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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-Müller 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, leaving 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.