DIVISION OF DAIRY RESEARCH LABOR.4TORIES, BUREAU OF DAIRY INDUSTRY, AGRICULTURAL RESEARCH ADMINISTRATION, UNITEDSTATES DEPARTMENT OF AGRICULTURE]
[CONTRIBUTION FROX
EVIDENCE OF THE EXISTENCE OF BISULFITE COMPOUNDS OF
SUGARS H. H. BROWNE Received March 6, 19&
I n connection with an investigation into the possibility of fermenting the lactose in cheese whey t o glycerol, in analogy t o the much publicized (at one time) molasses fermentation, it was considered desirable after certain preliminary experiments to investigate the reaction of sodium bisulfite upon lactose, and, incidentally, upon other sugars, since many of the investigators claimed t o have used alkali bisulfites to combine with acetaldehyde, the formation of which in addition t o glycerol had been postulated as a product of this fermentation. While the bisulfite compounds of some aldehydes have been investigated fairly thoroughly and in some cases have been put to use industrially, those of the sugars have received comparatively little attention except in connection with the wine industry. Rocques (1) seems to have been the first to report upon the glucose compound, which Kerp (2) claimed t o have isolated, and from solutions of it to have determined the dissociation constant for various concentrations by methods similar to those described in this paper. About the same time Farnsteiner (3) determined the bound bisulfite for some ten sugars in one concentration with bisulfite of approximately constant concentration. Years later Tomoda and Taguchi (4)repeated certain features of the work cf both of the last two mentioned workers, and showed also that raffinose, sucrose, and D-fructose did not react. Nothing was published on the sugar-bisulfites by the workers on the glycerol fermentation, and in fact some of them used the term “sulfite” and “bisulfite” interchangeably and as a rule did not specify what sugar they were trying to ferment. General information on these compounds would be of help in developing methods of control in following the fermentation. There is also the possibility that it would yield information relative to sugar structures in general. METHODS
The sodium bisulfite and sugars used were obtained through commercial channels, the purity of the sugars being determined polarimetrically. The manufacturers’ designations of purity for materials in Table VI were accepted; the “sodium bisulfite” was found upon analysis to be the meta-bisulfite, whose anion it will be recalled dissociates in solution according to the equation SzO, HzO 2HS03. All solutions were made up in sugar (1/10 dilution) flasks, generally by adding a “10% sugar solution” to the main volume mark, then using this volume to dissolve the required weights of “bisulfite” (the sugar and bisulfite must not be dissolved together), the resulting solution then being returned to the flask and
+
477
478
H. H. RROWNE
made up to the “10%” mark with water. The final solution would then contain 100/110 of the original sugar concentration, and 100/110 (of 1.10) of the bisulfite as weighed. The bisulfite was used in quantities varying from 0.5 g. t o 30 g. per 100 ml. of the final solutions. The maximum concentration of sodium bisulfite was limited to 30 g./100 ml. due to the fact that this quantity increases the volume of 100 ml. of the sugar solutions approximately by 10 ml. All solutions were stored at 20” for about 20 hours before titrations and polarimetric readings were made at this temperature, although equilibrium was apparently established in less than two hours. The analytical procedure adopted was to titrate the “free” bisulfite with 0.1 normal iodine solution, previously standardized against arsenious oxide, by running the solution into 50 ml. of the iodine solution (diluted with about 200 ml. of water). “Bound” bisulfite was computed from the difference between the bisulfite originally added and “free” bisulfite. It may be noted that the value so computed is somewhat high due to the fact that there is a slight loss of sulfur dioxide when the sugar solution is added to the dry bisulfite. Polarimetric readings were made in a s. & H. saccharimeter equipped with a dichromate filter and using a 200-mm. tube. Iodine is not known to oxidize aldoses in acid solution, and in fact in control tests it took generally about 24 hours for the sugar solutions to reduce 1 drop of the standard iodine solution. The titrations and rotations for lactose, D-glucose, maltose, D-galactose, and D-mannose for different concentrations of bisulfite are given in Tables I to V inclusive. It will be noted that with each sugar there is a progressive lowering of the rotation and a correspondingly lower reducing value per unit concentration with increasing added bisulfite, so that the logical assumption was that the sugar and bisulfite had combined. The proof of the existence of an equilibrium constant mould be evidence of the validity of this premise. The constant K for the monomolecular reaction on h E e A B, Le., K =
+
[B1,was first computed by using the values with lactose: [ill = concentra[ ABI tion of free bisulfite determined by titration, [ B ]= concentration of free lactose = original1 actose - bound bisulfite, [ A B ] = bound bisulfite = original bisulfite - free bisulfite. When the values of this so-determined [ B ]were plotted against the observed rotation a , a nearly straight line resulted whose slope was approximabely equal to .01, or [ B ]= k a, w-hich conforms to the quantitative expression for simple polarization of sugars. The resulting inference is that only part of the sugar is optically active. I n order to express [ B ]in terms of a and a constant, use was made of thestandard relations: [ a ] = -,a moles/liter = loooc, and de2c M grees S = - this last term used as if applicable to sugars other than sucrose, .3462’ and [ a ] was treated as a constant, independent of the sugar concentration. After properly combining, etc., the value of the constant k for any sugar is given by 173.1 - and for lactose
M [ffl’
=
.00913. All symbols have their customary meaning.
BISULFITE COMPOUNDS OF SCGARS
479
I n the case of [ A B ] ,since there was no suitable direct method for determining that, resort was had t o one of three indirect methods; lst, using the difference hritmeen the bisulfite added and the bisulfite found, which equals the bound biruifite; 2nd, using the difference between the sugar added and the sugar found, which equals the bound sugar; 3rd, using the arithmetical mean of the 1st and 2nd method. While each method has its own advantages or disadvantages, in any case it is desirable to compute the constant for all silgars in the same n.ay. The results of the 1st method should equal those of the 2nd in the ideal case where no sulfur dioxide is lost and where the bisulfite does not enter into any other reaction than the monomolecular one postulated. It may be said that in the 3rd method there is the apparent advantage that any losses of sulfur dioxide nil1 raise the [ A B ] computed from the titration and lower that computed from the optical rotation so that their effects are opposite and their average should closely approximate the true condition. Where the differences between the two terms are not too great, as in the lower bisulfite concentrations, this method would be best, but as mill be seen in the case of maltose particularly, both the differences ([bound sugar] - [bound bisulfite]) are too great to ignore. Where the first. method was used for both [ B ]and [ A B ] greater , variations in the K values were found than in the method adopted. The writer in all cases has used the 2nd method, with computed K's in a column in Tables I-V, each table representing a different sugar-the different colurniis are self-explanatory. This constant is, of course, a composite one, taking in the equilibria between the bisulfite constituents, between the different forms of the sugars, and that between all of them combined. As may be seen in Tables I-V, the values of K in most cases show remarkable agreement in spite of the many equilibria involved, but the value for glucose is not in agreement with that of Kerp (2) mho found various values for various concentrations of his solid compound in aqueous Polution, which this writer believes would indicate more than one reaction, i.c. the value was not that of a constant. As somewhat of a check upon this idea, values were computed fur other reacB, and A3Bz S 3A 2B with indifferect results, tions such as A2B $ 2A although it is very possible that other reactions do take place. More corroborative evidence that this is an equilibrium reaction with the aldehyde grouping of the sugars is as follows: since it may be calculated from dissociation data that the bisulfite ion ceases t o exist at about p H 9.0 the solutions were made alkaline (blue t o thymolphthalein) witchstrong sodium hydroxide, keeping them ice-cold, then made colorless with a drop or two of glacial acetic acid, and polarized. It was found that the polarization values always went back to approximately the equivalent of what they would have been if no bisulfite had been added. This is also evidence that the sugar hats not been irreversibly altered to any extent by the bisulfite. While the writer believes that it would be of some interest t o use this reaction on other sugars in some sort of systematic way, such as the comparison of epimers (touched upon with one pair in this paper); following down a D or L series; check-
+
+
TABLE I LACTOSEHYDRATE(U.S.P.) [a] = +52.6” = +151.9” SI (B. of S.). “Free” sugar = 0.00913 (L. “Free” HSO; = 2.4985/V. pH range on polarized solutions = 2.88-4.00. Author’s sample = +151.3” S. a
‘S.
27.5 26.9 26.5 26.2 25.7 24.9 22.3 18.8 16.1 13.9 12.5 9.3 8.2
SODIUM BISULFITE
TITRATION
=v
added
“free”
rnl .
Mil
kl /I
118.2 58.0 45.4 35.6 23.3 11.5 5.4 3.55 2.56 2.02 1.21 0.98
0.0 .0288 .0577 .0721 .0962 .1442 .2884 .5769 .8562 1.1538 1.4422 2.3076 2.8840
SUGAR
“bound”
Mi1
,0077 .0147 .0171 .0261 .0368 ,0702 ,1143 .1614 .1779 .206 .244 .335
“bound”
11/I
M/l
0.0
0.0211 .0430 .0550 .0701 .lo74 .2182 ,4626 ,7038 .9759 1.236 2.064 2.549
IC
i
“free”
’
0.2511 .2456 .2419 .2392 .2346 .2273 .2036 ,1716 .1470 .1269 .1141 ,0849 ,0749
0.0
0.1242 .1125 ,0995 .0831 ,0621 .0420 .0319
0.0119 ,0247 .0411 .0621 .0822 .0923
.0055 .0092
.om
.0165 ,0238 .0475 .0795 .lo41 .1242 .1370 .1662 .1762
0.946 1.135 1.100 1.001 1.025 0.935 0.999 0.993 0.997 1.029 1.053 1.083
TABLE I-A LACTOSEHYDRATE 13.6 12.3 10.9 9.1 6.8 4.6 3.5
20.37 10.02 4.92 2.35 1.14 0.73
0.0 .1442 .2884 .5769 1.154 2.037 3.461
0.1226 .2494 .5080 1.064 2.192 -
0.0 .0216 .0390 ,0689 .090 .116
-
1.161 1.005 1.025 1.064 1.121
-
TABLE I1 D-GALACTOSE [a]= $80.5” = f232.5” S (Mfgrs’). Author’s sample = +237” S. “Free” sugar = 0.01193 a -a
“S.
43.1 41.1 40.2 37.8 37.3 34.4 26.4 18.2 13.6 11.2
SODIUM BISULFITE
TITRATION
=v
. -
ml
257 162 117 74 26.4 9.9 5.0 3.0
added
“free”
1
hl/l
SUGAR
K “bound”
“free”
“bound”
M/I
M/1
X/l
0.0
.0240 .o481 .0721 ,0962 .1442 .28% .5769 .8652 1.1538
0.0097 .0154 .0213 .0337 .0946 .2523 .4997 .8328
0.0384 .0567 .0749 .1105 .1938 .3246 .3655 .3210 480
0.5141 ,4903 .4796 .4509 .4450 .41o4 .3150 .2171 .1622 .1336
0.0
,0238 ,0345 .0632 .0691 .lo37 ,1991 .2970 .3519 .3805
_____
0.1362 .110 .1371 .1336 .150 .184 .232 .292
48 1
BISULFITE COMPOUNDS OF SUGARS
TABLE I11 D-GLUCOSE (ANHYD.) [a]= $52.5 (Mfgrs’) = +151.6” S. Author’s sample =
“S.
ml.
27.8 26.9 25.6 23.1 22.9 21.7 21.2 16.1 12.2 9.3
added
“free”
M/l
M/1
0.0278 .0561 .OS52 .1146 .1444 ,1810 ,3785 .6575 .8615
0.0
N
.0481 .0962 .1442 .1924 .NO5 .2884 .5769 .8652 1.1538
89.6
44.5 29.3 21.8 17.3 13.8 6.6 3.8 2.9
4-152.9”s.
I
SODIUM BISULFITE TITPATION
-v
=
“Free” sugar
0.0183 Q
“bound”
MI1 0.0
.0203 ,0401 ,0590 .0778 .0961 .lo74 .1984 .2077 .2923
-1 , ~
~
~
’
~
~
I
SUGAR
K “free”
M/l 0.5087 .4922 .4685 .4227 .4190 .3971 .3879 .2946 ,2232 .1702
M/1
0.0
.0165 .0402 .OS60 ,0897 .1116 .1208 .2141 .2855 .3385
I ~
j
~
0.820 .655 .420 .530 .512 .577 .438 .516 .433
TABLE IV [a]= $131” =
a
1
T
I
MALTOSE HYDRATE +378.4”S (Mfgr’s). Author’s sample = $378.9” S. “Free” sugar
T
7
1
rnl.
OS.
34.0 17.0 10.9 5.1 3.4 2.5
68.9 67.5 65.9 64.7 63.9 62.6 61.7
= 0.00366 a SODIUM BISULFITE
SUGAR
K added
“free”
M/1 0.0 .0962 .1924 ,2884 .5769 .8652 1.1538
M/1 0.0734 .1469 .2292 .4899 .7348 .9994
“bound”
“free”
M/1
M/1 0.2521 .2470 .2412 .2368 .2339 .2291 .2258
0.0
.0228 .0455 .0592 .0870 .1304 ,1544
“bound”
MI1 0.0
.0051
.om
.0153 ,0182 .0230 .0263
3.55 3.25 3.55 6.30 7.33 8.14
TABLE V
D-MANNOSE [a]= f14.2”
(B. of S.) = $41.OoS. =
Author’s sample = +41.5’S. 0.0668 a
“Free” sugar
I
a
=v
4s.
7.55 6.6 6.3
SODIUM BISULFITE
TITPATION
mi.
20.7 13.0
added
M/1 0.0 .2884 .5769
1 I
K “free”
“bound”
“free”
“bound”
MI1 0.1205 .1922
MI1
MI1 0.5043 .4409 ,4208
0.0 .Of334
-
0.1679 .3847
MI1
.OS36
0.93 .97
482
H. H. BROWNE
ing paired hexoses of the same type, such as maltose, gentiobiose, and cellobiose (paired glucoses), etc., but due to their scarcity, it is not altogether feasible a t this time, although some carbohydrates have been tried against sodium bisulfite to see whether they did react. Results are tabulated in Table VI. Referring to Table VI, it will be seen that there is no evidence of addition to mannitol which has no aldehyde group, but there is this evidence in rhamnose, which has no primary alcohol grouping but does have the aldehyde group. TABLE VI EFFECT OF 30% CONCENTRATION OF N A H S O ~ SOLUTIONS UPON ROTATION OF CARBOHYDRATES AND DERIVATIVES IN 9.09 ~ . / 1 0 0ML. SOLUTION (UXLESS NAME
OTHERWISE
YFGR’S DESIGNATION
CHANGE
-
+0.7
NOTED) REMARKS
0s.
Amygdalin
Mannitol y-Galactolactone 6-Gluconolactone L-Rhamnose Melezitose Trehalose
-
0
C.P. anhyd.
+3.9
Practical
-0.9 -3.5 -1.0
-
Readings on 4.5 g./100 ml. solution. Deposited crystals of amygdalin when regular 9.09% solution was used. Readings on 4.5 g./100 ml. solution, and slowly changing with time.
0
DISCUSSION
I n the light of the above findings, and those of previous workers, it is to be regretted that many text book writers ignore these compounds, or even deny their existence, as did Pringsheim ( 5 ) . As will be seen from the tables, some values of the “constant” justify that term better than others, that of lactose being the best and that of mannose the worst. It would appear that the proportionality constant in the expression “free sugar = ka” plays a large part in this, but not the only part. On the whole, mannose and maltose, due to their small change in rotation with change in bisulfite concentration, the first probably because of its low [a], the latter because of its large dissociation constant, are the least informative when treated with bisulfite. Conversely, with galactose, with a concentration of 0.25 g./100 ml. of bilsulfite, there is a loss of rotation of 2” S. SUMMARY
Data are presented as evidence of the existence of sugar-bisulfite compounds; the rotation in sugar solutions containing the bisulfite-ion is taken to be directly
BISULFITE COMPOUNDS OF SUGARS
483
proportional t o the concentration of the “free” sugar. The sugars worked with were : lactose, D-glucose, D-galactose, maltose, and mannose. WASHINGTON, D. C.
REFERENCES (1) (2) (3) (4) (5)
ROCQUES, J . pharm. ehim., 7 , 605 (1898). KERP,Arb. kaiserl. Gesundh., 21, 141 (1904); 26,269 (1907). FARNSTEINER, Z. Untersuch. Nahr. u. Genussm., 7 , 449 (1907). TOMODA AND TAGUCHI, J . Soc. Chem. Ind. Japan, (Suppl.) 33,434 B. (1930). PRINQSHEIM, “Chemistry of the Monosaccharides and the Polysaccharides.” Hill, N. Y. City, 1932, page 17.
McGraw
