Anal. Chem. 1997, 69, 4842-4848
Separation and Pulsed Amperometric Detection of Alditols and Carbohydrates by Anion-Exchange Chromatography Using Alkaline Mobile Phases Modified with Ba(II), Sr(II), and Ca(II) Ions Tommaso R. I. Cataldi,* Diego Centonze, and Giovanna Margiotta
Dipartimento di Chimica, Universita` degli Studi della Basilicata, Via N. Sauro, 85, 85100 Potenza, Italy
The effect of some divalent nonelectroactive cations (DNCs) in the anion-exchange chromatographic separations with alkaline mobile phases of carbohydrates and alditols was investigated; the ions examined at a typical concentration of 1 mM were Ca(II), Ba(II), and Sr(II). The use of these cations in the eluent as their corresponding acetates or nitrates has been found to yield at least a twofold effect. First, the peak symmetry and concurrently the column efficiency is greatly improved. Second, the presence of Ba(II) or Sr(II) significantly enhances the response of all analytes investigated in pulsed amperometry at a gold working electrode. The action of the DNCs on the separation seems to be related to a very effective removal of carbonate ion from the alkaline eluent and, especially, their ability to complex cyclic and acyclic polyhydroxy compounds. Efficiency estimated from data calculated at 10% of peak height can increase by as much as 25% with a comparably lower RSD (15%, n ) 9). From the viewpoint of the separation efficiency and reproducibility of chromatographic data, all divalent inorganic ions employed were well-behaved. However, only the use of Ba(II) or Sr(II) is recommended since alkaline mobile phases containing Ca(II) ion negatively affect the gold electrode response of some analytes. In the last ten years, several papers have appeared concerning the pulsed amperometric detection (PAD) of sugars and alditols (also known as sugar alcohols) at gold electrodes in strongly basic solutions.1-12 The use of PAD is required because polyhydroxylated compounds are scarcely electroactive and their oxidation at * Corresponding author: (Fax) 39-971-474223; (E-mail)
[email protected]. (1) Hughes, S.; Johnson, D. C. Anal. Chim. Acta 1981, 132, 11. (2) Olechno, J.; Carter, S. R.; Edwards, W. T.; Gillen, D. G. Am. Biotechnol. Lab. 1987, 5, 38. (3) Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990, 62, 589A. (4) Johnson, D. C.; LaCourse, W. R. Electroanalysis 1992, 4, 367. (5) Johnson, D. C.; Dobberpuhl, D. A.; Roberts, R. A.; Vandeberg, P. J. J. Chromatogr. 1993, 640, 79. (6) Rocklin, R. D.; and Pohl, C. A. J. Liq. Chromatogr. 1983, 6, 1577. (7) Welch, L. E.; LaCourse, W. R.; Mead, D. A., Jr.; Johnson, D. C.; Hu, T. Anal. Chem. 1989, 61, 555. (8) Jackson, W. A.; LaCourse, W. R.; Dobberpuhl, D. A.; Johnson, D. C. Electroanalysis 1991, 3, 607. (9) LaCourse, W. R.; Jackson, W. A.; Johnson, D. C. Anal. Chem. 1991, 63, 134. (10) Lee, Y. C. Anal. Biochem. 1990, 189, 151. (11) LaCourse, W. R.; Johnson, D. C. Anal. Chem. 1993, 65, 50. (12) Johnson, D. C.; LaCourse, W. R. In Carbohydrate Analysis. High Performance Liquid Chromatography and Capillary Electrophoresis; El Rassi, Z., Ed.; Elsevier: Amsterdam, 1995; Chapter 10.
4842 Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
a Au electrode in alkaline solutions is characterized by fouling of surface by accumulated detection products.3-5 Moreover, the attractiveness of such a detection mode stems from the fact that carbohydrates and CHOH-bearing molecules, which are weakly ionizable compounds, can be successfully separated in highperformance anion-exchange chromatography (HPAEC) and capillary electrophoresis.12-14 Both these separation methods in alkaline media combine very well with PAD. Actually, HPAEC is particularly appropriate for the separation of sugars and alditols provided that the alkaline mobile phase is prepared by carbonate-free sodium hydroxide solutions.15 Of particular concern is the role that carbonate ion plays in limiting the column performances; its effects are sufficient to warrant removal from the alkaline eluent. Indeed, the presence of carbonate in the mobile phase poses some practical problems in terms of irreproducible effects on retention times and column efficiency when its concentration is unknown or frequently changed. A current strategy for minimizing the uptake of carbon dioxide, and thus of carbonate in the mobile phase, is to sparge the eluent and to pressurize the reservoir with an inert gas such as helium or nitrogen. Whereas such a strategy is apparently consistent to apply, it does not guarantee the complete absence of carbonate ion in the column. Even if commercially available carbonate-free 50% NaOH solutions are employed, the use of no well-degassed water in which carbon dioxide is present may significantly affect the separation of alditols and carbohydrates. Thus, the aim of this investigation was to explore the use of an innovative procedure for improving the chromatographic separation and detection of alditols and carbohydrates. The common knowledge that some divalent nonelectroactive cations (DNCs) such as Ca(II), Ba(II), or Sr(II) rapidly form insoluble carbonate salts has led us to consider a competitive, and more straightforward approach of minimizing the CO32- level in the mobile phase. Instead of preventing the introduction of atmospheric carbon dioxide during preparation, use, and storage of the eluent, our strategy is to affect the solubility of carbonate by the formation of sparingly soluble salts. There is currently no report of DNCs used for improving the separation of carbohydrates and alditols in HPAEC with PAD under any conditions. It is well-known, however, that cation-exchange supports loaded with Ag+, Pb2+, or Ca2+ counterions have been widely applied to (13) Lu, W.; Cassidy, R. M. Anal. Chem. 1993, 65, 2878. (14) O’Shea, T. J.; Lunte, S. M.; LaCourse, W. R. Anal. Chem. 1993, 65, 948. (15) Dionex Corp. Installation, Instructions and Troubleshooting Guide, 1992; Document 034752. S0003-2700(97)00374-0 CCC: $14.00
© 1997 American Chemical Society
separate mono- and disaccharides.16-18 Deionized water was used as the eluent in such chromatographic mode, in which the mechanism of separation acts through ligand-exchange interactions. The formation of relatively stable bi- or tridentate adducts with metal ions by hydroxyl groups of aldoses or alditols it is believed to influence the elution order of solutes.19 The stability of such solution complexes is indeed determined by the steric arrangement of the OH groups and by the metal cation involved, and the ability to orient the hydroxyl groups toward the metal ion are of major significance. Actually, the formation of complexes between sugars and metal ions has been the subject of investigation since 1825,20 but only in the last thirty years there has been more attention focused on the interactions of metal ions with polyhydroxylated compounds.21,22 We demonstrate here that not only it is possible to perform chromatographic separations in the presence of Sr(II), Ba(II), or Ca(II) without the need of sparging the alkaline mobile phase with inert gases but the simple strategy we have adopted presents unexpected benefits on column performances, i.e., improved column efficiency and better run-to-run reproducibility. The marked improvements were evaluated and discussed in terms of complex-forming ability of determined sugar conformers with Sr(II), Ba(II), and Ca(II) and the effect of such solution complexes on the capacity factors. EXPERIMENTAL SECTION Chemicals. Sodium hydroxide, 50% (w/w) solution in water (d ) 1.515 g/mL), Ca(CH3COO)2‚H2O 99+%, Sr(CH3COO)2 (water ∼3%), Sr(NO3)2, Sr(OH)2‚8H2O 96%, Ba(CH3COO)2 99%, xylitol, D-fructose, and D-ribose were purchased from Aldrich (Milan, Italy); myo-inositol, D-sorbitol, D-mannitol, i-erythritol, D-glucose, and sucrose were from Sigma (Milan, Italy) and were used as received. Stock solutions of sugars and alditols were prepared in pure water containing 0.1% sodium azide to prevent microbial growth. Just before use, samples to be injected were prepared from the stock solutions by dilution to the desired concentration with pure water. Other chemicals employed were of analytical grade and were used without further purification. Doubly distilled, deionized water was used throughout for preparing solutions. Sodium hydroxide solutions used as the mobile phases of the desired concentration were prepared by diluting a carbonate-free 50% (w/w) NaOH solution. The exact concentration of the hydroxide ion in the mobile phase was determined, by titration against a standard solution of hydrochloric acid using phenolphthalein as an indicator in the neutralisation reaction. Chromatographic System and Detection. All chromatograms were generated using a metal-free isocratic pump (Dionex, Sunnyvale, CA), Model IP20, a Dionex pulsed amperometric detector, Model ED40, and a Dionex metal-free rotary injection valve with 10-µL injection loop. A Dionex CarboPac MA1 column, 8.5-µm bead diameter (250 mm × 4 mm i.d.), coupled with a guard CarboPac MA1 column (5 mm × 4 mm i.d.) was used for the separations. The flow-through detection cell (Dionex) contained a 1.0-mm-diameter gold working electrode and a Ag/AgCl reference electrode with the titanium cell body serving as the counter (16) Goulding, R. W. J. Chromatogr. 1975, 103, 229. (17) Sasaki.; K.; Hicks; K. B.; Nagahashi, G., Carbohydr. Res. 1988, 183, 1. (18) Bonn, G. J. Chromatogr. 1985, 350, 381. (19) Honda, S.; Suzuki, S.; Kakehi, K. J. Chromatogr. 1984, 291, 317. (20) Colloud, F. Me` m. Soc. Acad. Savoie 1825, 1, 34; J. Pharm. 1825, 11, 562. (21) Angyal, S. J. Aust. J. Chem. 1972, 25, 1957. (22) Angyal, S. J. Adv. Carbohydr. Chem. Biochem. 1989, 47, 1.
electrode. Acquisition and processing of chromatographic data were done by a personal computer equipped with the Kontron PC Integration Pack software (Kontron Instruments, Milan, Italy). The chromatographic system was operated using column flow rates of 0.4 or 0.5 mL/min. Sodium hydroxide eluents were kept in plastic bottles. The same column was used in all experiments reported here. Column re-equilibration after modification of the alkaline mobile phase with the DNCs generally requires several hours, so we adopted a flush of the column overnight at a flow rate of 0.1 mL/min. All experiments were carried out at ambient temperature, 21 ( 2 °C. No problems were encountered by using DNCs in the alkaline mobile phases. All the experimental results presented here were deliberately performed without filtering out the mobile phase; the formation of insoluble precipitates, hydroxides, and/or carbonates do not cause any problems to the chromatographic system provided that the pump is extensively washed with water after each session of work. It is, however, good practice to filter the mobile phase upon addition of the divalent metal ions. As detection mode we have employed a modified version of pulsed amperometry that involves the electronic integration of current, integrated pulsed amperometric detection (IPAD).11 Unless otherwise stated, the pulsed amperometric detector settings were as follows: EOX ) +650 mV (tOX ) 190 ms), EDET ) +50 mV (tDEL ) 150 ms, and tINT ) 300 ms), and ERED ) -150 mV (tRED ) 340 ms). The response time was set to 1s. Calculations. Since measurement of theoretical plates (observed column efficiency), N, is significantly dependent on the peak asymmetry, such a value was calculated both from N ) 5.545(tR/w0.5)2 and, as recommended by Foley and Dorsey23 from N0.1 ) 47.1(tR/w0.1)2/(As + 1.25), where tR is retention time, w0.5 and w0.1 are peak widths at 50 and 10%, respectively, and As (>1) is the peak asymmetry factor evaluated as B/A and obtained at 10% peak height. The capacity factor, k′, was calculated according to the expression k′ ) (tR - tM)/tM, where tR is the retention time and tM is the column dead time, measured from the front disturbance in the chromatogram. An average of at least three replicates was used for the calculations. RESULTS AND DISCUSSION Anion-Exchange Separations. Based on the weak acidic properties of alditols, which are even weaker acids than carbohydrates, there is a need for strongly alkaline eluents where these compounds are ionized sufficiently to make possible their separation by an anion-exchange mechanism. The most used mobile phase is made by sodium hydroxide, but some examples of barium hydroxide solutions also exist.24,25 The most common alditols and carbohydrates were chosen to evaluate the effects of DNCs in HPAEC. The solubility products (pKs) in water at ambient temperature26 of Ca(OH)2, Sr(OH)2, and Ba(OH)2‚8H2O are 4.2, 3.8, and 1.6, respectively. Thus, relatively high concentrations of Ca(II), Ba(II), or Sr(II) in highly basic solutions cannot be obtained since metal hydroxide precipitation occurs. As a result, millimolar concentrations of these cations were tested as components of the sodium hydroxide mobile phase. (23) Foley J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730. (24) Johnson, D. C. Nature (London) 1986, 321, 541. (25) Johnson, D. C.; Polta, T. Z. Chromatogr. Forum 1986, 1, 37. (26) The Handbook of Chemistry and Physics, 67th ed.; Weast, R. C., Ed.; Chemical Rubber Co.: Cleveland, OH, 1986.
Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
4843
Figure 1. Separation of alditols and carbohydrates in HPAEC with IPAD in the (a) absence and (b) presence of Sr(II) ion in the mobile phase. Peaks and concentrations: (1) myo-inositol, 6.2 µM; (2) xylitol, 50 µM; (3) D-sorbitol, 20 µM; (4) D-mannitol, 20 µM; (5) D-glucose, 20 µM; (6) D-fructose, 25 µM; (7) D-ribose, 25 µM; and (8) sucrose, 50 µM. The eluents were (a) 0.50 M NaOH prepared with nondegassed water and (b) 1 mM Sr(CH3COO)2 in 0.50 M NaOH; flow rate, 0.5 mL/min. The column was a Dionex Carbopac MA1 (250 × 4 mm) plus guard column. Detection at a gold working electrode with the following wave form; EOX ) +650 mV (tOX ) 190 ms), EDET ) 0 mV (tDEL ) 150 ms, and tINT ) 300 ms), and ERED ) -150 mV (tRED ) 340 ms).
Sr(II)-Containing Alkaline Eluents. Usually, carbonate-free mobile phases are required for obtaining reproducible retention times and minimal chromatographic peak distortion. Practically, to minimize the effect of CO2 pickup from the atmosphere, NaOH solutions need to be continuously sparged with inert gases such as nitrogen or helium. A considerable improvement in terms of column efficiency and selectivity was simply achieved by using alkaline mobile phases (i.e., 480-620 mM NaOH) modified with strontium as acetate or nitrate salts.27 Sample solutions containing alditols and carbohydrates were examined. Figure 1 shows the striking difference observed in the separation of a mixture of myoinositol, xylitol, D-sorbitol, D-mannitol, D-glucose, D-fructose, Dribose, and sucrose using as the eluents (a) 0.50 M NaOH and (b) 1 mM Sr(CH3COO)2 in 0.50 M NaOH. In Figure 1a, it may be noted that especially D-sorbitol (peak 3) and D-ribose (peak 7) present the most pronounced asymmetrical peaks due to tailing, with D-ribose exhibiting more tailing than D-sorbitol. The chromatographic data capacity factor, k′, selectivity, R, and peak asymmetry factor, As, evaluated from both chromatograms a and b are compared in Table 1. The N0.1 parameter represents the number of theoretical plates calculated at 10% of the peak height as recommended by Foley and Dorsey23 when asymmetric peaks are involved. Interestingly, both peak symmetry and intensity of all compounds were significantly enhanced when the Sr(II)(27) Cataldi, T. R. I.; Centonze, D.; Margiotta, G.; Zambonin, C. G. Abstract of Euroanalysis IX, Bologna, Italy, September, 1996.
4844 Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
containing alkaline eluent was employed. This last aspect will be discussed later. Note that a better peak shape of D-ribose and D-sorbitol is also reflected in the higher number of theoretical plates. There is a noticeable (statistically significant) increase in plate counts for D-sorbitol and D-ribose by a factor approximately higher than 2, and 3, respectively. The mean plate number value ((SD) in the presence of strontium, 3100 ( 1000, is higher than that evaluated with the unmodified eluent, 2200 ( 1100. For comparison, the number of plates evaluated from the peak width at half peak height (N) is also reported. Although this value should be little affected by baseline tailing, the observed efficiency seems to be overestimated by more than 35-40% when the modified mobile phase was used with a marked dispersion of data, that is 33% as relative standard deviation (RSD). The background signals do not change significantly with the presence of strontium ion in the alkaline mobile phase, and column back pressure remains practically constant. Apparently, the beneficial effects of strontium on peak shape can be assigned to depletion of carbonate from the mobile phase. This is presumably due to formation of sparingly soluble SrCO3 (solubility, 0.0011 g/100 mL)26 and consequent precipitation in the eluent reservoir. Note that D-sorbitol, D-fructose, and D-ribose exhibit a noticeable decrease in the retention. Such a behavior is not fully justified because the removal of carbonate (a strong eluent ion) should lead to longer retention times. To prove whether the carbonate ion was really removed from the alkaline mobile phase, its content was evaluated by anion-exchange chromatography with suppressed conductivity detection. The residual carbonate present was, as expected, below the limit of detection of the adopted analytical method. These findings, hence, imply that an additional effect is actually involved in the reduced retention times, which is probably related to complexation between polyhydric compounds and divalent ions.21 Angyal has reported, in an excellent review, that the ions most firmly bound to sugar molecules are Ca(II), Sr(II), Ba(II), and La(III).22 The most effective of such arrangements is a sequence of an axial (ax), an equatorial (eq), and an axial (ax) hydroxyl group on a six-membered ring, or three consecutive cis hydroxyl groups on a five-membered ring. The common forms of D-ribose, the R-pyranose (Ia and Ib) and β-pyranose (IIa and IIb), and the R-furanose (III) and β-furanose (IV), are reported in Chart 1. Interconversion between all species of this aldopentose is possible through a series of tautomeric rearrangements. In aqueous solution, β-D-ribopyranose is a conformational mixture of the 4C1 (IIa) and the 1C4 (IIb) forms in the approximate proportion of 3:1. This is fully justified because the 4C1 form has three equatorial hydroxyl groups, whereas the alternative conformation 1C4 has only one, and the equilibrium composition is a function of their relative free energies.28 On considering the feasibility of complex formation with divalent cations, the 1C4 conformer contains an ax-eq-ax sequence of the oxygen atoms on C2, C3, and C4 and will form therefore a relatively more stable complex with free Sr(II) ions. Thus for the β-pyranose forms, the complexation should shift the equilibrium in favor of IIb, changing the conformational equilibrium, which accounts for the related change in the ion-exchange equilibrium with the stationary phase. On the basis of the ax-eq-ax rule, both the R-pyranose conformations, the 4C1 (Ia) and 1C4 (IIa), will form stable complexes. For the former, oxygen atoms on the C1, C2, and C3 are involved in the metal complex, while in the conformation IIa (28) Lenkinski, R. E.; Reuben, J. J. Am. Chem. Soc. 1976, 98, 3089.
Table 1. Effect of Sr(II) Ion on the Separation of Alditols and Carbohydrates in HPAECa xylitol
myo-inositol
D-sorbitol
k′c Rd Ne As f N0.1g
0.59 4500 1.17 2700
1500 1.45 1200
1000 1.38 900
k′c Rd Ne As f N0.1g
0.57
1.25
2.01
1.32 2.24
6000 1.00 4300
2.23 1.69
2.19
1.61 3200 1.21 2100
D-mannitol
1.19
D-glucose
0.50 M NaOH b 2.66 1.29 2800 1.15* 2100
D-fructose
3.44
D-ribose
4.31 1.25
5500 1.29* 3700
0.50 M NaOH + 1 mM Sr(CH3COO)2 h 2.54 3.41 1.26 1.34 1.23 2100 4600 6000 1.18 1.08* 1.15* 1700 3600 4200
5.16 1.20
4800 1.10* 3500
7.59 1.47
1000 1.32 900
4.20
4000 1.26* 2900
4.74 1.13
5000 1.12 3600
sucrose
7.57 1.60
3100 1.21 2200
3900 1.19* 3000
a Column, CarboPac MA1 plus guard column; flow rate, 0.5 mL/min; back pressure, 109 bar; E b DET ) 0.000 V. Alkaline mobile phase prepared by a 50% NaOH solution using nondegassed water. c Capacity factor evaluated with a dead time of 4.42 min. d Selectivity factor. e Number of theoretical plates evaluated using the tangent method. f Peak shape evaluated as asymmetric factor (As) at 10% of peak height. Values with asterisk refer to values evaluated as A/B instead of B/A. g Number of theoretical plates evaluated as N0.1 ) 41.7(tR/w0.1)2/(As + 1.25). h After the strontium salt was added at the sodium hydroxide mobile phase, the column was flushed overnigth.
Chart 1
the oxygens involved are bound to C2, C3, and C4. Note that the β-pyranose has to change into a higher energy conformer in order to complex the cation, so it will form a complex to a lesser extent than does the R anomer, which has an ax-eq-ax unit in its predominant conformation. The five-membered ring systems exist as R-ribofuranose and β-ribofuranose, III and IV forms, respectively. The R form (III) having an ax-eq-ax sequence should bind strongly to Sr(II), whereas the β form (IV) should not. The net effect should be the prevalence of D-ribopyranose and D-ribofuranose forms that are stabilized by formation of complexes with Sr(II) ions.
Also, monosaccharide alditols form complexes with cations, but the extent of complex formation varies considerably.29 The relative effectiveness of acyclic complexing sites follows the descending order22 threo-threo triol > threo pair adjacent to a primary hydroxyl group > erythro-threo triol > erythro pair adjacent to a primary hydroxyl group > erythro-erythro triol. If we consider the alditols investigated in this work, it appears that the complex ability is greater for D-sorbitol, which has a threothreo triol and an erythro-threo triol sequence, plus a threo and an erythro pair adjacent to primary hydroxyl groups, compared to D-mannitol, which possesses two erythro-threo triol sequences and two erythro pairs adjacent to primary hydroxyl groups. Xylitol is a pentahydroxylated compound, which has a threo-threo triol sequence and two threo pairs adjacent to primary hydroxyl groups. The interaction with divalent metal cations should increase with the number of favorable orientated hydroxyl groups present. Thus, the complex formation between Sr(II) and open-chain carbohydrates, notably D-sorbitol, is accompanied by a change of conformation that may be responsible for the considerable improvement of peak symmetry. Although the mobile-phase pH should control the charge on the sample molecules, the formation of adducts leads possibly to positively charged solutes, thereby shifting the equilibrium toward the eluent phase. A reduced retention is consistently observed because these complexes will be less retained by an anion-exchange mechanism. We wish to underline that we are not at all certain about the stoichiometry in basic solutions of sugar-divalent metal ion complexes, which is function of many variables, such as concentration of the free Sr(II) and concentration of the polyhydroxy compound and its conformation. The result is, however, a mobile phase that matches the carbonatefree requirement in HPAEC separations and ensures a better runto-run reproducibility. Ba(II)-Containing Alkaline Eluents. It must be emphasized that the alkaline mobile phase of chromatogram a in Figure 1 was prepared by diluting a 50% NaOH solution with nondegassed water and without degassing the eluent reservoir during the chromatographic runs. Figure 2a shows the chromatogram obtained for the detection and separation of a test solution in carbonate-free 0.58 M NaOH mobile phase, prepared and stored as recommended by the column manufacture. Whereas peak broadening (29) Dawber, J. G.; Hardy, G. E. J. Chem. Soc., Faraday Trans. 1 1984, 80, 2467.
Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
4845
Figure 2. Separation of alditols and carbohydrates in HPAEC with IPAD in the (a) absence and (b) presence of Ba(II) ion in the mobile phase. Peaks and concentrations: (1) myo-inositol, 12.5 µM; (2) i-erythritol, 50 µM, (3) xylitol, 50 µM; (4) D-sorbitol, 20 µM; (5) D-mannitol, 20 µM; (6) D-glucose, 25 µM; (7) D-fructose, 50 µM; (8) D-ribose, 25 µM; and (9) sucrose, 50 µM. Eluents: (a) 0.58 M NaOH was prepared with carbonate-free 50% NaOH using degassed water and was continuously sparged with purified N2; (b) 1 mM Ba(CH3COO)2 in 0.58 M NaOH. Flow rate, 0.4 mL/min. Other conditions as in Figure 1, but EDET ) +50 mV.
and a noticeable asymmetry are still evident on the D-sorbitol and especially on D-ribose peaks, their shape and symmetry are distinctly enhanced when the running mobile phase (0.58 M NaOH) contained 1 mM Ba(CH3COO)2, as demonstrated clearly in Figure 2b. Hence, under these experimental conditions and similarly to the mobile phase containing strontium ion, it was possible to obtain a more satisfactory separation of all components the mixture. To compare the inherent advantage of using barium ion as an eluent additive, chromatographic data collected for each compound are presented in Table 2. Although the tabulated data for the asymmetry factors of myo-inositol, i-erythritol, D-mannitol, D-glucose, D-fructose, and sucrose do not necessarily show great improvement using the mobile phase containing Ba(II) ions over the blank eluent because these peaks exhibit already good symmetry, the peak tailing of xylitol, D-sorbitol, and D-ribose show a marked enhancement. With D-ribose, for instance, a significant change of As from 1.90 to 1.07 was observed. The experimentally observed plate numbers for D-sorbitol, 3200, and D-ribose, 3600, are significantly higher compared to 1400 and 1200, respectively. Besides, the observed column efficiency was increased by as much as 25% with a greater precision of the plate counts (RSD 15%, n ) 9). Most significant is the observation that retention times are slightly decreased for nearly all compounds, with D-ribose being more affected, passing from 27.25 min in 0.58 M NaOH to 26.15 min using the modified eluent. Again, we suggest that the enhanced symmetry of D-ribose in the presence of Ba(II) may be explained as due to formation of relatively strong complexes, especially with those conformers having an ax-eq-ax arrangement of hydroxyl groups (Chart 1). As pointed out earlier, in the presence of a complexing cation in the mobile phase, some of 4846 Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
the R-pyranose will be removed from the equilibrium as a metal complex, and the equilibrium will shift in favor of the R-pyranose form. Accordingly, this effect leads to a marked shift of the complex toward the alkaline mobile phase. The improved symmetry may be then the result of a faster conversion from one conformer to another in comparison to the chromatographic time scale. The inclusion of strontium ion as nitrate or acetate salts (1 mM) in carbonate-free alkaline mobile phases was also investigated (results not shown). The resulting chromatographic data compare very well with those evaluated in the case of Ba(II) (see Table 2). The within-run precision values of retention times (n ) 3) were from 0.15 to 0.38% RSD. Using the same eluent for four days, even nonconsecutively, the values for the between-run precision of the retention times ranged from 0.52 to 0.84% RSD (n ) 8). The overall result is that the presence of divalent cations affects the separation in two ways: by lowering the carbonate content in the alkaline mobile phase and forming relatively stable complexes with certain alditols and carbohydrates. Table 3 provides a comparison of the DNCs used in terms of column efficiency, which was evaluated using all peaks present in each chromatogram (n ) 9). Although the average numbers of plates (N0.1) are not statistically different (P ) 0.01) as verified using the t-test30 when conventional and modified alkaline mobile phases were employed, the calculated F ratio of the F-test (12002/6002 ) 4.0) was significantly greater than the critical value (3.44 with ν1 ) ν2 ) 8) at the 95% confidence level, so it can be concluded that the null hypothesis is rejected and the proposed mobile phase containing Ba(II) is characterized by a higher precision in the column efficiency. As outlined earlier, the presence of Ba(II) or Sr(II) in alkaline electrolytes enhances the amperometric response of several sample molecules. To assess the impact of mobile-phase modification on sensitivity, the percentage increments in peak heights and areas were evaluated (Table 4). It can be inferred that both barium and strontium cation also contribute significantly to the response of alditols and carbohydrates. Indeed, for most of these compounds, the peak areas (heights) are considerably increased. For example, the increments obtained in peak areas of D-fructose were 80%, and 65% with Ba(II) and Sr(II), respectively. Such relative increments may be explained as due to specific interactions between the gold electrode surface and either the metal complexes or the DNCs, or both. Work is in progress to clarify these findings.31 Ca(II)-Containing Alkaline Eluents. The idea of modifying the alkaline mobile phase for use in HPAEC was expanded to include calcium as divalent cation; thus, its effect was investigated in another set of experiments. This because most ligand-exchange separations of aldoses are reported with cation-exchanger columns loaded with Ca(II).16,19,32 Figure 3 shows the chromatograms of a standard solution, recorded under analogous experimental conditions before (a) and after (b) the addition of calcium acetate in the mobile phase. As discussed earlier, when the regular alkaline eluent was used, the peak distortion and plate counts are considerably worse than subsequent separations obtained in the presence of Ca(II). Detailed quantitative data are summarized in Table 5, in which can be observed an enhanced symmetry and (30) Miller, J. C.; Miller, J. N. In Statistics for Analytical Chemistry; Ellis Horwood Ltd.: Chichester, U.K., 1988, pp 55-59. (31) Cataldi, T. R. I.; Casella, I. G.; Centonze, D. Anal. Chem. 1997, 69, 4849. (32) Wang, W.-T.; Safar; J.; Zopf, D. Anal. Biochem. 1990, 188, 432.
Table 2. Effect of Ba(II) Ion on the Separation of Alditols and Carbohydrates in HPAECa myo-inositol
i-erythritol
xylitol
k′c R N As N0.1
0.57
1.03
1.28
5000 1.04 3700
5300 1.02 4200
3200 1.35 2700
k′ R N As N0.1
0.55
0.99
1.23
1.81
1.24
1.80
D-mannitol
0.58 M NaOH b 2.46 1.24 1.17 4600 1.00 4100
1.98 1.55 2200 1.57 1400
D-glucose
2.88
5600 1.00 4500
D-fructose
D-ribose
sucrose
3.55
4.00
6.26
1.23 5600 1.19* 4300
0.58 M NaOH + 1 mM Ba(CH3COO)2d 1.87 2.35 2.77 1.52 1.26 1.18 4000 3700 5500 5500 1.19 1.14 1.08* 1.15* 3300 3200 4700 4400
1.24
5100 1.08 3500
D-sorbitol
1.13 4500 1.10* 3600
2300 1.90 1200
3.40 1.23
1.56
3.80 1.12
5500 1.13* 4400
4200 1.14* 3100 5.94 1.56
5000 1.07 3600
3700 1.12* 3200
a Flow rate, 0.4 mL/min; back pressure, 101 bar; E b DET ) +0.050 V. Alkaline mobile phase prepared by carbonate-free 50% NaOH solutions using freshly bidistilled water. c Capacity factor evaluated with a dead time of 5.45 min. d After the barium salt was added at the sodium hydroxide solution the column was flushed overnight with the modified mobile phase.
Table 3. Comparison of the Column Efficiency in HPAEC for Different Alkaline Mobile-Phase Compositions mobile phase
103 N ( SD (n ) 9)a
103 N0.1 ( SD (n ) 9)
0.58 M NaOH 0.58 M NaOH + 1 mM Sr(NO3)2 0.58 M NaOH + 1 mM Ba(CH3COO)2 0.58 M NaOH + 1 mM Ca(CH3COO)2
4.1 ( 1.4 4.4 ( 0.7 4.8 ( 0.8 4.7 ( 0.9
3.1 ( 1.2 3.6 ( 0.6 3.9 ( 0.6 3.8 ( 0.6
a
Number of peaks in the chromatogram.
Table 4. Relative Increment (%) of Response Observed for Alditols and Carbohydrates at a Au Electrode in HPAEC with Integrated Pulsed Amperometric Detection upon Adding Ba(II), Sr(II), or Ca(II) Ions at the Alkaline Mobile Phasea Ba(II)b
myo-inositol i-erythritol xylitol D-sorbitol D-mannitol D-glucose D-fructose D-ribose sucrose
Sr(II)c
Ca(II)d
peak heighte (%)
peak area (%)
peak height (%)
peak area (%)
peak height (%)
peak area (%)
+25 +12 +65 +95 +55 +30 +90 +120 +65
+25 +10 +40 +45 +40 +28 +80 +50 +55
+20
+20
+35 +80 +40 +20 +70 +90 +30
+25 +50 +30 +15 +65 +50 +30
+10 -35 +10 +45 -10 -25 -15 +60 -50
15 -30 -5 10 -15 -20 -10 +10 -50
a Column, CarboPac MA1 plus guard column; flow rate, 0.4 mL/ min. b Mobile phase, 0.58 M NaOH + 1 mM Ba(CH3COO)2. c Mobile phase, 0.58 M NaOH + 1 mM Sr(NO3)2. d Mobile phase, 0.58 M NaOH + 1 mM Ca(CH3COO)2. e Averaged values (5% RSD.
generally higher plate counts for all analytes examined. Although the greater ability of Ca(II) to complex sugars and alditols in aqueous solutions is recognized,33-35 the extent of reduction in capacity factors, observed for each analyte in the present experimental conditions, is comparably similar to that reported above (33) Briggs, J.; Finch, P.; Matulewicz, M. C.; Weigel, H. Carbohydr. Res. 1981, 97, 181. (34) Symons, M. C. R.; Benbow, J. A; Pelmore H. J. Chem. Soc., Faraday Trans. 1 1982, 78, 3671. (35) Symons, M. C. R.; Benbow, J. A; Pelmore H., J. Chem. Soc., Faraday Trans. 1 1984, 80, 1999.
Figure 3. Separation of alditols and carbohydrates in HPAEC with IPAD in the (a) absence and (b) presence of Ca(II) ion in the mobile phase. Peaks and concentrations: (1) myo-inositol, 12.5 µM; (2) i-erythritol, 20 µM; (3) xylitol, 75 µM; (4) D-sorbitol, 20 µM; (5) D-mannitol, 20 µM; (6) D-glucose, 25 µM; (7) D-fructose, 50 µM; (8) D-ribose, 25 µM; and (9) sucrose, 50 µM. Eluents: (a) 0.58 M NaOH prepared by carbonate-free 50% NaOH and degassed water; (b) 1 mM Ca(CH3COO)2 in 0.58 M NaOH. Flow rate, 0.4 mL/min. Other conditions as in Figure 1 except EDET ) +50 mV.
in the case of Sr(II)- or Ba(II)-containing mobile phases. Moreover, from the viewpoint of the separation efficiency, all divalent inorganic cations employed were well-behaved, providing virtually identical beneficial effects. Regarding peak intensities as well as peak areas in the presence of Ca(II), however, the prominent and easily recognizable feature observed is that some analytes such as i-erythritol, D-mannitol, D-glucose, D-fructose, and sucrose exhibit a noticeable decrease. As a result, the use of calcium for the analysis of carbohydrates in HPAEC-IPAD is not recommended. This inhibition effect of calcium ion on the gold electrode surface is under investigation.31 In addition, more fundamental work is still needed to understand the complexation Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
4847
Table 5. Effect of Ca(II) Ion on the Separation of Alditols and Carbohydrates in HPAECa xylitol
myo-inositol
i-erythritol
k′c R N As N0.1
0.54
0.95
4700 1.03 3800
5600 1.05 4200
2900 1.38 2300
k′ R N As N0.1
0.52
0.94
1.16
1.76
4600 1.04 4400
1.20 1.26
1.81
4500 1.20 2900
D-mannitol
0.58 M NaOHb 2.31 1.24 1.18 2300 5200 1.45 1.02* 1500 4400 1.86
1.55
1.23 6000 1.02 4400
D-sorbitol
D-glucose
2.72
D-fructose
D-ribose
sucrose
3.58
3.82
5.81
1.31 6000 1.20* 4200
0.58 M NaOH + 1 mM Ca(CH3COO)2d 1.76 2.24 2.65 1.52 1.27 1.18 4100 4900 5500 1.06 1.18* 1.15* 3300 4400 4300
1.07 5400 1.17* 4500
2200 1.60 1600
3.25 1.23
1.52 4200 1.17* 4000
3.60 1.11
5400 1.17* 3900
5.52 1.53
4800 1.04 3800
2700 1.10 3200
a Flow rate, 0.4 mL/min; back pressure, 101 bar; E b DET ) +0.050 V. Alkaline mobile phase prepared with carbonate-free 50% NaOH solutions using freshly bidistilled water. c Capacity factor evaluated with a dead time of 5.51 min. d After the calcium salt was added at the sodium hydroxide solution, the column was flushed overnight with the modified mobile phase.
chemistry in alkaline solutions between DNCs and aldoses and alditols as well. CONCLUSIONS The findings presented here suggest that, while strontium, barium, and calcium ions do not result in uniform improvements in all performance parameters with all solutes under anionexchange separations with integrated amperometry, the use specifically of Sr(II) or Ba(II) in the alkaline eluents is powerful for its simplicity, providing substantial benefits in terms of peak shape and attainable column efficiency. Both barium and strontium ions enhance the sensitivity when compared to the separations of unmodified eluents under otherwise identical chromatographic conditions. Their presence ensures that thoroughly carbonate-free mobile phase flows through the column. Moreover, the present results strongly support that metal ion binding to sugars and sugar alcohols also occurs in alkaline solutions. More detailed studies based on an extended examination of sugar complexes with Ca(II), Ba(II), and Sr(II) ions and other cations, including Pb(II) and La(III), should be of interest in much less alkaline mobile phases in order to affect the retention more effectively. Another important conclusion is that the enhanced sensitivity and selectivity observed with the modified alkaline mobile phases
4848 Analytical Chemistry, Vol. 69, No. 23, December 1, 1997
result in simpler chromatographic conditions. Mixtures of alditols and carbohydrates can be repeatedly separated without deterioration of column performances, also avoiding irreproducible quantitative results in certain situations where real samples contain unknown amounts of carbonate. The introduction of this novel procedure will considerably enhance the potential of analysis of alditols and carbohydrates by HPAEC with IPAD, rendering the control of carbonate content in the mobile phase much less problematic. ACKNOWLEDGMENT The Regione Basilicata provided partial funding for developing a laboratory of synthesis and characterization of innovative materials “LaMI”. This work was also supported by the National Research Council of Italy (CNR, Rome) and Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST, Rome).
Received for review April 8, 1997. 1997.X
Accepted July 29,
AC970374J X
Abstract published in Advance ACS Abstracts, October 15, 1997.
