Periodate oxidation analysis of carbohydrates

rlrltrlyrictr Clrivlictr Acta. 77

( 1975) 125 -131 (‘J Elscvicr ScknIific Publishing Company. Amsterdam .. Prinwd in The Ncthcrl;lnds

PERIODATE

OXIDATION

ANALYSIS

OF

125

CARBOHYDRATES

PART IV. SIMULTANEOUS DETERMINATlON OF ALdEHYDES ALDEHYDE FRAGMENTS AS 2.4-DINITROPHENYLHYDRAZONES THIN-LAYER OR LIQUID’CHROMATOGRAPWY

IN

DIBY

Periodate oxidation of carbohydrates produces a variety of aldchydes along with carboxylic acids. and the determination of such aldehydcs can give valuable structural information. Among these aldchydes. formaldehyde and acetaldchydc. which exist in unconjugated forms. may be determined by estublished methods’.“. whereas glyoxal and several kinds of hydroxyaldehydes must bc liberated from the oxidation products before their dcterminution. since such ~~ldcl~yclcs arc connected through hemiacetul linkages in these products. The libcrution process rccluires such drastic conditions that the products are partially decomposed. hence the determination of these aldehydes is difficult. Fortunately. this undesirable decomposition can be eliminated by modifying the Barry degradation3 with 2.4-dinitrophenylhydrazine hydrochloride. In a previous paper of this seriesJ. a spcctrophotometric method was reported for the selective detcrminution of plyoxal in the dialdehydc fragments formed from glycosides by periodate oxidation. In a conti’nuation of this work. a simple method for the simultaneous determination of glyoxal and hydroxyaldehydes has been developed; thinlayer chromatographic (t.1.c.) separation of their 2.4-dinitrophenylhydrazones is followed by spectrophotometric measurement. On the basis of this t.1.c. study. a convenient method of fragment analysis by liquid chromatography (l.c.) on a smull open column has also been developed. EXPERIMENTAL

A reagent-grade sample of 2.4-dinitrophenylhydrazine hydrochloride (Tokyo Kasei Kogyo Co.. Ltd) was used without further purification. All samples of aldehydes were also of reagent grade. All solvents were dehydrated by conventional methods before purification by distillation. An authentic specimen of the bis-hydrazone of glyoxal was prepared as reported previouslyJ. The hydrazones of D-glyceraldehyde. D-erythrose. D-arabinosc and D-glucose were synthesized by refluxing the aldehydes with twice the cquivalcnt remounts of 2.4-dinitrophenylhydrazine in ethanol. The hydrazones were rccrystallized from ethanol. The melting points and the analytical data for the purified hydrazones were as follows.

S. HONDA.

126

K. KAKEHI.

K. TAKIURA

D-glyceraldehyde 2.4-dinitrophcnylhydrazone: m-p. 174-l 75°C: CBH I ON406 40.0’%, C, 3.7:<, I-l. 20.7(x, N: found. 39.8’2; C. 3.7’%; H, 20.6(x, N. D-Erythrose 2.4-dinitrophenylhydrazone: m-p. 163-l 65°C: C1 0H I ZNJ07 rcquircs 40.0’%: C. 4.0’%, H. 18.7’%, N: found. 39.5’%, C. 3.8’Y0 H. 18.2’% N. D-Arabinose 2.4-dinitrophcnylhydrslzone: m.p. 183°C: C, , I-114NJ08 requires 40.0’%, C. 4.3’%, H, 17.0?:, N: found. 39.9’;/, C. 4.5:?:, l-1. 16.8’%, N. D-Glucose 2.4-dinitrophenylhydrazonc: m.p. 123-125°C: C, zH I hNjOg* Hz0 requires 38.1’;;; C. 4.8(x, H. 14.8’%, N: found. 38.8’,‘.<,C. 4.8’%, H. 14.9(x; N. The dialdehydes I and II were prepared from methyl SC- and /j-D-glucopyranoside. rcspcctively. by oxidation with periodic acid. followed by purification of the products on a column of silica gel with a 10: 1 chloroform-methanol eluant. The yields of the syrupy products of I and II were 74”’,,, and 68’%,. respectively. The analytical results were: 40.0’.!;, C. 6.714, H for 1. and 39.9’:,:, C. 7.O”A, H for II: C(,H, oOs * Hz0 requires 40.0:x, C. 6.7’::, H. The preparation of the dialdchydes. III and IV was reported previouslyj. requires

I

RI

I

II

RI

D OMe.

L-4, R2

:OMe R2-

H

Ph-CH

OH

The prcparcd ml).

alkali solution for developing the color of the glyoxal fraction by dissolving potassium hydroxide (12 g) in aqueous 80’%, ethanol

A Shimuzu UV-200 spcctrophotometer plates ( IO x 20 cm) prc-coated with silica No. 572 I /0025).

was used with I-cm gel were used (Merck

was (100

glass cells. Glass Silica Gel Plate

Heat the plates. before use. at 120°C for 10 min and cool to room temperature in a desiccator containing nnhydrous silica gel. Apply samples of hydrazones (50 pg). dissolved in 1.2-dimcthoxyethanc (0.1 ml). in narrow bands and develop with 10: 1 chloroform-methanol. Remove the solvent under a hood. and redevelop with 5: 1 chloroform-methanol until the solvent front reaches the zone of the hydrazone of glycolaldchyde. Strip off the individual zones and extract with ethanol (5 ml). Read the absorbanccs at the absorption maxima. except for the his-hydrazone of glyoxal. For this fraction. evaporate to dryness. and dissolve the residue in aqueous 2OIY, dimethylsulfoxidc (6.00 ml). Add the alkali solution (1.00 ml) to this solution. and read the absorbance at 576 nm. Calculutc the amounts of hydruzoncs from their molar absorptivities. To a 10M2 M solution

(5.00

ml) of ;1 glycoside

sample

add

lo-

* M sodium

SIMULTANEOUS

DETERMINATION

OF ALDEHYDES.

IV

127

metaperiodatc (5.00 ml). and maintain the solution I’or the rcquircd time at 25 C in a thermostated water bath. shielded from Ilght. Remove a 3.00.ml aliquot. dcionize by passing through a column of a mixture of Amberlite IR-120 (H+ form. 1 ml) and Amberlitc IRA-410 (HC03 ‘- form. I ml). and cvaporatc to dryness. Redissolve the residual syrup in water. and adjust the volume to IO.0 ml. Evaporate a 0.50.ml aliquot of this solution to dryness. and subject the residual syrup to fragment analysis by the standard procedure described below.

Dissolve in l.2-dimethoxycthane (0.1 ml) the syrup which was obtained from the carbohydrate sample (50-250 l(g) by oxidation. followed by deionization and evaporation. To this solution add 2.4.dinitrophcnylhydruzinc hydrochloride ( 1.2 mg). and keep the resultant solution at 25°C for 3 h. Evaporate the reaction solution to dryness, and dissolve the rcsiduc in chloroform (0.5 ml). Introduce the chloroform solution to a column ( 1.0 cm i.d.. 10 cm long) of silica gel (Mullinckrodt Chemical Works; 5.0 g. activated by heating at 120 ‘C for IO min. followed by storugc in ~1 desiccator containing anhydrous silica gel) impregnated with chloroform. and clutc the column with the following solvent systems in this order: chloroform (60 ml). 20: I chloroform-methanol (40 ml). IO: 1 chloroform--mcthtnol (80 ml). Collect the fractions as follows: the excess of 2.4.dinitrophenylhydrazinc hydrochloride. 16-21 ml; glyoxal bis-2.4.dinitrophenylhydrazonc. 27-35 ml; glycolalclehydc 2.4.dinitro74-91 ml: pcntosc phcnylhydrazone. 37-47 ml: triosc 2.4.dinitrophcnylhydrazonc. 1432.4.dinitrophenylhydrazonc. 108-123 ml: hcxose 2.4.dinitrophcnylhydrazonc. 162 ml. Dilute each fraction with methanol to 25.0 ml. and read the absorbanccs at the absorption maxima. except for the glyoxul fraction. Evaporate ;I 3.00.ml aliquot of the solution from the glyoxal fraction to dryness, and dissolve the rcsiduc in aqueous 20’?,; dimcthylsulfoxide solution (6.00 ml). Add the alkali solution (I 00 ml) to this solution, and read the ubsorbancc at 576 nm. Calculate the amount of each aldehyde from the molar absorptivity of its hydrnzonc. RESULTS

AND

DISCUSSION

1.2-Dimethoxycthanc was cmploycd as the reaction solvent for the formation of the hydruzones from the uldehydes. instead of dimethylsulfoxidc which had been the latter caused heavy used in the sclectivc determination of glyoxal ‘. bccuuse tailing of t.1.c. spots. On standing for 3 h in the solution of the hydrazinc hydrochloride. the aldehydcs in the conjugates wcrc converted quantitatively and directly to the corresponding hydrazones: no intcrmediatc procedure to liberate the aldchydes by acid hydrolysis was needed. Since the dissociation of the conjugates to their component aldehydes is cm equilibrium process. the removal of the aldchydcs by conversion to their hydrazones probably facilitates the dissociation process. Under the conditions used. osazonc Ibrmation was not observed. hydrazones being the only products in all cases. T.1.c. studies were performed under various conditions with stundurdizcd of prc-coated silica gel plates. Artificial mixtures of 2.4.dinitrophcnylhydrazoncs the aldehydes possibly formed from glycosidcs including oligo- and poly-saccharides.

S. HONDA.

128

K. KAKEHI.

K. TAKIURA

were well separated by double development with 10: 1. followed by 5: 1 chloroformmethanol, The only exception was that the zone of the his-hydrazone of glyoxal. the fastest moving dcrivativc, was superimposed on that of the hydrazinc reagent, Zones were extracted with ethanol. and individual hydrazones were dctermincd from their absorbances at the absorption maxima. except for the bis-hydrazone of glyoxal. This particular hydrazonc was converted to its ionized form by adding alkali. The absorption maximum was shifted bathochromically. so that this hydrazone could be determined without interference from the contaminating hydrazine reagent, Table I summarizes the mobilities and the optical data of these hydrazones, and gives the recoveries of individual hydrazones from their equimolur mixtures: good recoveries with high reproducibilities were obtained. TABLE T.L.C.

I SEPARATION

OF

EQUIMOLAR

MIXTURES

R,: cctlws _-_._ So/l*ertr

OF

-_.----

A”

Srhcrlf

(5 dctcrminnlions

._ . _

wcrc done

Ixtrltlcll.ldc~

for cxh

OF

4,w

I:“,“,

(Ill,l)

( * IO”)

438 (576)” 355 .IC? 354 353 345

40.2 (65.5)” 00.6 19.5 9Y.9 19.3 IO1 I H.9 OH.5 20.0 98.7 16.2 IO1 ----.--___---

13”

0.69 0.80 ClyOXOl 0.94 0.72 Glycolaldchydc 0.34 0.56 D-Glyccrnldchydc 0.20 0.42 D-Erythrosc 0.11 0.30 D-Arabinosc 0.04 0. I6 D-Glucose --_-_--..----__--..-----__--______-_-. “ Solvcn( A, IO: I cllloroform--mctllanol. ” Solvent L3. 5: I chloroform-mcthilnol. c 5 Dctcrminutions wcrc done for c;lch nldchydc. J After addition of alkali solution.

TABLE II T.L.C. AND L.C. l~ETERMINATION DIALDliI-IYDL-: COMPOUNDS

2.4-DINITROPHENYLHYDRA%ONES

COMPONENT

dinldchydc

ALDEHYDES

somplc.)

___,__ ._.__

.4lllcrrrllf.~

rl Ill~Jllllf

l?/’

(plllolr)

__...._

_- .

I,,

rllcdc~

‘I‘lllvJr*. )

IN MODEL

tlevirtrlo~t

.._

I-‘o1trrtl

Fo1trrtl

r.1.c.

l.c.

0.55 0.55 0.55 0.55 0.70 0.79 0.77 0.77

0.56 0.56 0.56 0.56 0.7x 0.77 0.76 0.77

._-

?'.I.c. _. _.-.-

I II Ill IV

Glyoxal D-glyccrnldchyrlc Qlyoxnl D-glyccrnldchydc Glyoxal D-crythroso GIyoxiIl D-cry!hrosc

(1.55 0.55 0.55 0.55 0.70 0.70 0.7Y 0.70

. .---.____._..._- ..._- . _. . . .._- .._. _._.._..... _

0.55 0.55 0.55 0.55 0.7Y 0.71) 0.79 0.70

_.___.._ ._ _

-

_ ._.....

__

_

__ __

SlWtltll~d

(?f’ cllrlcll~dL~

tlictltlc4ylc trtltlctl

0.97 0.55 0.63 0.4 I 0.62 0.71 ___I_.__

* JO- ‘) L.C. -_..___

0.70 0.32 0.7 I 0.30 I.1 1.4 0.73 I.2

.-...

(

an2 0.55

0.13 0.10 0.7x 0.35 I.3 2.3

- _._....._...

---.~--_-_____

OF: ALDEHYDES

--~-------_._-----

LtThe theoretical value is I in all UXS.

z-D-ducopyanoside Methyl 4.6-O-benzylidene/I-D-glucopynnosidr --.-_-~_-._----.~-

hleth~l4,6-O-benzylidene-

Methyl /I-D-_rlucopyrunoside

Methyl r-D-glucopynnoside

----_-__---..

G!\Tvsidr

_--_--._-.

LC. DETERMINATION

TABLE 111

D-glycmldehyde Glyoral D-erythrose Glyoul D-erythrose

GlpUl

Dglyce:cealdehyde

C$OXill

--._--.

fiirurrl/

Fo~rrrrl

0.98 0.97 0.98 0.96 0.3 I 0.29 0.30 0.30

3 If U.YS O.Y3 O.Y7 0.96 1.01 O.Y9 0.9Y 0.97

_

I

0.30

0.31

1.95

_-.-..-.--_--__.---_

I

7

I O.YY I 0.Y8

1 0.98

.‘J II _.

0.98 I 0.94 I I O.YS 1.00 ____-.-

I

I O.Y9

Jh 24 h ________.___

jorswt

o/ gl.lwsill~~p

--_-._-_-

dlobr prqwlif~n

.4uutirnrforml ( rrMlt+tlulr

FROM SELECTED GLYCOSIDES ---

rlkdrhyk @lKd

FORMED

0.99

1.03

1.11

P

S. HONDA.

Reaction

time

K.

KAKEHI.

K. TAKIURA

(h)

Fig. I. Pcriodatc oxidation of mcIltosc. Conccntrution of maltose. 5. IO-” M: initial conccntr;ltion sodium mctapcriodatc. 5 + IO- ’ M: reaction tcmpcroturc. X’C. ( x) Pcriodatc consumption. Glyoxal formution. (0) D-Glyccraldchydc formulion. ( A) D-Erythrosc formation.

of (0)

The uldchydes in the model dialdehydc compounds I-IV were determined by the proposed method: the results are given in Table II. The dialdehydes I and II which were prepared from anomeric methyl D-glucopyranosides by periodate oxidation. should form glyoxal and D-glyceraldehydc in equimolar proportions. Table II indicates that the amounts of both aldchydcs found wcrc in good agreement with the theoretical values. Similarly. glyoxal and D-erythrose which were formed from dislldehydes 111 and IV. prepared from the 4.6-0-benzylidenc derivatives of anomeric methyl D-glucopyranosides. were accurutcly determined without interference from benzaldehyde. Separation of hydrazones was also performed by column chromatography. since this simplilies the determination by eliminating the extraction procedure in the t.1.c. method. On the basis of the above results. the aldehydes formed from sclccted glycosidep were determined by the open column method. and the results were correlated to their periodate consumption. As can be seen from Table III. the pcriodate consumption of methyl z-D-glucopyranoside reached the theoretical value of 2 moles per mole of the glycoside after 3 IL Thereafter it increased gradually to give a value of 2.16 after 24 h. because of slight over-oxidation. The course of oxidation of the /&anomer of this glycoside was similar to that of the cr-anomcr, but slightly slower. The amounts of glyoxal and D-glyccruldehyde formed were consistent with the periodate consumption. taking into account the slight degradation caused by overoxidation. For the 4.6-0-benzylidine derivatives of the foregoing glycosides. the reaction was much slower than that of the parent glycosides. but the amounts of aldchydes (glyoxal and D-erythrosc) accorded well with the periodate consumption. It should be noted that the molar proportions of these aldehydes were approximately 1 :I without regard to the species and the reaction times. Figure 1 shows the course of the oxidation of maltose, This 1,4-linked glucobiose should produce glyoxul and D-glyccraldehyde from its non-reducing moiety. all in equimolar proportions. Up to 5 h. the molar proportion of these of D-erythrose aldehydes remuincd at this theoretical value. but the amount decreased thereafter to reach 68’;:, of the theoretical amount after 96 h. presumably because of the hydrolysis of the formyl ester. followed by over-oxidation of the resultant D-erythrose derivative. The amounts of glyoxal and D-glyceraldehyde

SIMULTANEOUS

DETERMINATION

OF

ALDEHYDES.

IV

131

also decrcascd. but the dccreusc was slower. and the proportion remained approximately I :I. The amount of pcriodatc con’surncd within 24 h supported this reasoning about over-oxidation. However. the rise in the periodate consumption curve after this reaction time cxceedcd that expected from the drop of the curves for aldchydc formation. Unexpcctcd non-Malaprade oxidation should be taken into account when such reactions proceed for many hours. Thus. the simultaneous dctcrminntion of glyoxul and hydroxyaldchydcs in diuldchyde fragments provides more reliable information concerning the linkages of carbohydrates. as well as the mode and the degree of over-oxidation. than that obtained from the classical method based on the pcriodate consumption alone. It is worthy of special mention that the absolute amounts of samples need not be known. since this analysis is based on comparison of the amounts of aldehydes formed. Although there is some similar work on the simultuncous dctcrmination ofoxidation fragments by gas chromatography. which is based on the Smith degradation of curbohydrutcs. followed by oximution and trimcthylsilylation of products. the present method is prefcrablc to this gas chromatographic analysis. because of its simplicity and accuracy. SUMMARY

A method for the simultuncous dctcrmination of aldchydes in dialdchydc fragments. obtained from carbohydrate silmplcs by pcriodate oxidation. is proposed. Rcuction mixtures obtained from the periodatc oxidation oLcrlrbohydratc samples are deionized by resins. and cvaporatcd to dryness. The syrupy rcsiducs arc treated with 2,4-dinitrophenylhydruzine hydrochloride in I .2-dimcthoxycthane at 25°C for 3 h. The resultant mixtures of hydrazoncs arc scpurated by t.1.c. or l.c.. and individual hydruzoncs arc determined by spectrophotomctric mcasuremcnt. Rcliablc analyses of carbohydrate linkages are possible with 50-250-/lg samples. REFERENCES I M. Lambcrt anal A. C. Ncish. Cctrr. J. Rcs., Xl3 ( 1950) X3. 2 D. Alnino[T and W. T. J. Morgan. Rloc~hw. J.. 4X ( I95 I) 74. 3 V. C. Barry. Ncrrrrw (Lorulor~). IS2 ( 1043) 537: V. C. I3urry and P. W. D. Mitchell. ( 1054) 4020. 4 S. I-Ioruh K. Kukchi. I-I. Yuki and K. Tukiura. Arrtrl. Chim ACIU. 1975.

.I. Chcru. S~JC../.~rt/orr.