10 days and measured for the intensity of fluorescence. I n no case was there a variation in the previously determined values, Table I1 contains a list of the amino acids which were observed to fluoresce under the conditions described. There is also given a comparison of the results obtained after irrigating the strips with n-butyl alcohol-acetic acid-potassium chloride-hydrochloric acid buffer, as carried out in actual chromatographic analysis, and then analyzed by the two methods of color absorption and intensity of fluorescence. In each case there was applied to the paper strip 2 GI. of solution containing 0.02 pmole of the amino acid. The ninhydrinsprayed chromatograms were heated for 5 minutes a t 60" C., the xylosesprayed chromatograms were heated for 10 minutes a t 120' C. The ninhydrin-treated chromatograms did not give any recorder response for the solvent front. Those chromatograms observed in the ultraviolet region gave a considerable response for the solvent front, although in none of the amino acid chromatograms analyzed did the solvent front and the deposit of the amino acid coincide exactly. Because the 570-mp narrow band pass filter was used in the case of the ninhydrin chromatograms, there was no
absorption for proline and hydroxyproline. It was necessary to read these chromatograms with a 485-mp filter. This difficulty was not experienced in the case of the ultraviolet range. The chromatograms developed and read in the ultraviolet region were not affected by exposure to atmospheric conditions for 2 weeks. The ninhydrin chromatograms fade considerably after this time (6). In both methods of analysis it is possible to observe suitable readings for 2 pl. of solution containing 0.02 pmole of the amino acid. In some cases it is possible to detect color for quantities as small as 0.002 pmole, but the recorder response for such amounts is below 6. No recorder response was observed in the ultraviolet region for quantities of the amino acids of the order of 0.002 pmole. In all cases it is possible to reproduce the results of the analysis in the ultraviolet to within 2 parts per 100. CONCLUSION
Certain precautions should be given consideration in the use of ultraviolet densitometry of amino acid chromatograms. The fluorescence of phenol, pyridine, and cresol on paper make such solvents unsuitable for this method of analysis. All grades of filter paper
cannot be used, or can be used in a limited way, because of irregular base line effects. Heating the chromatograms for 10 minutes a t 120" C. produces maximum fluorescence of the amino acids. The sensitivity of fluorescence is comparable to that of absorption in the visible range. Fluorescence is stable under atmospheric conditions. The reproducibility of amino acid analysis in the ultraviolet is compared t o that obtained by other methods. Twenty-seven amino acids were observed to fluoresce. ACKNOWLEDGMENT
Financial assistance from the American Academy of Arts and Sciences is gratefully acknowledged. LITERATURE CITED
(1) Graham, R. D., Hsu, P. Y., McGinnis, J., Science 110, 217 (1949). (2) Ffavrodineanu, R., Sanford, W. W., Hitchcock. A. E.. Contribs. Bouce Thomwson'Inst. 18/3\. 167 (1955). ' (3) Milier, G. D., Johhsonl'J. A.', ANAL. CHEY.28,884 (1956). (4) Shore, V. G., Pardee, A. B., ANAL. CHEM.28, 1479 (1956). (5) Ven Horst. Sr. H., Jurkovich. Ir., Carstens, Y.,'Zbid., 29; 788 (1957): (6) Woiwod, .4. J., Nature 166,272 (1950).
.,
'
RECEIVED for review November 8, 1957. Accepted August 11, 1958.
Quantitative Determination of Methylal in Formalin ODD HEGGELUND and KAREL RUZICKA Gullaug Kjemiske Fabrikker AIS, Lillesfrk, Subsidiary of Norsk SpraengsfofindustriAIS, lillestr&n, Noway
A new physical method for the determination of methylal (formal) in the presence of water, formaldehyde, and methanol is based on the difference in the refractive indices of methylal and methanol. The formaldehyde reacts first with sodium sulfite. The solution is then distilled in a fractionating column. All the methylal and part of the methanol are distilled over, the refractive index is measured, and the percentage of methylal is read off from a calibration curve. Analysis time is 60 minutes.
M
is an undesired byproduct in the manufacture of formaldehyde from methanol. It is therefore desirable to determine the amount of methylal at various stages of its preparation. No simple and accurate method exists 138
ETHYLAL
0
ANALYTICAL CHEMISTRY
for the determination of methylal in a mixture containing water, formaldehyde, and methanol, without use of expensive equipment. Chemical methods based on the hydrolysis of methylal to formaldehyde and methanol, and then determination of the total amount of formaldehyde, have been proposed (11).
CH,(OCH,),
+ HzO + CHzO + 2 CHIOH
However, the equilibrium constant (6, 8) for this reaction and the small
amount of methylal as compared with the large amounts of formaldehyde and methanol usually present in the samples to be analyzed, render this method unsuitable. Other methods, based on the conversion of methylal to methyl iodide with hydroiodic acid (9-4) and the reduction of the mercuric chloride to mercurous chloride (1) are described.
These are not suitable in the presence of methanol and formaldehyde. Methylal can also be determined by mass spectrophotometry (6, 9). BASIS OF METHOD
The mixture of water, formaldehyde, methanol, and methylal first reacts with powdered sodium sulfite, is diluted with methanol, and then distilled through a fractionating column. Formaldehyde reacts with sodium sulfite to make an alkaline solution in which methylal is stable. All the methylal and part of the methanol are distilled over into a tared flask, The distillate is weighed and its refractive index is measured. The percentage of methylal is read off, from a graph for methylalmethanol mixtures, and the amount of methylal in the sample is calculated.
EQUIPMENT
Table 1.
A 1-liter round-bottomed flask, with a fractionating column 50 em. long and 2.5 em. in diameter, is filled with ceramic beads 7 mm. in diameter. The column is connected to a water condenser 50 em. long and is mounted in a slanting position (ea. 40' with the vertical). This condenser is connected to another vertical one, 25 em. long, with insulation, which is cooled with a urea-water solution to -5' C. The distillate is collected in a 50-ml. flask, cooled to -15' C. with a mixture of ice and ammonium chloride. The apparatus has ground-glass joints, because methylal dissolves rubber. Silicone Dow Corning stopcock grease was used as a lubricant. PROCEDURE
Transfer to the round-bottomed flask 100 ml. of the sample, then add 50 grams of powdered sodium sulfite and 100 ml. of methanol. Heat the contents of the flask to boiling and hold the distillation rate a t 20 to 40 drops per minute. Count the drops after they pass the first condenser After 20 minutes draw o f f the cooling water from the first condenser and distill for 10 minutes, to wash down the walls of the condenser with methanol vapor. If the content of methylal in the sample is about a%, 30 minutes of distillation time is sufficient. If the content is higher, up to S%, it is necessary to distill for 60 minutes. The distillate contains all the methylal and much methanol, but not more than 0.1% water and 0.05% formaldehyde. It is impossible to obtain a distillate without water. The graph for the refractive index of methylal-methanol mixtures was therefore made so that it corresponds to a mater content of 1.00%. The distillate is rreighed exactly, and the water content determined in a sample of about 1 gram with Karl Fischer reagent. The water content in the distillate is then adjusted by use of a microburet to 1.00%. After mixing, the refractive index is measured a t 18' C. The per cent of methylal in the adjusted distillate is determined from the calibration curve and the per cent of methylal in the original 100-ml. sample is calculated. The following points can be used for construction of the calibration curve in the graph (Table I).
+++ ++
0.0% methylal 8.0% methylal 20 .O% methylal 28.0% methylal 40.0% methylal
Data for Construction of Calibration Curve
99.0% methanol 91.0% methanol 79.0% methanol 71 .O% methanol 59.0% methanol
reproducibility of the method are illustrated in Table 111. DISCUSSION
If small amounts of methylal are to be determined, it is advantageous to use a 200-mi. sample, 100 grams of sodium sulfite, and 200 ml. of methanol. If a sample with more than 50% methanol content is to be examined, no more methanol should be added. The above amount of sodium sulfite was found suitable for formalin with 3575 formaldehyde content. The amount of sodium sulfite should be increased or decreased according to formaldehyde content of the sample. It was found that a formaldehyde content of 0.10% in the distillate increased the refractive index by 0.00015. This error of analysis decreases with increasing methylal content in the sample. In the presence of exceedingly small amounts of methylal, the refractive index measurements should be corrected, according to the content of formaldehyde in the distillate. The remaining distillate, after the refractive index measurement, is then analyzed for formaldehyde, using the sulfite method. A value of about 0.03 to 0.04% formaldehyde was normally obtained by these determinations, and the correction was calculated by interpolation.
Table
II.
Physical Properties of Methylal (7)
iluthors' Marsden Product Density, 20°/4" C. 0.8601 0.8610 Boiling point, 760 mm. HE. C. 42.3 42.3 -, n'," 1.3534 1 ,35335 Water content, % 0 0.15 Formaldehyde content, % 0 0 A z e o t r o p e me t h y la 1-methanol. B.p. 41.82' C. with 92.15% methvlal Azeotrope methylal-watir. B.i_42.05"C. with 98.60% methylal
RESULTS
Methylal produced in the laboratory according to Fischer and Giebe (10) was used. The obtained methylal was distilled three times through a fractionation column. The first portions were discarded. A product with the properties shown in Table I1 was obtained. The methanol used contained 0.007% water and had a refractive index of 1.3295 at 20' C. The accuracy and
Table Ill. Accuracy and Reproducibility of Method
Weighed Oit Methylal, G.
0.172 1.03 1.82 5.26 8.70
-
I 0.19 0.98 1.88 5.22 8.53
Found I1 0.13 0.99
i.83
5.39 8.55
I11 0.15 I ni 1.84 5.18 8.58
++ + ++
1.00% water shows nL8 1.00% water shows n1,8 1.OO% water shows n1,8 1.OO% water shows n1,8
1.00% water shows n y
1.3299 1.3317 1.33455 1.3365 1.33945
LITERATURE CITED
( I ) Bourgom, A., Bull. SOC. chim. Belg. 33, 101-15 (1924). (2) Furter, M., Helv. Chim. Acta 21, 1144
(1938). (3) Hoffman, D. O., Wolfrom, M. L., ANAL.CHEM.19, 225 (1947). (4) Johnson, P. R., Barnes, H. M., McElvain, S. M., J . Am. Chem. Soc. 62, 969 (1940). (5) Langer, A., Fox, R. E., ANAL.CHEM. 21, 1032 (1949). (6) Long, F. A., McIntyre, D., J . Am. Chem. Soc. 76, 32$3 (1954). (7) Marsden, C., Solvents and Allied Substances Manual," p. 246, CleaverHume Press, London, 1954. (8) Skrabal, A,, Eger, H. H., 2. physik. Chem. 122, 349 (1926). (9) Smith D. M., Mitchell, J., Jr., ANAL. &HEM. 22, 750 (1950). (IO) Walker, F. I., "Formaldehyde," 2nd ed., p. 203, Reinhold, Kew Pork, 1953. (11) Zbid., p. 400. RECEIVED for review May 2, 1958. Accepted September 2, 1958.
Determination of Reducing Sugars and Reducing End Groups in Polysaccharides by Reaction with Carbon14-Labeled Cyanide -Correction In the article on "Determination of Reducing Sugars and Reducing End Groups in Polysaccharides by Reaction with Carbon-14-Labeled Cyanide" [J. D. Moyer and H. S. Isbell, ANAL.CHEM. 30, 1975 (1958)] the abstract a t the beginning should have read : ,A method is presented for the determination of reducing sugars in quantities of less than 0.2 mg. and for the estimation of carbonyl groups in polysaccharides. The material is allowed to react with carbon-14-labeled cyanide, excess cyanide is volatilized as hydrogen cyanide, and the nonvolatile residue is assayed for radioactivity. Monosaccharides and some disaccharides fix one mole of cyanide per mole of sugar. Alkali-labile polysaccharides and some sugars fix more than one equivalent of cyanide per reducing end group. Mechanisms are presented to account for the high cyanide combining power of the alkali-labile substances. VOL. 31, NO. 1, JANUARY 1959
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