December 15, 1943
'163
ANALYTICAL EDITION
acid. The calibration curves of glycolic acid (logarithmic) and of oxalic acid were both straight lines within the ranges used.
Determination of Glycolic Acid The solution to be analyzed should be 2 N with sulfuric acid and should contain not over 100micrograms of glycolic acid per ml. Iuto a 15-ml. conical g h - s t o pered centrifuge tube, 0.2 ml. of this solution is introduced wit\ a aerological pipet, and 2 ml. of 0.01 r cent solution of 2,74ihydroxpa hthalene in concentrategulfuric acid are added from a rmcroguret, the tube being kept in a cold-water bath during the addition of the reagent. The mixture is made homo eneous by rotatin the tube between the hands, and the tube is &en placed in a bo$ing water bath for 20 minutes, removed and allowed to cool to room tem rature. After cooling the sofution is diluted with 4 ml. of 2 hpesulfuric acid and shaken, lightly a t first, until the heat of reaction has subsided. The solution is made homogeneous by vigorous shaking and the color is then read. The method is very flexible; after the color has been formed, practically any dilution volume can be used if it is preferable to reduce the sensitivity of the method so that higher concentrations of glycolic acid can be determined. Even the volume of test solution can be changed within narrow limits.
Determination of Oxalic Acid This differs from the receding only in that to the original sample aa measured out 70.2 ml. of a 2 N sulfuric acid solution containing not more than 200 micrograms of oxalic acid) 5 mg. of powdered m y u m are added; after rotating between the alms of the ands, the tube is allowed to stand for an hour. %hen with the tube in cold water 2 ml. of the dihydroxynaphthalene solution are added, and aker being made homogeneous, the solution is heated on the boiling water bath for 20 mjnutes, removed, and allowed to cool to room temperature. It is then diluted with 4 ml. of 2 N sulfuric acid, mixed, and the color is measured. The calibration curve shows that Beer's law is obeyed from 0 to 200 micrograms of oxalic acid, and even with this range the method is sensitive a t low concentrations (0 to 20 micrograms).
Literature Cited Boraook, Henry, Davenport, H. W., Jeffreys, C. E. P., and Warner, R. C., J. BWZ. C h . ,117,237 (1937). Calkina, V. P., and Mattill, H. A., J . Am. C h m . ~ ' o c . ,in press. Eegriwe, E., 2. and. Chnn.,89, 12 (1932).
Micromethod for Estimation of Sulfonamides Semipermanent Color Standards S. W. LEE AND N. B. HANNAY Wallace Laboratories, Inc., New Brunswick, N. J.
A
S A supplement to the method for sulfonamide determination previously reported ( I ) , a simple procedure has been worked out, using color standards prepared from easily available, water-soluble dyes. It is felt that this modification will be of use especially in areas remote from laboratory facilities. Acceptable accuracy has been obtained to blood levele of 15 mg. per cent (for sulfathiazole). By averaging the reaults of several observers, it waa found that the error (for dathiazole) was *0.5 mg. per cent to levels of about 6 mg. per cent, and * 1mg. per cent a t higher levela to 15 mg. per cent. The advantages of the parent micromethod have been retained, though the procedure has been simplified. The number of manipulations and the required apparatus are minimal. The volume of blood needed (0.3 ml.) may be taken from the finger tip, if the hand of the atient has been vigorously washed to remove traces of the sulfa drug. Flabbottomed 10ml. vials (18 X 46 mm.), calibrated for the urpose of the method, are to be preferred to centrifuge or test tuges. The Standards of known sulfonsrmde concentration may be made from any of the sulfonamides, and the values for the others obtained by using molecular weight corrections. The values obtained for sulfathiazole may be multiplied by 0.68 to convert to values for sulfanilamide, while those for sulfapyridine are the same aa for sulfathiazole. PROCEDURE. The same reagents and rocedure are used aa in the parent method (I), but only the h o d and precipitating solution volumes need be measured with accuracy. The nitrite and dye solution (2 drops or 0.1 ml.) may be added from a dro per. Com arisons of the tubes from the determinations witB the stan&rds are made visually, always using vials of uniform sha e. I t was noted that the eye best measures the intensities of transmitted light if the absorbing substances he held 1 to 3 om. from the eye. One should look through the solution. COLORSTANDPOR COMPARISONS. Semipermanent fuchmn-methyl violet color standards were prepared as follows:
Acid fuchsin (0.100 ram) waa dissolved in 250 ml. of distilled water? and this waa difuted 1 to 8 with water, giving a solution containing O.ooOo5 gram in 1ml. Methyl violet (0.100gram) waa dissolved in 250 ml. of water, and 2 ml. of this solution were made up to 250 with water (0.0000032 gram in 1 ml.). These solutions were mixed and diluted aa shown in Table I to give color intensities corresponding to the indicated amounts of sulfathiazole.
TABLE I. PREPARATION OF COLOR STANDARDS Fuchsin
Standard
Solution
Meth 1 Violet SoLtion
Ma. 9i
MI.
M1.
M1.
2 3
0.9 0.9 0.9 0.9 0.9 0.9 0.9
9.1
36.0 25.3 19.0 14.7 11.2
4 5
6
7
8 9 10 11 12
0.9 0.9 0.9 0.9
9.1
9.1 9.1 9.1 9.1 9.1 9.1 9.1 9.1 9.1
Water
8.8 7.0 5.4 3.9 2.9 2.1
Several combinations of red and violet water-soluble dyes gave shadea almost identical to that of the azo color formed in the reaction. Colors were matched with the eye. The dye combination is, of course, bleached by sunlight, and those in use lasted for about a month. Standards should be kept in the dark when not in use. Stronger solutions of the dyes keep longer than the weak ones, and may be kept for subsequent diluting.
Literature Cited (1) Lee,S. W., Hannsy, N. B.. and Hand, W. C.. IND.ENG.CEEY.. AXNAL. ED.,15, 403 (1943).
