Table
I.
Reagent Isoquinaline
Is0 uinoline plus
Color of Derivatives Separating from lsoquinoline Reagents
Au
Yellow
Pt Yellow
Pd
...
Ru Ir Rh Amher Amher . . .
Os
... ... ...
Reddish orange Yellow Red-brown ... If room temperature is helow approximately 22’ C. clusters of short, stocky prisms of an iridium derivative may separate. Their color is a deep reddish amher. 3XS 0
1500-vielded tvnical crvstals in about “_ 2 minutes. Solutions of osmic acid and rhodium trichloride yielded no crystals with isoquinoline, regardless of the presence or absence of thiocyanate ion. The results of all the tests are summarized in Table I. Not only did osmium fail to yield any crystalline product when treated with the isoquinoline reagents, but it did not interfere in tests for other metals here considered. Rhodium, on the other hand, interfered in a few instances. In testing for platinum with the mixed reagent, the presrnce of 1 part of rhodium per 1000 decreased the yield of crystals produced. A similar inter-
ferencc mas observed in the test for palladium. Hovever, thc same concentration of rhodium did not seem to interfere with the tests for any other metals concerned in this paper. The composition and structure of most of the compounds produced in these tests remain to be investigated. There is a possibility, for example, that the isoquinoline reagent may form isoquinoline trirhlorogdd,
are prepared. With the thiocyanateisoquinoline reagent certain divalent ions, as copper and zinc, yield precipitates (4) with the composition .\I(CgH7N)2(CNS)2.It is not yet known XThether the palladium reaction product has a comparable formula. Because observations on micro quantities of some of the derivatives here described indicate that adhering moisture can he readily rpmoved, they may prove useful in gravimetric procedures. LITERATURE CITED
e.
M., Mason, C. W., “Handbook of Chemical Microscopy,” Vol. 2,pp. 67-9, Wiley, Xew York, 1040. ( 2 ) Schaeffer, H. F., ANAL. CHEM.23, 1674 (1051). (3) Schaeffer, H. F., Pioe. Indiana Acad. (1) Chamot,
Scz. 60, 162 (1950).
(4)Spakownki, A. E., Freiser, H., ANAL. C~~~.21,086(1949).
but this point remains to be checked when larger quantities of the products
A MicrocrystaIIine Test for Osmium HAROLD F. SCHAEFFER College of Emporio, Emporio, Kon.
b A test has been devised for the convenient detection of osmium when present as a dilute solution of the tetroxide. The lotter causes the seporation of characteristic crystals when treoted with an acid solution of lA3.4tetrohydro-6-methoxyquinoline.
A.
the difficulties encountered m the detection of osmium, especially in dilute solutions of its compounds, Chamot and Mason (1) have cited the highly volatile nature of its oxides and chlorides. A compound which has been found convenient for the detection of osmium present as the tetroxide is 1,2,3,4tetrahydro-6-methoxyquinoline(Eastman Kodak Co.), H H MONC
H The reagent consists of a 3% solution of the organic base in 2N hydrochloric acid. Although this solution deterie rates in storage, it yields satisfactory results for at least 1 month.
In performing a test, in the absence of interfering ions (gold and platinum), 1112
ANALYTICAL CHEMISTRY
Figure 1 . Representative crystals in tests for osmium
one drop of test solution is added to a drop of the reagent on an object slide. After the preparation assumes a dark green color, it is examined under a magnification of 50 to 100 diameters. A solution containing the equivalent of 0.2 of 1% of osmic acid will yield characteristic crystals (Figure 1). At this concentration one drop contains approximately 7 5 y of the metal. The presence of copper or iridium does not seem to inhibit crystal formation. Should the sample be too dilute to yield a positive test within 5 to 10 minutes, or if interfering ions are present, one can take advantage of the highly volatile nature of the osmic acid. In this case an object slide (more conveniently a “half-slide,” measuring 1.5 X 1 inch)
bearing a hanging drop of reagent solution is placed over a microcrucible or microbeaker containing one or two drops of test solution. TTTith a sample containing 20 y of osmium sufficient fumes reach the reagent to yield a positive test within 5 to 10 minutes a t room temperature. The reaction may be hastened by gently warming the crucible or beaker. If the element present is potassium osmate, for example, a positive test is obtained by dissolving the sample in normal hydrochloric acid, and then applying the reagent solution as described. The crystals exhibit a deep red-green color, and may be opaque unless they are small. The preparation is di-
chroic; if the crystals are sufficiently small (and thin), the colors are green and light tan. The composition of the compound has not yet been determined, but observation of slide preparations indicates that the dried crystals are stable under ordinary room conditions. LITERATURE CITED
(1) Chamot, E. M., RIason, C. W.,
“Handbook of Chemical Xcroscopy,” Vol. 11, 2nd ed., pp. 297-8, Wiley, New York, 1940. RECEIVEDfor review June 19, 1958. Accepted January 5 , 1959. Division of Chemistry, 19th Annual Meeting, Kansas Academy of Science, Ottawa, Kan., May 1958. Work made possible in part by a Cottrell grant from the Research Corp.
Ultramicromethod for Assay of Pteroylglutamic Acid (Folic Acid) Activity WILLIAM N. PEARSON, EILEEN R. BRODOVSKY, E. RAYMOND CARNES, and WILLIAM J. DARBY Division of Nutrition, Departmenfs o f Biochemistry and Medicine, Vanderbilf University School of Medicine, Nashville 5, Tenn.
b A modification of the usual microbiological assay for pteroylglutamic acid effects a 100-fold increase in sensitivity. It is possible to measure titrimetrically 0.01 to 0.10 m y of folic acid in a total volume of 0.20 ml., with S. fecalis as the test organism.
I
of studies of pteroylglutamic acid (folic acid, PGA) metabolism, the need arose for a method of assaying this vitamin in extremely small quantities of sample and in samples of extremely low potency. For example, the study of blood folic acid levels in the rat required a method utilizing only 5 to 10 pl. of serum, especially if serial determinations were to be made and the animal could not be sacrificed. Accordingly, the usual macromethod for the assay of folic acid, utilizing S. fecalis as the test organism, has been modified to permit a submicro level of analysis. The adaptation of the riboflavin assay (an L. casei assay) to a submicro level has been reported by Lowry and Bessey ( S ) , and the general method followed is similar to theirs. The procedure described below permits the quantitative assay of as little as 0.01 my of folic acid activity, l/loo of that which can be measured by the conventional macromethod. N THE COURSE
APPARATUS AND REAGENTS
Gilmont ultramicroburet, capacity 1.O ml. (Emil Greiner Co., Sew York). Test tube racks t o hold 6 X 50 mm. tubes (Sorthern Tool and Instrument Co., 50-23 192nd St., Flushing, S . Y.).
The nutrient broth used to culture the organism consisted of 2.0y0 yeast extract, 0.5% proteose peptone, 1.0% dextrose, and 0.2% potassium dihydrogen phosphate. The nutrient agar on which the culture was maintained with monthly transfer contained all the ingredients listed for the nutrient broth and 1.0% agar in addition. Both nutrient broth and agar mere sterilized by autoclaving a t 121” C. for 15 minutes. Charcoal-treated, vitamin-free casein hydrolyzate was prepared by adjusting the pH of 100 ml. of acid-hydrolyzed casein (loyocasein) (Nutritional Biochemicals Corp.) to pH 3.5 Rith sodium hydroxide. To this was added 2.0 grams of activated charcoal. The solution was shaken on a reciprocating shaker for 1 hour, filtered, and made to a volume of 200 ml. Solutions of purines, amino acids, vitamins, and salts were made as for ordinary microbiological assays. The basal medium used is given in Table I. It is similar to that used in the folic acid macromethod described by the Association of Official A.gricultura1 Chemists (1) except that charcoaltreated casein hydrolyzate was used, methionine was added ( d ) ,and potassium salts were substituted for the sodium salts employed in the macroassay. Folic acid standard solution was prepared by adding 50 ml. of 0.01N sodium hydroxide in 20% ethyl alcohol to 20.0 mg. of pteroylglutamic acid. As soon as the pteroylglutamic acid was dissolved, the pH was readjusted to 7 to 8 with hydrochloric acid, and the solution was made t o a final volume of 1000 ml. n-ith distilled water. This solution was then diluted to give working standard solutions containing 0.01, 0.02, 0.04, 0.06, and 0.10 my of pteroylglutamic acid per 100 pl, The solutions were
stored by freezing them in small portions, so that fresh aliquots could be thawed each time an assay was set up and the excess discarded. Desiccated chicken pancreas (Difco Laboratories Inc., Detroit 1, Mich.) was used as a source of folic acid conjugase. PROCEDURE
The assay was carried out in 6 X 50 nim. borosilicate glass test tubes in a total volume of 0.20 ml. The standard curve was set up by measuring in triplicate 100-PI. amounts of each of the five standards plus a distilled water blank with a Levy-Lang constriction pipet. The same pipet m-as used for all additions, but was thoroughly rinsed before a new standard was added. Each group of three tubes was covered with an aluminum cap and the rack of tubes was autoclaved for 10 minutes a t 121” C. along with a test tube containing 10 ml. of the double-strength medium. Addition of the basal medium and inoculation of the tubes were accomplished in a single step. The tube of sterile, doublestrength medium was inoculated with a single drop of a barely turbid, well washed suspension of a 24-hour culture of S.fecalis 29-21. (This wild strain gives excellent growth and has been used for routine macroassays in this laboratory for more than 4 years, but there is no reason to suspect that the usual organism specified for this assay, S. fecalis ATCC 8043, could not be used.) One-tenth milliliter of this inoculated medium was pipetted into each tube with a sterile, 1-ml. graduated pipet, the tip of which was bent and slightly constricted to permit better control of its contents. Usually one pipet was used for 18 to 20 tubes. Samples t o be assayed were prepared as recommended VOL. 31, NO. 6, JUNE 1959
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