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〈 621 〉 CHROMATOGRAPHY
INTRODUCTION
Chromatographic separation techniques are multistage separation methods in which the components of a sample are distributed
between two phases, of which one is stationary and the other is mobile. The stationary phase may be a solid or a liquid supported on
a solid or a gel. The stationary phase may be packed in a column, spread as a layer, distributed as a lm, or applied by other
techniques. The mobile phase may be in a gaseous or liquid form, or a supercritical uid. The separation may be based on adsorption,
mass distribution (partition), or ion exchange; or it may be based on differences among the physicochemical properties of the
molecules, such as size, mass, and volume. This chapter contains general procedures, de nitions, and calculations of common
parameters and describes general requirements for system suitability. The types of chromatography useful in qualitative and
quantitative analyses employed in USP procedures are column, gas (GC), paper, thin-layer (TLC) [including high-performance thin-layer
chromatography (HPTLC)], and pressurized liquid chromatography [commonly called high-pressure or high-performance liquid
chromatography (HPLC)].

GENERAL PROCEDURES
This section describes the basic procedures used when a chromatographic method is described in a monograph. These
procedures are followed unless otherwise indicated in the individual monograph.

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Paper Chromatography

STATIONARY PHASE
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The stationary phase is a sheet of paper of suitable texture and thickness. Development may be ascending, in which the solvent is
carried up the paper by capillary forces, or descending, in which the solvent ow is also assisted by gravitational force. The
orientation of paper grain, with respect to solvent ow, is to be kept constant in a series of chromatograms. The machine direction is
usually designated by the manufacturer.
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APPARATUS
The essential equipment for paper chromatography consists of a vapor-tight chamber with inlets for the addition of solvent and a
rack of corrosion-resistant material about 5 cm shorter than the inside height of the chamber. The rack serves as a support for
solvent troughs and antisiphon rods that, in turn, hold up the chromatographic sheets. The bottom of the chamber is covered with
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the prescribed solvent system or mobile phase. Saturation of the chamber with solvent vapor is facilitated by lining the inside walls
with paper wetted with the prescribed solvent system.

SPOTTING
The substance or substances analyzed are dissolved in a suitable solvent. Convenient volumes delivered from suitable
micropipettes of the resulting solution, normally containing 1–20 µg of the compound, are placed in 6- to 10-mm spots NLT 3 cm
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apart.

DESCENDING PAPER CHROMATOGRAPHY PROCEDURE

1. A spotted chromatographic sheet is suspended in the apparatus, using the antisiphon rod to hold the upper end of the sheet
in the solvent trough. [ NOTE— Ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber
without touching the rack, the chamber walls, or the uid in the chamber.]

2. The chamber is sealed to allow equilibration (saturation) of the chamber and the paper with the solvent vapor. Any excess
pressure is released as necessary.

3. After equilibration of the chamber, the prepared mobile phase is introduced into the trough through the inlet.
4. The inlet is closed, and the mobile solvent phase is allowed to travel the desired distance down the paper.
5. The sheet is removed from the chamber.
 6. The location of the solvent front is quickly marked, and the sheet is dried.
7. The chromatogram is observed and measured directly or after suitable development to reveal the location of the spots of the
isolated drug or drugs.

ASCENDING PAPER CHROMATOGRAPHY PROCEDURE

1. The mobile phase is added to the bottom of the chamber.
2. The chamber is sealed to allow equilibration (saturation) of the chamber and the paper with the solvent vapor. Any excess
pressure is released as necessary.
3. The lower edge of the stationary phase is dipped into the mobile phase to permit the mobile phase to rise on the
chromatographic sheet by capillary action.
4. When the solvent front has reached the desired height, the chamber is opened, the sheet is removed, the location of the
solvent front is quickly marked, and the sheet is dried.
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.

Thin-Layer Chromatography

STATIONARY PHASE
The stationary phase is a relatively thin, uniform layer of dry, nely powdered material applied to a glass, plastic, or metal sheet or
plate (typically called the plate). The stationary phase of TLC plates has an average particle size of 10–15 µm, and that of HPTLC
plates has an average particle size of 5 µm. Commercial plates with a preadsorbent zone can be used if they are speci ed in a
monograph. The sample applied to the preadsorbent region develops into sharp, narrow bands at the preadsorbent–sorbent
interface. The separations achieved may be based on adsorption, partition, or a combination of both effects, depending on the
particular type of stationary phase.

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A chromatographic chamber made of inert, transparent material and having the following speci cations is used: a at-bottom or
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twin trough, a tightly tted lid, and a size suitable for the plates. The chamber is lined on at least one wall with lter paper. Su cient
mobile phase or developing solvent is added to the chamber so that, after impregnation of the lter paper, a depth appropriate to the
dimensions of the plate used is available. The chromatographic chamber is closed and allowed to equilibrate. [ NOTE— Unless
otherwise indicated, the chromatographic separations are performed in a saturated chamber.]
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DETECTION/VISUALIZATION
An ultraviolet (UV) light source suitable for observations under short- (254 nm) and long- (365 nm) wavelength UV light and a
variety of other spray reagents, used to make spots visible, is often used.
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SPOTTING
Solutions are spotted on the surface of the stationary phase (plate) at the prescribed volume in su ciently small portions to
obtain circular spots of 2–5 mm in diameter (1–2 mm on HPTLC plates) or bands of 10–20 mm × 1–2 mm (5–10 mm × 0.5–1 mm
on HPTLC plates) at an appropriate distance from the lower edge and sides of the plate. [ NOTE— During development, the application
position must be at least 5 mm (TLC) or 3 mm (HPTLC) above 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 centers of spots or 4
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mm (2 mm on HPTLC plates) between the edges of bands, then allowed to dry.

PROCEDURE
1. Place the plate in the chamber, ensuring that the spots or bands are above the surface of the mobile phase.
2. Close the chamber.
3. Allow the mobile phase to ascend the plate until the solvent front has traveled three-quarters of the length of the plate, or the
distance prescribed in the monograph.
4. Remove the plate, mark the solvent front with a pencil, and allow to dry.
5. Visualize the chromatograms as prescribed.
6. Determine the chromatographic Retardation factor (RF) values for the principal spots or zones.
7. Presumptive identi cation can be made by observation of spots or zones of identical RF value and about equal magnitude
obtained, respectively, with an unknown and a standard chromatographed on the same plate. A visual comparison of the size
or intensity of the spots or zones may serve for semiquantitative estimation. Quantitative measurements are possible by
means of densitometry (absorbance or uorescence measurements).
 Column Chromatography

SOLID SUPPORT
Puri ed siliceous earth is used for normal-phase separation. Silanized chromatographic siliceous earth is used for reverse-phase
partition chromatography.

STATIONARY PHASE
The solid support is modi ed by the addition of a stationary phase speci ed in the individual monograph. If a mixture of liquids is
used as the stationary phase, mix the liquids before the introduction of the solid support.

MOBILE PHASE
The mobile phase is speci ed in the individual monograph. If the stationary phase is an aqueous solution, equilibrate with water. If
the stationary phase is a polar organic uid, equilibrate with that uid.

APPARATUS
Unless otherwise speci ed in the individual monograph, the chromatographic tube is about 22 mm in its inside diameter and 200–
300 mm long. Attached to it is a delivery tube, without stopcock, about 4 mm in its inside diameter and about 50 mm long.
Apparatus preparation: Pack a pledget of ne glass wool in the base of the tube. Combine the speci ed volume of stationary phase
and the speci ed amount of solid support to produce a homogeneous, uffy mixture. Transfer this mixture to the chromatographic
tube, and tamp using gentle pressure to obtain a uniform mass. If the speci ed amount of solid support is >3 g, transfer the mixture
to the column in portions of approximately 2 g, and tamp each portion. If the assay or test requires a multisegment column with a
different stationary phase speci ed for each segment, tamp after the addition of each segment, and add each succeeding segment
directly to the previous one. Pack a pledget of ne glass wool above the completed column packing. [ NOTE— The mobile phase
should ow through a properly packed column as a moderate stream or, if reverse-phase chromatography is applied, as a slow
trickle.]

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If a solution of the analyte is incorporated into the stationary phase, complete the quantitative transfer to the chromatographic
tube by scrubbing the beaker used for the 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 nal portion of glass wool.
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PROCEDURE
1. Transfer the mobile phase to the column space above the column packing, and allow it to ow through the column under the
in uence of gravity.
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2. Rinse the tip of the chromatographic column with about 1 mL of mobile phase before each change in composition of mobile
phase and after completion of the elution.
3. If the analyte is introduced into the column as a solution in the mobile phase, allow it to pass completely into the column
packing, then add the mobile phase in several small portions, allowing each to drain completely, before adding the bulk of the
mobile phase.
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4. Where the procedure indicates the use of multiple chromatographic columns mounted in series and the addition of mobile
phase in divided portions is speci ed, allow each portion to drain completely through each column, and rinse the tip of each
with mobile phase before the addition of each succeeding portion.

Gas Chromatography
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LIQUID STATIONARY PHASE

This type of phase is available in packed or capillary columns.

PACKED COLUMN GAS CHROMATOGRAPHY

The liquid stationary phase is deposited on a nely divided, inert solid support, such as diatomaceous earth, porous polymer, or
graphitized carbon, which is packed into a column that is typically 2–4 mm in internal diameter and 1–3 m in length.

CAPILLARY COLUMN GAS CHROMATOGRAPHY

In capillary columns, which contain no packed solid support, the liquid stationary phase is deposited on the inner surface of the
column and may be chemically bonded to it.

SOLID STATIONARY PHASE

This type of phase is available only in packed columns. In these columns the solid phase is an active adsorbent, such as alumina,
silica, or carbon, packed into a column. Polyaromatic porous resins, which are sometimes used in packed columns, are not coated
with a liquid phase. [ NOTE— Packed and capillary columns must be conditioned before use until the baseline and other
 characteristics are stable. The column or packing material supplier provides instructions for the recommended conditioning
procedure.]

APPARATUS
A gas chromatograph consists of a carrier gas source, injection port, column, detector, and recording device. The injection port,
column, and detector are temperature controlled and may be varied as part of the analysis. The typical carrier gas is helium,
nitrogen, or hydrogen, depending on the column and detector in use. The type of detector used depends on the nature of the
compounds analyzed and is speci ed in the individual monograph. Detector output is recorded as a function of time, and the
instrument response, measured as peak area or peak height, is a function of the amount present.

TEMPERATURE PROGRAM
The length and quality of a GC separation can be controlled by altering the temperature of the chromatographic column. When a
temperature program is necessary, the individual monograph indicates the conditions in table format. The table indicates the initial
temperature, rate of temperature change (ramp), nal temperature, and hold time at the nal temperature.

PROCEDURE
1. Equilibrate the column, injector, and detector with owing carrier gas until a constant signal is received.
2. Inject a sample through the injector septum, or use an autosampler.
3. Begin the temperature program.
4. Record the chromatogram.
5. Analyze as indicated in the monograph.

Liquid Chromatography
LC, as used in the compendia, is synonymous with HPLC (both high-pressure and high-performance). LC is a separation technique
based on a solid stationary phase and a liquid mobile phase.

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STATIONARY PHASE
Separations are achieved by partition, adsorption, or ion-exchange processes, depending on the type of stationary phase used.
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The most commonly used stationary phases are modi ed silica or polymeric beads. The beads are modi ed by the addition of long-
chain hydrocarbons. The speci c type of packing needed to complete an analysis is indicated by the “L” designation in the individual
monograph (see also Chromatographic Columns). The size of the beads is often described in the monograph as well. Changes in the
packing type and size are covered in System Suitability in this chapter.
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CHROMATOGRAPHIC COLUMN
The term “column” includes stainless steel, lined stainless steel, and polymeric columns, packed with a stationary phase. The
length and inner diameter of the column affects the separation, and therefore typical column dimensions are included in the
individual monograph. Changes to column dimensions are discussed in System Suitability. Compendial monographs do not include
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the name of appropriate columns; this omission avoids the appearance of endorsement of a vendor’s product and natural changes
in the marketplace. See Chromatographic Columns for more information.
In LC procedures, a guard column may be used with the following requirements, unless otherwise indicated in the individual
monograph: (a) the length of the guard column must be NMT 15% of the length of the analytical column, (b) the inner diameter must
be the same or smaller than that of the analytical column, and (c) the packing material should be the same as the analytical column
(e.g., silica) and contain the same bonded phase (e.g., C18). In any case, all system suitability requirements speci ed in the o cial
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procedure must be met with the guard column installed.

MOBILE PHASE
The mobile phase is a solvent or a mixture of solvents, as de ned in the individual monograph.

APPARATUS
A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to force the mobile phase through the system
at high pressure, an injector to introduce the sample into the mobile phase, a chromatographic column, a detector, and a data
collection device.

GRADIENT ELUTION
The technique of continuously changing the solvent composition during the chromatographic run is called gradient elution or
solvent programming. The gradient elution pro le is presented in the individual monograph as a gradient table, which lists the time
and proportional composition of the mobile phase at the stated time.
 PROCEDURE
1. Equilibrate the column and detector with mobile phase at the speci ed ow rate until a constant signal is received.
2. Inject a sample through the injector, or use an autosampler.
3. Begin the gradient program.
4. Record the chromatogram.
5. Analyze as directed in the monograph.

CHROMATOGRAPHIC COLUMNS
A complete list of packings (L), phases (G), and supports (S) used in USP–NF tests and assays is located in USP–NF, Reagents,
Indicators, and Solutions—Chromatographic Columns. This list is intended to be a convenient reference for the chromatographer in
identifying the pertinent chromatographic column speci ed in the individual monograph.

DEFINITIONS AND INTERPRETATION OF CHROMATOGRAMS

Chromatogram: A graphical representation of the detector response, concentration of analyte in the e uent, or other quantity used as
a measure of e uent concentration versus e uent volume or time. In planar chromatography, chromatogram may refer to the paper or
layer with the separated zones.
Figure 1 represents a typical chromatographic separation of two substances, 1 and 2. tR1 and tR2 are the respective retention times;
h is the height, h/2 is the half-height, and Wh/2 is the width at half-height, for peak 1. W1 and W2 are the respective widths of peaks 1
and 2 at the baseline. Air peaks are a feature of gas chromatograms and correspond to the solvent front in LC. The retention time of
these air peaks, or unretained components, is designated as tM.

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Figure 1. Chromatographic separation of two substances.

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Dwell volume (D): Also known as “gradient delay volume”, is the volume between the point at which the eluents meet and the top of
the column.

Hold-up time (tM): The time required for elution of an unretained component (see Figure 1, shown as an air or unretained solvent
peak, with the baseline scale in minutes).
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Hold-up volume (VM): The volume of mobile phase required for elution of an unretained component. It may be calculated from the
hold-up time and the ow rate, F, in mL/min:

VM = tM × F
In size-exclusion chromatography, the symbol VO is used.

Number of theoretical plates (N):1 A measure of column e ciency. For Gaussian peaks, it is calculated by:

N = 16(tR/W)2
where tR is the retention time of the substance, and W is the peak width at its base, obtained by extrapolating the relatively straight
sides of the peak to the baseline. The value of N depends upon the substance being chromatographed as well as the operating
conditions, such as the ow rate and temperature of the mobile phase or carrier gas, the quality of the packing, the uniformity of the
packing within the column, and, for capillary columns, the thickness of the stationary phase lm and the internal diameter and length of
the column.
 Where electronic integrators are used, it may be convenient to determine the number of theoretical 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 portion of the chromatographic recording of the detector response when a single component 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): p/v may be employed as a system suitability criterion in a test for related substances when baseline
separation between two peaks is not achieved. Figure 2 represents a partial separation of two substances, where Hp is the height above
the extrapolated baseline of the minor peak and Hv is the height above the extrapolated baseline at the lowest point of the curve
separating the minor and major peaks:

p/v = Hp/Hv

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Figure 2. Peak-to-valley ratio determination.

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Relative retardation (Rret): The ratio of the distance traveled by the analyte to the distance simultaneously traveled by a reference
compound (see Figure 3) and is used in planar chromatography.

Rret = b/c
 Figure 3. Typical planar chromatography.

Relative retention (r):1 The ratio of the adjusted retention time of a component relative to that of another used as a reference,
obtained under identical conditions:

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r = (tR2 − tM)/(tR1 − tM)
where tR2 is the retention time measured from the point of injection of the compound of interest; tR1 is the retention time measured
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from the point of injection of the compound used as reference; and tM is the retention time of a nonretained marker de ned in the
procedure, all determined under identical experimental conditions on the same column.

Relative retention time (RRT): Also known as the “unadjusted relative retention”. Comparisons in USP–NF are normally made in
terms of unadjusted relative retention, unless otherwise indicated.
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RRT = tR2/tR1

The symbol rG is also used to designate unadjusted relative retention values.

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Relative standard deviation in percentage (%RSD):

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Resolution (RS): The resolution is the separation of two components in a mixture, calculated by:

RS = 2 × (tR2 − tR1)/(W1 + W2)

where tR2 and tR1 are the retention times of the two components; and W2 and W1 are the corresponding widths at the bases of the peaks
obtained by extrapolating the relatively straight sides of the peaks to the baseline.
Where electronic integrators are used, it may be convenient to determine the resolution, by the equation:

RS = 1.18 × (tR2 − tR1)/(W1,h/2 + W2,h/2)

Retardation factor (RF): The 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 Also known as the “capacity factor (k′)”. De ned as:

or

The k of a component may be determined from the chromatogram:

k = (tR − tM)/tM

Retention time (tR): In LC and GC, the retention time, tR, is de ned as the time elapsed between the injection of the sample and the
appearance of the maximum peak response of the eluted sample zone. tR may be used as a parameter for identi cation.
Chromatographic retention times are characteristic of the compounds they represent but are not unique. The coincidence of retention
times of a sample and a reference substance can be used as a partial criterion in construction of an identity pro le, but may not be
su cient on its own to establish identity. Absolute retention times of a given compound may vary from one chromatogram to the next.

Retention volume (VR): The volume of mobile phase required for elution of a component. It may be calculated from the retention
time and the ow rate in mL/min:

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VR = tR × F
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Separation factor (α): The relative retention calculated for two adjacent peaks (by convention, the value of the separation factor is
always >1):

α = k2/k1
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Symmetry factor (AS):2 Also known as the “tailing factor”, of a peak (see Figure 4) is calculated by:

AS = W0.05/2f
where W0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak, the
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distance being measured at a point 5% of the peak height from the baseline.
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Figure 4. Asymmetrical chromatographic peak.

Tailing factor (T): See Symmetry factor.

 SYSTEM SUITABILITY
System suitability tests are an integral part of GC and LC methods. These tests are used to verify that the chromatographic system
is adequate for the intended analysis.
The tests are based on the concept that the equipment, electronics, analytical operations, and samples analyzed constitute an
integral system that can be evaluated as such.
Factors that may affect chromatographic behavior include the following:
Composition, ionic strength, temperature, and apparent pH of the mobile phase
Flow rate, column dimensions, column temperature, and pressure
Stationary phase characteristics, including type of chromatographic support (particle-based or monolithic), particle or
macropore size, porosity, and speci c surface area
Reverse-phase and other surface modi cation of the stationary phases, the extent of chemical modi cation (as expressed by
end-capping, carbon loading, and others)
RS is a function of the number of theoretical plates, N (also referred to as e ciency), α, and k. [ NOTE— All terms and symbols are
de ned in De nitions and Interpretation of Chromatograms.] For a given stationary phase and mobile phase, N may be speci ed to
ensure that closely eluting compounds are resolved from each other, to establish the general resolving power of the system, and to
ensure that internal standards are resolved from the drug. This is a less reliable means to ensure resolution, as opposed to direct
measurement. Column e ciency is, in part, a re ection of peak sharpness, which is important for the detection of trace components.
Replicate injections of a standard preparation or other standard solutions are compared to ascertain whether requirements for
precision are met. Unless otherwise speci ed in the individual monograph, data from ve replicate injections of the analyte are used
to calculate the relative standard deviation (RSD), if the requirement is ≤2.0%; data from six replicate injections are used if the RSD
requirement is >2.0%.
For the assay in a drug substance monograph, where the value is 100% for the pure substance, and no maximum RSD is stated, the
maximum permitted %RSD is calculated for a series of injections of the reference solution:

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%RSD = KB√n/t90%,n−1

where K is a constant (0.349), obtained from the expression K = (0.6/√2) × (t90%,5/√6), in which 0.6/√2 represents the required %RSD
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after six injections for B = 1.0; B is the upper limit given in the de nition of the individual monograph − 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 freedom.
Unless otherwise prescribed, the maximum permitted RSD does not exceed the appropriate value given in Table 1 of repeatability
requirements. This requirement does not apply to tests for related substances.
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Table 1. RSD Requirements

Number of Individual Injections

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3 4 5 6

B (%) Maximum Permitted RSD

2.0 0.41 0.59 0.73 0.85

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2.5 0.52 0.74 0.92 1.06

3.0 0.62 0.89 1.10 1.27

AS, a measure of peak symmetry, is unity for perfectly symmetrical peaks; its value increases as tailing becomes more pronounced
(see Figure 4). In some cases, values less than unity may be observed. As peak symmetry moves away from values of 1, integration,
and hence precision, become less reliable.
The signal-to-noise (S/N) ratio is a useful system suitability parameter. The S/N ratio is calculated as follows:

S/N ratio = 2H/h

where H is the height of the peak measured from the peak apex to a baseline extrapolated over a distance ≥5 times the peak width at
its half-height; and h is the difference between the largest and smallest noise values observed over a distance ≥5 times the width at
the half-height of the peak and, if possible, situated equally around the peak of interest (see Figure 5).
 Figure 5. Noise and chromatographic peak, components of the S/N ratio.

These system suitability tests are performed by collecting data from replicate injections of standard or other solutions as speci ed
in the individual monograph.
The speci cation of de nitive parameters in a monograph does not preclude the use of other suitable operating conditions.
Adjustments to the speci ed chromatographic system may be necessary in order to meet system suitability requirements.
Adjustments to chromatographic systems performed in order to comply with system suitability requirements are not to be made in
order to compensate for column failure or system malfunction. Adjustments are permitted only when suitable standards (including
Reference Standards) are available for all compounds used in the suitability test, and the adjustments or column change yields a

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chromatogram that meets all the system suitability requirements speci ed in the o cial procedure.
If adjustments of operating conditions are necessary in order to meet system suitability requirements, each of the items in the
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following list is the maximum variation that can be considered, unless otherwise directed in the monograph; these changes may
require additional veri cation data. To verify the suitability of the method under the new conditions, assess the relevant analytical
performance characteristics 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 implementation. In some circumstances, it may be desirable to
use an HPLC column with different dimensions to those prescribed in the o cial procedure (different length, internal diameter, and/or
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particle size). In either case, changes in the chemical characteristics (“L” designation) of the stationary phase will be considered a
modi cation to the method and will require full validation. Adjustments to the composition of the mobile phase in gradient elution
may cause changes in selectivity and are not recommended. If adjustments are necessary, a change in column packing (maintaining
the same chemistry), the duration of an initial isocratic hold (when prescribed), and/or the dwell volume are allowed. Additional
allowances for gradient adjustment are noted in the text below.
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pH of mobile phase (HPLC): The pH of the aqueous buffer used in the preparation of the mobile phase can be adjusted to within
±0.2 units of the value or range speci ed. Applies to both gradient and isocratic separations.

Concentration of salts in buffer (HPLC): The concentration of the salts used in the preparation of the aqueous buffer employed in
the mobile phase can be adjusted to within ±10% if the permitted pH variation (see above) is met. Applies to both gradient and isocratic
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separations.

Ratio of components in mobile phase (HPLC): The following adjustment limits apply to minor components of the mobile phase
(speci ed as ≤50%). The amounts of these components can be adjusted by ±30% relative. However, the change in any component
cannot exceed ±10% absolute (i.e., in relation to the total mobile phase). Adjustment can be made to one minor component in a ternary
mixture. Examples of adjustments for binary and ternary mixtures are given below.

Binary mixtures

SPECIFIED RATIO OF 50:50: 30% of 50 is 15% absolute, but this exceeds the maximum permitted change of ±10% absolute in
either component. Therefore, the mobile phase ratio may be adjusted only within the range of 40:60–60:40.
SPECIFIED RATIO OF 2:98: 30% of 2 is 0.6% absolute. Therefore, the maximum allowed adjustment is within the range of 1.4:98.6–
2.6:97.4.

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%–45%
absolute. For the third component, 30% of 5 is 1.5% absolute. In all cases, a su cient quantity of the rst component is used to give a
total of 100%. Therefore, mixture ranges of 50:45:5–70:25:5 or 58.5:35:6.5–61.5:35:3.5 would meet the requirement.

Wavelength of UV-visible detector (HPLC): Deviations from the wavelengths speci ed in the procedure are not permitted. The
procedure speci ed by the detector manufacturer, or another validated procedure, is used to verify that error in the detector wavelength
is, at most, ±3 nm.

Stationary phase

COLUMN LENGTH (GC): Can be adjusted by as much as ±70%.

COLUMN LENGTH (HPLC): See Particle size (HPLC) below.
COLUMN INNER DIAMETER (HPLC): Can be adjusted if the linear velocity is kept constant. See Flow rate (HPLC).
COLUMN INNER DIAMETER (GC): Can be adjusted by as much as ±50%.
FILM THICKNESS (CAPILLARY GC): Can be adjusted by as much as −50% to 100%.
Particle size (HPLC): For isocratic separations, the particle size and/or the length of the column may be modi ed provided that the
ratio of the column length (L) to the particle size (dp) remains constant or into the range between −25% and 50% of the prescribed L/dp
ratio. Alternatively (as for the application of particle-size adjustment to super cially porous particles), other combinations of L and dp
can be used provided that the number of theoretical plates (N) is within −25% to 50%, relative to the prescribed column. Caution should
be used when the adjustment results in a higher number of theoretical plates that generate smaller peak volumes, which may require
adjustments to minimize extra-column band broadening by factors such as instrument plumbing, detector cell volume and sampling
rate, and injection volume. For gradient separations, changes in length, column inner diameter, and particle size are not allowed.

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Particle size (GC): Changing from a larger to a smaller or from a smaller to a larger particle size GC mesh support is acceptable if the
chromatography meets the requirements of system suitability and the same particle size range ratio is maintained. The particle size
range ratio is de ned as the diameter of the largest particle divided by the diameter of the smallest particle.
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Flow rate (GC): The ow rate can be adjusted by as much as ±50%. [ NOTE— When the monograph speci es a linear velocity parameter,
the allowed velocity adjustment is between +50% and −25%, provided the carrier gas system can be maintained under control at the
desired set points.]

Flow rate (HPLC): When the particle size is changed, the ow rate may require adjustment, because smaller-particle columns will
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require higher linear velocities for the same performance (as measured by reduced plate height). Flow rate changes for both a change in
column diameter and particle size can be made by:

F2 = F1 × [(dc22 × dp1)/(dc12 × dp2)]

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where F1 and F2 are the ow rates for the original and modi ed conditions, respectively, dc1 and dc2 are the respective column
diameters, and dp1 and dp2 are the particle sizes.
When a change is made from ≥3-µm to <3-µm particles in isocratic separations, an additional increase in linear velocity (by
adjusting ow rate) may be justi ed, provided that the column e ciency does not drop by >20%. Similarly, a change from <3-µm to ≥3-
µm particles may require additional reduction of linear velocity ( ow rate) to avoid reduction in column e ciency by >20%. Changes in
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F, dc, and dp are not allowed for gradient separations.

Additionally, the ow rate can be adjusted by ±50% (isocratic only).
EXAMPLES: Adjustments in column length, internal diameter, particle size, and ow rate can be used in combination to give
equivalent conditions (same N), but with differences in pressure and run time. Table 2 lists some of the more popular column
con gurations to give equivalent e ciency (N), by adjusting these variables.

Table 2. Column Con gurations

Column Relative Values

Length (L, Diameter (dc, Particle Size
mm) mm) (dp, µm) L/dp F N Pressure Run Time

250 4.6 10 25,000 0.5 0.8 0.2 3.3

 Column Relative Values
Length (L, Diameter (dc, Particle Size
mm) mm) (dp, µm) L/dp F N Pressure Run Time

150 4.6 5 30,000 1.0 1.0 1.0 1.0

150 2.1 5 30,000 0.2 1.0 1.0 1.0

100 4.6 3.5 28,600 1.4 1.0 1.9 0.5

100 2.1 3.5 28,600 0.3 1.0 1.9 0.5

75 4.6 2.5 30,000 2.0 1.0 4.0 0.3

75 2.1 2.5 30,000 0.4 1.0 4.0 0.3

50 4.6 1.7 29,400 2.9 1.0 8.5 0.1

50 2.1 1.7 29,400 0.6 1.0 8.5 0.1

For example, if a monograph speci es a 150-mm × 4.6-mm; 5-µm column operated at 1.5 mL/min, the same separation may be
expected with a 75-mm × 2.1-mm; 2.5-µm column operated at 1.5 mL/min × 0.4 = 0.6 mL/min, along with a pressure increase of
about four times and a reduction in run time to about 30% of the original.

Injection volume (HPLC): The injection volume can be adjusted as far as it is consistent with accepted precision, linearity, and

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detection limits. Note that excessive injection volume can lead to unacceptable band broadening, causing a reduction in N and
resolution, which applies to both gradient and isocratic separations.
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Injection volume and split volume (GC): The injection volume and split volume may be adjusted if detection and repeatability are
satisfactory.

Column temperature (HPLC): The column temperature can be adjusted by as much as ±10°. Column thermostating is
recommended to improve control and reproducibility of retention time, which applies to both gradient and isocratic separations.
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Oven temperature (GC): The oven temperature can be adjusted by as much as ±10%.
Oven temperature program (GC): Adjustment of temperatures is permitted as stated above. When the speci ed temperature must
be maintained or when the temperature must be changed from one value to another, an adjustment of up to ±20% is permitted.
Unless otherwise directed in the monograph, system suitability parameters are determined from the analyte peak.
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Measured values of Rr, RF, or tR for the sample substance do not deviate from the values obtained for the reference compound and
mixture by more than the statistically determined reliability estimates from replicate assays of the reference compound. RRT may be
provided in monographs for informational purposes only to aid in peak identi cation. There are no acceptance criteria applied to RRT.
Suitability testing is used to ascertain the effectiveness of the nal operating system, which should be subjected to this testing.
Make injections of the appropriate preparation(s), as required, in order to demonstrate adequate system suitability (as described in
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the Chromatographic system section of the method in a monograph) throughout the run.
The preparation can be a standard preparation or a solution containing a known amount of analyte and any additional materials
(e.g., excipients or impurities) useful in controlling the analytical system. Whenever there is a signi cant change in the
chromatographic system (equipment, mobile phase component, or other components) or in a critical reagent, system suitability is to
be reestablished. No sample analysis is acceptable unless the suitability of the system has been demonstrated.

QUANTITATION
During quantitation, disregard peaks caused by solvents and reagents or arising from the mobile phase or the sample matrix.
In the linear range, peak areas and peak heights are usually proportional to the quantity of compound eluting. The peak areas and
peak heights are commonly measured by electronic integrators but may be determined by more classical approaches. Peak areas are
generally used but may be less accurate if peak interference occurs. The components measured are separated from any interfering
components. Peak tailing and fronting is minimized, and the measurement of peaks on tails of other peaks are avoided when
possible.
Although comparison of impurity peaks with those in the chromatogram of a standard at a similar concentration is preferred,
impurity tests may be based on the measurement of the peak response due to impurities and expressed as a percentage of the area
 of the drug peak. The standard may be the drug itself at a level corresponding to, for example, 0.5% impurity, assuming similar peak
responses. When impurities must be determined with greater certainty, use a standard of the impurity itself or apply a correction
factor based on the response of the impurity relative to that of the main component.

External Standard Method

The concentration of the component(s) quanti ed is determined by comparing the response(s) obtained with the sample solution
to the response(s) obtained with a standard solution.

Internal Standard Method

Equal amounts of the internal standard are introduced into the sample solution and a standard solution. The internal standard is
chosen so that it does not react with the test material and does not contain impurities with the same retention time as that of the
analytes, and is stable and resolved from the component(s) quanti ed (analytes). The concentrations of the analytes are determined
by comparing the ratios of their peak areas or peak heights and the internal standard in the sample solution with the ratios of their
peak areas or peak heights and the internal standard in the standard solution.

Normalization Procedure
The percent content of a component of the test material is calculated by determining the area of the corresponding peak as a
percentage of the total area of all the peaks, excluding those due to solvents or reagents or arising from the mobile phase or the
sample matrix and those at or below the limit at which they can be disregarded.

Calibration Procedure
The relationship between the measured or evaluated signal y and the quantity (e.g., concentration or mass) of substance x is
determined, and the calibration function is calculated. The analytical results are calculated from the measured signal or evaluated
signal of the analyte and its position on the calibration curve.
In tests for impurities for both the external standard method, when a dilution of the sample solution is used for comparison, and the

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normalization procedure, any correction factors indicated in the monograph are applied (e.g., when the relative response factor is
outside the range of 0.8–1.2).
When the impurity test prescribes the total of impurities or there is a quantitative determination of an impurity, choice of an
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appropriate threshold setting and appropriate conditions for the integration of the peak areas is important. In such tests the limit at or
below which a peak is disregarded is generally 0.05%. Thus, the threshold setting of the data collection system corresponds to at
least half of this limit. Integrate the peak area of any impurity that is not completely separated from the principal peak, preferably by
valley-to-valley extrapolation (tangential skim).
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1  The parameters k, N, r, and rG were developed for isothermal GC separations and isocratic HPLC separations. Because these terms
are thermodynamic parameters, they are only valid for separations made at a constant temperature, mobile phase composition, and
ow rate. However, for separations made with a temperature program or solvent gradient, these parameters may be used simply as
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comparative means to ensure that adequate chromatographic conditions exist to perform the methods as intended in the monographs.

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  It 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 interpolated baseline and the peak front, and the distance between that line and the peak back measured at 10% of
the peak height (see Figure 4), which would be (W0.10 − f0.10)/f0.10. However, for the purposes of USP, only the formula (AS) as presented
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here is valid.

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< 621 > CHROMATOGRAPHY Horacio N. Pappa GCCA2020 General Chapters - Chemical
Director, General Chapters Analysis 2020

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Pharmacopeial Forum: Volume No. 43(5)

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