Complete Solution for Achiral Reverse

Phase Chromatography using HPLC and

UHPLC

Kunal Kangane
1

Outline

• YMC History
• Parameters of method development
• Types of Silica and columns with different technologies
• Chemistries of the bonding phase and their interactions
• Online tools
• USP allowable changes
• Method development tips and tricks

1
History
Yamamura Chemical Research Institute established with the paid-up capital
1980
of 2.5 million yen in Yawata-city, Kyoto.
"YMC*GEL" packing material for high performance liquid chromatography
1981
developed for start of sales
1982 Sale of packed columns of "YMC- Pack" series started
1985 YMC Inc. established in New Jersey, USA
1987 "Separation Center" established in Kumiyama-cho,Kuze-gun, Kyoto
1989 Company name has changed to YMC CO., LTD.
1993 YMC Europe GmbH established in Germany
1996 Sale of packed columns of "Pro" series started
1997 ISO 9001 certification acquired
2006 Sale of "Microreactor" started
2008 YMC America, Inc. established in Pennsylvania, USA
2008 Sale of packed columns of "Bio Pro" series started
2008 YMC India Pvt. Ltd. established in India
2009 Sale of packed columns of "YMC-Triart" series started
2012 YMC Singapore Tradelinks Pte. Ltd. established in Singapore
2013 Sale of packed columns of "CHIRAL ART" series started
"The YMC CO., LTD. Chiral Technologies Laboratory" established in
2013 Iwakuraminami, Sakyo-ku, Kyoto.(An existing YMC CO., LTD. Kyoto
Research Laboratories)
2015 YMC Technologies Pvt. Ltd. established in India.
2015 "API Purification Plant" established in YMC CO., LTD. Komatsu Works. 3

Reverse Phase and Normal Phase

Chromatography
• Reversed-phase chromatography (also called RPC,
reverse-phase chromatography, or hydrophobic
chromatography) includes any chromatographic method that
uses a hydrophobic stationary phase.
• Normal-phase chromatography: this method separates
analytes based on their affinity for a polar stationary surface
such as silica

2
 Critical Parameters of Method Development

• Column Length

• Flow Rate

• Temperature

• Column Chemistries

• Mobile Phase

• Analyte Characteristics

Selecting the right column length

Column length selection depends majorly on the following

criteria:
• Goals of separation
• Particle Technology
• Particle Size
• Instrument model
• Application 6

3
Temperature

• The increase in column temperature decreases the retention

time.
• Bit frustrating if we try to operate at room temperature in a
laboratory that doesn’t have very good climate control.
• Retention is not the only thing that can change when the
temperature changes. It is common to see changes in peak
spacing with a change in temperature, too.
• Therefore, not only should it be considered as a tool for
method development but it should be taken special care
during method validations.

Types of Particle Technologies available today

• Monolith Silica

• Core-shell technology particles

• Traditional Fully porous particle

4
Core-Shell Technology

Columns packed with core-shell particles deliver significantly higher

efficiency (N) than columns packed with fully-porous particles of the same
diameter.
Benefits of Core-shell technology

• Improved Results

• Increased Productivity

• Significant Cost Savings

• Easy Transferability

Meteoric-Core from YMC

• Meteoric Core is core-shell column with outstanding resolution for

UHPLC & HPLC.
• Meteoric Core can be used across a wide pH range and provides
excellent peak shape for basic and coordination compounds compared
to conventional columns or competitors’.
• This feature enables smoother method development.
• Meteoric Core is ideal for ultra fast and high resolution analysis.
• Meteoric Core can reduce its backpressure by half compared to sub-2
µm columns with the same resolution as this.
• Meteoric Core can be used with conventional HPLC as well as UHPLC.

10

5
 Name Meteoric Core C18 Meteoric Core C18 BIO Meteoric Core C8

Base particle Core-Shell type silica gel

Particle size ( µm) 2.7
Pore size (Å) 80 160 80
Specific surface area (m2/ 150 90 150
g)
Bonding Trifunctional
Carbon content (%) 7 5 5
Endcapping Yes
pH range 1.5-10 1.5-9
USP Classification L1 L1 L7

11

Meteroric Core on the HPLC

• Ideal dimensions and particle size

• UHPLC Performance on HPLC
• System Enhancement
• Tubing
• Data Acquisition rate
• Flow Cell

12

6
 Excellent Peak Shapes for Basic Compounds

Column:50 X 2.1 mmI.D.

Eluent:20mM KH2PO4-K2HPO4(pH 6.9)
/acetonitrile (65/35)
Flow rate: 0.2 mL/min
Temperature: 40℃
Detection: UV at 235 nm
Sample
1. Chlorpheniramine
2. Dextromethorphan (Peak 2)
3. Propyl p-hydroxybenzoate (I.S.)

13

Peptide/Protein Separation

Meteoric Core C18 BIO with wider pore size: Appropriate for separation of peptides/proteins whose
molecular weight are up to 30,000
14

7
 TRIART

15

Triart Surface Chemistry

Polymerization

Alkaline  

Tetraethoxysilane   Silica  Sol-­‐Gel  

16

8
Triart Surface Chemistry

Siloxane   Polymer
Bridge  

Dissolu7on  at  pH  >  7.5   pH range 1-12

17

Practical Benefits

• Lower silanol activities leading to lesser tailing

• Uniform particle size.
• Polymeric bonding offers increased column stability,
particularly when highly aqueous mobile phases are used.
• Polymeric bonding also enables the column to accept higher
sample loading
• Spherical particles offer reduced back pressures and longer
column life when using viscous mobile phases like 50:50
MeOH:H2O.
• Available for UHPLC, HPLC and prep.

18

9
 YMC Triart

• Hybrid media made out of robust polymer along with sharpness of silica
• Longer lifetime than conventional silica
• 70% lower leachable level
• Cost effective than any other competitor product
• Hybrid Silica Based Columns : YMC Triart pH range1-12
• Available in 1.9u, 3u, 5u,10u analytical scale and also prep and loose media in
respective particle sizes.
• YMC Triart 1.9um comes with a waters end-fittings
19

YMC Triart Express C18

• High hydrophobicity due to the high carbon loading (25%).

• Suitable for hydrophobic isomers and structural analogs.
• Additional flexibility in choosing separation conditions.
• Improved shape recognition compared to Triart C18.

20

10
 Triart C18 Vs Triart Express C18

Specifications Triart C18 Triart Express C18

Base Organic/ inorganic hybrid silica Organic/ inorganic hybrid silica
Particle Sizes 1.9um, 3um, 5um 1.9um, 3um, 5um
Pore Size 120A 80A
Carbon Load 20% 25%
Usable pH range 1-12 1-12
USP Lisiting L1 L1
Bonding polymeric type Trifunctional
Sp Surface area 350 m sq./g 430 m sq./g

• High surface area generally provides greater retention, capacity and

resolution for separating complex, multi-component samples.
• Low surface area packings generally equilibrate quickly, especially
important in gradient analyses
21

Coordination compound Hinokitiol

1 2
Triart C18 Tf(1)=1.31
5 µm, 150X3.0mm

0 3 6 9 12 15 min
Triart C18 1 2
1.9 µm, 50X2.0mm Tf(1)=1.35

0 1 2 3 4 5 min
XBridge C18 2
Tf(1)=N.D.
5 µm, 150X4.6mm 1

0 3 6 9 12 15 min
ACQUITY BEH C18 2 Tf(1)=N.D.
1.7 µm, 50X2.1mm

0 1 2 3 4 5 min
Gemini NX C18 2 1. Hinokitiol
1 Tf(1)=1.24
5 µm, 150X4.6mm 2. Methyl benzoate

0 3 6 9 12 15 min Eluent : acetonitrile/0.1% H3PO4

(40/60)
Kinetex C18 1 2
Tf(1)=1.22 Flow rate : 1.0 ml/min for 4.6mmi.d.
2.6 µm, 50X2.1mm
0.425 ml/min for 3mmi.d
0.2 ml/min for 2mmi.d.
Kinetex0C18 1
2
2 3 4 5 min
for 2.1mmi.d.
1.7 µm, 50X2.1mm Tf(1)=N.D. Detector : UV at 254 nm
Temp. : 40℃
0 1 2 3 4 5 min

11
Comparison of Analysis of Basic Drugs

1. Chlorpheniramine 2. Dextromethorphan 3. Propyl paraben (I.S.)

Column : 50 X 2.0 mmI.D. or 2.1 mmI.D.
Eluent : 20 mM KH2PO4-KH2PO4 (pH 6.9)/acetonitrile (65/35)
Flow rate : 0.2 mL/min
Temperature : 40℃
Detection : UV at 235 nm

23

Method transfer between HPLC and UHPLC

24

12
YMC Triart Chemistries

• YMC-Triart Phenyl ■ Pore size : 120 Å

• Unique selectivity due π-π interaction ■ Carbon content : 17%
• Excellent resolution without adsorption and tailing ■ Usable pH range : 1.0~10.0 USP: L11
• Ideal for separations of aromatic compounds or compounds having long conjugated system

• YMC-Triart PFP
• Alternative selectivity to C18/C8 due to unique polar interaction ■ Carbon content : 15%
• Superior shape recognition ability / steric selectivity ■ Usable pH range : 1.0~8.0
• Ideal for separations of polar compounds or isomers ■ USP: L43

• YMC-Triart Diol-HILIC
• ■Ideal for separations of highly polar compounds, which are hardly retained on a reversed-phase column
• ■Superior durability and usable under wide range of mobile phase conditions ■ USP: L43
• ■Excellent reproducibility with less ionic adsorption ■ Carbon content : 12%
■ Usable pH range : 2.0~10.0
■ USP: L20
■ Pore size : 120 Å

25

Online Links

• www.chemicalize.org
• www.drugbank.com
• http://www.ymc.co.jp/en/products/
• http://www.ymc.co.jp/en/download/
• http://www.ymc.co.jp/en/columns/application/
• http://www.ymcamerica.com/applications/

26

13
Method Development

Common Mistakes in Method Development:

• Inadequate Formulation of Method Goals
• Little Knowledge of Chemistry of Analyte Mixture
• Use of the First Reversed Phase C18 Column Available
• Trial and Error with Different C18 Columns and Mobile Phases

These Mistakes Result In:

• Laborious, Time-consuming Development Projects
• Methods that Fail to Meet the Needs of the Analyst

27

Proposed Method

At Your Desk
• Define your knowledge of the sample
• Define your goals for the separation method
• Choose the columns to be considered

In the Laboratory
• Choose the initial mobile phase chemistry
• Choose the detector type and starting parameters
• Evaluate the potential columns for the sample
• Optimize the separation conditions (isocratic or gradient)
for the chosen column
" Validate the method for release to routine laboratories

28

14
Factors affecting reversed phase separations:

• Structures of analytes
• Properties of stationary phase materials
• Stationary phase selectivity
• Mobile phase conditions
- Buffer
- Organic modifier

29

•Improved understanding of factors

affecting column behavior
•There is more to HPLC than C18
•Mobile phase considerations

30

15
Structures of analytes

Case 1
Anthracene

(CH2)5CH3
3-Hexylanthracene

The structural difference between these two compounds is the hydrophobic hexyl side chain.
This suggests a non-polar C18 or C8 column would interact with this area of difference to
help provide separation of these two compounds.

What would you recommend in this case?

31

Case 2
H O H O

O O
O H O O H
H O

O O
Prednisolone Prednisone

Use the results of the structural comparison to select a bonded phase showing optimal
selectivity for these two molecules. In this case consider using a silica column (no bonded
phase) for its ability to retain polar solutes through hydrogen bonding.

32

16
Separation of cis-trans isomers

33

Properties of stationary phase materials

• Base material
• Ligand chemistry
• Silica purity
• Pore Size/Surface Area
• Carbon Load
• Inertness
• Particle Size

34

17
Allowable Changes

• Ratio of components in mobile phase: ± 30 % (relative)

• Mobile phase pH: ± 0.2 units (or ± 1.0 units for non-ionizable substances)
• Concentration of salts in buffer: ± 10 %
• Stationary phase: No change of the identity of the substituent permitted
• Particle size: Can be reduced as much as 50 %, but cannot be increased
• Column length: ± 70 %
• Column inner diameter: 150 x 4.6 mm column can be varied from 3.45 - 5.75
mm in diameter
• Flow rate: ± 50 %
• Column temperature: ± 10° C
• Wavelength of UV-visible detector: No deviations permitted
• Injection volume: Can be reduced as long as precision and detection limits are
achieved (no increase is permitted)

35

Conclusion

In this approach to HPLC column selection, the bonded phase

chemistry of the column is chosen on the basis of an analysis
of the sample component structures. The physics of the
column is chosen according to an analysis of the goals for the
separation method. This approach succeeds in predicting
unique, optimum bonded phase chemistries and particle bed
physical characteristics that are likely to meet the goals for the
separation method.
" None of these can completely explain all of the
observed retention in reversed phase HPLC

36

18
Method Development Kit

• Triart C18
• Triart C8/ Phenyl
• Triart PFP
• Meteoric Core C18

37

Thank You

38

19