The separation and identification of oligosaccharides

THE

SEPARATION

AND

IDENTIFICATION

OF OLIGOSACCHARIDES R. W. BAILEY*

ANDJ. B. PRIDHAM

Chemistry Department, Royal Holloway College, University of London, Englefield Green, Surrey (Great Britain)

CONTENTS I. Introduction

.....................

2. Paper chromatography.

‘14

................

a. General considerations.

. .

115

...............

115

b. Solvents. ..................... c. Spray reagents ................... d. Quantitative paper chromatography

. 1’5 I20

.........

.

3. Paper electrophoresis. ................. 4. Column chromatography ................ a. Partition on cellulose ................ b. Partition on celite ................. c. Adsorption on charcoal ............... d. Ion-exchange chromatography ............

.

124 125

.

127 127 129 129 131

I. INTRODUCTION The separation graphic

and identification

procedures

paper partition Identification nowadays

of various types of monosaccharides

has been extensively

chromatography

to the resolution

of monosaccharides

by modern

normally

reliable.

The

in examining enzymic

processes.

theoretically greater

oligosaccharide in elucidating

review

The aim of the present firstly

identification

of paper electrophoresis,

used for the isolation l

investigations

discusses

provides a preliminary technique

the identification

and chromatographic

raphy in oligosaccharide The

mixtures,

structures

of sufficient

is

is still

of which is of considerable and

in investigating

must,

a vast

therefore,

is to describe

many

number

are

be used with

the use of chromatog-

and to discuss its limitations.

paper

partition

chzromatography which

of the sugars in a mixture; and thirdly, material

secondly,

column chromatography

for confirmatory

Present address: Plant Chemistry Division, D.S.I.R.,

References p. 133.

methods

however,

is also used extensively

however,

methods article

of these compounds.

chromatographic

of enantiomers,

In the case of oligosaccharides,

possible

caution.

of mixtures

Paper chromatography

polysaccharide

by chromato-

since PARTRIDGE~~Ofirst applied

paper

determination

beyond the bounds of the technique. importance

developed

chemical

generally the related

which may be identification.

Palmerston North, New Zealand.

SEPARATION AND IDENTIFICATION OF OLIGOSACCHARIDES

II5

2. PAPER CHROMATOGRAPHY (a) General considerations The paper chromatography conditions

of oligosaccharides

to those used for separating

nique is normally equivalent)

is generally

monosaccharides.

used with, in the great majority

filter paper. The simple apparatus

a closed, gas-tight, Compounds or alcoholic

glass tank, is adequately under examination

solutions.

For the application

Glass capillary

of large volumes,

in the case of preparative

described

tubes

consisting

solvent techNo. I (or an

of glass troughs in

by LEDERER AND LEDERER~O’.

applied to the papers as aqueous

are commonly

a camel-hair

under similar

of cases, Whatman

required,

are normally

performed

A descending

used for this purpose.

brush is recommended,

paper chromatography

particularly

where the sugars are streaked

along

the origin. Strong acids and bases and high concentrations from solutions

of sugars prior to chromatographic

rials will interfere techniques,

with the resolution.

It should be remembered, Ion-exchange should

however,

that hot pyridine

be treated

safe provided first with

the necessary

precautions

the cation-exchange

and then with the anion-exchanger

epimerise

can epimerise

resin

under reduced or pyridinell6.

reducing

sugars.

sugar solutions.

are taken. IR-4B

This

The solution

(e.g. Amberlite

(e.g. Amberlite

mate-

by a number of

methanol

used for desalting

or a mixed bed resin can be used. If this sequence may

as these inorganic

may be achieved

of the sugars with anhydrous

resins are also commonly

is relatively

Dowex-2)

Desalting

the simplest being careful removal of water by distillation

pressure followed by extraction

method

of salts should always be removed analysis,

IR-120

or

or Dowex-go),

is followed, high pH values, which

or in other ways affect sugars, are avoided.

Sugar acids and amino-

sugars are of course adsorbed

by anion- and cation-exchangers

respectively.

base resins such as Amberlite

IRA-400

(OH- form) can de-

grade and adsorb neutral Other techniques

sugars and should always be used in the carbonate

for desalting

use of carbonso. The removal borohydride

reduction

nol% or methanolic treatment

particularly

an ion-exchange

sugar solutions

include electrolytic

Strong formll3.

methods4g and the

of borate ions from column washings (see pp. 131,133) or

reaction

mixtures

HC142 after removal

with carbor?.

complexes,

(OH- form) or Dowex-2

can be achieved by treatment

with metha-

of cations with an ion-exchange

resin%, or by

It should be noted, however, under alkaline

conditions,

that many sugars form borate

and these may be adsorbed

resin which is used to remove borate

on to

ions.

(b) Solvents Almost

all solvent

systems

employed

have water as the stationary one or two exceptions stead

chromatography

to be generally

Careful equilibration

applicable

or a salt solution in-

to the chromatography claimed. The method

BAYLY AND BouRNE~~, in which a sheet of paper wetted with the developing r33.

of

of papers with the vapour phase of the solvent

does not always appear to be as vital as some early authors References p.

of sugars

as the mobile phase. The

which employ either two organic solvents

of water do not appear

oligosaccharides.

for the partition

phase and an organic solvent

of

solvent is

R. W. BAILEY, J. B. PRIDHAM

116

hung alongside

the chromatograms

being developed,

appears to give suitable

bration

and was employed in the present work for evaluation

Precise

temperature

connection

control

is generally

is to avoid low temperatures

unnecessary;

the main precaution

the tank should be gas tight and of a construction Many of the solvents mixtures

In resolving ability

of mixtures

of sugars,

of oligosaccharides

of different

which have been reported

These have been tested isomaltose,

trehalose,

melibiose,

lactose,

-pentaose,

-hexaose,

maltose,

(Table I) for this

laminaribiose, panose,

range of

sophorose,

a,a-

isomalto-tetraose,

sucrose, raffinose, melezitose,

stachyose,

acid and the di- and tri-galacturonic

acids from

than report numerous RF and RG values, the 46 solvents which proved in Table I according

above. To give an indication

with each solvent its speed has been classified

(22 cm x 44.5 cm) sheet of Whatman

of the time required quarter

No. I paper. Likewise,

of each solvent to resolve oligosaccharides

in the isomaltose

the sugars which just move clear from the starting

to their ability

(see footnote

according to the time in which D-glucose moves to the bottom

for

to Table

I)

of a standard

in recording

the ability

series of increasing

line when D-ghCOSe

D.P.,

has reached the

of the paper have been noted. All of the solvents listed in Table I are

either single-phase is phenol-water acidic solvents

for paper

for oligosaccharide

and the following

maltotriose,

and -octaose,

D-galacturonic

to perform the tasks mentioned

quarter

particularly

to be suitable

and classified

to above,

cellobiose,

isomaltotriose,

-heptaose,

acid,

referred

useful have been listed in a more general manner,

bottom

on its

D.P. values; partic-

to be of value

by the authors,

apparatus

sugars : gentiobiose,

development

depends

of the same D.P.;

some 60 appeared

using the standard

Rather

of mono-

separations.

series.

chromatography

pecti9.

for the resolution

for oligosaccharide

of oligosaccharides

Of the many solvent systems

D-glucuronic

suitable

of these higher sugars the value of a system

of mixtures

ularly homologous

review,

are that

values of z or 3;

(b) Resolution

separations.

criteria

one or other, or both, of the following functions:

(a) Resolution those with D.P.

the essential

which enables it to be easily cleaned.

have been developed

and are not necessarily

mixtures

to perform

described

in this

which cause some solvent systems to separate

into two phases. From the point of view of the solvent,

saccharide

equili-

of the various solvents.

systems

solvent

or the top layers of two-phase

which is the bottom

were suitable

for resolving

systems:

the one exception

layer of a two-phase

the hexuronic

system.

Only the

acids and the acidic oligo-

saccharides. Inspection

of Table

sugars of different

I shows that

D.P. values,

lated sugars of the same D.P. basic

composition

Nevertheless,

are available

for most

(where all ratios are by volume) chromatography

References p. r33.

are useful for separating

sufficient purposes.

were also tested

solvents

of closely re-

of acidic, neutral

The following

solvent

and found to be unsuitable

and

systems for the

of oligosaccharides:

n-Butanol-formic isopropanol-acetic

while many solvents

only a few give really good separations

acid-water acid-water

(2 :I :z), ethyl

(I : 5.9: 3.2),

acetate-acetic

rt-butanol-ethanol-water

acid-water

(3 : I :3))

(4: I : 5)) ethyl

SEPARATION

AND IDENTIFICATION TABLE

SOLVENT

SYSTEMS

SUITABLE

OF OLIGOSACCHARIDES

117

1

FOR RESOLVING

OLIGOSACCHARIDE

MIXTURES

A. Acidic n-Butanol-acetic

acid-water

I. 2. 3. 4.

F

4:1:2 5:1:2

MS MS

moderate poor poor moderate

2: 7.

5:1:4 4:1:5 6.3:1:2

S VS

moderate moderate

*:I:1 5.2:

1.3:3.5

F

Ethyl acetate-acetic 8. 9. IO. II.

VF VF VF VS

2:1:2

10:5:6 3:3:1§ g:2:2

13.

MF

moderate good poor poor

52

very good

4

moderate

poor

6

good

moderate

5

poor

57

68

moderate moderate poor moderate

96 6

80

acid-water MF

4:1:2

Isopuopanol-acetic ‘4.

8 6 4 3

85 76 16

acid-formic acid-water

g:1.5:0.5:2

?a-Propanol-acetic

80 I29

acid-water

Ethyl acetate-acetic 12.

good good good good moderate moderate moderate

acid-water

7:1:2

MF

Benzyl alcohol-n-pvopanol-formic

acid-water

7.2:5:2:2

MS

good

4

good

Phenol-water Bottom layer 16.

MS

moderate

3

-

MS MS MS MS vs

good good moderate moderate moderate

4 6 5 4 4

5 II 76 55 57

MF MF MF MS

poor poor moderate very good

8 4 5 4

43

MF F

poor moderate

4 4

88 I14

‘5.

I20

B. _Veutral solvents vc-B&am-ethanol-water 77. 18. 19. 20. 21.

4:1.1:1.9 51212 5,7:1.3:3.2 5:1:4 10:1:2

Ethyl acetate-n-pvopanol-water 22.

23. 24. 25.

1:6:3 5.7:4.2:2 11712 5.7:3.2: I.3

2

57 2

n-Propanol-water 26. 27.

4:1§ 7.8:2.2§

References p. 133.

118

R. W. BAILEY, TABLE

J. B. PRIDHAM

1 (continued)

Isopropanol-water 28.

C.

912

MS

moderate

3

82

Basic solvents n-Butanol-ethanol-water-ammonia (used for separation of reducing sugars as N-benzylglycosylamine derivatives) 29. 30. 31.

4:1:4.g:o.r 4.5:o.5:4.g:o.I 4:1.2:2:0.1

VF VF VF

good very good moderate

4 4 7

31 3’ 3’

MF MS MS S S

moderate moderate poor moderate very good

6 6 6 5 5

141 93 93 85

MS

moderate

5

3

VF VF F MS S

moderate moderate good moderate poor

6 6 5 4 3

96

moderate

9

n-B&anal-pyridine-water 32. 33. 34. 35. 36.

3:2:1.3 6:4:3 3:1.3:1.5 10:3:3

5:3:2

II

n-Butanol-pyridine-benzene-water 37.

5:3:1:3

Ethyl acetat&+yridine-water 38. 39. 40. 41. 42.

2:1:2

10:5:6 IO:‘+:3

8:2:1 5:2:5

8 85 93

Ethyl acetate-pyvidine-water-acetone 43.

defined by S. G.

Ethyl acetate-pyridine-acetic 44.

5:5:1:3

VF

‘I5

acid-water VF

moderate

4

77

Co&dine-ethanol-water 45.

4 :3 :2

VF

poor

5

93

MF

poor

5

93

Collidine-pyvidine-water 46.

2:1:1s

* VF: very fast; glucose moved to bottom quarter of a standard 22 x 44.5 cm chromatogram in 18 h; F: fast; glucose moved to bottom quarter of a standard 22 x 44.5 cm chromatogram in 24-30 h. MF: moderately fast; glucose moved to bottom quarter of a standard 22 x 44.5 cm chromatogram in 36-40 h. MS : moderately slow; glucose moved to bottom quarter of a standard 22 x 44.5 cm chromatogram in 48-60 h. S: slow; glucose moved to bottom quarter of a standard 22 x 44.5 cm chromatogram in 72-80 h. VS: very slow; glucose moved to bottom quarter of a standard 22 x 44.5 cm chromatogram in over 96 h. ** Poor: all di- or tri-saccharides tend to move at the same rate. Moderate: only di- or trisaccharides of markedly different structures separate. Good : sugars of closely similar structures partially separate. Very good: sugars of closely similar structures (e.g. maltose and cellobiose) clearly separate. **’ Figures refer to D.P. values for the oligosaccharides, in the isomaltose series, which just move clear from the starting line when n-glucose has reached the bottom quarter of a 22 x 44.5 cm standard chromatogram. t References to some solvent systems could not be traced in the literature. %Sugar spots rather diffuse or streaking. References p. 133.

SEPARATION

acetate-ethanol-water dine-water

AND

IDENTIFICATION

OF OLIGOSACCHARIDES

(z : I :2), ethanol-methanol-water

(IO: I :2, g :5 :8, I :I :I

119

(g : g : 2), n-butanol-pyri-

and I :I :1.5). n-butanol-acetone-ammonia-water

(4: 5 : 0.5 : 1.5) and n-propanol-ammonia-water (16: 3 : I). These solvents were either too slow for sugars of D.P. 3, and at the same time gave very poor resolution of diand tri-saccharide mixtures, or they caused the sugar spots to streak. The movement in the various solvents of oligosaccharides containing amino sugar or sugar phosphate residues was not investigated for this review, as a suitable range of test sugars was not available. BARKER et aLB, however, have used solvent No. 2g (omitting the benzylamine) for the resolution of the glucosamine oligosaccharides obtained by the partial hydrolysis of chitin. Solvents used in the paper chromatography of amino sugar containing disaccharides obtained from various mucopolysaccharides include Iz-butanol-pyridine-water (3 : I :I~O and No. 33g3) and nbutanol-acetic acid-water (6.3 : I :2.7124 and 4.4: 1.6: 4117). Other nitrogen-containing oligosaccharides from human milk and mammary gland have been investigated with solvent Nos. Us, 38 and 43r15. Little work has been carried out on the paper chromatography of oligosaccharide phosphates. However, solvents described by HARRAP’~,RUNECKLESANDKROTKOV’~~, HESTRIN ANDAVIGAD” and HORECKER, SMYRNIOTIS,ANDSEEGMILLER*~for separatting monosaccharide phosphates may be of value for sugar phosphates of higher D.P. THOMAANDFRENCH~~have investigated the effect of changing the proportions of a solvent mixture on its ability to separate oligosaccharides. These authors describe a method for deducing the most suitable composition of a particular solvent system for separating the members of an homologous series of oligosaccharides.The method is demonstrated with n-butanol-pyridine-water and ethyl acetate-acetic acid-water solvent mixtures. Development with most systems is generally continuous for from 1-4 days. With the longer development times the solvent is allowed to drip off the serrated bottom edge of the paper. Multiple development in which the paper chromatogram is repeatedly developed for a short period, dried and then re-developed has been shown by JEANES, WISE AND DIMLER93 to give improved separation of many mono- and some di-saccharides with some solvent systems. This technique may be well worth considering when attempting to separate closely moving oligosaccharides. A study of the movement of an unknown oligosaccharide in a range of solvents can often provide useful information regarding its homogeneity, D.P. and in some cases, by comparison with authentic specimens, its exact identity. The movement of a sugar as a single component in at least three different solvents including acidic, basic and neutral systems, is a useful indication of homogeneity. While this often holds good for disaccharides it may not always hold for tri- and higher saccharides and exceptions have been reported. Thus BACON~~ found that two of the fructosylsucrose trisaccharides commonly produced in invertase-sucrose digests tended to move as a single discrete spot on paper chromatograms and were only 74 has also reported that two galacseparable on charcoal-celite columns. HATANAKA tosylsucrose tetrasaccharides could not be separated on paper chromatograms or References

p.

133.

120

R. W. BAILEY,

J. B. PRIDHAM

charcoal columns; the presence of two sugars was only deduced by chemical means. In general, disaccharides move at slower rates than their constituent monosaccharides. Exceptions to this rule are rhodymenabiose (3-O-~-D-xylanopyranosylD-XylOSe) which, in some solvents, moves in front of xylosesg and sucrose which moves at the same rate as an aldohexose in many solvents. Whilst disaccharides rarely move at the same chromatographic rate as pentose and hexose monosaccharides, confusion can arise in the case of the heptoses which have small RF values. This can often be avoided by the use of the appropriate spray reagents. Comparison of the movement of an unknown oligosaccharide with known oligosaccharides will generally give an indication of D.P. which must, however, be checked by other methods. In the case of the members of an homologous series, FRENCH AND WILDS’ have established that the relationship between the number of monosaccharide units per molecule and the logarithm of the partition function, RF/(I-RF), is linear. The use of this empirical relationship will indicate whether the sugars present in a mixture are in fact the members of an homologous series. It is often possible to establish the identity of an oligosaccharide, particularly a disaccharide, by comparing its movement with that of known sugars in a selection of solvents. Although this can be achieved by using published RF or RG values, it is preferable and safer wherever possible to compare the movement of the unknown sugar with authentic specimens on the same paper chromatograms. In general, however, the identification of an oligosaccharide should never be considered complete until a specimen of the sugar has been isolated and examined by chemical methods. (c) Sfway reagents In conjunction with the rates of movement of sugars in various solvents, reactions with spray reagents can generally provide a reliable identification of most monosaccharides. With oligosaccharides, the sprays can contribute useful, and sometimes in the case of disaccharides conclusive evidence of structure. The spray reagents are required to elucidate, if possible, the following points: I. Location of the oligosaccharide on the paper chromatogram. 2. Presence or absence of a reducing unit. 3. Identity of the monosaccharide reducing unit. 4. The point of attachment of the 0-glycosidic linkage to the reducing unit. 5. Position of other 0-glycosidic linkages. 6. Identity of non-reducing monosaccharide units. A large number of spray reagents which react with sugars are available. HOUGH~~ has reviewed in detail their reactions with monosaccharides. In Table 2 an attempt has been made to classify these reagents according to their ability to react with oligosaccharides and to provide evidence concerning the points listed above. Sensitivity has also been indicated on the basis of the amount of sugar on a chromatographic spot which will just allow the colour reaction in question to be observed clearly. The most widely used technique for applying reagents is by spraying their References p. r33.

SEPARATION

AND IDENTIFICATION TABLE SPRAY

OF OLIGOSACCHARIDES

121

2

REAGENTS

A. Locating reducing arad non-reducing oligosaccharides* * * I.

Silver nitrate-acetone/ NaOH-ethanol

VHS

2.

HS

5.

Periodate/+anisidine hydrochloride Periodate/potassium permanganate Periodate/potassium permanganate/benzidine Periodate/Schiff’s reagent

6.

Borate-phenol

7.

Vanillin-perchloric

3. 4.

HS MS MS MS

red acid

HS

R

and NR differentiated (not specific for carbohydrates) R and NR give same reaction R and NR give same reaction R and NR give same reaction R and NR give same reaction R and NR give same reaction Reacts with most R and NR giving various colours (also reacts with phenols)

142 40 108 ‘51 44 81 106

B. Reacting with specific sugar groups in oligosaccharidast 8.

p-Anisidine hydrochloride (or phosphate)

9.

3,5_Dinitrosalicylic acidNaOH IO. Triphenyltetrazolium chloride (see No. 36) I I. Aniline and diphenylamineHaPO, (see No. 37) 12. Phloroglucinol-HCl (or CCl,CO,H) 13. ,%NaphthylamineFe,(SO,) s-HCl 14. a-NaphthplamineCCl,CO,H 15. m-Phenylenediamine-HCl 16. 17. 18. 19. so. 21. 22.

Aniline hydrogen phthalate Aniline hydrogen oxalate Aniline hydrogen phosphate Benzidine-acetic acid (or CCl,CO,H) Ninhydrin (for N-benzyl glycosylamine derivatives) a,5-Diphenyl-3-P-styrylphenyltetrazolium chloride Urea-H,PO,(orHCl)

References p. 133.

VHS-HS

9, 47, 86

HS

TR; With hydrochloride aldohexoses and 6deoxyhexoses brown, ketohexoses yellow, aldoand keto-pentoses pink, hexuronic acids pink TR; brown

HS

TR;

32, 57, I43

MS-LS

TR

red

; various colours

19, 45, ‘35

MS-LS

35

MS HS

93

119

TR; various colours

86

HS J VHS-HS VHS-HS VHS-HS HS

TR; various colours; weak reaction with ketoses

HS

TR;

HS

TR;

VHS-HS

M; ketoses only, blue with HsPO,; black with HCl. Heptoses pink

TR; brown aldoses

only,

ketoses only,

48 9, 121 86 65, 86 16, 84

blue

3’

purple

‘4 86, 150 70

R. W. BAILEY,

122

TABLE

23. 24. 25. 26.

27. 28.

29. 30.

HS MS MS

M; ketoses blue M ; ketoses only, red M; ketoses red

3 86, 103

MS-LS

M;

59, rso

Anthrone-HsPO, and acetic acid Bromophenol blue Periodatejsodium nitroprusside/piperazine Periodate/p-nitroanilineHCl

MS

ketoses red, pentoses and hexuronic acids violet M; ketoses only, purpleyellow M; uranic acids Deoxy-sugars blue Deoxy-sugars deep yellow (not 6-deoxy derivatives) Sugar phosphates U.V. fluorescence M; sugar phosphates blue

56

Quinine

32.

Molybdate-perchloric

35.

37.

HS-MS MS MS

sulphate

HS acid/

H,S Ninhydrin Acetylacetone-NaOH/ P-dimethylaminobenzaldehyde-HCl Periodatc glycol-thiobarbiturate

C. Indicating 36.

2 (continued)

a-Naphthol-HsPO, Orcinol-HCl(or CClsCOsH) Resorcinol-HCl (or CCl,CO,H) Naphthoresorcinol-HCl (or CClsCO,H)

3’.

33. 34.

J. B. PRIDHAM

MS-LS

97 52 56

r3r 72

HS HS

sugars blue M; amino Amino sugars red, N-acetylhexosamine purple-violet

29,122.124,

VHS

M( ?) ; sialic acid and deoxysugars

I44

‘37

51. I34

specific0-glycosidic linkages

Triphenyltetrazolium chloride Aniline and diphenylamine-

HS MS-LS

HsPO,

38.

59, 86

Diazouracil

MS

I + 2; no reaction if C, of reducing unit substituted I + 4 ; deep blue if C, of reducing unit substituted (colours given by other reducing hexose disaccharides depend to some extent on linkage to reducing unit) “Sucrose linkage”; blue. Generally no reaction if fructose moiety is substituted

32, 57, 143 19, 45, ‘35

41, 130

Approximate sensitivity: VHS < 5 pg; HS, 5510 ,ug; MS, ro--20 pg; LS > so pg. R, reducing sugar; NR, non-reducing sugar; TR, terminal reducing unit; M, sugar present at any position in a chain. *** These reagents will not function with highly substituted mono- and oligo-saccharides. t Specificities may be determined in some cases by the strength of the associated acid and the degree of heating to which the chromatograms are subjected after spraying. l

l l

solutions benzidine,

on to the dried

chromatogram.

may be hazardous

a safer procedure.

In this case the reagent

in the case of oligosaccharides) References

p. 133.

and therefore

Spraying

with

certain

compounds,

dipping the paper in the reagent must be dissolved in a solvent

in which sugars are insoluble.

e.g.

may be

(e.g. acetone

A number

of special

SEPARATIONANDIDENTIFICATIONOF OLIGOSACCHARIDES

I23

dipping reagents have been developed by GORDON,THORNBURGAND WERUM~. Silver nitrate is probably the best locating spray. Although it suffers from the disadvantage that it is very unspecific and reacts with a number of other groups of compounds, e.g. phenols, it is highly sensitive and the sprayed papers, if washed with ammonia142, are permanent records. The reagent, as published, also distinguishes between reducing and non-reducing sugars by the rate at which the coloured spots develop, the non-reducing sugars reacting more slowly. Provided sufficient nonreducing sugar is on the paper (5-10 pg or more) this difference may be accentuated by using half-strength silver nitrate solution. In this case non-reducing substances take 15-20 min to appear as compared with I-Z min for reducing substances. Periodate sprays (reagent Nos. z-5) are also useful for locating non-reducing substances, the initial periodate reaction depending on the presence of adjacent hydroxyl groups in the sugar molecule. Reducing oligosaccharides can be located on paper by the use of a number of reagents, for example p-anisidine hydrochloride (No. S), aniline hydrogen phthalate (No. 20). In some instances (e.g. No. 8) an indi(No. 16) or benzylamine-ninhydrin cation of the type of monosaccharide at the reducing end of an oligosaccharide chain may also be obtained. It should be noted, however, that strongly acidic reagents such as p-anisidine hydrochloride, will give positive reactions with non-reducing oligosaccharides which are acid labile, e.g. sucrose. Monosubstitution at C-2 or C-4 of the terminal reducing unit can often be detected using the triphenyltetrazolium chloride (No. 36) and aniline/diphenylamine (No. 37) reagents, respectively, and the socalled “sucrose linkage” can be identified with diazouracil (No. 38). In addition, reagents Nos. 8@, II, 16-18 give varying colour reactions, which to some extent appear to be related to the type of substitution at the reducing terminal unit. With this latter case, provided care is used in interpreting such results, and the colours so obtained are compared with those given by authentic specimens, much useful information can often be obtained. The presence of ketose residues at any position in short oligosaccharide chains can be fairly easily demonstrated, e.g. with sprays Nos. 22-27. These reagents are strongly acidic and the colour reactions in all cases are almost certainly dependent on the prior conversion of the ketose, to a furfural derivative which then gives a coloured complex with the reagent. The acid may also have to hydrolyse glycosidic linkages before reaction can occur. When the acid used in the reagent is relatively weak, e.g. trichloroacetic acid, a positive reaction may only be obtained when the ketose unit is held in combination by an acid-labile linkage, such as in raflinose. When this linkage is not of the furanoside type, then the reagent should be made more acidic in order to detect the ketose. Thus leucrose (5-0-cc-D-glucopyranosyl-D-fructopyranose) gives a positive reaction with sprays Nos. 25 and 26 in the presence of HCI, but not when trichloroacetic acid is used. The urea reagent (No. 22) only gives a typical ketose colour reaction with leucrose after prolonged heatingl*. Tests for combined ketose should, therefore, always include a spray reagent sufficiently acidic to react with these more stably linked oligosaccharides. References

p.

r33.

R. W. BAILEY,

124

J. B. PRIDHAM

Uranic acid units in any position in a chain can often be detected suitable

acid-base

containing

indicator

amino

such as bromophenol

sugars may be located

acid reagent

reducing

with less reactive

units

possible

to prove unless the oligosaccharide

difficult

to obtain

bonds with spray

information reagents

regarding

without

by the use of a

(No. 28). Oligosaccharides

with ninhydrin

with the molybdate-perchloric monosaccharide

blue

(No. 33) and phosphates

(No. 32). The existence functional

is first hydrolysed. the nature

resorting

im-

it is very

“internal”

hydrolysis

non-

is almost

Similarly,

of the

to partial

of other

groups

glycosidic

of the oligosac-

charides. (d) Quantitative

paper

chromatography

Most of the methods

available

of monosaccharide

for the quantitative

mixtures

can

conveniently

determination

of the components

be used for oligosaccharides.

They

may be divided into two main groups: (i) where mixtures

are resolved

on chromatograms

from the paper and then determined (ii)

where the analytical

With streaked

reactions

are actually

the first group of methods

the solution

along the origin of the chromatogram

able solvent and dried. The positions then located

by spraying

from the centre

marker

and the components

eluted

and carried out on the paper. of the sugar mixture

is normally

which is then developed with a suit-

of the bands of the separated

components

are

strips which have been cut from both edges and

of the chromatograms.

The latter

strip is important

as the

sugar

bands may not be linear. The bands are then cut out, the sugars eluted, and portions of the eluates used for analysis. including

Elution

may be achieved

ratus%, or by elution in a chromatography solute values may be obtained weight of a “foreign”

with the other componentsa.

Numerous

micro-methods,

These

include

H,SO,

reagent54, benzidine%,

With carried spots

the use of the

the second

volumetric

SOMOGYI

reagentl%,

and the developed

cut out of the chromatogram

spectrophotometer. hydrochloridelB, of these

could,

References

p. 133.

The

reagents

aniline hydrogen no doubt,

instance,

and determining

this

the volume of solution

and calorimetric,

are available

from the chromatographic sodium

for

strips.

metaperiodate7s,

phenol-

and anthrone17~30~32~75.a3,S~.

group of methods,

which are calorimetric, Solutions

used

derivatives

and the colours for

the reactions

to be analysed

chromatogram

reagent which produces coloured or fluorescent are then

ab-

volumes of

accurately.

in the eluates

out on the paper chromatogram. to the paper

method

measured

under examination

In this latter

both

of the sugars

accurately

the marker stripsro5) or (b) by adding a known

sugar to the solution

applied to the paper need not be measured the determination

them in a reflux appa-

tank. Using this quantitative

by either (a) applying

sugar solutions to the paper (excluding together

by a number of techniques

shaking the strips in tubes with water132, extracting

is sprayed

with

a suitable

with the sugars. The spots eluted

monosaccharides

and measured include

for use with oligosaccharides.

in a

p-anisidine

phthalatela* 14s, and o-aminobipheny1125,

be adapted

are

can be applied as

and some

This

general

SEPARATION technique

is superior

regarding

the

AND IDENTIFICATION

to those

exact

using

positions

of a sugar solution

the technique

is that

of a “foreign” Related spraying

to this

latter

group

the chromatogram

togram tive,

volume

sugar is also necessary

and measuring,

or measuring

with

photometrically,

doubt

only

very

which

often

are being

to the paper

have

of

to be

analysed.

The

or the addition

this technique.

a suitable

are less common

reagent,

directly

procedures,

then photographing

the intensity

the spot intensity

any

and

amounts,

of solution

of methods

with

is never

The main disadvantage

in measured

as the sugar solutions

measured

there

for analysis.

compounds,

run on the same chromatogram

in that

12.5

on the chromatograms,

are required

standard

of an accurately

strips

of the sugars

small volumes

application

marker

OF OLIGOSACCHARIDES

such as

the chroma-

of the sugar spots on the nega-

on the chromatogram34r

111y1r2.

3. PAPER ELECTROPHORESIS In addition

to paper

can provide

usefu1 confirmatory

Wherever

possible,

tography retie

chromatography, electrophoresis

and the identity

rate of movement The

literature

the related

evidence should

up to

that

1958 on this of a simple

Most

electrophoretic

observations

carried rates

on the

out using an alkaline of

movement,

disaccharides

to

D-glucose

In the case of the raffinose the mobilities

increasing

galactosylsucrose.

(MG

With

the

mannose

values),

D-mannose <

by

FOSTER@

and

by FosTER~O. have

been

FOSTERED has recorded various

characteristic

stachyose

<

family (D.P.

the

molecular

of

2-5).

of the group,

verbascose

however,

increasing

the

glucopyranosyl

of the raffinose oligosaccharides

is highly

with

chroma-

its electropho-

apparatus

of

oligosaccharides,

rate of movement

reviews

the mobilities

raffinose

paper

of oligosaccharides

example,

series the behaviour

in the order:

of decreasing

includes

For

and a series of I + 4 p-linked

with

by comparing

electrophoresis

behaviour

buffer.

electrophoresis

of an oligosaccharide.

specimen.

subject

paper

and PRIDHAM 12* has examined

oligosaccharides

behaviour

borate

relative

checked

of an authentic

MICHL~~~ and a description

of paper

the structure

be used in conjunction

of an oligosaccharide

with

technique

regarding

<

tetra-

more

weight

usual

was ob-

served. Paper

electrophoresis,

terisation

of the

using a borate

various

sucrose

by enzymic

glucose,

resulting

transglycosylation from

the partial

and glycogen123g 152,153.Mo Other sodium

electrolyte

arsenite,

metavanadate

molybdate,

saccharides

and

by

ignored.

BOURNE,

Referelzces

their

has also been useful

derivatives

which

of an

Aspergillus

for some oligosaccharides

which

acetate, or

for the charac-

can be obtained

from

reactions47 13171112’ and for trisaccharides hydrolysis have

sodium sodium

however,

these

derivatives

and

A sodium

HUTSON

p. 133.

lead

ammonium

ammonium largely

values

systems

basic and

buffer,

fructosylsucrose

been

molybdate-sulphuric

are given for

hydroxide’j5, have

behaviour acid

AND WEIGEL 37, to distinguish

in Table

3.

this

technique

include

sodium

tungstate,

sodium

molybdate37*BB.

systems the

used

of D-

wiger polyglucan2’

mainly

With

the

exception

been used with

of oligosaccharides mixture between

(pH

mono-

has been

5) has been

disaccharide

of

used

alcohols

126

R. W. BAILEY, J. B. PRIDHAM TABLE MG VALUES

FOR

SOME

3

OLIGOSACCHARIDES

IN BORATE

BUFFER

Refewm

cr,a-Trehalose a$-Trehalose P,#?-Trehalose Sophorose Nigerose Laminaribiose Maltose Cellobiose Isomaltose Gentiobiose Maltotrioee Isomaltotriose Panose

a-G I-I a-G a-G I-I /?-G j?-G I-I /T-G

8-G I-Z G a-G 1-3 G

Fru = D-fructose;

possessing I + 3 <

{:E

::;

:

:I:

:I;

,”

B-G 1-6 G a-G 1-4 a-G 1-4 G a-G 1-6 a-G 1-6 G

a-G 1-6 a-G 1-4 G a-G I-4 a-G 1-3 G a-G I-3 a-G 1-4 G B-Gal 1-4 G a-Gal 1-6 G B-G I-I Fru a-G I-Z ,%Fru a-G 1-3 Fru a-G 1-4 Fru a-G 1-5 Fru a-G 1-6 Fru a-G 1-4 a-G 1-4 Fru cr-G 1-6 a-G 1-5 Fru a-G r-6 a-G 1-6 Fru x-G I-Z p-Fru 3-I a-G B-Man r-4 Man /&Man r-4 B-Man 1-4 Man B-Man r-14 B-Man I] s-4 Man j3-Man I- [4 /?-Man 11s‘4 Man U-Gal 1-6 a-G 1-2 p-Fru a-Gal 1-6 a-Gal 1-6 a-G 1-2 B-Fru a-Gal I-[6 a-Gal 1]~-6 a-G 1-2 j3-Fru a-Gal r-:6 a-Gal 1]~-6 cc-G 1-2 ,&Fru

Lactose Melibiose Sucrose Turanose Maltulose Leucrose Isomaltulose Maltotriulose Glucosylleucrose Isomaltotriulose Melezjtose Mannobiose Mannotriose Mannotetraose Mannopentaose Raffinose Stachyose Verbascose Tetragalactosylsucrose l

various I -+ 4 <

low mobilities.

G = n-glucose;

glycosidic

2;

of the D.P.

oligosaccharides linkages

the N-benzylglycosylamine their electrophoretic Silver

nitrate

References fi. 133.

(spray

are dependent estimates of reducing

under strongly

No. I; Table

II

91 II

36

0.69 0.67 0.63 0.59 0.3I 0.35

12s

128 128 128 128 128 128 128

0.43 0.50

order:

had very

have also studied

and other buffer systems. may be achieved

solution as the electrolytea.

Similar

derivatives

mobilities

38 38163 38 38 38 38

0.22

The same worker9

by the stereochemistry

in the molecule.

22

61 61

were in the following

in molybdate

under these conditions

are not influenced

present

22

reducing disaccharides

value for an oligosaccharide

using sodium bisulphite

61 61 61 61 61 I53 91 I53

can, of course, be readily reduced to the corres-

borohydride.3g)

of glucopyranosyl-fructoses

An estimate

63 63 63 38 61

0.23 0.1g--0.20 0.24 0.69 0.69 0.32 0.28 0.69 0.75 0.33 0.57 0.33 0.62 0.32 0.38 0.80 0.74 0.10,0.17 0.69 0.63 0.56 0.60 0.35 0.44 0.31

Man = D-mannose.

The mobilities

the corresponding

(Reducing disaccharides

electrophoresis in the main

Gal = D-galactose;

linkages.

I + 6 and I +

ponding alcohols with potassium the mobilities

0.19

by paper

The mobilities

on, the molecular or the types

of

weight and of glycosidic

may also be made by preparing oligosaccharides

and examining

acidic conditions2*.

2) is suitable

for locating

sugars and sugar

SEPARATION

AND IDENTIFICATION

alcohols

on paper

electrophoretograms.

its rate

of reaction

to some extent.

spray

reagents

claim

that

and

fibre

non-reducing

describe

glass

sugars

and the separation

The electrolytes FRAHN

suitable

sheet

BOURNE,

are superior

can be detected

more

similar

No.

of ions on

3 paper

processes

mobilities

slow down

FOSTER AND GRANTS”

absorption

absolute

may

the effect

to Whatman

readily,

127

on the paper

AND MILLS~~ discuss

modifications.

supports

of sugars with

OF OLIGOSACCHARIDES

in that

are reduced

is facilitated.

4. COLUMN CHROMATOGRAPHY After

tentative

identification

desirable

to isolate

chemical

and physical

raphy from

of the

methods.

to the carbohydrate complex

of an oligosaccharide

sufficient

mixtures

Prior

unless

they

been

prepared

saccharides dried,

have

ash-free

sufficiently The

in crystalline been

powders

were

chromatography

on charcoal

obtained

by preparative

solution

along

as described

processing

is often

and, in the when they

years

many

homogeneous, generally

line

workable required

can be effected

oligofreeze-

regarded

as

main

resin chromatography.

In addition,

This

can generally

is achieved

with

section

to obtain

three

(2) adsorption

of the paper, amounts

by

or celite,

of oligosaccharides

chromatography.

in the analytical

3 MM,

recent

on cellulose

and (3) ion-exchange

paper

No.

components

are now

on columns

chromatography

the starting

e.g. Whatman

during

Such preparations

oligosaccharides

characterised

as chromatographically

here that small amounts

and processing second

However,

by

chromatog-

investigations.

(I) partition

be mentioned

sugar

form.

or syrups.

it should

to isolate

as major

it is

identification

of preparative

impossible

present

chromatography

absolute

to the application

of oligosaccharides

these are:

paper

for

as pure and adequately

only

pure for structural separation

methods:

isolated

by

compound

field it was virtually

past, such sugars were only regarded had

pure

by

appropriate

streaking marker

(p. 124). By using a thick

of sugar

can readily

be the spots

paper,

be separated.

a chromatographically

A

pure compound.

(a) Partition on cellulose This

method

was first

monosaccharide mixtures same

a particular

pattern

The apparatus by LEDERER

solvent

system

column required

of a solvent

analysis

of the eluted

for cellulose

system,

to

for a given

partition

the

is somewhat

mixture

of sugars,

chromatogram

to achieve

of

separation

of

slower. give

the

as on a cellulose

the same degree

of separa-

column

The important packing

chromatography

practical

and loading

points

has been described

are concerned

of the column

with

and collection

the and

fractions.

with the sugar mixture

References p. 133.

should,

more difficult

Solvent systems. The system studies

applicable

as on paper.

AND LEDERER’O’.

selection

readily

in this case the procedure

on a paper

it is probably

tion on the cellulose

JONES AND WADMAN~~ for the resolution

is, however, although

of separation

However,

HOUGH,

It

of oligosaccharides,

In theory column.

used by

mixtures.

used should which

be selected

is to be resolved,

by paper

using various

chromatographic solvent

systems.

R. W. BAILEY, J. B. PRIDHAM

128 For the separation mixtures

of monosaccharides

alcohol

(e.g. n-butanol

have been used and in general simple binary

more complex

mixtures

are preferable

and loading the colzcmn. This is most important,

Packing

is to be avoided.

poor resolution

particularly

Columns may be packed

is to prepare the cellulose, by thorough

solvent

such as aqueous

(empty

or partially

under gravity

acetone

dry or wet. Dry packing

washed

acetone-resolving

solvent

and finally

which,

however,

the slurry

produces

to the column,

fine tip. In this way the formation very evenly

acetone)

packed

A simple

as a slurry in a

and the powder allowed to settle

and the solvent to drain to within approximately It is then

transfer

easier.

mixing in a blendor,

in a is de-

(50 %). The slurry is poured into the glass column

filled with aqueous

of the cellulose. technique

if undue

of closely moving components

scribed by HOUGH, JONES AND WADMAN86. Wet packing is probably technique

to the

often used for paper chromatography.

tailing of sugars with consequent mixture

or isopropanol)-water

systems

successively

with

resolving columns

one inch of the surface

mixtures

solvent.

of acetone-water,

A much

slower

packing

with high powers of resolution,

filled with solvent,

using a bulb pipette

is to with a

of small lumps of cellulose powder is avoided and a

column is obtained 21. The packing

can be checked

by allowing

suitable dyes to pass down the column with the resolving solventss. If the dye does not move in a reasonably

compact

also be used as markers, examined

band, the column should be repacked.

so that

unnecessary

fractions

Collection and analysis of eluted fractions. Suitable

or

eluted

fractions

quantitatively

useful reagents This reagent

may

be determined

by various for quantitative

qualitatively

analytical

procedures.

measurements

containing

doubt

that

D.P. by adsorption

Anthrone

could

(I) large numbers

in the fractions. hexoses32fg49 140

acids75 and sugar

also be adopted

danger of losing monosaccharides

of small fractions

must be collected

(2) oligosaccharides

phosphates3s.

for oligosaccharides

and (3) the resolution will, however,

of higher

chromatography.

charides

may be overcome

by developing

charides

have been eluted.

The column

extracted

use of this technique

The

p. r33.

for component

be poor.

can then

of the higher

column

be extracted,

oligosac-

until di- or tri-sacsectioned

and the

MALPRESS AND HYTTEN~‘~ describe

of the sugar mixture

and have been able to fractionate

the

Cellulose

which are not resolved easily by other

milk. THOMA, WRIGHT AND FRENCH 13s have operated temperatures,

may

poor resolution the cellulose

from each section.

for the fractionation

of low

of these columns are:

and analysed

oligosaccharides

often resolve mixtures

of column

sugars finally

or oligosaccharides

may take a long time to move through

methods

References

of

chromatography

is one of the most

sugars

on to the cellulose. The main disadvantages

sugars (cf. charcoal), columns

by paper

of free and combined

uranic

are avail-

the latter three sugars. There is little

column

the method

collectors

here. The composition

of the total

has been used for the determination

is little

fraction

and need not be described

and amino sugar@“, and for free pentosesl’, There

and

before the sugars start to come off the column,

able from many manufacturers the

The dyes may

need not be collected

cellulose

maltodextrins

obtained

the

from human

columns up to D.P.

at elevated 18.

SEPARATION AND IDENTIFICATION OF OLIGOSACCHARIDES A commercial improvement

129

of the cellulose column is the wound paper column

(made by L. K. B. Producta, Sweden). Packing problems are avoided and relatively large amounts of sugars can be resolved with precision comparable to that obtained by ordinary paper chromatography.

Sugar mixtures should first be treated with a

small part of cellulose to remove any substances that are likely to be irreversibly

ab-

sorbed on to the paper coil. Full details of the operation of this column are available from the manufacturers. (b) Partition on celite LEMIEUX, BISHOP AND PELLETIER~~ replaced cellulose with celite for the partition chromatography

of monosaccharides and their derivatives.

They claim that columns

are superior from the point of view of speed, ease of packing, and of operation and product purity. BACONS*in an investigation

of closely related plant trisaccharides has

used small celite columns for purifying the fractions isolated from a charcoal column. The relative

merits of celite and cellulose columns should be carefully considered

before attempting the fractionation of an oligosaccharide mixture by partition chromatography. (c) Adsorption

on charcoal

The separation

of oligosaccharides

on columns of charcoal was first described by

WHISTLER AND DuRso~*~. The method depends on their differential

adsorption

on to

the charcoal followed by fractional elution with aqueous solvents. Adsorbent. Generally the charcoal is mixed with one to two volumes of celite to aid flow. JERMYN~~recommends cellulose instead of celite; this gives faster flow rates and also avoids contamination of the eluates with colloidal silica. For most work ordinary grades of charcoal “suitable for decolourising”

are satisfactory.

The charcoal-

celite mixture may be cleaned by washing firstly with HCl then absolute ethanol%1ll”, or with citrate buffer146, and finally with distilled water. The washed charcoal is then dried at 80”. Columns of the mixture are best packed as a wet slurry; details of the packing of columns are given by WHELAN, BAILEY AND RoBER-&~~, BARKER, BOURNE AND THEANDER~, and LINDBERG AND WICKBERG~~O. Approximately

25 g of charcoal should be used per g of sugar placed on the

column. Although some workers have applied suction or pressure to the column to aid flow rates, elaborate charcoal satisfactory

arrangements

seem to be unnecessary. With most grades of

flow rates can be obtained by having the eluent reservoir from

6-8 ft. above the column. Simple solve& fractionation.

With this technique the sugars are adsorbed on to

the top of the column and, after removal of monosaccharides with water, oligosaccharides are eluted with water containing an alcohol, normally ethanol. Disaccharides are generally desorbed from the charcoal with 4-6 0/0aqueous ethanol, trisaccharides with 8-10 y. and so on up to heptaoses with approximately This method is ideal for fractionating

28-30 y0 aqueous ethanol.

an homologous series of oligosaccharides. With

the higher members of such series there tends to be some overlap of the sugars in the Hejerencesp. 133.

R. W. BAILEY, J. B. PRIDHAM

130

fractions. This can be avoided if after removal of a major fraction, the column is well washed with an ethanol-water mixture of a slightly higher ethanol concentration which will remove any traces of the already eluted compound, but will not elute the next higher oligosaccharide. It is not necessary to collect large numbers of samples, and, provided the column has been carefully packed, high flow rates (up to several drops per second) may be used. The technique is thus suitable for large scale fractionations. The method, for example, has been used for the preparation of members of the following homologous series; maltodextrins146, isomaltodextrins20 and xylan oligosaccharidesl&. Normally oligosaccharides up to a D.P. value of 6-7 can be eluted from the charcoal column; the hexaoses and heptaoses normally come off with 30 0/Oaqueous ethanol but with higher alcohol concentrations elution ceases. Using a charcoal of low adsorptive capacity, BAILEY ANDCLARKE~O were able to extend the fractionation of the isomaltodextrins up to the octaose and isomaltose was eluted from the column with only 1-2 0/O aqueous ethanol. Charcoal fractionation can often be speeded up by using the modification described by ANDREWS,HOUGHANDPOWELL’, in which the charcoal is packed as a short, wide column in a sintered glass Buchner funnel. This method is particularly useful for purifying individual oligosaccharides or for removing a very high concentration of a mono- or a disaccharide from a solution containing trace amounts of other sugars. We have found the method to be particularly useful for freeing commercial maltose from traces of glucose and maltotriose. Quite large quantities of maltose (10-15 g on a column 8 x 13 cm of charcoal) can be dealt with if half the column is packed and then the remainder of the charcoal stirred (1-2 h) with an aqueous solution of the maltose and this slurry finally poured on to the top of the packed charcoal. Gradient e&ion. It is evident that the simple fractionation technique is unsuitable for the resolution of a mixture containing, for example, several di- or trisaccharides. In this case modifications of the method are necessary. The simplest still uses aqueous ethanol but employs gradient elution as described by BACONANDBELLOW and by BARKER et al .25128.Briefly, instead of attempting complete elution of, for example, a disaccharide in a single aqueous ethanol fraction, the column is washedwith solvent of gradually increasing ethanol concentration over the range o-8 yO ethanol. Many small fractions must be collected and analysed for their constituent sugars. The method depends on the fact that all oligosaccharides of the same D.P. are not eluted by identical alcohol concentrations and thus some degree of separation may be achieved. In addition to the results reported by the above authors, JERMYN~~gives a detailed study of the method and describes the use of a number of other aqueous solvents as eluents. BACON~~describes the use of the technique for the resolution of closely related trisaccharides isolated from plant material. Apart from complete separations the method is often of value for the partial purification of sugars prior to partition chromatography on cellulose or celite. The technique has been used to resolve mixtures of sugars which moved as a single spot on paper chromatograms13. Fractionation on borate- and molybdate-treated charcoal columns. The ability of References

p. I33.

131

SEPARATION AND IDENTIFICATION OF OLIGOSACCHARIDES some sugars to form borate complexes was used by BARKER, BOURNE in another

modification

of charcoal

added to the column and eluent. complexes

chromatography

Disaccharides

are eluted at much lower ethanol

in which

which are capable concentrations

aqueous ethanol which desorbs the free disaccharides. using gradient elution, 0.8-1.2

4-6 o/Oaqueous

but the corresponding ethanol

to remove

borate

of forming

was

borate

than the normal

4-6 yO

Thus these authors found that,

I --j 3 and I + 6 linked glucose disaccharides

yO aqueous ethanol

required

AND THEANDER~, sodium

were eluted with

I --f 4 and I -+ 2 linked compounds

them from the borate-carbon

column.

(Details of the removal of borate ions from the eluates are given on p. 115). With complex mixtures

of oligosaccharides

lation of small amounts mixture

of isomaltose

the method

is generally

of the pure compounds. and maltose,

suitable

In certain

the method

only for the iso-

cases, however,

is suitable

for isolations

e.g. with a on a larger

scale. BARKER et dz3 have also shown that a molybdate-treated with aqueous general,

ethanol

molybdate

that

converts

treatment

developed

by BARKER, BOURNE

of disaccharides

charcoal-celite

aqueous ethanol, The furanosides

whereas the furanosides may readily

dilute acid. The method separation

enzymic destruction

treatment

preparations

it

to the disaccharides

temperature of the sugars “/6

that

by treatment

with a view to the

oligosaccharide

where only one component mixture

a more

with

and could be applied on a large scale.

of the other constituents

into

of

utilises the

are eluted with 46

above have been developed

of a maltose-isomaltose

converts

HCl at room After adsorption

disaccharides

of all of the sugars in a complex

ever, it is should be remembered

modification

AND O'MANT~

require 10-20 0/0aqueous ethanol for elution.

be reconverted

described

In

are those

from nigerose and cellobiose from laminaribiose.

has wide application

Most of the methods

required,

methanolic

column unreacted

Thus maltose can easily be separated

complete

with

column

complexes.

some, but not all, to their methyl furanosides.

on a standard

complexes

on charcoal columns. A further

of sugars as furanosides

chromatography

charcoal-celite

gives useful separations.

as molybdate

which do not form fast running borate

Separation charcoal

Thus

as eluent

the sugars which are eluted most readily

(e.g. maltose)

fact

containing

easily

may simplify with

maltase

resolvable

mixture.

How-

of a mixture

is

the separation. or glucamylase

mixture

of isomaltose

and glucose. (d) Ion-exchange Ion-exchange

chromatography

resins may be used either

ionised complexes) groups another exchange quately

(e.g. sugar phosphates, and from neutral resins together described

Fractionation sugars, References

or, more commonly,

of charged p.

133.

for the fractionation

for the separation

sugar carboxylic

components.

sugars

acids and amino sugars),

The technique

with descriptions

of neutral

of sugars possessing for preparing

(as

ionised

from one

and using ion-

of the main type of resins have been ade-

elsewhere46y92. of neutral sugars. The principle complexes

of this method

with ions such as borate.

is the formation

The complexes

by

may be ad-

R. W. BAILEY, J. B. PRIDHAM

132 sorbed

on to a resin,

e.g. Dowex-1

(borate

form)

and then selectively

eluted

with

borate buffers. The method

has been applied to the resolution

of monosaccharides

(e.g. KHYM,

ZILL AND COHN~~~)but does not appear to have been used extensively oligosaccharides.

It is probable

anion exchange

that

in future

resins, will be used effectively

other

forms,

such as molybdate,

for the separation

JONES, WALL AND PITTET loo have reported

for resolving of

of sugars.

a good, rapid separation

of D-glUCOse,

sucrose and raffinose on a column of Dowex-go W (Li+ form, using water as the eluent) ; resin size appeared value particularly than

2-3

to be important.

Extensions

for the fractionation

as elution

proceeds

in order

of decreasing

resins are also of value for the separation ionizable

functions

comparatively exchange

such as carboxyl,

simple;

the sugar solution Neutral

the other compounds

completely that

or fractionally

groups.

is passed down a column of a suitable depending

on the resin. The latter eluent.

is

ion-

on the components

sugars may be washed from the column adsorbed

with water

may then either

be

MACHELL~~, points out

sugar acids should be in the carbonate

of acidic and basic sugars. In a number

Fractionation few examples

ion-exchange

form, otherwise

chromatography

of separations

(i) Sugar carboxylic acids. Fractionations

of these sugars are generally

resin in the formate

or acetate

form) fractionated

the oligogalacturonate

series obtained

of pectic acid. WEISSMANN et a1.145 (using D owex-r, the oligoglucuronates Oligosaccharides the hydrolysis has generally

produced

by the enzymic

containing

been seperated

by fractional

Resolution

have recently

tained by the partial hydrolysis IRA-400

(acetate

Dowex-r

(acetate

containing

form; form;

oligosaccharides

(ii) Sugar phosphates. resolution

aqueous

acetic formic

obtained Separations

of monosaccharide

are often

of their salts or by cellulose may

in many

instances

of acidic oligosaccharides

ob-

Similarly

ADAMSI has used

to fractionate

hexuronic

acid-

from Spruce hemicellulose. in this field have been concerned and,

review of early work on the fractionation

occasionally,

disaccharide

of these compounds

mainly with

phosphates.

A

on resins is given by

BENSON~~. KHYM, ZILL AND COHN101,102 found that good separations p. 133.

by

of such sugars

gum, on a column of Amberlite

acid eluent). acid eluent)

acid. obtained

ASPINALL, HIRST AND MATHE-

a mixture

of Khaya grandifolia

aqueous

of hyaluronic

column

these compounds.

fractionated

using Dowex3

by the hydrolysis

In the past a mixture

precipitation

carried out

form) have also resolved

acid residue

on an ion-exchange

method for separating

~0~10, for example,

formate

hydrolysis

one hexuronic

of plant gums and hemicelluloses.

chromatography. offer a better

and a

form using aqueous

formic or acetic acids as eluents. DERUNGS AND DEUEL~~, for example, (formate

of acidic or

has proved advantageous

are given below:

on columns of an anion exchange

References

possessing

The method

sugars may be destroyed.

basic oligosaccharides

the

values greater

size. Ion-exchange

sugars from those

or amino

eluted with an appropriate

resins used to adsorb

neutral

of neutral

phosphate,

may prove to be of

with D.P.

molecular

resin (cation, anion or mixed resin exchanger,

in the sugar mixture). leaving

of this method

of oligosaccharides

of sugar phos-

133

SEPARATION AND IDENTIFICATION OF OLIGOSACCHARIDES

phates, adsorbed on Dowex-r (Cl- form), required the presence of small amounts of borate in the eluent (NH*OH-NH&l solution). Several workers30~82~104 have also fractionated sugar phosphates on Dowex-r (Cl- or formate form), by gradient elution with formic acid or HCl solutions. The methods used by these authors should prove satisfactory for phosphorylated oligosaccharides. (iii) Amino sugars. The fractionation of amino sugars can be effected on a cation exchange resin of the Dowex-so or Zeo-Karb 225 type. An example of such a fractionation is given by HOROWITZ, ROSEMAN AND BLUMENTHAL*~ who resolved the homologous series of p-glucosamine oligosaccharides obtained by the partial hydrolysis of chitin on a column of Dowex-go (H+ form) by gradient elution with dilute HCI. Apart from the collection of large numbers of fractions the main problem with ion-exchange resin chromatography lies in removing the eluting agent from the sugars. Procedures for the removal of HCl, formic acid and borate ions are described in a previous section (p. 115). In preliminary paper chromatographic analysis of the eluates the relatively low concentrations of acidic ions present in many eluting systems are not likely to cause gross interference. Cations should, however, be removed from borate eluates with a strong cation exchange resin. Spots of the acidic solutions should not be dried on the papers at high temperatures in order to avoid possible reversion*’ or destruction of labile sugars. Formic acid present in column effluents does not interfere with anthronem in the measurement of sugar concentrations. With this reagent a correction may be made for interference by HC130 or the acid first removed8s. The preceding review has illustrated the great value of the many varied chromatographic techniques which are available for the study of the chemistry and biochemistry of oligosaccharides. Much work has been done on these special analytical and preparative procedures but there is, of course, still room for improvements. For example, column chromatography, which is at present tedious, would benefit from more “automation”, perhaps on similar lines to that which has been applied to the fractionation of amino acid mixtures. More spray reagents with highly specific reactions for the various molecular groups and linkages found in these sugars are desirable and the possible use of cellulose ion-exchangers for fractionation, on a wide scale, should be borne in mind. REFERENCES 1 G. A. ADAMS, Can. J. Chem., 37 (1959) zg. 2 R. .4.AITKEN, B. P. EDDY, M. INGRAM AND C. WEURMAN, Biochem.J., 64 3 N. D. ALBON AND D. GROSS,A~UZ~~~, 75 (1950) 454. 4 P. J. ALLEN AND J. S. D. BACON, Biochena.J., 63 (1956) zoo. 5 P. ANDREWS, D. H. BALL AND J. K. N. JONES, J.Chem.Soc., (1953) 4090. B P. ANDREWS, L. HOUGH AND J. K. N. JONES, J.Chem. Sac., (1952) 2744. ’ P. ANDREWS,L. HOUGH AND D. B. POWELL, Chem. 62 Ind.(London),(rg56) 8 A. R. ARCHIBALD AND D. J. MANNERS, Biochem.J., 73 (1959) 292. s K. Aso AND F. YAMAUCHI, Tohoku J. Agr. Research, 5 (1955) 305. l”G. 0. ASPINALL, E. L. HIRSTAND N. K.MATHEsoN,J.C!Z++Z. 1' G. AVIGAD, Biochem. J., 73 (1959) 587. I2 S. BAAR, B&hem. J., 58 (1954) 175. I3 J. S. D. BACON, Biochem. J., 57 (1954) 320. 1d J. S. D. BACON, Biochem. J., 73 (1959) 507.

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SEPARATION

AND IDENTIFICATION

OF OLIGOSACCHARIDES

I33

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