8r‘24r'2015
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`General Ctraaters: <521> CHROMATOGRAPHY
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`us. PHARMACOPEIA
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`{ 621 3 CHROMATOGRAPHY
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`INTRODUCTION
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`This chapter defines the terms and procedures used in chromatography and provides general information. Specific
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`requirements for chromatographic procedures for drug substances and dosage forms, including adsorbent and developing
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`solvents, are given in the individual monographs.
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`Chromatography is defined as a procedure by which solutes are separated by a dynamic differential migration process in a
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`system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual
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`substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapor pressure, molecular
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`size, or ionic charge density. The individual substances thus separated can be identified or determined by analytical
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`procedures.
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`The general chromatographic technique requires that a solute undergo distribution between two phases, one of them fixed
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`(stationary phase), the other moving (mobile phase). It is the mobile phase that transfers the solute through the medium
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`until it eventually emerges separated from other solutes that are eluted earlier or later. Generally, the solute is transported
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`through the separation medium by means of a flowing stream of a liquid or a gaseous solvent known as the “eluant.” The
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`stationary phase may act through adsorption, as in the case of adsorbents such as activated alumina and silica gel, or it
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`may act by dissolving the solute, thus partitioning the latter between the stationary and mobile phases. In the latter
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`process, a liquid coated onto an inert support, or chemically bonded onto silica gel, or directly onto the wall of a fused silica
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`capillary, serves as the stationary phase. Partitioning is the predominant mechanism of separation in gas—liquid
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`chromatography, paper chromatography, in forms of column chromatography and in thin-layer chromatography designated
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`as liquid-liquid separation. In practice, separations frequently result from a combination of adsorption and partitioning
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`effects. Other separation principles include ion exchange, ion-pair formation, size exclusion, hydrophobic interaction, and
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`chiral recognition.
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`The types of chromatography useful in qualitative and quantitative analysis that are employed in the USP procedures are
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`column, gas, paper, thin-layer, (including high-perforrnance thin-layer chromatography), and pressurized liquid
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`chromatography (commonly called high-pressure or high-perfonnance liquid chromatography). Paper and thin-layer
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`chromatography are ordinarily more useful for purposes of identification, because of their convenience and simplicity.
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`Column chromatography offers a wider choice of stationary phases and is useful for the separation of individual
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`compounds, in quantity, from mixtures. Modern high-perforrnance thin-layer chromatography, gas chromatography, and
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`pressurized liquid chromatography require more elaborate apparatus but usually provide high resolution and identify and
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`quantitate very small amounts of material.
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`Use of Reference Substances in Identity Tests— In paper and thin-layer chromatography, the ratio of the distance (this
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`PARAGON — EXHIBIT 2010 1126
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`PARAGON - EXHIBIT 2010
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`8r‘24r'2015
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`General Chaaters: <621> CHROMATOGRAPHY
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`distance being measured to the point of maximum intensity of the spot or zone) traveled on the medium by a given
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`compound to the distance traveled by the front of the mobile phase, from the point of application of the test substance, is
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`designated as the R,_- value of the compound. The ratio between the distances traveled by a given compound and a
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`reference substance is the RR value. RR values vary with the experimental conditions, and thus identification is best
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`accomplished where an authentic specimen of the compound in question is used as a reference substance on the same
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`chromatogram.
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`For this purpose, chromatograms are prepared by applying on the thin-layer adsorbent or on the paper in a straight line,
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`parallel to the edge of the chromatographic plate or paper, solutions of the substance to be identified, the authentic
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`specimen, and a mixture of nearly equal amounts of the substance to be identified and the authentic specimen. Each
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`sample application contains approximately the same quantity by weight of material to be chromatographed. If the substance
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`to be identified and the authentic specimen are identical, all chromatograms agree in color and RR value and the mixed
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`chromatogram yields a single spot; i.e., RR is 1.0.
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`Location of Components— The spots produced by paper or thin-layer chromatography may be located by: (1) direct
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`inspection if the compounds are visible under white or either short-wavelength (254 nm) or long-wavelength (360 nm) UV
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`light, (2) inspection in white or UV light after treatment with reagents that will make the spots visible (reagents are most
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`conveniently applied with an atomizer), (3) use of a Geiger-Muller counter or autoradiographic techniques in the case of the
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`presence of radioactive substances, or (4) evidence resulting from stimulation or inhibition of bacterial growth by the placing
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`of removed portions of the adsorbent and substance on inoculated media.
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`In open-column chromatography, in pressurized liquid chromatography performed under conditions of constant flow rate,
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`and in gas chromatography, the retention time, t, defined as the time elapsed between sample injection and appearance of
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`the peak concentration of the eluted sample zone, may be used as a parameter of identification. Solutions of the substance
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`to be identified or derivatives thereof, of the reference compound, and of a mixture of equal amounts of these two are
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`chromatographed successively on the same column under the same chromatographic conditions. Only one peak should be
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`observed for the mixture. The ratio of the retention times of the test substance, the reference compound, and a mixture of
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`these, to the retention time of an internal standard is called the relative retention time RR and is also used frequently as a
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`parameter of identification.
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`The deviations of RR, RR, or t values measured for the test substance from the values obtained for the reference compound
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`and mixture should not exceed the reliability estimates determined statistically from replicate assays of the reference
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`compound.
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`Chromatographic identification by these methods under given conditions strongly indicates identity but does not constitute
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`definitive identification. Coincidence of identity parameters under three to six different sets of chromatographic conditions
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`(temperatures, column packings, adsorbents, eluants, developing solvents, various chemical derivatives, etc.) increases
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`the probability that the test and reference substances are identical. However, many isomeric compounds cannot be
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`separated. Specific and pertinent chemical, spectroscopic, or physicochemical identification of the eluted component
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`combined with chromatographic identity is the most valid criterion of identification. For this purpose, the individual
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`components separated by chromatography may be collected for further identification.
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`PAPER CHROMATOGRAPHY
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`In paper chromatography the adsorbent is a sheet of paper of suitable texture and thickness. Chromatographic separation
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`may proceed through the action of a single liquid phase in a process analogous to adsorption chromatography in columns.
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`Since the natural water content of the paper, or selective imbibition of a hydrophilic component of the liquid phase by the
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`paper fibers, may be regarded as a stationary phase, a partitioning mechanism may contribute significantly to the
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`separation.
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`Alternatively, a two-phase system may be used. The paper is impregnated with one of the phases, which then remains
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`stationary (usually the more polar phase in the case of unmodified paper). The chromatogram is developed by slow passage
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`of the other, mobile phase over the sheet. Development may be ascending, in which case the solvent is carried up the
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`paper by capillary forces, or descending, in which case the solvent flow is also assisted by gravitational force.
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`Differences in the value of RF have been reported where chromatograms developed in the direction of the paper grain
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`(machine direction) are compared with others developed at right angles to the grain; therefore, the orientation of paper grain
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`with respect to solvent flow should be maintained constant in a series of chromatograms. (The machine direction is usually
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`designated by the manufacturer on packages of chromatography paper.)
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`In descending chromatography, the mobile phase flows downward on the chromatographic sheet.
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`Apparatus— The essential equipment for descending chromatography consists of the following:
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`Descending Chromatography
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`A vapor-tight chamber provided with inlets for addition of solvent or for releasing internal pressure. The chamber is
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`constructed preferably of glass, stainless steel, or porcelain and is so designed as to permit obsen/ation of the progress of
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`the chromatographic run without opening of the chamber. Tall glass cylinders are convenient if they are made vapor-tight
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`with suitable covers and a sealing compound.
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`A rack of comosion-resistant material about 5 cm shorter than the inside height of the chamber. The rack serves as a
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`support for solvent troughs and for antisiphon rods which, in turn, hold up the chromatographic sheets.
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`One or more glass troughs capable of holding a volume of solvent greater than that needed for one chromatographic run.
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`The troughs must also be longer than the width of the chromatographic sheets.
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`Heavy glass antisiphon rods to be supported by the rack and running outside of, parallel to, and slightly above the edge of
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`the glass trough.
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`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
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`length approximately equal to the height of the chamber. A fine pencil line is drawn horizontally across the filter paper at a
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`distance from one end such that, when the sheet is suspended from the antisiphon rods with the upper end of the paper
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`resting in the trough and the lower portion hanging free into the chamber, the line is located a few centimeters below the
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`rods. Care is necessary to avoid contaminating the filter paper by excessive handling or by contact with dirty surfaces.
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`Procedure— The substance or substances to be analyzed are dissolved in a suitable solvent. Convenient volumes,
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`delivered from suitable micropipets, of the resulting solution, normally containing 1 to 20 pg of the compound, are placed in
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`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
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`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.
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`The spotted chromatographic sheet is suspended in the chamber by use of the antisiphon rod, which holds the upper end of
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`the sheet in the solvent trough. The bottom of the chamber is covered with the prescribed solvent system. Saturation of the
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`chamber with solvent vapor is facilitated by lining the inside walls with paper that is wetted with the prescribed solvent
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`system. It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber
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`without touching the rack or the chamber walls or the fluid in the chamber. The chamber is sealed to allow equilibration
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`(saturation) of the chamber and the paper with the solvent vapor. Any excess pressure is released as necessary. For large
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`chambers, equilibration overnight may be necessary.
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`A volume of the mobile phase in excess of the volume required for complete development of the chromatogram is saturated
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`with the immobile phase by shaking. After equilibration of the chamber, the prepared mobile solvent is introduced into the
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`trough through the inlet. The inlet is closed and the mobile solvent phase is allowed to travel the desired distance down the
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`paper. Precautions must be taken against allowing the solvent to run down the sheet when opening the chamber and
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`removing the chromatogram. The location of the solvent front is quickly marked, and the sheets are dried.
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`The chromatogram is observed and measured directly or after suitable development to reveal the location of the spots of
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`the isolated drug or drugs. The paper section(s) predetermined to contain the isolated drug(s) may be cut out and eluted by
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`an appropriate solvent, and the solutions may be made up to a known volume and quantitatively analyzed by appropriate
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`General Chaaters: <621> CHROMATOGRAPHY
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`chemical or instrumental techniques. Similar procedures should be conducted with various amounts of similarly spotted
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`reference standard on the same paper in the concentration range appropriate to prepare a valid calibration curve.
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`Ascending Chromatography
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`In ascending chromatography, the lower edge of the sheet (or strip) is dipped into the mobile phase to permit the mobile
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`phase to rise on the chromatographic sheet by capillary action.
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`Apparatus— The essential equipment for ascending chromatography is substantially the same as that described under
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`Descending Chromatography.
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`Procedure— The test materials are applied to the chromatographic sheets as directed under Descending Chromatography,
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`and above the level to which the paper is dipped into the developing solvent. The bottom of the developing chamber is
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`covered with the developing solvent system. If a two-phase system is used, both phases are added. It is also desirable to
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`line the walls of the chamber with paper and to saturate this lining with the solvent system. Empty solvent troughs are
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`placed on the bottom of the chamber, and the chromatographic sheets are suspended so that the end on which the spots
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`have been added hangs free inside the empty trough.
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`The chamber is sealed, and equilibration is allowed to proceed as described under Descending Chromatography. Then the
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`developing solvent (mobile phase) is added through the inlet to the trough in excess of the solvent required for complete
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`moistening of the chromatographic sheet. The chamber is resealed. When the solvent front has reached the desired height,
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`the chamber is opened and the sheet is removed and dried.
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`Quantitative analyses of the spots may be conducted as described under Descending Chromatography.
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`THIN-LAYER CHROMATOGRAPHY
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`In thin-layer chromatography, the adsorbent is a relatively thin, unifonn layer of dry, finely powdered material applied to a
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`glass, plastic, or metal sheet or plate, glass plates being most commonly employed. The coated plate can be considered
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`an “open chromatographic column” and the separations achieved may be based upon adsorption, partition, or a combination
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`of both effects, depending on the particular type of stationary phase, its preparation, and its use with different solvents.
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`Thin-layer chromatography on ion-exchange layers can be used for the fractionation of polar compounds. Presumptive
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`identification can be effected by obsenration of spots or zones of identical RF value and about equal magnitude obtained,
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`respectively, with an unknown and a reference sample chromatographed on the same plate. A visual comparison of the size
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`or intensity of the spots or zones may serve for semiquantitative estimation. Quantitative measurements are possible by
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`means of densitometry (absorbance or fluorescence measurements), or the spots may be carefully removed from the plate,
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`followed by elution with a suitable solvent and spectrophotometric measurement. For two-dimensional thin-layer
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`chromatography, the chromatographed plate is turned at a right angle and again chromatographed, usually in another
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`chamber equilibrated with a different solvent system.
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`Apparatus— Acceptable apparatus and materials for thin-layer chromatography consist of the following.
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`A TLC or HPTLC plate. The chromatography is generally carried out using precoated plates or sheets (on glass, aluminum,
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`or polyester support) of suitable size. It may be necessary to clean the plates prior to separation. This can be done by
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`migration of, or immersion in, an appropriate solvent. The plates may also be impregnated by procedures such as
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`development, immersion, or spraying. At the time of use, the plates may be activated, if necessary, by heating in an oven
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`at 120': for 20 minutes. The stationary phase of TLC plates has an average particle size of 10-15 pm, and that of HPTLC
`plates an average particle size of 5 pm. Commercial plates with a preadsorbant zone can be used if they are specified in a
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`monograph. Sample applied to the preabsorbant region develops into sharp, narrow bands at the preabsorbant-sorbent
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`interface. Alternatively, flat glass plates of convenient size, typically 20 cm X 20 cm can be coated as described under
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`Preparation of Chromatographic Plates.
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`A suitable manual, semiautomatic, or automatic application device can be used to ensure proper positioning of the plate
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`and proper transfer of the sample, with respect to volume and position, onto the plate. Alternatively, a template can be used
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`to guide in manually placing the test spots at definite intervals, to mark distances as needed, and to aid in labeling the
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`plates. For the proper application of the solutions, micropipets, microsyringes, or calibrated disposable capillaries are
`recommended.
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`For ascending development, a chromatographic chamber made of inert, transparent material and having the following
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`specifications is used: a flat bottom or twin trough, a tightly fitted lid, and a size suitable for the plates. For horizontal
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`development, the chamber is provided with a reservoir for the mobile phase, and it also contains a device for directing the
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`mobile phase to the stationary phase.
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`Devices for transfer of reagents onto the plate by spraying, immersion, or exposure to vapor and devices to facilitate any
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`necessary heating for visualization of the separated spots or zones.
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`A UV light source suitable for observations under short (254 nm) and long (365 nm) wavelength UV light.
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`A suitable device for documentation of the visualized chromatographic result.
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`Procedure— Apply the prescribed volume of the test solution and the standard solution in sufficiently small portions to
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`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
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`mm by 0.5 to 1 mm on HPTLC plates) at an appropriate distance from the lower edge—during chromatography the
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`application position must be 3 mm (HPTLC) to 5 mm (TLC) above the level of the developing solvent—and from the sides
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`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
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`HPTLC plates) between the centers of spots or 4 mm (2 mm on HPTLC plates) between the edges of bands, and allow to
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`dry.
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`Ascending Development— Line at least one wall of the chromatographic chamber with filter paper. Pour into the
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`chromatographic chamber a quantity of the mobile phase sufficient for the size of the chamber to give, after impregnation of
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`the filter paper, a level of depth appropriate to the dimension of the plate used. For saturation of the chromatographic
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`chamber, close the lid, and allow the system to equilibrate. Unless otherwise indicated, the chromatographic separation is
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`performed in a saturated chamber.
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`Place the plate in the chamber, ensuring that the plate is as vertical as possible and that the spots or bands are above the
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`surface of the mobile phase, and close the chamber. The stationary phase faces the inside of the chamber. Remove the
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`plate when the mobile phase has moved over the prescribed distance. Dry the plate, and visualize the chromatograms as
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`prescribed. For two-dimensional chromatography, dry the plates after the first development, and cany out a second
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`development in a direction perpendicular to that of the first development.
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`Horizontal Development— Introduce a sufficient quantity of the developing solvent into the reservoir of the chamber using a
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`syringe or pipet. Place the plate horizontally in the chamber, connect the mobile phase direction device according to the
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`manufacturer's instructions, and close the chamber. If prescribed, develop the plate starting simultaneously at both ends.
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`Remove the plate when the mobile phase has moved over the distance prescribed in the monograph. Dry the plate, and
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`visualize the chromatograms as prescribed.
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`For two-dimensional chromatography, dry the plates after the first development, and carry out a second development in a
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`direction perpendicular to that of the first development.
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`Detection— Observe the dry plate first under short-wavelength UV light (254 nm) and then under long-wavelength UV light
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`(365 nm) or as stated in the monograph. If further directed, spray, immerse, or expose the plate to vapors of the specified
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`reagent, heat the plate when required, obsewe, and compare the test chromatogram with the standard chromatogram.
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`Document the plate after each obsenration. Measure and record the distance of each spot or zone from the point of origin,
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`and indicate for each spot or zone the wavelength under which it was observed. Detennine the RF values for the principal
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`spots or zones (see Glossary of Symbols).
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`Quantitative Measurement— Using appropriate instrumentation, substances separated by TLC and responding to ultraviolet-
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`visible (UV-Vis) irradiation prior to or after derivatization can be detennined directly on the plate. While moving the plate or
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`the measuring device, the plate is examined by measuring the reflectance of the incident light. Similarly, fluorescence may
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`be measured using an appropriate optical system. Substances containing radionuclides can be quantified in three ways: (1)
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`directly by moving the plate alongside a suitable counter or vice versa; (2) by cutting the plates into strips and measuring
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`the radioactivity on each individual strip using a suitable counter; or (3) by scraping off the stationary phase, dissolving it in
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`a suitable scintillation cocktail, and measuring the radioactivity using a liquid scintillation counter (see Radioactivity ( g }
`).
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`The apparatus for direct quantitative measurement on the plate is a densitometer that is composed of a mechanical device
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`to move the plate or the measuring device along the x-axis and the y-axis, a recorder, a suitable integrator or a computer;
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`and, for substances responding to UV-Vis irradiation, a photometer with a source of light, an optical device capable of
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`generating monochromatic light, and a photo cell of adequate sensitivity, all of which are used for the measurement of
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`reflectance. In the case where fluorescence is measured, a suitable filter is also required to prevent the light used for
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`excitation from reaching the photo cell while permitting the emitted light or specific portions thereof to pass. The linearity
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`range of the counting device must be verified.
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`For quantitative tests, it is necessary to apply to the plate not fewer than three standard solutions of the substance to be
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`examined, the concentrations of which span the expected value in the test solution (e.g., 80%, 100%, and 120%).
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`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.
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`Preparation of Chromatographic Plates-
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`Apparatus-
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`Flat glass plates of convenient size, typically 20 cm x 20 cm.
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`An aligning tray or a flat surface upon which to align and rest the plates during the application of the adsorbent.
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`A storage rack to hold the prepared plates during drying and transportation. The rack holding the plates should be kept in a
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`desiccator or be capable of being sealed in order to protect the plates from the environment after removal from the drying
`oven.
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`The adsorbent consists of finely divided adsorbent materials, normally 5 to 40 pm in diameter, suitable for chromatography.
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`It can be applied directly to the glass plate or can be bonded to the plate by means of plaster of Paris [calcium sulfate
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`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
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`surface as will the starch, but it is not affected by strongly oxidizing spray reagents. The adsorbent may contain fluorescing
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`material to aid in the visualization of spots that absorb UV light.
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`A spreader, which, when moved over the glass plate, will apply a uniform layer of adsorbent of desired thickness over the
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`entire surface of the plate.
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`Procedure— [NOTE—|n this procedure, use Purified Water that is obtained by distillation.] Clean the glass plates
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`scrupulously, using an appropriate cleaning solution (see Cleaning Glass Aggaratus ( 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
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`that the plates be completely free from lint and dust when the adsorbent is applied.
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`Arrange the plate or plates on the aligning tray, place a 5- x 20-cm plate adjacent to the front edge of the first square plate
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`and another 5- x 20-cm plate adjacent to the rear edge of the last square, and secure all of the plates so that they will not
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`slip during the application of the adsorbent. Position the spreader on the end plate opposite the raised end of the aligning
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`tray. Mix 1 part of adsorbent with 2 parts of water (or in the ratio suggested by the supplier) by shaking vigorously for 30
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`seconds in a glass-stoppered conical flask, and transfer the slurry to the spreader. Usually 30 g of adsorbent and 60 mL of
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`water are sufficient for five 20- X 20-cm plates. Complete the application of adsorbents using plaster of Paris binder within 2
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`minutes of the addition of the water, because thereafter the mixture begins to harden. Draw the spreader smoothly over the
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`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
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`of the aligning tray. (Wash away all traces of adsorbent from the spreader immediately after use.) Allow the plates to remain
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`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
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`of plates in the rack. When the plates are dry, allow them to cool to room temperature, and inspect the uniformity of the
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`distribution and the texture of the adsorbent layer; transmitted light will show uniformity of distribution, and reflected light will
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`show uniformity of texture. Store the satisfactory plates over silica gel in a suitable chamber.
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`COLUMN CHROMATOGRAPHY
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`Apparatus— The apparatus required for column chromatographic procedures is simple, consisting only of the
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`chromatographic tube itself and a tamping rod, which may be needed to pack a pledget of glass wool or cotton, if needed, in
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`the base of the tube and compress the adsorbent or slurry uniformly within the tube. In some cases a porous glass disk is
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`sealed at the base of the tube in order to support the contents. The tube is cylindrical and is made of glass, unless another
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`material is specified in the individual monograph. A smaller-diameter delivery tube is fused or otherwise attached by a
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`leakproof joint to the lower end of the main tube. Column dimensions are variable; the dimensions of those commonly used
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`in pharmaceutical analysis range from 10 to 30 mm in uniform inside diameter and 150 to 400 mm in length, exclusive of
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`the delivery tube. The delivery tube, usually 3 to 6 mm in inside diameter, may include a stopcock for accurate control of
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`the flow rate of solvents through the column. The tamping rod, a cylindrical ram firmly attached to a shaft, may be
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`constructed of plastic, glass, stainless steel, or aluminum, unless another material is specified in the individual monograph.
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`The shaft of the rod is substantially smaller in diameter than the column and is not less than 5 cm longer than the effective
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`length of the column. The ram has a diameter about 1 mm smaller than the inside diameter of the column.
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`Column Adsorption Chromatography
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`The adsorbent (such as activated alumina or silica gel, calcined diatomaceous silica, or chromatographic purified siliceous
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`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
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`amount of solvent is added to the top of the column and allowed to flow into the adsorbent. The drug principles are
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`quantitatively removed from the solution and are adsorbed in a narrow transverse band at the top of the column. As
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`additional solvent is allowed to flow through the column, either by gravity or by application of air pressure, each substance
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`progresses down the column at a characteristic rate resulting in a spatial separation to give what is known as the
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`chromatogram. The rate of movement for a given substance is affected by several variables, including the adsorptive power
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`of the adsorbent and its particle size and surface area; the nature and polarity of the solvent; the hydrostatic head or
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`applied pressure; and the temperature of the chromatographic system.
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`If the separated compounds are colored or if they fluoresce under UV light, the adsorbent column may be extruded and, by
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`transverse cuts, the appropriate segments may then be isolated. The desired compounds are then extracted from each
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`segment with a suitable solvent. If the compounds are colorless, they may be located by means of painting or spraying the
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`extruded column with color-forming reagents. Chromatographed radioactive substances may be located by means of
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`Geiger-Muller detectors or similar sensing and recording instruments. Clear plastic tubing made of a material such as nylon,
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`which is inert to most solvents and transparent to short-wavelength UV light, may be packed with adsorbent and used as a
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`chromatographic column. Such a column may be sliced with a sharp knife without removing the packing from the tubing. If
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`a fluorescent adsorbent is used, the column may be marked under UV light in preparation for slicing.
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`A "flowing" chromatogram, which is extensively used, is obtained by a procedure in which solvents are allowed to flow
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`through the column until the separated drug appears in the effluent solution, known as the "eluate.” The drug may be
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`detennined in the eluate by titration or by a spectrophotometric or colorimetric method, or the solvent may be evaporated,
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`leaving the drug in more or less pure fonn. If a second drug principle is involved, it is eluted by continuing the first solvent
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`or by passing a solvent of stronger eluting power through the column. The efficiency of the separation may be checked by
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`obtaining a thin-layer chromatogram on the individual fractions.
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`A modified procedure for adding the mixture to the column is sometimes employed. The drug, in a solid form, and, as in the
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`case of a powdered tablet, without separation from the excipients, is mixed with some of the adsorbent and added to the
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`top of a column. The subsequent flow of solvent moves the drug down the column in the manner described.
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`Column Partition Chromatography
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`In partition chromatography the substances to be separated are partitioned between two immiscible liquids, one of which,
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`the immobile phase, is adsorbed on a Solid Support, thereby presenting a very large surface area to the flowing solvent or
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`8i24l2015
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`General Chaaters: <621> CHROMATOGRAPHY
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`mobile phase. The exceed