สารบัญ

Gas Chromatography​

SUPPORTS

          Diatomaceous Support (Diatomaceous Earth; Siliceous Earth) White or almost white, fine granular powder made up of siliceous frustules of fossil diatoms or debris of fossil diatoms. It may be identified by microscopic examination with a magnification of ×500.

          Diatomaceous Support, Acid-washed Diatomaceous support that has been purified by treatment with hydrochloric acid and washed with water to remove metallic impurities, and to reduce surface activity and peak-tailing.

          Diatomaceous Support, Alkali-washed Diatomaceous support that has been treated with potassium hydroxide solution to reduce peak-tailing of basic compounds.

          Diatomaceous Support, Silanized Diatomaceous earth for gas chromatography, silanized Acid-washed diatomaceous support that has been silanized with dimethyldichlorosilane or other suitable silanizing agents.

STATIONARY PHASES

          A wide range of chemical substances is used, including polyethylene glycols, high-molecular weight esters and amides, hydrocarbons, silicone gums and fluids (polysiloxanes often substituted by methyl, phenyl, nitrilo, vinyl, or fluoroalkyl groups, or mixtures of these), and microporous cross-linked polyaromatic beads. Care should be taken to select grades specifically intended for use in gas chromatography. In most cases reference is made to a particular commercial brand which has been found to be suitable for the determination in question, but such statements do not imply that a different but equivalent commercial brand may not be used.

INTERNAL STANDARDS

          Reagents used as internal standards should not contain any impurity which would produce a peak likely to interfere in the determination described in the monograph.

Thin-layer Chromatography

          The coating substances described below are used to prepare thin-layer chromatoplates in accordance with the procedure described in Appendix 3.1. Prepare suspensions of the coating substances as recommended by the manufacturer unless otherwise prescribed. Commercial pre-coated chromatoplates may be used for Pharmacopoeial tests provided they comply with the test for chromatographic separation described for the corresponding coating substance.

Kieselguhr G A fine, greyish white powder, the grey colour becoming more pronounced on triturating with water. The average particle size is between 10 and 40 μm. It consists of natural kieselguhr which has been extracted with hydrochloric acid and calcined, and to which about 15 per cent w/w of calcium sulfate hemihydrate has been added. It complies with the following requirements.

          CONTENT OF CALCIUM SULFATE Carry out the test described under Silica gel G.

          SEPARATING POWER Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using a mixture of 65 volumes of ethyl acetate, 23 volumes of 2-propanol and 12 volumes of water as the mobile phase. Prepare the chromatoplates using a slurry of the sample in 0.02 M sodium acetate. Apply to the plate, 5 μl of a solution in pyridine containing 0.01 per cent w/v of each of lactose, sucrose, fructose, Dglucose, and D-galactose. After removal of the plate, dry it at 105º to 110º, and allow to cool. Spray the plate with about 10 ml of anisaldehyde TS and heat at 105º to 110º for 5 to 10 minutes; the chromatogram shows five, welldefined, well-separated spots with no tailing.

          ALKALINITY pH of a suspension prepared by shaking 1 g with 10 ml of carbon dioxide-free water for 5 minutes, 7.0 to 8.0 (Appendix 4.11).

          Silica gel G A fine, white, homogeneous powder of an average particle size between 10 and 40 μm containing about 13 per cent w/w of calcium sulfate hemihydrate, and complying with the following requirements.

          CONTENT OF CALCIUM SULFATE To about 250 mg, accurately weighed, add 3 ml of 2 M hydrochloric acid and 100 ml of water and shake vigorously for 30 minutes. Filter, wash the residue with water and carry out the “Complexometric Titration of Calcium” (Appendix 6.3) on the combined filtrate and washings. Each ml of 0.1 M disodium edetate VS is equivalent to 14.51 mg of CaSO₄. ^1/2H₂O.

          SEPARATING POWER Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using toluene as the mobile phase. Apply to the plate, 10 μl of a solution in dichloromethane containing 0.1 mg per ml of each of indophenol blue, sudan red G and dimethyl yellow in toluene. Allow the mobile phase to ascend 10 cm. The chromatogram shows three clearly separated spots of the indophenol blue, sudan red G and dimethyl yellow in order of increasing Rf value.

          ALKALINITY pH of a suspension prepared by shaking 1 g with 10 ml of carbon dioxide-free water for 5 minutes, about 7 (Appendix 4.11).

          Silica gel GF254 A fine, white, homogeneous powder of an average particle size between 10 and 40 μm containing about 13 per cent w/w of calcium sulfate hemihydrate and about 1.5 per cent w/w of a fluorescent indicator having a maximum intensity at 254 nm. It complies with the tests for Content of Calcium Sulfate, Alkalinity and Separating Power stated under Silica gel G and with the following test.

          FLUORESCENCE Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using a mixture of 90 volumes of 2-propanol and 10 volumes of anhydrous formic acid as the mobile phase. Apply separately to the plate, increasing quantities from 1 to 10 μl of a 1 mg per ml solution of benzoic acid in the same solvent mixture. Develop the plate and dry in a current of warm air. Examine the chromatogram under ultraviolet light at 254 nm. The benzoic acid appears as dark spots on a fluorescent background in the upper third of the chromatogram at levels of 2 μg and greater.

          Silica gel H A fine, white, homogenous powder of an average particle size between 10 and 40 μm. It is free from calcium sulfate hemihydrate and complies with the tests for Separating Power and Alkalinity stated under Silica gel G.

          Silica gel HF254 A fine, white homogeneous powder of an average particle size between 10 and 40 μm containing about 1.5 per cent w/w of a fluorescent indicator having a maximum intensity at about 254 nm. It is free from calcium sulfate hemihydrate and complies with the tests for Alkalinity and Separating Power stated under Silica gel G, and with the test for Fluorescence stated under Silica gel GF254.

          Silica gel F254 A fine, white, homogeneous powder of an average particle size of about 15 μm containing a suitable binding agent and about 1.5 per cent w/w of a fluorescent indicator having a maximum intensity at 254 nm. It complies with the following requirement.
          FLUORESCENCE Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1), using a mixture of 90 volumes of 2-propanol and 10 volumes of anhydrous formic acid as the mobile phase, but allowing the solvent front to ascend 10
cm above the line of application. Apply separately to the plate, ten portions from 1 to 10 μL of a 1 mg per mL solution of benzoic acid in the mobile phase.  After removal of the plate, dry in a current of warm air.  Examine the chromatogram under ultraviolet
light at 254 nm. The benzoic acid appears as dark spots on a fluorescent background in the upper third of the chromatogram at levels of 2 μg and greater.

         Silica gel, Octadecylsilyl  A very finely divided silica gel, chemically modified at the surface by the bonding of octadecylsilyl groups.