Complexes of copper(II), calcium, and other metal ions with carbohydrates: Thin-layer ligand-exchange chromatography and determination of relative stabilities of complexes

Carbohydrate Re.search, 97 (1981) 181-188 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

COMPLEXES OF COPPER( CALCIUM, AND OTHER METAL IONS WITH CARBOHYDRATES: THIN-LAYER LIGAND-EXCHANGE CHROMATOGRAPHY AND DETERMINATION OF RELATIVE STABILITIES OF COMPLEXES JUNE BFUGGS,PAUL FINCH, IMAR~A CRISTINA ~~ATULEWICZ, The Bourne Laboratory, Royal Holloway CoIIege (University (Great Britain)

AND

HELhiUr

of London),

WEIGEL

Egham, Surrey TW20 OEX

(Received April lOth, 1981; accepted for publication, May 18th, 1981)

ABSTRACT

Thin-layer &and-exchange chromatography with sodium, magnesium, aluminium, calcium, chromium(III), iron(III), nickel, copper( zinc, strontium, cadmium, and barium as the central atoms has been investigated. With copper(I1) as the central atom, the method is a simple, inexpensive, and speedy means of resolution of mixtures of carbohydrates not easily achieved by other methods. The molar ratios of complexed to uncomplexed polyhydroxy compounds, which give an indication of the relative stabilities of the complexes, are calculated from the chromatographic migration rates. For a particular compound, complex with the copper(H) ion.

this ratio is, in general,

greatest

for the

INTRODUCTION

Polyhydroxy compounds have long been known to act as iigands in the formaand several polyvalent’*3 tion of co-ordination complexes of alkali’, alkaline-earth’, metal ions. The metal-centred structures may carry a negative3s4 or positive charge. Cationic complexes are formed in aqueous solutions of magnesium5, calcium’, copper(II)6,

and barium’ acetates. Complexes have been investigated by potentiostatic titration’, and ‘, n.m.r. spectroscopy’, 5*10*11. Recently, chromatography employing a column of an ionchromatography exchange resin in the lanthanum form has been described”. Some conclusions regarding the structure and the stability constants of compIexes have been drawn from these investigations. For a particular chelating moIecule, e.g., D-gluconic acid, the stability constants of transition-metal ion complexes generally seem to be greater than those of alkali and alkaline-earth metal ion compIexes13. The current interest in “metal conjugation”14-‘6 and in the role of trace elements in the pathogenesis and polarimetry

strontium5,

‘, eIectrophoresis”-

treatment of some diseases” with metal ions. In aqueous solutions, OOOS-6215/8l/oooo-oooo

prompts

us to report further work on cationic

the metal ions formrsu

/$02.50, @

1981 -

the octahedral

complexes

aqua ion, or aqua

Elsevier Scientilic Publishing Company

182

J.

complex, e.g., [CU(H~O),]*~.

BRIGGS,

P. FINCH,

M.

C. MATULEWICZ,

The process of complex formation in aqueous solution

is essentially a displacement of one set of ligands, i.e., water molecules complex, by another set, e.g., a diol (Eq. I).

[Cu(HzO),]‘+

H. WEIGEL

+ R(OH), =+ [CU(H,O),R(OH)~]~+

of the aqua

+ 2Hz0

(0

(1.e.c.) involves this type of ligand-exchange Ligand-exchange chromatography1g*20 reaction when such complex-forming ions as [CU(H,O),J~~ are immobilised, e.g., and R2(OH),, are on ion-exchange resins or silica gel. Potential ligands, R’(OH), then distributed between this stationary phase (s) and water, the mobile phase (m), stability constants (K)_ Potential in a ratio which is reIated2’ to their stoichiometric Iigands will migrate as sharpIy defined zones if the equilibrium of Eq. I is established rapidly when compared with the chromatography time-scale_ Applications of 1.e.c. in the carbohydrate field, although not always recognised as such, using either h.p.1.c. open-column method6*1’*22 are already apparatus1 O or the more time-consuming, recorded. We have used the latter method6 for the separation of selected pairs of poIyhydroxy compounds on Amberlite IR-120(Cu2*) resin. In our continuous effort23 to extend the range of simple, inexpensive, and speedy methods for the resolution of mixtures of polyhydroxy compounds, our present method of choice was thin-layer ligand-exchange chromatography employing copper as the central atom. For comparison, the complexes with sodium, magnesium, aluminium, calcium, chromium(III), iron(III), nickel, zinc, strontium, cadmium, and barium ions were also investigated. TABLE RF

I

VALUES

FORMS,

Metal iorr

Nat’= Mg”-’ AlCa% Cr3&

Fe3’ Ni’Y CIP&” Sr”-Cd3r Bazf

OF

D-GLUCOSE,

COMPLEXATION

D-GLUCITOL,

COEFFICIENTS,

AND AND

D-hiANt.TOL

PERCENTAGES

ON

THIN

LAYERS

OF COMPLEXED

RF

k

Percentage compked

RF

0.88 0.93 0.84 0.92 0.82 0.85 0.88 0.80 0.92 0.93 0.92 0.87

0.05 0 0.10 0.01 0.13 0.09 0.05 0.16 0.01 0 0.01 0.06

4.8 0 9.1 1.0 11.5 8.3 4.8 13.8 1.0 0 1.0 5.7

0.83 0.91 0.79 0.66 0.81 0.71 0.81 0.02 0.88 0.69 0.88 0.72

VARIOUS

METAL-ION

COMPOUNDS

D-JbfaJOlitOl

D-GklCitOl

D-mnzose

IN

POLYHYDROXY

k

0.12 0.02 0.17 O-40 0.14 0.30 0.14 45.30 0.05 0.34 0.35 0.29

Percentage conlplexed

RF

k

Percentage complexed

10.7 2.0 14.5 28.6 12.3 23.1 12.3 97.5 4.8 25.4 4.8 22.5

0.82 0.91 0.81 0.77 0.71 0.81 0.82 0.18 0.89 0.80 0.90 0.80

0.13 0.02 0.14 0.20 0.30 0.14 0.13 4.14 0.04 0.16 0.03 0.16

11.5 2.0 12.3 16.7 23.1 12.3 11.5 80.5 3.8 13.8 2.9 13.8

KThe RF values on thin layers in the Na+-form of all compounds listed in Table II are >0.75 talose).

(D-

THIN-LAYER, TABLE RF

LIGANJD MCHANGE

CHROMATOGRAPHY

183

II

VALUES

COEFFICIENTS,

OF POLYHYDROXY AND

PERCENTAGES

Compound

COhfPOUNDS

cRzT-

AND

Ct.+-FORhfS

POLYHYDROXY

0.86

0.86 0.60 0.92 0.64-0.84 0.90 0.92 0.49-0.92 0.91 0.62 0.85 0.89 0.84 0.79-0.94 0.93 0.94 0.94 0.94 0.94 0.95 O-78-0.93 0.90 0.91

OF THIN

LAYERS,

COMF’LEXATION

COMPOUNDS

Ca”-form

RF

D-Arabinose D-Lyxose D-Ribose D-Xylose D-Ahose D-Galactose D-Ghrcose D-Gtdose D-Mannose D-Tatose r-Gaiactose, 6-deoxyL-Mannose, 6-deoxyD-Fructose Kojibiose Laminaribiose Maltose Cellobiose Isomaltose Gentiobiose Lactose Lactulose /?-D-Galactopyranoside, methyl a-n-Glucowranoside. methyl methyl %~mino-4,6-0benzylidene-2-deoxymethyl 3-amino4,6-0benzylidene-3-deoxya-D-Mannopyranoside, methyl methyl 3-amine-4,6-0benzylidene-3-deoxyD-Galacturonic acid D-Ghrcuronic acid D-Mannuronic acid D-Ghrconate, sodium L-Ascorbic acid Glycerol Erythritol L-Threitol D-Arabinitol Ribitol Xylitol Allitol D-Ahritol l-deoxy-

ON

OF COMPLEXED

C&‘-form

k

Percentage camp Ie_ved

0.08 0.08 0.54 0.01 to.45 0.03 0.01 to.89 0.02 0.49 0.09 0.04 0.10 to.17 0 0 0 0 0 0 to.19

7.4 7.4 35.1 1.0 t31.0 2.9 1.0 t47.1 2.0 32.9 8.3 3.8 9.1 (14.5 0 0 0 0 0 0 ~16.0

0.03 0.02

2.9 2.0

0.83

0.12

10.7

0.82 0.86 0.74

0.13 0.08 0.25

11.5 7.4 20.0

0.77 0.78 0.73 0.80 0.86 0.71 0.87 0.78 0.79

0.20 0.19 0.27 0.16 0.08 0.30 0.06 0.19 0.17

16.7 16.0 21.3 13.8 7.4 23.1 5.7 16.0 14.5

RF

0.76 0.67 0.30 0.76 0.39 0.79 0.80 0.40-0.59 0.63 0.14 0.78 0.69 0.65 0.87 0.87 0.87 0.88 0.92 0.88 0.87 0.77 0.78 0.81

k

Percentage complexed

0.22 0.38 2.09 0.22 1.37 0.17 0.16 t1.31 0.47 5.61 0.19 0.34 0.42 0.06 0.06 0.06 0.05 0.01 0.05 0.06 0.20

18.0 27.5 67.6 18.0 57.8 14.5 13.8 t56.7 32.0 s4.9 16.0 25.4 29.6 5.7 5.7 5.7 4.8 1.0 4.8 5.7 16.7

0.19 0.14

16.0 12.3

0

>90

>99

0

>90

>99

0.75 0 O-O.15 0.02 0 0 0.01 0.70 0.56 0.37 0.28 0.56 0.05 0.42 0.23 0.30

0.23 >90 >90 45.30 >90 >90 91.59 0.32 0.65 1.50 2.31 0.65 17.52 1.20 3.03 2.09

18.7 >99 97.8 >99 >99 98.9 24.2 39.4 60.0 69.8 39.4 94.6 54.5 75.2 67.6

184

J. BRIGGS, P. FINCH, M. C. MATULEWICZ, H. WJZIGEL.

TAIILE II (continued)

Ca’+-form

Compound

Galactitol &&0Xy-b D-Glucitol 4-O-a-D-g!ucopyranosyE 4-O-j3-D-glucopyranosyID-Mannitol r-Mannitol,

l-deoxy-

D-Talitol, l-deoxy-

C@-form

RF

k

Percentage complexed

RF

0.67

0.38 0.32 0.40 0.08 0.38 0.20 0.20 0.10

27.5 24.2 28.6 7.4 27.5 16.7 16.7 9.1

0.13 0.28 0.02 0.30 0.46 0.18 0.31 0.49

0.70 0.66 0.86 0.67 0.77 0.77 0.84

k

6.12 2.31 45.30 2.09 1.01 4.14 1.99 0.89

Percentage complexed 86.0 69.8 97.8 67.6 50.2 80.5 66.6 47.1

RESULTS AND DISCUSSION Separation

of contpoztnds

Tables I and II show the observed RF values (see below for real migration-rates, R:) of compounds on a mixture of silica gel and IONEX-25 SA in the various metalion forms, layered onto POLYGRAM sheets. The H+-form of the thin layers furnished RF values in the absence of any metal ion. As RF CO.93 would indicate complex formation, the results (Tables 1 and II) show that complex formation can be readily detected by thin-Iayer 1.e.c. The method is therefore complementary to electrophoresis in the appropriate electrolyte. Of the metal ions listed in Table I, the copper ion complexes with D-glucose, D-@ucitol, and D-mannitol to the greatest extent, and these complexes have been compared with those of calcium. Although complex formation with calcium ions in these circumstances is not very selective, mixtures of selected compounds, e.g., talose and other, ribose and other, and some aldoses and their reduction products, can be satisfactorily resolved by this method. The results obtained also serve as more realistic indicators as to the separations that may be feasible on larger scale” 1.e.c. employing calcium as central atom than the suggested11*24V25electrophoretic mobilities. The R, values on the Cu2+-form of the thin layer, in contrast to those on the Cazf-form, have a much wider range. Therefore, complex formation with copper ions seems to be more selective than with calcium ions. The simplicity of thin-layer I_e-c. with copper as the central atom, which has the added advantage that water is the only solvent used, renders it a speedy and inexpensive means of resolving mixtures of polyhydroxy compounds not easily achieved by other methods. For example, the members within the groups of tetritols, pentitols, and hexitols can be distinguished. Monosaccharides, and in at least two cases also disaccharides, can be separated from their reduction products. Where the identity of a monosaccharide might be in doubt, e-g, arabinose (R, O-76) or xylose (R, O-76), it can be unambiguously identifkd after reduction, e.g., to arabinitol (RF 0.28) or xylitol (RF 0.05). Similarly, whereas

185

THIN-LAYER, LIGAND EXCHANGE CHROMATOGRAPHY

maltose and cellobiose cannot be distinguished by their RP values, their anomeric reduction products, 4-O-a- (maltitol) and 4-0-/?-D-glucopyranosyl-D-glucitol (cellobiitol), respectively, can be resolved. It is anticipated that the range of possible resolutions is far greater than is reported here. Further, and as indicated for 1.e.c. using calcium ions, the RF values of compounds in thin-layer 1.e.c. with copper ions serve as indicators for the separations that may be achieved in larger scale 1-e-c. Stabilities of complexes

The chromatographic definedz6 by Eq. 2.

distribution*

K’ = xp,lxp, i,

coefficient,

K’, of a polyol, P, can

be (2)

where x,,, and xPS are the mole fractions of the polyol in the mobile and stationary phase, respectively, i.e., xPm -I- xPs = 1, and where the phase ratio, C, accounts for the amount of the mobile phase (V,,,) and of the stationary phase (VS), i.e., 5 = V,JV,. In genera126, l/K’ = [(I/&

-

I),

(3)

and, if c and R; are known, K’ may be calculated. That this approach is justifiable here follows from a consideration ” of the RF values of a series of substituted styrenes (on thin Iayers of silica gel impregnated with silver nitrate) in terms ofdG = -RTlnK’ and the Hammett relationship IogK - lo@,, = op. The value for the reaction constant, p, thus obtained was in close agreement with that obtained from distribution measurements (i.e., complexed and uncomplexed styrenes) in free solution. Unfortunately, it is difficult to determine reliably the phase ratio, c, in thin layers. However, combining Eq. 2 (after rearranging) with Eq. 3 gives the ratio in Eq. 4, namely, a complexation coefficient, i.e., the molar ratio of the complexed to the uncomplexed polyol. The percentage of complexed polyol, lOOk/(k + l), can therefore be calculated without knowledge of the stoichiometry of the complexation reaction. k = xPJxPm = I/R; -

1

(4)

In Eqs. 3 and 4, R; represents the real migration rate relative to the solvent front, which is related26 to the observed migration rate, RF, by Eq. 5. R:=gR,

(5)

UsuallyZ6, 5 is - 1.1-1.2. From the observed R, values on the thin layers in the Hf-form, 5 was determined as 1.08. Tables I and II give the values of k calculated from Eq. 4, as well as the percentages of the complexed polyols. It is appreciated that the values of k would have a more significant meaning if the stoichiometry of the complexation reactions under the conditions of thin-layer 1-e-c. were known. *In ref. 26, the relationshipis developedfor the processof adsorption.The process of l&and exchange on an immobilised metal ion can be considered analogowly.

186

J. BRIGGS,

P. FINCH,

M. C. MATULEMCZ,

H. WEIGEL

However, the complexation coefficients, k, serve as reasonable measures of the relative stabilities of complexes. The following conclusions can be drawn. All of the compounds examined formed complexes to some extent with copper(H) ions. For a particular compound, the extent of complex formation is greatest with copper(H) ions. In general, alditols form stronger complexes than do monosaccharides. Amino sugars form very strong complexes with copper(H) ions, offering a means of separating them from other sugars. The results with D-ribose, D-gulose, and D-talose indicate that strongcopper(II)ion complexes are formed when the relative disposition of three hydroxyl groups can approximate structure 1, a tridentate arrangement which has already been noted to react with periodate”, molybdate”, tungstate30, and benzeneboronic acid31*32, as well as calcium’ and europiumz3 ions. In the series of alditols, there are similar trends in the sets of calcium and copper complexes, e.g., the complexation coefficients seem to be dependent on the number of t/zreo-1,2-diol groups (or the presence of a tlzreotlzreo-1,2,3-trio1 group). It is, however, unlikely that the polyol ligands in the calcium and copper complexes have identical conformations, as the dg configuration makes Cu(I1) subject to the Jahn-Teller distortion’8b. Also, the stoichiometry of the complexation reaction with copper(I1) ions* may be different from that with calcium ions, for which a 1: 1 complex has been proposed 25. Further work on thecomplexation reactions with copper(I1) ions and the structures of the copper complexes will be reported elsewhere. OH

EXPERIMENTAL

POLYGRAM IONEX-25 SA-Na sheets (Macherey-Nagel GmbH, Dliren) were immersed in deionised water until the thin layers were completely wet. Separate sheets were then immersed for 1 h in the following aqueous solutions: 0.1~ HCl, 0.05hr Mg(AcO)z, 0.1~ AlCl, - 6 H20, 5% Ca(AcO),, 0-1~ Cr(AcO),, 0.1~ FeCI,, 0.1~ Ni(AcO), - 4 HzO, 5 % Cu(AcO), - HzO**, 0.05~ ZnCl,, 0.1h1 SrCl,, 0.1~ *with Harjagbir Gill and Shirley Sylvester]. The Job plots 33.34 of changes of optical rotation and absorbance (at 765 nm) of solutions (pH 5.5) containing o-glucitol and copper(B) acetate (combined, constant concentration O.Z%r) showed maxima corresponding to a complex containing copper and o-glucitol in the ratio 3 : l_ A similar ratio was found for the copper complex3 of o-glucitol formed at pH 12. **Ready-for-use Carbohydrate TLC plates SLL/IONEX-25 Cu are available from MacheryNageI, P-0. Box 307, D-5160 Diiren, Germany, or its agents.

THIN-LAYER,

LIGAND

EXCHANGE

CHROMATOGRAPHY

187

CdCI, - 2.5 H,O, and 0.0% BaCI,. The sheets were washed several times with deionised water and then dried in air at room temperature for 24 h. Compounds were spotted and the chromatograms developed (-1-l-5 h). Except for the acids, compounds were located by treating the dried sheets with a saturated solution of potassium permanganate in acetone, when, within -2 min, bright-yellow spots appeared on a purple background_ As both colours fade after - 10 h, the position of the compounds was marked at once. Acids were detected with ethanolic 5 % H,SO, at - 100”.

On the layers in the H+-form, the following compounds

had the R, values

indicated:

D-arabinose, 0.92; D-ribose, 0.92; D-xylose, 0.94; D-galactose, 0.92; Dglucose, 0.92; D-mannose, 0.93; D-fructose, 0.93-, galactitol, 0.94; D-glucitol, 0.90; and D-mannitol, 0.93. Average R, = 0.93; i.e., e = l/O.93 = 1.08.

ACKNOWLEDGMENTS

The collaboration with Macherey-Nagel, GmbH, Diiren, in the preparation of various thin-layer plates is gratefully acknowledged. One of us (M-CM.) thanks the Consejo National de Investigaciones Cientificas y Tecnicas, Republic of Argentina, for the award of a research fellowship.

REFERENCES

I J.A. RENDLEhIAN,JR., Adv. C&m&&-. Chem.,21 (1966) 209-271. 2 R. E. REEVES, Adv. Curbohydr. Chenz., 6 (1951) 107-134. 3 E. J. BOURNE, R. NERY, AND H. WEICEL, CIzem. I?zd. (Lotzdo,z), (1959) 998-999, and references therein. 4 H. WEIGEL, Adv. Curbolzydr. Chem., 18 (1963) 61-97. 5 J. A. Mrrrs, Biochenz. Bioplzyx Rex Commtrzz., 6 (1961) 418421. 6 E. J. BOURNE, F. SEARLE,AND H. WEIGEL, Curbohydr. Res., 16 (1971) 185-187. 7 S. J. ANGYAL AND J.A. MILE, Asst. J. CIzem., 32 (1979) 1993-2001. 8 S. J. ANCYAL, Clzem. Sot. Rev., 9 (1980) 415-428. 9 H. L~NNBERG AND A. VESALA, Curbohydr. Res., 78 (1980) 53-59. 10 R. W. GOULDING, f. Chromutogr., :03 (1975) 229-239. 11 S. J. ANGYAL,G. SBETHELL, AND R.J.BEVERIDGE, Curbohydr.Res., 73 (1979)9-18. 12 L. PETRuS,V.B~LIK, L.KUNIAK, AND L.STANKO~I~, Chem.Zvesti, 34(1980) 530-536. 13 Stubiliry Corzstarztsof Metal-Ion Conzpkes, The Chemical Society, London, 2nd edn., 1964, p. 634. 14 Yu. A. ZHDANOV, 0. A.OsIeov,V. P. GRIGORIEV, A. D. GARNOVSKY,YU. E. ALEXEEV, V. G. ALEXEEV, N. M. GONT~~ACHER, P. A. PEROV, V. G. ZALIOTOV, V. N. FohIINA,T. A. UsmAN, 0. N. NECHAEVA, AND V_ N. MIRNY, Curbohydr_ Res.. 38 (1974) cl-c3.

15 M. J. LbAhf AND L. D. HALL, Chem. Conzman., (1979) 234-235. 16 L. D. HALL AND M. YALPANI, Carbohydr_ Rex, 83 (1980) c5-c7. 17 K. D. RAINSFORD, K. BRUNE, AND M. W. WHITEHOUSE (Eds.), Truce E(e,neIzts izzthe Pufhogenesis mzd Treutmefzt of I&mmation, Birkh%zser Verlag, Basel, 1981. 18 (a) F. A. COTTON AND A. WILKINSON, Advanced Izzorguzzic Cilenzistry, Wiley, New York and Chichester, 4th edn., 1980, p_ 66; (b) ibid., p. 811. 19 F. HELFFERICH, Natare (London), 189 (1961) lOOl-1002; F. HELFFERICH,J. Am. C/rem. Sot., 84 (1962) 3237-3242. 20 V. A. DAVANKOV AND A. V. SEhmxKI~, J. Chromatogr., 141 (1977) 313-353. 21 F. R. HARTLEY, C. BURGESS, AND R. M. ALCOCK, Solution Equilibria, Ellis Horwood, Chichester, 1980, pp_ 160-162. 22 J. K. N. JONESAND R. A. WALL, CUJZ.f. Chem., 38 (1960) 2290-2294.

188

J. BRIGGS, P. FINCH, M. C. MATULEWICZ,

23 J. BRIGGS, I. R. CHAMBERS,P. FINCH, I. R. &AIDING. AND H. WEIGEL, Curboh@_

365-367. 24 S. J. kGYAL,

H. WElGlX

Res., 78 (1980)

D. GREEVES,AND J. A. MILLS, h.sI. J. Chem., 27 (1974) 1447-1456. 25 S. J. ANGYAL AND J. A. MILLS. Aust. J. Chem., 32 (1979) 1993-2001. 26 M. BRL~, A. N~ERWIESER, G. PATAKI, AND R. WEBER, in E. STAHL (Ed.), DkmschichtChromutographie, Springer, Berlin, 1962, pp_ 79-137. 27 A. P. G. Kt~~ooht, N. DE KRUYF, AND H. VAN BEKKUM, J. Chromatogr., 95 (1974) 175-180. 28 R. G. BARKER AND D. F. SHAW, J. Gem. Sot., (1959) 584-592. 29 E. J. BOURNE, D. H. HUTSON, AND H. WEIGEL, C/rem. Ind. {London), (1959) 1047-1048. 30 H. WEIGEL, Arrgew. Chem., 73 (1961) 766. 31 E. J. BOURNE, E. M. LEES, AND H. WEIGEL, J. Chromafogr., 11 (1963) 253-257. 32 R. J. FERRIER, W. G. O~EREND, G. A. RAFFERTY, H. M. WALL, AND N. R. WILLIAMS, Proc. Chem. SOL, (1963) 133. 33 P. JOB, Ann. Chim., 9 (1928) 113-203. 34 A. E. MARTELL AND M. CALVIN, Chemistry of the Metal Chelate Cornpout&, Prentice-Hall, New York, 1952, p_ 29_