Bile acids paper chromatography

To recover bile acids or their conjugates from biological fluids, such as bile or intestinal contents, several different procedures are available. When the bile acid concentration is sufficiently high (above 1 meq/liter), as, e.g., in bile, 5-100 p. of the fluid may be directly applied to the starting line for paper or thin-layer chromatography. When low concentrations of bile acids are present, bile may be added drop by drop into 10-20 volumes of ethanol... 

TABLE III. Solvent Systems for Quantitative Analysis of Conjugated Bile Acids by Paper Chromatography... 

Although separation factors between conjugated bile acids are not as good in thin-layer as in paper chromatography, the thin-layer method has been much more used in recent years because of its greater simplicity and speed. The mobilities of common conjugated bile acids in different solvent systems are given in Table IV. It should be observed that several systems can be used in two-dimensional development. 

When mixtures of bile acids are hydrolyzed at an early stage of the analytical procedure, the free bile acids formed may have to be further purified. Extraction, counter-current distribution, reversed phase partition, and paper chromatography have been described (Section III.A) and these methods can also be used for free bile acids. 

To perform a systematic TLC analysis of a mixture of free bile acids, the solvents, techniques, and adsorbents presented in Table Vl-X can be used. Table XI lists some systems for chromatography on adsorbent impregnated glass fiber paper (GFP). An analysis of bile acids with GFP may require only 10-30 min on the other hand, this method is very sensitive to overloading and is used for bile acids in the 0.1-5 /ig range (84, 85). 

The separation of bile acids by gas-liquid chromatography is determined by the choice of stationary phase and bile acid derivative. Data that permit the selection of stationary phase and bile acid derivative to suit most separation problems are shown in Tables XII-XIV. As in the original papers relative retention times have been used in the tables instead of the more acceptable retention index (112) which permits better interlaboratory comparisons. Relative retention times are subject to variation, mainly due to temperature differences and, to a lesser extent, to differences in column preparation. In our experience the temperature-dependence is most pronounced with trimethylsilyl ether derivatives on Hi-Eff-8B (cyclohexanedimethanol succinate) columns. Temperature differences do not affect relative retention times on QF-1 (a trifluoropropyl, methyl siloxane) to the same extent. 

During chromatography the ion-exchange papers are suspended in suitable glass containers so that the free ends just dip into the developing solution. Depending on the solvent, up to 2 hr may be required for the solvent front to ascend 20 cm. The paper is air-dried and the bile acids are located by spraying with phosphomolybdic acid. 

Because of its speed and simplicity thin-layer chromatography on silica gel is often used to follow the course of elution and separation of solutes from ion-exchange columns. It provides an excellent substitute for the paper-chromatographic monitoring of bile acids described by Gordon et al. (2). 

Ganshirt et al. 1960). Paper chromatography with various solvent mixtures enabled separation of free and conjugated bile acids (Sjovall 1959). 

Since free and conjugated dihydroxy cholanic acids cannot be separated completely by paper and thin-layer chromatography they must be differentiated by specific reactions. The following procedures are suitable for quantitative determination of free bile acids reaction with concentrated sulfuric acid for determination of cholic acid (Hammarsten 1925), reaction with salicylic aldehyde and sulfuric acid for determination of deoxycholic acid (Szalkowski and Mader 1952), reaction with ethyl acetate, concentrated sulfuric acid and acetic acid anhydride for chenodeoxycholic acid (Isaksson 1954). 

The determination of bile acids in bile and duodenal contents by quantitative paper chromatography. Clin. chim. Acta 4, 652 (1959). 

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