Triple-pulse amperometric detection of carbohydrates after chromatographic separation

Analytica Chimica Acta, 149 (1983) l-10 Blsevier Science Publishers B.V., Amsterdam -Printed

in The Netherlands

TRIPLE-PULSE AMPEROMETRIC DETECTION OF CARBOHYDRATES AFTER CHROMATOGRAPHIC SEPARATION

SCOTT HUGHESa and DENNIS C. JOHNSON* Department

of Chemistry,

Iowa State

University,

Ames, IA 50011

(U.S.A.)

(Received 2nd August 1982)

SUMMARY Triple pulse amperometry at a platinum wire electrode is demonstrated for chromatographic separations of carbohydrate mixtures on two commercially available “carbohydrate columns” and the performances of the columns are compared. The effect of detector temperature is described; an increase from 35 to 85°C resulted in an increase of sensitivity by a factor of 1.5-2.1. A consideration of errors is presented and dilution is recommended for samples of high concentration to minimize relative error. Results are given for determination of dextrose, fructose, glycerol, and ethanol in three wines with relative uncertainties of approximately 10% or better, calculated at the 90% confiddnce level.

Moderately efficient columns have been developed for the liquid chromatographic separation of complex mixtures of carbohydrates [l] . Because of the low molar absorptivities of carbohydrates in the ultraviolet-visible region of the spectrum, the measurement of refractive index has been the standard method of chromatographic detection. Conventional refractive index detection is sometimes characterized by a poor detection limit [2], however, a value of 0.04 mg ml-’ has been reported [3]. Recently, we described the development of a very sensitive method for the amperometric detection of organic compounds containing the C-OH moiety at platinum electrodes in alkaline media [4, 51. The technique is based on the application of a triple-pulse potential waveform which achieves detection of the organic analyte as well as reactivation of the platinum electrode within each waveform executed in a time period of 0.5-2 s. Application of triple pulse amperometry (t.p.a.) is especially promising for detection of simple alcohols, polyols, and simple carbohydrates in flow-through detectors applied for liquid chromatography as well as for flow-injection determinations [4-6]. A detection limit of about 5 pg ml-’ was obtained for dextrose in a 100-111sample by t.p.a. in 0.1 M NaOH. The mechanism for oxidation of alcohols and carbohydrates on a platinum electrode is concluded to involve adsorption of the analyte on the electrode aPresent address: Utopia Instrument Co., P.O. Box 863, Joliet, IL 60434, 0003-2670/83/$03.00

o 1983 Elsevier Science Publishers B.V.

U.S.A.

2

surface followed by the surface-catalyzed anodic dehydrogenation of the adsorbed molecule [4, 51. The hydrocarbon products of the surface-controlled reaction remain adsorbed on the electrode surface, thereby inhibiting adsorption of unreacted molecules, and the anodic current observed at the detection potential quickly decays to zero. If the electrode potential is made sufficiently large and positive, corresponding approximately to the anodic breakdown of the aqueous solvent, the adsorbed hydrocarbons are oxidatively removed from the electrode, perhaps as CO?, with concomitant formation of platinum oxide, PtO. Oxidation of the organic analyte in the solution will not occur on the oxide-covered surface. The PtO is reduced by the subsequent application of a negative potential, corresponding approximately to the cathodic breakdown of the solvent, resulting in “reactivation” of the electrode surface, and analyte molecules are again adsorbed. The waveform for t.p.a. corresponds to the sequential application of three electrode potentials corresponding to anodic detection (E,), anodic cleaning (E,), and cathodic reactivation (E3). The current is sampled near the end of the period for application of El and a proportional voltage is retained in analog or digital memory until updated during the succeeding application of the waveform. Because the mechanism of the anodic detection of alcohols and carbohydrates involves prior adsorption, the shape of the response curve is strongly influenced by the adsorption isotherm for the analyte. Under conditions for which the extent of adsorption is at an equilibrium value prior to application of E,, the absolute value of faradaic signal (I) can be related satisfactorily to the concentration of the electroactive species (C) by I = C/(b + UC)

(1)

Equation (1) has the form of the Langmuir isotherm and is expected to be valid for relatively low surface coverages, for which the lateral interaction of adsorbed molecules is minimal. The constants a and b for the detection of alcohols and carbohydrates are determined experimentally by linear regression analysis of the linear plot of l/1 vs. l/C [7] : I-’ = a + b(C)-‘. Furthermore, a and b are concluded to correspond to fundamental chemical and electrochemical constants according to the equations a = (nFAkr,,,)-’

and b = (nFAkKl?,,J1

(2)

where K is the equilibrium constant for adsorption, k is the heterogeneous rate constant (s-l) for the faradaic reaction, I’,,, is the maximum surface coverage (mol cmW2)of adsorbed analyte at high concentration in the solution, and n, F, and A have their usual electrochemical significance. There is virtually no selectivity afforded by the triple pulse technique and application of t.p.a. to mixtures of carbohydrates and alcohols is predicated on successful a priori separation. Use of h.p.1.c. for separation of carbohydrates has numerous advantages over other types of chromatography [ 8-121. Two classes of column materials are used primarily for separation of carbo-

. 3

hydrates by h.p.1.c. : silica-based materials and ion-exchange. For the former, some workers have used pure silica columns with a small amount of an amine added to the eluent which then coats the silica by simple adsorption [2, 131. More commonly, commercially available, chemically modified columns with alkylamine or octadecylamine functional groups are utilized. The mobile phase in the case of the silica columns is usually a mixture of water and acetonitrile with the water content between 10% and 40%. A retention mechanism based on hydrogen bonding between the sugar hydroxyl groups and the amine groups of the stationary phase can satisfactorily explain the observed retention orders, with longer retention times observed for sugars having a greater number of hydroxyl groups [ 14,151. Anion-exchange columns in the sulfate form have been utilized with excellent resolution of complex mixtures of carbohydrates [ 16-211. The weakly acidic hydroxyl groups of the sugar are considered to play a part in the separation; however, steric influences are probably also important [20]. Reports of long separation times, ranging from 4 h [21] to 15 h [17], have hindered the acceptance of anion-exchange columns for routine use. The separation of sugars with cation-exchange resins is steadily gaining in acceptance [9, 20, 22-241. Separations have the advantage that pure water can be used as the eluent and separation times are well under an hour. Several factors are considered to be responsible for the separation; however, the most important is thought to be the partitioning of the carbohydrate between the mobile phase and the stagnant water phase within the resin matrix. Increased resolution is obtained when the column is operated at elevated temperature, because of the increased diffusion of the analyte between the mobile and stagnant phases [9]. Size exclusion also plays a role in the separation, with the larger saccharides having the shorter retention times [24]. The degree of crosslinking of the resin can be altered to optimize this effect, with less crosslinking (i.e. larger pore size) being used for the separation of oligosaccharides. Results are included here for two commercially available cation-exchange columns in the Ca(I1) form operated at 80°C with water as eluent. EXPERIMENTAL

Instrumentation Potentiostatic control was achieved with a Model 173 potentiostat, with a Model 176 current-to-voltage converter (EG & G Princeton Applied Re search, Princeton, NJ). Generation of the triple-pulse waveform was by a device built in this laboratory from timing modules [4]. Values of potential and their duration were as follows: El = -0.40 V (182 ms) with the faradaic signal sampled for 1 ms after 173 ms, Ez = +0.80 V (185 ms), and E3 = -1.00 V (223 As). All electrode potentials were measured vs. the SCE reference. All chromatographic data were obtained with a data system [ 251 based on the HP-85 computer (Hewlett-Packard, Corvallis, OR). Separations were achieved with calcium(II)-loaded, cation-exchange

. 4

columns from Hamilton (Model HC-75, Rainin Instrument Co., Wobum, MA; lo-15 pm particle size, 7.5% cross-linked, 30-cm length) and Dionex Corp. (Sunnyvale, CA; 7.5 I.trn size, 7% cross-linked, 25-cm length). The chromatographic columns were maintained at 80°C during separations by means of a thermostated water jacket. The eluent was water, pumped at a flow rate of 0.50 ml min-’ by a Milton Roy Model CK miniPump with a Model 709 pulse dampener (Laboratory Data Control, Riviera Beach, FL). The sample-injection valve was a Rheodyne Model 7125 (Larry Bell and Assoc., Minneapolis, MN) with a sample loop of 100 ~1. Detection of carbohydrates by triple pulse amperometry (t.p.a.) is most sensitive at high pH. Hence a stream of 6.1 M NaOH pumped at 0.01 ml min-’ by a Gilson Minipuls-2 peristaltic pump (Middleton, WI) equipped with 0.25-mm i.d. polyvinyl chloride manifold tubing (Rainin Instrument Co.) was mixed with the effluent stream in a tee-connexion of low dead volume (Unimetrics, Anaheim, CA). The mixing tee was maintained at 60°C (except for the temperature study) by circulating thermostated water through a surrounding water jacket. The net concentration of sodium hydroxide in the detector was 0.1 M. All tubing was stainless-steel with compression fittings used for all connections. An adjustable needle valve constructed locally from Kel-F was connected in the flow system after the detector to generate sufficient back-pressure (about 15 psi) to eliminate bubble formation in the detector. The platinum, wire-tip, flow-through detector has been described [6]. Chemicals and procedures All chemicals were reagent grade. All water had been distilled, demineralized, passed through a 12-m. X 1.5-in. o.d. column of activated carbon, and filtered through a 0.45~pm membrane. Dissolved oxygen was removed from the eluent by saturation with nitrogen. To extend the life of the chromatographic columns, all samples were passed through a mixed bed of ion-exchange resin (Amberlite MB-3; Mallinckrodt). RESULTS

AND DISCUSSION

Chromatography The resolutions of the Hamilton and Dionex columns were compared. Synthetic samples containing 0.40 mg ml-’ each of eight carbohydrates were injected onto each column and typical chromatograms are shown in Fig. 1. The retention times (t,), number of theoretical plates (N), and heights equivalent to theoretical plates (H) are listed for each compound in Table 1. Even though the Dionex column is 5 cm shorter than the Hamilton column, it exhibited much better resolution. The smaller size of the resin particles in the Dionex column undoubtedly is a factor contributing to the higher resolution. To illustrate the applicability of the technique for a complex matrix, a

9

I 9

1i 5uA

t

10mrn ’ il

a

5’ I

dx

1 ,

J

Fig. 1. Comparison of separations for eight carbohydrates (each at 0.40 mg ml-‘) by Hamilton (I) and Dionex (II) h.p.1.c. columns: m, maltotriose; s, sucrose; d, dextrose; x, xylose; f, fructose; g, glycerol; a, arabitol; s’, sorbitol. Sample volume, 100 ~1.

TABLE 1 Comparison of performance of Hamilton* and Dionexb h.p.1.c. columns Carbohydrate=

Maltotriose sucrose Dextrose Xylose Fructose Glycerol Arabitol Sorbitol

Hamilton

Dionex

tr (min:s)

Nd

He

rr

N

H

lo:52 11~42 13~42 14:47 16:12 18 :58 20:32 23~37

1600 1100 1000 1100 1400 1400 1200 1600

0.19 0.27 0.30 0.27 0.21 0.21 0.25 0.19

lo:06 lo:56 13:OB 14:24 16:ll 19:50 21:35 25:33

1000 600 1400 1900 2100 3200 3100 3900

0.25 0.42 0.179 0.132 0.119 0.078 0.081 0.064

*Hamilton column: lo-15 I.cm particle size, 7.5% cross-linking, 30 cm long. bDionex column: 7.5 pm particle size, 7% cross-linking, 25 cm long. cO.4O mg ml-l. dN = 16 (tr/W)’ where W is the peak width. eH = L/N where L isthe column length (mm).

6

chromatogram of a human urine sample is shown in Fig. 2. Peaks were identified by comparison of retention times to standards. Peaks corresponding to dextrose, xylose, arabinose, glycerol, and urea were identified. No attempt was made to quantify the carbohydrates in this sample. The application of t.p.a. detection for quantitative purposes was demonstrated for three varieties of wine. Chromatograms are shown in Fig. 3 for 1:12.5 dilutions of the three wines, a “sweet” white, a “dry” red, and a “light” burgundy. Peaks were observed for dextrose, fructose, glycerol and ethanol. A calibration curve was made for each of these compounds. The concentrations of the compounds in the wine varieties are given in Table 2. Even though many other carbohydrates than detected here are found in grapes, they are broken down in the fermentation process and, therefore, are not observed in the final product [ 261. The absence of a peak for sorbitol is consistent with the fact that this carbohydrate, though commonly found in many fruits, is not observed in grapes. The presence of sorbitol in wines

Fig. 2. Chromatographic separation of human urine components. Separation achieved with the Dionex column. Peaks: D, dextrose; X, xylose; A, arabinose; G, glycerol; U, urea.

I

20 pA E

-,I IO

R-II”

G

F

D

-

Fig. 3. Chromatographic data for wine on the Dionex column. Wines: I, sweet white; II, dry red; III, light burgundy. Peaks: D, dextrose; F, fructose; G, glycerol; E, ethanol.

7 TABLE 2 Results of the wine analysis Carbohydrate content (mg ml-‘)

Liebfraumilch, “sweet” white Premium red, “dry” red Light burgundy, “sweet” red

Dextrose

Fructose

Glycerol

5.67 * 0.12 0.049 f 0.005 7.7 f 0.2

15.4 f 1.5 1.30 f 0.03 9.5 f 0.7

5.2 f 0.3 7.6 * 0.6 6.2 f 0.4

Ethanol (% by vol) 7.4 f 0.4 9.7 f 0.4 4.60 f 0.14

has been used to indicate adulteration of the grape wine with fermentation products of other fruits [ 261. The sweetness of a wine is a function of the carbohydrate concentration. This is readily observed by an examination of Table 2 with the dry red wine having very low dextrose and fructose concentrations compared to the sweet white. The light Burgundy is marketed as a “diet wine”, lower in calories than regular wines. The results indicate that the caloric savings are due not to the lowering of carbohydrates but to the reduction of the ethanol content of the product. Detector temperature Because the chromatographic column in this work is operated at an elevated temperature, it is convenient to operate the detector at an elevated temperature. Chromatograms obtained with the Dionex column at 80°C were recorded as a function of detector temperature in the range 35--85°C for a synthetic mixture of six carbohydrates, which included two sugar alcohols, three aldoses and a disaccharide. The results are shown in Fig. 4 plotted as log(&) vs. T-’ where Ip is the absolute value of the anodic detection peak corresponding to detector temperature T (K). Increasing the temperature over the range 35--85°C resulted in higher detector sensitivity by a factor of from 1.5 for arabitol and sorbitol to 2.1 for lactose. Without thermostatic control of the detector, the average temperature of the solution entering the detector was approximately 60°C and this temperature was chosen for all qualitative and quantitative work. Plots of the type shown in Fig. 4 are useful in estimating the overall energy of activation for kinetically controlled processes. For the range of concentrations used to obtain the data in Fig. 4, plots of I, vs. C were nearly linear, i.e., b S a for Eqn. (1) and & was proportional to C/b. On the basis of the substitution for b from Eqns. (2) we can write d ln(l,)/d(T-‘)

= [d ln(I’,,)/d(T-‘)I

+ [d ln(K)/d( T-l)]

+ [d ln(k)/d(T-‘)I (3)

It is assumed that ln(P,,) varies insignificantly. The substitution for an irreversible electron-transfer reaction is made so that

Fig. 4. Results of temperature study. A, Arabitol (0.12 mg ml”); B, sorbitol (0.12 mg ml-‘); C, arabinose (0.12 mg ml-‘); D, xylose (0.12 mg ml-‘); E, glucose (0.12 mg ml-‘); F, lactose (0.14 mg ml-‘). Fig. 5. Calibration curve and estimated relative error. (----) extrapolation of ZP-Cs plot; (-) calculated on the basis of data in Table 3.

d ln(k)/d(T-‘)

= [d ln(A,&/d(T-‘)I

+ [%lodF(E -

of linear portion

- [AG&/R]

~“)/~l

(4)

where A_,., is the preexponential term in the Arrhenius rate equation and AGLti is the activation energy for the anodic process at E = E”. The number of electrons, n, for the anodic reaction is included in the coefficient (Y,,; the value of n is not known for these reactions. Following the formulation of an expression for K based on the Arrhenius equations for the adsorption and desorption processes, K = k,/kh, d ln(K)/d(T-‘)

= [d ln(A~/A~)/d(P)J

- [AG&/R]

+ [AG&/R]

(5)

where A, and Ades are the Arrhenius preexponential terms for adsorption and desorption, respectively, and AG& and AG& are the respective activation energies with A G& = AG& - AG&. The preexponential terms are assumed to be independent of temperature, so that d ln(l,)/d(P)

= [-AGkti/R]

- [AG&/R]

+ [a,,F(E

- EO)/R]

(6)

In Eqn. (6), the first term must be negative because, for a reaction of finite rate, AGkti is taken to have a positive value. The second and third terms, however, are probably positive because AGA is negative for spontaneous adsorption and E 3 E” for an irreversible anodic reaction. Hence, depending on the relative values of the three terms in Eqn. (6), the slope of the plot of In(&) vs. T-’ can be expected to be negative, zero, or positive. The slopes of the six plots in Fig. 4 are consistently negative and of similar value. We

9

TABLE

3

Linear regression statistics for the calibration curve for dextrose plotted in Fig. 5 (N = 5) Parameter Slope Intercept Standard error of estimate Correlation coefficient

Symbol

Value

b SYX

(4.048 i: 0.049) x lo-’ mg ml-’ uA-* (9.3 * 1.2) x lo+ PA-’ 5.4 X 1O-4 &A-’

r

0.99998

a

conclude that the term, A G&/R, in Eqn. (6) dominates and, furthermore, that the compounds studied react by a similar electrochemical mechanism, i.e., similar values of A G.&&. Variation of results

The slope of the nonlinear plot of I vs. C for carbohydrates detected by t.p.a. decreases with increasing C. Hence, it is expected that the relative error for determination of an unknown concentration will be inversely proportional to C. Calibration data for the measurement of dextrose was obtained for standard samples of concentration, C,, in the range 0.2-1.5 mg ml-’ and the plot of Ii, vs. C, is shown in Fig. 5. The regression statistics for the linear plot of l/I, vs. l/C, are summarized in Table 3 with uncertainties computed at the 90% confidence level. The fit of the Ip vs. C, data shown in Fig. 5 was made based on Eqn. (1) using values of a and b from Table 3. Of primary concern is the minimization of the relative uncertainty for determination of an unknown concentration C,, A,/&. In Fig. 5 is shown the estimated value of AX/C, computed on the basis of single values for measured anodic currents over the range of values of C, = C, included in the figure. It is quite apparent that unknown samples of high concentration should be diluted to minimize relative error of the results. The support of this research by Dionex Corp., Sunnyvale, CA, is gratefully acknowledged. REFERENCES 1 K. Aitzetmiiller, J. Chromatogr., 156 (1978) 354. 2 S. I. M. Johncock and P. J. Wagstaffe, Analyst, 105 (1980) 581. 3 K. Brunt, Potato Processing Research Institute, Groningen, personal communication, 1982. 4 S. Hughes, P. L. Meschi and D. C. Johnson, Anal. Chim. Acta, 132 (1981) 1. 5 S. Hughes and D. C. Johnson, Anal. Chim. Acta, 132 (1981) 11. 6 S. Hughes and D. C. Johnson, J. Agric. Food Chem., 30 (1982) 712. 7 M. G. Natrella, Experimental Statistics, Nat. Bur. Stds. Handbook 91, U.S. Dept. Commerce, Washington, 1963, Ch. 5. 8 J. A. Elvidge and P. G. Sammes, A Course in Modern Techniques of Organic Chemistry, 2nd edn., Butterworths, London, 1966.

10 9 J. K. Palmer and W. B. Brandes, J. Agric. Food Chem., 22 (1974) 709. 10 P. E. Shaw, C. W. Wilson III and R. J. Knight, Jr., J. Agric. Food Chem., 28 (1980) 379. 11 G. J. Dickes and P. V. Nicholas, Gas Chromatography in Food Analysis, Butterworths, London, 1976, Ch. 8 and 15. 12 J. C. Linden and C. L. Lawhead, J. Chromatogr., 105 (1975) 125. 13 K. Aitzetmuller, J. Chromatogr., 156 (1978) 354. 14 H. Binder, J. Chromatogr., 189 (1980) 414. 15 M. D’Amboise, D. Noel and T. Hanai, Carbohydr. Res., 79 (1980) 1. 16 S. Katz, S. R. Dinsmore and W. W. Pitt, Jr., Clin. Chem., 17 (1971) 731. 17 R. L. Jolley and M. L. Freeman, Clin. Chem., 14 (1968) 538. 18 K. Mopper, Anal. Biochem., 87 (1978) 162. 19 E. Martinson and 0. Samuelson, J. Chromatogr., 50 (1970) 429. 20 P. Jondera and J. Churacek, J. Chromatogr., 98 (1974) 55. 21 J. Havlicek and 0. Samuelson, J. Inst. Brew., 81 (1975) 466. 22 J. C. Kuo and E. S. Yeung, J. Chromatogr., 223 (1981) 321. 23 J. S. Hobbs and J. G. Lawrence, J. Chromatogr., 72 (1978) 311. 24 H. D. Scobell, K. M. Brobst and E. M. Steele, Cereal Chem., 54 (1977) 905. 25 S. Hughes, Ph.D. Thesis, Iowa State University, Ames, IA, 1982. 26 J. F. Gallander, in A. D. Webb (Ed.), Chemistry of Winemaking, American Chemical Society, Washington DC, 1974, Ch. 2.