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 gasliquid chromatography, paper chromatography, in forms of column chromatography and in thin-layer chromatography designated as liquid-liquid separation. 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 RF
value of the compound. The ratio between the distances traveled by a given compound and a reference substance is the RR
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 RF value and the mixed chromatogram yields a single spot; i.e., RR is 1.0.
Location 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-Müller 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 RR and is also used frequently as a parameter of identification.
The deviations of RR, RF, 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.