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3.3 COLUMN CHROMATOGRAPHY

3.3 COLUMN CHROMATOGRAPHY

         Column chromatography offers a wider choice of stationary phases and is useful for the separation of individual compounds, in quantity, from mixtures.

         When the stationary phase is a solid, the process is termed adsorption chromatography, while if the stationary phase is liquid, the process is termed partition chromatography. The difference between partition and adsorption chromatography lies in the nature of the forces that determine distribution of the solute between the two phases. In ion-exchange chromatography the stationary phase is an ion-exchange material, usually called ion-exchange resin.

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 adsorbant or slurry uniformly within the tube. In some cases a porous glass disc 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 smallerdiameter delivery tube is fused or otherwise attached by a leak-proof 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 15 to 40 cm 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 aluminium,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.

Adsorption Chromatography

         The adsorbant (such as activated alumina, silicic acid, etc.) as a dry solid or as a slurry is packed into a tube made of glass, quartz, or other material, of suitable dimensions having a restricted out-flow orifice. 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 adsorbant. The drug principles are quantitatively removed from the solution and are adsorbed in a narrow transverse band at the top of the column. As further amounts of solvent are 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 adsorbant, the nature of the solvent, and temperature of the chromatographic system.

         If the separated compounds are coloured or if they fluoresce under ultraviolet light, the adsorbant 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 colourless, they may be located by painting or spraying the extruded column with colour-forming reagents.

         The “flowing chromatogram” is used quite extensively. With this type, 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.

         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 adsorbant and added to the top of a column. The subsequent flow of solvent moves the drug down the column in the usual manner.

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 (acid-washed Celite 545 or equivalent is suitable). In reverse-phase partition chromatography the adsorbed immobile phase is less polar than the mobile phase and the solid adsorbant is rendered nonpolar by suitable 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 adsorbant.

         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 favourable 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 method.

         Solid support Use purified siliceous earth. Use silanized chromatographic siliceous earth for reversephase 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 20 to 30 cm in length, without porous glass disc, to which is attached a delivery tube, without stopcock, about 4 mm in inside diameter and about 5 cm 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 multi-segment 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.

Ion-exchange Chromatography

         Ion-exchange is defined as the reversible interchange between the ion present in the solution and the counterion of the resinous polymer, modified cellulose, or bonded silica gel support; it may be exemplified for the H+ /Na+ exchange of a strongly acidic cation exchange resin as:

RSO3H + Na+ RSO3Na + H+

and for a Cl /OH strongly basic anion-exchange resin as:

RN(CH3)3OH + Cl–  RN(CH3)3Cl + OH

         The selection of strong or weak resins, of either type, will largely depend on the pH at which the exchange is to be carried out and on the types of cation or anion that are to be exchanged. However, the strongly acidic and basic exchange resins will serve in most analytical applications. Their specific capacity may vary from 2 to 5 millimoles per gram (dry basis). In practice, a large (200 to 300 per cent) excess of resin is used over the calculated stoichiometric requirement.

         The laws governing the exchange reaction are complete, being in part described by mass action, ionic charge, and activity relationships. The selectivity coefficient is used to indicate the preference of the ionexchange resin for the uptake of 2 (or more) ions from solution. Generally speaking, the resin will take up divalent (or higher) ions in preference to monovalent ions, and in the case of a choice between ions of the same valence, the resin will take up the heavier ion preferentially.

Treatment of the ion-exchange resin and preparation of the column Usually the ion-exchange resin is immersed in water and allowed to swell for 24 hours; it is then packed into a suitable column and, in the case of an anion-exchange resin, converted to the basic form by passing 2 M sodium hydroxide through the column at a rate of about 3 ml per minute until the effluent is free of chloride, followed by carbon dioxide-free water, to remove alkalinity. In the case of a cation-exchange resin, conversion to the acidic form is achieved by passing 2 M hydrochloric acid through the column, followed by carbon dioxide-free water until the washings are neutral.

         The prepared column is used in a similar manner to that described for adsorption column chromatography except that there is usually no need to monitor the effluent; according to the type of resin chosen and the type of material being determined the volume ofeffluent detailed in the particular application is collected and titrated with acid or base as appropriate, using a suitable indicator.

         After the determination has been completed, the ion-exchange column may be regenerated by washing either with 2 M sodium hydroxide for an anion-exchange column, or 2 M hydrochloric acid for a cation-exchange column, followed by water until a neutral reaction is obtained.

APPENDICES • 3.3 COLUMN CHROMATOGRAPHY
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หมายเหตุ / Note : TP II 2011 PAGE 392-394