 7@SDQR½!#15)49½50,# System and tunable UV detector (Optional
as important, our solution provides performance-qualified methodolo-
fluorescence and PDA detectors are also fully supported)
gies whic h are designed to be rugged and reliable, assuring reproduc-
ible results day-to-day, instrument-to-instrument, lab-to-lab, around ½ !BB14@F Ultra derivatization chemistries including column,
the world—with the expert support that scientists have come to expect reagents, and eluents (all quality control tested)
from Waters. ½ %LONVDQ™ 2 pre-configured projects, methods, and report templates
½ )MRS@KK@SHNM½@MC½@OOKHB@SHNM½SQ@HMHMF½@MC½RTOONQS½HMBKTCDC½
½ !OOKHB@SHNM RODBHEHB½0DQENQL@MBD½1T@KHEHB@SHNM
½ #NMMDBSHNMR½).3)'(4 ™ Intelligent Services

36 Amino Acid Analysis www.waters.com

 AMINO ACID ANALYSIS

Amino Acid Analysis Methods for the Analysis of #GDLHRSQX½NE½SGD½!BB14@F½$DQHU@SHY@SHNM½2D@BSHNM

Hydrolysates and of Cell Culture Media
O O
AMQ

Hydrolysates O OH 1° Amino Acid:

H
OH
H NH 2 t1/2<<1s N NH
N O
N

Lys
O O
N O O N

NVa
Deriv Peak
AQC Reagent

Phe
Leu
Lle
Tyr

Val
Met
OH

Cys
NH

Pro
Derivatized Amino Acids
Thr

Ala
Glu

2° Amino Acid:
Asp
Arg
Gly
Ser

t1/2<<1s
NH3

His

AMQ H
N N
H2O O
O
t1/2~15s N HO
Lys
Cell Culture
Orn

H H
Hy Lys 1

Deriv Peak

N-Hydroxy NH2 N N

Lle

Trp
Phe
Leu
Tyr

NVa
Val
Cys

Met

+
ABAA

Succinimide O
Hy Lys 2
GABA

+ CO2 N N N
Pro
Ala
Glu

Thr
Asp

6-Aminoquinoline (AMQ) bis-aminoquinoline urea (derivatization peak)

Hy Pro
NH3

Tau

Gln
Arg

AE
Gly
Ser
Asn
His

!BB14@F½$DQHU@SHY@SHNM½2D@BSHNM
1.50 3.00 4.50 6.00 7.50 Min.

The UPLC Amino Acid Analysis solution can be used for the analysis of protein hydrolysates or for
½ 5SHKHYDR½!BB14@F½5KSQ@½2D@FDMS½0NVCDQ
the larger set of amino acids found in cell culture media. The only differences in the methods are a
– 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
different dilution of the supplied eluent A concentrate and operation at a different temperature.
No method development or buffer reformulation is required. – US Patent 5,296,599 and European
Patent EP 0 533 200 B1

!BB14@F½5KSQ@½#GDLHRSQX½ ½ 2D@BSR½Q@OHCKX½VHSG½ANSG½OQHL@QX½@MC½RDBNMC@QX½@LHMDR

4 GD½!BB14@F½5KSQ@½BGDLHRSQX½OQNCTBSR½@QD½@½BNLOQDGDMRHUD ½ETKKX SDRSDC½ ½ %WBDRR½QD@FDMS½QD@BSR½LNQD½RKNVKX½VHSG½V@SDQ½SN½ENQL½@½OQNCTBS

set of reagents, columns, and eluents optimized for use with the UPLC that can be separated chromatographically from the derivatized
Amino Acid Analysis Solution. T his c hemistry is based on Waters amino acids
VHCDKX TRDC½@MC½VDKK TMCDQRSNNC½!BB14@F½CDQHU@SHY@SHNM½LDSGNC½0QHL@QX½ ½ 2DPTHQDR½MN½U@BTTL½CQXHMF ½R@LOKD½OQDO ½NQ½DWSQ@BSHNM
and secondary amino acids are derivatized before separation with a single
QD@FDMS ½!BB1&KTNQ™, in a high-throughput batch process resulting in
exceptionally stable derivatives. High resolution separations are achieved
TRHMF½SGD½OQD PT@KHEHDC½!BB14@F½5KSQ@½50,#½BNKTLMR½@MC½LNAHKD½OG@RDR½
Derivatized amino acids are quantified to sub-pmol levels with single
wavelength UV detection.
Literature
!BB14@F½½5KSQ@½#GDLHRSQX References

Description Part No. HPLC and UPLC Amino Monitoring Cell Culture Media with the Waters
Acid Analysis Brochure, Amino Acid Analysis Solution,
UPLC AAA Application Add-on Kit* 176001279 Literature Reference 720001946EN Literature Reference 720002381EN
!BB14@F½5KSQ@½2DEHKK½0@BJ@FD ½ 176001235
UPLC Amino Acid UPLC Amino Acid Analysis Solution,
Refill Package Includes: Analysis Solution Brochure, Literature Reference 720001683EN
½ !BB14@F½5KSQ@½$DQHU@SHY@SHNM½+HS ½½!M@KXRDR½ 186003836 Literature Reference 720001837EN
ACQUITY UPLC for the Rapid
½ !BB14@F½5KSQ@½#NKTLM½½W½½LL½ 186003837
Amino Acid Analysis for Monitoring Cell Analysis of Amino Acids in Wine,
½ !BB14@F½5KSQ@½%KTDMS½! ½#NMBDMSQ@SD½½L, ½ 186003838 Culture Media and Protein Structure Poster, Literature Reference 720002044EN
½ !BB14@F½5KSQ@½%KTDMS½"½½L, ½ 186003839 Literature Reference 720002035EN
Sample Tubes, 4 x 72/pkg WAT007571 Determination of Amino Acids in Beers Using
Amino Acid Analysis for Monitoring the UPLC Amino Acid Analysis Solution,
Amino Acid Standard, Hydrolysate, 10 x 1 mL ampules WAT088122 Protein Structure and for Measuring Protein Literature Reference 7200002158EN
Total Recovery Vials, 3 x 100 vial/pkg 186000384C Concentration Poster,
Literature Reference 720001993EN Application Solutions for Biopharmaceuticals,
Literature Reference 720002487EN
* This kit is intended to enable existing ACQUITY UPLC systems for AAA applications. Amino Acid Analysis of Pure Protein
4GD½!CC½NM½+HS½BNMS@HMR½SGD½!BB14@F½½5KSQ@½#GDLHRSQHDR½@MC½#NKTLM ½$NBTLDMS@SHNM Hydrolysates with Application of Waters UPLC
and additional hardware accessories needed for AAA applications. Amino Acid Analysis Application,
½ 4GD½2DEHKK½+HS½HR½HMSDMCDC½SN½QDBG@QFD½SGD½!BB14@F½½5KSQ@½BGDLHRSQHDR½SG@S½@QD½@½O@QS½NE½ Literature Reference 720002404EN
the Application Add on Kit. This kit should not be purchased as part of an initial system.

www.waters.com Amino Acid Analysis 37

AMINO ACID ANALYSIS

!BB14@F½!LHMN½!BHC½!M@KXRHR½5RHMF½(0,#
!BB14@F½!M@KXRHR½NE½(XCQNKXR@SD½!LHMN½!BHC

Sample: AQC-Derivatized Amino Acid Standards 19

#NKTLM½ !BB14@F½#NKTLM ½½W½½LL
Temperature: 37 °C
%KTDMS½!½ !BB14@F½%KTDMS½! 17
Eluent B: Acetonitrile 18
Eluent C: HPLC grade water
14
Flow Rate: 1 mL/min
'Q@CHDMS½ !BB14@F½-DSGNC
15
Detection: Fluorescence: lex = 250 nm, lem = 395 nm
13
1 AMQ 16
2 Asp 8 10
3 Ser 6 9
4 Glu A. 50 pmol Standard 3 7 11
4 5
5 Gly 2
1
4 GD½(0,#½!BB14@F½LDSGNC½TSHKHYDR½OQD BNKTLM½CDQHU@SHYDC½QD@FDMSR½ 6 His 12
7 NH3
SG@S½XHDKC½D@RHKX½CDSDBSDC½EKTNQDRBDMBD½@CCTBSR½4GD½!BB1&KTNQ½QD@FDMS ½ 8 Arg
30 Min.
9 Thr
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatizes primary and 10 Ala 19
11 Pro 17
7
secondary amines in a simple, single-step reaction to yield highly stable, 12 Cys
18
13 Tyr
EKTNQDRBDMS½@CCTBSR½7D½NEEDQ½SGD½!BB14@F½LDSGNC½@R½@½RXRSDL½O@BJ@FD½ 14 Val 14
15 Met 1 15
consisting of prepackaged reagents and extensive documentation. 16 Lys
13
17 Ile
18 Leu 16
4 GD½!BB14@F½#GDLHRSQX½0@BJ@FD½BNMS@HMR½SGD½HSDLR½XNT½MDDC½ENQ½TO½SN½ 19 Phe B. 1 pmol Standard
9
10
3
56 11 12
250 analyses of protein and peptide hydrolysate amino acids. 2 4
8

!BB1&KTNQ½2D@FDMS½+HS½&HUD½"NSSKDR½%@BG ˜
30 Min.

½ !BB1&KTNQ½"NQ@SD½"TEEDQ½
!OOKHB@SHNM½NE½SGD½!BB14@F½-DSGNC½SN½SGD½@M@KXRHR½NE½GXCQNKXR@SD½@LHMN½@BHCR½HR½HKKTRSQ@SDC
½ !BB1&KTNQ½2D@FDMS½0NVCDQ 4GD½GHFG½OTQHSX½QD@FDMSR½OQNUHCDC½HM½SGD½!BB14@F½#GDLHRSQX½0@BJ@FD½DM@AKD½GHFG½RDMRHSHUHSX
analysis by minimizing background amino acid content.
½ !BB1&KTNQ½2D@FDMS½$HKTDMS * AQC-6-aminoquinolyl-N-hydroxysuccinimidyl, NHS-N-hydroxysuccinimidyl, AMQ-6-aminoquinoline

!BB14@F½!LHMN½!BHC½!M@KXRHR½#NKTLM˜
3DO@Q@SDR½SGD½@LHMN½@BHC½CDQHU@SHUDR½OQNCTBDC½AX½SGD½!BB1&KTNQ½CDQHU@SH- Injection Volume Guidelines
Y@SHNM½QD@BSHNM½4 GD½!BB14@F½BNKTLM½HR½@½GHFG DEEHBHDMBX½#NKTLM½RODBHEH-
B@KKX½BDQSHEHDC½ENQ½TRD½VHSG½SGD½!BB14@F½LDSGNC½#@QD½@MC½TRD½NE½SGD½ Sample Estimated Sample Quantity* Injection Volume
BNKTLM½HR½CDRBQHADC½HM½SGD½7@SDQR½!BB14@F½!LHMN½!BHC½!M@KXRHR½#NKTLM½ Proteins 0.1 to 1.0 μg 4 to 40 picomole 10 to 20 μL
Care and Use. 0.1 to 5.0 μg 4 to 200 picomole 5 to 10 μL
Peptides 0.02 to 0.20 μg 20 to 200 picomole 10 to 20 μL
!BB14@F½%KTDMS½!½#NMBDMSQ@SD˜ 0.02 to 1.0 μg 200 to 1000 picomole 5 to 10 μL
A premixed concentrated aqueous buffer. Standard 50 picomole 5 μL
*Based on protein average molecular weight=25,000, and peptide average molecular weight=1,000.
!LHMN½!BHC½(XCQNKXR@SD½3S@MC@QC˜
Ten 1 mL ampules of the Amino Acid Hydrolysate Standard. Each ampules
Column and Accessories Dimensions/Qty. Part No.
contains a 2.5 mM mixture of the 17 hydrolysate amino acids with the
exception of cystine (1.25 mM). !BB14@F½#GDLHRSQX½0@BJ@FD½ENQ½½ ½ WAT052875
up to 250 analyses. Includes:
½W½½LL½3@LOKD½4TADR˜ ½ !BB1&KTNQ½2D@FDMS½½ ½W½½L,½UH@KR
½ !BB1&KTNQ½2D@FDMS½!½ ½W½½LF½UH@KR
Used for preparing samples and standards. ½ !BB1&KTNQ½2D@FDMS½"½ ½W½½L,½UH@KR
!BB14@F½#GDLHRSQX½0@BJ@FD½)MRSQTBSHNM½-@MT@K˜ ½ !BB14@F½#NKTLM½ ½W½½LL
½ !BB14@F½%KTDMS½! ½#NMBDMSQ@SD½½½½ ½W½½KHSDQ
$DRBQHADR½SGD½!BB14@F½!LHMN½!BHC½!M@KXRHR½-DSGNC Sample tubes 4 x 72/pkg
Amino Acid Standard, Hydrolysate 10 x 1 mL ampules WAT088122
½ !BB14@F½5RDQ½'THCD½ ½ WAT052874
!BB1&KTNQ½2D@FDMS½+HS ½ ½ WAT052880
)MBKTCDR½!BB1&KTNQ½2D@FDMS½ ½½W½½L,½UH@KR ½!BB1&KTNQ
2D@FDMS½! ½½W½½LF½UH@KR ½!BB1&KTNQ½2D@FDMS½" ½½W½½L,½UH@KR
!BB14@F½#NKTLM½ ½W½½LL½ WAT052885
!BB14@F½%KTDMS½! ½#NMBDMSQ@SD½ ½W½½KHSDQ½ WAT052890
!BB14@F½%KTDMS½" ½ ½W½½KHSDQ½ WAT052895
* The components of this kit are not available separately.
** For use with multi-pump gradient systems.

38 Amino Acid Analysis www.waters.com

 AMINO ACID ANALYSIS

0HBN4@F½(0,#½-DSGNC
A widely used technique for HPLC amino acid analysis is the Waters 0K@RL@½!LHMN½!BHC½0QNEHKD½5RHMF½SGD½0HBN4@F½-DSGNC
0HBN4@F½LDSGNC½"@RDC½NM½@M½NOSHLHYDC½RXRSDL½BNMEHFTQ@SHNM ½OQDO@BJ-
@FDC½QD@FDMSR½@MC½DWSDMRHUD½CNBTLDMS@SHNM ½SGD½0HBN4@F½LDSGNC½OQNUHCDR½

Val
Gln

3 Nitro Tyr (I.S.)

a turnkey, guaranteed approach to modern HPLC amino acid analysis.

Pro
Precolumn derivatization relies on the coupling reaction of the well known

Lys
Met SO2 (I.S.)
Edman Degradation, the reaction of phenylisothiocyanate (PITC) with both

Ala

Reagent
primary and secondary amino acids to form phenylthiocarbamyl (PTC) deriv-

Leu
atives. The method is applicable to any sample including protein hydro-
lysates, physiologic fluids, feeds, foods, and pharmaceutical preparations.

Gly

Tyr

Trp
Tau

Ile
Arg

Orn
Cys

Phe
Ser
0HBN4@F½$DQHU@SHY@SHNM½2D@BSHNM

T hr
Glu

1 Me His
NH3

Met
His
Cit
Asn
R S R

N = C = S + NH2 CH COOH NH C NH CH COOH

66 Min.

2DOQNCTBHAKD½@MC½QDKH@AKD½OK@RL@½@LHMN½@BHC½OQNEHKDR½@QD½NAS@HMDC½HM½½LHMTSDR½TRHMF½7@SDQR½0HBN4@F½-DSGNC
In this analysis, 100 μL plasma was diluted with an internal standard, deproteinized by centrifugal ultrafiltration,
The derivatization of amino acids with PITC is the first step of the well- and derivatized. The methionine sulfone (internal standard) peak represents 25 picomoles.
known Edman degradation reaction. The PTC-amino acid adducts are Courtesy of A.S. Feste, R.W. Drummond, and S.J. Dudrich, Nutritional Support Service,
stable and easily separated by reversed-phase HPLC. A single product is St. Luke Episcopal Hospital, Houston, Texas.

formed for each amino acid. Most reaction by-products and all derivati-
zation reagents are volatile, so they may be removed from the sample
0HBN4@F½!LHMN½!BHC½!M@KXRHR½NE½0QNSDHM½(XCQNKXR@SDR
by vacuum drying.

Column and Accessories Dimensions/Qty. Part No.

Peptide Hydrolysate Amino Acid Analysis
0HBN4@F½#GDLHRSQX½0@BJ@FD½HMBKTCDR½ ½ WAT007360
column, reagent kit, eluents, diluent, manual,
A. Hydrolysate Standard and column heater inserts)
B. 16 Amino Acid Peptide, 40 pmol injected
0HBN4@F½#NKTLM½½ ½W½½LL½ WAT088131
Lys

Peptide Reagent Kit (contains PITC, TEA, and Standards) WAT088123

Composition
Asp 0.99 T hr 0.89
0HBN4@F½%KTDMS½!½½ ½W½½KHSDQ½ WAT088108
Tyr

0HBN4@F½%KTDMS½"½½ ½W½½KHSDQ½ WAT088112

Pro

Glu 0.94 Pro 2.11

Val
Met

Gly 5.14 Val 2.00 0HBN4@F½$HKTDMS½½ ½L,½ANSSKD½ WAT088119

Ile
Ala

Leu

His 0.86 Lys 2.05

Phe
T hr

Cys
Asp

Arg
Glu

0HBN4@F½!LHMN½!BHC½!M@KXRHR½NE½0GXRHNKNFHB½!LHMN½!BHCR
Ser
Gly
His

Column and Accessories Dimensions/Qty. Part No.

Free Amino Acid Chemistry Package (includes WAT091681
column, reagent kit, eluents, diluent, manual,
Rgt

column heater inserts, and sample tubes)

A Free Amino Acid Analysis column 3.9 x 300 mm WAT010950
Reagent Kit (contains PITC, TEA and
B Standards A/N and B) WAT010947
0HBN4@F½%KTDMS½½½ ½W½½KHSDQ½ WAT010960
0HBN4@F½%KTDMS½½½ ½W½½KHSDQ½ WAT010965
12 Min.
0HBN4@F½$HKTDMS½½ ½L,½ANSSKD½ WAT088119

4GHR½½LHMTSD½@M@KXRHR½TRHMF½7@SDQR½0HBN4@F½!LHMN½!BHC½!M@KXRHR½-DSGNC
provides identification and accurate quantitation of the amino acid composition.

www.waters.com Amino Acid Analysis 39

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

Oligonucleotide Purification and Analysis

Synthesis Synthetic Oligonucleotide Length Compared to
T heoretical Yield at Various Coupling Efficiencies
Oligonucleotides are employed in an ever-expanding host of applications,
including use as primers or hybridization probes, to adoption as therapeutic 100

90
agents. In brief, their synthesis is frequently performed via a series of step- 80

wise reactions that result in the addition of specific nucleotides (protected 70

% Overall Yield
at their 5’ hydroxyl end with a dimethoxytrityl group [DMT]) to the solid 60
99.5
50
phase support containing the growing chain. Variations in choice of incom- 40 99
.0
ing nucleotide monomers (both protected ribose and deoxyribose nucle- 30
98
98
.5
20 97 .0
otides), as well as modifications after synthesis, can yield a final product 97
.0
.5
10
containing atypical bases, sugars, or backbone composition. Some common 0
0 40 80 120 160 200
modifications include substitution of sulfur atoms or methyl groups for Length of Oligonucleotide

oxygen on the phosphate backbone (creating phosphorothioates and methyl-

phosphonates) or post-synthesis labeling to create molecular probes used
for various diagnostic investigations.
Isolation
A variety of methods exist for the lab-scale purification (25-500 nmole) of
To keep pace with the ever increasing demand for high-quality oligo-
oligonucleotides. T he advantages as well as disadvantages of techniques are
nucleotides, automated synthesizers capable of producing 0.05 to
outlined in the table below.
thousands of micromoles of product in a single synthesis exist. T hese
instruments and methods frequently possess high coupling-efficiency Analysis
capability with multi-strand synthesis flexibility. Yet, even with
constant tec hnology advancements, the coupling efficiency in eac h W hile polyacrylamide slab gel techniques can resolve long oligonucleotide
synthesis cycle is less than 100% resulting in contamination of the sequences, impurity detection of analyzed samples frequently involves
target oligonucleotide product (e.g., 60 mers) with undesired impuri- use of radio labeled nucleotides or gel staining techniques. T he advent of
ties. T he total yield of full length product is a function of the length of capillary gel electrophoresis (CGE) with on-line detection helped overcome
the desired sequence and the degree of synthesis coupling efficiency some post-run detection issues. However, precise c hemical composition
as shown (right). Most “failure products” (typically labeled N-1, N-2..., determination of each separated oligonucleotide species cannot to deter-
N-x) are shorter sequences generated by a premature coupling failure mined using UV absorption detection. Furthermore, in some environments
of an incoming nucleotide monomer to the growing oligonucleotide the routine use of CGE for oligonucleotide analysis has been problem-
c hain. Some failures contain a missing nucleotide(s) in the middle atic due to method robustness and ruggedness concerns. HPLC, Waters
of sequence, rather than at the end. “Mismatc hed failure sequences” UltraPerformance LC® Technology, and Mass Spectrometry have overcome
having a greater molecular weight (often labeled N+x) than the desired many challenges related to the separation, quantitation, and identification
oligonucleotide product can also be produced from either incomplete of synthetic oligonucleotides. Rugged and robust separation and analysis
post-synthesis deprotection or from branc hing of the growing oligo- methods can frequently be performed in minutes rather than in hours. In
nucleotide c hain during synthesis. addition, quantitation with oligonucleotide composition confirmation is
possible using MS or hyphenated-MS methods.

Advantages Disadvantages
Polyacrylamide Gel Well-established and efficient method. It separates Low mass-loading capacity. Gels are typically overloaded for purification and the resolution is com-
Electrophoresis (PAGE) long oligonucleotides (>50-60 mer). promised. PAGE does not separate N+x sequences. Manual band cutting. Excision is based on markers
without detailed knowledge of target oligo retention. Samples need to be extracted from the gel and
desalted, recovery of target oligonucleotides is low. Method is laborious; it is typically utilized only
when no other technique is suitable for the task.
Ion-Exchange Liquid Trityl-off method. Separation of failure sequences is IEX-LC is efficient only for relatively short oligos (<20-25 mers); longer oligos are poorly resolved.
Chromatography (IEX-LC) due to the backbone charge. Sample is contaminated with high concentration of salts; further desalting is required. IEX columns
packed with non-porous sorbent offer improved resolution, but suffer with low mass load capacity.
When loading exceeds 10-20 nmoles (for 4.6 mm i.d. columns), the resolution is compromised. IEX-LC
does not separate N+x sequences.
Trityl-on Liquid Chromatography Elegant, fast, and universal method for oligos of various length and Does not adequately remove mismatch failure sequences (similarly as the target oligo they carry DMT
(Trytil-on LC; DMT-on LC) sequence. RP columns used with this method have sufficient mass group). DMT group is labile, part of the product may be lost due to the spontaneous detritylation. DMT
load capacity. residue and remaining acid have to removed after the detritylation.
Trityl-off Liquid Chromatography Effectively removes practically all types of failure products. Uses Method requires efficient columns packed with small particle size sorbent. Oligonucleotide retention
(Trityl-off LC; DMT-off LC) volatile solvents; samples do not have to be further desalted. and resolution partially depends on the sequence. Method development for different oligonucleotide
Collected fractions are simply lyophilized and ready for use. RP sequence and length probes is necessary.
columns used with this method have sufficient mass load capacity.
Labeled and dually-labeled oligonucleotide probes can be also
purified. Method is suitable for LC-MS analysis (with MS compatible
ion-pairing buffers).

40 Oligonucleotide Separation Technology www.waters.com

 OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

Oligonucleotide Separation Technology

Waters Oligonucleotide Separation Tec hnology (OST ) columns contain
second-generation hybrid silica BEH Tec hnology particles functionalized
with C18. T he separation of detritylated synthetic oligonucleotide samples
is based on the well-established method of ion-pair, reversed-phase
chromatography. T he availability of 1.7 μm UPLC particles or 2.5 μm HPLC
particles in various column dimensions provides flexibility to meet various
lab-scale isolation or analysis needs and delivers exceptional sample
resolution and superior column life. In addition, Waters manufacturing and
quality control testing procedures help ensure consistent batch-to-batch and
column-to-column performance regardless of application demands.
½ 3DO@Q@SHNM½DEEHBHDMBHDR½DPTHU@KDMS½NQ½ADSSDQ½SG@M
PAGE, CGE, or ion-exchange HPLC methods
½ 2DRNKUD½E@HKTQD½RDPTDMBDR½EQNL½CDSQHSXK@SDC½ETKK½KDMFSG½OQNCTBSR
½ 3B@KD@AKD½BNKTLM½NEEDQHMFR½ENQ½K@A RB@KD½HRNK@SHNM½MDDCR
½ %WBDOSHNM@K½BNKTLM½KHED½ENQ½QDCTBDC½BNRS½ODQ½@M@KXRHR
½ 1#½4DRSDC½VHSG½-@RR02%0½/34
Standard to help ensure
performance consistency

Exceptional Resolution of Separation of Detritylated Oligodeoxythymidine Ladders by Capillary

Gel Electrophoresis (CGE) vs. Ion-Pair, Reversed-Phase Chromatography
Oligonucleotide Mixtures
ACQUIT Y UPLC OST C18, 1.7 m (designed or use with an ACQUIT Y System: Capillary Gel Electrophoresis System
CGE Column: PEG sieving matrix (BioCap 75 μm x 27.5 (to detector) / 34.5 cm (total length)
UPLC System) and XBridge OST C18, 2.5 m columns are well suited for Injection: 45 injection at 5 kV
Running: 15 kV 30
the analysis of detritylated oligonucleotides using ion-pair, reversed- Temp: 30 °C
phase c hromatography. As indicated (see figure on right), separations 15
10

are comparable to that obtained by capillary gel electrophoresis (CGE)

in terms of component resolution yet analyses times are significantly
decreased using Waters UPLC Tec hnology. T he ability to resolve large UV 260 nm

12
22 Min.
oligonucleotide sequences (e.g., N from N-1) is possible due to the
enhanced resolving power obtained using sub-3 m, BEH Tec hnology LC system: Waters ACQUITY UPLC System
Column: ACQUITY OST C18, 1.7 μm (2.1 x 50 mm)
particles. In addition, quantitation with molecular weight c haracter- Mobile Phase: A: 15 mM TEA, 400 mM HFIP, pH 7.9
ization of the separated target oligonucleotide product from failure B: 50% A, 50% MeOH
Flow Rate: 0.4 mL/min 25
sequences is possible using Waters OST columns with hyphenated-Mass Column Temp: 60 °C
Gradient: 40 to 48% B in 4 minutes
Spectrometry methods and MS friendly eluents. (20-24% MeOH) 20
30

Detection: 260 nm 35

0
4 Min.

www.waters.com Oligonucleotide Separation Technology 41

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

Separation of a 15-60 mer Detritylated Impact of Different Ion-Pairing Agents on Varying

Oligodeoxythymidine Ladder Oligonucleotide Sequence Separations

LC System: Waters ACQUITY UPLC System Column Temp.: 60 °C

with PDA and QTof micro MS Gradient: 19 to 26.5.5% B in 60 minutes
Column: ACQUITY UPLC OST C18, Detection: UV: 260 nm, 9-11 % 28-39 %
1.7 μm (2.1 x 50 mm) 10 scans per second ACN in 10 min ACN in 10 min
Mobile Phase: A: 15 mM TEA - 400 mM HFIP, MS: 1 scan per second,
B: Methanol 0.1 sec interscan
Flow Rate: 0.1 mL/min

UV 260 nm

15.5-23.5 %
MeoH in 10 min 23-37 %
ACN in 10 min

MS (TIC)
Improved Oligo separations can
12-20 % be achieved using alternative
ACN in 10 min
IP agents compared to use of
traditional TEAA

0 45 Min. 0 10 Min.

UPLC/MS Analysis Of Interfering Outstanding Column Life

RNA Oligonucleotides Waters OST columns packed with BEH Tec hnology particles have shown
Discovery of the RNA interference (RNAi) mechanism now broadly used remarkable column longevity, under these demanding separation condi-
for silencing of target gene expression has prompted a need for the tions, while maintaining outstanding separation performance. By compari-
analysis of small interfering RNAs (siRNA) molecules. To satisfy the son, significantly reduced column life results when traditional silica-based
need for a robust, fast, and sensitive analysis of 20-25 nucleotides of columns are used under these same demanding separation conditions.
small interfering RNA (siRNA), a UPLC/MS method has been developed
utilizing UPLC OST columns and Synapt HDMS mass spectrometer. Separation of 5-25 mer Detritylated Oligodeoxythymidine Ladder

T he acquisition of the accurate masses allowed for an assignment of the HPLC System: Alliance Bio 2796 with PDA Column temp: 60° C
peaks of 5’-truncated oligomers (failed sequences generated during oligo- Column: XBridge OST C18, 2.5 μm (2.1 x 50 mm) Gradient: 0 – 100% B in 30 min
Mobile Phase: A: 10% MeOH / 90% (10-25 % MeOH).
nucleotide synthesis), as well as some other impurities. T he mass of each (385mM HFIP + 14.3mM TEA) Flow Rate: 1.0 mL/min
B: 25% meOH / 75% Detection: 260 nm, 5 scans per second
peak in the MS chromatogram was deconvoluted using MaxEnt 1 software. (385mM HFIP + 14.3mM TEA)
T he tentative 5’-end failure products are assigned in Figure 2. Nearly the
entire sequence of the parent oligonucleotide was elucidated. MS analysis


also revealed a presence of an extra uridine mononucleotide added to the
target 21-mer RNAi sequence.

LC/MS Analysis of RNA (21 mer)

TIC MS: Synapt MS LC System: Waters ACQUITY UPLC

Capillary: 2000 V Column: ACQUITY UPLC OST C18, 2.1 x 50 mm, 1.7 μm
Sample Cone: 35 V Column Temp.: 60 °C 

Extraction Cone: 3V Sample Injected: 2.5 μL (100 pmole of 21 nt RNAi)
Desolvation Temp.: 200 °C Flow Rate: 0.2 mL/min
Source Temp.: 120 °C Mobile Phase A: 15 mM TEA. 400 mM HFIP
Cone Gas Flow: 50 L/ hr Mobile Phase B: 50% A, 50% methanol
Desolvation Gas Flow: 600 L/ hr Gradient: 20-40% B in 10 min. RNAi
Detection: PDA, 260 nm UV 21-mer         

20 nt
TEA

8.73 min
TEA

Description Particle Size Pore Size Dimension Part No.

U

5800 680 0
ACQUITY UPLC OST C18 * 1.7 m 135Å 2.1 x 50 mm 186003949
m/z

G
U ACQUITY UPLC OST C18 * 1.7 m 135Å 2.1 x 100 mm 186003950
C
C U U
U U G U U Custom ACQUITY UPLC OST C18 * — — — 186003951
C
U A
C U A
XBridge OST C18 2.5 m 135Å 2.1 x 50 mm 186003952
XBridge OST C18 2.5 m 135Å 4.6 x 50 mm 186003953
1 10 Min. XBridge OST C18 2.5 m 135Å 10 x 50 mm 186003954
Custom XBridge OST C18 — — — 186003955
* For use on Waters ACQUITY UPLC Systems

42 Oligonucleotide Separation Technology www.waters.com

 LIFDLKR@IBLQFABPBM>O>QFLKQB@EKLILDV

Scalable DNA and RNAi Separations Purification of siRNA Duplex from Impurities

with Good Product Recovery LC System: Waters Alliance Bio™ Target siRNA
85 nmol duplex
Column: XBridge OST BEH C18
XBridge OST C18 columns are the preferred offering for detritylated oligo- 4.6 x 50 mm, 2.5 μm
nucleotide purifications due to the availability of column sizes designed Column Temp:
Flow Rate:
20 °C
1.0 mL/min
to meet lab-scale isolation requirements. As indicated in the table below, Mobile Phase A: 0.1 M TEAA, pH 7.0
Mobile Phase B: 20% ACN in A 15 nmol
the c hoice of XBridge OST C 18 column dimension and operating flow Gradient: 25 - 75% B in 30.0 min
Detection: PDA, 260 nm
rate depends primarily on the scale of the synthesis reaction mixture.
For example, a 4.6 x 50 mm column containing XBridge OST C 18, RNA
Impurities
siRNA
Impurities
2.5 m material is an excellent selection when oligonucleotide mass
loads are less than or equal to 0.2 mol. Selection of the appropriate
column size for the amount of oligonucletide sample loaded is recom- 1.5 nmol
0.15 nmol
mended to maximize component resolution and recovery of the target
product from non-desired failure sequences. 0 25 Min.

For researchers involved in gene silencing it is often necessary to work

with RNA of high purity. Crude synthetic oligonucleotides used for gene
knockout are typically purified. T he figure below illustrates a a lab-scale XBridge OST C18 Column Selection Guide
purification of 21 mer RNA at various column loads. Using OST column for Detritylated Oligonucleotide Purification
chemistry and a Alliance System, large quantities of crude single stranded
Dimensions Approx Mass Load** mg*** Flow Rate
RNA can be successfully purified yielding material of high purity, ca. 95%,
with an estimated yield of 55% based on collected peak area to the total 2.1 x 50 mm 0.04 μmoles 0.2 mg 0.2 mL/min
4.6 x 50 mm 0.20 μmoles 1.0 mg 1.0 mL/min
peak area of the sample.
10 x 50 mm 1.00 μmoles 4.5 mg 4.5 mL/min
In addition, OST columns are well suited for analysis and purification of 19 x 50 mm* 4.00 μmoles 16.0 mg 16.0 mL/min
30 x 50 mm* 9.00 μmoles 40.0 mg 40.0 mL/min
siRNA. As shown in the figure to the right siRNA is well resolved from
50 x 50 mm* 25.00 μmoles 110.0 mg 110.0 mL/min
single stranded RNA and truncated duplexes.
* OST Custom Column
** Values are only approximates and vary depending on oligonucleotide length,
Purification of Single Stranded RNA base composition, and “heart-cutting” fraction collection method used.
*** Estimated for average oligonucleotide MW and synthesis yield.

LC System: Waters Alliance Bio

Column: XBridge OST BEH C18, 4.6 x 50 mm, 2.5 μm
Column Temp: 60 °C
Flow Rate: 1.0 mL/min
Mobile Phase A: 0.1M TEAA, pH 7.5
Mobile Phase B:
Gradient:
20% Acetonitrile in A
30 – 52.5% B in 10.0 min (0.15% ACN/min)
Purified Oligonucleotide Literature
Purity > 95%
Detection: PDA, 260 nm References
140 nmol
RNAi Duplex Analysis and UPLC/MS Analysis of Interfering RNA
N-1
Purification Application Note, Oligonucleotides Application Note,
* Literature Reference 720002800EN Literature Reference 720002412EN
Purity ≈ 78% 0 6
84 nmol Optimization of LCT Premier XE MS Settings UPLC Analysis of Phosphorothioate
97.8% UPLC purity (area)
for Oligonucleotide Analysis Application Note, Oligonucleotides: Method
Literature Reference 70002798EN Development Application Note,
Literature Reference 720002405EN
UPLC Separation of DNA
28 nmol Duplexes Application Note, UPLC/MS Separation of Oligonucleotides
Literature Reference 720002741EN in Less than Five Minutes: Method
Development Application Note,
UPLC/UV-MS Analysis of Phosphorothioate Literature Reference 720002387EN
Oligonucleotides Application Note,
1.4 nmol
Literature Reference 720002621EN Oligonucleotide Separation Technology:
0 18 Min.
Synthesis Challenges and HPLC Isolation
Semi-Preparative Scale Single-Stranded RNA Options Application Note,
Purification Application Note, Literature Reference 720002386EN
Literature Reference 720002602EN
UPLC Separation of Oligonucleotides: Method
Real-Time Analysis of RNAi Development Application Note,
Duplexes Application Note, Literature Reference 720002383EN
Literature Reference 720002573EN
HPLC and UPLC Columns for the Analysis of
UPLC/UV-MS Analysis of Oligonucleotides Application Note,
Application Note Oligonucleotides, Literature Reference 720002376EN
Literature Reference 720002413EN

www.waters.com Oligonucleotide Separation Technology 43

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

Columns for Large DNA/RNA Species

In general, molecular biology methods for manipulation of DNA rely on Separation of Duplex DNA Fragments: HaeIII and MspI
restriction enzymes, polymerase-c hain reaction (PCR), and sequencing Restriction Enzyme Digests of pBR322 Plasmid
techniques. Using these methods, genomic DNA is typically converted into
shorter double stranded (ds)DNA sequences, typically 100-1000 base LC System: Waters ACQUITY
Column: ACQUITY UPLC PST BEH300 C18 , 2.1 x 50 mm, 1.7μm
pairs (bp) in length. T he shorter dsDNA molecules are often analyzed or Column Temp: 50 °C
isolated by methods suc h as slab gel or capillary electrophoresis. Use Flow Rate: 0.2 mL/min
Mobile Phase A: 0.1M TEAA, pH 7.0
of Waters ACQUITY BEH300 C18 reversed-phase or GenPak FAX anion- Mobile Phase B: 20% ACN in A
Gradient: 57.5 – 84.5 % B in 20.0 min
exchange columns offer alternatives to more traditional electrophoretic Detection: PDA, 260 nm
methods and are particularly well suited for various analytical and small-
scale purification applications.

Description Dimension Particle Size Part No.

1 20 Min.
ACQUITY UPLC BEH300 C18 2.1 x 50 mm 1.7 μm 186003685

Gen-Pak FAX Anion-Exchange Column

Waters Gen-Pak FAX columns offer the highest resolution available in Separation of DNA Restriction Fragments
anion-exchange HPLC of nucleic acids. T he Gen-Pak FAX column contains
a weak anion exchanger based on DEAE functionalized non-porous resin. Sample: 3.0 μg of BstN1 digest of pBR322 1. 13 bp
Column: Gen-Pak FAX, 4.6 x 100 mm column 2. 121 bp
It contains 2.5 μm particles and is well suited for analytical and micro- Eluent A: 25 mM Tris/Cl, 1 mM EDTA, 3. 383 bp
preparative applications. Eluent B:
pH 8.0
25 mM Tris/Cl, 1 mM EDTA,
6
4. 929 bp
5. 1060 bp
1.0 M NaCl, pH 8.0 6. 1857 bp
Gradient: 25-75% B in 30 min, linear
Chromatography of a PCR Amplification Mixture Generated Flow rate: 0.75 mL/min
Temperature: 30 °C
using 3 ng and 1 fg of HBV S-gene Template Detection: 260 nm
45

Sample: PCR amplification mixture using 3 ng HBV-S gene template Gradient: 40 - 75% B in 30 min., Linear 3
Column: Gen-Pak FAX (4.6 x 100 mm) Flow Rate: 0.75 mL/min.
Eluent A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0 Temperature: 30 °C 2
Eluent B: 25 mM Tris/Cl, 1 mM ESTA, 1.0M NaCl, pH 8.0 1

dNTPs and Taq enzyme 465 bp PCR product 35 Min.

0.040
Absorbance 260 nm

Nonspecific DNA sequences

Description Dimensions Part No.
0.000
0 15 30 Min.
Gen-Pak FAX Column 4.6 x 100 mm WAT015490

44 Oligonucleotide Separation Technology www.waters.com

 OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

MassPREP OST Standard

½ #NMS@HMR½@½B@QDETKKX½CDEHMDC½LHWSTQD½NE½RXMSGDRHYDC
oligodeoxythymidine fragments 15 nt
20 nt
½ 5RDETK½HM½SDRSHMF½@MC½BNMEHQLHMF½(0,#50,# ½,#-3 30 nt
25 nt 35 nt
and column performance for oligo applications
½ %@BG½1#½SDRSDC½@MC½RGHOODC½VHSG½@½BDQSHEHB@SD½NE½@M@KXRHR
T he pre-packaged MassPREP Oligonucleotide Separation Technology (OST)
Standard is designed for verification of HPLC/UPLC instrument and column
performance for analysis of synthetic oligonucleotides. Approximately
equimolar amounts of 15, 20, 25, 30, and 35 nucleo-tide (nt) long
oligodeoxythymidines are lyophilized and packaged in 1.5 ml LC vials.
T hese vials are vacuum-sealed in foil pouches to reduce degradation that
0 10 Min.
can occur by excessive exposure to light and air. Approximately 1 nmole of
each oligonucleotide is present in the vial. Waters ACQUITY UPLC analysis of MassPREP OST Standard on an ACQUITY UPLC OST C18, 1.7 μm column.
The main components are labeled. Small peaks eluting between labeled oligonucleotides are N-1, N-2, etc.
failure sequences generated during the oligonucleotide syntheses. The ACQUITY UPLC system is equipped
Description Qty. Part No. with 50 μL standard mixer and PDA detector (260 nm).

MassPREP OST Standard 1/pk 186004135

Oasis μElution Plates

Oligonucleotide Desalting by Solid-Phase Extraction Effective Use of Oassis HLB for Oligonucleotide Desalting Prior to MALDI-TOF MS

½ 2DLNUDR½R@KS½OQHNQ½ ½ (HFG½RDMRHSHUHSX
to MS analysis MALDI-TOF Spectrum of 16 mer Prior to Desalting
½ 3@LOKD½BNMBDMSQ@SHMF
½ ,NV½DKTSHNM½UNKTLDR
½ (HFG½SGQNTFGOTS 900

800

700
Matrix Solution: 70 mg/mL 3-hydroxypicolinic acid
and 40 mM Ammonium Acetate
600 Mode: Negative Ion
Abundance

500

400

300

200

100

0
4000 4500 5000 5500
M/Z

Desalting of synthetic oligonucleotides is essential for MS analysis (QC,

MALDI-TOF Spectrum of 16 mer After Desalting
genotyping applications and SNP analysis). Waters new μElution plate is
using Oasis HLB μElution Plate
an excellent choice for high throughput analysis with minimal amount of
sample. T he Oasis® μElution plate combines patented plate design, proven
Oasis c hemistries, and generic protocols enabling elution volumes 1800

Matrix Solution: 70 mg/mL 3-hydroxypicolinic acid

1600
and 0.04 M Ammonium Acetate
as low as 25 μL. Now for the first time you can perform SPE clean- Mode: Negative Ion
1400

up and concentration of very small sample volumes. T he Oasis HLB 1200

sample extraction products incorporate a patented copolymer made

Abundance

1000

from a balanced ratio of two monomers; the lipophilic divinylbenzene and 800

the hydrophilic N-vinylpyrolodone that is ideally suited for this application. 600

400

200

Description Part No.

0
4000 4500 5000 5500
M/Z

Oasis HLB μElution Plate (for oligonucleotides) 186001828BA

www.waters.com Oligonucleotide Separation Technology 45

 [ EXCELLENCE ]

BETTER RESULTS FROM YOUR

MS OR LC/MS SYSTEM

GREATER ACCURACY
INCREASED SENSITIVITY
REPRODUCIBLE DATA

MASSPREP™ SAMPLE PREP FOR MASS SPECT ROMET RY AND LC/MS

You need high quality results from your research and have made a significant
investment in your mass spectrometry equipment. Obtain the best possible
results by using the highest quality MS consumables from Waters.

The MassPREP™ family of products includes conveniently packaged

standards, sample prep plates, and application specific kits.

To discover how Waters consumables can help you, visit

www.waters.com/biosep

© 2009 Waters Corporation. Waters, MassPREP and The Science of What’s Possible
are trademarks of Waters Corporation.
 MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

MS and LC/MS Consumables for Biomolecules

The use of mass spectrometry is increasing in laboratories involved with
proteomics and the characterization of biopharmaceuticals. Sample prepara-
tion, the testing of system performance with simple standards, the use high
quality reagents and matrices, and the LC process are critical to the quality
of results.
Only when all these elements are considered can the highest quality results
can be obtained. Waters has developed MS and LC/MS consumables specifi-
cally designed to ensure the best results from MS and LC/MS systems.

LC/MS The MassPREP family of products includes conveniently packaged,

highly-purified standards, specialty plates, and kits and reagents.

Protein
Proteomics
Characterization/QC
RapiGest SF RapiGest SF
MassPREP Protein Digestion Standards MassPREP OST Standard
MassPREP Digestion Standards Mixtures MassPREP Protein Digestion Standards
MassPREP Peptide Reference Standards MassPREP Peptide Reference Standards
MassPREP Phosphopeptide Standards MassPREP Desalting Cartridges
NanoEase Traps, Capillary, and Nano Columns NanoEase Traps, Capillary, and Nano Columns
MassPREP Phosphopeptide nanoACQUITY UPLC Traps, Capillary,
Enrichment Kit and Nano Column and 2D Kit
nanoACQUITY UPLC Traps, Capillary,
and Nano Column and 2D Kit
The NanoEase family contains high performance
capillary, nano-scale, and trapping columns.

nanoACQUITY UPLC columns consist of a complete line of columns

specifically designed for the nanoACQUITY UPLC system.

www.waters.com MS and LC/MS Consumables for Biomolecules 47

MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

RapiGest SF and MassPREP Oligonucleotide,

Peptide, and Protein Digest Standards
Prepackaged MassPREP synthetic oligonucleotide peptide and protein digest
standards eliminate the need to prepare, test, and store these materials
for use in LC or LC/MS applications. In addition, RapiGest™ SF Surfactant is
valuable in the enzymatic digestion of proteins for faster, higher quality,
and more reproducible sample preparation.

MassPREP Peptide Standards

T he MassPREP peptide standard mixture contains a void volume (V O) Baseline HPLC Resolution of Nine Peptides
column marker and nine carefully selected peptides with a broad range of Contained in MassPREP Standard Mixture
polarities and isoelectric points. T he MassPREP standard is useful to test
UPLC and HPLC columns and systems dedicated to peptide separations.

Angiotensin I
Angiotensin II

Enolase T35
Renin substrate
Angiotensin frag. 1-7
0.5

Components contained in MassPREP Peptide Standard Mixture

Bradykinin

Enolase T37
Component Molecular weight **
RASG-1
AU

Peak # name (g/mol) pKa Peptide sequence

Melittin
Allantoin

1 Allantoin (V0 marker) 158.0440 – –

2 RASG-1 1000.4938 9.34 RGDSPASSKP
3 Angiotensin frag.1-7 898.4661 7.35 DRVYIHP
0.0
4 Bradykinin 1059.5613 12.00 RPPGFSPFR
0 10 20 30 Min.
5 Angiotensin II. 1045.5345 7.35 DRVYIHPF
6 Angiotensin I. 1295.6775 7.51 DRVYIHPFHL Chromatography of MassPREP Peptide Standard Mixture obtained on a BioSuite C18,
7 Renin substrate 1757.9253 7.61 DRVYIHPFHLLVYS 3 μm Peptide Analysis – A Column (2.1 x 150 mm) performed on a Waters Alliance HPLC System.

8 Enolase T35 1871.9604 7.34 WLTGPQLADLYHSLMK

9 Enolase T37 2827.2806 3.97 YPIVSIEDPFAEDDWEAWSHFFK
10 Melittin 2845.7381 12.06 GIGAVLKVLTTGLPALISWIKRKRQQ Description Qty./Pack Part No.
MassPREP Peptide Standards 1 vial 186002337
* BioSuite C18 3 μm, Peptide Analysis - A Column (2.1 x 150 mm). Eluent A: 0.02% TFA MassPREP Peptide Standards 5 vials 186002338
in water, Eluent B: 0.016% TFA in acetonitrile. Gradient: 0-50% B in 30 minutes. Flow:
Note: The peptide mixture contains approximately 1.5 μg (~1 nmole) of each peptide
0.20 mL/min. Temp: 40 °C.
** Monoisotopic molecular weight

48 MassPREP Standards and RapiGest www.waters.com

 JP>KAI@,JP@LKPRJ>?IBPCLO?FLJLIB@RIBP

MassPREP Protein Digestion Standards

T he MassPREP protein digestion sandards are prepared under strict quality
Description Qty./Pack Part No.
control and contain no undigested standard proteins, trypsin, or other
MassPREP Digestion Standard Kit 1 vial from each digestion 186002330
hydrophilic components. Test results from each batch of digestion standards
Standard (5 vials total) Kit Consists of:
are provided on the included Certificate of Analysis report. MassPREP Phosphorylase b Digestion Standard 1 vial 186002326
MassPREP Bovine Hemoglobin Digestion Standard 1 vial 186002327
MassPREP ADH Digestion Standard 1 vial 186002328
MassPREP BSA Digestion Standard 1 vial 186002329
MassPREP Enolase Digestion Standard 1 vial 186002325

MassPREP Phosphopeptide Standards

½ #NMS@HMR½OGNROGNRDQHMBD ½OGNROGNSXQNRHMD T he MassPREP phosphopeptide standard-enolase contains four phosphopep-
and phosphothreonine peptides tides based on tryptic yeast enolase peptides. T he MassPREP enolase digest
with phosphopeptide mix contains equimolar mixture of enolase digest and
½ 5RD½SN½NOSHLHYD½OGNROGNODOSHCD½CDSDBSHNM½HM
four phoshopeptides.
LC/MS, LC/UV, and MALDI-MS
½ #@M½AD½TRDC½@R½BNMSQNK½R@LOKDR
Phosphorylated protein and peptides are difficult to detect and character- Each new phosphopeptide standard contains
ize due to their low abundance and low ionization efficiency. Using these the following four purified peptides:
standards, scientists have greater control over sample preparation, with the Synthetic Amino Acid
option to use pure peptides or to define phosphopeptides to unmodified Phosphopeptides Sequence Residue [M + H]+ [M + 2H]2+
peptide ratios. Enolase T18 1P NV PL(pY) K 126-131 813.3912 407.1995
Enolase T19 1P HLADL (pS)K 132-138 863.4028 432.2053
Enolase T43 1P VNQIG (pT)LSES IK 346-357 1368.6776 684.8428
LC MS MassPREP Phosphopeptide Standard - Enolase
Enolase T43 2P VNQIG TL(pS)E(pS) IK 346-357 1448.6439 724.8259
T18 1P
T43 1P
T43 2P T hese standards can be used on many Waters instrument systems, including the MALDI micro
T19 1P
MX™, ACQUITY UPLC, Alliance , ZQ™, Q-Tof™, LCT Premier, and nanoACQUITY UPLC systems.
Either UV or MS detection is possible.

10 15 20 25 30 35 40 45 Min.

MassPREP Enolase Digest with Phosphopeptides Mix

T19 1P
MassPREP Phosphopeptide Standards
T18 1P T43 2P T43 1P w/enolase peptides
w/enolase
peptides
Description Qty./Pack Part No.
MassPREP Phosphopeptide Standard Enolase 186003285
MassPREP Enolase Digest with Phosphopeptides Mix 186003286
10 15 20 25 30 35 40 45 Min.
MassPREP Phosphopeptide Sample Kit - Enolase 2 vials - 186003287
Kit Consists of:
MassPREP Phosphopeptide Standard
Enolase 1 nmol/vial, 1 vial 186003285
MassPREP Enolase Digestion Standard 1 vial 186002325

www.waters.com MassPREP Standards and RapiGest 49

MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

MassPREP Digestion Standard Mixtures

½ 5RDC½SN½DMRTQD½@M½@M@KXSHB@K½RXRSDLR½@AHKHSX½SN½OQNUHCD½ANSG MPDS Mix 1 and MPDS Mix 2 Separation Using ACQUITY UPLC BEH
qualitative protein analysis and relative protein quantification C18, 1.7 μm, 75 μm x 100 mm nanoACQUITY UPLC Column
½ 9HDKCR½GHFG½BNMEHCDMBD½@MC½BNUDQ@FD½CTQHMF½DWOQDRRHNM½OQNEHKHMF
of complex protein samples (by spiking the standards)
½ $DRHFMDC½@R½RS@MC@QCR½ENQ½SGD½7@SDQR½0QNSDHM½%WOQDRRHNM½3XRSDL
MassPREP Digestion Standards consist of two standard mixtures (MPDS MPDS Mix 2
Mix 1 and MPDS Mix 2), which were prepared by individually digesting
Yeast Alcohol Dehydrogenase (ADH, SwissProt P00330), Rabbit Glycogen
Phosphorylase b (GPB, SwissProt P00489), Bovine Serum Albumin (BSA,
SwissProt P02769), and Yeast Enolase I (ENO, SwissProt P00924) with
sequencing grade trypsin, and producing mixtures in the indicated molar
ratios. The MPDS mixtures are purified, and do not contain undigested
MPDS Mix 1
target protein, trypsin, other hydrophilic components or salts.

MPDS Mix 1 and Mix 2 Contents

Sample: Column: nanoACQUITY UPLC BEH C18,
MPDS Mix 1 MPDS Mix 2 MPDS Mix 1: Alcohol Dehydrogenase 1.7 μm, 75 μm x 100 mm
Protein [Molar ratio, Amount (pmol)] [Molar ratio, Amount (pmol)] 25 fmol/μL, PhosphorylaseB 25 fmol/ Mobile Phase A: 0.1% FA in H2O
μL; Enolase 25 fmol/μL; Bovine Serum Mobile Phase B: 0.1% FA in ACN
Albumin 25 fmol/μL MPDS Mix 2: Alcohol Gradient: Linear Gradient from 3 to 40% B in 90 min.
ADH 1.0, 50 pmol 1.0, 50 pmol Dehydrogenase 25 fmol/μL; Phosphorylase Injection Volume: 2 μL
B 12.5 fmol/μL; Enolase 50 fmol/μL; Detection: MS on Q-Tof Premier (m/z 50-1990)
GPB 1.0, 50 pmol 0.5, 25 pmol Bovine Serum Albumin 200 fmol/μL Ionization: ESI
Mode: Positive
ENO 1.0, 50 pmol 2.0, 100 pmol
BSA 1.0, 50 pmol 8.0, 400 pmol
Description Qty. Part No.
MassPREP Digestion Standard Mix 1 1/pk 186002865
MassPREP Digestion Standard Mix 2 1/pk 186002866

MassPREP OST Standard

½ #NMS@HMR½@½B@QDETKKX½CDEHMDC½LHWSTQD½NE½RXMSGDRHYDC
oligodeoxythymidine fragments 15 nt
20 nt
½ 5RDETK½HM½SDRSHMF½@MC½BNMEHQLHMF½(0,#50,# ½,#-3 30 nt
25 nt 35 nt
and column performance for oligo applications
½ %@BG½1#½SDRSDC½@MC½RGHOODC½VHSG½@½BDQSHEHB@SD½NE½@M@KXRHR
T he pre-packaged MassPREP Oligonucleotide Separation Technology (OST)
Standard is designed for verification of HPLC/UPLC instrument and column
performance for analysis of synthetic oligonucleotides. Approximately
equimolar amounts of 15, 20, 25, 30, and 35 nucleotide (nt) long
oligodeoxythymidines are lyophilized and packaged in 1.5 ml LC vials.
T hese vials are vacuum-sealed in foil pouches to reduce degradation that
0 10 Min.
can occur by excessive exposure to light and air. Approximately 1 nmole of
each oligonucleotide is present in the vial. Waters ACQUITY UPLC analysis of MassPREP OST Standard on an ACQUITY UPLC OST C181.7 μm column.
The main components are labeled. Small peaks eluting between labeled oligonucleotides are N-1, N-2, etc.
failure sequences generated during the oligonucleotide syntheses. The ACQUITY UPLC system is equipped
with 50 μL standard mixer and PDA detector (260 nm).
Description Qty./Pack Part No.

MassPREP OST Standard 1 vial 186004135

50 MassPREP Standards and RapiGest www.waters.com

 MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

RapiGest SF Protein Digestion Surfactant

RapiGest SF, a novel Surfactant improves protein enzymatic digestion Numerous published scientific articles and presentations have documented
in terms of speed and peptide recovery. T he unique features of this the benefits of RapiGest SF use to improve the protein sequence coverage
product are: and reduce the sample preparation time. T he areas of application are
diverse, ranging from proteomic research to therapeutic protein character-
½ )LOQNUDR½RNKTAHKHSX½NE½GXCQNOGNAHB½OQNSDHMR
ization and is effective for in-solution digestion protocols.
for improved enzymatic digest
½ #NLO@SHAKD½VHSG½U@QHNTR½DMYXLDR How RapiGest SF works

½ 5MKHJD½BNMUDMSHNM@K½CDM@STQ@MSR ½RapiGest SF
does not inhibit enzyme activity
RapiGest SF
½ 2DCTBDR½SGD½CHFDRSHNM½SHLD ½QDPTHQDR½KDRR½DMYXLD
to achieve optimum digestion
Cleavage Sites
Cleavage Sites
½ )LOQNUDR½SGD½CHFDRSHNM½NE½DMYXLD½QDRHRS@MS
proteins such as membrane proteins
½ #NLO@SHAKD½VHSG½BTQQDMS½OQNSDHM½R@LOKD½OQDO½OQNSNBNKR
RapiGest shows significant advantage over other denaturants
½ $DBNLONRDR½@S½KNV½O(½@MC½CDFQ@C@SHNM½OQNCTBSR½CN½
since it is not disruptive to endoprotease activity.
not interfere with LC/MS or MALDI MS analysis
Trypsin * Trypsin Trypsin * Trypsin
½ $NDR½MNS½B@TRD½OQNSDHM½LNCHEHB@SHNMR Solution Activity (%) ** Solution Activity (%) **

No additive 100 0.1% SDS/0.1% RapiGest 67

1 hour Proteolysis of Myoglobin Using Various Endoproteases 0.1% RapiGest 100 50% Methanol 29
0.5% RapiGest 100 50% Acetonitrile 87
A) 0.1% (w/v) RapiGest B) Control
0.1% SDS 24 1M Urea 97
Intact Myoglobin 0.5% SDS 1 2M Urea 83
Asp-N

* .5 μg of trypsin in 50 mM ammonium bicarbonate, pH 7.9; 0.2 mM of BEAA

** Measured as delta BEAA absorbance @ 253 nm (slope within 5 min)
Lys-C

O
(CH2)3–SO3-Na+ CH3(CH2)10
O Low pH
Glu-C
O O
(CH2)3–SO3-Na+
CH3(CH2)10 O
5 10 15 20 25 30 Min. 5 10 15 20 25 30 Min.
HO OH

RapiGest hydrolyzes under acidic condition (half life time = 7.6 minutes at pH 2).
Therefore, it is compatible with LC/MS and MALDI MS analysis.
Use of RapiGest SF to Assist in Protein Deglycosylation

A) Control
Description Part No.
Deconvoluted Mass
Rapi Gest SF 1 mg vial 186001860
Rapi Gest SF 1 mg vial (5 pack) 186001861
Rapi Gest SF 10 mg vial 186002123
B) 0.1% OG
Rapi Gest SF 50 mg vial 186002122
Rapi Gest SF Custom 186002118

C) 0.1% RapiGest SF

Literature
References
800 1000 1200 1400 1600 1800 2000 42000 43000 44000 45000 46000
m/z mass
Enzyme-Friendly, Mass A complete peptide mapping A rapid sample prepara-
LC/MS spectra of deglycosylated ovalbumin are shown. A) Ovalbumin was solubilzed without the use of denatur- Spectrometry-Compatible of membrane proteins: a tion method for mass
ant, and was not deglycosylated. B) Ovalbumin was denatured using 0.1% n-octyl-b-glycopyranoside (OG) and Surfactant for In-Solution novel surfactant aiding the spectrometric characteriza-
deglycosylated. C) Ovalbumin was denatrued in 0.1% RapiGest SF solution and deglycosylated. The MS scans Enzymatic Digestion of enzymatic digestion of bacte- tion of N-linked glycans,
were deconvoluted to the MW of the protein. Complete deglycosylated was observed after 2 hours deglycosylation Proteins, Yu YQ, Gilar M, Lee riorhodopsin, Yu YQ, Gilar M, Ying Qing Yu, Martin Gilar,
for the RapiGest SF solubilized ovalbumin. PJ, Bouvier ES, Gebler JC, Gebler JC, Rapid Commun Jennifer Kaska and John C.
Anal Chem. 2003 Nov 1; Mass Spectrom. 2004; 18 Gebler, Rapid Commun Mass
75(21):6023-8. (6):711-5. Spectrom. 2005;
19: 2331-2336.

www.waters.com MassPREP Standards and RapiGest 51

MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

MassPREP On-Line Desalting Devices

½%EEDBSHUDKX½CDR@KSR½OQNSDHMR½XHDKCHMF½HLOQNUDC½,#-3½QDRTKSR Excellent Recovery with No Detectable Carryover
½&@RS½NM KHMD½LDSGNC½ENQ½GHFG½SGQNTFGOTS½@OOKHB@SHNMR Competitor’s On-line Desalting Cartridge
½%WBDKKDMS½OQNSDHM½QDBNUDQHDR½@MC½MN½CDSDBS@AKD½B@QQXNUDQ
Inject #4 = 10 μL PBS Blank
½½HMIDBSHNMR½EQNL½@½RHMFKD½B@QSQHCFD

Inject #3 = 10 μL mAb
(1 μg/μL PBS)

Inject #2 = 10 μL PBS Blank,

following a 5 μg mAb injection

T he MassPREP on-line desalting column can effectively desalt proteins

prior to LC/MS analyses. Because non-volatile salts (e.g., NaCl) can Waters MassPREP On-line Desalting Cartridge

suppress ionization of intact proteins leading to poor detection sensitiv-

ity, it is important to remove or significantly minimize the introduction Inject #3 = 10 μL PBS Blank

of these compounds into the mass analyzer. T he reversed-phase, phenyl

material contained in MassP REP on-line column successfully “traps” Inject #2 = 10 μL mAb
(1 μg/μL PBS)
proteins, allowing the salts to be washed to waste prior to protein elution
into the mass spectrometer. With an optimized LC/MS method, cycle
Inject #1 = 10 μL PBS Blank
times as low as 4 minutes (for intact antibody) and 10 minutes (for
reduced antibody) are achievable.

Description Qty. Part No. “Column-related carryover” from previous protein sample injections can
compromise the integrity of collected LC/MS data. Compared to results obtained
MassPREP Micro Desalting Column 1/pk 186004032 on a competitive on-line desalting cartridge (top), excellent sample recovery is
MassPREP On-line Desalting Cartridge (2.1 x 10 mm)* 2/pk 186002785 obtained with Waters MassPREP on-line desalting cartridge (bottom).
UPLC Intact Mass Analysis Application Kit** (Includes MassPREP
Micro Desalting Column and ACQUITY Tubing Kit) 1/pk 176001519
Sentry 2.1 x 10 mm Guard Cartridge Holder.* Waters MassPREP Micro Desalting Column (2.1 x 5 mm)
Required for use of MassPREP On-line DesaltingCartridge 1/pk WAT097958
Experimental
LC System: Waters ACQUITY UPLC system
** See: UPLC Intact Mass Analysis Application Kit Manual (715001664) MS System: Waters LCT Premier™ ESI-TOF MS/Synapt HDMS
Ionization Mode: ESI Positive, V mode
Eluent A: 0.1% Formic Acid (H2O)
Waters MassPREP On-Line Desalting Cartridge (2.1 x 10 mm) Eluent B: 0.1% Formic Acid (ACN)
Column temp: 80 ˚C

Experimental
LC System: Alliance 2796 Separation Module Pre-run Blank Max:2 Counts
MS System: Q-Tof micro™, ESI Positive
Eluent A: 0.1%Formic Acid (H2O) Eluent B: 0.1% Formic Acid (ACN)
0.5 μg Intact IgG1 754 Counts
Inject #100 = 5 μL PBS Blank
Post-run Blank 5 Counts

Inject #99 = 5 μL mAb Combined ESI-TOF mass spectra of an intact IgG1 antibody from a 4 minutes LC/MS analysis.
(1 μg/5 μL PBS) The results reveal no detectable carryover following a 0.5 μg injection of the antibody.

Inject #98 = 5 μL BSA MS MS

(1 μg/5 μL PBS) Light Chain Heavy Chain

Inject #97 = 5 μL PBS Blank

TIC
(1.3 μg load)

Over a series of 100 injections, satisfactory results were obtained for BSA and a mAb,
as shown for injections #97-100 on a MassPREP on-line desalting cartridge.
Total ion chromatogram (TIC) from UPLC/MS analysis of light and heavy chains
Reference Desalting of Proteins Using MassPREP On-line Desalting Cartridges Prior to from a reduced IgG1 antibody. A 10 min LC/MS run largely resolved the earlier
Mass Spectrometry. 2005 Waters Applications Note 720001077EN eluting light chain from the later eluting glycosylated heavy chains.

52 Traps, Columns, and Cartridges www.waters.com

 MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

NanoEase Trap, Capillary, and Nano Columns

Waters NanoEase Trapping, Capillary, and Nano columns, contain carefully-
selected, reversed-phase and ion-exchange chemistries for bioseparations.
T he column offerings are developed and intended for use on various tradi-
tional low pressure, capillary, and nano flow LC Systems.

NanoEase Trapping Columns

½ .@MN%@RD½SQ@O½šCHQDBS½BNMMDBS›½CDRHFM½LHMHLHYDR BSA Digest Using NanoEase Symmetry300 C18 Trap Columns
band broadening
½ 3#8½NEEDQHMF½ENQ½$½@OOKHB@SHNMR½ Column: NanoEase Symmetry300 C18
180 μm x 150 mm
½ 20½NEEDQHMFR½ENQ½R@LOKD½CDR@KSHMF½NQ½BNMBDMSQ@SHNM Trapping Columns: NanoEase Symmetry300 C18 Trap
Mobile Phase: A: 98% H2O/2% ACN/0.1% FA
B: 98% ACN/2% H2O/0.1% FA
Gradient Conditions: Time %A %B
0.00 95.0 5.0
30.00 50.0 50.0
31.00 20.0 80.0
41.00 20.0 80.0
Flow Rate: 3.6 μL/min
Injection Volume: 1 μL
Temperature: 20 °C

MS Conditions:
Ion Source: Electrospray positive
Source Temperature: 80 °C
Desolvation
Temperature: 150 °C
Instrument: Waters CapLC with
Q-tof micro technology

Symmetry300 C18, Atlantis dC18, and strong cation-exc hange (SCX)

NanoEase trap columns are robust devices that can effectively separate
complex samples or remove buffers and high concentrations of salts through
many injections. T he reversed-phase trap design minimizes peak band
broadening while the design of the SCX device provides increased binding
capacity. T hese devices generate low backpressure and yield good
recovery to the subfemtomole peptide level. In addition, NanoEase
0 10 12.5 15 17.5 20 22.5 25 27.5 30 Min.
trap column “direct connect” design allows for easy configuration and
replacement in a six or a ten-port valve for either one-dimensional or
two-dimensional LC/MS analyses.
Description Particle Size Dimension Part No.
Symmetry300 C18 Trap Column 5/pack 5 μm 0.18 x 23.5 mm 186002622
Atlantis dC18 Trap Column 5/pack 5 μm 0.18 x 23.5 mm 186002574
SCX 300Å Trap Column 5/pack 5 μm 0.50 x 23.5 mm 186002623
Symmetry C18 Trap Column 5/pack 5 μm 0.18 x 23.5 mm 186002808
0.18 i.d. Custom Trap Column 5/pack Custom 0.18 x 23.5 mm 186002687
0.50 i.d. Custom Trap Column 5/pack Custom 0.50 x 23.5 mm 186002806
NanoEase Trap Column hardware obtained from Optimize Technologies Inc., Oregon City, OR, 97045-0009, USA

www.waters.com Traps, Columns, and Cartridges 53

MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

NanoEase Nano and Capillary Columns

NanoEase columns contain high performance, small particle sorbents. NanoEase LC/MS Nano Column Reproducibility
Exacting manufacturing and QC production procedures ensure consistent
column performance. Superior column longevity is ensured by a novel 500 fmol Enolase Digest
Gradient: 5-50% B in 60 min
frit tec hnology that minimizes bed disturbance throughout the life of Column: NanoEase Symmetry C18, Flow Rate: 400 nL/min
3.5 μm, 75 μm x 100 mm System: Waters CapLC™ operated in
the column. Eluent A: 0.1% FA in water split flow mode, Q-Tof micro™
Eluent B: 0.1% FA, 2% water in acetonitrile
½ 3TODQHNQ½BGQNL@SNFQ@OGHB½ODQENQL@MBD
100 TOF MS ES+
½ !U@HK@AKD½HM½U@QHDSX½NE½7@SDQR½RS@SHNM@QX½OG@RDR Inj #4
TIC
9.78e4
%

½ 2NATRS½@MC½D@RX½SN½G@MCKD 0 -

½ #@M½AD½TRDC½VHSG½@MX½B@OHKK@QX½,#½RXRSDL 100 TOF MS ES+

TIC
Inj #3 1.48e5
%

100 TOF MS ES+

TIC
Inj #2 1.71e5
%

100 TOF MS ES+

TIC
Inj #1 1.69e5
%

0
10 20 30 40 50 60 Min.

Description Inner Diameter Length Particle Size Part No. Description Inner Diameter Length Particle Size Part No.
Symmetry C18 75 μm 50 mm 3.5 μm 186002188 Custom 75 μm Custom Custom 186002213
Symmetry C18 75 μm 100 mm 3.5 μm 186002189 Custom 100 μm Custom Custom 186002214
Symmetry C18 75 μm 150 mm 3.5 μm 186002190 Custom 150 μm Custom Custom 186002472
Symmetry300 C18 75 μm 50 mm 3.5 μm 186002191 Symmetry C18 300 μm 50 mm 3.5 μm 186002581
Symmetry300 C18 75 μm 100 mm 3.5 μm 186002192 Symmetry C18 300 μm 100 mm 3.5 μm 186002582
Symmetry300 C18 75 μm 150 mm 3.5 μm 186002193 Symmetry C18 300 μm 150 mm 3.5 μm 186002583
Atlantis dC18 75 μm 50 mm 3 μm 186002194 Symmetry C18 300 μm 50 mm 5 μm 186002584
Atlantis dC18 75 μm 100 mm 3 μm 186002195 Symmetry C18 300 μm 100 mm 5 μm 186002585
Atlantis dC18 75 μm 150 mm 3 μm 186002197 Symmetry C18 300 μm 150 mm 5 μm 186002586
Symmetry C18 100 μm 50 mm 3.5 μm 186002201 Symmetry300 C18 300 μm 50 mm 3.5 μm 186002587
Symmetry C18 100 μm 100 mm 3.5 μm 186002202 Symmetry300 C18 300 μm 100 mm 3.5 μm 186002588
Symmetry C18 100 μm 150 mm 3.5 μm 186002203 Symmetry300 C18 300 μm 150 mm 3.5 μm 186002589
Symmetry300 C18 100 μm 50 mm 3.5 μm 186002204 Symmetry300 C18 300 μm 50 mm 5 μm 186002590
Symmetry300 C18 100 μm 100 mm 3.5 μm 186002205 Symmetry300 C18 300 μm 100 mm 5 μm 186002591
Symmetry300 C18 100 μm 150 mm 3.5 μm 186002206 Symmetry300 C18 300 μm 150 mm 5 μm 186002592
Atlantis dC18 100 μm 50 mm 3 μm 186002207 Atlantis dC18 300 μm 50 mm 3 μm 186002593
Atlantis dC18 100 μm 100 mm 3 μm 186002208 Atlantis dC18 300 μm 100 mm 3 μm 186002594
Atlantis dC18 100 μm 150 mm 3 μm 186002209 Atlantis dC18 300 μm 150 mm 3 μm 186002595
Symmetry C18 150 μm 50 mm 3.5 μm 186002459 Atlantis dC18 300 μm 50 mm 5 μm 186002596
Symmetry C18 150 μm 100 mm 3.5 μm 186002460 Atlantis dC18 300 μm 100 mm 5 μm 186002597
Symmetry C18 150 μm 150 mm 3.5 μm 186002461 Atlantis dC18 300 μm 150 mm 5 μm 186002598
Symmetry300 C18 150 μm 50 mm 3.5 μm 186002462 XBridge BEH130 C18 300 μm 50 mm 5 μm 186003682
Symmetry300 C18 150 μm 100 mm 3.5 μm 186002463 Custom 300μm Custom Custom 186002605
Symmetry300 C18 150 μm 150 mm 3.5 μm 186002464
Atlantis dC18 150 μm 50 mm 3 μm 186002466
For Peptide Separation Technology NanoEase
Atlantis dC18 150 μm 100 mm 3 μm 186002467
Atlantis dC18 150 μm 150 mm 3 μm 186002468 and Capillary Columns, See Page 10

54 Traps, Columns, and Cartridges www.waters.com

 MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

nanoACQUITY UltraPerformance LC Columns

½Novel frit technology and design for extended column life Separation of Enolose Digest
 Capable of operation up to 10,000 PSI
200 fmol enolase digest, 5% B - 60% B in 30 minutes, 250 nL/min flow rate
½Easily interfaces with LC/MS/MS systems
½Available with ACQUITY UPLC C18, 1.7 μm BEH Technology particles E041110mds04
23.42 24.98
TOF MS ES+
TIC
7.93e3
20.02 20.48

22.44 28.21
nanoACQUITY UPLC System
Peak widths: 3.6 to 4.8 sec
17.59 column backpressure ~ 3,300

16.41 19.18
27.62

32.36
14.73 15.63 25.54
29.46 29.85

15 20 25 30 35 Min.

The ability to operate at higher pressures enables the use of longer columns
(up to and including 250 mm in length) and further leverage the separation
power of 1.7 μm particles. Thus, the nanoACQUITY UPLC chemistry
offering now includes the Peptide Separation Technology nanoACQUITY
UPLC columns, which incorporate the 1.7 μm BEH130 material.

Increased Peak Capacity Separations

Waters nanoACQUITY Trapping, Capillary, and Nano columns are designed
100 fmoles of a mixture of 5 MassPREP Digest Standards* 3 μm conventional column
specifically for use on Waters nanoACQUIT Y UPLC System. As with our 75 μm i.d x 300 mm (custom length) Pressure 3,500 PSI
3-50% B 120 min Peak Capacity 297
line of NanoEase columns, these offerings containing carefully selected 250 nL/min
reversed-phase and ion-exchange chemistries for bioseparations.

1.7 μm BEH
Pressure 9,500
Peak Capacity 458

30 40 50 60 70 80 90 Min.
Column i.d. Flow Rate
300 μm 4 μL/min * Mixture includes: Part Number
MassPREP Enolase Digestion Standard 186002325
150 μm 1 μL/min MassPREP Phosphorylase b Digestion Standard 186002326
MassPREP Bovine Hemoglobin Digestion Standard 186002327
MassPREP ADH Digestion Standard 186002328
75 μm 250 nL/min
MassPREP BSH Digestion Standard 186002329

Flow Rates and Column Internal Diameters for Capillary and Nanoflow. Capillary-scale separations
are done in a flow rate range of 1 μl/min to 10 μl/min on columns with internal diameters (i.d.)
ranging from 300 μm to 100 μm. Nanoflow is <1 μl/min on columns with i.d. ≥ 75 μm.

www.waters.com Traps, Columns, and Cartridges 55

MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

nanoACQUITY UPLC Columns (10,000 psi)

nanoACQUITY UPLC 2D Kit
Particle Inner
Description Size Diameter Length Part No. T he two-dimensional (2D) LC/MS Kit for the nanoACQUITY UPLC System
provides high efficiency on-line 2D-LC/MS analysis for complex proteomics
Symmetry C18 3.5 μm 75 μm 100 mm 186003491
Symmetry C18 3.5 μm 75 μm 150 mm 186003492 samples, such as protein global digests.
Symmetry C18 3.5 μm 100 μm 100 mm 186003493 T he first and second dimensions are strong cation exchange (SCX) and
Symmetry C18 3.5 μm 100 μm 150 mm 186003494
Symmetry C18 3.5 μm 150 μm 100 mm 186003495
reversed phase (RP), respectively.
Symmetry C18 3.5 μm 150 μm 150 mm 186003496
Symmetry C18 3.5 μm 300 μm 100 mm 186003497
Symmetry C18 3.5 μm 300 μm 150 mm 186003498
Atlantis dC18 3 μm 75 μm 100 mm 186003499
Atlantis dC18 3 μm 75 μm 150 mm 186003500
Atlantis dC18 3 μm 100 μm 100 mm 186003501
Atlantis dC18 3 μm 100 μm 150 mm 186003502
Atlantis dC18 3 μm 150 μm 100 mm 186003503
Atlantis dC18 3 μm 150 μm 150 mm 186003504
Atlantis dC18 3 μm 300 μm 100 mm 186003505
Atlantis dC18 3 μm 300 μm 150 mm 186003506
nanoACQUITY UPLC Trap, SCX (std fitting) 180 μm 20 mm 186003507
nanoACQUITY UPLC Trap,
Symmetry C18, (std fitting) 5μm 180 μm 20 mm 186003514
The nanoACQUITY UPLC on-line 2D-LC/MS kit contains the tubing to configure the system for
nanoACQUITY UPLC Trap 2-pump traping in conjuction with online 2D operations, the nanoACQUITY UPLC SCX,
Symmetry C18, (v/v fitting) 5μm 180μm 20mm 186004630 trapping and RP analytical columns, and MassPREP Protein digest standards.
Custom 186003513
Peptide Separation Technology Columns First-Dimension Separation
BEH130 C18 1.7 μm 75 μm 100 mm 186003542 T he first-dimension separation employs the Waters nanoACQUITY UPLC
BEH130 C18 1.7 μm 75 μm 150 mm 186003543
180 μm x 23 mm SCX column. Two step gradients, a salt gradient and
BEH130 C18 1.7 μm 75 μm 200 mm 186003544
BEH130 C18 1.7 μm 75 μm 250 mm 186003545 then an organic gradient, are applied to the SCX column. W hile the salt
BEH130 C18 1.7 μm 100 μm 100 mm 186003546 gradient separates peptides that have different c harge properties, the
BEH130 C18 1.7 μm 150 μm 100 mm 186003550 organic gradient gradually elutes the hydrophobic peptides that remain on
BEH300 C4 1.7 m 75 m 100 mm 186004639 the SCX column after the salt gradient is applied. A nanoACQUITY UPLC
BEH300 C4 1.7 m 100 m 100 mm 186004640
trapping column collects the peptides that elute.
BEH300 C4 1.7 m 150 m 100 mm 186004641
For use with nanoACQUITY UPLC systems rated to 10,000 psi only.Not for use with nanoACQUITY UPLC systems rated to 5,000 psi.
Second-Dimension Separation
T he second-dimension separation employs a RP gradient to separated each
nanoACQUITY UPLC Columns (5,000 psi) SCX fraction with a nanoACQUITY UPLC 75 μm x 100 mm reversed-phase
Particle Inner analytical column. T he reversed-phase analytical column is used to separate
Description Size Diameter Length Part No. the peptides collected on the trapping column. T he separated peptides are
Symmetry C18 3.5 μm 75 μm 100 mm 186002821
then directed to an on-line MS system for characterization
Symmetry C18 3.5 μm 75 μm 150 mm 186002822
Symmetry C18 3.5 μm 100 μm 100 mm 186002823 nanoACQUITY UPLC 10,000 psi On-Line 2D Kit
Symmetry C18 3.5 μm 100 μm 150 mm 186002824
Symmetry C18 3.5 μm 150 μm 100 mm 186002825
Description Part No.
Symmetry C18 3.5 μm 150 μm 150 mm 186002826
Symmetry C18 3.5 μm 300 μm 100 mm 186002827 nanoACQUITY UPLC (10K psi) on-line 2D-LC/MS kit 176001367
Symmetry C18 3.5 μm 300 μm 150 mm 186002828 Kit consists of:
Atlantis dC18 3 μm 75 μm 100 mm 186002829 Assembly, 25 μm, capillary, BSM (binary solvent manager)
Atlantis dC18 3 μm 75 μm 150 mm 186002830 to trap valve 430001575
Atlantis dC18 3 μm 100 μm 100 mm 186002831 Assembly, 40 μm capillary, ASM (auxiliary solvent manager)
Atlantis dC18 3 μm 100 μm 150 mm 186002832 to injection valve 430001576
Atlantis dC18 3 μm 150 μm 100 mm 186002833 Assembly, capillary tubing, injection valve to trap valve 430001577
Atlantis dC18 3 μm 150 μm 150 mm 186002834 Assembly, capillary tubing, injection to trap valve 430001629
Atlantis dC18 3 μm 300 μm 100 mm 186002835 nanoACQUITY UPLC SCX column, 5 μm, 180 μm x 23 mm 186003507
Atlantis dC18 3 μm 300 μm 150 mm 186002836 nanoACQUITY UPLC trapping column, 5 μm Symmetry C18,
BEH130 C18 1.7 μm 75 μm 100 mm 186002837 180 μm x 20 mm 186003514
BEH130 C18 1.7 μm 100 μm 100 mm 186002838 nanoACQUITY UPLC analytical column, 1.7 μm BEH C18,
BEH130 C18 1.7 μm 150 μm 100 mm 186002839 75 μm x 100 mm 186003542
Custom 186002840 MassPREP 5 Protein Digest Standard Kit 186002330
Symmetry C18 Trap Column 5 μm 180 μm 20 mm 186002841 nanoACQUITY UPLC on-line 2D-LS/MS User’s Guide 715001357
For use with nanoACQUITY UPLC systems rated to 5,000 psi only. Not for use with nanoACQUITY UPLC systems rated to 10,000 psi.

56 Traps, Columns, and Cartridges www.waters.com

 MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

MassPREP Kits
Water MassPREP Gylcoanalysis, Phosphopeptide Enric hment, and Protein ½ 3HLOKD½OQNSNBNKR½@MC½D@RX½SN½TRD
Expression Kits were developed to address important sample preparation needs
½ %M@AKDR½GHFG SGQNTFGOTS½R@LOKD½OQDO@Q@SHNM
required prior to subsequent LC, MS, or LC/MS analyses. Each application specific
kit contains a comprehensive, fully-tested set of reagents and documentation ½ (HFGKX½QDOQNCTBHAKD½@MC½BNMRHRSDMS½R@LOKD½OQDO½QDRTKSR
regarding effective use.

Sample Preparation from Glycoprotein

MassPREP Glycoanalysis Kit to Purified 2-AB Labeled Glycans

Glycoproteins

Denaturation with MassPREP RapiGest

1 DTT Reduction (if necessary),
Deglycolisation by PNGase F

Protein Removal
2 MassPREP HILIC μElution Plate

3 2-AB Labelling of Purified Glycans

Excess Labelling Agent Removal

4 MassPREP HILIC μElution Plate
½ /OSHLHYD½OQNSDHM½CDFKXBNRXK@SHNM½QD@BSHNMR½VHSG
RapiGest SF surfactant
½-HMHLHYD½R@LOKD½L@MHOTK@SHNMR Purified 2-AB Labeled Glycans

½%EEDBSHUD½CDR@KSHMF½R@LOKD½BKD@M TO½LDSGNC
MALDI MS LC-FLR
½#NLO@SHAKD½VHSG½-!,$)½-3½@MC½NSGDQ½FKXB@M½@M@KXRHR½SDBGMHPTDR
½!RRHRS½HM½HRNK@SHNM½NE½ !"½K@ADKDC½FKXB@MR
MALDI MS Mass Profiling of Native and 2AB-Labeled IgG Glycans
Glycosylation is one of the most important types of post-translational
modification (PTM) in eukaryotic proteins. Efficient sample deglycosylation N-Acetylglucosamine Fucose (M+Na) +
m/ z
Manose Galactose G0 1339.47
and sample preparation is a key requirement for successful and sensitive
glycan analysis. In addition, the preparation and purification 2-AB-labeled A) G0F 1485.53
G1F
glycans released from glycoproteins can also be an important step in their 10 0
G0 F G1F 1647.58
successful analyses.
G2F 1809.63
T he MassPREP Glycoanalysis kit provides simple and robust sample prepa-
%

ration without compromising sample recovery. As shown (right), unlabeled G2F Underivatized glycans after
or 2-AB labeled glycans can be successully analyzed by MALDI-MS or by G0 protein removed by HILIC plate

LC with fluorescence detection following MassPREP Glycoanalysis Kit use. 0

B)
2AB - G0F 2AB - G1F
Description Dimensions/Qty. Part No. 10 0

MassPREP Glycoanalysis Kit

(includes, MassPREP HILIC μElution Plate,
%

RapiGest SF, and MassPREP MALDI Matrix DHB) 186002817 Labeled glycans (+12.1 Da)
2AB - G2 F after excess labeling reagents
Kit Consists of: 2AB - G0
removed by HILIC plate
MassPREP HILIC uElution Plate 96-well 186002780
0
RapiGest SF 1 mg vial 186001860 1200 1400 1600 1800 2000 2200 2400 2600 2800 m /z

MassPREP MALDI Matrix DHB 10 mg vial 5/pk 186002333 MALDI-TOF MS spectra of: (A) 8 pmol of the unlabeled IgG1glycans after first HILIC cleanup prior
to labeling. (B) 3 pmol of the 2-AB-labeled IgG1 glycans after second HILIC cleanup.
For more information see RapiGest SF Page 51 Each spectrum shows well purified glycans with no inferences from impurities.

www.waters.com Kits 57
MS AND LC/MS CONSUMABLES FOR BIOMOLECULES

MassPREP Phosphopeptide Enrichment Kit

Phosphopeptide Analysis without (top) and with (bottom)
MassPREP Phosphopeptide Kit Enrichment

Column: Symmetry C18 nanoACQUITY Trapping Column, 5 μm, 180 μm x 20 mm.

Trapping Mode: 5 μl/min for 3 minutes (100% aqueous).
Column: nanoACQUITY Atlantis dC18, 3 μm, 75 μm x 100 μm
Solvent A: 0.1% formic acid in 100% Milli Q water
Solvent B: 0.1% formic acid in 100% acetonitrile
Flow Rate: 300 nl/min
Gradient: 2% -40% B, 1% B per minute
Injection Volume: 2 μl

A) Control T18_1P
T19_1P
Enolase peptides :
4 phosphopeptide stdandard T43_1P
in 1:1 molar ratio T43_2P

½ 3DKDBSHUD½DMQHBGLDMS½NE½OGNROGNODOSHCDR½EQNL½BNLOKDW
proteomic samples
½ (HFGKX½DEEHBHDMS½@MC½QDOQNCTBHAKD½@EEHMHSX A@RDC½DMQHBGLDMS
B) After Enrichment
½ %@RX½SN½TRD
No Non-Specific
½ !OOKHB@SHNM½HM½GHFG SGQNTFGOTS½@M@KXRHR Binding

MassPREP Phosphopeptide Enrichment Kit, developed for selective enrich-

ment of phosphopeptides from complex samples, includes a 96-well micro- 20 30 40 50 Min.

scale, solid-phase extraction (SPE) plate packed with an affinity sorbent.

T he kit also includes a unique chemical (Enhancer™) that can be added to
Description Units/Kit Amt/Unit Part No.
further improve the selectivity of phosphopeptides. In contrast to currently
popular immobilized metal affinity chromatography (IMAC) technology, MassPREP Phosphopeptide Enrichment Kit 186003864
Kit Consists of:
this isolation method does not require pre-loading the sorbent with metal
MassPREP Phosphopeptide Enrichment μElution Plate 1 1 plate 186003820
chelator. T his phosphopeptide enrichment protocol is simplified. T he robust- MassPREP Enolase/Phosphopeptide Standard 1 1 nmole 186003286
ness of this method is provided by the sorbent that has high native affinity MassPREP Enhancer 10 500 mg 186003821
towards phosphopeptides and the Enhancer which yields superior selectivity MassPREP Enhancer 5 500 mg 186003863
of the sorbent towards phosphopeptides.
For more information see Phosphopeptide Standards on page 49

Waters Protein Expression System Kits

T he Protein Expression System consists of nanoACQUITY UPLC system, MassPREP Chemistry Consumable Kits for the Protein Express System
Q-Tof Premier, MassLynx™ ProteinLynx™ Global Server bioinformatics include nanoACQUIT Y UPLC columns, MassP REP Protein Digestion
software, and Waters MassPREP chemistry consumables and kits. Standards Mix 1 and 2, MassPREP Enolase Digest Standard, and RapiGest
SF Surfactant. Both Microscale and a Nanoscale Kits are available.

Microscale Kit Nanoscale Kit

Description Qty. Part No. Description Qty. Part No.

Microscale Kit 186003903 Nanoscale Kit 186003904
Kit Contains : Kit Contains :
nanoACQUITY UPLC 300 μm x 150 mm nanoACQUITY UPLC 75 μm x 150 mm
Atlantis dC18 3 μm Column 1 186003506 Atlantis dC18 3 μm Column 1 186003500
MPDS Mix 1 1 186002865 nanoACQUITY UPLC 180 μm x 20 mm
MPDS Mix 2 1 186002866 Symmetry C18 5 μm Trap Column 1 186003514
MPDS Enolase 1 186002325 MPDS Mix 1 1 186002865
RapiGest SF, 1 mg 1 186001860 MPDS Mix 2 1 186002866
MPDS Enolase 1 186002325
RapiGest SF, 1 mg 1 186001860

58 Kits www.waters.com
 UPLC TECHNOLOGY FOR BIOMOLECULES

UPLC Technology for Biomolecules

Waters offers a collection of powerful tools for c haracterizing biophar- To gain the benefits of the small particles columns, the system must
maceuticals. T hese solutions are designed specifically to provide more generate exceptionally accurate and precise gradients at relatively high
accurate, reliable, and robust results, simplify your analyses and thereby operation pressures. T he sample manager must provide accurate, small-
increase productivity. Our system solutions combine Waters market-leading volume injections with minimum cycle time. Furthermore, the detectors
instrumentation, column chemistries, and software and informatics products, must truly minimize band-broadening while providing high signal-to-
that leverage the sensitivity and resolution of Ultra Performance LC noise ratios for peaks that are narrow in both time and volume.
(UPLC) Tec hnology.
Based on chromatographic principles, Waters synergistically developed the
UPLC Technology was introduced by Waters as a novel chromatographic columns and the instrumentation that combine to make UPLC technology a
technique that takes full advantage of sub-2 μm column chemistries used reality–and now a proven success in laboratories around the world. W hile
in a system optimized for pressures necessary to produce best-in-class the first successful application of UPLC technology was in small molecule
separations. By packing columns with very small particles, band-broadening pharmaceuticals, Waters dedication to developing impactful laboratory
during separation is minimized. In addition, since distances are reduced in innovations quickly saw the benefits of UPLC tec hnology applied to the
small particles, separations can be optimized using higher linear velocities separation of biological and bioc hemically-significant macromolecules.
(i.e., flow rates), while maintaining chromatographic equilibrium. T he result
UPLC tec hnology brings a new level of separation and detection perfor-
of these chromatographic developments is separations with dramatically
mance to the analysis of biomolecules, whether from complex peptide
increased resolution, sensitivity, and speed.
mixtures or from a potentially life saving monoclonal antibody biothera-
T he properties of these columns places a new level of demand on the peutic. Today, Waters UPLC tec hnology provides improvements in the
c hromatographic system, and Waters has holistically developed the resolution, sensitivity, separation, and detection efficiency for amino
ACQUIT Y UPLC and nanoACQUIT Y UPLC systems to take advantage of acids, peptides, proteins, and synthetic DNA and RNA oligonucleotides
these c hromatographic principles. that help scientists and c hromatographers ac hieve desired objectives.

www.waters.com UPLC Technology for Biomolecules 59

UPLC TECHNOLOGY FOR BIOMOLECULES

Peptide Mapping Synthetic Oligonucleotides Analysis

Waters UPLC Peptide Analysis Solution, including UPLC Peptide Separation T he UPLC Oligonucleotide Analysis Solution and UPLC Oligonculeotide
Technology columns, delivers maximum resolution and sensitivity. UPLC Separation Tec hnology (OST ) columns provide higher resolution in less
chromatographic improvements have lead to greater confidence in protein time of synthetic oligonucleotide target products from contaminating
identification with improved determination of modified peptides, including failure sequences. T his Waters tec hnology leads to better resolution
glycopeptides. In the comparison of separations using 3.5 μm particles and increased sample throughput compared to use of traditional
and 1.7 μm particles, peaks are clearly sharper, and more small peaks are HPLC methods.
resolved using UPLC technology.

Protein Analysis Amino Acid Analysis

UPLC/MS tec hnology is available for rapid, efficient mass profiling of T he UPLC Amino Acid Analysis Solution is a total application solution
protein samples, including intact antibodies and their associated variants. optimized for an accurate, reliable, and reproducible analysis of amino
Samples can be successfully desalted on-line using the UPLC Intact Mass @BHCR½#NLAHMHMF½7@SDQR½MDV½!BB14@F½5KSQ@½B GDLHRSQX½@MC½!#15)4 9½
Analysis Kit, which ensures a high resolution spectrum with good signal- UPLC system, performance is assured in the areas of protein character-
to-noise. T he resulting total ion chromatograms (TICs), combined ESI-TOF ization, cell culture monitoring, and the nutritional analysis of foods and
mass spectra, and MaxEnt1 deconvolution mass spectrum of the intact IgG1 feeds. T he enhanced resolution and sensitivity of the separation ensures
quickly confirm the identity of the molecule while providing a measure of that the analysis yields accurate and precise qualitative and quantitative
the degree of modification of the protein in this sample. results. T his complete solution includes the instrument, derivitization and
separation chemistry, software, and comprehensive support for a true amino
In addition, reversed-phase separations of proteins are improved with
acid analyzer solution.
UPLC Protein Separation Tec hnology columns, used with ACQUIT Y
UPLC. T he BEH300 C4 column gives good peak shape for wide range of
proteins with minimized carryover. T his column c hemistry is available
in 3.5 and 1.7 μm particles for UPLC separations and can be effectively
used to analyze reduced and alkylated monoclonal antibodies.

60 UPLC Technology for Biomolecules www.waters.com

 HOW TO ORDER

On-line at www.waters.com

by Telephone by Fax by Mail

To place an order, request price and availability, delivery status/ 508-482-4820 (U.S.) Waters Corporation
expediting an order, locate your local sales representative and all other Mail Stop: CM
905-678-2350 (Canada)
customer service, 1-800-252-4752 5 Technology Drive,
Milford, MA 01757

1. Can I order by phone or fax? 4. How will my order be shipped?

Yes, Monday through Friday from 8:00 a.m. – 6:00 p.m. E.S.T., just You select the transportation best suited to your needs. Waters offers
call our toll free number, 1-800-252-4752, press 1, press 1, or fax FedEx and UPS as our standard carriers offering 3-day ground saver,
your order to 508-482-4820 (U.S.) or 905-678-2350 (Canada). Priority 1, 10:30 a.m. Next Day; Standard, 3:00 p.m. Next Day; and
W hen ordering, please have your purc hase order number or credit card Economy Two-Day Service.
number, the catalog number and description for eac h product. We will
also need your shipping address, including an attention line if applica- 5. W hat are the payment terms?
ble, and a complete billing address. In addition, please be sure to give
us your daytime telephone number so that we can reac h you promptly Net 30 days. Shipment on U.S. orders is FOB, Franklin, MA.
it there’s a follow-up question about your order.
6. How do I return an item?
2. How do I release product(s) against my
Chemical Products Standing Order? A return authorization number (RA#) must be obtained to return an item.
If an item is being returned for a warranty reason, please contact Technical
To release product(s) from your c hemical products standing/blanket
Support at 1-800-252-4752, extension 8360. If the return is being
order, call 1-800-252-4752, press 1, then press 5 for a direct line or
requested due to an incorrect order, shipping error, or damage claim, please
for an alternate who will make arrangements to release the product(s)
contact Customer Support at 1-800-252-4752, extension 8365.
from your order or answer any questions that you may have. You can
also fax your request to 508-482-2672. Outside the U.S.A., please refer to page 312 for the address and telephone
number of your local Waters office, or visit www.waters.com
3. W hen will I receive my order?

T he majority of items in this catalog are in stock and will normally be

shipped within 24 hours of receipt of your order. We can also arrange
blanket or standing orders to meet your needs.
Sales Offices

Austria and European Export The Netherlands 31 76 508 7200

(Central South Eastern Europe, CIS
Norway 47 6 384 60 50
and Middle East) 43 1 877 18 07
Poland 48 22 833 4400
Australia 61 2 9933 1777
Puerto Rico 1 787 747 8445
Belgium 32 2 726 1000
Singapore 65 6273 7997
Brazil 55 11 5094-3788
Spain 34 93 600 9300
Canada 1 800 252 4752 x2205
Sweden 46 8 555 11 500
China 86 21 6879 5888
Switzerland 41 56 676 70 00
CIS/Russia +497 727 4490/290 9737
Taiwan 886 2 2543 1898
Czech Republic 420 2 617 1 1384
United Kingdom 44 208 238 6100
Denmark 45 46 59 8080
All other countries:
Finland 09 5659 6288
Waters Corporation U.S.A.
France 33 1 30 48 72 00 1 508 478 2000
1 800 252 4752
Germany 49 6196 400600
www.waters.com
Hong Kong 852 29 64 1800

Hungary 36 1 350 5086

India and India Subcontinent

91 80 2837 1900

Ireland 353 1 448 1500

Italy 39 02 265 0983

Japan 81 3 3471 7191

Korea 82 2 820 2700

Mexico 52 55 5524 7636

©2008 Waters Corporation. Waters, The Science of What’s Possible, BioSuite, Symmetry, Symmetry300,
Delta-Pak, Accell, AccQ-Tag, Pico-Tag, UPLC, Oasis, Gen-Pak, MassPREP, NanoEase, UltraPerformance LC,
nanoACQUITY UPLC, Atlantis, XTerra, RapiGest, ACQUITY UPLC, BEH Technology, PrepLC, Radial-Pak, Guard-
Pak, Alliance, Sentry, Empower, AccQ-Fluor, XBridge, BioAlliance, Gen-Pak, Enhancer, MassLynx, Protein-pak,
MALDI micro MX, ZQ, Q-Tof, LCT Premier, ProteinLynx, Global Server, OBD, Enhancer and Q-Tof Premier are
trademarks of Waters Corporation.
All other trademarks are acknowledged.
The quality management system of Waters’ manufacturing facilities
in Taunton, Massachusetts and Wexford, Ireland complies with the International
Standard ISO 9001:2000 Quality Management and Quality Assurance Standards. 720002148EN January 2009 IH-FP
Waters’ quality management system is periodically audited by the registering body
to ensure compliance.