Reaction of Sodium Chlorite with Various Polysaccharides Rate Studies and Aldehyde Group Deterrninations HERBERT F. LAUNER and YOSHIO TOMIMATSU Western Regional Research laboratory, Albany, Calif.
Aldehyde contents and degree of polymerization values of polysaccharides were calculated from chlorite results obtained under various conditions and using the corresponding stoichiometric ratios determined for substances of low molecular weight. The polysaccharides were three native dextrans, a series of hydrolyzed dextrans including clinical dextran, a periodate dextran, a series of araban fractions, and starches including pea, wheat, and rice amylopectin, and a series of periodate cornstarch products. The results with various chlorite procedures were self-consistent and agreed well with those of other chemical methods, and fairly well with those of light-scattering for fractionated polymers. For native dextrans, where no agreement was to b e expected, the large divergence demonstrated inhomogeneity of substance. Agreement with osmotic pressure results was fair to poor.
T
oxidation by sodium chlorite in phosphate buffer of a variety of mono- and disaccharides and benzaldehyde was recently studied (12) with respect to the chlorite-aldehyde stoichiometry as affected by buffer strength, reaction rate, and concentration of reactants. Various experimental conditions employing sodium chlorite were dweloped for application t o t h e determination of aldehyde groups in polysaccharides. This communication describes the study of the rates of oxidation under those conditions and others of a series of periodate cornstarches; of a series of dextrans, including native, periodate, clinical, and more extensively acidhydrolyzed; and of a series of arabans. It describes the determination of degree of polymerization of these (except periodate dextran and periodate starch) and of amylopectins by determining aldehyde end groups with chlorite. HE
SUBSTANCES STUDIED
The pol>saccharides, with one exception, were produced and made
Substances Studied
Substance Dextran A [NRRL B-512 XIV.2144-1,2] Hydrolyzed dextrans [111 B 2500-14, I11 A 250013, I11 A 2500-22, IA 2500-21, 1.4 2500-21 Expts. 1 to 5, resp. Clinical dextran [P.P.
Prepared by Prepared from Jeanes, Wilham, Miers Leuconostoc mesenteroides
Dextran-B [NRRL B-512-E, S-R,3795-23]
Jeanes et al. Wilham et al.
Dextran C [XRRL B-1254, S-R,3795-501
Jeanes et al. Wilham et al.
Periodate dextran [3793-42-B]
Sloan et al. Jeanes and Wilham
Periodate starch [series 39'781 [parent starch SD149, 1, 5, 10, 20, 40, 80, 100, 100-R, 100-0,] EX ts. 13 to 22, resp. +able 111 Pea amylopectin Wheat amylopectin Rice amylopectin
Sloan et
58-R. R-2-hl
Jeanes, Schielta, Wilham Wilham and Jeanes
Dextran A by controlled treatment with dilute HzSOl and fractionation
Rolff et al.
Dextran comparable to dextran A _.
aZ.a
Ref. (10)
(8) (26) (28)
(7)
Fraction of undegraded (26) enzvmaticallv" synthesized " macerial Fraction of undegraded product of Streptobacterium ( 7 ) dextranicum
Oxidation of dextran B512 F with sodium metaperiodate Cornstarch
Potter et a1.b Smooth Seeded Alaska peas Potter and Hassida Launer and Tomimatsub hlochi-Come rice starch by method of Schoch Goodban and Owensb Sugar beets
Araban fractions [7-11, 4-6, 31 Expts. 23,24,25, resp. Figure 3 A similar product was discussed by Jeanes and Wilham (9). Prepared at this laboratory.
(25) (21)
(22)
(16)
(15) (20)
(4)
5
available by others. The materials listed in the table were prepared at the Northern Regional Research Laboratory, Peoria, Ill. Notaticns in brackets are the original ones used by the workers who synthesized or produced the materials. I n all but Experiments 21 and 22 (Table 111) stock "solutions" were prepared of the polysaccharides and then samples were pipetted for rate studies or analysis. Clear solutions were obtained in water at 25" C. of the arabans, hydrolyzed dextrans, clinical dextran, dextran C, and periodated cornstarches containing less than 0.5 mole of aldehyde per anhydroglucose unit. Wheat and rice amylopectins gave clear solutions after a few minutes' immersion in a boiling water bath. D e d r a n B gave a turbid dispersion in water at 25" C., and periodate dextran
at 50" C. in spite of very low concentrations. Pea amylopectin gave a turbid dispersion even after 15 minutes' immersion in the boiling water bath. Dextran A at 5.7 and 23 mg. per ml. gave turbid dispersions in treatments described later. Polysaccharide concentrations in the reacting media covered a wide range according to aldehyde content expected eithrr from other data or from preliminary experiment (mg. per ml.) : periodate dextran 0.004 to 0.02, periodate starch 0.009 to 2, hydrolyzed dextrans 0.1 to 0 . 7 , clinical dextran 2 to 6, amylopectins 2 to 4 , arabans 5 , and native dextrans 6 to 23. Of periodate dextran, for example, only 0.6 to 0.8 mg. was consumed in establishing an entire rate curve of 8 to 10 points. Other amounts were proportionately higher. hIoisture contents were determined by VOL. 31, NO. 9, SEPTEMBER 1959
1569
0 0
E
-020
-
Figure 1. Rate of reaction of chlorite with polysaccharides and glucose under various conditions
16 15 14
1 to 5. Hydrolyzed dextran of various short chain lengths 6. Clinical dextran 7. Slightly hydrolyzed native dextran A 1 2 and 12’. Periodate dextran 8. Glucose 26. Pea amylopectin Ordinates of curves 7, 8, 12, 1 2 I , and 2 6 changed to permit inclusion in this figure. Numerical results given in Tables I, 11, and I V Conditions. 3M for curves 1,2,3,, 4, 12; 0.5M 50’ for curves 5, 6 , 2 6 ; 0.5M for curves 7, 8, 1 2
vacuum desiccation for a week or more at room temperature over anhydrous magnesium perchlorate. I n this communication the term experiment denotes one or more experimental procedures applied to a given substance. Curve numbers refer to experiments of the same number. EXPERIMEHTAL PROCEDURE
All determinations of aldehyde groups, except in arabans, were made with chlorite methods previously developed by the authors (11-13). Rate experiments were conducted either by removing aliquots from the reaction vessel or, when feasible, by automatically transferring vessels containing individual aliquots from the bath of reaction temperature to an ice bath. I n the rate experiments one test and one control were analyzed a t each time interval; in the others four to six replicates of test and control were usually analyzed. The rate data were treated as previously described: The reciprocals of titration volumes (milliliters of thiosulfate of normality A‘) were plotted against time. Points were taken from the smoothed curves of test and control t o calculate with Equation 1 the millimoles of chlorite per gram of polysaccharide, which, plotted against time, gave the rate curves of Figures 1 to 3 . Millimoles of chlorite per gram of poly-
1570
ANALYTICAL CHEMISTRY
I o
HOURS
2 c
Figure 2. Rate of reaction of chlorite with periodate cornstarch of various aldehyde contents under various conditions Ordinates of curve 1 9 ’ were divided by 2. Numerical results a r e given in Table Ill, including that for parent unoxidized cornstarch. Conditions. 3M for oIi curves excepting O.5M 2 5 ’ for curve 19’
where V,, V T ,and V , are the titration volumes corresponding to initial and subsequent test and control aliquots, and W is the grams of polysaccharide per aliquot. Various conditions were used (1%’) : “3M” indicates 3M phosphate buffer, pH 3.2, 50’ C., 0.0020M sodium chlorite, 0.0001M aldehyde groups; “ 0 . 5 M , 50”” indicates 0.5M buffer, pH 2.4, 50” C., 0.00080M sodium chlorite, 0.0001M aldehyde groups; “0.5M, 25’” indicates 0.5M buffer, p H 3.1, 25’ C., 0.010M sodium chlorite, 0.0005M aldehyde groups. A modified 3M procedure (%‘S), “3M, 2 5 O , ” indicates 3M phosphate buffer, p H 3.2, 25” C., 0.035M sodium chlorite, 0.0002 to 0.0006M aldehyde groups. As the stoichiometry varies with conditions, various chlorite-aldehyde ratios or factors were used to calculate degree of polymerization from the values of Equation 1. I n the 3M procedure the aldehyde content was calculated in all cases by dividing the 16-hour chlorite value, taken either from rate curves of Figures 1 and 2 or from replicate determinations after a 16-hour reaction time, by the factor 2.53. This was the principal procedure used. I n the 0.5M procedures the previously
determined chlorite-aidehyde ratio of an aldehyde or aldose n a s used whose rate of oxidation most nearly equaled that of the polymer being studied, :on the basis of the observation that the ratio is related to the rate pnder these experimental conditions ( I d ) . Thus, for a given experiment, a plot of Equation 1 values against time was usually drawn, and its maximum value was then divided by the R, value (maximum value of the chlorite-aldehyde ratio) from the most nearly appropriate R US. time curve, Not all curves for all experiments mentioned are shown and in a few experiments only thrge points around the maximum were determined. ‘CTnder the 0.5M, 25” conditions, curve 12’ for periodate dextran reached maximum in 1 to 1.5 hours, resembling that of benzaldehyde whose R, value, 3.78, was therefore used to calculate aldehyde content. For periodate starch curve 19’ reached maximum in approximately 8 hours, resembling that for xylose \\hose R,, 3.56, was used. For dextran A curve 7 reached maximum in 16 hours and therefore the R , for mannose, 3.50, n as used. Under the 0 . 5 M . 50” conditions, periodate dextran was oxidized in less than 1 hour at approximately the same rate as benzaldehyde, so that the factor for the latter, 3.5, \?asused to calculate the corresponding results in Table I. For the end-group oxidatlons of high polymers curves 5,6, and 26 reached maxima
ntxar 16 hours, resembling rhamnose, whose R,, 3.3, F a s thus used for this group. These R, values were given in the previous paper ( l a ) . I n the case of the arabans somewhat different experimental conditions, 3M, 2 5 O , were used, as explained in the section dealing with these unusually acid-sensitive substances, and the factor of 2.53 was applied to the 6-hour chloritr values. DP values (anhydroglucose units or AGU's per aldehyde group) mere calculated from Equation 2
+
yC DP = R 162 C
whrre R is the chlorite-aldehyde ratio, C is the moles of chlorite per gram of polysaccharide from Equation 1, and 162 is the weight of the AGU (132 for the anhydroarabinose unit). Y is the wight-loss per aldehyde upon periodation: 16 for dextran, 1 For starch, and zern for no periodation S o attempt was made to correct any results for residual aldehyde (IZ),which ranges from 0.2 to approximately 1.5% nearly proportional to the rate of oxidation of the polysaccharide. Light-scattering measurements were made using a modified Brice-type photometer. The calibration of this instrument and the experimental details for thc araban fractions have been drscribed (23) Water solutions of the de,xtrans were clarified b y centrifugation, followed bv filtration through sinterctl-glass filters directly into cylindrical scattrring cells. Angular scattering measurements n r r e made with incident light of wave length 436 mp. Molecular weights were calculated from the intercept of Zimm plots. A measured value of the refractive increment, dn/dc, of 0.1505 was used. Depolarization of scattered light was negligible for the deutrans. RESULTS OF THREE CHLORITE PROCEDURES
Pr:icticallv all of the subqtancw n-cre studied with the 0 5-11 procedures before the 3 M procedurc was developed and reapplied to as many substances as feasible. For most applications the 3M procedure was preferred because no rate ciirves but onlv 16-hoiir points arc requircd and because of the fundamental advantage that much lower acidity is requirrd for a given rate of oxidation at the higher phosphate concentration. An attempt hac hern made to compare the results of the three procedures. although the experiments were not undertnken with such a comparison in mind and are thuq fragmentary. The results for four substances studied with two or more of the procedures are given in Table I For this purpose the results m e eupresseri as monomer units per
6
10
nouus
Figure 3. Rate of reaction of chlorite with various araban fractions under ordinary 3M (curve 24') and modified 3M, 25' conditions DP values corresponding to 6-hour points on curves 23, 24, and 25 a r e 67, 1 13, and 181, respectively
aldehyde group corresponding to degree of polymerization for polymers having only aldehyde end groups. The slightly lower values at 3M shown b y some of the results may be due to either slightly incorrect chlorite-aldehyde factors or more complete reaction at 3 M . Hydrolysis in the 3M systems appears unlikely as an explanation because the rate curves give no evidence of continued reaction for any of the substances. The spread in values for periodate dextran under the 0.5M 50" conditions is not typical. Each stock solution of periodate dextran was made u p with some 30 mg. from the original 1-gram sample. This imposes a severe test of homogeneity and it is possible that the sample was inhomogeneous on this small scale. Also, these experiments were conducted over a period of 2l/2 years and only one experiment (giving the result 0.540) was designed for maximum precision of points under 1 hour, the critical region for this rapidly oxidized substance, whereas the other experiments required more or less extrapolation. The 0.5M
Table I. Values for Anhydroglucose" Units per Aldehyde Group by Three Chlorite Proceduresb
Material Hydrolyzed dextran (1)c
331
0.5M, 50" C.
05M,
25" C
13 19 22
(2) (3) (4)
53
Clinical dextran ( 6 ) Periodate dextran (12)
...
367
0 . 564d
0 562 0.572 RIean 0.567
337 0.592 0,540 0.522 0 594
Mean 0.562
Periodate starch (17) (18)
2.w 1 13
2.47 ... 1.28 ... 0,600 0.615 ... (19) ... (20) 0.546 0.562 a Monomers of highly modified polymers are referred to as "anhydroglucose" units for convenience, although they are no longer glucose units. Symbols designating three chlorite procedures are described under "Experimental Procedure." Numbers in parentheses refer t o experiments listed in this and other tables. Reciprocals of more usual "aldehyde groups per PIGU." Table 11.
Degree of Polymerization of Dextrans
-
Expt. 1 2 3 4
5 6
9 10
Dextran Hydrolyzed Hydrolyzed Hydrolyzed Hvdrolvzed Hkdrol' zed ~~inica?' A
B
Chlorite Procedure 351 3M
3.11 3M 0 5A1, 50" 0 5M, 50" 0 5M,25O 0 5M,25O 0 5M,25O
11 C Light-scattering cannot be expected to group methods for these substances. a
Degree of Polvmerization Copper Sodium tartrate Jighta chlorite (26) scat teririg 13 13 ... 16 18 ... 20 19 ... 47 44 *.. 74 66 ... 337 263 10,700 2250 600 600 5,400 90.000 ... 2,800 ... give results comparable with those of end-
:
VOL. 31, NO. 9, SEPTEMBER 1959
1571
25" value for clinical dextran was derived from analyses at only two periods and is probably too high. In spite of the only moderate suitability of the results for comparative purposes, it is apparent that no large errors resulted from the assumptions made in the three procedures. EXPERIMENTS A N D DISCUSSION
Hydrolyzed Dextran. When dext r a n undergoes hydrolysis at t h e 1,6 main-chain or 1,4 and 1,3 sidechain linkages, a reducing end group appears at the liberated 1-position. Curves 1 t o 6, Figure 1, representing the rates a t which chlorite reacted with a series of controlled-hydrolysis products of dextran, show no difference in rate, judging by the position of the maxima when the relative rates of oxidation under the various conditions (12) are taken into account. For comparison with the others the ordinates of curves 5, 6, and 26 must be multiplied by 2.53/3.3, the R, values pertaining to the conditions given in the legend of Figure 1. The degree of polymerization values calculated from these curves are given in Table I1 and may be compared with those obtained with a copper tartrate (Somogyi) method by Wilham and Jeanes (26). Discrepancies between the two end-group methods become pronounced for the higher polymers, probably reflecting hydrolysis in the alkaline medium of the copper tartrate reagent, which occurs in the case of the higher polymeric, and therefore more alkali-sensitive, polysaccharides. Dextrans A, B, and C. Dextran A, comparable with t h e native parent substance of t h e hydrolyzed dextrans just discussed, contains extremely large molecules, D P = 6 X lo6, as determined from light scattering. When determined by measuring end groups with the 0.5M, 25" chlorite method (Experiment 9, Table 11) D P = 1.07 X lo4 was found. The divergence between the two values arises from the molecular-size heterogeneity of dextran A, light-scattering responding mainly to the large molecules and end-group methods mainly to the small molecules. These end-group results, heretofore not applicable to such high polymers because of insufficient sensitivity of previous end-group methods, may be considered to establish a minimum DP value, assuming each molecule to have an aldehyde end group. Such heterogeneity was previously indicated by fractional precipitation procedures applied to this same type of dextran (2b). Dextran A, carefully hydrolyzed and converted to clinical dextrans with acid and then fractionated with ethyl alcohol, showed dif1572
ANALYTICAL CHEMISTRY
ferences between number and weightaverage molecular weights, thus demonstrating heterogeneity (28). Furthermore, a light-scattering technique applied to various fractions of a native dextran gave a wide range of D P values (1) from 7.8 to 370 ( X lo4), demonstrating heterogeneity. A residue of "low molecular weight impurities," constituting 6% of the total, was not measured ( 1 ) but it can be calculated that if only 1% of the total had a DP = 100, differences in degree of polymerization by light-scattering and end-group methods would be accounted for. Glucose as an impurity of 0.01% would account for the difference but for the fact that curve 7, Figure 1, for Dextran A reached a maximum in 16 hours, whereas that for glucose (curve 8) required 50 hours under the same conditions. A rather low D P value, 4400, derives from curve 7, which shows the similarity with the curves for the much lower polymers. Experiment 7 was, for the sake of precision, conducted with 'a high concentration of dextran A, 23 mg. per ml., which was heated for 1 hour in the boiling water bath in an unsuccessful effort to obtain a water-clear solution. I t is probably that some hydrolysis resulted, leading to the low DP value for experiment 7. I n experiment 9, on the other hand, the dispersion with 5.7 mg. per ml. was not heated, and the D P value was calculated from three sets of quadruplicate tests and controls on three different days. obtained from the same solution over 12 days. During this period there was no evidence of hydrolysis of dextran A a t room temperature. Table I1 also shows D P values for dextran B, an enzymatic preparation from the same organism, and for dextran C, produced by a different microorganism. Dextran B was found to have a light-scattering DP of 9 X lo4, which, compared with the degree of polymerization by the chlorite method, demonstrates heterogeneity. Periodate Dextran. Most of t h e glucose units of this highly oxidized polymer have aldehyde groups on the 2,4 carbon atoms, carbon atoms 3 having been converted t o formic acid b y the periodate. Curve 12, Figure 1, for periodate dextran shows t h a t t h e dialdehyde groups in dextran react some ten times as rapidly as end-group aldehydes with chlorite under these conditions, with, no apparent difference in rate of oxidation between the aldehyde groups in the 2 and 4 positions, in contrast to the conclusions of others (3, 27) for periodate cellulose. Curve 12' for periodate dextran was obtained under different conditions, giving ordinates higher than those of curve 12 (both reduced to facilitate inclusion in Figure l ) , but the
corresponding chlorite-aldehyde ratios 3.78 and 2.53, gave good agreement, 1.78 and 1.77 aldehydes per anhydroglucose unit. These values appear reasonable on the basis of periodation (21) and methylation studies (24) in which it was established that this dextran has about 5% 1,3 linkages, incapable of dialdehyde formation, so that the theoretical maximum for complete periodation is 1.9. Periodate Cornstarch. T h e rate curve6 for t h e oxidation of periodate starch are shown in Figure 2. T h e curves for the more highly oxidized polymers, 19 and 20, closely resemble curve 12, Figure 1, for dextran dialdehyde in time of reaching maximum. This is in contrast to the results under 0.5M, 25" conditions (curve 19'), for example, under which the periodate starches react much more slowly than periodate dextran. The different conditions did not affect the final results, however, which, because of the application of corresponding chlorite-aldehyde factors, were found to be 1.63 and 1.67 aldehyde groups per anhydroglucose unit, for curves 19' and 19, respectively. The less highly oxidized periodate starches react more slowly, the curves reaching maxima in 3 to 5 hours, but in no experiment is there evidence of any difference in rate of oxidation of the aldehydes in positions 2 and 3 in agreement with the authors' findings for the ring aldehydes in periodate dextran. The values for aldehyde content calculated from the rate curves of Figure 2 are given in Table 111, which also includes aldehyde values obtained by other m-orkers by other methods for these same samples. Oxidation of these same samples with chlorite under very different conditions (6) yielded upn ard of 95% conversion of dialdehyde to dicarboxyl groups. The samples containing less than 0.5 mole of aldehyde r e r anhydroglucose unit dispersed easily in water at room temperature; the otLers required a week with occasional agitation, or 10 to 30 minutes in a boiling water bath. Both hot and cold dispersion gave the same results in experiments 18 and 19, for example, which also agreed with the results of experiments in heterogeneous systems, wherein the samples n ere initially undispersed. However, two samples (experiments 21 and 2 2 ) , M hich in preparation had been treated with periodate for long periods (22), gave aldehyde values above 2 per anhydroglucose unit when dispersed in the boiling water bath. As these samples would not disperse a t all at room temperature, heterogeneous systems mere resorted to. The sigmoid curves (21 and 22, Figure 2) show the effect of slow dispersion of the solid material at the 50' C. reaction temperature. Under these conditions experiment 22 yielded a
~
value which agreed with the other results for this sample. However, even under these conditions the value 2.3 was obtained in experiment 21, corresponding to a large amount of aldehyde, 0.3 mole per anhydroglucose unit, in excess of the dialdehyde groups. The value 2.3 was obtained in experiment 21, in both the homogeneous system, in which the sample was dispersed a t 100" and the heterogeneous system, in which the material was not previously dispersed, indicating that the excess aldehyde existed before dispersion (which seems unlikely because of the other results of Table III), or that decomposition occurred in the reacting medium. If the latter is a function of the severe periodation nrocess (22) of this sample, it is difficult to understand why the sample of experiment 22, e-iposed during synthesis to the sodium nietaperiodate almost three times as long, did not show the same or worse behavior. This apparent contradiction may be due to experimental error, but the writers have not bpen in a position to investigate this point further. Arabaus. This polvmer of arabinose is unusual in t h a t it is considered t o he very stable in alkali but subject t o hydrolysis in weak acids. I n a study (23) of the correlation between molecular weight valiies obtained with light-scattering and end-group methods it was found that araban fraction (4-6) probably tvpical, hydrolyzed approximately 18% during the 16hour period under the 3 M conditions, and no definite end point was reached. An attempt was made to use an alkaline hypoiodite method (Z), but this gave very low inconstant values. The 3.M chlorite procedure was therefore modified to the 3 M , 25" conditions: By lowering the temperature to 25" the rate of hydrolysis was considerably decreased. The likewise-decreased oxidation rate was overcompensated for hv using 0.035M instead of 0.002M chlorite, thus reducing the time for oxidation from 16 to 6 hours, during which period hydrolysis was rendered negligible. This manner of modifying the conditions for the chlorite reaction could be resorted to for any acid-sensitive substance. The rate of o-iidation of the arahan fractions bv this chlorite procedure especially designed for acid-sensitive materials is shown by ciirves 23, 24, and 25, Figure 3. The result of using the unmodified 3M conditions (curve 24') is also shown, demonstrating acid hvdrolysis for conditions under which all of the other substanccs studied were very stable. Although the higher chlorite concentration resulted in lower precision, the precision was still adequate, and probably higher than the accuracy in
c.,
Table 111.
____
_________
~~
Aldehyde Content of Periodate Starches'
Aldehyde Groups per AGU HydroxylPeriodate SaOH Borohyamine ( 2 2 ) used ( 2 2 ) (5) dride ( 1 7 )
Expt.
Chlorite
13b 14
0 00091 0 0256
0 04
0 02
0 028
20 21 22
1 83 2 3OC 1 90
1 74 1 90 1 80
1 84 2 00 2 04
1 72 1 92 1 86
Reperiox tion ( 1 7 )
0 01
1 94 1 90 1 94
1 94 1 92 1 94
Chlorite values are original data obtained with 3M procedure: all other values were obtained for same samples by other workers identified nith literature references at column headings. Parent starch not treated with periodate. c See text. this case. The chlorite-aldehyde factor, 2.53, was applied to the 6-hour values on the curves, although the experimental conditions for the arabans differed somewhat from those under which the factor had been established. Whereas it was found that the buffer concentration essentially determines the value of the factor (12) and an experiment with glucose gave results in agreement with those under the 50" conditions, application of the factor to modified conditions must be considered an approximation. Obviously, however, studies with known sugars could be used to establish a factor for these conditions, the important point being that hydrolysis was avoided. In contrast to the results with hydrolyzed dextrans, the rate of glucose oxidation was higher than that of the arabans, owing perhaps to the differing ring structure. The DP values of the arabans given in the legend of Figure 3 mav be compared with those found by light-scattering, which were 139, 183, and 280 (23) in the same order. The relative similarity of the results of number and weight average methods indicates that while the individual fractions were not homogeneous they were considerably more so than the native dextrans, but similar to the clinical, ethyl alcoholfractionated dextrans (28). Amylopectins. These carbohvdrates reacted with chlorite at about t h e same rate as did the hydrolyzed devtrans under the same conditions (curves 5 and 6. Figure 3 , which also includes curve 26 for pea amylopectin, typical of this group). As these substances represent greatly different chain lengths and chemical properties, it appears that the rate of oxidation of the aldehyde end group is fairly independent thereof, except glucose. m-hose rate of ovidation is less than one half as great. Anal.ytical data and degree of polymerization calculated from curve 26 and others not shown are given in Table IV. The value for pea amylo-
Table IV.
Degree of Polymerization of Amylopectins
Experiment 26 Smoothpea 27 Wheat ?8 Rice
llillimole Chlorite/'Grani Carbohydrate
DP
0 0063 0 0103 0 0077
5900 2Ooo 2600
pectin, 3900, is in rough agreement with 6700, obtained from osmotic pressure measurements on the same sample (16). On the other hand, the value for wheat amylopectin, 2000, differs considerably from the DP of 25,000 b y osmotic pressure measurements of the same sample (15). The osmotic pressure measurements were made on acetylated specimens in chloroform, whereas the chlorite measurements were made on the untreated material in aqueous solution. It is conceivable that aggregation and loss of low-polymeric material during acetylation may have caused the difference in results. CONCLUSIONS
Aldehyde contents and D P values of high polymers were calculated from chlorite results using stoichiometric ratios previously determined for substances of low molecular R eight. Chlorite procedures inrolving various R values were used. The results could be checked in several ways. The results of three chlorite procedures involving considerably different R values especially for periodate dextran were self-consistent. Chlorite results agreed with thoso of other chemical methods. The chlorite result for periodate dextran, 1.8 aldehyde groups per anhydroglucose unit, was compatible with the upper limit, 1.9, established by entirely different principlrs. The agreement between D P values of dextrans using chlorite and copper tartrate methods n a s good to DP = 50. ThPreafter, incrmsing VOL. 3 1 , NO. 9, SEPTEMBER 1959
1573
chain length, accompanied by increasing alkali sensitivity, caused low copper tartrate DP values. The agreement between the values for aldehyde groups per anhydroglucose unit in periodate starch by the chlorite and a variety of other methods, including the acidic hydroxylamine and four alkaline methods, was very good. The agreement with the alkaline methods was contrary to expectations, as the alkali sensitivity of periodate starch is the basis for a method (5) of determining aldehyde groups. Furthermore, periodate cellulose has been found to be very sensitive to alkali (14, 18, 19). A discussion of the failure of other a k a line methods, copper tartrate and hypoiodite, for periodate starch, is intended for future publication. Comparison of chlorite values with those of physical methods showed only limited agreement, as could be expected. The agreement with light-scattering values ranged from fair, for moderately homogeneous fractions of arabans, to no agreement a t all for inhomogeneous native dextrans. The agreement with osmotic pressure values of amylopectins ranged from fair to very poor, the latter probably due to differences in pretreatment of samples. The application of the R values of substances of low molecular weight to high polymers is encouraged not only by these cases of agreement and selfconsistency, but also by rate considerations. The rate curves for the oxidation of the aldehyde groups in benzaldehyde and mono- and disaccharide pentoses and hexoses ( I d ) are indistinguishable in shape from those of the polysaccharides. This can be seen by comparing the behavior with time of the R function of the previous work with the present AC/W, the two being proportional,
AC/W = R X millimoles of aldehyde per gram of polysaccharide. Also, the times required to reach curve maxima were comparable. The aldehyde end groups in more or less hydrolyzed dextrans and in amylopectins were actually oxidized at a more uniform rate than the various aldoses, when the rate differences among the chlorite conditions were taken into account ( I d ) . Periodated products reacted much more rapidly with chlorite than the parent dextran or cornstarch, demonstrating the difference between aldehyde groups at positions 1, 2, 3, and 4, although no difference between the two members of a dialdehyde group was observed. The rate of oxidation of periodate cornstarch decreased a-ith the extent of periodation, indicating a hindrance to the chlorite by unoxidized chain members. The aldehyde groups of the extensively periodated dextran and starch were oxidized at nearly the same rate as in benzaldehyde. ACKNOWLEDGMENT
The authors acknowledge helpful discussions with R. M. McCready, and also the cooperation of Allene Jeanes, I. A. Rolff, A. L. Potter, E. B. Kester, and A. E. Goodban in furnishing samples. LITERATURE CITED
(1) Arond, L. H., Frank, H. P., J . Phys. Chem. 58,953 (1954). (2) Blom, J., Rosted, C. O., dcta Chem. ' Scund. 1,32(1947).'
(3) Davidson, G. F., Nevell, T. P., J . Textile Inst. 46. T407 11955). (4) Goodban, A4.' E., Owens,'H. S., J . Polymer Sci. 23, 825 (1957). (5) Hofreiter, B. T., Alexander, B. H., Wolff. I. A,, ANAL. CHEM.27, 1930 (1955). ' (6) Hofreiter, B.
T.,Wolff, I. A,, Mehltretter, C. L., J . Am. Chem. Soe. 79, 6457 (1957).
( 7 ) Jeanes, A., Haynes, W.C., Wilham C. A., Rankin, J. C., Jfelvin, E . H., Austin, M. J., Cluskey, J. E., Fisher, B. E., Tsuchiva, H. M., Rist, C. E., Ibid., 76, 504i (i954). (8) . , Jeanes. A.. Schieltz. N. C.. Wilham.' C. A., J : B i d . Chem. 176, Slf(1948). (9) Jeanes, A,, Wilham, C. A., J . Am. Chem. SOC.72, 2655 (1950). (10) Jeanes, A,, Wilham, C. A,, Irliers, J. C., J . Biol. Chem. 176, 603 (1948). (11) Launer, H. F., Tomimatsu, Y . ,ANAL. CHEM.26, 382 (1954). (12) Ibid., 31, 1385(1959). (13'1 Launer. H. F.. Tomimatsu. Y..' ' J . A m . ChemrSoc.26, 2591 (1954): (14) Meller, A,, Tappi 34, 171 (1951). (15) Potter, rl. L.,Hassid, W. Z., J . Am. Chem. SOC.76.3488. 3774(1948). (16) Potter, A,' L., 'Silveira, V., McCready, R. M.,Owens, H. S., Ibid., 75, 1335 (1953). (17) Rankin, J . C., Mehltretter, C. L., ANAL.CHEM.28, 1012 (1956). (18) Reeves, R. E., Ind. Eng. Chem. 35, 1281 (1943). (19) Rutherford, H. A., Minor, F. W., Martin, A. R., Harris, iM.,J . Research Xatl. Bur. Standards 29, 131 (1942). (20) Schoch, T. J., Advances in Carbohydrate Chem. 1 , 247-77 (1945). (21) Sloan, J. W., Alexander, B. H., Lohmar, R. L., Wolff, I. A,, Rist, C. E., J . Am. Chem. SOC.76, 4429 (1954). (22) Sloan, J. W., Hofreiter, B. T., hfellies, R. L., Wolff, I. A., Ind. Eng. Chem. 48, 1165 (1956). (23) Tomimatsu, Y . ,Palmer, K. J., Goodban, A. E., Ward, W. H., J . Polymer Sci. 36,129 (1959). (24) Van Cleve, J . W., Schaefer, W. C., Rist, C. E., J . Am. Chem. SOC.78, 4435 (1956). (25) Wilham, C. A., Alexander, B. H., Jeanes, Allene, Arch. Biochem. Biophys. 59, 61 (1955). (26) Wilham, C. A., Jeanes, Allene, un-
published results.
(27) Wilson, W. K., Padget, A. A., Tappi 38, 292 (1955). (28) Wolff, 1. A., blehltretter, C. L.,
Mellies, R. L., Watson, P. R., Hofreiter, B. T., Patrick, P. L., Rist, C. E., Ind. Eng. Chem. 46, 370 (1954).
RECEIVEDfor review March 20, 1959. Accepted April 27, 1959.
Rapid Determination of Organically Bound Fluorine E. Z. SENKOWSKI, E. G. WOLLISH, and E. G. E. SHAFER Analytical Research Laboratory, Hoffmann-la Roche hc., Nufley, ,An uncomplicated method for the determination of organically bound fluorine was desired. The procedure described is rapid and simple and requires minimum equipment. The sample is burned in the presence of a small quantity of sodium peroxide in an atmosphere of oxygen, in a Schoniger borosilicate glass flask. In the resulting solution fluorine is determined photometrically by Megregian's procedure. Small quantities of phos-
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ANALYTICAL CHEMISTRY
N. 1.
phates do not interfere, but larger proportions require a Willard-Winter distillation. The method is applicable to a variety of organic fluorine compounds and can b e carried out with satisfactory accuracy and precision in less than 1 hour.
T
determination of organically bound fluorine has presented a problem for a considerable number of years. The fact that many methods HE
have been published indicates that the need for a simple and rapid routine procedure has not been satisfied. The literature pertaining to fluorine determinations in organic microanalysis has recently been reviewed in detail by M a (4, 6). Most of the investigators have decomposed the sample by oxidative fusion in a peroxide bomb or by reduction using sodium, potassium (4), or sodium biphenyl ( 1 ) . However, dissolution and
