Detection of minute traces of blood by thin-layer chromatography

MOTHS

156

Failure to wait for solvent evaporation results in an increase in the width of the sample band. The rate of solvent evaporation may be increased by heating the plate during sample application and by blowing warm air over the surface of the layer. The method of sample application described has been usecl successfully for over one year with 20 cm x 20 cm plates of silica gel G of z mm thickness. An example is the separation of the naturally occurring pigments javanicin and fusarubin’ which involved a difficult problem of sample application because of the lack of solubility of the pigments. Six milliliters of a saturated solution of IOO mg of pigments in chloroform was applied to each plate. A further example is the separation on one plate of a zoo mg mixture of equal amounts of (a) azobenzene, (b) +methoxyazobenzene, (c) Sudan Yellow, and (d) p-aminoazobenzene, The time required to apply the solution (2 ml) using a gauge 24 needle was 5 min. The sample band was 15 cm lnnm rvrrh

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ascending development using 2 passes of benzene horizontal bands in cm from the starting lint was 12.3, (c) 4.3-7.3, (d) 1.6-4.3. It is suggested that the technique described streak preparative thin-layer cl~roniatograms with visualize colorless bands. A tindO Halifax,

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for sample application be used to an appropriate reagent in order to

z. :i?. ARSENAULT

Laboraiogv,

J. Cltr~~w~to~.,

I M. I?.BACON, a J, M. BOBBITT, 3 J, 1’. CONNOLLY.

IG (1964) 552. TltiwLqw CIl,~oztzatoji~~~lry, licinl~olcl, New York, 1963, P, J. FLANAGAN, Ii. 0. DoRcHAf AND J. l3. THOMSON,.~.

pp. 53-57. Clwo~~z~log.,15 (1964)

4 ~~,5R~~~~~~~~, T1zi.wLayw C?tr.onlato,nrlapl~y, Academic Press, New York:, 1963, -2 y. I<, SEIKET, M. A. MILLETT AND J. F. SAEMAN, J. CIwomatog., 15 (1964) 115. G I?.J. RITTER ~~7x3G. 3.4.MEYER, Naizcre, rg3 (IgGzj 94.1. 7 G. P. ARSENAULT. Cum. J. Chem., 43 (1965) 2423,

Received

July Izth,

J, Ciwomatog.,

Detection

!y

as developer. The position of the as follows: (a) 13.7-15.2, (b) g.g-

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21 (1966)

p. 41.

1965 155-156

of minute

traces

of blood

by thin-layer

chromatography

A thin-layer chromatographic method has been developed for the rapicl detection of traces of blood in the microgram range. The method is described below. The material under test and suspected of containing blood is eluted with distilled water or an o.S5 yO solution of NaCl. 0.005-0.01 ml of this aqueous solutio$ is applied directly on to the thin layer. Lix$eGnmta.I d&ails Adsorbent : Kieselgel G (Merck). Layer thickness: 250 p. J. Chosnatog.,

21 (IgGG).IsG-157

NOTES

Distance of development :.I o cm. Apparatus: Grundausrustung Desaga, No. 600. Temperature: 20 to 24”. Solvent : methanol-glacial acetic acid-water (go : 3 : 7). No previous saturation of chamber is required. Duration of run: about 25 min. Control: 0.01 ml of a I v0 solution of human blood. Reagent A. 0.05 g of benzidine, analytical quality, dissolved in IO ml of ethanol. The pH is adjusted to 4.5 ,with glacial acetic acid. Reap& B. 3 y0 aqueous H,O,. The reagents must be freshly prepared. To render the highly sensitive, but not specific benzidine test specific for blood by the thin-layer chromatography, the following rules must be adhered to: I. A control blood sample must be run parallel with the material under test. 2. The plate must be dried in an incubator for 5 min at 100' after running. At that temperature any peroxidases present on the plate still saturated with the acetic acid are inactivated. 3. After drying, the plate is examined in U.V. light, and the fluorescent spots (e.g. from mineral oils) are marked. 4. The plate is sprayed with reagent A and then left for 5 min. Blood does not react, and the spots of any oxidising substances reacting directly with be&dine can be marked at this stage. Such spots are never blue, but brown, white, green, or red, and differ significantly from blood in RR value. 5. Five minutes later the plate is sprayed with reagent B. The presence of an intensely blue spot at the level of the spot seen in the control sample (Rp 0.79, S.D. ho.02) indicates the presence of blood. Limit of sensitivity: 3 to 4 pg of blood. According to the control tests, dyes, oxidising agents, biological contaminants, including benzidine-positive substances, do not interfere with the detection of blood. Because of its rapidity, the method may be used particularly advantageously in forensic medical work. Further investigations concerning the method are in progress, and will be reported at a later date. lnstitzcte Received

of Formsic

Chemistry,

Bzcdapest (Hungary)

A. FARAGd

July zest, 1965 J. Chromalog.,21 (x966) 156457