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Errata to Second Supplement to USP 36–NF 31 Physical Tests / 〈621〉 Chromatography 1

(2) The chamber is sealed to allow equilibration (satura-

tion) of the chamber and the paper with the solvent
〈621〉 CHROMATOGRAPHY vapor. Any excess pressure is released as necessary.
(3) After equilibration of the chamber, the prepared mo-
bile phase is introduced into the trough through the
inlet.
(4) The inlet is closed, and the mobile solvent phase is
INTRODUCTION allowed to travel the desired distance down the
paper.
Chromatographic separation techniques are multistage (5) The sheet is removed from the chamber.
separation methods in which the components of a sample (6) The location of the solvent front is quickly marked,
are distributed between two phases, of which one is station- and the sheet is dried.
ary and the other mobile. The stationary phase may be a (7) The chromatogram is observed and measured directly
solid or a liquid supported on a solid or a gel. The station- or after suitable development to reveal the location of
ary phase may be packed in a column, spread as a layer, the spots of the isolated drug or drugs.
distributed as a film, or applied by other techniques. The Ascending Paper Chromatography Procedure
mobile phase may be gaseous or liquid or supercritical fluid. (1) The mobile phase is added to the bottom of the
The separation may be based on adsorption, mass distribu- chamber.
tion (partition), or ion exchange; or it may be based on (2) The chamber is sealed to allow equilibration (satura-
differences among the physicochemical properties of the tion) of the chamber and the paper with the solvent
molecules, such as size, mass, and volume. This chapter vapor. Any excess pressure is released as necessary.
contains general procedures, definitions, and calculations of (3) The lower edge of the stationary phase is dipped into
common parameters and describes general requirements for the mobile phase to permit the mobile phase to rise
system suitability. The types of chromatography useful in on the chromatographic sheet by capillary action.
qualitative and quantitative analysis employed in USP proce- (4) When the solvent front has reached the desired
dures are column, gas, paper, thin-layer (including high-per- height, the chamber is opened, the sheet is removed,
formance thin-layer chromatography), and pressurized liquid the location of the solvent front is quickly marked,
chromatography (commonly called high-pressure or high- and the sheet is dried.
performance liquid chromatography). (5) The chromatogram is observed and measured directly
or after suitable development to reveal the location of
the spots of the isolated drug or drugs.
GENERAL PROCEDURES
This section describes the basic procedures used when a Thin-Layer Chromatography
chromatographic method is described in a monograph. The
following procedures are followed unless otherwise indicated Stationary Phase: The stationary phase is a relatively
in the individual monograph. thin, uniform layer of dry, finely powdered material applied
to a glass, plastic, or metal sheet or plate (typically called
Paper Chromatography the plate). The stationary phase of TLC plates has an aver-
age particle size of 10–15 µm, and that of high-performance
TLC (HPTLC) plates has an average particle size of 5 µm.
Stationary Phase: The stationary phase is a sheet of pa- Commercial plates with a preadsorbent zone can be used if
per of suitable texture and thickness. Development may be they are specified in a monograph. Sample applied to the
ascending, in which the solvent is carried up the paper by preadsorbent region develops into sharp, narrow bands at
capillary forces, or descending, in which the solvent flow is the preadsorbent–sorbent interface. The separations
also assisted by gravitational force. The orientation of paper achieved may be based on adsorption, partition, or a com-
grain with respect to solvent flow is to be kept constant in a bination of both effects, depending on the particular type of
series of chromatograms. (The machine direction is usually stationary phase.
designated by the manufacturer.)
Apparatus: A chromatographic chamber made of inert,
Apparatus: The essential equipment for paper chroma- transparent material and having the following specifications
tography consists of a vapor-tight chamber with inlets for is used: a flat-bottom or twin trough, a tightly fitted lid, and
addition of solvent and a rack of corrosion-resistant material a size suitable for the plates. The chamber is lined on at
about 5 cm shorter than the inside height of the chamber. least one wall with filter paper. Sufficient mobile phase or
The rack serves as a support for solvent troughs and for developing solvent is added to the chamber that, after im-
antisiphon rods that, in turn, hold up the chromatographic pregnation of the filter paper, a depth appropriate to the
sheets. The bottom of the chamber is covered with the pre- dimensions of the plate used is available. The chromato-
scribed solvent system or mobile phase. Saturation of the graphic chamber is closed and allowed to equilibrate.
chamber with solvent vapor is facilitated by lining the inside [NOTE—Unless otherwise indicated, the chromatographic
walls with paper wetted with the prescribed solvent system. separations are performed in a saturated chamber.]
Spotting: The substance or substances analyzed are dis- Detection/Visualization: An ultraviolet (UV) light
solved in a suitable solvent. Convenient volumes, delivered source suitable for observations under short- (254 nm) and
from suitable micropipets, of the resulting solution, normally long- (365 nm) wavelength UV light and a variety of other
containing 1–20 µg of the compound, are placed in 6- to spray reagents used to make spots visible are often used.
10-mm spots not less than 3 cm apart.
Spotting: Solutions are spotted on the surface of the
Descending Paper Chromatography Procedure stationary phase (plate) at the prescribed volume in suffi-
(1) A spotted chromatographic sheet is suspended in the ciently small portions to obtain circular spots of 2–5 mm in
apparatus, using the antisiphon rod to hold the upper diameter (1–2 mm on HPTLC plates) or bands of 10–20 mm
end of the sheet in the solvent trough. [NOTE—Ensure × 1–2 mm (5–10 mm × 0.5–1 mm on HPTLC plates) at an
that the portion of the sheet hanging below the rods appropriate distance from the lower edge of and sides of
is freely suspended in the chamber without touching the plate. [NOTE—During development, the application posi-
the rack, the chamber walls, or the fluid in the tion must be at least 5 mm (TLC) or 3 mm (HPTLC) above
chamber.] the level of the mobile phase.] The solutions are applied on
a line parallel to the lower edge of the plate with an interval
of at least 10 mm (5 mm on HPTLC plates) between the

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2 〈621〉 Chromatography / Physical Tests Errata to Second Supplement to USP 36–NF 31

centers of spots, or 4 mm (2 mm on HPTLC plates) between (2) Rinse the tip of the chromatographic column with
the edges of bands, then allowed to dry. about 1 mL of mobile phase before each change in
Procedure composition of mobile phase and after completion of
(1) Place the plate in the chamber, ensuring that the the elution.
spots or bands are above the surface of the mobile (3) If the analyte is introduced into the column as a solu-
phase. tion in the mobile phase, allow it to pass completely
(2) Close the chamber. into the column packing, then add mobile phase in
(3) Allow the mobile phase to ascend the plate until the several small portions, allowing each to drain com-
solvent front has traveled three-quarters of the length pletely, before adding the bulk of the mobile phase.
of the plate, or the distance prescribed in the (4) Where the procedure indicates the use of multiple
monograph. chromatographic columns mounted in series and the
(4) Remove the plate, mark the solvent front with a pen- addition of mobile phase in divided portions is speci-
cil, and allow to dry. fied, allow each portion to drain completely through
(5) Visualize the chromatograms as prescribed. each column, and rinse the tip of each with mobile
(6) Determine the chromatographic retardation factor (RF) phase before the addition of each succeeding portion.
values for the principal spots or zones.
(7) Presumptive identification can be made by observa- Gas Chromatography (GC)
tion of spots or zones of identical RF value and about
equal magnitude obtained, respectively, with an un-
known and a standard chromatographed on the same Liquid Stationary Phase: This type of phase is available
plate. A visual comparison of the size or intensity of in packed or capillary columns.
the spots or zones may serve for semiquantitative esti- Packed Column GC: The liquid stationary phase is de-
mation. Quantitative measurements are possible by posited on a finely divided, inert solid support, such as dia-
means of densitometry (absorbence or fluorescence tomaceous earth, porous polymer, or graphitized carbon,
measurements). which is packed into a column that is typically 2–4 mm in
internal diameter and 1–3 m in length.
Capillary Column GC: In capillary columns, which con-
Column Chromatography tain no packed solid support, the liquid stationary phase is
deposited on the inner surface of the column and may be
Solid Support: Purified siliceous earth is used for nor- chemically bonded to it.
mal-phase separation. Silanized chromatographic siliceous Solid Stationary Phase: This type of phase is available
earth is used for reverse-phase partition chromatography. only in packed columns. In these columns the solid phase is
Stationary Phase: The solid support is modified by the an active adsorbent, such as alumina, silica, or carbon,
addition of a stationary phase specified in the individual packed into a column. Polyaromatic porous resins, which
monograph. If a mixture of liquids is used as the stationary are sometimes used in packed columns, are not coated with
phase, mix the liquids before the introduction of the solid a liquid phase. [NOTE—Packed and capillary columns must
support. be conditioned before use until the baseline and other char-
Mobile Phase: The mobile phase is specified in the in- acteristics are stable. The column or packing material sup-
dividual monograph. If the stationary phase is an aqueous plier provides instructions for the recommended condition-
solution, equilibrate with water. If the stationary phase is a ing procedure.]
polar organic fluid, equilibrate with that fluid. Apparatus: A gas chromatograph consists of a carrier
Apparatus: Unless otherwise specified in the individual gas source, injection port, column, detector, and recording
monograph, the chromatographic tube is about 22 mm in device. The injection port, column, and detector are tem-
inside diameter and 200–300 mm long. Attached to it is a perature controlled and may be varied as part of the analy-
delivery tube, without stopcock, about 4 mm in inside diam- sis. The typical carrier gas is helium, nitrogen, or hydrogen,
eter and about 50 mm long. depending on the column and detector in use. The type of
APPARATUS PREPARATION: Pack a pledget of fine glass wool detector used depends on the nature of the compounds
in the base of the tube. Combine the specified volume of analyzed and is specified in the individual monograph. De-
stationary phase and the specified amount of solid support tector output is recorded as a function of time, and the
to produce a homogeneous, fluffy mixture. Transfer this instrument response, measured as peak area or peak height,
mixture to the chromatographic tube, and tamp, using gen- is a function of the amount present.
tle pressure, to obtain a uniform mass. If the specified Temperature Program: The length and quality of a GC
amount of solid support is more than 3 g, transfer the mix- separation can be controlled by altering the temperature of
ture to the column in portions of approximately 2 g, and the chromatographic column. When a temperature program
tamp each portion. If the assay or test requires a multiseg- is necessary, the individual monograph indicates the condi-
ment column with a different stationary phase specified for tions in table format. The table indicates the initial tempera-
each segment, tamp after the addition of each segment, ture, rate of temperature change (ramp), final temperature,
and add each succeeding segment directly to the previous and hold time at the final temperature.
one. Pack a pledget of fine glass wool above the completed Procedure
column packing. [NOTE—The mobile phase should flow (1) Equilibrate the column, injector, and detector with
through a properly packed column as a moderate stream or, flowing carrier gas until a constant signal is received.
if reverse-phase chromatography is applied, as a slow (2) Inject a sample through the injector septum, or use
trickle.] an autosampler.
If a solution of the analyte is incorporated into the sta- (3) Begin the temperature program.
tionary phase, complete the quantitative transfer to the (4) Record the chromatogram.
chromatographic tube by scrubbing the beaker used for the (5) Analyze as indicated in the monograph.
preparation of the test mixture with a mixture of about 1 g
of Solid Support and several drops of the solvent used to
prepare the sample solution before adding the final portion Liquid Chromatography (LC)
of glass wool.
Procedure The term liquid chromatography, as used in the compen-
(1) Transfer the mobile phase to the column space above dia, is synonymous with high-pressure liquid chromatogra-
the column packing, and allow it to flow through the phy and high-performance liquid chromatography. LC is a
column under the influence of gravity.

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Errata to Second Supplement to USP 36–NF 31 Physical Tests / 〈621〉 Chromatography 3

Figure 1. Chromatographic separation of two substances.

separation technique based on a solid stationary phase and for the chromatographer in identifying the pertinent chro-
a liquid mobile phase. matographic column specified in the individual monograph.
Stationary Phase: Separations are achieved by parti-
tion, adsorption, or ion-exchange processes, depending on
the type of stationary phase used. The most commonly used DEFINITIONS AND INTERPRETATION OF
stationary phases are modified silica or polymeric beads. The CHROMATOGRAMS
beads are modified by the addition of long-chain hydrocar-
bons. The specific type of packing needed to complete an Chromatogram: A chromatogram is a graphical repre-
analysis is indicated by the “L” designation in the individual sentation of the detector response, concentration of analyte
monograph (see also the section Chromatographic Columns, in the effluent, or other quantity used as a measure of efflu-
below). The size of the beads is often described in the mon- ent concentration versus effluent volume or time. In planar
ograph as well. Changes in the packing type and size are chromatography, chromatogram may refer to the paper or
covered in the System Suitability section of this chapter. layer with the separated zones.
Chromatographic Column: The term column includes Figure 1 represents a typical chromatographic separation
stainless steel, lined stainless steel, and polymeric columns, of two substances, 1 and 2. tR1 and tR2 are the respective
packed with a stationary phase. The length and inner diam- retention times; and h is the height, h/2 the half-height,
eter of the column affects the separation, and therefore typi- and Wh/2 the width at half-height, for peak 1. W1 and W2
cal column dimensions are included in the individual mono- are the respective widths of peaks 1 and 2 at the baseline.
graph. Changes to column dimensions are discussed in the Air peaks are a feature of gas chromatograms and corre-
System Suitability section of this chapter. Compendial mono- spond to the solvent front in LC. The retention time of
graphs do not include the name of appropriate columns; these air peaks, or unretained components, is designated as
this omission avoids the appearance of endorsement of a tM.
vendor’s product and natural changes in the marketplace. Dwell Volume (D): The dwell volume (also known as
See the section Chromatographic Columns for more informa- gradient delay volume) is the volume between the point at
tion. which the eluents meet and the top of the column.
Mobile Phase: The mobile phase is a solvent or a mix- Hold-Up Time (tM): The hold-up time is the time re-
ture of solvents, as defined in the individual monograph. quired for elution of an unretained component (see Figure 1,
Apparatus: A liquid chromatograph consists of a reser- shown as an air or unretained solvent peak, with the base-
voir containing the mobile phase, a pump to force the mo- line scale in min).
bile phase through the system at high pressure, an injector Hold-Up Volume (VM): The hold-up volume is the vol-
to introduce the sample into the mobile phase, a chromato- ume of mobile phase required for elution of an unretained
graphic column, a detector, and a data collection device. component. It may be calculated from the hold-up time
Gradient Elution: The technique of continuously and the flow rate F, in mL/min:
changing the solvent composition during the chromato-
graphic run is called gradient elution or solvent program- VM = tM × F
ming. The gradient elution profile is presented in the indi-
vidual monograph as a gradient table, which lists the time In size exclusion chromatography, the symbol VO is used.
and proportional composition of the mobile phase at the Number of Theoretical Plates (N)1: N is a measure of
stated time. column efficiency. For Gaussian peaks, it is calculated by:
Procedure
(1) Equilibrate the column and detector with mobile N = 16(tR/W)2
phase at the specified flow rate until a constant signal
is received. where tR is the retention time of the substance, and W is the
(2) Inject a sample through the injector, or use an peak width at its base, obtained by extrapolating the rela-
autosampler. tively straight sides of the peak to the baseline. The value of
(3) Begin the gradient program. N depends upon the substance being chromatographed as
(4) Record the chromatogram. well as the operating conditions, such as the flow rate and
(5) Analyze as directed in the monograph. temperature of the mobile phase or carrier gas, the quality
of the packing, the uniformity of the packing within the
1The parameters k, N, r, and rG were developed for isothermal GC
CHROMATOGRAPHIC COLUMNS separations and isocratic HPLC separations. Because these terms are
thermodynamic parameters, they are valid only for separations made at
constant temperature, mobile phase composition, and flow rate. However, for
A complete list of packings (L), phases (G), and supports separations made with a temperature program or solvent gradient, these
(S) used in USP–NF tests and assays is located in USP–NF parameters may be used simply as comparative means to ensure that
and PF, Reagents, Indicators, and Solutions—Chromatographic adequate chromatographic conditions exist to perform the methods as
Columns. This list is intended to be a convenient reference intended in the monographs.

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4 〈621〉 Chromatography / Physical Tests Errata to Second Supplement to USP 36–NF 31

column, and, for capillary columns, the thickness of the sta- tance simultaneously traveled by a reference compound (see
tionary phase film and the internal diameter and length of Figure 3) and is used in planar chromatography.
the column.
Where electronic integrators are used, it may be conven- Rret = b / c
ient to determine the number of theorical plates, by the
equation:

where Wh/2 is the peak width at half-height. However, in the

event of dispute, only equations based on peak width at
baseline are to be used.
Peak: The peak is the portion of the chromatographic
recording of the detector response when a single compo-
nent is eluted from the column. If separation is incomplete,
two or more components may be eluted as one unresolved
peak.
Peak-to-Valley Ratio (p/v): The p/v may be employed Figure 3. Typical planar chromatography.
as a system suitability criterion in a test for related sub-
stances when baseline separation between two peaks is not
achieved. Figure 2 represents a partial separation of two sub- Relative Retention (r)1: Is the ratio of the adjusted re-
stances, where Hp is the height above the extrapolated tention time of a component relative to that of another
baseline of the minor peak and Hv is the height above the used as a reference obtained under identical conditions:
extrapolated baseline at the lowest point of the curve sepa-
rating the minor and major peaks: r = tR2 − tM/tR1 − tM
p/v = Hp/Hv where tR2 is the retention time measured from the point of
injection of the compound of interest; tR1 is the retention
time measured from the point of injection of the compound
used as reference; and tM is the retention time of a
nonretained marker defined in the procedure, all deter-
mined under identical experimental conditions on the same
column.
Relative Retention Time (RRT): Also known as unad-
justed relative retention. Comparisons in USP are normally
made in terms of unadjusted relative retention, unless other-
wise indicated.
RRT = tR2/tR1
The symbol rG is also used to designate unadjusted relative
retention values.
Relative Standard Deviation in Percentage

Figure 2. Peak-to-valley ratio determination.

Relative Retardation (Rret): The relative retardation is Retardation Factor (RF): The retardation factor is the
the ratio of the distance traveled by the analyte to the dis- ratio of the distance traveled by the center of the spot to
the distance simultaneously traveled by the mobile phase
and is used in planar chromatography. Using the symbols in
Figure 3:
RF = b/a

Retention Factor (k)1: The retention factor is also

known as the capacity factor (k′). Defined as:

or

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Errata to Second Supplement to USP 36–NF 31 Physical Tests / 〈621〉 Chromatography 5

The retention factor of a component may be determined Tailing Factor (T): See Symmetry Factor.
from the chromatogram:
k = (tR − tM)/tM Change to read:

Retention Time (tR): In liquid chromatography and gas

chromatography, the retention time, tR, is defined as the SYSTEM SUITABILITY
time elapsed between the injection of the sample and the
appearance of the maximum peak response of the eluted System suitability tests are an integral part of gas and
sample zone. tR may be used as a parameter for identifica- liquid chromatographic methods. These tests are used to
tion. Chromatographic retention times are characteristic of verify that the chromatographic system is adequate for the
the compounds they represent but are not unique. Coinci- intended analysis.
dence of retention times of a sample and a reference sub- The tests are based on the concept that the equipment,
stance can be used as a partial criterion in construction of electronics, analytical operations, and samples analyzed con-
an identity profile but may not be sufficient on its own to stitute an integral system that can be evaluated as such.
establish identity. Absolute retention times of a given com- Factors that may affect chromatographic behavior include
pound may vary from one chromatogram to the next. the following:
Retention Volume (VR): The retention volume is the • Composition, ionic strength, temperature, and apparent
volume of mobile phase required for elution of a compo- pH of the mobile phase
nent. It may be calculated from the retention time and the • Flow rate, column dimensions, column temperature,
flow rate in mL/min: and pressure
• Stationary phase characteristics, including type of chro-
VR = tR × F matographic support (particle-based or monolithic),
particle or macropore size, porosity, and specific surface
Resolution (RS): The resolution is the separation of two area
components in a mixture, calculated by: • Reverse-phase and other surface modification of the sta-
tionary phases, the extent of chemical modification (as
RS = 2(tR2 − tR1)/(W1 + W2) expressed by end-capping, carbon loading, etc.)
The resolution, RS, is a function of the number of theoreti-
where tR2 and tR1 are the retention times of the two compo- cal plates, N (also referred to as efficiency), the separation
nents; and W2 and W1 are the corresponding widths at the factor, α, and the capacity factor, k. [NOTE—All terms and
bases of the peaks obtained by extrapolating the relatively symbols are defined in the preceding section Definitions and
straight sides of the peaks to the baseline. Interpretation of Chromatograms.] For a given stationary
Where electronic integrators are used, it may be conven- phase and mobile phase, N may be specified to ensure that
ient to determine the resolution, by the equation: closely eluting compounds are resolved from each other, to
establish the general resolving power of the system, and to
RS = 1.18(tR2 − tR1)/(W1,h/2 + W2,h/2) ensure that internal standards are resolved from the drug.
This is a less reliable means to ensure resolution than is di-
Separation Factor (α): The separation factor is the rela- rect measurement. Column efficiency is, in part, a reflection
tive retention calculated for two adjacent peaks (by conven- of peak sharpness, which is important for the detection of
tion, the value of the separation factor is always >1): trace components.
Replicate injections of a standard preparation or other
α = k2/k1 standard solutions are compared to ascertain whether re-
quirements for precision are met. Unless otherwise specified
Symmetry Factor (AS)2: The symmetry factor (also in the individual monograph, data from five replicate injec-
known as the tailing factor) of a peak (see Figure 4) is calcu- tions of the analyte are used to calculate the relative stan-
lated by: dard deviation, %RSD, if the requirement is 2.0% or less;
data from six replicate injections are used if the relative
AS = W0.05/2f standard deviation requirement is more than 2.0%.
For the Assay in a drug substance monograph, where the
where W0.05 is the width of the peak at 5% height and f is value is 100% for the pure substance, and no maximum
the distance from the peak maximum to the leading edge relative standard deviation is stated, the maximum permit-
of the peak, the distance being measured at a point 5% of ted %RSD is calculated for a series of injections of the refer-
the peak height from the baseline. ence solution:
%RSD = KB√n/t90%, n − 1
where K is a constant (0.349), obtained from the expression
K = (0.6/√2) × (t 90%,5/√6), in which 0.6/√2 represents the
required percentage relative standard deviation after six in-
jections for B = 1.0; B is the upper limit given in the defini-
tion of the individual monograph minus 100%; n is the
number of replicate injections of the reference solution
(3 ≤ n ≤ 6); and t90%, n − 1 is the Student’s t at the 90%
probability level (double sided) with n − 1 degrees of free-
dom.
Figure 4. Asymmetrical chromatographic peak. Unless otherwise prescribed, the maximum permitted rela-
tive standard deviation does not exceed the appropriate
2It is also a common practice to measure the Asymmetry Factor as the ratio
of the distance between the vertical line connecting the peak apex with the value given in the table of repeatability requirements. This
interpolated baseline and the peak front, and the distance between that line requirement does not apply to tests for related substances.
and the peak back measured at 10% of the peak height (see Figure 4), would
be (W0.10 − f0.10)/f0.10. However, for the purposes of USP, only the formula (As)
as presented here is valid.

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6 〈621〉 Chromatography / Physical Tests Errata to Second Supplement to USP 36–NF 31

Relative Standard Deviation Requirements pH of Mobile Phase (HPLC): The pH of the aqueous
Number of Individual Injections
buffer used in the preparation of the mobile phase can be
adjusted to within ±0.2 units of the value or range specified.
3 4 5 6
Concentration of Salts in Buffer (HPLC): The concen-
B (%) Maximum Permitted RSD
tration of the salts used in the preparation of the aqueous
2.0 0.41 0.59 0.73 0.85 buffer employed in the mobile phase can be adjusted to
2.5 0.52 0.74 0.92 1.06 within ±10% if the permitted pH variation (see above) is
3.0 0.62 0.89 1.10 1.27 met.
Ratio of Components in Mobile Phase (HPLC): The
The symmetry factor, AS, a measure of peak symmetry, is following adjustment limits apply to minor components of
unity for perfectly symmetrical peaks; and its value increases the mobile phase (specified at 50% or less). The amounts of
as tailing becomes more pronounced (see Figure 4). In some these components can be adjusted by ±30% relative. How-
cases, values less than unity may be observed. As peak sym- ever, the change in any component cannot exceed ±10%
metry moves away from values of 1, integration, and hence absolute (i.e., in relation to the total mobile phase). Adjust-
precision, become less reliable. ment can be made to one minor component in a ternary
The signal-to-noise ratio (S/N) is a useful system suitability mixture. Examples of adjustments for binary and ternary
parameter. The S/N is calculated as follows: mixtures are given below.
S/N = 2H/h Binary Mixtures
SPECIFIED RATIO OF 50:50: 30% of 50 is 15% absolute, but
where H is the height of the peak measured from the peak this exceeds the maximum permitted change of ±10% abso-
apex to a baseline extrapolated over a distance ≥5 times the lute in either component. Therefore, the mobile phase ratio
peak width at its half-height; and h is the difference be- may be adjusted only within the range of 40:60 to 60:40.
tween the largest and smallest noise values observed over a SPECIFIED RATIO OF 2:98: 30% of 2 is 0.6% absolute. There-
distance ≥5 times the width at the half-height of the peak fore the maximum allowed adjustment is within the range
and, if possible, situated equally around the peak of interest of 1.4:98.6 to 2.6:97.4.
(see Figure 5). Ternary Mixtures
SPECIFIED RATIO OF 60:35:5: For the second component,
30% of 35 is 10.5% absolute, which exceeds the maximum
permitted change of ±10% absolute in any component.
Therefore the second component may be adjusted only
within the range of 25% to 45% absolute. For the third
component, 30% of 5 is 1.5% absolute. In all cases, a suffi-
cient quantity of the first component is used to give a total
of 100%. Therefore, mixture ranges of 50:45:5 to 70:25:5
or 58.5:35:6.5 to 61.5:35:3.5 would meet the requirement.
Wavelength of UV-Visible Detector (HPLC): Devia-
tions from the wavelengths specified in the procedure are
not permitted. The procedure specified by the detector
manufacturer, or another validated procedure, is used to
verify that error in the detector wavelength is, at most,
Figure 5. Noise and chromatographic peak, components of ±3 nm.
the S/N ratio. Stationary Phase
COLUMN LENGTH (GC, HPLC): Can be adjusted by as much
These system suitability tests are performed by collecting as ±70%.
data from replicate injections of standard or other solutions COLUMN INNER DIAMETER (HPLC): Can be adjusted if the lin-
as specified in the individual monograph. ear velocity is kept constant. See Flow Rate (HPLC) below.
The specification of definitive parameters in a monograph
COLUMN INNER DIAMETER (GC)—Can be adjusted by as much
does not preclude the use of other suitable operating condi-
tions. Adjustments are permitted only when as ±50% for GC.
• Suitable standards (including Reference Standards) are FILM THICKNESS (CAPILLARY CG)—Can be adjusted by as much
available for all compounds used in the suitability test; as −50% to 100%.
and Particle Size (HPLC): The particle size can be reduced
• Those standards show that the adjustments improved by as much as 50%, but cannot be increased.
the quality of the chromatography with respect to the Particle Size (GC): Changing from a larger to a smaller
system suitability requirements. or from a smaller to a larger particle size GC mesh support
Adjustments to chromatographic systems performed in or- is acceptable if the chromatography meets the requirements
der to comply with system suitability requirements are not of system suitability and the same particle size range ratio is
to be made in order to compensate for column failure or maintained. The particle size range ratio is defined as the
system malfunction. diameter of the largest particle divided by the diameter of
If adjustments of operating conditions are necessary in or- the smallest particle.
der to meet system suitability requirements, each of the Flow Rate (GC): The flow rate can be adjusted by as
items in the following list is the maximum variation that can much as ±50%.
be considered, unless otherwise directed in the monograph;
these changes may require additional validation data. To Flow Rate (HPLC): When column dimensions have
verify the suitability of the method under the new condi- been modified, the flow rate can be adjusted using:
tions, assess the relevant analytical performance characteris-
tics potentially affected by the change. Multiple adjustments
can have a cumulative effect on the performance of the
system and are to be considered carefully before implemen- •
tation. Adjustments to the composition of the mobile phase
in gradient elution are not recommended. If adjustments are in which F1 is the flow rate indicated in the monograph, in
necessary, only column changes (same packing material) or mL/min; F2 is the adjusted flow rate, in mL/min; d1 is the
dwell volume adjustments are recommended.

Official from December 1, 2013

Copyright (c) 2014 The United States Pharmacopeial Convention. All rights reserved.
Accessed from 117.198.192.193 by amneal on Sun Mar 16 03:09:43 EDT 2014

Errata to Second Supplement to USP 36–NF 31 Physical Tests / 〈621〉 Chromatography 7

column inner diameter indicated in the monograph; and d2 measured are separated from any interfering components.
is the internal diameter of the column used. Additionally, Peak tailing and fronting is minimized, and the measure-
the flow rate can be adjusted by ±50%.• (ERR 1-Feb-2013) ment of peaks on tails of other peaks are avoided when
Injection Volume (HPLC): The injection volume can be possible.
reduced as far as is consistent with accepted precision and Although comparison of impurity peaks with those in the
detection limits; no increase is permitted. chromatogram of a standard at a similar concentration is
Injection Volume and Split Volume (GC): The injec- preferred, impurity tests may be based on the measurement
tion volume and split volume may be adjusted if detection of the peak response due to impurities and expressed as a
and repeatability are satisfactory. percentage of the area of the drug peak. The standard may
be the drug itself at a level corresponding to, for example,
Column Temperature (HPLC): The column tempera- 0.5% impurity, assuming similar peak responses. When im-
ture can be adjusted by as much as ±10°. Column thermo- purities must be determined with greater certainty, use a
stating is recommended to improve control and reproduc- standard of the impurity itself or apply a correction factor
ibility of retention time. based on the response of the impurity relative to that of the
Oven Temperature (GC): The oven temperature can main component.
be adjusted by as much as ±10%. External Standard Method: The concentration of the
Oven Temperature Program (GC): Adjustment of tem- component(s) quantified is determined by comparing the
peratures is permitted as stated above. When the specified response(s) obtained with the sample solution to the re-
temperature must be maintained or when the temperature sponse(s) obtained with a standard solution.
must be changed from one value to another, an adjustment Internal Standard Method: Equal amounts of the in-
of up to ±20% is permitted. ternal standard are introduced into the sample solution and
Unless otherwise directed in the monograph, system suit- a standard solution. The internal standard is chosen so that
ability parameters are determined from the analyte peak. it does not react with the test material, is stable, is resolved
Measured values of Rr or RF or tR for the sample substance from the component(s) quantified (analytes), and does not
do not deviate from the values obtained for the reference contain impurities with the same retention time as that of
compound and mixture by more than the statistically deter- the analytes. The concentrations of the analytes are deter-
mined reliability estimates from replicate assays of the refer- mined by comparing the ratios of their peak areas or peak
ence compound. Relative retention times may be provided heights and the internal standard in the sample solution
in monographs for informational purposes only to aid in with the ratios of their peak areas or peak heights and the
peak identification. There are no acceptance criteria applied internal standard in the standard solution.
to relative retention times.
Suitability testing is used to ascertain the effectiveness of Normalization Procedure: The percentage content of a
the final operating system, which should be subjected to component of the test material is calculated by determining
this testing. Make injections of the appropriate prepara- the area of the corresponding peak as a percentage of the
tion(s) as required in order to demonstrate adequate system total area of all the peaks, excluding those due to solvents
suitability (as described in the Chromatographic system sec- or reagents or arising from the mobile phase or the sample
tion of the method in a monograph) throughout the run. matrix and those at or below the limit at which they can be
The preparation can be a standard preparation or a solu- disregarded.
tion containing a known amount of analyte and any addi- Calibration Procedure: The relationship between the
tional materials (e.g., excipients or impurities) useful in con- measured or evaluated signal y and the quantity (e.g., con-
trolling the analytical system. Whenever there is a significant centration, mass) of substance x is determined, and the cali-
change in the chromatographic system (equipment, mobile bration function is calculated. The analytical results are cal-
phase component, or other components) or in a critical rea- culated from the measured signal or evaluated signal of the
gent, system suitability is to be reestablished. No sample analyte and its position on the calibration curve.
analysis is acceptable unless the suitability of the system has In tests for impurities for both the External Standard
been demonstrated. Method, when a dilution of the sample solution is used for
comparison, and the Normalization Procedure, any correction
factors indicated in the monograph are applied (e.g., when
QUANTITATION the response factor is outside the range 0.8–1.2).
When the impurity test prescribes the total of impurities
During quantitation, disregard peaks caused by solvents or there is a quantitative determination of an impurity,
and reagents or arising from the mobile phase or the sam- choice of an appropriate threshold setting and appropriate
ple matrix. conditions for the integration of the peak areas is important.
In the linear range, peak areas and peak heights are usu- In such tests the limit at or below which a peak is disre-
ally proportional to the quantity of compound eluting. The garded is generally 0.05%. Thus, the threshold setting of
peak areas and peak heights are commonly measured by the data collection system corresponds to at least half of this
electronic integrators but may be determined by more class- limit. Integrate the peak area of any impurity that is not
ical approaches. Peak areas are generally used but may be completely separated from the principal peak, preferably by
less accurate if peak interference occurs. The components valley-to-valley extrapolation (tangential skim).

Official from December 1, 2013

Copyright (c) 2014 The United States Pharmacopeial Convention. All rights reserved.