Stable Colorimetric Reagent for Chromium J. F. EGE, JR., AND LESLIE SILVERhl4N D e p a r t m e n t of Industrial H y g i e i e , Harvard School of Public Health, Boston, .Mass. HE s-diphenyl carbazide reagent has been used for many Tyears in the colorimetric microdetermination of hexavalent chromium. Several modifications have been made (1, 3-7) in an effort to improve the analytical procedure and obtain rapid color development, increased sensitivity, and stability. Rowland (6) recently reported such refinements, and a discussion of various methods is given by Sandell ( 7 ) . One disadvantage, however, is the fact that the reagent itself develops color fairly rapidly, and for this reason must' be prepared fresh. In the course of an investigation (8)for the rapid determination of chromic acid mists in air, a reagent with considerably more stability \vas developed, which makes possible a simpler analytical procedure, since a single mixed reagent is added t o the neutralized or aqueous solution of the unknown. Air-borne mists are collected by drawing them through a dry filter paper Tvhich has previously been impregnated with the reagent and a humectant, glycerol. .1stain develops which is proportional to the quantity of chromic acid mists in the air. The actual air-borne concentrations are determined by Comparison with a set of artificial color standards.
weeks the phthalic anhydride reagent vdue was 627,, that of the acetone reagent 43%, and the others ran less than 20%. The phthalic anhydride reagent could still be used after 5 months of standing a t room temperature in the laboratory. Storage i n I refrigerator in a brown bottle also increases it's stability and retards color development. SPECTRAL ABSORPTION CURVE AND SENSITIVITY
In Figure 2 it may be observed that the maximum light a h o r p tion for the chromium complex occurs at 540 mp. This is ideiitical with that obtained with other reagents. The variation of light absorption of the reagent alone, with wave length, is also shown in Figure 2. The reagent itself does not have an apprecinble light absorption a t 540 mp, and its absorption is much less than the usual acetic acid reagent'. The fact that the reagent is added in proportions of 1 part in 10 in making an analysis, further reduces the color of the h h n k Figure l indicates that diir-
PREPARATlON OF REAGENT
The reagents for other methods have been prepared in alcoholic or acetone solutions and added to the unknown after adjustment of acidity ITith sulfuric, hydrochloric, or acetic acid. I n the new reagent, phthalic anhydride is used in approximately the same concentration. The reagent is made by adding 4.0 grams of powdered phthalic anhydride to 0.25 gram of s-diphenyl carbazide in 100 ml. of 95% ethanol. For paper impregnation, a crystalline acid is preferable and phthalic anhydride was selected after testing many other feasible acids. RESULTS e w
Figure 1 presents a comparison of the stability of the various reagents. A 75% transmission was found at the end of one week for the phthalic anhydride reagent, compared with a distilled water blank and measured with a Model, 11 Coleman spectrophotometer at 540 mp. The reading for the acetone reagent was 65% and the other reagents m-ere below 45%. At the end of 6
a
D
ACETIC REAGENT
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PHTHALIC R E A G E N T
ACETIC R E A G E N T
I I
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HOURS AFTER P R E P A R A T I O N O F REAGENT
Figure 1.
Stability Curves, 540 m p
A . New phthalic anhydride reagent B . Acetic acid reagent similar to that described in (1, 3-5) C. Acetone reagent described by Sandell (7) D . 0.2570 s-diphenyl carbazide in methyl alcohol (dilution of F ) E . Saturated s-diphenyl carbazide in 9570 ethyl alcohol as recommended by Rowland (6) F. 1% s-diphenyl carbazide similar to that used as spot test reagent for chromium (2) Wave length, 540 m p
MICROGRAMS OF C r O l PER I i ML.
Figure 3. Transmission Curies, for New Phthalic i Anhydride Reagent Compared to i c e t i c i c i d Reagent Note that sensitivity is identical with the two reagents. length, 540 rnw
693
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694
V O L U M E 19, NO. 9
ing the first 7 hours, distilled water can be employed for a reference blank. Figure 3 shows no difference in the sensitivity of the reagent as compared to the acetic acid reagent, which is widely used for chromium determination. Maximum color develops within 5 minutes after addition of the reagent. The prepared reagent is added in the proportion of 1 ml. to each 10 ml. of the unknown in neutral or slightly acid solution. The pH is not critical. DEVELOPMENT OF
REAGENT
In a series of experiments which led to the selection of the final reagent it was found that the concentration of phthalic anhydride was the most critical factor in color development. Decreasing the phthalic anhydride concentration from 10 to 0.2 gram per 100 ml. of solution progressively retarded color development in the reagent. Four grams of phthalic acid were used since, with concentrations below 4 grams per 100 ml., there was an increasing lag in the formation of the chromium complex. The concentration of s-diphenyl carbazide was not so critical; however, amounts of s-diphenyl carbazide exceeding 0.25% produce a faint color development in the reagent. The final reagent produced a chromium complex which was stable for several hours. Calculated on the basis of molarity of chromium, the ex-tinction coefficient of the complex is 3.14 X lo4. This value agrees well with that reported by Sandell (7').
AND
EVELYN K. RhTLIFF, The I n s t i t u t e of Paper Chemistry, Appleton, Vis.
N 1945, Breddy and Jones (1) showed that xylose could be identified and determined proximately in the presence of other sugars as the insoluble crystalline dibenzylidene dimethyl acetal by the action of dry methanolic hydrogen chloride and benzaldehyde on xylose, over a period of 7 days. Whereas their qualitative findings were fully confirmed, it x a s at first found impossible t o substantiate their quantitative data. This led to correspondence nith Jones (S), who stated that two points had been omitted from their original communication: ( a ) during the last 18 hours of the 7-day period the reaction mixture was kept in an ice chest and (b) their equation for computing xylose from the weight of the diacetsl derivative had been changed from the original 21 = 0.482s 0.055 to y = 0.526s 0.046 (where = the weight of xylose and z = the weight of derivative). Even a-hen these modifications were applied to the estimation of xylose, the results fluctuated over a wide range, and could not be considered as proximately quantitative. After a series of experiments, it was shown that, with the following additional precautions, reasonably satisfactory determinations of xylose could be obtained. The reagent (2.5 K hydrogen chloride in anhydrous methanol) containing purified benzaldehyde must be used shortly after its preparation. The reagent deteriorates very rapidly a t laboratorv temperatures and the titer falls quickly, owing to the chemica1"interaction of methanol with hydrogen chloride. The water formed in this reaction disturbs the equilibrium and prevents the complete separation of the dimethyl acetal.
+
+
Table I.
D-Xylose Determined as Its Dibenzylidene Dimethyl Acetal Derivative
Xylose Taken MQ.
a
Xylose Founda
MQ.
Recovery
%
81.6 163.1 87.9 176.7 200 100.9 504.4 500 91.2 456.9 600 99.6 696.9 700 99.3 7nn 695.0 . _. Values calculated from the more recent Breddy-Jones equation. 200
LITERATURE CITED
(1) Cazeneuve, A., Bull. SOC. chim., (3) 23, 701 (1900); 25, 781 (1901). (2) Feigl, F.,"Laboratory Manual of Spot Tests," New York, Academic Press, 1943. (3) Hodgman, C.D.,and Holmes, H. N., "Handbook of Chemistry and Physics," 25th ed., p. 1263,Cleveland, Ohio, Chemical Rubber Publishing Go., 1941. (4) Loginov, M. E., Leningrad.Znst. Gig.Truda i Tekh. Bezopasnoati, T ~ u d iy MatWialy Byull., NOS.7-8, 42 (1931). ( 5 ) Moulin, A., Bull. SOC. chim., 31,295 (1904). (6) Rowland, GI. P., Jr., IND.EXQ. CHEM.,ANAL. ED.. 11, 442 (1939). (7) Sandell, E. B., "Colorimetric Determination of Traces of Metals,'' New York, Interscience Publishers, 1944. (8) Silverman, L., and Ege, J. F., Jr., J. I d . Hug. Tosicol. (March 1947).
Identification and Determination of o-Xylose
I
LOUIS E. WISE
SUMMARY
In a modification of the s-diphenyl carbazide reagent for the colorimetric determination of hexavalent chromium, the major change is the substitution of phthalic anhydride for the acidic constituent. The resulting reagent is stable for weeks. It compares very well with other analytical reagents, and results in a simplified analytical procedure. The reagent is well adapted, upon the addition of glycerol, for impregnating filter papers, which are used in a direct field method for the estimation of concentrations of chromic acid mists in air.
The reaction must be carried out at 20" C. for 6 days and finally a t 4' C. for 18 to 24 hours. Provided no crystallization has occurred within the first 24 hours, the mixture must be seeded with the derivative. After filtration, the xylose derivative should be washed with 200 cc. of ice water, follon-ed by washing with methanol saturated with the derivative. The washed precipitate should be dried no more than 1 hour a t 100" to 105' (despite the fact that this drying period may not ensure constant weight). When these precautions avere observed, the results given in Table I were obtained. The importance of seeding the xylose-methanolic hydrogen chloride-benzaldehyde mixture within 48 hours (provided crystallization has not occurred spontaneously) is illustrated in the following experiment.
A 500-mg. sample of xylose was treated with 10 cc. of the Breddy-Jones reagent a t 20" C. and seeded within 48 hours. In this case, the xylose found after 7 days was 97.2% of the theoretical (based on the Breddy-Jones equation). A similar mixture, not seeded, remained liquid for 6 days. Crystallization was then induced by scratching the sides of the flask. In this case only 23% of the original xylose was recovered. Breddy and Jones have shown that the following carbohydrates form no crystalline deposit with the xylose reagent: glucose, mannose, galactose, fructose, sorbose, maltose, sucrose, methyl a-mannoside, methyl a-glucoside, rhamnose, and arabinose. To this list may be added D-glucurcme and D-galacturonic acid. On the other hand, Irxylose also gives a crystalline derivative. The method thus furnishes an excellent, highly specific qualitative test for xylose, which has been used in identifying the sugar separated from the hydrolysis products of the hemicelluloses of overcup oak and slash pine. Breddy and Jones recrystallized their dimethyl acetal of dibenzylidene xylose from chloroform-ligroin, reporting a melting 9" (chloroform), It was found more point of 211" and satisfactory to recrystallize the product from anhydrous methanol, thus obtaining silky needles, melting a t 211-12" (corrected), - 6.65" (chloroform).
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