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Tipo de Documento: Capítulo General
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DOI: https://doi.org/10.31003/USPNF_M99380_05_01
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〈 621 〉 CHROMATOGRAPHY
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▲
INTRODUCTION
Chromatographic separation techniques are multistage separation procedures in
which the components of a sample are distributed between
two phases, one of which
is
stationary while 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, or distributed as a film, etc. The mobile phase may be gaseous or liquid or
supercritical fluid. The separation may be based on adsorption, mass distribution
(partition), ion exchange, etc., or may be based on differences
in the physicochemical
properties of the molecules such as size, mass, volume, etc.
Portions of the present general chapter text that are national USP–NF text, and therefore not part of the harmonized text, are marked with
symbols
(♦♦) to specify this fact.
♦This chapter describes general procedures, definitions, and calculations of
common parameters and generally applicable requirements for
system suitability. AL
The types of chromatography useful in qualitative and quantitative analysis
employed in USP procedures are column, gas, paper, thin-layer
(including high-performance
thin-layer chromatography), and pressurized liquid chromatography (commonly called
high-pressure or high-
performance liquid chromatography).
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GENERAL PROCEDURES
This section describes the basic procedures used when a chromatographic method
is described in a monograph. The following procedures
are followed unless otherwise
indicated in the individual monograph.

Paper Chromatography
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stationary phase
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 flow is also assisted by
gravitational force. The orientation of paper grain with
respect to solvent flow is
to
be kept constant in a series of chromatograms. (The machine direction is usually
designated by the
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manufacturer.)

apparatus
The essential equipment for paper chromatography consists of a vapor-tight
chamber with inlets for addition of solvent and a rack of
RÁ

corrosion-resistant material
about 5 cm shorter than the inside height of the chamber. The rack serves as a support
for solvent troughs and for
antisiphon rods that, in turn, hold up the chromatographic
sheets. The bottom of the chamber is covered with 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 micropipets of the
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resulting solution,
normally containing 1–20 µg of the compound, are placed in 6- to 10-mm spots not less
than 3 cm 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 fluid in the chamber.]
2.
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The chamber is sealed to allow equilibration (saturation)

of the chamber and the paper with the solvent vapor. Any excess pressure is
3. After
released
equilibration
as necessary.
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, finely
powdered material applied to a glass, plastic, or metal sheet or plate
(typically called
the plate). The stationary phase of thin-layer chromatography (TLC) plates has an
average particle size of 10–15 µm, and that
of high-performance TLC (HPTLC) plates has
an average particle size of 5 µm. Commercial plates with a preadsorbent zone can be used
if

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they are specified in a monograph. 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.

apparatus
A chromatographic chamber made of inert, transparent material and having
the following specifications is used: a flat-bottom or twin trough,
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a tightly fitted
lid, and a size suitable for the plates. The chamber is lined on at least one wall
with
filter paper. Sufficient mobile phase or
developing solvent is added to the chamber
so
that, after impregnation of the filter 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 spray
reagents to
make
spots visible are often used.
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spotting
Solutions are spotted on the surface of the stationary phase (plate) at
the prescribed volume in sufficiently 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
RÁ

(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
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.
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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.

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Presumptive identification 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 fluorescence
measurements). Column Chromatography

solid support
Purified 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 modified by the addition of a stationary phase
specified 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 specified in the individual monograph. If the
stationary phase is an aqueous solution, equilibrate with water. If the
stationary
phase
is a polar organic fluid, equilibrate with that fluid.

apparatus
Unless otherwise specified in the individual monograph, the
chromatographic tube is about 22 mm in inside diameter and 200–300 mm in
length.
Attached to it is a delivery tube, without stopcock, about 4 mm in inside diameter and
about 50 mm in length.
Apparatus preparation: Pack a pledget of fine glass wool in the base of the tube.
Combine the specified volume of stationary phase and the
specified amount of solid
support to produce a homogeneous, fluffy mixture. Transfer this mixture to the
chromatographic tube, and tamp
using gentle pressure to obtain a uniform mass. If
the
specified amount of solid support is more than 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

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specified for each segment,
tamp after the addition of each segment, and add each succeeding segment directly
to
the previous one. Pack a
pledget of fine glass wool above the completed column
packing. [Note—The mobile phase should flow through a properly packed
column as a
moderate stream or, if reverse-phase chromatography is applied, as a
slow trickle.]
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 final 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 flow through the column under the influence
of
gravity.
2. Rinse the tip of the chromatographic column with about 1
mL of mobile phase before each change in composition of mobile phase and
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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 mobile phase in several small portions, allowing each to drain
completely, before adding the bulk of the mobile phase.
4. Where the procedure indicates the use of multiple
chromatographic columns mounted in series and the addition of mobile phase in
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divided portions is specified, 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 (GC)

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liquid stationary phase

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

packed column GC
The liquid stationary phase is deposited on a finely 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.
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capillary column GC
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

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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 specified
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), final temperature, and
hold
time at the final temperature.

procedure
1. Equilibrate the column, injector, and detector with
flowing 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

AL
The term "liquid chromatography" (LC), as used in the compendia, is synonymous
with high-pressure liquid chromatography and high-
performance liquid chromatography.
LC is
a separation technique based on a solid stationary phase and a liquid mobile phase.

stationary phase
Separations are achieved by partition, adsorption, or ion-exchange
processes, depending on the type of stationary phase used. The most
commonly used
stationary phases are modified silica or polymeric beads. The beads are modified by the
addition of long-chain hydrocarbons.
CI
The specific type of packing needed to complete an
analysis is indicated by the “L” designation in the individual monograph (see also the
section 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
the System Suitability section of 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 the System Suitability section of this chapter. Compendial monographs do
not include the
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name of appropriate columns; this omission avoids the appearance of

endorsement of a vendor’s product and natural changes in the
marketplace. See the
section Chromatographic Columns for more information.
In LC procedures, a guard column may be used with the following
requirements, unless otherwise is 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
RÁ

bonded phase (e.g., C18). In any case, all system suitability

requirements specified in the official procedure must be met with the guard column
installed.

mobile phase
The mobile phase is a solvent or a mixture of solvents, as defined in the
individual monograph.
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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 profile is presented in the individual monograph as a gradient table, which
lists the time and proportional

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composition of the mobile phase at the stated time.

procedure
1. Equilibrate the column and detector with mobile phase at
the specified flow 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.
Analyze as directed in the monograph.
5.
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 specified in the individual monograph.♦

DEFINITIONS
The system suitability and acceptance criteria in monographs have been set
using parameters as defined below. With some equipment,
certain parameters, such as
the
signal-to-noise ratio and resolution, can be calculated using software provided by
the
manufacturer. It is the
responsibility of the user to ensure that the calculation
methods
used in the software are equivalent to the requirements of the US
Pharmacopeia and to make any necessary corrections if this is not the case.

Chromatogram: A graphical or other representation of detector response,

effluent concentration, or other quantity used as a measure of effluent
concentration
versus time or volume. Idealized chromatograms are represented as a sequence of Gaussian
peaks on a baseline (Figure 1).

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Figure
1.
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VM = hold-up volume

tM = hold-up time

VR1 = retention volume of peak 1

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tR1 = retention time of peak 1

VR2 = retention volume of peak 2

tR2 = retention time of peak 2

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Wh = peak width at half-height

Wi = peak width at the inflection point

h = height of the peak

h/2 = half-height of peak

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Distribution constant (K0): In size-exclusion chromatography, the elution characteristics of

a component in a particular column may be given
by the distribution constant (also
referred to as distribution coefficient), which is calculated using the following
equation:

tR− t0
K0 =
tt −t0

tR = retention time

t0 = retention time of an unretained compound

tt = total mobile phase time

Dwell volume (D) (also referred to as VD): The dwell volume (also known as gradient delay volume) is the
volume between the point at which
the eluents meet and the inlet of the column. It
can be
determined using the following procedure.
column: Replace the chromatographic column by an appropriate capillary
tubing (e.g., 1 m × 0.12 mm).

mobile phase: See Table 1.

mobile phase A: Water

mobile phase B: 0.1% v/v solution of acetone in water

Table 1

Time
Mobile phase A
Mobile phase B

(min) (% v/v) (% v/v)

0–20

20–30
AL 100→0

0
0→100

100
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flow rate: Set to obtain sufficient back-pressure (e.g., 2 mL/min).

detection: Spectrophotometer at 265 nm

Determine the time (t0.5), in
minutes, when the absorbance has increased by 50% (Figure 2).
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D = tD × F

tD = t0.5 — 0.5tG (min)

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tG = pre-defined gradient time, 20 min

F = flow rate (mL/min)

RÁ
SE

Figure
2.

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[Note—where applicable, this measurement is performed with the autosampler

in the "inject" position so as to include the injection loop
volume in the dwell
volume.]

Hold-up time (tM): Time required for elution of an unretained component (see Figure 1, baseline scale being in minutes or seconds).
In size-exclusion chromatography, the term retention time of an unretained
compound (t0) is used.

Hold-up volume (VM): Volume of the mobile phase required for elution of an unretained
component. It may be calculated from the hold-up time
and the flow rate (F) in milliliters per minute using the following equation:

VM = tM × F

In size-exclusion chromatography, the term "retention volume" of an

unretained compound V0 is used.

Peak: Portion of a chromatogram recording the detector response when a

single component (or two or more unresolved components) is eluted
from the column.
The peak response may be represented by the peak area or the peak height (h).

Peak-to-Valley Ratio (p/v): The peak-to-valley ratio may be employed as a system suitability
criterion when baseline separation between two
peaks is not achieved (see Figure 3).

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Figure 3.

Hp
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p/v =
Hv

Hp = height above the extrapolated baseline of the minor

peak

Hv = height above the extrapolated baseline at the lowest point

of the curve separating the minor and major peaks
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Plate height (H) (height equivalent to one theoretical plate [HETP]): Ratio of the column length (L), in
micrometers, to the plate number
(N):

L
H =
N

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Plate number (N) (number of theoretical plates): A number indicative of column performance (column efficiency) can
only be calculated
from data obtained under either isothermal, isocratic, or isodense
conditions, depending on the technique, as the plate number, using the following
equation,
the values of tR and Wh being expressed in the same units:

2
tR
N = 5.54  ( )
Wh

tR = retention time of the peak corresponding to the

component

Wh = peak width at half-height (h/2)

The plate number varies with the component as well as with the column, the
column temperature, the mobile phase, and the retention time.

Reduced plate height (h): Ratio of the plate height (H), in micrometers,
to the particle diameter (dp) in micrometers:

H
h =
dp

Relative retardation (Rrel): The relative retardation, used in thin-layer chromatography, is

calculated as the ratio of the distances travelled by the
spot of the compound of
interest
and a reference compound (Figure 4).

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Figure 4.
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Rr el   = b/c

a = migration distance of the mobile phase

b = migration distance of the compound of interest

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c = migration distance of the reference compound

Relative retention (r): Relative retention is calculated as an estimate using the

following equation:

tRi −tM
r =
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tRst −tM

tRi = retention time of the peak of interest

tRst = retention time of the reference peak (usually the peak

corresponding to the substance to be examined)

tM = hold-up time

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Relative retention, unadjusted (rG)♦or (RRT)♦

The unadjusted relative retention is calculated using the following

equation:

tRi
rG =
tRst

Unless otherwise indicated, values for relative retention stated in

monographs correspond to unadjusted relative retention.

♦Relative retention time (RRT): See Relative retention,

unadjusted♦
Resolution (Rs): The resolution between peaks of two components (Figure 1) may be calculated using the following equation:

1.18(tR2 −tR1 )
RS =
W h1 +W h2

tR 2 > tR 1

tR1, tR2 = retention times of the peaks

Wh1, Wh2 = peak widths at half-height

In quantitative thin-layer chromatography, using densitometry, the migration

distances are used instead of retention times and the resolution
between peaks of
two
components may be calculated using the following equation:

1.18a(RF 2 −RF 1 )
RS =
W h1 +W h2

RF 2 > RF 1

RF1, RF2 = retardation factors of the peaks

Wh1, Wh2

a
= peak widths at half-height
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= migration distance of the solvent front

Retardation factor (RF): The retardation factor, used in thin-layer chromatography, is the
ratio of the distance from the point of application to
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the center of the spot and
the
distance simultaneously travelled by the solvent front from the point of application
(Figure 4).

b
RF =
a

b = migration distance of the component

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a = migration distance of the solvent front

Retention factor (k): The retention factor (also known as mass distribution ratio (Dm) or capacity factor (k′))
is defined as:
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amount   of  c omponent  i n s t at i onar y  phas e VS

k = = Kc
amount  of  c omponent   i n  mobi l e phas e VM

KC = distribution constant (also known as equilibrium distribution

coefficient)
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VS = volume of the stationary phase

VM = volume of the mobile phase

The retention factor of a component may be determined from the

chromatogram using the following equation:
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tR −tM
k =
tM

tR = retention time

tM = hold-up time

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Retention time (tR): Time elapsed between the injection of the sample and the
appearance of the maximum peak response of the eluted
sample zone (Figure 1, baseline scale being in minutes or seconds).

Retention volume (VR):

Volume of the mobile phase required for elution of a component. It may be

calculated from the retention time and the flow rate (F), in
milliliters per minute, using the following equation:

V R = t R ×  F

Retention time of an unretained compound

(t0): In size-exclusion chromatography, retention time of a component
whose molecules are
larger than the largest gel pores (Figure 5).

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Figure
5.
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Retention volume of an unretained

compound (V0): In size-exclusion chromatography, retention volume of a component
whose molecules
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are larger than the largest gel pores. It may be calculated from the
retention time of an unretained compound and the flow rate (F), in
milliliters per
minute, using the following equation:

V 0 = t0 × F
RÁ

Separation factor (α): Relative retention calculated for two adjacent peaks (by
convention, the value of the separation factor is always >1):

α = k 2 /k 1

k1 = retention factor of the first peak

k2 = retention factor of the second peak

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Signal-to-noise ratio (S/N): The short-term noise influences the precision and accuracy of
quantitation. The signal-to-noise ratio is calculated
using the following equation:

2H
S /N =
h

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H = height of the peak (Figure 6) corresponding to the component concerned, in the
chromatogram obtained with the prescribed reference
solution, measured from the
maximum of the peak to the extrapolated baseline of the signal observed over a
distance equal to 20 ti-
mes the width at half-height

h = range of the noise in a chromatogram obtained after

injection of a blank (Figure 7), observed over a distance equal to 20 times the
width
at half-height of the peak in the chromatogram obtained with the prescribed
reference solution and, if possible, situated equally around
the place where this
peak would be found

Figure 6.
Chromatogram of the reference solution.

AL
CI

Figure 7.
Chromatogram of a blank.
FI

If a baseline of 20 times the width at half-height is not obtainable because

of peaks due to the solvents or reagents, or arising from the mobile
phase or the
sample
matrix, or due to the gas chromatographic temperature program, a baseline of at least
5
times the width at half-height is
permitted.

Symmetry factor (AS): The symmetry factor of a peak (also known as

the asymmetry factor or tailing factor) (Figure 8) is calculated using the
O

following equation:

W 0.05
As =
2d
RÁ

W0.05 = width of the peak at one-twentieth of the peak height

d = distance between the perpendicular dropped from the peak maximum

and the leading edge of the peak at one-twentieth of the peak
height

An As value of 1.0 signifies symmetry. When As > 1.0, the

peak is tailing. When As < 1.0, the peak is fronting.
SE

Figure
8.

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System repeatability: The repeatability of response is expressed as an estimated

percentage relative standard deviation (%RSD) of a
consecutive series of measurements
for
not fewer than three injections or applications of a reference solution, and is calculated
using the
following equation.
−−−−−−
2
100 Σ(y −ȳ )
%R S D = √ i

ȳ n−1

yi = individual values expressed as peak area, peak height, or

ratio of areas by the internal standardization method

¯
y = mean of individual values

n = number of individual values

Total mobile phase time (tt): In size-exclusion chromatography, retention time of a component
whose molecules are smaller than the smallest
gel pores (Figure 5).

Total mobile phase volume (Vt): In size-exclusion chromatography, retention volume of a component
whose molecules are smaller than the
smallest gel pores. It may be calculated from
the
total mobile phase time and the flow rate (F), in milliliters/minute,
using the following equation:

V t = tt × F

SYSTEM SUITABILITY
[Note—This section only covers liquid chromatography and gas
chromatography]

AL
The various components of the equipment employed must be qualified and be
capable of achieving the performance required to conduct the
test or assay. The system
suitability tests represent an integral part of the analytical procedure and are used
to
ensure adequate performance of
the chromatographic system. Column plate number, retention
factor (mass distribution ratio), system repeatability, signal-to-noise, symmetry
factor,
and resolution/peak-to-valley ratio are the parameters that may be employed in assessing
the
performance of the chromatographic
system. When it is indicated in the individual
monograph,
in case of complex chromatographic profiles (e.g., for biotechnological and biological
CI
products) visual comparison of the profiles can be used as a system suitability test.
Factors that may affect the chromatographic behavior
include:
Composition and temperature of the mobile phase
Ionic strength and pH of the aqueous component, of the mobile
phase
Flow rate, column dimensions, column temperature, and
pressure
FI

Stationary phase characteristics including, type of

chromatographic support (particle-based or monolithic), particle or pore size, and
porosity, and specific surface area
Reverse phase and other surface modification of the
stationary phases, the extent of chemical modification (as expressed by end-
capping,
carbon loading, etc.)
O

Retention times and relative retentions may be provided in monographs for

information purposes only, unless otherwise stated in the
monograph. There are no
acceptance
criteria applied to relative retentions.
Compliance with the system suitability criteria is required throughout the
chromatographic procedure. No sample analysis is acceptable
unless the suitability
of the
system has been demonstrated.
RÁ

The following requirements are to be fulfilled, in addition to any other

system suitability criteria stated in the monograph. When specific
requirements are
stated
in the monograph, they supersede the requirements mentioned in this chapter.

Change to read:

System
Repeatability▲▲ (ERR 1-Dec-2022)

♦When a relative standard deviation requirement is specified in an

individual monograph, if the requirement is 2.0 or less the calculation is
SE

based
on data from five replicate injections of the analyte, if the requirement is
more than 2.0% data from six replicate injections are used.♦
In an assay of an active substance or an excipient, where the target
value is 100% for a pure substance, and a system repeatability
requirement is
not specified, the maximum permitted relative standard deviation (%RSDmax) for the defined limits is calculated for a series
(n = 3
to 6) of injections of the reference solution. The
maximum permitted relative standard deviation of the peak response does not
exceed the
appropriate value given in Table 2.

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K B√n
%R S Dmax =
t90%,n−1

K = constant (0.349), obtained from the

expression K =
0.6
×
t90%,n−1
in which
0.6
represents the required percentage relative stan-
√2 √6 √2

dard
deviation determined on 6 injections for B =
1.0

B = upper limit given in the definition of the

individual monograph minus 100%

n = number of replicate injections of the

reference solution (3 ≤ n ≤ 6)

t90%,n = Student’s t at the 90%

probability level (double sided) with n−1
degrees of freedom

–1

Table 2. Maximum
Permitted Relative Standard Deviation of Peak Response (Assay)

Number of Individual
Injections (n)

3 4 5 6

B
(%) Maximum
Permitted Relative Standard Deviation (%)

2.0 0.41 0.59 0.73 0.85

2.5 0.52 0.74 0.92 1.06

3.0 0.62 0.89 1.10 1.27

B AL
= upper limit of content given in the
individual monograph minus 100%

System Sensitivity
The signal-to-noise ratio is used to define the system sensitivity. The
limit of quantitation (corresponding to a signal-to-noise ratio of 10) is
equal to
or less
than the reporting threshold.
CI
Peak Symmetry

Unless otherwise stated, in a test or assay, the symmetry factor (tailing

factor) of the peak used for quantification is 0.8–1.8.

Change to read:
FI

ADJUSTMENT OF CHROMATOGRAPHIC
CONDITIONS
The chromatographic conditions described have been validated during the
elaboration of the monograph.
The extent to which the various parameters of a chromatographic test may
be adjusted without fundamentally modifying the pharmacopeial
O

analytical procedures
are listed below. Changes other than those indicated require revalidation of the
procedure.
Multiple adjustments can have a cumulative effect on the performance of
the system and are to be properly evaluated by the users. This is
particularly
important in cases where the separation pattern is described as a profile. In those
cases, a risk assessment has to be carried out.
Any adjustments must be made on the basis of the pharmacopeial
procedure.
If adjustments are made to a pharmacopeial procedure, additional
verification tests may be required. To verify the suitability of the adjusted
RÁ

pharmacopeial procedure, assess the relevant analytical performance characteristics

potentially affected by the change.
When a pharmacopeial procedure has been adjusted according to the
requirements stated below, no further adjustments are allowed without
appropriate
revalidation.
Compliance with the system suitability criteria is required to verify
that conditions for satisfactory performance of the test or assay are
achieved.
Adjustment of conditions with gradient elution (HPLC) or temperature
programming (GC) is more critical than with isocratic (HPLC) or
SE

isothermal (GC)
elution, since it may shift some peaks to a different step of the gradient or to
different elution temperatures, potentially causing
partial or complete coelution
of
adjacent peaks or peak inversion, and, thus leading to the incorrect assignment of
peaks and to the masking of
peaks or a shift such that elution occurs beyond the
prescribed elution time.
For some parameters the adjustments are explicitly defined in the
monograph to ensure the system suitability.

Thin-Layer Chromatography

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Composition of the mobile phase: the amount of

the minor solvent components may be adjusted by ±30% relative or ±2% absolute,
whichever
is the larger; no other component is altered by more than 10%
absolute. A minor component comprises less than or equal to (100/n)%, n being
the total number of components of the
mobile phase. For a minor component at 10% of the mobile phase, a 30% relative
adjustment allows a
range of 7%–13% whereas a 2% absolute adjustment allows a
range of 8%–12%, the relative value therefore being the larger; for a minor
component at 5% of the mobile phase, a 30% relative adjustment allows a range of
3.5%–6.5% whereas a 2% absolute adjustment allows a
range of 3%–7%, the absolute
value being the larger in this case.
pH of the aqueous component of the mobile phase:
±0.2 pH units, unless otherwise prescribed
Concentration of salts in the buffer component of a
mobile phase: ± 10%
Application volume: 10%–20% of the prescribed
volume if using fine particle size plates (2–10 µm)

Liquid Chromatography: Isocratic

Elution

column parameters and flow

rate
Stationary phase: No change of the identity of
the substituent (e.g., no replacement of C18 by C8); the other
physicochemical characteristics
of the stationary phase, i.e.,
chromatographic support, surface modification and extent of chemical
modification must be similar; a change
from totally porous particle (TPP)
columns to superficially porous particle (SPP) columns is allowed provided
the above-mentioned
requirements are met.
Column dimensions (particle size, length):
The particle size and/or length of the column may be modified, provided that
the ratio of the
column length (L) to the particle size
(dp) remains constant or in the range between −25% to
+50% of the prescribed L/dp ratio.
Adjustments from totally porous to superficially
porous particles: For the application of particle-size adjustment
from totally porous to
superficially porous particles, other combinations of
L and dp can be used, provided
that the plate number (N) is within −25% to +50%,
relative
to the prescribed column. These changes are acceptable, provided
that system suitability criteria are fulfilled, and selectivity and elution
order
of the specified impurities to be controlled are demonstrated to be
equivalent.
Internal diameter: In absence of a change in
particle size and/or length, the internal diameter of the column may be
adjusted.
Caution is necessary when the adjustment results in smaller peak
volumes due to a smaller particle size or smaller internal column
AL
diameter,
a situation that may require adjustments to minimize extra-column band
broadening by factors such as instrument connections,
detector cell volume
and sampling rate, and injection volume.
When the particle size is changed, the flow rate requires
adjustment, because smaller-particle columns will require higher linear
velocities for
the same performance (as measured by reduced plate height).
The flow rate is adjusted for both the change in column diameter and
particle
size using the following equation:
CI
2 2
F2 = F1 × [(d c × d p )/ (d c × d p )]
2 1 1 2

F1 = flow rate indicated in the monograph

(mL/min)

F2 = adjusted flow rate (mL/min)

FI

dc1 = internal diameter of the column

indicated in the monograph

dc2 = internal diameter of the column used

(mm)
O

dp1 = particle size indicated in the monograph

(µm)

dp2 = particle size of the column used

(µm)

When a change is made from ≥3-µm to <3-µm particles in

isocratic separations, an additional increase in linear velocity (by
adjusting the
RÁ

flow rate) may be justified, provided that the column

performance does not drop by more than 20%. Similarly, when a change is made
from <3-
µm to ≥3-µm particles, an additional reduction of linear velocity
(flow rate) may be justified to avoid reduction in column performance by
more
than 20%.
After an adjustment due to a change in column dimensions, an
additional change in flow rate of ±50% is permitted.
Column temperature: ± 10°C, where the
operating temperature is specified, unless otherwise prescribed
SE

Further adjustments in analytical procedure conditions (mobile

phase, temperature, pH, etc.) may be required, within the permitted ranges
described under System Suitability and Adjustment of Chromatographic Conditions in this chapter.

mobile phase
Composition: The amount of the
minor components of the mobile phase can be adjusted by ±30% relative.
However, the change in any
component cannot exceed ±10% absolute. A minor
component comprises less or equal than (100/n) %, n being
the total number of
components of the mobile phase:

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♦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 70:25:5: For the second
component, 30% of 25 is 7.5% absolute. Therefore, the second component may
be adjusted within
the range of 32.5%–17.5% absolute. For the third
component, 30% of 5 is 1.5% absolute. In all cases, a sufficient quantity of
the first
component is used to give a total of 100%. Therefore, mixture
ranges of 62.5: 32.5: 5 to 77.5: 17.5: 5 or 68.5: 25: 6.5 to 71.5: 25: 3.5
would meet
the requirement.♦
pH of the aqueous
component of the mobile phase: ±0.2 pH units, unless
otherwise prescribed
Concentration of salts in
the buffer component of a mobile phase: ±10%
Flow rate: In absence
of a change in column dimensions, an adjustment of the flow rate
by ±50% is permitted

detector wavelength
No adjustment permitted.

injection volume
When the column dimensions are changed, the following equation
may be used for adjusting the injection volume:

▲V 2 2
i nj 2 = V i nj 1 (L 2   dc 2 ) / (L 1  dc 1 ) ▲ (ERR 1-Dec-2022)

Vinj1 = injection volume indicated in the

monograph (µL)

Vinj2

L1
= adjusted injection volume (μL) AL
= column length indicated in the monograph
(mm)

L2 =
▲new column
length
(mm)▲ (ERR 1-Dec-2022)
CI
dc1 =
▲column
internal diameter indicated in the monograph
(mm)▲ (ERR 1-Dec-2022)

dc2 =
▲new column
internal diameter
(mm)▲ (ERR 1-Dec-2022)

This equation may not be applicable to changes from TPP

columns to SPP columns.
FI

Even in the absence of any column dimension change, the

injection volume may be varied provided System Suitability criteria remain
within
their established acceptability limits. When the injection volume is
decreased, special attention is given to (limit of) detection and
repeatability
of the peak response(s) to be determined. An increase is
permitted provided, in particular, linearity and resolution of the peak(s)
to be
determined remain satisfactory.
O

Liquid Chromatography: Gradient

Elution
Adjustment of chromatographic conditions for gradient systems
requires greater caution than for isocratic systems.

column parameters and flow

rate
RÁ

Stationary phase: No change of the identity of

the substituent (e.g., no replacement of C18 by C8); the other
physicochemical characteristics
of the stationary phase i.e.,
chromatographic support, surface modification, and extent of chemical
modification must be similar; a change
from totally porous particle (TPP)
columns to superficially porous particle (SPP) columns is allowed provided
the above-mentioned
requirements are met.
Column dimensions (particle size, length):
The particle size and/or length of the column may be modified provided that
the ratio of the
SE

column length (L) to the particle size

(dp) remains constant or in the range between −25% to
+50% of the prescribed L/dp ratio.
Adjustments from totally porous to superficially
porous particles: For the application of particle-size adjustment
from totally porous to
superficially porous particles, other combinations of
L and dp can be used provided
that the ratio (tR/Wh)2 is within
−25% to +50%, relative to
the prescribed column ♦for all the peaks used to
determine the system suitability parameters♦. These changes are acceptable
provided
system suitability criteria are fulfilled, and selectivity and
elution order of the specified impurities to be controlled are demonstrated
to be
equivalent.
Internal diameter: In absence of a change in
particle size and/or length, the internal diameter of the column may be
adjusted.

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Caution is necessary when the adjustment results in smaller peak

volume resulting from smaller particle size or smaller internal column
diameter, a situation which may require adjustments to minimize extra-column
band broadening by factors such as instrument connections,
detector cell
volume and sampling rate, and injection volume.
When the particle size is changed, the flow rate requires
adjustment, because smaller-particle columns will require higher linear
velocities for
the same performance (as measured by reduced plate height).
Flow rate is adjusted for both the change in column diameter and particle
size
using the following equation:

▲F 2 2
2 = F1 × [(d c2 × d p )/ (d c1
1
× d p )]
2 ▲ (ERR 1-Dec-2022)

F1 = flow rate indicated in the monograph

(mL/min)

F2 = adjusted flow rate (mL/min)

dc1 = internal diameter of the column indicated

in the monograph (mm)

dc2 = internal diameter of the column used

(mm)

dp1 = particle size indicated in the monograph

(µm)

dp2 = particle size of the column used

(µm)

A change in column dimensions, and thus in column volume,

impacts the gradient volume which controls selectivity. Gradients are
adjusted
to the column volume by changing the gradient volume in proportion
to the column volume. This applies to every gradient segment volume.
Since
the gradient volume is the gradient time, tG, multiplied by the flow rate, F,
the gradient time for each gradient segment needs to be
adjusted to maintain
a constant ratio of the gradient volume to the column volume (expressed as
L × dc2). Thus, the new
gradient time, tG2 can
be calculated from the original gradient time, tG1, the
flow rate(s), and the column dimensions as follows:
AL t G 2 = t G 1 × (F1 /F2 )   [(L 2 × d c2

Thus, the change in conditions for gradient elution requires

three steps:
2
)/ (L 1 × d c1
2
)]

1. Adjust the column length and particle size

according to L/dp.
CI
2. Adjust the flow rate for changes in particle
size and column diameter.

3. Adjust the gradient time of each segment for

changes in column length, diameter, and flow rate. The example below
illustrates this process.
FI

Table 3

Variable Original Conditions Adjusted Conditions Comment

O

Column length (L), in

mm 150 100 User’s choice

Column diameter (dc),

in mm 4.6 2.1 User’s choice

Particle size (dp),

in µm 5 3 User’s choice
RÁ

L/dp 30.0 33.3 (1)

Flow rate, in mL/min 2.0 0.7 (2)

Gradient adjustment factor —

SE

(tG2/tG1) 0.4 (3)

Gradient conditions — — —

Time
Time

B
(%) (min) (min)

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Variable Original Conditions Adjusted Conditions Comment

30 0 0 —

30 3 (3 × 0.4)
= 1.2 —

70 13 [1.2 + (10
× 0.4)] = 5.2 —

30 16 [5.2 + (3
× 0.4)] = 6.4 —

1. An 11% increase within allowed L/dp change of −25% to +50%

Calculated using F2 = F1 [(dc22 × dp1)/(dc12 × dp2)]
2.
3. Calculated using tG2 = tG1 × (F1/F2)
[(L2 × dc22)/(L1 × dc12)]
Column temperature: ±5° C, where the operating
temperature is specified, unless otherwise prescribed
Further adjustments in procedure conditions (mobile phase,
temperature, pH, etc.) may be required, within the permitted ranges
described
under System Suitability and Adjustment of Chromatographic Conditions in this chapter.

mobile phase
Composition/gradient: Adjustments of the
composition of the mobile phase and the gradient are acceptable provided
that:
The system suitability criteria are fulfilled.
The principal peak(s) elute(s) within ±15% of the
retention time(s) obtained with the original conditions; this
requirement does not apply
when the column dimensions are
changed.
The composition of the mobile phase and the gradient
are such that the first peaks are sufficiently retained and the
last peaks are
eluted.

AL
pH of the aqueous component of the mobile
phase: ±0.2 pH units, unless otherwise prescribed
Concentration of salts in the buffer component
of a mobile phase: ±10%
Where compliance with the system suitability criteria cannot be
achieved, it is often preferable to consider the dwell volume or to change
the
column.

dwell volume
CI
The configuration of the equipment employed may significantly
alter the resolution, retention time, and relative retentions described.
Should
this occur, it may be due to a change in dwell volume. Monographs
preferably include an isocratic step before the start of the gradient
program
so that an adaptation can be made to the gradient time points to
take account of differences in dwell volume between the system used for
analytical procedure development and that actually used. It is the user’s
responsibility to adapt the length of the isocratic step to the analytical
FI

equipment used. If the dwell volume used during the elaboration of the
monograph is given in the monograph, the time points (t
min) stated in
the gradient table may be replaced by adapted time points (tc min), calculated using the
following equation:

(D−D0 )
tc = t −
O

D = dwell volume (mL)

D0 = dwell volume used for development of the

method (mL)

F = flow rate (mL/min)

RÁ

The isocratic step introduced for this purpose may be omitted if

validation data for application of the analytical procedure without this
step
is available.
Detector wavelength: No adjustment permitted
Injection volume: When the column dimensions are changed, the
following equation may be used for adjusting the injection
volume:

2 2
SE

V i nj 2 = V i nj 1  (L 2 d c )/ (L 1 d c )
2 1

Vinj1 = injection volume indicated in the

monograph (µL)

Vinj2 = adjusted injection volume (µL)

L1 = column length indicated in the monograph

(cm)

= new column length (cm)

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L2

dc1 = column internal diameter indicated in the

monograph (mm)

dc2 = new column internal diameter

(mm)

This equation may not be applicable to changes from TPP

columns to SPP columns.
Even in the absence of any column dimension change, the
injection volume may be varied provided system suitability criteria remain
within
their established acceptability limits. When the injection volume is
decreased, special attention is given to (limit of) detection and
repeatability
of the peak response(s) to be determined. An increase is
permitted provided, in particular, linearity and resolution of the peak(s)
to be
determined remain satisfactory.

Gas Chromatography

column parameters
Stationary phase
Particle size: Maximum reduction of 50%;
no increase permitted (packed columns)
Film thickness: −50% to +100% (capillary
columns)
Column dimensions
Length: −70% to +100%
Internal diameter: ±50%
Column
temperature: ±10%
Temperature
program: Adjustment of temperatures is permitted as stated
above; adjustment of ramp rates and hold times of up to ±20% is
permitted.
Flow rate: ±50%
The above changes are acceptable provided system suitability
criteria are fulfilled and selectivity and elution order of the
specified impurities
AL
to be controlled are demonstrated to be
equivalent.
Injection volume and split
ratio: May be varied provided system suitability
criteria remain within their established acceptability limits. When
the
injection volume is decreased, or the split ratio is increased special
attention is given to (limit of) detection and repeatability of the peak
response(s) to be determined. An increase in injection volume or
decrease in split ratio is permitted provided, in particular, linearity
and
resolution of the peak(s) to be determined remain satisfactory.
CI
Injection port temperature
and transfer-line temperature in static headspace conditions: ±10° C, provided no decomposition or condensation
occurs

QUANTITATION
The following quantitation approaches may be used in general texts or
monographs.
FI

External Standard Method

Using a calibration function: Standard solutions with several graded amounts
of a reference standard of the compound to be analyzed are
prepared in a range that
has
been demonstrated to give a linear response, and a fixed volume of these standard
solutions is injected. With the
O

chromatograms obtained, a calibration function is

prepared
by plotting the peak areas or peak heights on the ordinate against the amount of
reference
standard on the abscissa. The calibration function is generally obtained by linear
regression. Then, a sample solution is prepared
according to the procedure specified
in
the individual monograph. The chromatography is performed under the same operating
conditions as
for the preparation of the calibration function, the peak area or peak
height of the compound to be analyzed is measured, and the amount of the
RÁ

compound
is read
out or calculated from the calibration function.
Using one-point calibration: In an individual monograph, generally one of
the standard solutions with a concentration within the linear range of
the calibration
function and a sample solution with a concentration close to that of the standard
solution
are prepared, and the chromatography
is performed under fixed conditions to obtain
the
amount of the component by comparing the responses obtained. In this method, all
procedures, such as the injection procedure, must be carried out under constant
conditions.

Internal Standard Method

SE

Using a calibration function: In the internal standard method, a stable

compound is chosen as an internal standard which shows a retention
time close to that
of
the compound to be analyzed, and whose peak is well separated from all other peaks
in the
chromatogram. Several
standard solutions containing a fixed amount of the internal
standard and graded amounts of a reference standard of the compound to be
analyzed
are
prepared. Based on the chromatograms obtained by injection of a fixed volume of individual
standard solutions, the ratio of peak
area or peak height of the reference standard
to
that of the internal standard is calculated. A calibration function by plotting these
ratios on the
ordinate against the amount of the reference standard or the ratio of
the
amount of reference standard to that of the internal standard on the

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abscissa is prepared.
The calibration function is generally obtained by linear regression. Then, a sample
solution containing the internal
standard in the same amount as in the standard solutions
used for the preparation of the calibration function is prepared according to the
procedure specified in the individual monograph. The chromatography is performed
under
the same operating conditions as for the preparation
of the calibration function.
The
ratio of the peak area or peak height of the compound to be analyzed to that of the
internal standard is
calculated, and the amount of the compound is read out or calculated
from the calibration function.
Using one-point calibration: In an individual monograph, generally one of the
standard solutions with a concentration within the linear range of
the calibration
function and a sample solution with a concentration close to that of the standard
solution, both containing a fixed amount of the
internal standard, are prepared, and
the
chromatography is performed under fixed conditions to determine the amount of the
compound
to be
analyzed by comparing the ratios obtained.
Normalization procedure: Provided linearity of the peaks
has been demonstrated, individual monographs may prescribe that the percentage
content
of
a component of the substance to be examined 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 disregard limit or reporting threshold.

OTHER CONSIDERATIONS
Detector response: ♦The detector sensitivity is the signal output per unit
concentration or unit mass of a substance (also known as response
factor) in the mobile
phase entering the detector. The relative detector response factor, commonly referred
to
as relative response factor,
expresses the sensitivity of a detector for a given substance
relative to a standard substance.
In tests for related substances, any correction factors indicated in the
monograph are applied.♦
Interfering peaks: Peaks due to solvents and reagents or arising from the
mobile phase or the sample matrix are disregarded.
Measurement of peaks: Integration of the peak area of any impurity that is
not completely separated from the principal peak is preferably
performed by tangential
skim (Figure 9).

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Figure
9.
RÁ

reporting threshold
When the related substances test prescribes a limit for the total of
impurities or a quantitative determination of an impurity, it is important to
choose
an
appropriate reporting threshold and appropriate conditions for the integration of
the
peak areas.
In such tests the reporting threshold, i.e., the limit above which a peak
is reported, is defined generally
0.05%.▲ (USP 1-Dec-2022)
SE

Información auxiliar
-
Por favor
visite la sección de preguntas más frecuentes
antes de comunicarse con la USP.

Tema/Pregunta Persona de contacto Comité de expertos

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11/10/22, 08:56 USP-NF <621> Cromatografía

Tema/Pregunta Persona de contacto Comité de expertos

< 621 > Cromatografía Horacio N. Pappa

GCCA2020 General Chapters - Chemical
Director, General Chapters
Analysis 2020

Consultas científicas en español:

[email protected]

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 43(5)

Page Information:

USP43-NF38 - 6853
USP42-NF37 - 6781
USP41-NF36 - 6363

Current DocID: GUID-6C3DF8B8-D12E-4253-A0E7-6855670CDB7B_5_en-US

DOI: https://doi.org/10.31003/USPNF_M99380_05_01
DOI ref:
8x7r6

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