V O L U M E 2 4 , NO. 8, A U G U S T 1 9 5 2 chloride and 1.0 gram of C.P. anhydrous sodium acetate are dissolved in the solution. The pH is adjusted to 4.6 with either concentrated hydrochloric acid or sodium hydroxide and the solution is diluted to 100 ml. with distilled water. An aliquot poition of the prepared solution containing from 2 to 8 mg. of fluoride is transferred to the titration cell and deaerated with nitrogen for 10 to 15 minutes. After the deaeration the titration is carried out a t an applicd potential of -0.370 volt vs. S.C.E. Increments of aluminumdye complex reagent are added and the diffusion current is obtained after the addition of each increment It is necessary to stir the solution with nitrogen after the addition of each increment in order to ensure complete mixing and deaeration of the added reagent. The decomposition of the aluminum-dye complru reaches completion after 4 to 5 minutes. The observed values of the diffusion current are corrected for the volume change (lue to the addition of reagent The volume correction (3) 1s
where 1' denotes the original volume of the solution and the volume of reagent added a t an)- point. The corrected values of the diffusiori current are plotted against the quantity of reagent added and the lines on each side of the equivalence point are extrapolated to the point of intersection, which is the equivalence point of the titration. The equivalence point is found to correspond to the stoichiometric point for the formation of ..ZIFs---. DISCUSSION O F METHOD
T h e fluoride concentration present in the final polarographic solut,ion should be between 2 and 8 mg. of fluoride ion per 100 ml. The upper limit of 8 mg. per 100 ml. of solution is imposed by the solubility of the aluminum-dye complex in the reagent solution. By selection of a sample weight between 0.1 and 1.0 gram and rithout exceeding a dilution factor of 10, the method may be used
1371 over a range of 0.2 to 807, fluoride. The lower limit of 2 mg. is baaed upon the particular concentration of the reagent used. By decreasing the reagent concentration, the method might be extended downward into the microgram range. All observat,ions made during this work indicate that this extension would be satisfactory. The noninterference of many of the common cations and anions makes this a potentially useful method. Sulfate and silicate ions do not show significant interference up to 20 mg. per 100 ml. Up to 10 mg. of phosphate ion per 100 ml. can be tolerated, but above this concentration the interference becomes significant. Large amounts of sodium, potassium, chloride, and acet'at'e ions are permissible, as shown by the fact that each 100-ml. sample contains 11.4 grams of potassium chloride and 1.0 gram of sodium acetate. The serious interference of cobalt, iron, nickel, thorium titanium, vanadium, and zirconium requires that these elements hp removed completcly. These ions can be removed eit,her by electrolysis a t a mercury cathoclr 01' hh- a Wllard-Winter distillation LITERATURE CI'rEI)
(1) Fredrickson, L. D., Jr., ANAL.CHEhf., 23, 813 (1951). (2) Haul, V. R., and Greiss, W., Z.anorg. Chem., 259, 42 (1949). (3) Kolthoff, I. hl., and Lingane, J. J., "Polarography," New Torli, Interscience Publishers, 1946. (.4) Langer, A., IND. ESG. CHEM.,A 4 ~ . 4 ED., ~ . 12, 511 (1940). ( 5 ) Luzina, G. S., Zavodskaya Lab., 15, 1412 (1949). (6) Parker. 11,E., 1Zf.A. thesis, Duke University, 1946. (7) Petrow, H. G., and Xash, L. K., ANAL. CHEM.,22, 1274 (1950). (8) Willard, H. H., and Dean, J. A , , Ibid., 22, 1264 (1950). R K C E I V Efor O review February 2 5 , 1952. Accepted April 30, 1952. Taken in part from t h e thesis submitted by C. R. Castor t o the G r a d u a t e School of Duke University, Durham, S . C., in partial fulfillinent of t h e requirements for the degree of Inaeter of arts, June 1952.
Benzidine Reagent in Paper Chromatography HOWARD MILLER AND D. &I. KRAEMER State C-niwrsity Medical Center, College qf Medicine, Brooklyn, N. Y .
E S Z I D I N E has been used as a reagent for the detection of various inorganic substances ( 3 ) as well as in the paper chromatography of reducing substances ( 1 , 4). Use of this reagent in the identification of some of the constituents of protein hydrolyzates has necessitated further study on the reactions of this reagent in the paper chromatography of various inorganic and organic substances. I t is particularly important to know the behavior of inorganic compounds on benzidine when i t is impractical to remove them. This paper is an attempt t o supply some of the needed data in this field. PROCEDURE
Samples of approximately 0.001 ml. of a 1 to 5% solution mere placed 2 cm. apart on 28 by 41 cm. Whatman No. 1 filter papers and subjected to "ascending," one-dimensional chromatography. Butanol-acetic acid-water (40: 10: 50) ( 5 ) \vas used as solvent, and benzidine-acetic acid-ethanol (4) as spraying reagent. The procedure described by Horrocks and Manning ( 4 ) was tollowed, with the exception that the paper v-as placed in an oven at 85' C. for 30 minutes after spraying ITith the benzidine reagent. A glucose standard was run on each paper; all Rj values were calculated with reference to this sugar, assuming that the Rt value of glucose is 0.18. Depending upon the results, each sample was run from 2 to 10 times. RESULTS
In general, duplicate determinations of the R f values of most inorganic compounds showed a greater variability than those of
most organic compounds. Since the background is light brown in color, the presence of well 'defined colorless areas (white spots) is indicative of many compounds (Table I). Inorganic compounds which reacted with the benzidine reagent are listed, n ith their respective Rf values, in Table I. The inor_.
'Table I.
Reactions of Inorganic Conipounds with Benzidine Reagent
Compound Ammonium sulfate Cerium ammonium sulfate Cerium nitrate Cobalt acetate Cobalt nitrate Copper sulfate Ferric chloride Hydrochloric acid Lithium sulfate Magnesium chloride Manganese nitrate Nitric acid Potassium dichromate Potassium ferricyanide Potassium periodate Potassium permanganate Phosphoric acid Silver nitrate Sodium sulfate Sulfuric acid Uranium acetate Zinc sulfate a
Ir
~
~~
~~
R/ 0.0 0.0
0.20 0.28 0.23 0.0 0 . O , 0.30 0 . 0 , o .24 0.0 0.11 0.26 0.0, 0.24
0.0,0,15 0.0 0.0 0.0 0.45 0.0, 0.25 0.0
0.0.0.37 0.0 0.0
Description White Brown (white cap)" White Red Blue-green Gray (brown cap) Brown brown Brown,' red-brown White White White Brown, red-brown Brown, gray (gray cap) Blue Brown (gray cap) Brown Brown (white halo) h Brown, purple White Brown, white (brown tail) Brown White
A cap is a colored area extending from,major spot into higher Ri range. A halo is a colored area encircling malar spot.
ANALYTICAL CHEMISTRY
1372 ganic substances which failed to react with the benzidine reagent are acetic acid, aluminum sulfate, ammonium chloride, arsenic acid, barium hydroxide, barium acetate, cadmium chloride, calcium phosphate, chromium fluoride, lead nitrate, lead acetate, mercuric chloride, potassium chloride, potassium carbonate, potassium hydroxide, sodium chloride, sodium hydroxide, and stannous chloride. Reacting organic compounds are classified in Table 11. Though some of the reducing substances listed have been previously described ( 2 , 4,6 ) they are included because most of the published data are the result of “descending” paper chromatography, a method which gives slightly different R, values from the “ascending” technique employed in this investigation. The method utilized here for the calculation of the K! is also *lightly different. The benzidine reagent did not react with the following carbohydrates: adonitol, arabin, glycogen, i-inositol, inulin, mannitol, raffinose, sorbitol, starch, and sucrose. Thirty-eight amino acids and amino acid derivatives were also nonreactive. Miscellaneous compounds that were tested and did not react \yere: adenosine triphosphate (dibarium salt), atropine sulfate, betaine, betaine hydrochloride, choline chloride, acetylcholine chloride, citric acid, creatine, creatinine zinc chloride, epinephrine, folic acid, gelatin, glutathione, sodium heparin, histamine dihydrochloride, lactic acid, e o d i m malonate, nicotinamide, phlorizin, pyruvic acid, sa1cosine, sulfadiazine, sulfathiazole, tartaric acid, thiamine hydrochloride, thiourea, tyramine hydrochloride, urea, and uric acid.
bait nitrate, and niaiiganese nitrate, for example, have been aided by this procedure. At 85’ C. none of the amino acids reacts with the benzidine reagent, a factor of importance in the analysis of glycoprotein hydrolyzate~. The characteristic color and R! reactions of alloxan (Table 11) may be of value in the determination of this Pubstance in biological fluids and tissues.
DISCUS SIOk
ACKNOWLEDGMENT
Six of the inorganic compounds tested yielded R/ values a t the origin as well as a second spot in a higher R f range (Table I). It is difficult to explain these results, as well as the apparent discrepancy between the R, values of cations wch as cerium and cobalt, and anions such as nitrate and chloride. It seems important that the R f values of nianv of these compounds are close to those of a number of carbohydrates, necessitating the elimination or identification of inorganic impurities in carbohydrate samples. Many inorganic substances form benzidine-blue a t room temperature when ammonium hydroxide is added to the benzidine reagent ( 3 ) . Thus, positive identifications of cerium nitrate, co-
The authors wish to express their thanks to Jean Wright for her technical assistance and to Sam Seifter for his interest and advice concerning thie work.
I
Tahle 11. Reactions of Organic Compounds with Benzidine Reagent
a
Compound Rf Description 0.28 Brown S-Acetyl glucosamine 0.48 Violet (violet t a i l ) “ Alloxan 0.24 Brown hrabinose 0.46 Brown Ascorbic acid 0.19 Brown Dextrin 0.33 Brown Fucose 0.16 Brown Galactose 0.14 Brown Glricosamine hydrochloride 0.18 Brown Red-brown Glucose 0.38 Glucuronic acid 0.07 Brown Lactose 0.25 Brown Levulose 0.08 Brown Maltose 0.22 Brown Mannose 0.72 White Oxalic acid 0 . 8 5 Brown Phloroglucine 0.39 Brown Rhamnose 0 32 Brown Ribose 0 28 Rrow-n Xylose a,!’ea. A tail is a colored area extending f r o m inajor s p o t into Glnrose was standard for determination of all Rf valupa
LITERATURE CITED
(1) Bacon, J. S. D., and Edelman, J., Biochem. J., 48, 114 (1981). (2) Evans, E. E., and Mehl, J. W., Science, 114, 10 (1951). (3) Feigl, F., “Qualitative Analysis by Spot Testa,” New Yolk, Elsevier Publishing Co., 1946. (4) Horrocks, R. H., and Manning, G. B., Lancet, 1, 1042 (1949). (5) Partridge, S. M., Biochem. J., 42, 238 (1948). RECEIVED for review February 20, 1452.
Accepted M a y 26, 1952.
Microdetermination of Phosphorus GEORGE R. NAKAMURA, University of Caliifornia, Berkeley, Calif.
RAPID method has been developed for the estimation of
A phosphorus in biological materials and is used routinely in this laboratory. It is an adaptation of King’s colorimetric determination of phosphorus in a micro range of 1 to 10 micrograms of phosphorus (1).
To a measured amount of sample in a small borosilicate glass tube, 10 X 75 mm., which had been calibrated to a 1-ml. mark, was added 0.1 ml. of 60 to 70% perchloric acid. The tube was placed in a sand bath (as many as 20 tubes have been run simulTable I. Phosphorus Recovery from Phosphoric Esters by Micromethod P Present, Compound Disodium glycerophosphate. 5H20
Calcd.,
P P Found, Theoret.,
P Found,
Y
Y
%
5.07
5.10 5.05 5.05
10.13
10.20 10.10 10.10
%
Barium phosphoglyceric acid
4.83
4.80 4.80 4.78
9.65
9.60 9.60 9.56
Disodium phenyIphosphate2H,O
6.11
6.10 e. 10 6.05
12.21
12.20 12.20 12.10
taneously) a t about 190’ to 200” C.; excessive heating beyond this range caused undesirable splattering. The length of diges tion period depended on the nature of the sample; the phosphoric esters listed in Table I required a digestion time of only 2 minutes. If the sample contained a large amount of organic material, the content of the tube became brown and then discolored. If the discoloration did not occur within 15 minutes, a drop of 30% hydrogen peroxide was added and the tube was reheated for an additional 5 minutes. The cooled content was then diluted with about 0.5 ml. of water, and washed down the wall of the tube. To the digestion mixture, 0.1 ml. of 5% ammonium molybdate and 0.1 ml. of sulfonic acid (an aqueous solution containing 0.2% 1,2,4-aminonaphthol sulfonic acid, 12% sodium bisulfite, and 2.4y0 sodium sulfite) were added and brought to the 1-ml. mark with water. The resulting blue solution was transferred to a matched cell and compared with a standard in a Jr. Coleman spectrophotometer a t 700-mr wave length using an adapter for 10 X 75 mm. cuvets. Micropipets, such as obtained from &licrochemicalSpecialties, 1834 University Ave., Berkeley, Calif., were extremely useful in transferring 1to 100 PI. of samples. LITERATURE CITED
(1) King, E. J., Biochemical J., 26, 292 (1932). RECEIVED for review March 13, 1952.
Accepted M a y 26, 1952.
