Spray detection of bile acids on thin-layer chromatograms

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:be::followed: by..a ‘desalting ‘procedure, with consequent loss of material, if the pure’ com,pound:, is’,&@.$red~ :We have’ found. that. this difficulty can be obviated by the ,use ’ orthe completely. volatile solvent system IV,’ be&-amyl alcohol-pyridine‘,$cetic:*adid-water .(IIO :I .o:0.08 :25). ‘Descending development on washed Whatman No. 3MMlpaper for 30 h gave good resolution, without tailing, of DNP-amino acids ‘-with’:the. exception of the DNP-glycine and DNP-serine pair (Table I). : &&t

S&b01 of M$crobioZogy, University of New So&h ,R.c; B?x,i, ‘K&s+agto~n, N.S.W. 2033 (A+radia)

WaZes,

W.D. GRANT A. J. WICKEN

i,'TV. D. GRANT AND A...J. WICKEN, Biochem. J,, submitted for publication 2 M. BRENNER, A. NIEDERWI~SER AND’G. PATAICI, in E. STAHL (Editor), Thin-layer CiJvomato,grapky-A ‘Labovatoyy Hcin,dbook, Springer-Verlag, Berlin, Gbttingen, Heidelberg, 1965, p. 391. 3 J.kM. .,GHu+EN, ?..J. TIPPER AND J. L. STROMINGER, Metho& _EnzymoZ,, 8 (1966) 685. 4 IL H.’ SCHLE~PER AND 0. KANDLER. Arck. Mikvobidl., 57 (1967) 335. ~5’ 'J.-M. GWUYSEN; D. J. TIPPER, C. M. BIRGE AND J. L. STROMINGER, Biochemistry, 4 (1965) 2245. 6, A. ‘L. LEVY, ‘Nqtzcre, 174 (1954) 126. $..S.:B~ACKBURN AND A.,G. &OWTHER, Biochem. J., 48 (1951) 126. ..’

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Received

,

November, &th,

; ,, ‘_’

1969

:

,$. ~C@&ato~.~~47

(1970) i24-126

:,cWRoM.’ 4521. ,’ : .,.., I : (‘,,

;

dete&ion

Spra$ I ..,

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of bile acids on thin-layer

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‘,’

chromatograms

The detection of ‘bile .,acids in thin-layer chromatography (TLC) has been ac&omplished by means of spray reagents: namely, phosphomolybdic acidl, antimony .,+.richloride2.or concentrated sulfuric acids. But the usefulness of color detection in :bile, acid ‘identification. has been demonstrated by ,KRITCHEVSKY et aZ.4 and by AN..+I~NY. AND’ B&IER~. The present study expands on the use of color detection, thus ‘afding ‘in the’ identification of bile acids. ‘),

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~._Materials and methods ,,,,.’

,: ; ‘: The bile,acids were obtained from ,Applied Science Laboratories, Inc., Pa., and isooctane, isopropyl alcohol arid ,acetic acid were all Baker Analysed Reagent grade. ::+ thinYlayerplate precoated ‘to a thickness of b.5 mm with Silica Gel F,,, (Merck) ,e$nd..a commercial chromatographic chamber (Gelman Instrument Co., Ann Arbor, Mich;) were used. Tlieplates were heated at IOOO for I h and the chamber was saturated ‘With,’ the ,‘,solvent “is,ooctaneLisopropyl alcohol-acetic acid (40 :. IO :I) prior to the :*chromatogramphy at ‘room ‘temperature (23-25 “), When chromatograms were run, :,:.+o%:X ,‘zo.cm plates’were used and the solvent front was permitted to rise 135 mm from “‘the .,,origin.‘Th,e .chromatoplate ‘.was then removed from the chamber, air dried for :several minutes .and then dried thoroughly at roe’ for IO min. After cooling, the plate ,)‘+a~ expos_ed to iodine,,vapor for 15 min,‘sprayed with tap water and the color was : ,‘,:, .,: “, ‘, ‘. .b ‘..., ‘.’ .Ijl; Cltvomqlog., 47 (1970)

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,(

12*127

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127 ,. noted. This color was found to disappear within IO-IS min when left exposed to air or dried at 100~ for 5 min. The plate was next sprayed with the benzoic acid spray reagent (0.15 g of benzoic acid, 0.15 ml of concentrated sulfuric acid and 20 ml of acetic acid) and placed in an oven at 100' for 15-20 min and the color was noted. Five to twenty micrograms of each acid in ethanol were sufficient for color detection_ NOTES

and discussion Color reactions of the bile acids are shown in Table I. The methyl ester of cholic acid (not shown in Table I) gives a navy blue color with iodine water. The special interest of benzoic acid spray reagent is that it gives a different color with chenodeoxy- and deoxycholic acids, two compounds whose Rp values do not always permit

Results

TAULE COLOR

I REACTIONS

OF I31LE ACIDS

(IO/A&

Detcctin~ -A

Cholic ncicl Chcnoclcoxycholic Dcoxycholic acid Hyocholic acid Lithocholic acid

acid

reapntsa

Brown . Yellow Yellow Light yellow Light yellow

-

-----

B

Brownish yellow Brown Deep yellow Light brown Light brown

n A = Exposed to iodine vapor for 15 min and sprayed with water. B = Uenzoic acid spray reagent (0.15 g of bcneoic acid, 15 ml of conccntratecl sulfuric acid ancl zo ml of acetic acid).

unequivocal identification. One other distinct advantage is the lack of background. color. The reagent reacts only with the test compounds and the resultant color stands out clearly from the white background. Lastly it is suggested that each investigator should prepare his own color standards. This work was supported in full by the National Health Service Grant AM 11315. De$artnmat of General Surgery, University of Michiga9~ Medical Ccntcv, Agaqt Arbor, Mida. 48ro4 (U.S.A.)

Institutes of Health, Public

SATINDRA

I<. GOS~AMI

CHARLES

F. FREY

I D. I~RITCHEVSICY ANI) M. R. KIRK, Arch. Bioclrctw. Biophys., 35 (1952) 340. 2 M. D. SIPERSTEIN, IT. M. HAROLD, J. L. CIIAIKOI~FANIJ W. G. DAUBEN, J. Biol. Chw-rt., ZOI: (1954) ISI* 3 I?.. ENEROTM, J. L,i$id Rcs., 4 (1963) I I. 4 D. KRITCHEVSICY, D. S. MARTAK AND G. I-1.~C
Received

November

IIth,

rgGg J. C~L?'O?~tatog., 47 (1970) 126-127