Determination of Hydroxymethyl Groups in 1,2-GIycols and Related Substances 1. C. SPECK,
JR.,
and A. A. FORIST'
Kedzie Chemical Laboratory, Michigan State College, East Lansing, M i c h .
A procedure has been devised for determining hydroxyinethyl groups in substances in which this group is convertible to formaldehyde by periodate scission. Formaldehyde thus produced is estimated in situ by reaction with chromotropic acid after reducing iodate and excess periodate with sulfite. Reduction of the formaldehyde-chromotropic acid dye by either sulfite ion or sulfur dioxide is reversed by simple aeration of the samples. The effect of acidity of the periodate oxidation mixture on the rate of formaldehyde production has been investigated with three types of reducing sugars, and i t has been observed that formaldehyde formation is more rapid at lower acidities.
T
HE inherent usefulness of periodate oxidation for determining hydroxymethyl groups in 1,2-glycols and related substances has led to development of several improved procedures which do not require physical separation of formaldehyde production in these oxidation mixtures. Thus, for example, Reeves's method ( 7 ) involves reduction of iodate and periodate ions by arsenite followed by precipitation of formaldehyde as the dimedon ( 515-dimethyl-1,3-cyclohexanedione) derivative, whereas in the scheme devised by Corcoran and Page ( 2 ) stannous chloride is utilized for reduction of iodate and periodate before estimating formaldehyde by reaction with rhromotropic acid (4,5-dihydroxy2,7-naphthalenedisulfonic acid). Lambert and Seish ( 5 ) in developing a method for glycerol determination have combined arsenite reduction and the chromotropic acid reaction into what appears to be an excellent method for determining formaldehyde formed in periodate oxidation mixtures. The present paper describes an alternate procedure for determining hydroxymethyl groups by periodate oxidation in xhich the oxidation mixture is reduced by sodium sulfite prior to formation of the formaldehyde-chromotropic acid dye. PROCEDURE
To a mixture of 2.0 ml. of 0.3M periodic acid and 2.0 ml. of 1.OM sodium bicarbonate in a 100-ml. volumetric flask is added 1.0 ml. of a solution which is equivalent to 0.01 to 0.10 millimole of formaldehyde (after periodate Oxidation) and which is capable of reducing no more than 1.0 meq. of periodate. The oxidation is allowed to proceed at 26" C. for 1 hour. At the end of this time 5.0 ml. of 0.55Jf sodium sulfite are added to the oxidation mixture, and the resulting mixture is diluted to 100 ml. A blank solution is also prepared a t this time by adding 5.0 ml. of 0.55M sodium sulfite to a mixture of 2.0 ml. of 0.3.M periodic acid and 2.0 ml. of 1.0M sodium bicarbonate and then diluting this mixture to 100 ml. One-milliliter aliquots of the blank solution and the solution prepared from the unknown are placed in 50-ml. volumetric flasks. To each sample are added 0.5 ml. of 10% chromotropic acid and 5 ml. of 14M sulfuric acid. The flasks are then placed in a bath of boiling water and are heated a t 100" C. for 30 minutes (the stoppers are inserted in the flasks after the first moment of heating). At the end of this time the flasks are cooled, and the contents are diluted, with cooling, to 50 ml. Air, saturated with water vapor by passing it through a gas-washing tower containing water, is then bubbled through each sample for 30 minutes a t a rate of 750 to 1000 ml. per minute. Finally, the absorbance of the unknova is determined against that of the blank at 5 i 0 mp.
ples of D-glucose by the above procedure. I t may also be prepared from standard formaldehyde solutions. The curve should extrapolate to zero absorbance a t zero glucose concent,ration and it should be strictly linear over t'he recommended concentration range. EXPERl&IENTAL
Materials. D-Glucose used for these experiments was Sational Bureau of Standards dextrose (lot 4560). D-xylOSe, D-mannitol, and maltose were C.P. qualitj- obtained from the Pfanstiehl Chemical Co. Dihydroxyacetone was a I\utritional Biochemicals product, whereas d2-glyceraldehyde was prepared by the "Organic Syntheses" procedure (9). Matheson Co. practical grade chromotropic acid was purified by recrystallization from 50% (by volume) aqueous ethanol. Victor Chemical Works glycolic acid was recrystallized from methpl ethyl ketone. Periodic acid was obtained from t,he G. Frederick Smith Chemical Co. All other reagents were either C.P. or reagent grade. Apparatus. Beckman Model B and DC spectrophotometers and 1-em. Cores cells nere used for all ahsorbance measurements. Identity of the Dye. The spectrum of the dye produced from 0.1 millimole of dihydrosyacetone by the recommended procedure was compared with that formed from formaldehyde. The two spectra were nearly identical. Stability of the Dye. The dye produced from 0.1 millimole of glucose was stored in the dark. The ahsorbance of this solution measured a t different times is shown in Table I. Stability of Formaldehyde-Sulfite Solutions. The stability of the formaldehyde-sulfite solutions resulting from the sulfite reduction of the periodate oxidation mixtures was tested by preparing such a solution from 0.1 millimole of glucose according to the recommended procedure. Aliquots of this solution were removed at the times indicated in Table I1 and were then carried through the procedure for development of the chromotropic: acid dye. Effect of Experimental Conditions. Except for the variable under consideration, the recommended procedure was followed in testing the effects of variation in the aeration time, sulfuric acid concentration, and acidity of the osidation mixture. T h e results of these experiments are given in Tables 111, IV, and V. Periodate consumption values given in Table V were obtained b y the following plight modification of the method of Fleury and Lange (3). The oxidation mixture was neutralized by addition of an escess of solid sodium bicarbonate and to this misture were added 2 ml.
Table I.
Table 11.
Absorbance a t 570 m b 0.323 0.325 0.323
Stability of Formaldehyde-Sulfite Solution .Ige of Solution, Hours 0 24 48
Absorbance a t 570 mu 0 . 3 2 3 , O .324 0.325, 0.325 0.323. 0 . 3 2 4
Table 111. Effect of Aeration Time on Intensity of Dye Aeration Time, Minutes
n 10 20 30 45 60 180
A cali5ration curve is conveniently prepared by determining the absorbance of solutions obtained from the treatment of sam1
Stability of Dye Solutions
Age of Solution, Hours 0 24 48
Present address, T h e Upjohn Co., Kalamazoo, Mioh.
1942
Absorbance a t 570
n.. _ 17.5 .. 0.270 0.289 0.301 0.305 0.302 0.301
iufi
V O L U M E 26, NO. 1 2 , D E C E M B E R 1 9 5 4
1943
Table IV. Effect of Sulfuric Acid Concentration on Intensity of Chromotropic Acid-Formaldehyde Dyeo Absorbance a t 570 mpb dl-Glyceraldehyde ___. Oxidized, Me. 18M HiSOa 14M HzSOa 9.0 0.300 0.301 7.2 0.239 0,238 5.4 0.179 0.179 3.6 0.120 0.121 a Concentration given is t h a t of sulfuric acid added for developing dye. b Average of three samples. ~~~
Table V. Effect of Acid Oxidation on Formaldehyde Production and Periodate Consumption Oxidation Time, Hours
D-Glucose, % HCHO" 104-b
1
63 63
99.0 99.2
The present method has given good results with a variety of polyhydroxy compounds, as is shown by the data in Table VII. The precision in the analysis of 38 glucose samples reported here is within i1.1%. Maltose gave high values (Table VIII), based on the production of 1 mole of formaldehyde per mole of sugar, which increased upon extending the oxidation time. Hence, hydrolysis of the maltose scission product ( I ) must proceed under these conditions (periodic acid-sodium bicarbonate mixture) so as to permit further oxidation by periodate with production of a second mole of formaldehyde. CHO
D-Xylose, % DL-Glyceraldehyde, % HCHO" IO4-a HCHOa 1 0 1 - b 59
98.4 98.4 98.1
88 99.2 .. ... 88 99.3 97 99.4 97 99.4 4 100 99.4 .. 100 100.0 P e r w r i t of theoretical forinsldcl.vil~iornled. b Per rent of theoretical prriodnte bonsumed. 2
... ...
3
91
... 100 100
HbO, I
I
