LITERATURE CITED
Am. SOC.Testing Materials, Standards, Pt. V, D 1158-55T, p. 595. Braae, B., ANAL.CHEM.21, 1461 (1949). CPlta, F., KuEera, Z.,Chem. Listy 47, 1166 (1953). DeFord, D. D., Record Chent. Progr. 16, 165 (1955). DuBois, H. D., Skoog, D. A., ANAL. CHEW 20, 624 (1948).
(6) Kaufmann, H. P., 2. Untersuch. Lebensm. 51 , 3 (1926). (7) Leisey, F. A., Grutsch, J. F., ANAL. CHEM.28, 1553 (1956). (8) Lucas, H. J., Pressman, D., IND. ENQ. CHEM.,ANAL. ED. 10, 140 (1938). (9) Rdsenmund, K. W., Kuhnhenn, W., Ber. 56B, 1262 (1923). (10) Siggia, S., “Quantitative Organic Analysis via Functional Groups,”
2nd etf., pp: 69-71, Wiley, New York, 1954. (11) Sweetser., P. B., Bricker, C. E., ANAL.’CHEM. 24, 1107 (1952). (12) Wilson. (2. E.. J . Inst. Petroleum 36 25 (i9!50). ’ RECEIVEDfor review August 18, 1956. Accepted December 28, 1956. Pittsburgh Conference OIL Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa. February-hlaich 1956. \
I
De rivative Polarography of Carbohydrates The Aldopentose Hydrazones JOHN
W. HAAS, Jr., and CECIL C. LYNCH
Brown laboratory, University of Delaware, Newark, Del.
b A polarographic procedure for the qualitative and quantitative determination of the aldopentoses in the concentration range from 1 X to 2 10-2M involves introduction of the sugar into a hydrazine sulfate solution buffered at pH 2.3. A single well-formed hydrazone wave i s obtained. Reduction currents are directly proportional to concentration, the ik/C constant differing with the hydrazone of each sugar. The sugars can be determined singly and in pairs with an error of less than 2% for the individual species, and from 2 to 10% for the pairs under certain limiting conditions.
x
D
URING investigations on the polar-
ography of the aldosugars, the hydrazone derivative was found to provide a rapid, accurate means of determination. As this method is extremely sensitive t o impurities, its application to pairs of sugars is definitely limited in scope to the determination of pure sugars. The work reported herein deals with the pentoses. The small reduction imves ( 2 ) obtained for the sugars are responsible for the several derivative methods which have been devised for determining these compounds. Glucose can be determined in small amounts by titrating with potassium ferricyanide and following the change in concentration of ferricyanide polarographically ( 1 ) . Sugars in tannins are determined by treating with excess Fehling’s solution, filtering off the cuprous oxide, and polarographically determining the excess copper in the filtrate (4). Also, the pentosans in cellulose have been estimated by converting them to furfural with hydro-
chloric acid and determining the furfural polarographically (6). Lupton and Lynch (7) have used the hydrazone derivative to determine a number of aliphatic aldehydes. Recently, the polarographic behavior of benzaldehyde derivatives of hydrazine, 1,l-dimethylhydrazine, and monomethylhydrazine has been investigated (9). APPARATUS AND REAGENTS
A Sargent Model XXI Polarograph was used throughout this investigation. pH measurements were obtained with a Beckman Model H-2 pH meter. The analyses were performed in a number of different conventional cells. An H-type cell similar to that described by Lingane and Laitinen (6),containing a reference saturated calomel electrode, mas used to determine El/z us. S.C.E. All experiments except those concerned with temperature coefficients were carried out a t 25 i 0.1”C. Dissolved oxygen v a s removed from the experimental solution by bubbling prepurified nitrogen through the cell for 10 to 20 minutes before measurement. The dropping mercury electrode used of had a capillary constant, m2/3t1‘6, 1.23. All reagents used mere of the best grade obtainable. Based on optical rotation data, the purity of the sugars mas estimated to be no less than 96%. The supporting electrolyte solution was 0.0528M dibasic sodium phosphate and 0.lM hydrazine sulfate. The pH of this solution was 2.3 and was maintained a t this value, inasmuch as the i k / C relationships vary with pH. Other p H values were obtained by the addition of sulfuric acid or dibasic sodium phosphate METHOD
Estimation of Individual Sugars. For all the aldopentoses the half-wave
potential is practically the same a t a given p H value. However, the ih/C values are quite different for the four sugars. Thus, the measurement of G / C for a pure mmple identifies the aldopentose, and for a known sugar, the measurement of i k permits evaluation of its concentration in a sample. Estimation of Sugar Pairs. If the i k / c values are known for two aldopentoses a t a given pH value, the amount of each can be estimated by the differenhl method in a pure mixture from nieasurement of the reduction current. The following relation must exist in the system: C = CA AI
=
ik
=
+ C E for sugars A and B
MA =: M E (All aldopentoses have the same molecular weight.)
+
-F ( i k ) ~= ( ~ M / C A ) C A (~LB/CB)CE
RESULTS AND DISCUSSION
Polarographic Wave. Well-defined and reproducible polarographic curves, stable over a period of several weeks, mer3 obtained for the hydrazone derivatives. Standard curves obtained from solutions containing the sugars in the concentration range from 1 x 10-3 to 2 x 10-2M showed a linear relalionship between diffusion current and concentration for each isomer a t p H 2.3. Table I shows in detail the polarographic data for the hydrazones Wave heights obtained for the D and L forms of each sugar mere identical. Based on relative wave heights, using the same sugar concentration hydrazone formation occurred VOL. 251, NO. 4, APRIL 1957
479
Table 1. Sugar
Polarographic Behavior of Hydrazones at pH 2.3 and 25" C. Concn., hIdI/Liter ib, pa. ik/C E1/zus. S.C.E., Volts
&Ribose
20
10
7.5 5 2.5 1
D-Lyxose
20
10
i.5
2.5 1
n-Arabinose
20
10 7.5 5 2.5 I
D-Xylose
20 10 7'.5
5 2.5 I
in the ratio of 1 to 1.48 to 2.00 to 4.34 for xylose, arabinose, lyxose, and ribose. These variations, coupled with the small values for the i k / C constants, seem to indicate that the amount of hydrazone being reduced in the equilibrium mixture is small, depending on the spatial configuration of the sugar and its hydrazone. Effect of pH. Table I1 shows the effect of p H on ib and Elit. Although the diffusion current greatly increased with increasing pH, the wave height
Table II.
Effect of pH on ik/C and € 1 1 2 of 0.01M D-Ribose pH E, 2 us. S.C.E. ik/C 1.40 -1.24 0.420 1.03 -1.26 0 .si2 2.30 -1.26 0.320 2.60 -1.1s 0.327 3.25 -1.13 0,373 4.00 -1.12 0.616 4.9s -1.16 1.76 5.30 -1.15 2.46 5.80 -1.17 3.54 6.63 -1.18 3.88
Table 111.
Comparison of i k / C Values at Two pH Values (0.OlM sugar originally present) Sugar
D-XylOSe D-Arabinose D-LyxOSe D-Ribose 480
i k / C __ - _ _
pH 2.3 0 073
0.107 0.144 0.320
pH 6.15 2.48 2.55 2.84 3.61
ANALYTICAL CHEMISTRY
6.30 3.20 2.35 1.61
0.315 0.320 0.313 0.322 0.80 0.320 0.322 0.322 Av. 0.319 2.39 0.145 1.44 0.144 1.08 0.144 0.743 0.148 0.376 0.150 0.149 0.149 Av. 0.147 2.10 0.105 1.07 0.107 ... 0.80 0.107 0.550 0.110 0.269 0.108 0.116 0.116 Av. 0.109 1.46 0.0730 0.732 0.0732 0.543 0.0724 0.0740 0.370 0.184 0.0730 0.075 0.078 Av. 0.0734
-1.26
-1.20
-1.23
~
-1.22
ratios ( i k / C ) between the hydrazones are reduced to such small differences that qualitative jifferentintions were impracticable (Table 111). A p H of 2.3 was found to give the optimum separation and was u;ed throughout these studies.
Tab1 3 IV.
Sugar Pr,irs Ribose Lyxose Ribose Lyxose Ribose Lysose Lyxose Arabinose Lyxose Arabinose Lyxose Arabinose Lyxose Arabino:3e Lyxose Arabino.;e Xylose Arabinose Xylose Arabinotie Xylose Arabino:ie Xylose Arabinor>e
Effect of Mercury Column Height and Temperature Variations. I n order to determine the electrode process, the height of the mercury column was varied between 55 and 105 cm. and the effect of these changes on ik was studied. I n all cases the diffusion current was found to be independent of the height of the mercury column, indicating that the reduction process was kinctically controlled. The effect of temperature on ik was determined over the temperature range from 16.7' to 36.7" C. The temperature coefficient computed by means of the compound interest formula was 6.8% for the four hydrazones, further demonstrating a kinetically controlled reduction process. Determination of Sugar Pairs. Because the E112 values for the hydrazones are approximately the same, mixtures of two sugars may be quantitatively estimated from the differential method described. Table IT1 presents data obtained from a number of such mixtures. It appears that the method is best utilized in situations where the i, obtained is above 1.0 pa, and the sugars are in no greater than a 4 to 1 ratio. The rather large errors found in some of the xylose-arabinose mixtures are due to the small deviation from linearity of the it/C constants in the region of lower concentration. If this is taken into account and better constants for i k / C are applied in cases of
Determination of Two-Component Mixtures
1.84
Present, hI3I 2 50 10.0 2.50 5.00 2.50 2.50 10.0 10.0 5.0
1.4G
10.00
il, pa.
2.26 1.54 1.20 2.53
0.649 0.481
IO. 00
2.50 2.50 2.50 2.50 1.oo
1.85
10.0 10.0
1.16
10.0 10.0
0.860 0.641
1.o
1 .oo
1.00 5.00
Found, hZ3f 2 40 10 0
2.54 4.00 2.47 2.53 10.3 9.7 5.39 9.6 2.57 9.9
2.24 2.74 2.62 0.88 9.5 10.5 0.99
10.1
10.6 0.41 0.42 5.58
lower concentration, accuracy is substantially improved. Reduction Process. AI cister and Major (8) have shown the amount of ring or chain form of a number of aldohexose hydrazones. B y varying the mercury column height on the hydrazones which they report, kinetic currents were found in cases of ring structure and mixed kinetic and diffusion currents in situations where both ring and chain forms were found. The hydrazones of the aldopentoses seem t o exist primarily in a nonreducible form in equilibrium with only a minute amount of the reducible chain form. During the reduction of the reducible form, equilibrium is disturbed a t the dropping electrode and new reducible form is produced. The height of the wave is thus a function of the rate constant for the kinetic process:
hydrazone(ringform)
LITERATURE CITED
+
hydrazone(chainform). This concept parallels the conclusions of Wiesner (10) and Delahay and Strassner (5) on the sugars themselves. The ease of transformation of the cyclic hydrazone based on relative wave heights a t the same concentration of sugar is similar to the ease of transformation found by Delahay and Strassner ( 3 ) and Cantor and Peniston (2) for the cyclic form of the sugar to the open aldehyde form. The results of Cantor and Peniston have to be reinterpreted according to the concept of kinetic currents. The reduction process is probably the addition of 2 hydrogen ions to the C=N- bond, a two-electron process.
,4dams, €1. N., Reilley, C. N., Furman, IT. H., ANAL. CHEW 24, 1200 (1952). Cantor, 1,. bl.,Peniston, Q. bl., J . Am. Clrem. SOC.62, 213 (1940). Delahay, P., Strassner, J. E., Ibid., 74, 893 (1952). Dlezelr, J., Xbornik dlezinbrod. Polarag. Sjezdu Prme, 1st Congr. 1951, €It. 1, 740. DomansltJf, R., Ibid., p. 730. Lingane, J. J., Laitinen, H. A., IND. ESG. (>HEM., ANAL. E D . 11, 504 (1939). Lupton, J. AI., Lynch, C. C., J . Am. Chem. SOC.66, 697 (1944). hIeister. L.. Maior. " , A.., Ibid.. 77. I
.
4297(1996).
WhitnacE:, G. C., Young, J. E., Sisler, H. H., Gantz, E. S., AXAL. CHEX 28, 833 (1956). Wiesner, I
