The International Pharmacopoeia - Ninth Edition, 2019 1.14.

5 Gas chromatography

1.14.5 Gas chromatography

Gas chromatography may be regarded as a form of column chromatography in which the mobile phase is a gas (referred to as
the carrier gas) rather than a liquid solvent. The stationary phase may be an active adsorbent such as alumina, silica gel, or
carbon (gas-solid chromatography) or it may be a liquid that is coated as a thin film on a finely divided inert solid support such as
diatomaceous earth, firebrick, glass beads, or other suitable material (gas-liquid chromatography); if the chromatographic column
is of very small diameter the stationary phase may be coated on its inner wall to provide the so-called "open tube" or "capillary"
column. Certain materials are available that do not require coating with a liquid phase, for example, polyaromatic porous beads,
and these are of great value for specific applications.

The substance is introduced in a vaporized state into the carrier gas stream at the head of the column and it undergoes
distribution between the gas and liquid or solid stationary phase in a similar manner to that in other forms of chromatography. The
partition coefficient (K), which is defined as

will depend upon the nature of the solute, the nature and amount of the stationary phase, the temperature, and the carrier gas
flow rate. It will be clear then that the value of K will depend upon the particular column and precise operating conditions used
and, since these would be impossible to reproduce exactly from one laboratory to another and from one make of instrument to
another, it is necessary to treat the operating conditions set down for pharmacopoeial purposes with a degree of flexibility that is
not accorded to other, nonchromatographic, test procedures.

The essential features of the apparatus for effecting gas-liquid chromatography (much more widely used for pharmaceutical
applications than adsorption chromatography on a solid) are a source of carrier gas (usually contained in compressed form in a
cylinder fitted with a pressure-reducing valve), a flowmeter through which the gas passes, and an injection port that may be
heated to a suitable temperature to volatilize but not decompose the substance and through which the test solution is introduced
into the flow of carrier gas, preferably directly into the column packing. The constant flow of carrier gas is then maintained as the
chromatographic process takes place, the components of the test solution being separated according to the value of K for each
under the particular conditions being used.

As the components emerge from the column they enter a suitable differential-type detector, which usually depends on changes of
ionization in a flame or on changes of thermal conductivity. Many other types of detectors exist and some of these are useful for
specific pharmaceutical applications, for example, the electron capture detector that is of special value in the sensitive detection
of halogenated compounds. Electrical signals from the detector are passed to an amplifier connected to a suitable recording
device, such as a strip-chart recorder, where signals may be plotted against time. A powerful though very expensive means of
detection is to use a mass spectrometer coupled to the gas chromatograph. This is very sensitive and provides information that
enables unambiguous identification of substances issuing from the column.

The column is made of glass or metal (in the latter case care has to be taken since certain organic substances undergo metal-
catalysed degradation reactions at elevated temperatures) and is contained in a temperature-controlled oven capable of being
maintained at temperatures ranging from just above ambient temperature to about 300 °C according to the particular application.
Ovens may also be controlled in such a way that a steady rise of temperature may be maintained over a given period of time;
such "temperature programming" is frequently of value when complex mixtures of compounds having widely different
volatilization characteristics are being examined. Columns of different lengths are commonly employed, varying from as little as
0.5 m to almost 3 m for packed and from 10 to 100 m for capillary columns; for many pharmaceutical applications a column of
about 1.5 m is generally used and it may be from 2 to 5 mm in internal diameter.

The packing contained within columns for gas-liquid chromatography has a profound effect on the quality and effectiveness of
separations. The solid support, which may vary in particle size from about 75 μm to 250 μm (60-200 mesh; although in any given
column it should be closely defined within a narrow range), must be as inert as possible, particularly when polar drugs are to be
chromatographed on supports coated with low concentrations of a liquid of low polarity. Active sites on the solid support may
result in peak tailing of the solute, or even in its decomposition or rearrangement. Reactivity of the support may be minimized by
treatment with a silanizing reagent before it is coated with the stationary phase. Injection residues might render the inlet part of
the column ineffective.

Liquid phases commonly used include macrogols (polyethylene glycols) and esters, high molecular weight amides, silicone gums
and fluids, and hydrocarbons. The silicone gums are substituted polysiloxanes and are a particularly valuable series of stationary
phases. The highest temperature at which the stationary phase is designed to be used should be carefully noted and respected
since "column bleeding" may occur and vitiate results if excessive temperatures are used. Prior to use a new column should be
conditioned by maintaining it for several hours with a stream of carrier gas passing through it at a temperature somewhat higher
than the temperature at which the column is subsequently to be used but certainly not above the highest recommended
temperature.

Page 1 of 4
The International Pharmacopoeia - Ninth Edition, 2019 1.14.5 Gas chromatography

As carrier gas it is necessary to choose one that is inert and for flame-ionization detection (the most commonly used method in
pharmaceutical analysis), nitrogen is very satisfactory. Though helium is also suitable and is actually to be preferred for work with
a thermal conductivity detector because of its high thermal conductivity, it is expensive and not readily available in all parts of the
world, whereas nitrogen is.

For quantitative applications of gas-liquid chromatography in the pharmacopoeia an internal standard is usually used since the
comparison of one chromatogram with another resulting from a second injection on to the column could be subject to error. The
addition of a suitable internal standard to the test solution and to a standard solution eliminates this error since the ratio of area or
height (see below) of the peak due to the substance to be determined and of that due to the internal standard is compared from
one chromatogram to another. In other applications it may be more convenient to use a process of normalization (particularly
where an impurity is being assessed). In this case, the area of the peak due to the sought-for impurity is expressed as a
percentage of the total area of all peaks derived from the substance being examined and its attendant impurities. Since the peak
due to the principal component will usually be at least two orders of magnitude greater than that due to a minor impurity, it is
necessary for such determinations to use a reliable automatic integrator and a wide-range amplifier that will respond in a linear
fashion to both the major and the minor components. Peak areas may also be determined by planimeter, graphically, or by
comparing the weights of paper cut from underneath the various peaks. In certain circumstances, it may be valid to use
measurements of peak height rather than peak area, although the latter is considered to be more accurate for quantitative
determinations. When the measurement of peak width is required, it is defined as the distance along the baseline that is between
the two points of intersection of lines drawn tangentially to the sides of the peak.

When the internal standard method is used the detailed procedure given below is applicable. It will be noted that reference is
made to 3 solutions. The first of these (solution 1) contains an internal standard and an appropriate quantity of the substance to
be determined (in the case of an impurity determination this might be the impurity itself, if available, or a suitably low loading of
the substance in which impurities are to be sought). Chromatogram A thus obtained permits the relationship of response to the
internal standard and to the substance being determined to be established. The second solution (solution 2) consists only of the
substance being examined; this enables the analyst to confirm that there is no impurity present that will elute with the same
retention time as the internal standard or to make allowance for the quantity present if there is a coincident peak. The third
solution (solution 3) consists of the substance being examined and the internal standard (the latter present at the same
concentration as in solution 1). Data derived from chromatograms A and C, corrected if necessary for observations made on
chromatogram B, enable the quantities of minor constituents present in the substance being examined to be determined.

When a normalization procedure is used, a single solution may suffice since the total area of all minor peaks is to be expressed
as a proportion of the total peak area. If a specific minor peak is to be evaluated, a second solution containing the sought-after
material will be necessary so that the appropriate peak on the chromatogram of the substance being examined may be identified.

Recommended procedure

The length of the chromatographic column, the stationary phase, the solid support, the temperature, the carrier gas, the detector,
and any other relevant details that are required for the determination are specified in the monograph. Where a non-volatile
material is to be injected on to the column, a suitable interchangeable pre-column may be used.

In certain monographs a minimum column efficiency may be required. This is defined by the expression 16 tR 2/Ly2, where

tR is the distance (in mm) along the baseline between the point of injection and a perpendicular dropped from the
maximum of the peak produced by the internal standard specified in the monograph;

L is the length (in m) of the column;

y is the peak width (in mm) of the peak produced by the internal standard.

When aqueous solutions are examined and a flame ionization detector is used, the results are invalid if the water is eluted at the
same time as any of the required components.

Method

Using solution 1, determine experimentally suitable sensitivity settings of the instrument and the volumes of solutions to be
injected to produce an adequate response. Adjustment of the concentration of the internal standard should be made, if
necessary, so that the recorder response produced by the internal standard is approximately the same as the response of the
substance being determined. Prepare a differential chromatogram by injecting the selected volume of solution 1 through the
sample injection port on the chromatographic column, maintained at a suitable temperature, and eluting with the carrier gas.
Repeat the determination twice more. In the same manner, prepare graphs using the same volumes of solutions 2 and 3.
Measure the peak areas or, where the symmetry factor lies between 0.95 and 1.05, the peak heights produced by the substance
or substances being determined and the internal standard. If an impurity peak has the same retention time as the internal
standard, allowance is made in assessing the responses of these constituents for the contribution of each. From the values
obtained calculate the proportion of the substance or substances being determined.

Page 2 of 4
The International Pharmacopoeia - Ninth Edition, 2019 1.14.5 Gas chromatography

The symmetry factor of a peak is calculated from the expression yx /2A, where:

yx is the width of the peak at one-twentieth of the peak height;

A is the distance between the perpendicular dropped from the peak maximum and the leading edge of the peak at
one-twentieth of the peak height.

The results of the determination are usually not valid unless the resolution between measured peaks on the chromatogram is
greater than 1.0. The resolution is calculated from the expression 2(tRb - tRa )/(Ya + Yb ), where:

tRa and tRb are the distances along the baseline between the point of injection and perpendiculars dropped from
the maxima of two adjacent peaks;

Ya and Yb are the respective peak widths.

Static Head-space Gas Chromatography

Static head-space gas chromatography is a technique particularly suitable for separating and determining volatile compounds
present in solid or liquid samples. The method is based on the analysis of the vapour phase in equilibrium with the solid or liquid
phase.

Static head-space injection systems include a thermostatically controlled sample heating chamber in which closed vials
containing solid or liquid samples are placed for a fixed period of time to allow the volatile components of the sample to reach
equilibrium between the non-gaseous phase and the vapour phase. After equilibrium has been established, a predetermined
amount of the head-space of the vial is flushed into the gas chromatograph.

Apparatus

The apparatus consists of a gas chromatograph provided with a device for introducing the sample that may be connected to a
module that automatically controls the pressure and the temperature. If necessary, a device for eliminating solvents can be
added.

The sample to be analysed is introduced into a container fitted with a suitable stopper and a valve-system which permits the
passage of the carrier gas. The container is placed in a thermostatically controlled chamber at a temperature set according to the
substance to be examined.

The sample is held at this temperature long enough to allow equilibrium to be established between the solid or liquid phase and
the vapour phase.

The carrier gas is introduced into the container and, after the prescribed time, a suitable valve is opened so that the gas expands
towards the chromatographic column taking the volatilized compounds with it.

Instead of using a chromatograph specifically equipped for the introduction of samples, it is also possible to use airtight syringes
and a conventional chromatograph. Equilibration is then carried out in a separate chamber and the vapour phase is carried onto
the column, taking the precautions necessary to avoid any changes in the equilibrium.

Recommended procedure

Using the reference preparations, determine suitable instrument settings to produce an adequate response.

Method 1: Direct calibration

Separately introduce into identical containers the preparation to be examined and each of the reference preparations, as
prescribed in the monograph, avoiding contact between the sampling device and the samples.

Close the containers hermetically and place in the thermostatically controlled chamber set to the temperature and pressure
prescribed in the monograph; after equilibration, carry out the chromatography under the prescribed conditions.

Method 2: Standard additions

Add to a set of identical suitable containers equal volumes of the preparation to be examined. Add to all but one of the containers,
suitable quantities of a reference preparation containing a known concentration of the substance to be determined so as to
produce a series of preparations containing steadily increasing concentrations of the substance.

Close the containers hermetically and place in the thermostatically controlled chamber set to the temperature and pressure
prescribed in the monograph; after equilibration, carry out the chromatography under the prescribed conditions.

Calculate the linear equation of the graph using a least-squares fit, and derive from it the concentration of the substance to be
determined in the preparation to be examined.

Page 3 of 4
The International Pharmacopoeia - Ninth Edition, 2019 1.14.5 Gas chromatography

Alternatively, plot on a graph the mean of readings against the added quantity of the substance to be determined. Extrapolate the
line joining the points on the graph until it meets the concentration axis. The distance between this point and the intersection of
the axes represents the concentration of the substance to be determined in the preparation to be examined.

Page 4 of 4