visually detectable color with tiron. Spectrophotometric measurements, however, showed slight positive absorbances by 500 and 1000 pg of the metal. Ammonium citrate acted more effectively than ammonium phosphate, ammonium tartrate, or tartaric acid in controlling tungsten interference. Vanadium at the 400-118 level caused no interfering absorbance. However, 1000 pg of vanadium produced an absorbance equal to that developed by approximately 1 pg of titanium. Fluoride reportedly ( I ) interferes in the titanium-tiron reaction. The present method excludes fluoride as a reagent, and thus avoids a contaminating source of interference. Stability. Solutions of samples prepared in dilute oxalic acid have remained clear and free of precipitate for 3 years with apparently indefinite stability. Test solutions of standard titanium tironates in stoppered cuvettes showed constant absorbances during periods of 2 weeks. Titanium test solutions containing diverse metallic ions remained color stable at least 1 day after development with tiron. From 2 to 100 pg of titanium can be determined in 50-ml
test solutions. Depending on the aliquot taken, the sensitivity and range permit determination of 0.02 to 10.0% of titanium in 0.1000-gram samples, or 0.004 to 2.00% of titanium in 0.5000-gram samples. The precision of the spectrophotometric procedure, only, was estimated from the absorbances of a series of test solutions containing 50 pg of titanium. The standard deviation between measurements was calculated to be +0.3 pg of titanium, equal to a relative deviation of h0.6 percent. The accuracy of the overall method is shown by comparative results in Table I11 on eleven Standard Reference Materials certified by the National Bureau of Standards. The titanium determinations by the tiron method agree favorably with the NBS analytical values. Applications. Although developed primarily for highiron samples, this spectrophotometric method can be used to determine titanium in a wide variety of inorganic materials including metals, alloys, minerals, and rocks. RECEIVED for review November 7,1969. 1970.
Accepted March 18,
SpectrophotometricDetermination of Sulfite with 4,4‘-Dithiodipyridine and 5,5’-Dithiobis(2-Nitrobenzoic Acid) Ray E. Humphrey, Melbourne H. Ward, and Willie Hinze Department of Chemistry, Sam Houston State University, Huntsvilk, Texas 77340 Sulfite ion reacts essentially quantitatively at room temperature in aqueous buffers with 4,Q-dithiodipyridine and 5,5r-dithiobis(2-nitrobenzoic acid) to displace the thiol and form an organic thiosulfate. Sulfite is determined by measurement of the absorption of the thiol. The reaction is useful for the determination of sulfite over a concentration range of approximately 0.2 to 6.0 pg/ml as SOz. The pH value is not critical and can be varied over a range of 3-4 units. The disulfide and reaction solutions are reasonably stable. Some possible metal ion interferences, except for Hg(ll), can be eliminated by use of EDTA. Other thiols, thiosulfate, sulfide, and cyanide ion will interfere. Nitrite ion does not appear to cause any difficulty. Sulfite solutions were quite stable when EDTA was present.
SULFITE ION reacts with many organic disulfides to displace a thiol anion and form the organic thiosulfate, commonly called a “Bunte” salt. This reaction proceeds as shown in Equation 1.
RSSR
+ S03-’
@
RSS03-
+ RS-
(1)
This reaction was recently reviewed by Klayman and Shine ( I ) . In almost all instances, a large excess of sulfite ion is used in order to have quantitative reaction of the disulfide. The thiol anion is then titrated with silver nitrate ( 2 ) or (1) D. L. Klayman and R. J. Shine, Quart. Rep. Sulfur Chem., 3, 190 (1968). (2) R. Cecil and J. R. McPhee, Biochem. J . , 59, 234 (1954). 698
ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
mercuric chloride (3). Some nitro-substituted aromatic disulfides have been reported to react readily with sulfite ion, using a rather small excess of sulfite ( 4 ) or having equimolar amounts of sulfite and disulfide (5, 6). Room temperatures are generally used for the sulfite-disulfide reactions although in one study ( 4 ) , several substituted aromatic disulfides were refluxed with excess sulfite in aqueous methanol. There appear to be no detailed studies of the reaction of aromatic disulfides with sulfite ion in the literature. Certain disulfides, notably 5,5’-dithiobis(2-nitrobenzoic acid), “Ellman’s” reagent (7), DTNB, and 4,4’-dithiodipyridine (8), 4-PDS, are used frequently for the determination of thiol groups in a variety of biochemical samples. The disulfide is reduced to the corresponding thiol which absorbs at a different wavelength than the disulfide. Since the disulfide is present in excess when thiol groups are determined by this method, presumably a mixed disulfide is formed and the absorbing thiol displaced as shown in Equation 2. R’SSR’
+ RSH F? R’SSR + R’SH
(2)
(3) W. Stricks, I. M. Kolthoff, and N. Tanaka, ANAL.CHEM., 26, 299 (1954). (4) H. Z. Lecher and E. M. Hardy, J . Org. Chem., 20, 4751 (1955). ( 5 ) A. J. Parker and N. Kharasch, J . Amer. Chem. SOC.,82, 3071 ( 1960). (6) A. J. Parker, Acfa Chem. Scand., 16,855 (1962). (7) G. L. Ellman, Arch. Biochern. Biophys., 82, 70 (1959). (8) D. R. Grassetti and J. F. Murray, Jr., ibid.. 119, 41 (1967).
t
Mole Fraction SO5
Figure 1. Continuous variation plot for the reaction of 4,4'-dithiodipyridine with sulfite ion Concentration at 1 :1ratio was about 5 X 10-6M
Moles SOvMole DtnB Figure 2. Mole ratio plot for the reaction of 5 3 ' dithiobis(2-nitrobenzoic acid) with sulfite ion. The concentration of disulfide was about 5 x 10-5~
This reaction has been found to be quite sensitive for the determination of rather low amounts of thiols (7,8). Both 4,4'-dithiodipyridine (4-PDS) and 5,5'-dithiobis(2nitrobenzoic acid) (DTNB) have been found to react rapidly and quantitatively at room temperature with sulfite ion yielding an equimolar amount of the absorbing thiol. This reaction has been evaluated as a possible spectrophotometric procedure for sulfite. Apparently the most generally accepted colorimetric method for sulfite, or sulfur dioxide, is that involving the use of pararosaniline and formaldehyde, referred to as the West-Gaeke procedure ( 9 ) . A comprehensive evaluation of this method for determination of atmospheric sulfur dioxide was reported recently (IO). The reduction of Fe(II1) to Fe(I1) by sulfur dioxide with 1,lO-phenanthroline present to form the tris(1 ,lo-phenanthroline) Fe(I1) complex has also been found to be a sensitive colorimetric method for the determination of
so2 (11).
EXPERIMENTAL Reagents. The disulfides, 4,4'-dithiodipyridine and 5,5 'dithiobis-(2-nitrobenzoic acid), were obtained from Aldrich Chemical Co., Milwaukee, Wis., and were used without further purification. Sodium sulfite was Baker Analyzed reagent grade, 98% minimum Na2S03. All other chemicals used, for preparation of buffers and evaluation of interferences, were the best-available reagent grade materials. Solutions. Sodium sulfite solutions were generally in the range of 0.0005-0.001M and were prepared by dissolving an accurately weighed amount of the anhydrous compound in a O.OO1M EDTA solution, diluting to volume in a volumetric flask. The EDTA solution was prepared using the disodium salt of ethylenediamine tetraacetate. These solutions were quite stable for periods as long as two weeks. Disulfide solutions were also approximately 0.001M and were prepared by dissolving a weighed amount of the compound in 5-10 ml of 95 % ethanol, diluting to volume with distilled water in the case of 4-PDS or pH 7 buffer for DTNB. The pyridine disulfide appeared to react slowly with the buffer so was generally made up to volume with distilled water. Buffer solutions were 0.025M Na2HP04 and KH2P04-pH 6.86, 0.01M Na2B40,-pH 9.18, a mixture of 0.1M K H 2 P 0 4 and 0.1MNaOH for pH 6 or 8, and 0.05M KHCsHaOl for pH 4. -
(9) P. W. West and G. C. Gaeke, ANAL.CHEM., 28, 1816 (1956). (10) F. P. Scaringelli, B. E. Saltzman, and S. A. Frey, ibid., 39, 1709 (1967). (11) B. G. Stephens and F. Lindstrom, ibid., 36, 1309 (1964).
Spectrophotometers. Absorption measurements were made with a Beckman Model DB-G spectrophotometer. Spectra were recorded with a Beckman DK-2A spectrophotometer. Procedure. Reactions were conducted by simply adding a measured volume of a standard sulfite solution to a solution of the disulfide in a buffer. For the quantitative studies of sulfite recovery and establishing Beer's law plots, the disulfide was present in at least a twofold excess for the highest concentration of sulfite to be used. Either the disulfide or sulfite solution can be added last. The reaction is essentially complete in two to three minutes for both DTNB and 4-PDS when either reactant is present in a slight excess. The final volume was IO ml in most of the experiments. Usually 1 ml of the disulfide solution was used with the volume of the sulfite solution being from 0.1 to 1.0 ml. The required amount of buffer solution was used to achieve the final volume. All readings were taken against a blank solution that contains all reagents except sulfite. RESULTS AND DISCUSSION Study of the Disulfide-Sulfite Reaction. Although sulfite ion has been used extensively to reduce disulfide groups, this reaction apparently has never been used for determination of the sulfite. Sodium sulfite is added in considerable excess and the displaced thiol titrated with standard silver nitrate (2) or mercuric chloride (3). In one instance, complete reaction between sulfite ion and p-nitrophenyl disulfide in 95 % ethanol was indicated but few details were given and the interaction apparently was not studied in detail (6). Both DTNB and 4-PDS react essentially quantitatively with sulfite ion at room temperature in pH 7 buffer. The DTNB-sulfite reaction appears to be almost complete over a pH range of 6 to 9. The reaction of sulfite ion with 4-PDS appears to proceed in the same manner over the pH range of 4 to 7. At pH 8 reaction occurs but the absorption peak for the reduced species is shifted to shorter wavelengths and overlaps with the absorption due to the disulfide. Hence, when using 4-PDS the pH value should not be much higher than 7. The reaction in the case of both disulfides is quite rapid and complete. The 1 :1 stoichiometry, shown in Equation 1, to produce the thiol and organic thiosulfate was confirmed for both disulfides by measuring absorbances at various ratios of reactants and constructing mole ratio and continuous variations plots. A continuous variations study for the 4-PDS-sulfite system is shown in Figure 1 and a mole ratio plot for the reaction of DTNB with sulfite is presented in Figure 2. The ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
699
Table I. Beer’s Law Data for Determination of Sulfite with DTNB Molar SOZ,wdml AbsorbanceQ absorptivity* 0.60 0.13 13,900 1.80 0.37 13,100 3.00 0.61 13,000 4.20 0.85 13,000 5.40 1.11 13,200 4 Wavelength 412 nm, 1-cm cells. * Molar absorptivity values are based on the molar concentration of sulfite ion. The concentration of DTNB was 1.0 X lW*M. The range of sulfite concentrations was 0.94-8.4 X 10‘+M.
X,nm
Figure 3. Absorption spectrum of 5’5 ’-dithiobis(Znitrobenzoic acid) and reaction solution containing a small excess of sulfite ion at pH 7
X,nm Figure 4. Absorption spectrum of 4,4’-dithiodipyridine and reaction solution containing a small excess of sulfite ion at pH 7 practical completeness of each reaction is indicated by the lack of any appreciable curvature in these plots. Absorbances were measured at the wavelength maximum for the thiol anion in each case. No evidence was found for further reaction when either reactant was present in excess. Absorption Spectra. Absorption spectra for DTNB and a reaction solution with a slight excess of sulfite are shown in Figure 3. Similar spectra for the 4-PDS system are shown in Figure 4. The results with DTNB, an absorption maximum at 325 nm with e = 18,500 for the disulfide and a maximum a t 412 nm with B = 15,500 for the thiol anion, agree reasonably well with the reported values for the analogous compounds without the carboxyl group (12). Similarly, results for 4-PDS, a wavelength maximum a t 247 nm with E = 15,800 for the disulfide and a maximum at 324 nm with E = 21,500 for the reduced species, agree with the reported figures (8). (12) G. L. Ellman, Arch. Biochem. Bioplrys., 74, 443 (1958). 700
ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
Table 11. Beer’s Law Data for Determination of Sulfite with CPDS Cell path Molar SOn,pg/ml length, cm Absorbancea absorptivityb 0.058 10 0.24 26,000 0.117 10 0.44 24,000 0.174 10 0.64 23,200 0.232 10 0.93 22,800 0.58 1 0.21 23,000 1.17 1 0.44 24,000 1.74 1 0.61 22,400 2.32 1 0.79 21,600 2.90 1 1.00 22,000 a Wavelength 324 nm. b Molar absorptivity values are based on the molar concentration of sulfite. The concentration of 4-PDS was 9.3 X 10-5M where 1-cm cells were used and 9.3 X 10-6M where 10-cm cells were used. Sulfite concentrations ranged from 9.1 X lO-7M to 4.5 x 10-6M.
There are differences in the spectra of solutions where disulfides reacted with sulfite ion and where the thiols were produced by reduction in that the sulfite solutions also contain the organic thiosulfate species. The DTNB reaction solution has a n absorption peak a t 325 nm, E = 8300, the same wavelength a t which the disulfide shows a maximum. Also, the 4-PDS solution containing sulfite exhibits a n absorption maximum at 265 nm with E = 6000, which is probably due to the RSS03- species. The 4-mercaptopyridine produced by reduction of 4-PDS is believed to exist predominantly in the thione form, Structure I, shown below (8). It has also been suggested that p-nitrothiophenolate anion possibly exists in a similar resonance form as shown in Structure I1 for the reducedDTNB(6).
-0, + ,oN
I
H Structure I
Structure I1
Neither disulfide shows appreciable absorption a t the wavelength maximum for the thiol so that the correction for absorption due to the excess disulfide in reaction solutions is not very large. Concentration Range for SOz Determination. Beer’s law data for the determination of sulfite with DTNB are shown in Table I and corresponding data for 4-PDS with sulfite are shown in Table 11. In both instances a straight line plot
is obtained. Absorbance readings are quite stable for 1-2 hours for both DTNB and 4-PDS reduced with sulfite. Beyond that time readings decrease very slowly probably because of air oxidation of the thiol anion. The range of concentrations of sulfite which can be determined by use of DTNB is approximately 0.5 to 6.0 pg/ml SOz while with 4-PDS the range is somewhat lower, 0.2 to 4.0 pg/ml SOZ. The range was extended down t o less than 0.1 pg/ml SOz using 4-PDS and absorption cells with 10-cm path length. These results are also shown in Table 11. Stability of Sulfite Solutions. Considerable difficulty was encountered in the early part of this study because of the instability of the sulfite solutions. Oxidation is reported to be quite rapid even when the solutions are made up in doubledistilled water which has been deaerated (10). Glycerol a t the 5 % level has been found t o be somewhat effective in stabilizing sulfite solutions, presumably because metal ions present in trace amounts which catalyze the oxidation of sulfite ion are complexed by the glycerol (13). In the WestGaeke procedure, sulfite is stabilized by formation of a complex with tetrachloromercurate ion (9). Glycerol was found to be helpful in this work but was not entirely satisfactory. The mercurate complex could not be used as the p H range which was necessary did not release the sulfite ion t o react with the disulfides. Sodium sulfite solutions made up in 0.001M EDTA were quite stable for as long as two weeks. Sulfur dioxide loss from solutions containing trace amounts of Fe(I1) or Fe(II1) due to oxidation is reported t o be lessened considerably by the presence of EDTA (14). In the present work, for solutions 0.001M in sodium sulfite and 0.001M in EDTA, there was no detectable loss of sulfite in one week at room temperature. After two weeks the loss was less than S%, after three weeks about lo%, and approximately 15% loss in four weeks. The sulfite solutions in EDTA are also quite stable to aeration. No loss was detectable by use of the DTNB procedure after passing air at a rate of 250 ml per minute through a 0.001Msulfite solution for 24 hours. Interferences. There are three likely categories of substances which might be expected to cause difficulty in this procedure. These are metal ions which might precipitate the thiol, substances which can reduce the disulfide, and oxidants which could oxidize the thiol anion or the sulfite ion. The effect of various metal ions, Cd(II), Cu(II), Fe(III), Hg(II), Pb(II), and Zn(II), at a level of 20 pg/ml, on the determination of sulfite using both of the disulfides is presented in Table 111. These solutions were also approximately 2 X 10-4M in EDTA. Each metal was tested individually. The only metal ions which showed any interference were Cu(I1) and Hg(I1). The interference due to Cu(I1) was eliminated by increasing the concentration of EDTA to about 0.005M. The presence of Hg(I1) completely prevents any reaction between sulfite and DTNB even when the EDTA concentration is of the order of 0.01M. This is probably due to the very great stability of the Hg(l1)-sulfite complex. If a solution containing Hg(I1) is added after the sulfite-DTNB reaction has occurred, the color is removed. When Hg(I1) was added to a solution containing the thiol of DTNB, produced by reduction with dithiothreitol, the yellow color of the thiol disappeared and the solution remained clear. Experiments t o determine the stoichiometry of the reaction of Hg(I1) with a solution containing the thiol and the organic thiosulfate were inconclusive. -
(13) P.Urone and W. E. Boggs, ANAL.CHEM., 23, 1517 (1951). (14) N. Zurlo and A. M. Griffini, Med. Lao., 53, 330 (1962).
Table 111. Effect of Metal Ions on Determination of Sulfite with DTNB and CPDS Metal iona A, DTNBb A, 4-PDSC None 0.62 0.66 Cu(I1) 0.25,O. 61d 0.28,O. 67d Cd(I1) 0.62 0.66 Fe(II1) 0.63 0.65 Tlgm 0.02 0.36 Pb(W 0.62 0.65 Zn(I1) 0.61 0.66 a The concentration of metal ion was 20 pg/ml. The concentration of EDTA was 2 X 10-4M. * Wavelength 412 nm. Concentration of SOt was 3.1 pg/ml. c Wavelength 324 nm. Concentration of SOn was 2.1 pg/ml. EDTA concentration was 5 X 10-sM. Table IV. Recovery of Sulfite from Synthetic Samples at pH 7 DTNB SOS,,ug/ml Absorbancea Found Present Error, 0.13 0.65 0.69 6.2 0.35 1.75 1.73 1.1 0.63 3.10 3.10 0 4.85 3.1 1 .oo 5.00 1.20 6.00 5.90 1.7 4-PDS 0.22 0.67 0.69 2.9 0.55 1.70 1.73 1.7 0.80 2.48 2.43 2.1 2.95 2.1 0.92 2.86 1 .oo 3.10 3.10 0 a 1-cm cells. Wave length for DTNB was 412 nm. Wavelength for 4-EDS was 324 nm.
The presence of thiols or sulfide ion leads to serious interference as would be expected since both disulfides are commonly used for the determination of -SH groups. Cyanide ion will react slowly with these disulfides so that some interference would occur if the absorbance is not read within 5 t o 10 minutes. Thiosulfate ion also produces some color with DTNB, 56 pg/ml S203-2corresponding to about 1 pg/ml of Sot. N o study was made of the effect of the presence of oxidizing substances on the determination of sulfite with these disulfides. It would be expected that any oxidant which could oxidize the thiol produced would be a serious interference. A brief investigation was made of the effect of nitrite ion on the determination of sulfite with DTNB. N o interference was noted on the color produced with DTNB when nitrite ion was present at a level of 35 pg/ml. Comparison with the West-Gaeke Method. The disulfide which affords the highest sensitivity for sulfite analysis is 4-PDS with a molar absorptivity of approximately 22,000 as compared to about 13,000 for DTNB. The value with 4-PDS is considerably lower than those reported (37,000, 48,000) for two methods using pararosaniline (IO). However, the sensitivity is probably high enough for many applications. Recovery of sulfite at low concentrations is reasonably good as shown in Table IV. In the West-Gaeke procedure, the p H is critical and must be adjusted to within about 0.2-0.4 unit. Also, the development of color and fading are affected considerably by temperature so that a constant temperature bath is necessary for highest precision. Development of color using pararosaniline requires about thirty minutes while the disulfide-sulfite reaction is complete within about two minutes. ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
701
It is also necessary to carefully purify the pararosaniline dye to obtain reproducible results while the disulfide reagents can be used as purchased (10). The disulfide method may have advantages in terms of simplicity of procedure, speed of reaction, pH range allowable, known stoichiometry, stability of the reaction products, and simplicity and reproducibility in preparation of the disulfide solutions. The absorbance values are linear over a reasonably large range of concentrations and the absorption due to the reagents is negligible. Determination of sulfite by use of these disulfides would probably be applicable to
many types of samples after necessary modifications are made as required by each individual situation. The use of EDTA to stabilize sulfite solutions should also be useful in about any application of such solutions. From the limited amount of work reported here, it appears that EDTA might be as effective in preventing loss of sulfite as the tetrachloromercurate ion. RECEIVED for review January 19, 1970. Accepted March 13, 1970. Research supported by the Robert A. Welch Foundation of Houston, Texas.
New Spectrophotometric Method for the Determination of Proline in Tissue Hydrolyzates Imanuel Bergman and Roy Loxley Safety in Mines Research Establishment, Ministry of Technology, Shefield, England
A number of methods for determining proline depend on its reaction with ninhydrin to give a red pigment. Such methods have so far given erratic results owing to the instability of the pigment in solution in the hot acid reaction medium. A new method is described in which an aqueous reaction medium contains sodium chloride to salt-out the pigment as it is produced, and thus protect it from decomposition. When reaction is complete, the suspension is cooled and diluted with acetic acid, in which the pigment is soluble and the color is stable at room temperature. Interferences from primary amino acids are eliminated by an improved nitrous acid treatment in which the excess nitrous acid is removed by heating with ammonium chloride-hydrochloric acid. These methods have been tested several hundred times on hydrolyzates of formalin-fixed lungs, and have given reproducible results and accurate recoveries of added proline.
As PART OF FUNDAMENTAL studies in pneumoconiosis, methods have been developed by the authors for the determination of the major known components of lung tissue: collagen, elastin, and blood. Most of the hydroxyproline in lung tissue is contributed by collagen although some is contributed by elastin. Proline is contributed mainly by collagen and elastin, and to some extent by blood. If the amounts of blood, hydroxyproline, and proline in the tissue are known, the amounts of collagen and elastin can be calculated. Two improved methods for hydroxyproline determination have already been developed ( I ) , and the optimum conditions of hydrolysis of lung tissue have been studied (2). The present paper describes a new method for the spectrophotometric determination of proline in hydrolyzates. There have been a number of publications concerned with the spectrophotometric determination of proline, each altering the procedures or adding steps in an effort to remove the interfering effects of other tissue constituents, and to make the results more reproducible. Most of the recent methods have been based on that of Chinard (3), who heated an aqueous
____
(1) I. Bergman and R. Loxley, ANAL.CHEM.,35, 1961 (1963). (2) I. Bergman and R. Loxley, Analyst, 94, 575 (1969). (3) F.P. Chinard, J. Biol. Chem. 199, 91 (1952). 702
ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
solution containing proline with a mixture of acetic and concentrated phosphoric acids containing ninhydrin, to give a red solution. Chinard proposed to remove interfering amino acids by chromatography. Schweet ( 4 ) found that Chinard’s method gave results that were variable owing to the instability of the colored product. His modification was sensitive to sodium chloride and phosphate buffer, but he proposed the nitrozation procedure based on that of Hamilton and Ortiz ( 5 ) to remove the interfering primary amino acids. Troll and Lindsley ( 6 ) modified Chinard’s method (3) by using benzene to extract the colored product. They removed three interfering basic amino acids by adsorption on Permutit. Messer (7) made a thorough investigation of the effect of twenty amino acids on Chinard’s method. Seven of these gave a color themselves and either depressed or enhanced the proline color yield, Ten enhanced this yield but gave no color. Messer’s modification removes this effect, but not the color production of seven amino acids and proline color yield depression of three of these. Wren and Wiggall (8) introduced a performic acid oxidation of protein prior to hydrolysis to remove cystine and cysteine. They varied the parameters in a procedure developed from those of Troll and Lindsley (6) and Messer (7). Their conclusion was: “These investigations revealed a more complicated pattern of interference than was previously recognized.” In the present work, the disadvantages of these methods have been overcome by the use of an aqueous reaction medium with a high electrolyte content to salt out the red reaction product and thus protect it from decomposition. The few primary amino acids that interfere with this procedure are removed by an improved nitrous acid treatment. (4) R. S . Schweet, ibid., 208, 603 (1954). (5) P.B. Hamilton and P. J. Ortiz, ibid., 187,733(1950). (6) W. Troll and J. Lindsley, ibid., 215,655 (1955). ( 7 ) M.Messer, Anal. Biochem., 2, 353 (1961). (8) J. J. Wren and P. H. Wiggall, Biochem. J . , 94, 216 (1965).
