ANALYTICAL CHEMISTRY
1024 mediate green. Therefore, trichloroacetic acid must be rated a weaker acid than aluminum bromide. The differences among aluminum bromide, stannic chloride, and ferric chloride are small, and the same is true for the strong bases. Therefore, the order of the reagents is a very general one. However, this qualitative rating of acid strength serves to indicate the higher acid strength of aluminum bromide, ferric chloride, and stannic chloride compared to the hydrogen acids hydrogen chloride and trichloroacetic acid. LITERATURE CITED
(1) Davis, ,M. M., and Schuhmann, P. J., J . Research S a t l . Bur. Standards, 39, 247 (1947);RP 1825. ( 2 ) Fritz, J. S., and Lisicki, N. hI., ANAL.CHEM.,23, 589 (1951).
(3) Hubbard, R. A.,2nd, and Luder, W. F., J . A m . Chem. Soc., 73: 1327 (1951). (4) Kolthoff, I. M.,“Acid-Base Indicators,” New York, Macmillan Co., 1937. (5) La Mer, V. K., and Downes, H. C., J . Am. Chem. SOC.,53, 895 (1931). (6) Lewis, G.N., J . Franklin Inst., 226, 293 (1938). (7) Lewis, G. N., and Biegeleisen, J., J . Am. Chem. Soc., 65, 1147 (1 943). (8) Luder, W. F., and Zuffanti, Saverio, “The Electronic Theory of Acids and Bases,” New Pork, John Wiley & Sons, 1946. (9) SearnaqWilliam,and .4llen,Eugene,ANAL.CHEM.,23,592(1951). RECEIVEDfor review September 29, 1961. Accepted January 1, 1952. Presented a t the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. March 1951. Paper VI1 in a series on acids and bases. Previous paper is (3).
Detection of Ultraviolet- Absorbing Substances on Paper Chromatograms ALEJANDRO C. PALADINI’ AND LUIS F. LELOIR Znstituto de Znvestigaciones Bioquimicas, Fundacibn Campomar, J. Alvaret 1719, Buenos Aires, Argentina
of purine and pyrimidine derivatives by paper DETECTIOY chromatography has been carried out by several procedures, such as the formation of mercury salts followed by treatment with hydrogen sulfide ( I I ) , by cutting out small portions of the paper followed by extraction and measurement of the ultraviolet absorption of the solutions ( 7 ) , or by photographic or direct examination of the paper using a special light source with strong emission at 240 to 260 mp (6,6, 9).
A arocedure hasbeen in usein thislaboratory ( 4 ) in which a standard Beckman spectrophotometer Model DU is used with an accessory with which the paper is passed along the p h o t o t u b e e n trance. The main difficulty is the opaqueness of the paper, and this was overcome bv making the paper mote r , , , , u IL“’. transparent by impregnstion with paraffin Figure 1. Paper-Carrying oil. Accessory The accessory that carries the paper, usually called the ‘kpotometer” (Figure l), is placed in the spectrophotometer instead of the cell holder. Two knobs, K1and K2, serve for moving the paper along in either direction, and the movement of the paper is registered by dial D. The apparatus in Figure 1 carries paper strips 30 mm. wide. As the light beam of the spectrophotometer is only 8 X 3 mm., it has been possible to construct another apparatus for 10-mm. strips. After the chromatography, the dry paper bands are impregnated with paraffin oil and the excess is removed by pressing between filter papers. The strips are then put in position in the paper holder, so that the light beam crosses a portion of the paper lying before the starting line of the chromatogram. The spectrophotometer is then adjusted so that the galvanometer reads zero with the density knob set a t 0,100 and the selector switch,at the 1 position. The paper is then moved along and the extinction readings as well as the distances are registered. I
I
I
I
This procedure is less laborious than the method of Hotchkiss ( 7 ) ,but the results are very similar, as shown in Figure 2. Using 10-mm. paper strips, amounts of adenine as low as 0 . 0 2 ~ kf can be detected. Even when the Rp values of two substances are very similar, their presence can be detected by the asymmetry of the peaks (Figure 3). This is a distinct advantage of the “spotometer” over the ultraviolet lamps ( 5 ) . In cases similar to the one represented in Figure 3, inspection with ultraviolet light 1
Present address, Rockefeller Institute for Medical Research, New York
2 1 . N Y.
shows only one dark spot. Readings a t several wave lengths can be taken on one chromatogram, thus providing a criterion of the homogeneity of the peaks ( I ) . Furthermore, the paraffin oil can
10 20 CENTIMETERS
30
Figure 2. Identical Chromatograms of Nucleotide Mixture from Yeast Developed with “spotomcter” A and by method of Hotchkiss, B. 1-cm. bands e h a i t e d 3 hours with 3.5 ml. of water
5
10 CENTIMETERS
15
Figure 3. Mixture of Adenosine Triphosphate, Adenosine Diphosphate, and Adenosine Monophosphate Run with solvent made by mixing 75 ml. of 95% ethyl alcohol with 30 ml. of 1 M ammonium acetate
V O L U M E 2 4 , NO. 6, J U N E 1 9 5 2
1025
0.9 -
10
20
20
30
30
40 5 0
MICROGRAMS Figure 4.
Quantitative Results
Log & / I is absorbancy of peaks; microgram scale is logarithmic. Adenosine run in ethyl alcohol-ammonium acetate, Readings at 260 mp. Glucose and threonine run in butanol-water, and developed as described by Partridge ( 1 0 ) and Bull e t al. (3). respectively. Readings at 400 mp (glucose) and 570 mp (threonine)
The use of the “spotometer” to obtain quantitative results is rather obvious. In Figure 4 it is shown that the absorbancy of the peaks is proportional, within certain limits, to the logarithm of the amount of substance, provided that the samples were initially deposited on the paper over equal areas. This relationship was shown by Block ( 2 ) to hold for amino acids developed with ninhydrin and by McFarren et al. (8) for sugars detected with ammoniacal silver nitrate. The applicability of the present technique to amino acids and sugars is also shown in Figure 4. One of the difficulties in obtaining quantitative results in direct photometry on filter paper chromatograms is the inhomogeneity of the paper, which introduces a certain amount of uncertainty in the measurements. Whatman No. 1 paper was used throughout. LITERATURE CITED
(1) Berosa. AI.. ANAL.CHEM..22. 1507 (1950) ~. (2j Block, R. J.; Ihid.,22, 1327(1950). 1,8) Bull, H. B., Hahn, J. W., and Baptist, V. H., J . Am. Chem. Soc., 71,550 (1939). (3) Caputto, R., J. Bid. Chem., 189, 801 (1951). (5) Carter, C. E., J . Am. Chern. Soc., 72, 1466 (1950). 16) Holiday, E. R., and Johnson, E. A., Nature, 163, 216 (1949). (7) Hotchkiss, R. D., J . Bid. Chem., 175, 315 (1948). 18) McFarren, E. F., Brand, K., and Rutkowsky, H. R., A N A L . CHEM.,23,1146 (1951). (9) Markham, R., and Smith, J. D., Nature, 163,250 (1949). (10) Partridge, S. M., Ibid., 164,443 (1949). (11) l-ischer, E., and Chargaff, E., J . Bid. Chem., 168, 781 (1947).
be removed with ether and the paper can be used for other chemical estimations. For instance, measurements can be made before and after treating the chromatogram with bromine vapors, thus distinguishing between uridine and adenosine derivatives. The use of solvents absorbing in the ultraviolet does not interfere with the procedure as long as they can be removed by evaporation or extraction. The authors experienced no difficulties in carrying on chromatograms with phenol, if they extracted the last traces of solvent with ether prior to examination of the paper in the spectrophotometer.
RECEIVEDfor review September 4, 1951.
Accepted December 12, 1931.
Specific Spot Test for Antimony PHILIP W. WEST
AND
WILLIAhl C. HAMILTON
Coates Chemical Laboratories, Louisiana State Uniwersity, Baton Rouge, La.
HE procedures heretofore reported for the spot test detection antimony have in most c ‘dses lacked sensitivity, specificity, or both. Rhodamine B, firfit used by Eegriwe ( 1 ) for the detection of antimony, was satisfactorily sensitive, but suffered from a number of interferences. Fluorone (9-methg1-2,3,7-trihydroxy-6fluorone), originally proposed by Wenger, Duckert, and Blancpain (S),proved somewhat superior to Rhodamine B both in sensivity and selectivity, but n-as very difficult to prepare and unstable in solution. Recently JVest and Conrad ( 5 ) have utilized gossypol for the detection of antimony. Their procedure, while superior to any reported prior to it, has qeveral shortcomings: the reagent is not yet commercially available, the acidity of the test solution requires careful control, and certain interferences exist. The authors, in search of a solvent suitsble for the extraction oi the tetraiodoantimonate (111)ion, observed that the yellow color of this complex disappears when acidic solutions of it are shaken with benzene. Further investigation showed that antimony could thus be extrdctcd quantitatively and exclusivelv, and that the benzene extract would react directly with Rhodaniine B to give a sensitive and sperific teht for antimony. REAGEATS
Rhodamine B, 0.2% aqueous solution. Potassium iodide, 10% aqueous solution. Sulfuric acid, 1 to 3 aqueous solutions. EXPERIMENTAL
The general technique followed for the detection of antimony was to perform a benzene extraction of the antimony (111) iodidc, after which Rhodamine B was added to th2 benzene layer as the.
color-developing reagent. The details of the method a w given in the procedure. The Concentrations of the aeveral reagents were varied over wide ranges without adversely affecting the test. Rhodamine B concentrations of 0.02, 0.05, 0.1, and 0.5% were tested. The potassium iodide concentration was varied between 2 and 20Yc. The acidity was varied from 4 N to 14 X without adverse effect. For acidities belop7 4 1%’ the sensitivity of the procedure was lessened, presumably because of poorer extraction, and at acidities much above 14 N the air-oxidation of potassium iodide was so much accelerated that the benzene layer became colored with free iodine, making interpretation of the test difficult. Similarly, higher concentrations of potassium iodide are more unstable and less convenient on that account. Sitrite ion and oxidizing agents int,erfered v i t h the test as clescribed, the former by- virtue of the fact that it gave a strongly fluorescent bluish color on extraction, the latter by the liberation of free iodine, which colored the benzene layer so deeply that the test color was hidden. The interference of nitrite ion was initially eliminated by evaporating the test solution to fumes of sulfur trioxide, then diluting with water to the initial volume and proceeding with the test as usual. Later it was found that this interlerence could be more conveniently circumvented by the addition of a few milligrams of solid urea t o the test solution before addition of the potassium iodide. The interference of oxidizing agents was obviated by the addition of solid sodium sulfite to the acidic solution just prior to the extraction, thus reducing any free iodine which had been liberated. The limit of identification and concentration limit of the test were measured as prescribed by Feigl ( 2 ) . The interference studies followed the procedure of West (4).The ions studied were
