A simple technique for scanning thin layer chromatograms

ANALYTICAL

BIOCHEMISTRY

4, 418421

(1962)

SHORT COMMUNICATIONS

A Simple Technique for Scanning Thin layer Chromatograms

Although thin-layer chromatography offers several advantages as a means of separating mixtures of biological compounds, its application to tracer studies is hampered by the lack of a convenient method for detecting closely adjacent labeled substances on the developed chromatoto be grams. The following technique enables such chromatograms scanned with an efficiency and resolution comparable to that obtained using paper chromatograms. The method involves an adaptation of a procedure described for removal and preservation of thin-layer chromatograms (1). The developed chromatogram is sprayed thoroughly with Neatan, an aqueous dispersion of polyvinyl propionate developed by E. Merck (Germany) and distributed in the United States by Brinkmann Instruments, Inc. The saturated layer is dried for a few minutes with the aid of a hair dryer and then a piece of colorless transparent tape (1.) is applied firmly to the surface. If the chromatogram is subjected to prolonged drying (for example, by heating at 105°C in an oven) it must be immersed in water before the chromatographic material can be removed cleanly from the glass plate. This procedure is satisfactory only for water-insoluble compounds. If not overdried the adsorbent can be readily peeled off along with the tape. At this stage the chromatogram is unsuitable for scanning owing to the loss during handling of small particles of adsorbent which contaminate the scanner. This problem can be obviated by a light application of Neatan to the underside of the chromatogram, with subsequent drying. The resulting chromatogram is completely dust-free, the emulsion surface having the appearance and texture of filter paper. Strips may be prepared for scanning by use of a sharp knife, cutter, or scissors. The scanning technique is identical to that used for paper chromatograms. For weak emitters like carbon-14, however, only 2 r geometry is obtain418

SHORT

419

COMMUNICATIONS

able, the emissions on one side being absorbed by the adhesive tape. It is therefore necessary to place the strip in the scanner with the emulsion side adjacent to the detector. The scanning sensitivity with respect to carbon-14 has been found to be at least 80% of that obtained under comparable conditions using untreated No. 1 Whatman paper. A sample scanogram is shown in Fig. 1.

lin.

.

Fro. 1. Photograph of scanogram obtained from a thin-layer the technique described. Total activity 5500 disintegrations per Vanguard Model 800 Autoscmer, time constant D, range 100, chart speed 6 in./hr, 2~ geometry. Chromatogram attached to

strip prepared by minute. Scanned in medium slit width, top of chart paper.

420

SHORT COMMUNICATIONS ACKNOWLEDGMENT

This Science

investigation Foundation

was carried (G-21374).

out

with

the

aid

of a grant

from

the

National

REFERENCES 1. Brinkmann Instruments, Inc., 115 Cutter Mill Road, Great Neck, N. Y., “Removal and Preservation of Chromatogrnms in Thin Layer Chromnt,ography,” April 15, 1962.

A. SAARI CSALLANY H. H. DRAPER DilGion of Animal Nutrition Iinivcrsity of Illinois lirbana. Illinois Receilled July 19, 1062

Citrate

Interference

in Inorg,anic

Phosphate

Analysis1

The determination of inorganic phosphate is a rout.ine analysis in biological investigations. A standard optical procedure is the FiskeSubbaRow method (l), which is based on the formation first of phoaphomolybdate followed by reduction to a blue color. We have recently found that the presence of citric acid interferes with the formation of phosphomolybdic acid and consequently the observed values for phosphate are lower than the actual values. The Fiske-SubbaRow method for determining inorganic phosphate was carried out on known quantities of phosphate with varying amounts of citrate. Reduction was generally accomplished with 1,2,4-aminonaphtholsulfuric acid in a sulfite-bisulfite solution ; however, other reducing agents gave the same results. The results obtained are given in Table 1. The effect of the order of addition of citrate was also investigated: these results are given in Table 2. The additions were 100 pmoles of citrate. All optical density measurements were made at 6660 A in a Beckman DU spectrophotometer. The results in Table 1 show that citrat.e interferes with the determination of phosphate and, at relatively high concentrations, citrate ‘This Society

investigation was aided by and The U. S. Public Health

grants Service.

from

the

National

Multiple

Sclerosis