(621)CHROMATOGRAPHY《色谱法》USP31

621

CHROMATOGRAPHY

INTRODUCTION

This chapter defines the terms and procedures used in chromatography and provides

general information. Specific requirements for chromatographic procedures for drug

substances and dosage forms, including adsorbent and developing solvents, are

given in the individual monographs.

Chromatography is defined as a procedure by which solutes are separated by a

dynamic differential migration process in a system consisting of two or more phases,

one of which moves continuously in a given direction and in which the individual

substances exhibit different mobilities by reason of differences in adsorption, partition,

solubility, vapor pressure, molecular size, or ionic charge density. The individual

substances thus separated can be identified or determined by analytical procedures.

The general chromatographic technique requires that a solute undergo distribution

between two phases, one of them fixed (stationary phase), the other moving (mobile

phase). It is the mobile phase that transfers the solute through the medium until it

eventually emerges separated from other solutes that are eluted earlier or later.

Generally, the solute is transported through the separation medium by means of a

flowing stream of a liquid or a gaseous solvent known as the ―eluant.‖ The stationary

phase may act through adsorption, as in the case of adsorbents such as activated

alumina and silica gel, or it may act by dissolving the solute, thus partitioning the latter

between the stationary and mobile phases. In the latter process, a liquid coated onto

an inert support, or chemically bonded onto silica gel, or directly onto the wall of a

fused silica capillary, serves as the stationary phase. Partitioning is the predominant

mechanism of separation in gas

–

liquid chromatography, paper chromatography, in

forms of column chromatography, and in thin-layer chromatography designated as

liquid- liquid chromatography. In practice, separations frequently result from a

combination of adsorption and partitioning effects. Other separation principles include

ion exchange, ion-pair formation, size exclusion, hydrophobic interaction, and chiral

recognition.

The types of chromatography useful in qualitative and quantitative analysis that are

employed in the

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). Paper

and thin-layer chromatography are ordinarily more useful for purposes of identification,

because of their convenience and simplicity. Column chromatography offers a wider

choice of stationary phases and is useful for the separation of individual compounds,

in quantity, from mixtures. Modern high-performance thin-layer chromatography, gas

chromatography, and pressurized liquid chromatography require more elaborate

apparatus but usually provide high resolution and identify and quantitate very small

amounts of material.

Use of Reference Substances in Identity Tests

—

In paper and thin- layer

chromatography, the ratio of the distance (this distance being measured to the point of

maximum intensity of the spot or zone) traveled on the medium by a given compound

to the distance traveled by the front of the mobile phase, from the point of application

of the test substance, is designated as the

R

F

value of the compound. The ratio

between the distances traveled by a given compound and a reference substance is

the

R

R

value.

R

F

values vary with the experimental conditions, and thus identification is

best accomplished where an authentic specimen of the compound in question is used

as a reference substance on the same chromatogram.

For this purpose, chromatograms are prepared by applying on the thin-layer

adsorbent or on the paper in a straight line, parallel to the edge of the

chromatographic plate or paper, solutions of the substance to be identified, the

authentic specimen, and a mixture of nearly equal amounts of the substance to be

identified and the authentic specimen. Each sample application contains

approximately the same quantity by weight of material to be chromatographed. If the

substance to be identified and the authentic specimen are identical, all

chromatograms agree in color and

R

F

value and the mixed chromatogram yields a

single spot; i.e.,

R

R

is 1.0.

Location and Identification of Components

—

The spots produced by paper or

thin-layer chromatography may be located by: (1) direct inspection if the compounds

are visible under white or either short-wavelength (254 nm) or long-wavelength (360

nm) UV light, (2) inspection in white or UV light after treatment with reagents that will

make the spots visible (reagents are most conveniently applied with an atomizer), (3)

use of a Geiger- Mü

ller counter or autoradiographic techniques in the case of the

presence of radioactive substances, or (4) evidence resulting from stimulation or

inhibition of bacterial growth by the placing of removed portions of the adsorbent and

substance on inoculated media.

In open-column chromatography, in pressurized liquid chromatography performed

under conditions of constant flow rate, and in gas chromatography, the retention time,

t,

defined as the time elapsed between sample injection and appearance of the peak

concentration of the eluted sample zone, may be used as a parameter of identification.

Solutions of the substance to be identified or derivatives thereof, of the reference

compound, and of a mixture of equal amounts of these two are chromatographed

successively on the same column under the same chromatographic conditions. Only

one peak should be observed for the mixture. The ratio of the retention times of the

test substance, the reference compound, and a mixture of these, to the retention time

of an internal standard is called the relative retention time

R

R

and is also used

frequently as a parameter of identification.

The deviations of

R

R

,

R

F

, or

t

values measured for the test substance from the values

obtained for the reference compound and mixture should not exceed the reliability

estimates determined statistically from replicate assays of the reference compound.

Chromatographic identification by these methods under given conditions strongly

indicates identity but does not constitute definitive identification. Coincidence of

identity parameters under three to six different sets of chromatographic conditions

(

temperatures, column packings, adsorbents, eluants, developing solvents, various

chemical derivatives, etc

.) increases the probability that the test and reference

substances are identical. However, many isomeric compounds cannot be separated.

Specific and pertinent chemical, spectroscopic, or physicochemical identification of

the eluted component combined with chromatographic identity is the most valid

criterion of identification. For this purpose, the individual components separated by

chromatography may be collected for further identification.

PAPER CHROMATOGRAPHY

In paper chromatography the adsorbent is a sheet of paper of suitable texture and

thickness. Chromatographic separation may proceed through the action of a single

liquid phase in a process analogous to adsorption chromatography in columns. Since

the natural water content of the paper, or selective imbibition of a hydrophilic

component of the liquid phase by the paper fibers, may be regarded as a stationary

phase, a partitioning mechanism may contribute significantly to the separation.

Alternatively, a two-phase system may be used. The paper is impregnated with one of

the phases, which then remains stationary (usually the more polar phase in the case

of unmodified paper). The chromatogram is developed by slow passage of the other,

mobile phase over the sheet. Development may be ascending, in which case the

solvent is carried up the paper by capillary forces, or descending, in which case the

solvent flow is also assisted by gravitational force.

Differences in the value of

R

F

have been reported where chromatograms developed in

the direction of the paper grain (machine direction) are compared with others

developed at right angles to the grain; therefore, the orientation of paper grain with

respect to solvent flow should be maintained constant in a series of chromatograms.

(The machine direction is usually designated by the manufacturer on packages of

chromatography paper.)

Descending Chromatography

In descending chromatography, the mobile phase flows downward on the

chromatographic sheet.

Apparatus

—

The essential equipment for descending chromatography consists of the

following:

A

vapor-tight chamber

provided with inlets for addition of solvent or for releasing

internal pressure. The chamber is constructed preferably of glass, stainless steel, or

porcelain and is so designed as to permit observation of the progress of the

chromatographic run without opening of the chamber. Tall glass cylinders are

convenient if they are made vapor-tight with suitable covers and a sealing compound.

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 for antisiphon rods

which, in turn, hold up the chromatographic sheets.

One or more

glass troughs

capable of holding a volume of solvent greater than that

needed for one chromatographic run. The troughs must also be longer than the width

of the chromatographic sheets.

Heavy glass antisiphon rods

to be supported by the rack and running outside of,

parallel to, and slightly above the edge of the glass trough.

Chromatographic sheets

of special filter paper at least 2.5 cm wide and not wider than

the length of the troughs are cut to a length approximately equal to the height of the

chamber. A fine pencil line is drawn horizontally across the filter paper at a distance

from one end such that, when the sheet is suspended from the antisiphon rods with

the upper end of the paper resting in the trough and the lower portion hanging free

into the chamber, the line is located a few centimeters below the rods. Care is

necessary to avoid contaminating the filter paper by excessive handling or by contact

with dirty surfaces.

Procedure

—

The substance or substances to be analyzed are dissolved in a suitable

solvent. Convenient volumes, delivered from suitable micropipets, of the resulting

solution, normally containing 1 to 20 ?

g of the compound, are placed in 6- to 10-mm

spots not less than 3 cm apart along the pencil line. If the total volume to be applied

would produce spots of a diameter greater than 6 to 10 mm, it is applied in separate

portions to the same spot, each portion being allowed to dry before the next is added.

The spotted chromatographic sheet is suspended in the chamber by use of the

antisiphon rod, which holds the upper end of the sheet in the solvent trough. The

bottom of the chamber is covered with the prescribed solvent system. Saturation of

the chamber with solvent vapor is facilitated by lining the inside walls with paper that is

wetted with the prescribed solvent system. It is important to ensure that the portion of

the sheet hanging below the rods is freely suspended in the chamber without touching

the rack or the chamber walls or the fluid in the chamber. 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. For large chambers, equilibration

overnight may be necessary.

A volume of the mobile phase in excess of the volume required for complete

development of the chromatogram is saturated with the immobile phase by shaking.

After equilibration of the chamber, the prepared mobile solvent is introduced into the

trough through the inlet. The inlet is closed and the mobile solvent phase is allowed to

travel the desired distance down the paper. Precautions must be taken against

allowing the solvent to run down the sheet when opening the chamber and removing

the chromatogram. The location of the solvent front is quickly marked, and the sheets

are dried.

The chromatogram is observed and measured directly or after suitable development

to reveal the location of the spots of the isolated drug or drugs. The paper section(s)

predetermined to contain the isolated drug(s) may be cut out and eluted by an

appropriate solvent, and the solutions may be made up to a known volume and

quantitatively analyzed by appropriate chemical or instrumental techniques. Similar

procedures should be conducted with various amounts of similarly spotted reference

standard on the same paper in the concentration range appropriate to prepare a valid

calibration curve.

Ascending Chromatography

In ascending chromatography, the lower edge of the sheet (or strip) is dipped into the

mobile phase to permit the mobile phase to rise on the chromatographic sheet by

capillary action.

Apparatus

—

The essential equipment for ascending chromatography is substantially

the same as that described under

Descending Chromatography.

Procedure

—

The test materials are applied to the chromatographic sheets as directed

under

Descending Chromatography,

and above the level to which the paper is dipped

into the developing solvent. The bottom of the developing chamber is covered with the

developing solvent system. If a two-phase system is used, both phases are added. It

is also desirable to line the walls of the chamber with paper and to saturate this lining

with the solvent system. Empty solvent troughs are placed on the bottom of the

chamber, and the chromatographic sheets are suspended so that the end on which

the spots have been added hangs free inside the empty trough.

The chamber is sealed, and equilibration is allowed to proceed as described under

Descending Chromatography.

Then the developing solvent (mobile phase) is added

through the inlet to the trough in excess of the solvent required for complete

moistening of the chromatographic sheet. The chamber is resealed. When the solvent

front has reached the desired height, the chamber is opened and the sheet is

removed and dried.

Quantitative analyses of the spots may be conducted as described under

Descending

Chromatography.

THIN-LAYER CHROMATOGRAPHY

In thin-layer chromatography, the adsorbent is a relatively thin, uniform layer of dry,

finely powdered material applied to a glass, plastic, or metal sheet or plate, glass

plates

being most commonly employed. The coated plate can be considered an ―open

chromatographic column‖ and the separations achieved may be based upon

adsorption, partition, or a combination of both effects, depending on the particular

type of stationary phase, its preparation, and its use with different solvents. Thin-layer

chromatography on ion- exchange layers can be used for the fractionation of polar

compounds. Presumptive identification can be effected by observation of spots or

zones of identical

R

F

value and about equal magnitude obtained, respectively, with an

unknown and a reference sample 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), or the spots may be

carefully removed from the plate, followed by elution with a suitable solvent and

spectrophotometric measurement. For two-dimensional thin-layer chromatography,

the chromatographed plate is turned at a right angle and again chromatographed,

usually in another chamber equilibrated with a different solvent system.

Apparatus

—

Acceptable apparatus and materials for thin-layer chromatography

consist of the following.

A

TLC or HPTLC plate.

The chromatography is generally carried out using

precoated

plates

or

sheets

(on glass, aluminum, or polyester support) of suitable size. It may be

necessary to clean the plates prior to separation. This can be done by migration of, or

immersion in, an appropriate solvent. The plates may also be impregnated by

procedures such as development, immersion, or spraying. At the time of use, the

plates may be activated, if necessary, by heating in an oven at 120

for 20 minutes.

The

stationary phase

of TLC plates has an average particle size of 10

–

15 ?

m, and

that of HPTLC plates an average particle size of 5 ?

m. Commercial plates with a

preadsorbant zone can be used if they are specified in a monograph. Sample applied

to the preabsorbant region develops into sharp, narrow bands at the

preabsorbant-sorbent interface. Alternatively, flat

glass plates

of convenient size,

typically 20 cm ×

20 cm can be coated as described under

Preparation of

Chromatographic Plates.

A suitable

manual, semiautomatic, or automatic application device

can be used to

ensure proper positioning of the plate and proper transfer of the sample, with respect

to volume and position, onto the plate. Alternatively, a

template

can be used to guide

in manually placing the test spots at definite intervals, to mark distances as needed,

and to aid in labeling the plates. For the proper application of the solutions,

micropipets, microsyringes, or calibrated disposable capillaries

are recommended.

For ascending development, a

chromatographic chamber

made of inert, transparent

material and having the following specifications is used: a flat bottom or twin trough, a

tightly fitted lid, and a size suitable for the plates. For horizontal development, the

chamber is provided with a reservoir for the mobile phase, and it also contains a

device for directing the mobile phase to the stationary phase.

Devices for transfer of reagents

onto the plate by spraying, immersion, or exposure to

vapor and devices to facilitate any necessary heating for visualization of the

separated spots or zones.

A

UV light source

suitable for observations under short (254 nm) and long (365 nm)

wavelength UV light.

A suitable

device for documentation

of the visualized chromatographic result.

Procedure

—

Apply the prescribed volume of the test solution and the standard

solution in sufficiently small portions to obtain circular spots of 2 to 5 mm in diameter

(1 to 2 mm on HPTLC plates) or bands of 10 to 20 mm by 1 to 2 mm (5 to 10 mm by

0.5 to 1 mm on HPTLC plates) at an appropriate distance from the lower

edge

—

during chromatography the application position must be at least 3 mm (HPTLC)

or 5 mm (TLC) above the level of the developing solvent

—

and from the sides of the

plate. Apply the solutions 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, and allow to dry.

Ascending Development

—

Line at least one wall of the chromatographic chamber

with filter paper. Pour into the chromatographic chamber a quantity of the mobile

phase sufficient for the size of the chamber to give, after impregnation of the filter

paper, a level of depth appropriate to the dimension of the plate used. For saturation

of the chromatographic chamber, close the lid, and allow the system to equilibrate.

Unless otherwise indicated, the chromatographic separation is performed in a

saturated chamber.

Place the plate in the chamber, ensuring that the plate is as vertical as possible and

that the spots or bands are above the surface of the mobile phase, and close the

chamber. The stationary phase faces the inside of the chamber. Remove the plate

when the mobile phase has moved over the prescribed distance. Dry the plate, and

visualize the chromatograms as prescribed. For two-dimensional chromatography,

dry the plates after the first development, and carry out a second development in a

direction perpendicular to that of the first development.

Horizontal Development

—

Introduce a sufficient quantity of the developing solvent

into the reservoir of the chamber using a syringe or pipet. Place the plate horizontally

in the chamber, connect the mobile phase direction device according to the

manufacturer's instructions, and close the chamber. If prescribed, develop the plate

starting simultaneously at both ends. Remove the plate when the mobile phase has

moved over the distance prescribed in the monograph. Dry the plate, and visualize the

chromatograms as prescribed.

For two-dimensional chromatography, dry the plates after the first development, and

carry out a second development in a direction perpendicular to that of the first

development.

Detection

—

Observe the dry plate first under short-wavelength UV light (254 nm) and

then under long- wavelength UV light (365 nm) or as stated in the monograph. If

further directed, spray, immerse, or expose the plate to vapors of the specified

reagent, heat the plate when required, observe, and compare the test chromatogram

with the standard chromatogram. Document the plate after each observation.

Measure and record the distance of each spot or zone from the point of origin, and

indicate for each spot or zone the wavelength under which it was observed.

Determine the

R

F

values for the principal spots or zones (see

Glossary of Symbols

).

Quantitative Measurement

—

Using appropriate instrumentation, substances

separated by TLC and responding to ultraviolet-visible (UV-Vis) irradiation prior to or

after derivatization can be determined directly on the plate. While moving the plate or

the measuring device, the plate is examined by measuring the reflectance of the

incident light. Similarly, fluorescence may be measured using an appropriate optical

system. Substances containing radionuclides can be quantified in three ways: (1)

directly by moving the plate alongside a suitable counter or vice versa; (2) by cutting

the plates into strips and measuring the radioactivity on each individual strip using a

suitable counter; or (3) by scraping off the stationary phase, dissolving it in a suitable

scintillation cocktail, and measuring the radioactivity using a liquid scintillation counter

(see

Radioactivity

821

).

The apparatus for direct quantitative measurement on the plate is a densitometer that

is composed of a mechanical device to move the plate or the measuring device along

the

x

-axis and the

y

-axis, a recorder, a suitable integrator or a computer; and, for

substances responding to UV-Vis irradiation, a photometer with a source of light, an

optical device capable of generating monochromatic light, and a photo cell of

adequate sensitivity, all of which are used for the measurement of reflectance. In the

case where fluorescence is measured, a suitable filter is also required to prevent the

light used for excitation from reaching the photo cell while permitting the emitted light

or specific portions thereof to pass. The linearity range of the counting device must be

verified.

For quantitative tests, it is necessary to apply to the plate not fewer than three

standard solutions of the substance to be examined, the concentrations of which span

the expected value in the test solution (e.g., 80%, 100%, and 120%). Derivatize with

the prescribed reagent, if necessary, and record the reflectance or fluorescence in the

chromatograms obtained. Use the measured results for the calculation of the amount

of substance in the test solution.

Preparation of Chromatographic Plates

—

Apparatus

—

Flat

glass plates

of convenient size, typically 20 cm ×

20 cm.

An

aligning tray

or a flat surface upon which to align and rest the plates during the

application of the adsorbent.

A

storage rack

to hold the prepared plates during drying and transportation. The rack

holding the plates should be kept in a desiccator or be capable of being sealed in

order to protect the plates from the environment after removal from the drying oven.

The

adsorbent

consists of finely divided adsorbent materials, normally 5 to 40 ?

m in

diameter, suitable for chromatography. It can be applied directly to the glass plate or

can be bonded to the plate by means of plaster of Paris [calcium sulfate hemihydrate

(at a ratio of 5% to 15%)] or with starch paste or other binders. The plaster of Paris will

not yield as hard a surface as will the starch, but it is not affected by strongly oxidizing

spray reagents. The adsorbent may contain fluorescing material to aid in the

visualization of spots that absorb UV light.

A

spreader,

which, when moved over the glass plate, will apply a uniform layer of

adsorbent of desired thickness over the entire surface of the plate.

Procedure

—

[

NOTE

—

In this procedure, use Purified Water that is obtained by

distillation.] Clean the glass plates scrupulously, using an appropriate cleaning

solution (see

Cleaning Glass Apparatus

1051

), rinsing them with copious

quantities of water until the water runs off the plates without leaving any visible water

or oily spots, then dry. It is important that the plates be completely free from lint and

dust when the adsorbent is applied.

Arrange the plate or plates on the aligning tray, place a 5- ×

20-cm plate adjacent to

the front edge of the first square plate and another 5- ×

20-cm plate adjacent to the

rear edge of the last square, and secure all of the plates so that they will not slip

during the application of the adsorbent. Position the spreader on the end plate

opposite the raised end of the aligning tray. Mix 1 part of adsorbent with 2 parts of

water (or in the ratio suggested by the supplier) by shaking vigorously for 30 seconds

in a glass- stoppered conical flask, and transfer the slurry to the spreader. Usually 30 g

of adsorbent and 60 mL of water are sufficient for five 20- ×

20-cm plates. Complete

the application of adsorbents using plaster of Paris binder within 2 minutes of the

addition of the water, because thereafter the mixture begins to harden. Draw the

spreader smoothly over the plates toward the raised end of the aligning tray, and

remove the spreader when it is on the end plate next to the raised end of the aligning

tray. (Wash away all traces of adsorbent from the spreader immediately after use.)

Allow the plates to remain undisturbed for 5 minutes, then transfer the square plates,

layer side up, to the storage rack, and dry at 105

for 30 minutes. Preferably place the

rack at an angle in the drying oven to prevent the condensation of moisture on the

back sides of plates in the rack. When the plates are dry, allow them to cool to room

temperature, and inspect the uniformity of the distribution and the texture of the

adsorbent layer; transmitted light will show uniformity of distribution, and reflected light

will show uniformity of texture. Store the satisfactory plates over silica gel in a suitable

chamber.

COLUMN CHROMATOGRAPHY

Apparatus

—

The apparatus required for column chromatographic procedures is

simple, consisting only of the chromatographic tube itself and a tamping rod, which

may be needed to pack a pledget of glass wool or cotton, if needed, in the base of the

tube and compress the adsorbent or slurry uniformly within the tube. In some cases a

porous glass disk is sealed at the base of the tube in order to support the contents.

The tube is cylindrical and is made of glass, unless another material is specified in the

individual monograph. A smaller-diameter delivery tube is fused or otherwise attached

by a leakproof joint to the lower end of the main tube. Column dimensions are variable;

the dimensions of those commonly used in pharmaceutical analysis range from 10 to

30 mm in uniform inside diameter and 150 to 400 mm in length, exclusive of the

delivery tube. The delivery tube, usually 3 to 6 mm in inside diameter, may include a

stopcock for accurate control of the flow rate of solvents through the column. The

tamping rod, a cylindrical ram firmly attached to a shaft, may be constructed of plastic,

glass, stainless steel, or aluminum, unless another material is specified in the

individual monograph. The shaft of the rod is substantially smaller in diameter than the

column and is not less than 5 cm longer than the effective length of the column. The

ram has a diameter about 1 mm smaller than the inside diameter of the column.

Column Adsorption Chromatography

The adsorbent (such as activated alumina or silica gel, calcined diatomaceous silica,

or chromatographic purified siliceous earth) as a dry solid or as a slurry is packed into

a glass or quartz chromatographic tube. A solution of the drug in a small amount of

solvent is added to the top of the column and allowed to flow into the adsorbent. The

drug principles are quantitatively removed from the solution and are adsorbed in a

narrow transverse band at the top of the column. As additional solvent is allowed to

flow through the column, either by gravity or by application of air pressure, each

substance progresses down the column at a characteristic rate resulting in a spatial

separation to give what is known as the

chromatogram.

The rate of movement for a

given substance is affected by several variables, including the adsorptive power of the

adsorbent and its particle size and surface area; the nature and polarity of the solvent;

the hydrostatic head or applied pressure; and the temperature of the chromatographic

system.

If the separated compounds are colored or if they fluoresce under UV light, the

adsorbent column may be extruded and, by transverse cuts, the appropriate

segments may then be isolated. The desired compounds are then extracted from

each segment with a suitable solvent. If the compounds are colorless, they may be

located by means of painting or spraying the extruded column with color-forming

reagents. Chromatographed radioactive substances may be located by means of

Geiger-Mü

ller detectors or similar sensing and recording instruments. Clear plastic

tubing made of a material such as nylon, which is inert to most solvents and

transparent to short-wavelength UV light, may be packed with adsorbent and used as

a chromatographic column. Such a column may be sliced with a sharp knife without

removing the packing from the tubing. If a fluorescent adsorbent is used, the column

may be marked under UV light in preparation for slicing.

A ―flowing‖ chromatogram, which is extensively used, is obtained by a procedure in

which solvents are allowed to flow through the column until the separated drug

appears in the effluent solution, known as the ―eluate.‖ The drug may be determined

in the eluate by titration or by a spectrophotometric or colorimetric method, or the

solvent may be evaporated, leavi

ng the drug in more or less pure form. If a second

drug principle is involved, it is eluted by continuing the first solvent or by passing a

solvent of stronger eluting power through the column. The efficiency of the separation

may be checked by obtaining a thin-layer chromatogram on the individual fractions.

A modified procedure for adding the mixture to the column is sometimes employed.

The drug, in a solid form, and, as in the case of a powdered tablet, without separation

from the excipients, is mixed with some of the adsorbent and added to the top of a

column. The subsequent flow of solvent moves the drug down the column in the

manner described.

Column Partition Chromatography

In partition chromatography the substances to be separated are partitioned between

two immiscible liquids, one of which, the immobile phase, is adsorbed on a

Solid

Support,

thereby presenting a very large surface area to the flowing solvent or mobile

phase. The exceedingly high number of successive liquid-liquid contacts allows an

efficiency of separation not achieved in ordinary liquid-liquid extraction.

The

Solid Support

is usually polar, and the adsorbed immobile phase more polar than

the mobile phase. The

Solid Support

that is most widely used is chromatographic

siliceous earth having a particle size suitable to permit proper flow of eluant.

1

In

reverse-phase partition chromatography the adsorbed immobile phase is less polar

than the mobile phase and the solid adsorbent is rendered nonpolar by treatment with

a silanizing agent, such as dichlorodimethylsilane, to give silanized chromatographic

siliceous earth.

The sample to be chromatographed is usually introduced into the chromatographic

system in one of two ways: (a) a solution of the sample in a small volume of the

mobile phase is added to the top of the column; or, (b) a solution of the sample in a

small volume of the immobile phase is mixed with the

Solid Support

and transferred to

the column as a layer above a bed of a mixture of immobile phase with adsorbent.

Development and elution are accomplished with flowing solvent as before. The mobile

solvent usually is saturated with the immobile solvent before use.

In conventional liquid-liquid partition chromatography, the degree of partition of a

given compound between the two liquid phases is expressed by its partition or

distribution coefficient. In the case of compounds that dissociate, distribution can be

controlled by modifying the pH, dielectric constant, ionic strength, and other properties

of the two phases. Selective elution of the components of a mixture can be achieved

by successively changing the mobile phase to one that provides a more favorable

partition coefficient, or by changing the pH of the immobile phase

in situ

with a mobile

phase consisting of a solution of an appropriate acid or base in an organic solvent.

Unless otherwise specified in the individual monograph, assays and tests that employ

column partition chromatography are performed according to the following general

methods.

Solid Support

—

Use purified siliceous earth. Use silanized chromatographic siliceous

earth for reverse-phase partition chromatography.

Stationary Phase

—

Use the solvent or solution specified in the individual monograph.

If a mixture of liquids is to be used as the

Stationary Phase,

mix them prior to the

introduction of the

Solid Support.

Mobile Phase

—

Use the solvent or solution specified in the individual monograph.

Equilibrate it with water if the

Stationary Phase

is an aqueous solution; if the

Stationary Phase

is a polar organic fluid, equilibrate with that fluid.

Preparation of Chromatographic Column

—

Unless otherwise specified in the

individual monograph, the chromatographic tube is about 22 mm in inside diameter

and 200 to 300 mm in length, without porous glass disk, to which is attached a

delivery tube, without stopcock, about 4 mm in inside diameter and about 50 mm in

length. Pack a pledget of fine glass wool in the base of the tube. Place the specified

volume of

Stationary Phase

in a 100- to 250-mL beaker, add the specified amount of

Solid Support,

and mix 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

specified for each segment, tamp after the addition of each segment, and add each

succeeding segment directly to the previous one.

If a solution of the analyte is incorporated in 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 test solution.

Pack a pledget of fine glass wool above the completed column packing. The

Mobile

Phase

flows through a properly packed column as a moderate stream or, if

reverse- phase chromatography is applied, as a slow trickle.

Procedure

—

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. 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. 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.

Where the assay or test requires the use of multiple chromatographic columns

mounted in series and the addition of

Mobile Phase

in divided portions is specified,

allow each portion to drain completely through each column, and rinse the tip of each

with

Mobile Phase

prior to the addition of each succeeding portion.

GAS CHROMATOGRAPHY

The distinguishing features of gas chromatography are a gaseous mobile phase and a

solid or immobilized liquid stationary phase. Liquid stationary phases are available in

packed or capillary columns. In the packed columns, the liquid 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 to 4 mm in internal

diameter and 1 to 3 m in length. In capillary columns, which contain no packing, the

liquid phase is deposited on the inner surface of the column and may be chemically

bonded to it. In gas-solid chromatography, 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.

When a vaporized compound is introduced into the carrier gas and carried into the

column, it is partitioned between the gas and stationary phases by a dynamic

countercurrent distribution process. The compound is carried down the column by the

carrier gas, retarded to a greater or lesser extent by sorption and desorption on the

stationary phase. The elution of the compound is characterized by the partition ratio,

k

?

,

a dimensionless quantity also called the capacity factor (see

Glossary of Symbols

for the definition of symbols). It is equivalent to the ratio of the time required for the

compound to flow through the column (the retention time) to the elution time of an

unretained compound. The value of the capacity factor depends on the chemical

nature of the compound, the nature, amount, and surface area of the liquid phase, the

column temperature, and the gas flow rate. Under a specified set of experimental

conditions, a characteristic capacity factor exists for every compound. Separation by

gas chromatography occurs only if the compounds concerned have different capacity

factors.

Apparatus

—

A gas chromatograph consists of a carrier gas source, an injection port,

column, detector, and recording device. The injection port, column, and detector are

temperature-controlled. The typical carrier gas is helium, nitrogen, or hydrogen,

depending on the column and detector in use. The gas is supplied from a

high-pressure cylinder or high-purity gas generator and passes through suitable

pressure-reducing valves and a flow meter to the injection port and column.

Compounds to be chromatographed, either in solution or as gases, are injec

ted into

the gas stream at the injection port. Depending upon the configuration of the

apparatus, the test mixture may be injected directly into the column or be vaporized in

the injection port and mixed into the flowing carrier gas prior to entering the column.

Once in the column, compounds in the test mixture are separated by virtue of

differences in their capacity factors, which in turn depend upon vapor pressure and

degree of interaction with the stationary phase. The capacity factor, which governs

resolution, retention times, and column efficiencies of components of the test mixture,

is also temperature-dependent. The use of temperature-programmable column ovens

takes advantage of this dependence to achieve efficient separation of compounds

differing widely in vapor pressure.

As resolved compounds emerge separately from the column, they pass through a

differential detector, which responds to the amount of each compound present. The

type of detector to be used depends upon the nature of the compounds to be

analyzed and is specified in the individual monograph. Detectors are heated to

prevent condensation of the eluting compounds.

Detector output is recorded as a function of time, producing a chromatogram, which

consists of a series of peaks on a time axis. Each peak represents a compound in the

vaporized test mixture, although some peaks may overlap. The elution time is a

characteristic of an individual compound; and the instrument response, measured as

peak area or peak height, is a function of the amount present.

Injectors

—

Sample injection devices range from simple syringes to fully programmable

automatic injectors. The amount of sample that can be injected into a capillary column

without overloading is small compared to the amount that can be injected into packed

columns, and may be less than the smallest amount that can be manipulated

satisfactorily by syringe. Capillary columns, therefore, often are used with injectors

able to split samples into two fractions, a small one that enters the column and a large

one that goes to waste. Such injectors may be used in a

splitless mode

for analyses of

trace or minor components.

Purge and trap injectors are equipped with a sparging device by which volatile

compounds in solution are carried into a low-temperature trap. When sparging is

complete, trapped compounds are desorbed into the carrier gas by rapid heating of

the temperature-programmable trap.

Headspace injectors are equipped with a thermostatically controlled sample heating

chamber. Solid or liquid samples in tightly closed containers are heated in the

chamber for a fixed period of time, allowing the volatile components in the sample to

reach an equilibrium between the nongaseous phase and the gaseous or headspace

phase.

After this equilibrium has been established, the injector automatically introduces a

fixed amount of the headspace in the sample container into the gas chromatograph.

Columns

—

Capillary columns, which are usually made of fused silica, are typically 0.2

to 0.53 mm in internal diameter and 5 to 60 m in length. The liquid or stationary phase,

which is sometimes chemically bonded to the inner surface, is 0.1 to 1.0 ?

m thick,

although nonpolar stationary phases may be up to 5 ?

m thick. A list of liquid phases in

current use is given in the section

Chromatographic Reagents.

Packed columns, made of glass or metal, are 1 to 3 m in length with internal

diameters of 2 to 4 mm. Those used for analysis typically are porous polymers or solid

supports with liquid phase loadings of about 5% (w/w). High-capacity columns, with

liquid phase loadings of about 20% (w/w), are used for large test specimens and for

the determination of low molecular weight compounds such as water. The capacity

required influences the choice of solid support.

Supports for analysis of polar compounds on low-capacity, low-polarity liquid phase

columns must be inert to avoid peak tailing. The reactivity of support materials can be

reduced by silanizing prior to coating with liquid phase. Acid-washed, flux-calcined

diatomaceous earth is often used for drug analysis. Support materials are available in

various mesh sizes, with 80- to 100-mesh and 100- to 120-mesh being most

commonly used with 2- to 4-mm columns. Supports and liquid phases are listed in the

section

Chromatographic Reagents.

Retention time and the peak efficiency depend on the carrier gas flow rate; retention

time is also directly proportional to column length, while resolution is proportional to

the square root of the column length. For packed columns, the carrier gas flow rate is

usually expressed in mL per minute at atmospheric pressure and room temperature. It

is measured at the detector outlet with a flowmeter while the column is at operating

temperature. The linear flow rate through a packed column is inversely proportional to

the square of the column diameter for a given flow volume. Flow rates of 60 mL per

minute in a 4-mm column and 15 mL per minute in a 2-mm column give identical

linear flow rates and thus similar retention times. Unless otherwise specified in the

individual monograph, flow rates for packed columns are about 30 to 60 mL per

minute. For capillary columns, linear flow velocity is often used instead of flow rate.

This is conveniently determined from the length of the column and the retention time

of a dilute methane sample, provided a flame-ionization detector is in use. At high

operating temperatures there is sufficient vapor pressure to result in a gradual loss of

liquid phase, a process called bleeding.