Carbohydrate Research, 113 (1983) 291-299 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

A SIMPLE AND RAPID PREPARATION MONOSACCHARIDE ANALYSIS

ANTHONY

OF ALDITOL

ACETATES

FOR

B.BLAKENEY, PHILIP J. HARRIS *,ROE%RTJ.HENRY,

ANDBRUCEA,STONE~

Department of Biochemistry, La Trobe University, Bwrdoora, Victoria 3083 (Australia)

(Received April 7th, 1982; accepted for publication in revised form, July 4th, 1982)

ABSTRACT

A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using I-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 1OC. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.
INTRODUCTION

Gas-liquid chromatography of alditoi acetates is widely used for determining the composition of monosaccharide mixtures, especially those resulting from the hydrolysis of polysaccharides. Alditol acetates are better resolved than other commonly used derivatives. However, current methods for preparing alditol acetates involve relatively long acetylation-times at elevated temperatures (100-120). We have therefore attempted to improve the method for preparing alditol acetates. In the preparation of alditol acetates, monosaccharides are first reduced to alditols with sodium borohydride and then acetylated. Borate, formed from sodium borohydride, complexes with the alditols and interferes with subsequent acetylation. Borate is usually removed as the volatile trimethyl borate by evaporation with methanol. This method is effective only under completely anhydrous conditions, as water converts methyl borate into methanol and boric acid. Repeated slow and tedious evaporations are necessary to remove all of the borate. Acetylation requires the presence of such catalysts as pyridine3 or sodium acetate, which are effective only in the absence of borate. Acid-catalysed acetylation, although apparently successful

*Permanent address: The Grassland Research Institute, Hurley, Maidenhead, Berks., SL6 5LR, U.K. The Grassland Research Institute is financed through the Agricultural Research Council, U.K. tTo whom correspondence should be addressed. 000%6215/83/0000-0000/$03.00, @ 1983 Elsevier Scientific Publishing Company

292
in the presence of boratci acetates. Connors for the rapid analytical

A. B.
produces acetylation

BLAKENEt,

P. I. HARRIS.

R. J.

I7EIR1.

R. A. STOXI

artifacts

having

retcntlon compounds

tlmcs similar at -15

to alditc>l

and Pandit

introduced of aldito!~. use of

the USCof I-mct!~ylimidazolr fol!o\vrng the rcmo\al

:I$ acatalyst -Phi< catalyst and the of borate, for

of polyhydroxy

has been used in the acet~latlon for the acetylation We now quantitative acetates for analysis
MATFRIAI,S

of aldonon~trile\. the successful of a!ditoli by capillary I -mct!~yli!nid:~zole of borate. as a catalyst The resulting in the prcscncc alditol

report

acetylntion

were scparatcd

g.1.c. on Solar ICC. The method of :I plant cell-wall

has been used preparation.

of mono~3cch:li-irlc~
AND METlTOIX

in acid hydroly~atcs

Reqcnts. stadt, Louis. Germany MO,

~-~ Dichloromcthane over molecular I-Meth),limidazoIc and alditols M~S the kind

(cat.

no. 6050)

and dimethyl from from Sigma

sulphoxide Merck. Chemical wurccs olcrudc

(cat. Darm-

no. 801912, stored U.S.A.

sieve type 4.4) \vere obtained was obtained were obtained gift of Prnfcssnr

Co . St. except ot

Monosaccharides for I.-sorbose. Melbourne. with and glucitol (50 mL) methane (200 mL) under which

finm

commercial

V. M. Trihojus. by hydrolysis from hexa-acctati r?l~wino\itol

IJniversltY

A sample containrn, 0 apiose was obtained acid for 30 min at 100 them in I-metliylir!~idazolc After heua-acetate with

apiin .I I

0.05b1 sulphuric

/?lj,o-Inositol

(n1.p. 71 7 b and glucitol

(111.p. 102 ) WOK prepared

(5 g) by suspending slowly.

mluing. and

(5 mL_ ) and adding acetic anhydridc 15 min. water (200 mL) \\a\ ;;dcicd to dccnmpnsc alditols \vcrc extracted with carbon \\ere into dichlc~roin acctonc (X00 IIIL). was dccolourised by the addition dissolved grade of endosperm Whites cells of Italian medium ryegrass -I,,
sub-

the excess of acetic anhydride. (200 mL), and the filtrate The rcprecipitated vacuum. All other reagents evaporated

The acctylatcd

the extract to drqnos precipitated

(5 g)_ filtered.

(SO ). The residues

dissc>lv:d

and the acetates

of could \+ntcr

acetates were dried,

in dichlor~~mcth;~ne.

and crystallized

were of analytical Suspension

Pk711t

w/l-WN//.V. --

cultures

(Frown in modified (Loliw~7 n~~rlt~fkmrt~~ Lam. ) wi-c c (w/v) of sucrose The x , Cultures cells lvcrc methanol, c\ere used at mid-log brohen by two culturing. at 0.62 water,

containing 5 days after wcccssivcly IO //m 1. ~itfi 7:!,,

phase of growth, and washod site. with

passages through mexh (port fitted

a French

pressure-cell with (\\,:\i. ) ccrcw mixer.

IO Nm

and the cell walls and pcntane ~-Cell argon under

\\ere collected on a nylon wall\

ethanol, acid

/f~~&~r/~~si.soj (,c/l ,~u//,Y. sulphuric After and

M

(IO mg) were trcatccl bq agitation with \\atcr \~crc made solul~on

(125 /lL)

in gla>s tubes

T&on-lined on a I ortcu

caps. Dissolution by sulphuric with

of the \~:~il\ in the acid was aided tcmpcrature. under After argon: (N) for

15 min at room

the acid was diluted the solutions

( I._35 mL) to gicc

neutral and made

acid and heated

I h at 121 . (h) for 7 h at 100 _ (0,71 mL. i. I~~j~o-lnositc~l

(~c.)for 3 h at IO0 respect to ammonia

cooling,

by adding

15~ :tmmonia

MONOSACCHARIDE

ANALYSIS

293

(0.05 mL of a 20 mg/mL solution) was added as an internal standard. Aliquots (0.1 mL) were reduced and acetylated as described. Reduction of monosaccharides. - Monosaccharides were reduced with a solution of sodium borohydride in dimethyl sulphoxide prepared by dissolving sodium borohydride (2 g) in anhydrous dimethyl sulphoxide (100 mL) at 100. Monosaccharides were routinely reduced for 90 min at 40 by adding 1 mL of the sodium borohydride solution to 0.1 mL of the monosaccharide mixture in M ammonia. After reduction, the excess of sodium borohydride was decomposed by the addition of 18~ acetic acid (0.1 mL). Acetylation. 1-Methylimidazole (0.2 mL), followed by acetic anhydride (2 mL), were added to the reduced monosaccharides and the components were mixed. After 10 min at room temperature, water (5 mL) was added to decompose the excess of acetic anhydride. When cool, dichloromethane (1 mL) was added and the mixture was agitated on a vortex mixer. After the phases had separated, the lower one was removed with a Pasteur pipette and stored in a I-mL, septum-cap vial at -20. Gas-liquid clwomatograph~~. - The alditol acetates were separated on a SCOT glass-capillary column (28.5 m x 0.5 mm, i.d., Silar IOC) S.G.E. Pty. Ltd., Melbourne, Australia) fitted to a Hewlett-Packard 571 OA chromatograph equipped with a flame-ionization detector, and a S.G.E. Unijector capillary injection-system used in the split mode. High-purity hydrogen was used as the carrier gas at a flow rate of 77 cmjsec (determined by using dichloromethane). Routinely, 2-PL samples were injected. The oven temperature was kept for 4 min at 190 following injection and then raised at 4/min to 230, where it was kept for 8 min. The injection port and detector were heated to 250 and 300, respectively.
RESULTS AND DISCUSSION

Separation of alditol acetates. - Separations of alditol acetates by g.1.c. using packed columns have utilized polar stationary-phases (such as ECNSS-M, SP2330, OV-275 has also been used for separations of alditol SP2340, and OV-275)l.l; a new polar phase, acetates on a capillary column3. Hirase et al. l4 introduced Silar lOC, for the separation of alditol acetates on packed columns, and partially methylated alditol acetates have recently been separated on a glass-capillary column coated with this phase15. Silar 1OC is reported to have excellent thermal stability and good resolving power for the g.1.c. analysis of alditol acetates. We have found that a capillary column of Silar 1OC gives baseline resolution of 13 alditol acetates (Fig. 1). Each peak in Fig. 1 corresponds to 3 pg of monosaccharide. The order of elution (Table I) was the same as that reported by Okahira et 01.~ using a column packed with Silar lOC/Chromosorb W. Reduction of monosaccharides ,vith sodium horohydride. - The use of dimethyl sulphoxide as the solvent in the reduction of monosaccharides has two advantages. Firstly, solutions of sodium borohydride in anhydrous dimethyl sulphoxide are stable, whereas aqueous solutions must be freshly prepared. Secondly, dimethyl

A.

13. I3LAKEN[-\I~,

P. J. HARKIS.

II. J. lif NR'I., 13. A. Sl-Oh1

56 9 76

-i_____LJ

5 Time
Fig. I. Separation of alditol

IO
(min)

I5

~lcetate~ hy gas--liyud

chromatography

on a Solar lO(. gla>wxprllary

column. The temperature ~vas kept at 190 for 4 mm and then mcrcrtsed to XI at 4 .min. I. crythrltol; 2, 2-cieo~y-r,.?.t/z/,~~-rcntltol, 3. rhamnltol; 1, fuatol; 5. ribitol: 6. arahinitol: 7, xylltoi; S. 7denxy-N~crhirlo-ho\Itul. 1. all1to1: IO. mdnnitol;

I I. galaclltol:

I?. glucfrol:

and I!,

nr~~ft-~iinvl~~i.

sulphoxide. tlon. A high attempt unrcacted addition tion. Most

unlike

water.

does not readily of sodium

consume Although

acetic anhydrjde (30 mgmL) it was necessaq acid

during

acctylain an the

concentration

bornhydride

was chosen

to increase sodium

the rate of reduction. borohydride by adding

to decompose (0.1 ml_). v ~lh acr~ylabut glucose \\as faster

of glt~cosc

an CYCCSS acetic of

of too much acid (for instance, monosaccharides were rcduccd temperature B maximum vigorous (0.5 min.

I .O mL.) was found to interfcrc

rapidly (Table at room lr). tempcrrtturc.

sw-e reduced more slowly

and rhamnose than at room

at

Reduction alter

a~ 40 a further After

and no epimctktion in 90 min and \\a\ solution Table II). ofgas miuturc additional

KXS observed. unchanged \ca

Rcd~~ct~vn

30

reached

90 mtn. ver) short

LIII-

At W , there \?a reduction-times reduced

and r-cductlnn peaks

texs ~ucccssful. pcwibl> to :tcctylntcd,

because of instnbillty

of the reduction

at this tempcraturc. attributed

monosaccharides

\~crc ohscrvcd,

These pcahs ucrt not prcsnl extent of reduction.

aftor longcl reduced to

timcs of reduction. In an attempt to an internal to determine the abboiutc with a standard $ucose for 90 min at -40 was compared standard of glucitol hc\a-acctatc. hoth rvlati\c

01 111.1 wlnositol

hexa-acctatc.

If ai1 Icwc~ arc ;lttrlbutctf

MONOSACCHARIDE

ANALYSIS

295

TABLE I
RETENTION TIMES OF ALDITOL ACETATES ON A SILAR ioC GLASS-CAPILLARY COLUMNa

Relative retention time (myo-inositol = 1.00)

Glycerol Erythritol 2-Deoxy-erythro-pentitol Rhamnitol Fucitol Ribitol Arabinitol Xylitol 2-Deoxy-arabino-hexitol Allitol Apiitol Mannitol Talitol Galactitol Glucitol Inositol Iditolb

0.91 4.66 6.70 7.08 7.45 9.12 9.68 11.40 11.90 13.0 13.8 14.2 14.3 15.1 16.2 18.1 18.6

0.08 0.26 0.31 0.39 0.41 0.50 0.53 0.63 0.66 0.72 0.76 0.78 0.79 0.83 0.90 1.00 1.03

uTemperature program as in Fig. 1. bBy reduction of L-sorbose.

TABLE II
REDUCTION OF MONOSACCHARIDES WITH SODIUM BOROHYDRIDE

Sugars

Relative peak heights Reduction 0.5 time (mill)

at 23

.__
Reductiorr time (mini at 40 I5 30 60 90 Redrrciiou time (min) at 60 15 30 60 90

~~

I5

<O

60

90

Rhamnose Fucose Arabinose Xylose Allose Mannose Galactose Glucose

0.47 0.69

1.10
0.91 0.75 0.68 0.64 0.26

0.59 0.84 1.16 1.05 0.82 0.74 0.72 0.38

0.71 0.95 1.19 1.13 0.87 0.79 0.77 0.50

0.77 0.98 1.07 1.07 0.86 0.72 0.66 0.55

0.98 1.13 1.26 1.22 0.86 0.75 0.74 0.73

0.86

0.85

1.02

1.05

1.01 1.13 1.12 0.85 0.74 0.70 0.58

1.04 1.15 1.17 0.97 0.84 0.80 0.69

1.17 1.19 1.28 1.30 1.27 1.32 0.95 1.06 0.81 0.91 0.76 0.85 0.65 0.74

0.79 0.91 0.99 0.99 0.76 0.66 0.63 0.51

0.70 0.82 0.95 0.94 0.75 0.65 0.62 0.52

0.77 0.93 1.07 1.07 0.86 0.74 0.71 0.58

0.80 0.94 1.07 1.05

0.81
0.71 0.67 0.55

A mixture of monosaccharides (total concentration 20 mg/mL) was reduced with sodium borohydride for various times at three different temperatures. The acetylated alditols were determined by gas-liquid chromatography following the addition of mvo-inositol hexa-acetate (0.2 mg) as the internal standard. DRelative peak height is the ratio of peak height to the peak height of the internal standard (mean of two determinations).

96 incotnplete
study

:\. 13.BL.AKENEY, reduction, 87 I),, of the glucose slow acetylation Maltbq

P. J. HARRIS,

R.

1. H[:VRI.

H. A. STOht,

was reduced.

Glucose

~a\

chosen

for

this

because

it was the sugar rcduccd

at the lowest

rate (Table

II). reported reduction by Ruchaln reduction of partialI> reduction is a highly \vc routt nelb when the

The apparent

of glucitol

and rhatnnitol
that, whcrcas

t>t 01. 7 and Selvcndran

of glucose and rhamnnsc. methylated alditol was apparently efficient catalyst acetylation acetylation
of glucitol

et ~1. tnay bc due to slow. and hence incomplete. ct r/l. reported was incomplete.

acetates for 60 min at rootn temperature in 90 min al 37 ofhydroxycstnpounds. temperature. No further

complete

iI ~~tf~~l~~tiot~of ~~Iditols it? the fwwm~t~ of tvwNt~~. ~- i-h~ethylimidaz(~le

for theacetylation at room of For alditol4. rcattron used a IO-tnin acctylation was continued of glucitol The concentratton

occurred

for 90 min. I-mcthyiimrdazolc reduction and acetic anhltiridc For the complete Bittncr mixture), (0.2 mL), inHuences acctjlalion of I tng

in the presence of borate. \fas found with

(in I.1 mL of acidified,

0.2 mL of I-metl~~litnidarole it tll. -- ttsed much fhcsc conditions i-tncth~lttntd~tzol~ (for inhtance. glucitoi The c\tcnt routtncly and inndtt. we or but under either

and 3 mL of acetic anhydrtdc lower found concentrattons that borate I! mL. that uylttol,
same

to be sut?icient. When

of acetic anhydridc interfered

acetylation. IO niL) with

acetic anh>dride, imidazole indicated ribitol,

in the

or both, acetic

\vcre LISXI at high of catalyst not intcrferc

concentrations less acetylatrd acctylattnn.

l-tncthylu~tf, p.1.c.

anhydrtdc.

t\:tc rccnvcred. of acetylation Glucitoi. b\, reagents

At the concentration\ borate did was determined

and acettc anhydride method of Connors

by using the tttritnetrtc and /?/_tGnositol

as used

were acetylatcd
in acetyiation

in the presence of horatc for g.1.c. Acclylatton (I00 *?I,,)

proportion5

\\\:ts complete. acet>lation if

to the limits Water

of precision

of the titrimetrtc M,tth the is prebcnt.

proccdurc Interfcrcnce

does not interfere or methanol, ts neccssarq,

an obvtous

I-tn~tliyiimidazolc-cataloged

an excess of acetic anhydridc such as ethanol of the sample

acetates. are to be reduced

b1y other hydt ox> cornpounds. that no c\,aporation

alditol sugars methqlated

may be overcotnc
thus obviattng

by addtn, 0 tnorc acetic anh\drtdc.

Ioksc\ nl mot-c-\ol;itiIe

Acetl lation
It is also

in the presence of borate

advantage

has the advantage

selective volatile. partially

where

and acetylated. reported

they

The selccti\,e loss of components in methylation

glass. The

during C\ aporation an;tl\si\. tncthod Dalvzon to and a\,oids c\:aporatton

has limited and this

dryness,

the quantitative

recc>\ery ofcotnpnnents selcctt\,e

to adsorption on

Mopper
which

lo5scs of tnonosacchartdci of the mixture

r?ioilo.Fac,c,linlitlt

during prcscnt

attributed

problem.
C,sc

as no concentration

is rcquircci.

and rccluctton

acetylatton

occur in one tube.

f!f the r~irtlwcl iti ywtititrrriw

monosaccharide

crtru(r~si.c. ~~ Equal

anioun ts of
The rrlatton-

I3 monosaccharides
ship bet\veen

were reduced and acctylated

concentration and

at five concentrattons. detector response

\bas Itnear

(Table

IIT). Tht: recovery

~?ljwinositol ofalditol acetates by partitioning tnto dichloromethanc extractton. Tht-ce containing ionsecutivc hexa-acctatc was investigated by repcatcd

MONOSACCHARIDE ANALYSIS TABLE III

RELATIVE PEAK HEIGHTS OF ALDIML ACETATESa

297

Alditol acetate

Relative peak height b 0.354 0.441 0.438 0.479 0.499 0.425 0.280 0.259 0.311 0.443 0.369 0.447

Correlation coefficient of standard curve 0.999 0.999 0.953 0.999 0.998 0.996 1.000 0.999 0.998 0.999 0.998 0.999

Allitol Arabinitol 2-Deoxy-arabino-hexitol 2-Deoxy-er:vrhro-pentitol Erythritol Fucitol Galactitol Glucitol Mannitol Ribitol Rhamnitol Xylitol

QMixtures of 12 monosaccharides at five concentrations up to a total of 20 mg/mL were reduced and then acetylated (in triplicate) and 2 mg of myo-inositol hexa-acetate was added as the internal standard. Slope of standard curve (peak height relative to internal standard plotted against monosaccharide concentration in mg/mL).

U-

. I5 (min)

I
5 Time

I
IO

Fig. 2. Separation of alditol acetates produced by reducing and then acetylating the monosaccharides in an acid hydrolysate of the cell walls of rye-grass. The cell walls were dissolved in 720/G(w/w) sulphuric acid at room temperature for 45 min and the acid diluted to M by the addition of water. Hydrolysis was continued at 100 for 2 h under argon. 1, arabinitol; 2, xylitol; 3, galactitol; 4, glucitol; and 5, myo-inositol (internal standard).

298
TABLE IV

$2. H. RLAKENLY. P. J. IK4KRIS. R. J. tlF\iR\.

I$. 2. STONI-

extracts Different

contained alditol

80. 19, and I I,,, respectively, acetates \vcre not extracted acetates

of the total selectively.

alditol Correction

acetate rcco~ered. for incomplete standard in the acctatc~ corrc-

partitioning

of alditol

may be made by including

--~The

an internal

dichloromethsnc. -I~ztr/~~si.s ofnphr7t derived sponding from to arabinose.

cr/l-~~~rl/ l7_wb_o/~~.va/c~.

5cparation 01 the alditol mayor peahk are present, dcrivati\w. II~c

a hydrolysatc

IS shown determined

in Fig. 2. Four and glucose

xylosc,

galactosr.

proportions but these do :I retentron

prtxnt.

of these four components by Anderson not necessarily time

wen

by the present method of small Ltcetata.

One of

agree \\ ith those reported

and Stone. correspond less than containing

A number
to alditol

pcahs are also present, these peaks. having

iiimno5~. \V:IS

slightly
in

mannosc,

but resolved and attributed In lower

from

always

samples time

no m~~~~osaccharides.

A contaminant to phthalic egcr\

having widely

;t sinlila used as

retention

has been reported at I31 resulted

plasticlsers. Hydrolysis hydrolysis walls at 100 protein the ratio5 at 100 , accounted contain total expected procedure recoveries of arabinosc after and ~qlose than hydrolyris arc close for 3 h to the but the content. The total and tnonosaccIi;lridcs, minor rccovcrcd

for 93*, of the dry wright other

of the cdl ~\alls (Tahlc those values

IV ). Aa the cell
used onI>

components.

carbohydrate-content

of the walls. Alditol

acctatcs are often hydrol>satcs(. total monclsacch~lridc

to determine

of mono~acchar~dcs

in polysaccharide

described

here olfers a means ofdctcrmining

4CKNOWLEDGMt:NTS We thank for a Royal the Australian Committee and Nufield Research for iinanaal Foundation Grants Committee and the New South S&et\

Wales Rice Research Society

support.

PJH thanhs

the Royal

Commonwealth

Rursary.

We thank

MONOSACCHARIDE

ANALYSIS

299

Mr. Trevor Norris of Scientific Glass Engineering Pty. Ltd., Melbourne, Australia, for his help and advice with capillary g.1.c. and Dr. C. M. Roxburgh, CSIRO, Division of Protein Chemistry, reading the manuscript.
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
18

and Dr. A. Bacic, University

of Melbourne,

for critically

19 20 21 22 23 24

G. G. S. DUTTON, Adv. Carbohydr. Chem. Biochem., 28 (1973) 1 l-160. L. P. ZILL, J. X. KHYM, AND G. M. CHENIAE,J. Am. Chem. Sot., 75 (1953) 1339-1342. J. D. BLAKE AND G. N. RICHARDS, Carbohydr. Rex, 14 (1970) 375-387. P. ALBERSHEIM, D. J. NEVINS, P. D. ENGLISH, AND A. KARR, Carbohydr. Res., 5 (1967) 340-345. J. M. OADES, J. Chromatogr., 28 (1967) 246-252. K. A. CONNORSAND N. K. PANDIT, Anal. Chem., 50 (1978) 1542-1.545. A. S. BITTNER,L. E. HARRIS, AND W. F. CA~IPBELL, Agric. Food Chem., 28 (1980) 1242-1245. J. C. C. CHEN AND G. D. MCGINNIS, Carbohvdr. Rex. 90 (1981) 127-130. E. VONGERICHTEN, Justus Liebigs Atzn. Chem., 318 (1901) 121-136. H. GRISEBACH AND W. BILHUBER,2. Natnrforsch., Teil B, 22 (1967) 736-75 I, M. M. SMITH AND B. A. STONE, Aust. J. Biol. Sci., 26 (1973) 123-133. A. G. W. BRADBURY, D. J. HALLIDAY, AND D. G. MEDCALF. J. Chromatogr., 213 (1981) 146-150. J. KLOK, E. H. NIEBERG-VANVELZEN, J. W. DE LEEUW, AND P. A. SCHENCK, J. Chromatogr., 207 (1981) 273-275. S. HIRASE, K. WATANABE, AND R. TAKANO, Agric. Biol. Chem., 42 (1978) 1065-1066. N. SHIBUYA, J. Chromatogr., 208 (1981) 96-99. A. OKAHIRA, H. KOBATAKE, AND H. KUSHIDA, Bunseki Kagaku, 30 (198 I) 154-l 59. A. .I. BUCHALA, C. G. FRASER,AND K. C. B. WILKIE, fhytochcmistry, 10 (1971) 1285-1291. R. R. SELVENDRAN, F. MARCH, AND S. G. RING, Anal. Biochem., 96 (1979) 282-292. J. D. MALTBY, N. C. CARPITA, D. MONTEZINOS,C. LUDLOW, AND D. P. DELMER, Plant Physiol., 63 (1979) 1158-1164. R. WACHOWIAK AND K. A. CONNORS,Anal. Chem., 51 (1979) 27-30. R. DAWSON AND K. MOPPER, Anal. Biochem., 84 (1978) 186-190. R. L. ANDERSONAND B. A. STONE, Aust. J. Biol. Sci., 31 (1978) 573-586. W. F. DUDMAN AND C. P. WHITTLE, Carbohydr. Rex., 46 (1976) 267-272. K. W. TALMADGE, K. KEECSTRA, W. D. BAUER, AND P. ALBERSHEIM, Plant Physiol., 51 (1973) 158-173.