Deoxycholate plate

Also streak samples onto two blood agar plates (Appendix 4) and two deoxycholate plates. 

Figure 12. Thin layer chromatography of base-hydrolyzed C-labeled deoxy-cholate-soluble peptidophosphogalactomannan. C-Labeled deoxycholate-soluble peptidophosphogalactomannan was treated with 4N KOH for 5 hr at 110°C in a sealed evacuated tube. The solution was extracted with CHClg. methanoh 2 1 (v/v). Fifty fiL sample was applied to a silica-gel, thin layer plate and chromatographed in benzene-.diethyl ethenethyl acetate. acetic acid, 80 10 10 2 (vlvjvlv). Reference compounds lysine, ethanolamine, monomethylethanolamine, dimethylethanolamine, and choline were chromatographed in a similar manner. The mobility of sphingosine is depicted in the upper...

Fig. 1 Densitometric profiles obtained for the investigated bile acids on aluminum plate precoated with silica gel 6OF254 (E. Merck, 1.05554) using n-hexane-ethyl acetate-acetic acid (22 21 5, v/v). Glycocholic acid (GC), glycodeoxycholic acid (GDC), cholic acid (C), glycohthocholic acid (GLC), cheno-deoxycholic acid (CDC), deoxycholic acid (DC), lithocholic acid (LC). (Camag densitometer, wavelength A = 250 nm slit dimensions 6 x 0.2 mm scanning speed 40 nm/sec).

Fig. 3 The densitograms of bile acids investigated (C, cholic acid GC, glycocholic acid GLC, glycolithocholic acid DC, deoxycholic acid CDC, chenodeoxycholic acid GDC, glyco-deoxycholic acid LC, lithocholic acid) at wavelengths 380, 400, 420, 440, and 460 nm after their separation using a n-hex-ane-ethyl acetate-methanol-acetic acid, 20 20 5 2 (v/v/v/v) as mobile phase and after the application of sulfuric acid in methanol (1 19, v/v the plate was immersed in dipping the solution of sulfuric acid for 15 sec, and it was then heated to 90°C for 20 min) as visualizing reagent.

With this solvent, polyamide (Miramid FP) is used as adsorbent, and the plates are developed 12 cm utilizing the wedge technique [see Table IV, Footnote e and (132, 133)]. With this system lithocholic, hyocholic, and cholic acids have a mobility relative to that of deoxycholic acid of 0.30, 0.97, and 1.24, respectively. 

Fig. 3. Schematic chromatogram of the ion-exchange thin-layer chromatography of free and conjugated bile acids. Ion exchanger polyethylenimine-cellulose (250-/ -thick layer, 20 X 20 mm plate) solvent 100 ml 1 iV acetic acid, 50 ml absolute ethanol development time 120-150 min. Amount applied 5-10 fxg of each acid detection 0.05% 2,7-dichlorofluorescein in 50% methanol, under UV light. Bile acid conjugates identified as in Fig. 2 C, D, and L free cholic, deoxycholic, and lithocholic acids, respectively FA common fatty acids.

When the separations of the acids within the free and glycine conjugated groups are considered, it is seen that on both columns and thin layers, the cholic acid derivatives are eluted ahead of the derivatives of the deoxycholic and chenodeoxycholic acids, and presumably other dihydroxy bile acids. The derivatives of the lithocholic acid are eluted last. Complete separations of the mono-, di-, and trihydroxy bile acids are not realized even on the thin-layer plates of ion exchangers, and there is no discernible resolution of the various taurine conjugates. This order of elution of the bile acids is opposite to that expected on the basis of their pK values (Table II). Free cholic acid (pK 5.29) would have been expected to be retained longer than the dihydroxy acids (pK 6.18-6.29) which should have been retained... 

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