zones, and compounds which do not respond to the treatment can be regenerated by adding stronger mineral acid and a solubilizing agent such as nitrobenzene. APPLICATIONS
The fact that carbonyl compounds ranging in properties from those of simple aliphatic to highly conjugated aromatic (cinnamaldehyde) carbonyls were easily regenerated with levulinic acid-hydrochloric acid suggests that the method should have wide general application. The regeneration of 2,3butanedione from its bishydraxone suggests that the method may be extended t o conjugated dicarbonyl derivatives by tlie addition of various solvents. The ZJ4-dinitrophenylhydrazones of the carbonyl compounds formed in oxidized fat, such as saturated aldehydes, 2enals, and 2,4-dienals have been successfully regenerated. Spots of these fat derivatives, cut from paper chromatograms, yielded detectable odors when regenerated. The method has been useful in study of unknown carbonyl flavor compounds in dairy products. I n a study of the off-flavor compounds formed during the storage of dry milk, 2,4-dinitrophenylhydrazones were prepared from vacuum
steam distillates of the reconstituted milk. The hydrazones were separated by chromatographic procedures and then regenerated for organoleptic evaluation. The observations demonstrated the importance of carbonyls as contributors t o the characteristic flavor of dry milk and indicated which of the chromatographic fractions contained the most potent flavor compounds. Regeneration of the major flavor fraction and addition of the steam distilled carbonyl to fresh milk permitted a rough estimation of the flavor threshold of the fraction in milk. The estimate indicated that the reconstituted dry milk contained between 20 and 50 p.p.b. of the potent flavor carbonyl. Information on the flsvor potency is a valuable aid for identification purposes and of great value in indicating how much product must be worked up in order to obtain a desired amount of derivative. I n the case of the dry milk, it was estimated that approximately 300 pounds of powder mould have to be processed in order t o isolate l b t o 15 mg. of 2,4-dinitrophenylhydrazone of the major flavor fraction. LITERATURE CITED
(1) A4nchel, M., Schoenheimer, R., J . Biol. Chem. 114, 539 (1936).
Braddock, L. I., Garlow, K. Y . ,Grim, L. I., Kirkpatrick, A. F., Pease, S. )V., Pollard, A. J., Price, E. F., Reissmann, T. L., Rose, H. .2., Willard, M. L., ANAL. CHEY.25, 301 (1953).
Braude, F. .4., Jones, E. R. H , J . Chem. SOC.1945, 498.
Demaecker. J., Martin, R. H , Nature 173, 266 (1954).
Djerassi, C., J . A m . Chem. SOC.71, 1003 (1949).
Forss, D. -1.,Dunstone, E. .4 , Stark, IT., Chern. & Ind. 1954, 1292.
Heulin, F. E., Australzan J . Sci Research 5B, 328 (1052).
Jones, L. A , , Holmes, J. C., Seliyman. R. B., ASAL. CHERZ.28, 191 (1956).
Mattox, V. R., Kendall, E. C.,
.I. Am. Chern. SOC.70. 8S2 (1948). Ibid., 72, 2290 (1950). ' Meister, A , , Abendschein, P. .I., h A L . CHERf.
28, 171 (1956).
Neuberg, C., Strauss, E., Arch. Bzochem. 7, 211 (1945).
Roberts, J. D., Green, C., J . Am.
Chem. SOC.68.214 (1946 1. (14) Robinson, R., A-utwe 173,541 (1954). (15) Strain, H. H., J . -4m. Chem. SOC. 57,758 (1935). (16) White, J. IT.,ASAL. CHEM. 20, 726 (1948).
RECEIVEDfor review June 29, 1956. Accepted June 6, 1957. Scientific Article A-567. Contribution 2725 of the Masyland Agricultural Experiment Station, Dairy Department.
Interferences with Biuret Methods for Serum Proteins Use of Benedict's Qualitative Glucose Reagent as a Biuret Reagent RICHARD
J. HENRY, CHARLES SOBEL, and SAM BERKMAN
Bio-Science laboratories, 10s Angeles 64, Calif.
b The biuret determination of serum proteins was studied employing Benedict's qualitative glucose copper reagent. The ratio of nitrogen to color produced was the same for human albumin and human y-globulin. The biuret method also agreed within experimental error with the Kjeldahl analysis in a series of sera in which the paper electrophoretic patterns varied widely. Various proposed corrections for turbidity were studied, with the conclusion that the most universally successful correction was ether extraction. Bilirubin did not interfere as long as turbidity was not present. Hemolysis interfered, but when the hemoglobin concentration was independently determined, a correction could b e made. interferences produced b y ammonium ion and high salt concentrations varied with the particular biuret technique employed.
K
(IO) simplified the biuret procedure for determination of serum proteins by using a reagent relatively low in copper and high in alkalinity, so that the reaction could be carried out directly on serum without precipitation of cupric hydroxide. The high alkalinity kept the excess copper in solution. It has been claimed that this reagent is unstable because of its high alkalinity and several modifications haye been proposed, all aiming a t stabilization of the reagent a t low concentrations of alkali without the formation of insoluble cupric hydroxide. Thus, Nehl (16) used ethylene glycol as a complexing agent, and Weichselbaum (23) used sodium potassium tartrate for complexing and potassium iodide to prevent autoreduction. Gornall ( 5 ) retained the tartrate but believed it safe to omit the potassium iodide. Goa (3) has proposed, and the present authors INGSLEY
have also used for approximately 15 years, Benedict's qualitative glucme reagent as a biuret reagent. The citrate present complexes the excess copper in the reaction and the reagent is stable a t room temperature because of its low alkalinity. There are a t least four possible sources of interference with the biuret determination : 1. Turbidity. Four solutions to this problem have been proposed-namely, extraction with ether ( 8 ) , subtraction of a serum blank (18), preliminary precipitation of protein by trichloroacetic acid (ZO), and subtraction of residual absorbance after the biuret color has been dispelled by cyanide (7'). 2. Presence of Salts. Salts used in protein fractionation, such as sodium sulfate and sodium sulfite, have been reported to increase the color intensity (22). Ammonium ion has been observed to affect results but the error is VOL. 29,
NO. 10, OCTOBER 1957
1491
said not to be serious if the ammonia nitrogen does not exceed the protein nitrogen-Le., about 0.8;11 in the original sample (16 ) . 3. Bilirubin. It has been stated (.5, 10) that total bilirubin levels up to 2,; mg. per 100 ml. do not interfere. 4. Hemolysis. This paper presents a study of the use of Benedict’s qualitative glucose reagent in the biuret determination of serum proteins and the means of overcoming the above interferences. I n addition, the behavior of other biuret reagents toward these interferences is reported. METHOD
Reagents. BIURET REAGEKT (BEKEDICT’SQUALITATIVE GLUCOSE REAGENT). Dissolve 17.3 grams of cupric sulfate pentahydrate in about I00 ml. of hot water. Dissolve 17.3 grams of sodium citrate and 100 grams of anhydrous sodium carbonate in about 800 ml. of water n ith heating. When cool, pour the second solution into the first while stirring, and dilute to 1 liter a t room temperature. This reagent is stable at room temperature. The white precipitate which usually forms does not appear to alter the characteristics of the reagent in the biuret reaction. Sodium hydroxide, 0.75 S. Sodium hydroxide, 1.505. Protein Standard. Determine the protein concentration of a clear serum by Kjeldahl analysis, correcting for the nonprotein nitrogen and using the factor 6.25. Dilute t h e remainder of the serum 1 t o 25 with physiological saline (0.85 or 0.90% sodium chloride). It can also be diluted so t h a t it is the equivalent of a 1 t o 25 dilution of a 7.0(r, standard-for example, if the scrurn contains 8.0% protein, dilute 088 to 25. Saturate with benzoic acid. (The use of chloroform as a preservative is not so satisfactory.) This standard is stable in the refrigerator for several months. Several stable protein standards are also available commercially. Procedure. Set u p the following in separate tubes: REAGENT BLANK, 5 ml. of 0.75N sodium hydroxide. STANDARD, 2.5 ml. of protein standard plus 2.5 ml. of 1.5-V sodium hydroxide. UNKNOWN.Rinse 0.1 ml. of the serum sample from a Folin 0.1-ml. T C pipet into 4.9 ml. of 0.75N sodium hydroxide (alternatively, dilute 1 ml. of serum or plasma to 25 ml. with physiological saline and mix 2.5 ml. of this dilution with 2.5 ml. of 1.5N sodium hydroxide). Proceed with the next step without delay. Add 1 ml. of biuret reagent to each tube and mix. ]Trait a t least 15 minutes and examine for turbidity by looking for Tyndall effect in front of a bright spot lamp. If negative or only a trace, read standard and unknown against the reagent blank a t 545 mp or with a filter with a nominal wave length in this region (except where 1492
ANALYTICAL CHEMISTRY
otherwise noted all readings in this study were made with a Beckman Model D U spectrophotometer). If turbid, proceed as outlined below in the study on interferences. Calculation:
A. x
% protein in standard
=
n-idely differing protein patterns by comparing the protein determined by Kjeldahl nitrogen and protein determined by the biuret method standardized with human albumin. Table I shows the results on 12 sera, together with the electrophoretic distribution of component proteins determined by paper electrophoresis ( 6 ) . The precisions of the biuret and Kjeldah1 determinations were 1 0 . 2 gram per 100 ml. (95% limits). ,4ccording to the t test, therefore, the t n o methods must disagree by more than 0.3 gram per 100 ml. before the difference can be considered significant. No difference in Table I exceeds this figure. Interferences. TURBIDITY AND BILIRUBIN. Table I1 lists the results obtained with clear, turbid, and icteric sera, both with the technique eniploying Benedict’s qualitative glucose reagent and with Reinhold’s technique (18) employing Weichselbauni’s reagent ($2). Four procedures were used in measuring the absorbances:
% protein
A1in unknown EXPERIMENTAL
Absorption Curve and Beer’s Law. Peak absorbance, obtained by reading a standard or unknown against a blank, occurred a t about 545 mp, which is in agreement with that obtained with other biuret reagents (16, 20). The a t 545 mp and 24’ C., read against a reagent blank, was 3.27. The color was found to obey Beer’s law up to a protein concentration of 15y0, when a Beckman Model DU, a Bausch & Lomb Spectronic 20, and a Klett-Summerson filter photometer with a KO. 54 filter were used, having halfband widths of 1.3, 20, and 45 mp, respectkely. Development and Stability of Color. The color was found t o reach a maximum in 15 minutes and remained stable for varying periods of time. Between 30 minutes and several hours after addition of reagents, a turbidity or flocculent precipitate developed occasionally. There is evidence that this is due to a phospholipide which is protein-bound in the native serum or plasma (3,14). Biuret Equivalents of Various Protein Fractions. Armour’s bovine albumin (13-531, No. 53, Lab. 235), human albumin (lot 940 C2B, Hyland Laboratories, Los Angeles), and human 7-globulin (Lederle’s polio immune globulin, lot 2175-249B) checked within 2% with respect to the ratio of nitrogen (Kjeldahl-Gunning) to biuret color produced. As isolated CYI-, CYZ-, and @-globulins were not available, these were checked indirectly on sera with
Table I.
Serum
1. Direct. The unknown, whether clear or not, was read against a reagent blank. 2. Direct, Corrected by Serum Blank. A serum blank was set up employing a biuret reagent devoid of the copper sulfate. This was read against water and the reading subtracted from the direct reading. 3. Potassium Cyanide. Approximately 100 nig. of potassium cyanide was added to the unknown from Procedure 1 after reading and then read again against water after waiting about 2 minutes. This reading was then subtracted from the direct reading. 4. Ether Extraction. *4pproximately 3 ml. of ethyl ether was added to a duplicate of the unknown from Procedure 1, the tube was shaken vigorously for 30 to 60 seconds and centrifuged for 5 minutes in an Adams angle-head centrifuge, and the biuret color was read against reagent blank. The solubility of ether in the aqueous phase resulted in about a 4% increase
Comparison of Biuret and Kjeldahl Results in Sera of Widely Varying Electrophoretic Distributions J‘% of Total Protein Total Protein, Globulins G. per 100 MI.
KO,
h/G
Albumin
011
a2
la
0 19 0 62 0 25
15 9 38 3 20 2
3 6 2 1 17
10 2
5 2
37
177
3 9
117 17 9 173 2 i 9 9 6 4
6 4 710 150 124 11 2
2a
3b 4 5 6 7 8
9 10
055 173 1 03 088 029
228 4 1
355 634 50 9
470
10 2 6 7
695
3 6
_2_2 9
803
1 6
2 2
5 3 5 1
3
128 4 9 8 4 101 162
Y
651
479 20 211 8 6 9 8 162 679 8 6 1 0 121
Kjeldahl 9 8
102 119 6 8 6 2 5.3 6 3
70
6 1 3 7 2 6
Biuret 9 5 101 122 7 0 6 3 5 4
6 5
T O 6 0 3 7
2 8 555 5 4 012 108 85 8 5 123 340 6 3 430 4 4 075 a.Sera from cases of multiple myeloma with typical homogeneous protein fractions in region. * Serum from case of multiple myeloma with homogeneous fraction in 9, region. 11 12
y
Table II.
Serum
Type of Serum No. Clear, nonicteric 1
Turbid, nonicteric
Icteric
a
Turbid
2 3 4 5 6 7
8
9 10 11 12 13 14 15 16 17 18
Comparison of Correction Techniques for Biuret Reaction Total Protein by Biuret, 7' Benedict Weichselbaum Total Direct, Direct, Protein corcorrected Ether rected by by serum extracby serum Bjeldahl, . % Direct blank KCN tion Direct blank KCN 6 5 6 7 7. ..9
7.1 6.9 6.5 7.5 7.1 5.5 4.7 7.4 7.3 7.8 8 6 6.9 6.8 7.5 7.6
1.9 4.9 16.0 4.8 0 '75 0.91 3 4 9 13 11
28
Table 111.
6 5 6 8 7 9 7.2 7.0 6.5 10.4a 8.45 3O.Ei" 9.0a
8.P 8.7" 8.0 8.7 6.9 6.9 7.6 7.8
6 4 6 8 7. . 9-
7.1 7.0 6.5 8.9 7.4 4.8 5.8 8.0 8.0 7.9
8.7
6.7 6.8 7.4 7.4
NaCl, 0.85y0 0.184 Xa&Oa, 26% 0.192 NazS04, 23% 0.191
(NHdLSO, 0 Lii 0 2111 0 431 0 6M 0.8M
0 197 0 212 0 222 0 231 0,361" Intense hlue. ~
a
6 6 7 0 7 9 7.3 7.1 6.5 7.7 7.9~ 5.5 4.4 7.4 7.2 8.4a 8.9 7.6.
7.3a 7.9a 8.0"
6 6 6 8 8 0 6.9 6.8 6.4 9.0= 8.6~ 47.0a 10.7a 9.7~ 9.20 7.9 8.7 6.8 6.9 7.4 7.6
Standard - Standard own reagent KaCl reagent blank blank
6.3 6.5 7.7 6.6 6.5 6.2 6.4 7.1 -7.5 6.3 6.5 6.5 7.5 8.4 6.4 6.4 6.9 6.5
Weichselhaum Absorbance against Water Standard Reagent o m reagent blank Standard blank
0.528 0.537 0.526
0.344 0.345 0 335
0:353 0.342
0.060 0.066 0.063
0.293 0.313 0.294
0 530 0 522 0 508 0 408 0,363"
0 333 0 310 0 286 0 177 0.002
0 0 0 0
0.074 0.114 0 .251a 0 . 28Qa 0.310'
0.289 0.292 0 . 250a 0,290= 0 . 310a
in volume and reagent blanks shaken with ether showed a decrease in absorbance, about 4% for Benedict's reagent and about 2% for Weichselbaum's reagent. I n spite of this dilution effect,albumin and ?-globulin standards shaken with ether showed an increase in absorbance of 1 to 2'%, Clear sera showed an increase varying from 0 to 4% with an average of about 2%. Accordingly, the biuret color of unknowns after ether extraction was read against an unextracted reagent blank and a correction of -2% applied. I n some instances of very lipemic sera, a second ether extraction was required for clearing. Two other possible techniques of eliminating interference from lipemia or other possible causes of turbidity were tried-namely, preliminary precipitation of the proteins with trichloroacetic acid and n i t h organic solvents such as acetone, acetone-alcohol, and alcoholether, followed by solution of the protein precipitate in 0.75;V sodium hydroxide. Precipitation with trichloroacetic acid failed to yield clear solutions. Precipitation by organic solvents in most cases gave solutions free of turbidity. This procedure appeared t o
6 5 6 7 7 9 6.8 6.8 6.3 7.2 7.3 2.6 6.3 6.9 6.4 7.8 8.6 6.5 6.6 7.3 7.1
6.6 6.8 8.0 7.0 6.9 6.4 8.1" 7.70 5.5 4.7 7.4 7.1 8.oa 8.6 7.6O 7.1a 7.7a 7.7*
Effect of Salts on Biuret Reaction
Benedict
Absorbance against Rater Reagent blank Standard
6 2 6 5 7 7 6.9 6.9 6.3 7.1 7.3 4.4 5.5 6.3 6.3 7.7 8.4 6.5 6.5 7.2 7.0
Ether extraction
346 338 324 224
have no advantage over simple extraction of the biuret solution with ether, When the Kjeldahl analysis is used as reference, the results in Table I1 reTeal several points of interest. Correction for turbidity by subtraction of a serum blank (direct, corrected by serum blank) did not yield satisfactory results. Results corrected by a cyanide blank showed a distinct tendency to run low. This is attributed to a residual yellow color usually remaining after treatment with cyanide and the development of turbidity which increases with time. Results with nonicteric sera after ether extraction were satisfactory if complete clarity was obtained. If this was not accomplished (sera 7 and 8), the results mere still high. I n the case of icteric sera, saturation with ether frequently produced turbidity, giving spuriously high results. I n many instances the turbidity appearing with icteric sera could be removed by prolonged centrifugation (30 minutes), a white scum appearing a t the aqueous-ether interface. That the copper itself was in some way connected with this phenom-
Standard XaC1 reagent blank
0.230 0.247 0.231 0.215 0.178 -0.001 0.001 0
0 253 0.234
0.229 0.232 , .
..
enon was indicated by the turbidity's failing to develop when the serum blank was saturated with ether. Icteric sera 13, 15, 16, 17, and 18 were also studied with Kingsley's (10) and Gornall's ( 5 ) biuret reagents. Following ether extraction no turbidity developed with the former reagent and only slight turbidity developed with the latter. The presence of bilirubin up to a concentration of 29 mg. per 100 ml. did not appear to interfere significantly with the direct determination. This also held true when the biuret color was read with a wide band filter. Values for direct, corrected by blank in general a p peared to be slightly low when the bilirubin was elevated. Bilirubin in a concentration of 25 mg. per 100 ml. in a serum containing 7% protein had an ahsorbance a t 545 mp of about 4y0of that of the protein-biuret complex when essentially monochromatic light was used. When a wide band filter was employed, the bilirubin had an absorbance of 5 to 10% of that of the complex. This results from the fact that the absorption curve for bilirubin in serum begins to rise abruptly at about 530 rnp. VOL. 29, NO. 10, OCTOBER 1957
1493
Table IV.
Hemoglobin, % 0 *
0.25 0.5 1.0 2.0 4.0
Correction
Biuret Absorbance 0.470 0.502 0.520 0.600 0,725 0.960
It was observed that upon the addition of the biuret reagent in such an instance the color had a distinct greenish hue which disappeared in a few minutes. Furthermore, when cyanide was added to dispel the blue color, the yellow color of bilirubin was not found, whereas the addition of cyanide to the serum blank had no effect on the bilirubin color. It appears, therefore, that in the presence of the copper the bilirubin was destroyed before readings were made. The results obtained lead to the following suggested course of action in performing the biuret determination of total serum proteins. Examine each unknown for turbidity (Tyndall effect). If no more than a barely perceptible Tyndall effect is observed, proceed without any treatment. If appreciable turbidity is present, extract with ether. If the turbidity persists, try re-extraction with fresh ether. If it is obvious that ether extraction will not work, start over, read, add cyanide, read again against water, and subtract from original reading. Addition of the cyanide to the biuret mixture following saturation with ether results in troublesome bubble formation.
PRESENCE OF SALTS. Table I11 lists the effect of sodium sulfate, sodium sulfite, and ammonium sulfate on the absorbance of reagent blanks and an albumin standard, all read against water. I n the technique employing Benedict’s reagent, the reaction mixtures consisted of 2.5 ml. of the salt solution containing 1.0 mg. of protein nitrogen, 2.5 ml. of 1.50.V sodium hydroxide, and 1.0 ml. of biuret reagent. Neither sodium sulfite nor sodium sulfate altered the readings more than 37, when square cuvettes were used, and the absorption peak was not shifted. Readings made in round cuvettes in a Klett-Summerson photometer against mater were slightly higher owing to the difference in refractive indices of the solutions. Reading against reagent blanks containing the same salt concentration, however, eliminated this difference. That the difference in refractive index was responsible for this observation was indicated by the fact that blanks devoid of copper sulfate but containing either of these salts gave an appreciable absorbance reading against water. 1494
0
ANALYTICAL CHEMISTRY
parent serum protein concentration, it is seen that satisfactory results are obtained up to a hemoglobin concentraSerum Protein, yo tion of 4%. The factor 1.9 was deterUncorrected Uncorrected mined by measuring the relative absorbfor Hb (1.9) (% Hb) ances of the biuret reaction on stand6.8 6.8 ardized protein and hemoglobin solu6.8 7.3 7.6 6.7 tions. The correction was valid with 8.8 6.9 the Beckman Model DU and Bausch & 10.6 6 8 Lomb Spectronic 20 spectrophotometers 14.0 6.4 and the KlettrSummerson filter photometer. From comparison of the absorption curve of the biuret reaction applied to a solution of hemoglobin with the I n the technique employing Keichselcurve of the hemoglobin alone in the baum’s reagent, the reaction mixture same concentration of alkali, it was deconsisted of 2 ml. of the salt solution duced that the factor 1.9 results from containing 1 mg. of protein nitrogen and additivity of the absorbance of alkaline 5 ml. of biuret reagent. Although, hematin to that of the biuret reaction with square cuvettes, there appeared with the protein moiety of the hemoto be no significant change in absorbglobin. ance in the presence of sodium sulfate, I n practice, the hemoglobin concensodium sulfite consistently produced a tration is determined by any method of significant increase in absorbance. The sensitivity sufficient for the degree of findings of Gornall and coworkers (j), hemolysis present. Thus, for 4% hemothat, with their biuret reageht and techglobin either the oxyhemoglobin or cynique, sodium sulfate produces no anmethemoglobin methods would be effect and sodium sulfite produces a satisfactory. For low concentrations negligible one, were confirmed. Rosenthe benzidine method would be required thal and Kawakami (Wg), using Gor(1). nall’s reagent, observed a considerable increase in color with both salts, but DISCUSSION they altered the technique so that the ratio of salt t o biuret reagent concenThe determination of protein nitrotration was greater than that employed gen is generally employed as the referby Gornall and con-orkers. ence method for serum protein analysis. With the addition of ammonium ion It is convenient to have one method unito the reaction mixture, there is a proversally adopted for reference, but such gressive decrease in absorbance of the a choice is entirely arbitrary. The nibiuret-protein complex as the concentratrogen content of the various protein tion of ammonium ion is increased, unfractions is known to vary significantly. til a point is reached where apparently The biuret method is not absolute, the copper is completely complexed by having to be standardized by another the ammonium ion. The lowest conmethod which is absolute. The one centration, 0.1M, appeared to have but ordinarily employed is the Kjeldahl a slight effect on standards with either determination of nitrogen. The occathe Benedict or Weichselbaum copper sional discrepancy between the two reagent, Since 2.5 ml. of 0.lM ammomethods, such as reported by Reinhold nium sulfate contains 7 mg. of ammonia and associates (19), does not mean that nitrogen, it seems safe to conclude that the results of one or the other of the no significant error would be introduced methods are incorrect. if the ammonia nitrogen in a sample is I t has been reported that human no greater than about five times the serum albumin and globulins produce protein nitrogen present The greater the same amount of color in the biuret reresistance of Benedict’s reagent to the action (a,5 15,aO). On the other hand, effects of ammonium ion is presumably significant differences between the varidue to the greater final alkalinity with ous protein fractions have been reported this technique than with Weichselby some workers (4, 1’7). Conflicting baum’s (6). data are also encountered in reports both Table IV gives the reHEMOLYSIS. of agreement (21) and disagreement (9, sults of a typical experiment in which 11-14, 19) between results obtained by increasing amounts of hemoglobin were biuret and Kjeldahl analysis for total added to a serum in Khich the serum serum protein. The data reported protein concentration was kept conherein show that, a t least with those stant. The example given was obtained sera tested, the results obtained by the with the technique employing Benedict’s biuret technique employing Benedict’s reagent. Identical results were obqualitative glucose reagent and by tained using Weichselbaum’s reagent. Kjeldahl analysis were in close agreeIf the serum protein equivalent of the ment and, for most purposes, could be hemoglobin is calculated by multiplying considered identical. A prerequisite for this agreement is the absence of interthe hemoglobin concentration by 1.9 ference from turbidity, and high concenand then subtracting this from the ap-
for Presence of Hemolysis
trations of certain salts. Turbidity can usually be removed by ether extraction. If hemolysis is present, a correction can be applied if the hemoglobin concentration is known. Interferences produced by ammonium, sulfate, and sulfite ions apparently vary with the particular biuret technique employed, presumably because of different final concentrations of copper and alkali. LITERATURE CITED
(1) Bing, F. C., Baker, R. K., J . Biol. Chem. 92,589 (1931). 12) Fine, J.. Biochem. J . 29. 799 119351. (3) Goa,' J:, Scand. J . &in. & Lab. Inaest. 5 , 218 (1953). (4) Ibid., 7,Suppl. 22 (1955).
( 5 ) Gornall, 9.G., Bardawill, C. J., David, PVI. bI., J . Biol. Chem. 177, 751 (1949). (6) Henry, R. J., Golub, 0 J., Sobel, C., Clin. Chem., 3,49 (1957). (7) Keyser, J. W., Vaughn, J., Biochem. J . 44, xxii (1949). (8) Kingsley, G. R., J . Biol. Chem. 131, 197 (1939). 19) Ibid.. 140. lxix 11941). (10) Kingsley,'G. R:, J . Lab. Clin. X e d . 27,840 (1942). (11) Kingsley, G. R., Behrend, A . A , , Zbid., 34, 11178 (1949). 112) Kinaslev, G. R.. Machella.' T. E., 1&d.,"34, 1183 '( 1949). (13) Kingsley, G. R., Terzian, L. A,, Zbid., 34, 1175 (1949). (14) Levin, R., Brauer, R. W.,Ibid., 38,474 (1951). (15) Narver, I-L., S c a d J . Clin. & Lab. Invest. 7,Suppl. 21 (1955).
(16) Mehl, J. W.,J . Bid. Chem. 157, 173 (1945). (17) Llehl, J. R., Pacovska, E., Winzler, R. J., Zbid., 177, 13 (1949). (18) Reinhold. J. G.. "Standard hlethods of' Clinicil Chemistry," 5'01. I, p. 88, rlcademic Press, New 1-ork, 1953. (19) Reinhold, J. G., Bluemle, L. W., Jr., Campoy, F., Gilman, L., Gould, E. F., Martens, V. L., Shuman, R., Federation Proc. 8, 242 (1949): (20) Robinson, H. W,,Hogden, C. G., J . Bid. Chem. 135,707 (1940). (21) Ibid., p. 727. (22) Rosenthal, H. I,., Kawakami, T., Am. J . Clin. Pathol. 26. 1169 (1956). (23) Weichselbaum, T. E., Zbid., 16, 40 (1946).
RECEIVEDfor review October 29, 1956. Accepted 3Iay 9, 1957.
Determination of Sulfoxides R. R. LEGAULT and KERMIT GROVES Department of Agricultural Chemisfry, State College of Washington, Pullman, Wash.
b A modified Barnard and Hargrave method has been developed for determination of sulfoxides that yield water-soluble sulfides upon reduction with the titanium trichloride reagent. The interfering sulfides are removed b y extraction with 1 -butanol after partial saturation of the aqueous phase with ammonium sulfate. Final titration with potassium dichromate is carried out as in the original method.
B
Hargrave ( 1 , 2) have developed a method for the determination of sulfoxides based on the reduction of the sulfoxide to sulfide by titanium trichloride. The method involves heating a mixture of the sulfoxide, acetic acid, and a standard solution of titanium trichloride under nitrogen, adding a measured amount of ferric alum to oxidize the excess titanium trichloride, and titrating the ferrous ion with potassium dichromate. Carbon tetrachloride is added before the titration to remove the formed sulfide from the aqueous layer and thus prevent its reoxidation to sulfoxide. This method gives good results with many sulfoxides, but failed when applied to 2-hydroxydiethyl sulfoxide (3) and dimethyl sulfoxide. These sulfoxides gave low and erratic results, and seemed to interfere with the indicator, diphenylaminesulfonic acid. The carbon tetrachloride did not remove the sulfide completely from the aqueous phase before the titration with dichromate. Several extraction procedures for the removal of the sulfide were tried. The
method was modified by eliminating the acetic acid from the reduction step, as acetic acid tended to make the sulfides more water-miscible. Partial saturation of the mixture with ammonium sulfate and extraction with 1-butanol, followed by carbon tetrachloride, successfully removed the sulfides. The modified method gave good results with 2-hydroxydiethyl sulfoxide and dimethyl sulfoxide. REAGENTS
A R ~ A R Dand
Titanium trichloride solution, 0.1N. Mix 100 ml. of 15% titanium trichloride solution with a previously boiled mixture of 50 ml. of concentrated hydrochloric acid and 850 ml. of water. The solution is stored in a bottle protected by a chromous acid solution in a washing tower, and connected to dispense directly to a buret. The solution is standardized by the procedure used for sulfoxide analysis. Ferric alum solution. Dissolve 200
Table 1.
Determination of Sulfoxides
Sulfoxide. R Barnard PVTdm method method I
Compound
I
Y
2-Hydroxy-
diethyl sulfoxide 88zk6 100.4zk0.7 Dimethyl sulfoxide S8zk6 99.7f0.5 Diethyl sulfoxide 99.8&0.7 Dipropyl sulfoxide 94.910.6 92.110.7 Dibutyl sulfoxide
9S.OzkO.7 9 7 . 7 d 0 . 7
grams in 1 liter of water containing 60 ml. of concentrated sulfuric acid. Potassium dichromatesolution, 0.05A'. Dissolve 2.4518 grams of the reagent grade salt in water and make to 1 liter. Phosphoric acid solution. Dissolve 75 ml. of orthophosphoric acid (85%) in 350 ml. of water and 75 ml. of concentrated sulfuric acid. Diphenylaminesulfonic acid indicator, 0.25% aqueous solution. Nitrogen. Pass through a solution of chromous acid (4). Ammonium sulfate solution, saturated a t room temperature. PROCEDURE
Weigh a sample of 0.7 t o 1.0 meq. into a 250-ml. flask equipped with a standard ground joint and a stopper fitted with a three-way stopcock assembly which permits evacuation and filling with nitrogen. Evacuate the flask to about 20-mm. pressure and then fill with nitrogen that has been washed with chromous acid solution to remove tracesof oxygen. Add 15 nil. of 0.1N titanium trichloride solution to the nitrogen-filled flask under a stream of nitrogen. Then evacuate the flask and fill with nitrogen three times in the manner described above. Heat the flask and contents in a bath a t 80" C. for 1 hour. At the end of the hour, add to the flask a boiling mixture of ferric alum and ammonium sulfate solutions, prepared by adding 5 ml. of ferric alum solution to 40 ml. of saturated ammonium sulfate and heating the mixture to boiling. Allcw the solution to stand 30 seconds; then cool rapidly. Add 10 ml. of phosphoric acid solution to the flask. Then transfer the contents of the flask to a separatory funnel, extract the VOL. 29, NO. 10, OCTOBER 1957
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