Anal. Chem. 1988, 58,3203-3207
3203
Determination of Carbohydrates by Liquid Chromatography with Oxidation at a Nickel(I I I) Oxide Electrode Robert E. Reim* a n d R i c h a r d M. V a n Effen Analytical Laboratories, The Dow Chemical Company, 1602 Building, Midland, Michigan 48674
Mane, dC, and oligosaccharides are separated as ions by ion exchange chromatography using an alkaline moblle phase. At an appiled potential of 0.45 V vs. Ag/AgCI, a film of active nickei(II1) oxide (NIOOH) is generated in Situ at the surface of a tubular nickel electrode. Carbohydrates are detected by catalytic oxidation at the active electrode surface. Detection limits approach 20 ppb (1 ng) for monosaccharldes and 100 ppb (5 ng) for oligosaccharides.
Liquid chromatography has become a well-established technique for separation of carbohydrates. Unfortunately, the most generally used detection mode, refractive index, often lacks sensitivity. Recently, improvements in sensitivity and selectivity have been shown using anion exchange chromatography and triple-pulse amperometic detection a t a gold electrode (1). In this report, the combination of anion exchange chromatography and a sensitive dc amperometric detector based on oxidation at an active nickel(II1) oxide electrode is described. The anodic oxidation of alcohols, amines, carbohydrates, and other compounds using a nickel electrode in alkaline solution has been studied by a number of research groups (2-7). Reports by Fleischmann and others have established the mechanism as catalytic oxidation of the organic compound, e.g., glucose, by an active nickel(II1) oxide (NiOOH) formed in situ on the electrode surface at potentials near 0.45 V vs.
SCE:
-
+ OH- NiO(0H) + H20 + eNiO(0H) + RH -% Ni(OH), + R' NiO(0H) + R' Ni(OH)2 + products
Ni(OH)2
(1)
(3)
The nickel(II1) surface acts as a strong oxidant and reacts with the organic compound in the rate-limiting step by hydrogen atom abstraction to yield a radical. Further reaction of the radical with additional surface sites is possible. Applications of the nickel(II1) oxide electrode to the determination of amines, amino acids, ethanol, and glucose by flow injection analysis (8-12)and the determination of amino acids by reversed-phase chromatography (13) have been reported. Since carbohydrates have a number of electroactive hydroxy groups available for reaction, it is possible to detect low concentrations of these compounds at a nickel(II1) oxide electrode; however, the electrode response is not selective since the applied potential is used to create the active oxide surface and not to carry out direct oxidation reactions. A chromatographic separation is needed if resolution is to be attained. Because carbohydrates have pK values ranging from 12 to 14, retention is possible on a hydroxide-form exchange column with alkaline mobile phase. The advantages of anion exchange chromatography for the separation of carbohydrates have been reviewed by Rocklin and Pohl (I). Since alkaline media is also required for the formation of the active nickel(II1) oxide electrode surface, the use of the nickel electrode with anion 0003-2700/86/0358-3203$01.50/0
exchange chromatography offers advantages of selectivity and sensitivity for carbohydrate determination and requires no postcolumn pH adjustment of the mobile phase as has been reported in an earlier chromatographic application of the nickel(II1) oxide electrode (14). EXPERIMENTAL SECTION Apparatus. Chromatography was performed with a Waters M-45 pump with a LiChroDamp I11 pulse dampener, a Brownlee 7125 injector (50 pL), and a Dionex AS-6 analytical column. For some determinations,a Dionex AS-G guard column was also used. The detector is shown in Figure 1. The working electrode was a nickel tube of dimensions 1.59 mm i.d. X 5.0 mm 0.d. X 4.76 mm. Electrodes were machined from stock nickel rods of 99.998% purity obtained from Alfa Products, Danvers, MA. The reference electrode was Ag/AgCl (1M LiC1) and was separated from the flow stream by either a Vycor (Corning Glass Works) or porous polypropylene frit which was not attacked by the alkaline mobile phase. The auxiliary electrode was a 25.4-mm length of 316 stainless-steeltubing (1.76 mm id.) which also served as the outlet for the electrochemical cell. The dimensions of the lower part of the detector housing were such that it and the analytical column could be placed inside a SSI dual-columnheater. Potential control and current measurement were provided by an E.G.& G. Princeton Applied Research 174A polarographicanalyzer. Data were output to a Linear Model 1801 strip chart recorder and/or a HewlettPackard 33906 integrator. Reagents. Mobile phase was prepared daily from VWR reagent grade NaOH. During use mobile phase was protected from carbon dioxide uptake by an Ascarite I1 (A. H. Thomas Co.) trap on the mobile-phase reservoir. Carbohydrate standards were prepared in water from chemicals supplied by Sigma Chemical Co. without further purification. Formation of Active Electrode Surface. Following machining, the electrodewas degreased with methylene chloride. The bore was then successively polished with 5-pm and 0.5-pm alumina using a moistened pipe cleaner. The electrode was rinsed with deionized water and methanol and then air-dried prior to assembling the detector cell. The active electrode surface was formed by equilibrating the chromatographic system with the mobile phase and then applying a potential, e.g., 0.45 V, to the nickel electrode. After about 30 min, a stable base-line current was achieved typically in the range of 0.5-1.5 pA. If base-line currents in excess of 5 pA were observed, the reference electrode solution was refied and the Vycor frit replaced. If nickel electrode performance had deteriorated during use or on standing inactive overnight, it was restored by either disassembling the detector, removing the electrode, and polishing the bore with 0.5-pm alumina or cycling the electrode potential between h1.45 V before equilibrating at 0.45 V. RESULTS AND DISCUSSION Effect of Flow Rate. The effect of flow rate on detector response was tested by flow injection analysis using injections of 1ppm glucose solution. In agreement with the mechanism given in eq 1-3, as flow rate was increased, peak area decreased apparently due to the relatively slow reaction between analyte and the active oxide surface (the rate-limiting step for the electrode process). For example, in the flow range of 1-4 mL/min, peak area was found to be linearly proportional to 1/Fas shown by the data in Table I. The observed flow rate effect is in contrast with conventional amperometric detectors, 0 1986 American Chemical Society
3204
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14,DECEMBER 1986 27 rnm O.D.
7
IR A - 1.3 ppm xylitol B - 2.0 ppm sorbitol C - 3.9 ppm rhamnose D - 1.8 ppm arabinose E - 1.1 ppm glucose F - 2.0 ppm fructose G - 10 ppm lactose H - 22 ppm sucrose I - 45 ppm raffinose J - 21 ppm maltose
0
2
4
6
8 10 12 14 16 Time (Minutes)
18 20
Figure 2. Chromatogram showing detector response for various sugars: mobile phase, 0.15 M NaOH; flow rate, 0.9 mL/min; temperature, 20 O C .
-
Table 11. Effect of Temperature on Detector Response Flgure 1. Schematic diagram of nickef amperometric detector. ~
peak area (A), counts x
31 "C
35 "C
39 "C
slope, nAj0C
Aa
lactose arabinose sorbitol
190 178 172 161 117
320 310 298 268 195
473 460 445 370 289
6.1 17.2 15.2 19.9 9.2
2.7 2.9 3.0 2.4 2.7
~~~
Table I. Effect of Flow Rate on Detector Response flow rate (F), mL jmin
Deak current, nA compd
AXF
glucose
fructose 4.0 3.0 2.0 1.0
7.65 10.1 14.4 28.1
30.6 30.3 28.8 28.1
which operate under conditions of mass transport control where detector response increases as a function of flow rate (15). A similar effect was seen in the variation of response with electrode length. Molar response factors for analyte decreased rapidly as electrode length (residence time) was shortened. The nickel(II1) oxide electrode is therefore most beneficially used under conditions of low flow rate. Effect of Hydroxide Concentration. The overall effect of hydroxide concentration on the system performance was determined by observing the individual effects of hydroxide on detector response and chromatographic separation. The presence of hydroxide is necessary for formation of the active NiO(0H) surface. The effect of concentration on response was determined by measuring the oxidation current of CYlactose a t a nickel disk electrode in stirred solution. In the range of 0.01-0.5 M NaOH, peak potential corresponding to formation of NiO(0H) shifted in the cathodic direction with increasing hydroxide concentration by about 60 mV/ pH, and net current due to lactose oxidation increased linearly with the logarithm of hydroxide concentration. For a 10-fold increase in hydroxide concentration, net analytical current increased by a factor of 8.5; however, background current due to surface oxidation also increased so that little SIN advantage was gained with strongly alkaline solution. In general, hy-
'Current ratio for a 10 "C increase, ianiqn. droxide concentrations less than 0.5 M NaOH were optimal for detector performance. Also, because the analytical column is a strong anion exchange column, its performance is affected by hydroxide concentration in the mobile phase. The effect of hydroxide concentration on the capacity factor for selected sugars and sugar alcohols has been reported ( I ) . The optimum hydroxide concentration for separation of mono- and disaccharides was found to be 0.08-0.25 M NaOH. The separation of a number of sugars is shown in Figure 2 using 0.15 M NaOH mobile phase. Oligosaccharideswere eluted by using 1 M NaOH or a mobile phase containing a non-electroactive-displacing anion such as acetate. A chromatogram showing the separation of the maltose oligomers DP2-DP7 is given in Figure 3. Carbonate is a strong displacing anion for sugars, and care must be taken to minimize the uptake of carbon dioxide by the alkaline mobile phase. Low concentrations of carbonate, which can slowly accumulate on the column with use, can adversely affect column performance, especially the separation of glucose and fructose. With 1% Na2C03in the mobile phase, no resolution of sugars was observed. Degraded column performance was restored by flushing the column for 45 min with 0.6-1.0 M NaOH, which was carbonate free, followed by the mobile phase of choice. Effect of Temperature. Temperature has a significant effect on detector response as shown by data given in Table
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
3205
Table 111. Effect of Mode of Operation on Peak Responsen
- 4.5 ppm DP2 - 3.3 ppm DP3 - 5.9 ppm DP4 D - 3.7 ppm DP5 E - 5.0 ppm DP6 F - 15.0 ppm DP7
A
B C
F
compd
peak height, pA pulseb pulseC
dc
102 ppm sorbitol
7.08
0.24
0.24
55 ppm glucose 400 ppm lactose
2.24
0.32 0.24
0.40
3.08
0.32
nConditions: dc, 0.45 V; pulse, Ei = 0 V, Ef = 0.50 V. b0.5-s pulse. 2.0-9 pulse.
A
I
C AB
E
E
D
i
1 0 0 nA
E
L 1
2
3
4
5
6
7
S
9
A B C D
10
Time (Minutes)
Flgure 3. Chromatogram showing separation of maltose oligomers: mobile phase, 0.15 M NaOH-0.15 M CH,COONa; flow rate, 1.0 mL/min; temperature, 34 OC.
11. Between 30 and 40 OC,net analytical current increased linearly with temperature by an average factor of 2.7 for the sugars tested. Under the same conditions, the background current and chromatographic base-line noise increased by only a factor of 1.2. Therefore, the S I N ratio of the detector was increased by increasing the operating temperature of the detector. Qualitatively, the effect can be understood in terms of the relative effect of temperature on the rate of a chemical process (reaction of nickel(II1) oxide with analyte) vs. an electron-transfer process (electrooxidation of nickel(I1) hydroxide to form active nickel(II1) oxide). For a chemical process that is first order in reactant, a temperature increase of 10 O C might be expected to increase the reaction rate by about a factor of 2. For an electron-transfer process, the same temperature increase would increase the transfer rate 0.99. At low concentrations, and over a limited analytical range, the response can be approximated as a linear function of concentration. The precision of the method using 0.15 M NaOH mobile phase was estimated by making repetitive injections of a test solution containing 2.0 ppm sorbitol, 1.8 ppm arabinose, 1.1 ppm glucose, 2.0 ppm fructose, and 10 ppm lactose. The relative precision for seven determinations a t the 95% con-
- 0.18 ppm xylitol - 0.20 ppm sorbitol - 0.39 ppm rhamnose - 0.18 ppm arabinose
E - 0.1 1 ppm glucose F - 0.20 ppm fructose G - 1.0 ppm lactose H - 2.2 pprn sucrose
E
knA
a
H
I
0
2
4
6
.
8 1 0
Time (Minutes)
Figure 4. Chromatogram showing detector response for trace sugar determination. Conditions are given in Figure 2.
fidence level was found to be sorbitol, 7.4%;arabinose, 3.0%; glucose, 4.5%;fructose, 4.9%; and lactose, 4.6%. In another precision study using 0.60 M NaOH mobile phase and a test solution containing 2.2 ppm glucose, 3.0 ppm fructose, 10 ppm lactose, 15 ppm sucrose, and 2 1 ppm maltose, the relative precision for five determinations was found to be glucose, 2.7%; fructose, 6.3%; lactose, 4.0%; sucrose, 3.1%; and maltose, 8.7%. In an attempt to form the active electrode surface more repeatably, pulse experiments were carried out using an initial potential of 0 V and a final potential of 0.50 V. Peak currents for several injected sugars using pulse and dc detection are compared in Table 111. As compared to dc response, pulsed response was characterized by lower sensitivity, higher background current, larger peak-to-peak noise, and base-line drift. Apparently the kinetics of nickel oxide
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
3206
A
E
f
1
100 nA
0
2
4
6
8
Time IMinuteil
1 0 1 2 1 4
0
2
4
6
8 1 0 1 2 1 4
T me lMlnutePl
Fbure 5. Chromatograms showing response for sugars in (A) commercial soft drink, 1:2000, and (B) standard (22 ppm glucose (a),20 ppm fructose (b), 88 ppm sucrose (c));mobile phase, 0.15 M NaOH; flow rate, 0.8 mL/min; temperature, 23 O C . formation are slow relative to the pulse times used here. Detection limits for several sugars were estimated from the chromatogram shown in Figure 4. At S I N = 3, detection limits were calculated to be 23 ppb xylitol, 26 ppb sorbitol, 80 ppb rhamnose, 23 ppb arabinose, 16 ppb glucose, 29 ppb fructose, and 120 ppb lactose. These limits are comparable to those obtained by pulsed amperometric detection ( 1 ) and are about 40-fold lower than those previously reported for nickel electrode detection of sugars (14). Applications. The utility of the catalytic nickel(II1) oxide electrode for the determination of sugars is shown in several example applications that follow. In the first example, the sugar content of commercial soft drinks was readily determined. Samples were diluted 2000-fold with deionized water and filtered through 0.45-wm Millex filter prior to injection. Chromatograms showing the typical response for glucose, fructose, and sucrose in a commercial soft drink are given in Figure 5 . From the relative ratio of glucose and fructose to sucrose, it can be inferred that the soft drink was made by using a high-fructose corn syrup sweetener. Another application relates to the area of reagent chemical testing. The American Chemical Society specifications for reagent grade sucrose require that invert sugar, typically a mixture of 50% glucose and 50% fructose formed on hydrolysis of sucrose, be less than 0.1% (17). The present gravimetric procedure detects reducing sugars and is therefore nonspecific for glucose and does not detect fructose since it is not a reducing sugar. The feasibility of determining invert sugar in reagent grade sucrose using liquid chromatography and catalytic oxidation at a nickel(II1) oxide electrode is shown by the chromatogram in Figure 6. As compared to the present ACS method, the chromatographic method offers specificity and sensitivity. Under isocratic elution conditions, glucose and fructose are separated from sucrose in less than 10 min and each can be detected a t concentrations approaching 0.002%. Calibration curves for the determination show linear response in the range of 0.01-0.4% sugar, which spans the present specification limit of 0.1% invert sugar. A third application relates to the determination of oligomers, in particular the maltose oligomers DP2-DP7 (maltose through maltoheptaose). Because oligosaccharides are strongly retained on the analytical column, >1M NaOH is required to displace some oligomers from the column. An alternate approach t o displacing oligomers from the column is to use
a b
-it 0
I
\ 2
4
6
0
8 1 0 1 2
0 2
6
4
Time (Minutes1
8 1 0 1 2
Time (Minutes1
Flgure 6. Chromatograms showing response for (A) reagent grade sucrose and (B) A + 0.032% glucose (a) 0.0548% fructose (b); mobile phase, 0.075 M NaOH; flow rate, 1.0 mL/min; temperature, 32
+
OC.
1.5
-
1.0
-
0.5
-
0.0 Maltotetraose -0.5
-2.0
I
l
-1.8
l
I
-1.6
1
I
-1.4
I
I
-1.2
I
1 I
-1.0
1
1
-0.8
Log Acetate Concentration (MI
Figure 7. Effect of acetate concentration on separation of maltose oligomers: mobile phase, 0.15 M NaOH with added acetate; flow rate, 1.5 mL/min; temperature, 34 O C . an additional displacing anion added to the NaOH mobile phase. The effect of acetate concentration on the capacity factors for maltose oligomers is shown in Figure 7. Maltose and maltotriose were eluted without added acetate; however, the uncorrected retention time for maltotriose was in excess of 30 min. The addition of 0.15 M CH3COONa to the mobile phase decreased the uncorrected retention time to 1.8 min. The uncorrected retention time for maltoheptaose under the same conditions was 4.7 min. An example chromatogram showing the separation of maltose oligomers has been shown previously in Figure 3. The individual oligomers could be determined at concentrations below 1 ppm with detection limits approaching 0.1 ppm (5 ng). Typical nonlinear calibration curves are given in Figure 8. From multiple injections of a test solution containing 6 ppm maltose and 26 ppm maltotriose, the respective relative precisions were estimated to be 4.9% and 7.2% for seven determinations. The method has been successfully applied
3207
Anal. Chem. 1986,58,3207-3215 8
7.2 6.4
1
.
0
7
-
rT---I
-
r -~--r-r
1
DP3
-
4
5.6 4.8
-
4.0
-
0
20
40
60
100 120 140 Concentration Ipprn)
80
160
180
200
220
FIgure 8. Calibration curves for maltose oligomers.
to the determination of trace concentrations of oligomers in resin leachates.
ACKNOWLEDGMENT We thank H. D. Woodcock for machining the electrodes and cells and V. A. Stenger for suggestions on reagent chemical testing.
LITERATURE CITED (1) Rocklln, R. D.; Pohl, C. A. J . Li9. Chromatogr. 1083, 6, 1577-1590. (2) Fleischman, M.; Korinek, K.; Pletcher, D. J . €kcfroanal. Chem. 1971, 3 1 , 39-49. (3) Fleischman, M.; Korinek, K.; Pletcher, D. J. Chem. SOC., Perkln Trans. 2 1072, 1396. (4) Lu, P. W. T.; Srinivasan, S. J. Electrochem. SOC. 1078, 125, 1416. (5) Van Effen, R. M.; Evans, D. H. J. Electroanal. Chem. 1078, 703, 383-397. (6) Robertson, P. M. J . Electroanal. Chem. 1980, 7 1 7 . 97. (7) Vertes, G.; Horanyi, G. J. Electroanal. Chem. 1074, 52, 47. (8) Schick, K. G.; Magerearu, V. G.; Huber, C. 0. Clin. Chem. (WinstonSalem, N.C .) 1078, 24, 448-450. (9) Morrison, T. N.; Schick, K. G.; Huber, C. 0. Anal. Chlm. Acta 1080, 120, 75-80. (10) Yuan, C. J.; Huber, C. 0. Anal. Chem. 1085, 5 7 , 180-184. (11) Hui, B. S.; Huber, C. 0. Anal. Chim. Acta 1082, 134, 211-218. (12) Schick, K. G.; Huber, C. 0. U S . Patent 4183791. (13) Kafil, J. B.; Huber, C. 0. Anal. Chim. Acta 1085, 775, 275-280. (14) Buchberger, W.; Winsauer, K.; Breitwieser, Ch. Fresenius' 2.Anal. Chem. 1083, 315, 518-520. (15) Hanekamp, H. B.; Bos, P.; Frel, R. W. TrAC 1082, 7, 135-140. (16) Bockris, J. 0. M.; Reddy, A. K. N. Modern Nectrochemistfy; Plenum: New York, 1970; Voi. 2, Chapter 8. (17) Reagent Chemicals, 6th ed.; American Chemical Society Specifications, ACS Publications: Washington, DC, 1981.
RECEIVED for review May 19,1986. Accepted August 28,1986.
Properties and Applications of the Concentration Gradient Sensor to Detection of Flowing Samples Janusz Pawliszyn
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300
A concentratlon gradlent sensor based on light beam deflection measurement (Schlieren optics) has been used to analyze samples InJectedInto flowing streams. This detector In Its nonselective mode of operation responds llnearly to an Increase In sample concentration. Its linear dynamic range extends from about 0.1 M to 5 X lod M sucrose (n = 10-s/lOd R I units) which Is the concentration detectlon ilmR for a slngie injection experlment. This iimlt can be decreased by an order of magnitude via muitlpie Injectlons and a lock-In detectlon technlque. The concentration gradlent sensor had a detectlon volume In the nanollter range, whlch resulted In the detectlon of a few picrograms of sucrose in a single injection experhnent. The concentration gradient sensor Is able to effectively distinguish between broad and sharp peaks. The enhancement In sensitivity of this technlque can be accomplished by expandlng the effluent from the capillary coiumn Into the larger diameter sample cell. Broadening of the peak can be prevented by the sheath flow method. A slmple optical arrangement In such a sensor allows for a convenlent and inexpensive design. Application of this technique to flow InJectkn and chromatographic eluant analysis is demonstrated and dlscussed.
Recent developmental trends in flow injection analysis and liquid chromatography have led to more efficient, small-dim e t e r chromatographic columns ( I , 2) and miniaturization of flow systems (3). These advancements stimulate search
for low-volume, highly sensitive detection methods ( 4 ) . One way this task can be accomplished is by scaling down known techniques; however, this is not always successful. Another approach is to develop new methods specifically suited for small-volume systems. Recently such a small-volume detection technique based on observations of concentration gradients has been proposed (5). The gradients are generated during the injection of the sample into the solvent. The magnitude of these gradients depends on many factors including environmental conditions and experimental design. The ability to maintain a concentration gradient during transport of the sample toward the detector depends on the internal diameter of the tubes used. For smaller tube diameters less loss in the gradient magnitude occurs (6). Therefore, the method of detection described here is specifically designed for the microflow systems. The concentration gradient of the sample in the solvent produced during injection creates a refractive index gradient. The magnitude of this gradient can be measured by allowing a nonadsorbed light beam (probe beam) to pass through the detector volume. The physical reason for light deflection when passing through this gradient lies in the relationship between the refractive index and light propagation velocity. Different parts of the light advance to a different degree with time, which generates the phase shift. This results in a tilted light beam path illustrated in Figure l a (Schlieren Optics (7)). The light path through the concentration gradient can be calculated by using the Fermat principle (the light path through the medium is such that the time necessary for its traversal is minimum). The relationship between the angle of deflec-
0003-2700/86/0358-3207$01.50/0 0 1986 American Chemical Society
