- Email: [email protected]
Ion-exchange chromatography
ION-EXCHANGE
CHROMATOGRAPHY
Column efficiency and differences in adsorbabilities at equilibrium determine tile success of a chromatographic separation on a column. If the ratio between the equilibrium distribution coefficients, called the separation factor, is large, very primitive columns can give a cluantitativc separation. If, on the other hand, the distribution coefficients differ by only 10% or less, very cmcient columns must be used to achieve a satisfactory separation. In ion-cschangc cllromatograplly the distribution coeficicnts and separation factors can be varied within wide limits. Only in favorable cases can the influcncc of changes in working conditions be predicted theoretically. With cxamplcs from metal separations, separations of organic acids by ion eschangc, and partition chromatography of sugars, the influence of some variables will bc discussed.
In the first successful metal separations on cation-cschange resins the separation factors were increased to intinity by a transformation of .onc of the metal cations itlto a non-adsorbable anion. The first application of such a separation, publishccl in I<)Jc), was the determination of alkali metals in the presence of vanadium, converted before the separation into vanadntc anions’. In other applications of the method complcxing agents were used to convert metal ions such as cobalt and iron into nonadsorbable anions to effect a quantitative separation by selective sorption2. In separations of very similar species this technique cannot be used. Here the elution method is of predominant importance. The great potentialities of this technique became generally known in 1947 after the publication of the results of investigations, carried out as part of the Plutonium Project, on the separation of the fragments formed during fission of the heavy elements 3. Striking results were the separations of rare earths and the first isolation of element 61 (Promethium). Again, aclvantage was taken of complcsing agents such as citric acid to increase the separation factors of the elements to be separated. Under certain conditions the order of elution is largely determined by the stability constants of the compleses. Simple theories, which account reasonably well for changes of complesing agent, eluant concentration, and 1~13during the elution, and by KETELLE AND UOYD~. These were presented by TOMPKINS AND MAYEIZQ changes affect the fraction of the sorbable ions in the esternal solution and can be accounted for by the calculation methods used in classical solution chemistry. Although the application of complesing agents is a most convenient way to Anal.
C/rim.
Acta,
38 (19G7) rG3-1G8
0.
164
SAMUELSON
increase the separation factors, it must be stressed that many separations of importancc in analytical clicmistry, such as the separation of alkali metaW, can lx2 carried out in ctuants ~vhicti give no c0m!~tes formation. The selectivity in such systems is depcnclent mainly upon the interaction forces inside the resin, interionic forces and hydration forces, ancl is also directly influenced by the size of the individual inns. Since ttle resin phase is a concentrated electrolyte solution a rigorous t!ieorctical treatment is difficult. Practical calculations of the influence of eluant concentration and etuant composition can be made by approsimate methods. Published selectivity coefficients dctcrrninccl in cc1uilibriuni esperimcnts can lx used to aclvantagc. Interaction forces with the resin mntris have often been neglected but can sometimes lx of predominant im!x~rtancc. The uptake of ncgativcly charged iron( I I I) comptcses by cation-exch;mge resins from concentrated t~yctroct~toric acid’ is an csample \vlicre the importance of such forces has been welt cstal~tislicdH~u. The aVailiLbi!ity of commercial anion-csctiangc resins with quatcrnary ammonium groups gave rise to new applications of both sclcctive sorption and various elution tcchnic!ucs. Of great importance are the esceltcnt metal separations in hydrochloric acid ilt high concentration first dcmonstratcd by KRAUS ANT:, MOORE’“, and similar separations in various other comptcsing agents. In separations by selective sorption with an anion-cschange resin in the ED’I‘A-form, it was found tllat ttle separation factors could be increased by working with a water-ethanol solution insteact of aclueous solutions ll. The csptanation given was that the chelates wit11 certain metals (c.g. calcium) are more stable in the misccl solvent. ivloreover, the sc!>arution factors in tnisccl solvents are affcctcc! by tllc differctlcc in the solvent composition of the resin phase and the csternat solution 12*1:1. ‘!‘!lis diffcrcnce causes a kind of partition ctiromatogr;~pliy to be supcrimposcc! upon the ion-eschange ctironiato~ra!~tiy. Systematic investigations by ~
01:
ORGANIC
ACIDS
ON
ANION-ISXCHANCE
RESINS
In separations of organic acids on anion-cschangc resins, the separation factors arc infinite or approacti infinity in some itn!)ortant ty!x.s of separations. One e.xamptc of this, first demonstrated in esperimcnts with sulfite waste liquor, is the separation of high-niolccutnr-weight acids from toes-tiiotccular-\\tci~tlt ilCiClS*“. This separation is based upon the fact that the “mesh size” in the network structure of the resin is smatter than the dimensions of the high-molecular-weight ions. These ions, therefore, are c.sctucled from the resin phase and pass directly into the eifiucnt. Sometimes a sorption on the surface of the resin particles interferes nncl this is one of the very few ion-exchange separations in which it is rccomn~cnc~ect that coarse resin particles be used. Separation of strong acids from weak ones on a strongly basic resin in its sulfate form is another csamptc where the separation factors aplxoach infinity”. Otller simple separations whcrc large differences in sorbabilitics occur are those of anions of different charges. The tremendous progress made in gas chrotnatograptly during the last decade Asal.
Cl&n. Ach,
38
(x9G7) x63.-1cjg
.
ION-EXCHANGE
CHI10BIATOGI~Al’llY
1%
is probably the main reason why comparatively little time has been devoted to ionexchange chromatography of carbosylic acids (other than amino acids). With stable volatile acids and with samples containing only acids which are easily converted into derivatives of sufficient stability, gas chromatography is the m&hod of choice. In analyses of complicated misturcs containing various types of llydrosy acids, together with large amounts CJf sugars a;,.cl other non-electrolytes, it is believed that a combination of ion-cschange chromatography and gas chromatography gives better results. Such analyses arc necessary in various branches of carbohydrate chemistry. The success of the Stein and Moore technique for ion-eschange chromatography of amino acids’“, and the development of monitors for analyzing protein hyclrolyantcs encouraged continued work in this laboratory on ion-cschange separations of interest in carbohydrate cllemistry. Separations whicll LO years ago rccluircd several ~vceks with tedious manual work are now cffcctcd ovcrnigllt. The new tcclmicluc with recorcled chromatograms sho\vs an in~proved reproducibility. Even separations of vcrv similar acids can be accomplished within a fc\v hours. Tllc monitor was recently described in this journal~“. In most of the work chromic acid osidation \vns eml~loyecl to detcrminc the acids in the eluate. For tllis reason tile cluants used in tllc runs must not contain compounrls which are osiclizcd by cllromic acid under the conditions usccl in tile iln;\lyxing system. In the chromatogram rcpr(.)duccti in l?g. I the chrcmlic acid osidation
200 -
g g100L 5 u
I
fi II II I' ,I ; I I I \ -_--____ 1 ---
k_______,:
200
P 1; ' I :_____.
300 Eluatc
Wm.___-*
400 volume, ml
500
600
I:iK. 1 . SctxLration of ~~-C’)-(P-1,-~lucot~~r~~“os\‘li~ronic ~rcic\)-I,-~:nl;lctosc (I), ccllr~l~iouronir: xitl (I I), .)-<)-rrlcttiyl-r)-Rlucuronic ncitl (I I I). I,-~alncturonic ilcitl (IV), crvtlirc3nic acicl (V), I_-~illiironic acid (VI), I,-gluc~lronic acitl (VI I) ;mtl wmannuro!lic acid (VI I I). &rlmaolc mcthorl: broken line: tlichromatc nicthotl: full lint. C.oturnn : G x 8.\(0 mm; I~0wcx 1 S-X, Is--r 7 11. Elu;rnt:: 0.05 M rtottiumxetntc. Flow rntc: 1.1 ml/min.
as well as the carbaaolc reaction were rccordccl in different channels. It can bc seen that all acids were recorded in the chromic acid channel wllcrcas only uranic acids were recorded with carbaisole. Sodium acctnte used as eluant in this run is suitable for separations of carboxylate ions corresponding to several monoprotic acids. As a rule saccharinic, aldonic, and uranic acids appear in order of decreasing molecular weight. Comparisons of the elution behavior of acids with the same number of carbon atoms but a different number of hydrosy groups, indicate that the interaction forces with the resin matrix incrca
166
0.
SAMUELSON
with a decre,asisg number of such groups. Many stereoisomers are well separated, a fact which is ascribed to differences in hydration and interionic forces, Some isomcrii: acids show almost identical distribution coefficients. No theory has been forwarded which permits a prediction of the elution position of various species, but some empirical rules can be traced. For many purposes acetic acid gives more favorable separation factors than sodium acetate. Here the cliffcrence in acid strength of the acids to be separated is the most important factor. Expectedly, weak acids appear earlier Gn the chromatogram than stronger acids, The interaction forces with tile resin matrix increase. On a resin with a polystyrene-divinylben~cn~ matris tf;esd forces become very large with aromatic acids, and arc of very great importance with aliphatic acids of low polarity alsci. ‘I’hesc interaction forces arc not taken into consideration in the simple theory (cf. ref. 20) which accounts for the fact that the agreement between calculated and ol~servecl peak positions is somctimcs very poor. In clutions of hydroxy acids with mixtures of sodium acetate and acetic acid in varying proportions, the influence of changes in cluant comI~osition can be predicted rather well from the fraction of anions in the external solution calcuIated by the mass action law. A typical chromatogmm showing a separation of carboxylic acids by clution with acetic acid is reproduced in Fig. 2.
200
400
Eluote
volume, ml
600
Fig. 2. Scpariltion of Icvulinic (I), mclibionic (I I), lnctobionic (I I I), tnlonic (IV), m:iltoi.)ionic (V), ribonic (VI), gnlactonic (VI I). ~hicnnic (VI II), gdonic (1X). arrrbinonic (S), xylonic (XI). mannonic (Xl I), crythronic (S I I I) ant1 galacturonic acid (XIV). Carbazolc mcthocl: broken line; dichromntc mctitocl: full tine. Column: G x x3.50 mm; Dnw-cx I X-8, aG--32 p. IZluant: 0.50 M acetic acid. Flow mtc: x.5 ml/ruin. (0. Sabrum_.sor4 AND I,. 't‘rrtma: Unpublislwcl work).
Attempts to speed up the separ;itions by using eluants containing a cation which complexes with the anions to be scparatcd have met with some success. Copper acetate and zinc acetate were used and theoretical espressions for the influence of the complex constants upon the elution behavior were deriveda’. The uncertainty of the stability constants reported in the literature for the actual systems where the central ion is present at large concentration and the ligands in only trace amounts is large and, therefore, only predictions of a qualitntivc kind can be made. Especteclly, species which give rise to non-sorbable complexes appear very early in the eluate. Aldonic acids belong to this group and are separated very easily from other acids such as the common uranic acids, Large individual differences exist between some adsorbable species. T+ese cluants arc valuable complements to sodium acetate and acetic acid. The well-known formation of complex ions between borate and polyhydrosy
ION-EXCHANGE
CHROMATOGRhPHY
I67
compounds was first applied by KHynf AND ZILL 22 to separations of sugars on anionexchange resins. The anions of polyhydroxy acids obtain an increased charge in borate medium which explains why many polyhydroxy acids are held very strongly by a borate resin. Fortunately, the separation factors of many species which are difficult or impossible to separate in other eluants are improved. The application of this technique to the separation of the aldonic acids present in sulfite waste liquor (arabinonic, .xylonic, mannonic, galactonic and gluconic acid) has met with success. Here, other methods have failed 23.The relation between the structure of the acid and its elution position seems to be complicated, but from the theory of borate compleses with polyhydroxy compounds the order of elution in some systems can be predicted. The observations that aldonic acids appear in order of increasing molecular weight and that methylated uranic and biouronic acids appear much earlier than the corresponding non-methylated species arc cxplaincd by esisting theories, but these cannot explain the individual behavior of all stcreoisomcrs. Despite technical improvements and automation, separations in borate medium are in most cases more tedious than in other media. With many complicated systems, however, the method cannot be abandoned. In recent investigations on uranic acids, seven biouronic acids were studied. In sodium acetate (0.08 M) only three well separated bands were recorded. Acetic acid offered a separation into five bands while in sodium tctraborate all seven species were scparatcd. In this medium one of the peaks had the same position as the most common monouronic acid in wood chemistry (4-0-methylglucuronic acid). In acetic acid the peak elution volumes of these two compounds differed by a factor of 3, Separations in various cluants arc, thcreforc, necessary to obtain a complete resolution of complicated mixtures. As in metal separations, the application of mixed solvents can greatly improve the separation factors of various organic acids. In mixtures of water and organic solvents the interaction forces with the resin matrix decreasea, and activity coefficients, dissociation, and complcs constants are altered. With the polyhydrosy acids partition effects become predominant. In aqueous ethanol, aldonic acids and uranic acids can be retained effectively by a resin in its sulfate form under conditions where less polar acids such as acetic acid pass directly into the eflluent2G. Very little work has been done on separations based upon partition effects of very similar acids. PARTITION
CHROMATOGRAPHY
OF SUGARS
Among the 16 monosaccharides available from common commercial sources, 14 can be separated from one another on a column with a strongly basic anion eschanger in its sulfate form. Aqueous ethanol is used as eluantsO. An early study of the sorption mechanism revealed that the sorption of strongly polar solutes is largely due to the higher water concentration inside the resin compared with that of the external solution. Interaction forces between the resin and the polar solutes cannot be neglected, howevera’. This was a hindrance to a simple thermodynamic treatment, but has recently been shown to be of practical importance. In this separation method the distribution coefficients of all sugars increase with an increased ethanol concentration within the interval of interest. The order of elution is unaffected, which facilitates a qualitative discussion of the influence of other variations upon the separations. Two examples are worth mentioning, Anal.
C/rim Acta, 38 (1967)
x63-168
168
0.
SAMUELSON
In cxpcriments with a strongly basic resin with a matris of cross-linked destran, fructose and tagatose, which shelved almost identical distribution coefficients on the styrcne-divinylbcnzenc resin, tverc well separated. Rhamnose and z-deoxyglucose wcrc the only sugars which did not scparatc on either of the two resins. With the cllloridc form of the dcstran resin, ;L misturc of the two sugars was rcsolvcd into two elution bands. In many other systems the chloride form is less satisfactory than the sulfate form. ‘l’tlcsc results, achicvcd by the trial and error method, demonstrate that intcraction forces hottl wittl the resin matris and the counter ions inside the resin arc of great importance In separ;ltions based upon partition chromatography. More fundamcntnl research is necessary txforc the influence of such variations upon the scparatiori factors can be predicted.
A rcvicw is given of variahlcs which affect the separation filctors in ion-cxchange ctiromuto~rr~l,liy. In favorable cases the influence of complcs formation, pH, and cluant concentration Cim be prctlicted by simple calculations, wlicreas the intcrxc:tion forces inside tllc resin phase arc still largely unc_xplored.
CHROMATOGRAPHY
Column efficiency and differences in adsorbabilities at equilibrium determine tile success of a chromatographic separation on a column. If the ratio between the equilibrium distribution coefficients, called the separation factor, is large, very primitive columns can give a cluantitativc separation. If, on the other hand, the distribution coefficients differ by only 10% or less, very cmcient columns must be used to achieve a satisfactory separation. In ion-cschangc cllromatograplly the distribution coeficicnts and separation factors can be varied within wide limits. Only in favorable cases can the influcncc of changes in working conditions be predicted theoretically. With cxamplcs from metal separations, separations of organic acids by ion eschangc, and partition chromatography of sugars, the influence of some variables will bc discussed.
In the first successful metal separations on cation-cschange resins the separation factors were increased to intinity by a transformation of .onc of the metal cations itlto a non-adsorbable anion. The first application of such a separation, publishccl in I<)Jc), was the determination of alkali metals in the presence of vanadium, converted before the separation into vanadntc anions’. In other applications of the method complcxing agents were used to convert metal ions such as cobalt and iron into nonadsorbable anions to effect a quantitative separation by selective sorption2. In separations of very similar species this technique cannot be used. Here the elution method is of predominant importance. The great potentialities of this technique became generally known in 1947 after the publication of the results of investigations, carried out as part of the Plutonium Project, on the separation of the fragments formed during fission of the heavy elements 3. Striking results were the separations of rare earths and the first isolation of element 61 (Promethium). Again, aclvantage was taken of complcsing agents such as citric acid to increase the separation factors of the elements to be separated. Under certain conditions the order of elution is largely determined by the stability constants of the compleses. Simple theories, which account reasonably well for changes of complesing agent, eluant concentration, and 1~13during the elution, and by KETELLE AND UOYD~. These were presented by TOMPKINS AND MAYEIZQ changes affect the fraction of the sorbable ions in the esternal solution and can be accounted for by the calculation methods used in classical solution chemistry. Although the application of complesing agents is a most convenient way to Anal.
C/rim.
Acta,
38 (19G7) rG3-1G8
0.
164
SAMUELSON
increase the separation factors, it must be stressed that many separations of importancc in analytical clicmistry, such as the separation of alkali metaW, can lx2 carried out in ctuants ~vhicti give no c0m!~tes formation. The selectivity in such systems is depcnclent mainly upon the interaction forces inside the resin, interionic forces and hydration forces, ancl is also directly influenced by the size of the individual inns. Since ttle resin phase is a concentrated electrolyte solution a rigorous t!ieorctical treatment is difficult. Practical calculations of the influence of eluant concentration and etuant composition can be made by approsimate methods. Published selectivity coefficients dctcrrninccl in cc1uilibriuni esperimcnts can lx used to aclvantagc. Interaction forces with the resin mntris have often been neglected but can sometimes lx of predominant im!x~rtancc. The uptake of ncgativcly charged iron( I I I) comptcses by cation-exch;mge resins from concentrated t~yctroct~toric acid’ is an csample \vlicre the importance of such forces has been welt cstal~tislicdH~u. The aVailiLbi!ity of commercial anion-csctiangc resins with quatcrnary ammonium groups gave rise to new applications of both sclcctive sorption and various elution tcchnic!ucs. Of great importance are the esceltcnt metal separations in hydrochloric acid ilt high concentration first dcmonstratcd by KRAUS ANT:, MOORE’“, and similar separations in various other comptcsing agents. In separations by selective sorption with an anion-cschange resin in the ED’I‘A-form, it was found tllat ttle separation factors could be increased by working with a water-ethanol solution insteact of aclueous solutions ll. The csptanation given was that the chelates wit11 certain metals (c.g. calcium) are more stable in the misccl solvent. ivloreover, the sc!>arution factors in tnisccl solvents are affcctcc! by tllc differctlcc in the solvent composition of the resin phase and the csternat solution 12*1:1. ‘!‘!lis diffcrcnce causes a kind of partition ctiromatogr;~pliy to be supcrimposcc! upon the ion-eschange ctironiato~ra!~tiy. Systematic investigations by ~
01:
ORGANIC
ACIDS
ON
ANION-ISXCHANCE
RESINS
In separations of organic acids on anion-cschangc resins, the separation factors arc infinite or approacti infinity in some itn!)ortant ty!x.s of separations. One e.xamptc of this, first demonstrated in esperimcnts with sulfite waste liquor, is the separation of high-niolccutnr-weight acids from toes-tiiotccular-\\tci~tlt ilCiClS*“. This separation is based upon the fact that the “mesh size” in the network structure of the resin is smatter than the dimensions of the high-molecular-weight ions. These ions, therefore, are c.sctucled from the resin phase and pass directly into the eifiucnt. Sometimes a sorption on the surface of the resin particles interferes nncl this is one of the very few ion-exchange separations in which it is rccomn~cnc~ect that coarse resin particles be used. Separation of strong acids from weak ones on a strongly basic resin in its sulfate form is another csamptc where the separation factors aplxoach infinity”. Otller simple separations whcrc large differences in sorbabilitics occur are those of anions of different charges. The tremendous progress made in gas chrotnatograptly during the last decade Asal.
Cl&n. Ach,
38
(x9G7) x63.-1cjg
.
ION-EXCHANGE
CHI10BIATOGI~Al’llY
1%
is probably the main reason why comparatively little time has been devoted to ionexchange chromatography of carbosylic acids (other than amino acids). With stable volatile acids and with samples containing only acids which are easily converted into derivatives of sufficient stability, gas chromatography is the m&hod of choice. In analyses of complicated misturcs containing various types of llydrosy acids, together with large amounts CJf sugars a;,.cl other non-electrolytes, it is believed that a combination of ion-cschange chromatography and gas chromatography gives better results. Such analyses arc necessary in various branches of carbohydrate chemistry. The success of the Stein and Moore technique for ion-eschange chromatography of amino acids’“, and the development of monitors for analyzing protein hyclrolyantcs encouraged continued work in this laboratory on ion-cschange separations of interest in carbohydrate cllemistry. Separations whicll LO years ago rccluircd several ~vceks with tedious manual work are now cffcctcd ovcrnigllt. The new tcclmicluc with recorcled chromatograms sho\vs an in~proved reproducibility. Even separations of vcrv similar acids can be accomplished within a fc\v hours. Tllc monitor was recently described in this journal~“. In most of the work chromic acid osidation \vns eml~loyecl to detcrminc the acids in the eluate. For tllis reason tile cluants used in tllc runs must not contain compounrls which are osiclizcd by cllromic acid under the conditions usccl in tile iln;\lyxing system. In the chromatogram rcpr(.)duccti in l?g. I the chrcmlic acid osidation
200 -
g g100L 5 u
I
fi II II I' ,I ; I I I \ -_--____ 1 ---
k_______,:
200
P 1; ' I :_____.
300 Eluatc
Wm.___-*
400 volume, ml
500
600
I:iK. 1 . SctxLration of ~~-C’)-(P-1,-~lucot~~r~~“os\‘li~ronic ~rcic\)-I,-~:nl;lctosc (I), ccllr~l~iouronir: xitl (I I), .)-<)-rrlcttiyl-r)-Rlucuronic ncitl (I I I). I,-~alncturonic ilcitl (IV), crvtlirc3nic acicl (V), I_-~illiironic acid (VI), I,-gluc~lronic acitl (VI I) ;mtl wmannuro!lic acid (VI I I). &rlmaolc mcthorl: broken line: tlichromatc nicthotl: full lint. C.oturnn : G x 8.\(0 mm; I~0wcx 1 S-X, Is--r 7 11. Elu;rnt:: 0.05 M rtottiumxetntc. Flow rntc: 1.1 ml/min.
as well as the carbaaolc reaction were rccordccl in different channels. It can bc seen that all acids were recorded in the chromic acid channel wllcrcas only uranic acids were recorded with carbaisole. Sodium acctnte used as eluant in this run is suitable for separations of carboxylate ions corresponding to several monoprotic acids. As a rule saccharinic, aldonic, and uranic acids appear in order of decreasing molecular weight. Comparisons of the elution behavior of acids with the same number of carbon atoms but a different number of hydrosy groups, indicate that the interaction forces with the resin matrix incrca
166
0.
SAMUELSON
with a decre,asisg number of such groups. Many stereoisomers are well separated, a fact which is ascribed to differences in hydration and interionic forces, Some isomcrii: acids show almost identical distribution coefficients. No theory has been forwarded which permits a prediction of the elution position of various species, but some empirical rules can be traced. For many purposes acetic acid gives more favorable separation factors than sodium acetate. Here the cliffcrence in acid strength of the acids to be separated is the most important factor. Expectedly, weak acids appear earlier Gn the chromatogram than stronger acids, The interaction forces with tile resin matrix increase. On a resin with a polystyrene-divinylben~cn~ matris tf;esd forces become very large with aromatic acids, and arc of very great importance with aliphatic acids of low polarity alsci. ‘I’hesc interaction forces arc not taken into consideration in the simple theory (cf. ref. 20) which accounts for the fact that the agreement between calculated and ol~servecl peak positions is somctimcs very poor. In clutions of hydroxy acids with mixtures of sodium acetate and acetic acid in varying proportions, the influence of changes in cluant comI~osition can be predicted rather well from the fraction of anions in the external solution calcuIated by the mass action law. A typical chromatogmm showing a separation of carboxylic acids by clution with acetic acid is reproduced in Fig. 2.
200
400
Eluote
volume, ml
600
Fig. 2. Scpariltion of Icvulinic (I), mclibionic (I I), lnctobionic (I I I), tnlonic (IV), m:iltoi.)ionic (V), ribonic (VI), gnlactonic (VI I). ~hicnnic (VI II), gdonic (1X). arrrbinonic (S), xylonic (XI). mannonic (Xl I), crythronic (S I I I) ant1 galacturonic acid (XIV). Carbazolc mcthocl: broken line; dichromntc mctitocl: full tine. Column: G x x3.50 mm; Dnw-cx I X-8, aG--32 p. IZluant: 0.50 M acetic acid. Flow mtc: x.5 ml/ruin. (0. Sabrum_.sor4 AND I,. 't‘rrtma: Unpublislwcl work).
Attempts to speed up the separ;itions by using eluants containing a cation which complexes with the anions to be scparatcd have met with some success. Copper acetate and zinc acetate were used and theoretical espressions for the influence of the complex constants upon the elution behavior were deriveda’. The uncertainty of the stability constants reported in the literature for the actual systems where the central ion is present at large concentration and the ligands in only trace amounts is large and, therefore, only predictions of a qualitntivc kind can be made. Especteclly, species which give rise to non-sorbable complexes appear very early in the eluate. Aldonic acids belong to this group and are separated very easily from other acids such as the common uranic acids, Large individual differences exist between some adsorbable species. T+ese cluants arc valuable complements to sodium acetate and acetic acid. The well-known formation of complex ions between borate and polyhydrosy
ION-EXCHANGE
CHROMATOGRhPHY
I67
compounds was first applied by KHynf AND ZILL 22 to separations of sugars on anionexchange resins. The anions of polyhydroxy acids obtain an increased charge in borate medium which explains why many polyhydroxy acids are held very strongly by a borate resin. Fortunately, the separation factors of many species which are difficult or impossible to separate in other eluants are improved. The application of this technique to the separation of the aldonic acids present in sulfite waste liquor (arabinonic, .xylonic, mannonic, galactonic and gluconic acid) has met with success. Here, other methods have failed 23.The relation between the structure of the acid and its elution position seems to be complicated, but from the theory of borate compleses with polyhydroxy compounds the order of elution in some systems can be predicted. The observations that aldonic acids appear in order of increasing molecular weight and that methylated uranic and biouronic acids appear much earlier than the corresponding non-methylated species arc cxplaincd by esisting theories, but these cannot explain the individual behavior of all stcreoisomcrs. Despite technical improvements and automation, separations in borate medium are in most cases more tedious than in other media. With many complicated systems, however, the method cannot be abandoned. In recent investigations on uranic acids, seven biouronic acids were studied. In sodium acetate (0.08 M) only three well separated bands were recorded. Acetic acid offered a separation into five bands while in sodium tctraborate all seven species were scparatcd. In this medium one of the peaks had the same position as the most common monouronic acid in wood chemistry (4-0-methylglucuronic acid). In acetic acid the peak elution volumes of these two compounds differed by a factor of 3, Separations in various cluants arc, thcreforc, necessary to obtain a complete resolution of complicated mixtures. As in metal separations, the application of mixed solvents can greatly improve the separation factors of various organic acids. In mixtures of water and organic solvents the interaction forces with the resin matrix decreasea, and activity coefficients, dissociation, and complcs constants are altered. With the polyhydrosy acids partition effects become predominant. In aqueous ethanol, aldonic acids and uranic acids can be retained effectively by a resin in its sulfate form under conditions where less polar acids such as acetic acid pass directly into the eflluent2G. Very little work has been done on separations based upon partition effects of very similar acids. PARTITION
CHROMATOGRAPHY
OF SUGARS
Among the 16 monosaccharides available from common commercial sources, 14 can be separated from one another on a column with a strongly basic anion eschanger in its sulfate form. Aqueous ethanol is used as eluantsO. An early study of the sorption mechanism revealed that the sorption of strongly polar solutes is largely due to the higher water concentration inside the resin compared with that of the external solution. Interaction forces between the resin and the polar solutes cannot be neglected, howevera’. This was a hindrance to a simple thermodynamic treatment, but has recently been shown to be of practical importance. In this separation method the distribution coefficients of all sugars increase with an increased ethanol concentration within the interval of interest. The order of elution is unaffected, which facilitates a qualitative discussion of the influence of other variations upon the separations. Two examples are worth mentioning, Anal.
C/rim Acta, 38 (1967)
x63-168
168
0.
SAMUELSON
In cxpcriments with a strongly basic resin with a matris of cross-linked destran, fructose and tagatose, which shelved almost identical distribution coefficients on the styrcne-divinylbcnzenc resin, tverc well separated. Rhamnose and z-deoxyglucose wcrc the only sugars which did not scparatc on either of the two resins. With the cllloridc form of the dcstran resin, ;L misturc of the two sugars was rcsolvcd into two elution bands. In many other systems the chloride form is less satisfactory than the sulfate form. ‘l’tlcsc results, achicvcd by the trial and error method, demonstrate that intcraction forces hottl wittl the resin matris and the counter ions inside the resin arc of great importance In separ;ltions based upon partition chromatography. More fundamcntnl research is necessary txforc the influence of such variations upon the scparatiori factors can be predicted.
A rcvicw is given of variahlcs which affect the separation filctors in ion-cxchange ctiromuto~rr~l,liy. In favorable cases the influence of complcs formation, pH, and cluant concentration Cim be prctlicted by simple calculations, wlicreas the intcrxc:tion forces inside tllc resin phase arc still largely unc_xplored.