Determination of Sugars in Food Products Using HPLC and Electrochemical Detection at a Cu Electrode Peifang LUO; Matthew 2. Luo, and Richard P. 6aldwin2 University of Louisville, Louisville, KY 40292 One way to increase interest in laboratory courses is to incorporate into them features that draw upon common life experiences and thereby encourage students to see conactivities involved in a oarnections between ~ ~ ~ the ~ chemical ~ ticular lab exercise and some real and independently meaningful objective. In line with this philosophy, we have begun to organize our introductory laboratory courses a t the University of Louisville around the use of instrumental techniques to characterize some aspect of the composition of student-supplied, "real life" samples. In the course of this work, we have had particular success with a novel experiment involving the determination of sugars in food products by a simple liquid chromatography/electrochemical detection (LCEC) procedure. Most approaches for the identification and quantitation of individual sugars in commercial products make use of high performance liquid chromatography (HPLC). Amajor oroblem occurs in the detection step because simple sugars iack a chmmophore that absorbs .&ronfilyut a readily accessible wavelenmh and thus are poorly suited for the customary W-visitjle monitoring. A-ltern&ivel", approaches involving refractwe index detenlon ( 1 , or, possihly, formation of a-colored derivative could be employed. However, these methods are likely to suffer from one or more of the following disadvantages: diminished sensitivity, increased complexity, and poor selectivity. I n the past decade, electrochemical techniques have gained prominence i n s u g a r analysis, with pulsed amperometric detection a t platinum and gold electrodes now well accepted for this purpose (2,3).Continual pulsing of the electrode to extreme oxidizing and reducing ootentials is necessary for stable respons&th these m%rials, because of their gradual passivation due to adsorption by products of the sugar oxidation. Recently, copper surfaces have been shown to permit the electm-oxidation of a wide range of carbohydrate compounds without surface fouling and rapid loss of response. As a result, a n effective, reliable, and straightforward LCEC method for sugar detection a t constant potential is possible with Cu electrodes. The analytical capabilities of this approach have been characterized in several earlier studies (4-7).We have found the methodology to be well suited for use in the undergraduate analytical laboratory to determine the sugar content of a variety of common food products. ~
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Experimental Apparatus
Any conventional HPLC instrument can be used for this experiment. We have found i t instructional to assemble the instrument compnent-style by linking together separate pump, injector, column, and detector units. But integrated HPLC devices common to the research laboratory can be -
'C~rrentaddress. Shmaozu-Kansas Research Lawratory, Center for Btoanalyl~calResearcn. Un versQ of Kansas. 2095 Constant Aven~e.Lawrence, KS 66047. 2Towhom correspondence should be addressed,
used equivalently. The only special equipment required is an electrochemical detector unit cons~stingof a flowthrough cell assembly and a simple potentio&at for, controlling the applied potential and amplifying the small currents associated with the analyte redox process (8). I n these experiments, we used a Beckman Model llOB pump with an SSI (State College, PA) Model LP-21 pulse dampener, a Rheodyne (Berkeley, CAI Model 7125 injector eouimed with 20-uL samole looo. a 25-cm lone. *. -. 4-mm i.d. Dionex CarboPac PA1 anion-exchange column, and a Bioanalvtical Svstems (West Lafavette. IN) Model LC-4B amperoketric dktector. '&/&$1(3~ Nacl) reference and stainless steel counter electrodes were emoloved. . " The copper working electrode was constructed by sealing a 2-mm diameter comer wire into a Teflon block machined so as to be compatibie'with the Bioanalytical Systems thinlaver flow-cell desim (7).A fresh electrode was oreoared its surface with emery paper, for initial use by rinsing it with deionized water, placing i t in the HPLC flowstream, and applying the desired detection potential until a stable background was obtained. (Do not be alarmed to see a large background current when the p t e n tial is initially applied to a fresh Cu electrode. A period of approximately 30 min is typically required for the Cu surface to be fully oxidized and ready for use). An applied potential of +0.55V versus AglAgCl was optimum for detection when the mobile phase was 0.10 M NaOH. If desired, this value, which may vary somewhat if the NaOH concentration is changed, can be determined independently by the student by means of separate cyclic voltammetry experiments under the operative mobile phase conditions.
a
Reagents
Carbohydrate compounds were purchased from Sigma and Aldrich Chemical Companies and were used as received without further puri%cation. Mobile phases were prepared from carbonate-free NaOH and deionized water and then filtered and vacuum-degassed. It is important for reproducible retention with the CarboPac PA1 column that thk mobile phase contains no anionic species other than OH-; COs" from dissolved COz must be removed by thorough deaeration with Nz or He and then kept out by continuously blanketing the mobile phase with one of these eases durine the exoeriment. A broadlv aoolicable mobile phase consFsted of.0.10 M NaOH, b i t useful responses could be obtained for mobile ohases with OH concentrations as low as 10 ".These hj'ghly alkaline mobile phases were alwavs oreoared and introduced to the IIPLC system by experie%d s'taff. Although exposure to these so1;tions in oractice is minimal if the exoeriment eoes as ulanned, concerning the-undesirsuitable warning should be ability of allowing the mobile phase or column effluent to come into contact with skin or clothing. Samoles used in this work ordinarily consisted of food produ& purchased from grocery stores or pharmacies. In student experiments, the samples were supplied by the students themselves according to their individual interests. In most cases, no sample preparation was required Volume 70 Number 8 August 1993
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other than fdtering, usually through a 0.45-pm membrane, and dilution. Samples of high viscosity (such as fruit juices, milk, syrups, etc.) were diluted immediately tenfold with water before filtration and then diluted sequentially until their responses fell into the linear calibration ran&, usuallv lo4-104 M. AU standards and s a m ~ l ewere s prepared d&just before use. Procedure
It is expected that, prior to the start of the experiment, the students have had some introduction to the theory and practice of HPLC. In our experience, a profound understanding of HPLC is neither needed nor desired by the student a t this point. Rather, the most useful information is of a practical nature and can be supplied by a brief, interactive demonstration of the HPLC instrument that the student himself will be using for the sugar determination. In order for the experiment to fit into a convenient time frame, it is necessary that the HPLC instrument should already be set up at the beginning of the lab period, with the mobile phase, column, and Cu electrode already equilibrated and ready to go. After each unknown injection, a period of up to 10-15 min usually is required to be sure that all sample components have been eluted completely. As a result, there will be time in a 3-h lab period for, a t most, about 15 chmmatograms. Therefore, it is necessary to restrict the determination to onlv a few specific swars. Normally, such simple measures as reading the prod;ct's list of inmedients and consultation with the instructor are all that are needed to insure that the most suitable sugars are selected for study. Also, it is suggested that a standard
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Table 1. LCEC Response of Sugars at the Cu Electrodea Compound
Retention Sensitivity Time (min) (nA/pmoi)
Detection Limit
Fucose
2.8
1.3
0.5
3.1
Methylglucose
3.5
0.4
1.9
10.0
Arabinose
4.0
1.1
0.5
3.6
Gluwse
5.0
1.1
0.6
3.6
Fructose
5.6
0.7
1O .
6.7
Lactose
9.2
0.6
2.4
6.7
Sucrose
10.1
0.5
2.7
8.0
Cellobiose
14.6
0.4
3.4
10.0
0.2
7.2
20.0
Maltose 24.1 'Mnstant potential amperomelric detection at +0.55V versus AgIAgCI. Stationary phase: CarbaPac PA1 anion-exchange mlumn: mobile phase: 0.1 M NaOH; flow rate: 1.0 mlimin. solution consisting of a mixture of the selected sugars a t known concentrations is prepared and made available a t the beginnine of the lab. Dilution of this solution bv the &deG allo& calibration data for several different &gars to be obtained all at the same time with a minimum number of injections. Shown in part A of the figure (and summarized in Table 1)is a chromatogram obtained for such a mixture containing nine common mono- and disaccharides. A typical experiment might wnsist of the determination of the glucose, fructose, and sucrose content of a commercial beverage or, better, a comparison of these concentrations in two different beverages (e.g., regular and diet soft drinks). Such an experiment requires that chromatograms be obtained for the following: (1)Solutionscontaining each sugar by itself in order to establish the retention time for each under the prevailing HPLC conditions. It isconvenient at this mint for the student to reoeat one or more of his iniections to verifyldemonstrate system reproducibility. (2) Solutions containing all three sumrs for four unknown concentrations over-the linear range of response (generally 10~-10"M). (3) Sample solutions diluted sufficiently to bring the sugar concentrationswithin the calibrated range in (2) above. If two sugars of interest occur at widely differentconcentration levels, then different dilution factors may need to be applied to achieve quantitation of both. Begin by diluting the sample tenfold (at least) and then use these results to estimate the extent of any further dilution needed. As in all quantitative exercises, it is important to perform duplicate measurements.
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,-~-
min Chmmatograms of sugars with CarboPac PA1 column and 0.10 M NaOH mobile phase. (A) Standard mixture wnsisting of (1) fUCOSe, (2) methylgluwse, (3) arabinose. (4) glucose, (5) fructose,(6) lactose, (7)sucrose, (8)cellobiose, and (9) maltose; (6) Bud Dry beer diluted by a factorof 100 with deionized water. 0
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Journal of Chemical Education
It is usual that the dilutions and associated operations are computed and carried out by the students themselves during.the periods oftime spent waiting. - for sample elution to be &mpiete. Data work-up includes the measurement and comparison of retention times of unknown and standard peaks as the principal means of qualitative identification, plotting by hand or by computer the calibration curves for the standard sugar solutions, and calculation of the concentrations of the target sugars in the unknown sample(s). Care should be taken to have the students report these values in a format that clearly expresses their significance (e.g., weight percent or total weight in a serving rather thanmo-
Table 2. Sugar Contents in the Food Samples Used in Student LCEC Experiment
Sugar Concentration (g/L) Food Sample
Dilution Factor
Fuwse
Arabinose
0.05
0.09 0.04 0.03
Glucose
Fructose
Lactose
0.26 0.29 0.33
0.84 0.46 0.58
Sucrose
Maltose
Beers Budweiser Coors Draft
100 100 100
Vitamin D Evaporated
100-1 0000 100-1 0000
Bud Dry
0.54 0.14 0.50
2.05 0.26
Milks 0.04
. . .a
0.01 0.01
32.8 50.4
...a
Soft Drinks Coca-Cola 100-1 0000 Pepsi 100-1 0000 Diet Pepsi 10 Sprite 100-1 0000 Mello Yello 100-1 0000 Gatorade 100-10000 0.01 Grape Soda 100-1 0000 Diet Mountain 100-1 0000 Dew 'peak present at this retention time, possible galactose.
0.03
0.02 0.01
larity) and, if possible, compare their results with known or expected values. Discussion The anion-exchange chromatographyICu electrode a p pmach to sngar analysis is applicable, with few adjustments, to the determination of virtually any carbohydrate and to a wide variety of different kinds of samples. Previous studies (4-7) have explored in depth the range of carbohydrate wmpounds that can be oxidized usefully a t the Cu electrode. These include both monosaccharides and oligosaccharides, reducing and non-reducing sugars, and many related wmpounds such as alditols, acidic sugars, and amino sugars. The primary feature needed for response appears to be the presence of multiple aliphatic hydmxyl groups. Of course, identification and quantitation of any of these compounds in an authentic sample requires that the chromatography system is effective in separating them &om one another and from other oxidizable swcies present in the sample matrix. Fortunatelv. this has not prove* to be a serious d h d t y with the anion: exchange system used here, although some instances of sugars possessing very similar retention characteristics have been noted (9).The mobile phase must be strongly alkaline (lo3 M OH-or higher)both to maintain the activityof the Cu electrode and to maintain the sugars in anionic form. A typical chromatogram obtained for a wmmercial beer, a sample which contains appreciable levels of several of the simple sugars in Table 1,is shown in part B of the figure. Several of the sample peaks can be assigned by comparing their retention times to those of the sugars in the known mixture. Of course, such retention time comparisons do not represent an infallible method of peak identification; but this criterion can be used with success in many cases where the sugar composition of the sample is not too complex. Quantitation of the assigned peaks by calibration based on the injection of solutions containing known concentrations of the selected sugars is then a straightforward process. For most sugars, the Cu electrode provides extremely sensitive detection, with minimum detectable quantities in the ng range. In addition, the electrode response is quite stable. During the course of a 5 4 h work period, hourly injections showed relative standard deviaAlthough there was a gradual decrease tions of only 13%.
45.1 44.0 0.03 44.0 54.0 30.6 40.0 0.64
68.4 42.9 0.01 69.0 71.0 34.2 64.0 0.28
1.04 1.06
41.1
1.55 1.19
0.30
in electrode response from day to day, the same Cu surfaces typically were used for periods of 1-2 weeks without removal from the HPLC flowstream by continuing to maintain the applied potential and a slow flow of mobile phase. As shown in Table 2, sugar determinations can be carried out for numerous other carbohydrate-containingbeverages. In addition to the soft drinks, milks, and beers shown, our students have successfully applied the technique to fruit juices, candies, syrups, wine, diet drinks, and bottled water, all within the framework of a 3-h laboratory exercise. Note that the only required sample treatment in all of these cases consisted of simple filtration and dilution steps. In our applications a t the University of Louisville, an experiment based on sugar determination bv the LCECICu electrode approach ha; been employed for"23 years in our second-year analytical wurse. The experiment has proven to be successful as a means of introducing modem separations techniaues (also included in the course are expenments utilizAg gas chromatography and gas chromat&raphy-mass spectrometry) to a student audience that is "nsophistic&ed with respect to scientific instrumentation. The students particularlv seem tn appreciate the hands-on exposure to modern analytical equipment that it affords and the opportunity to select and characterize samples that have practical significance to them. At the same time, the experiment also could be employed usefully in a more advanced instrumental analysis setting to illustrate finer points of HPLC and electroanalytical applications. Acknowledgment This work was supported by the National Science Foundation through EPSCoR Grant EHR-9108764 Kentucky EPSCoR Advanced Development Program. Literature Cited 4. Prabhu,S. V.; Baldwh,R.P A n d Chem. l9€3,61,852456. 5. P1abhu.S. V:. Ba1dwin.R.PAnol. Chem. 1989.61.2258-2263. I . iwo,62,752-755. 6, LUO, P.:~ a b h " ,S. v.;Baldwin,R. P . A ~ ~cham. 7. Lua, P; Zhang,F;Baldurn, R P A m l Chim. h f o 1991,244,169-178. 8. Kissinger, P T. J Cham. Edue. 1883,60,30&311. 9. Prabhu, S. V; Baldwin, R. P. J. Chr0moU.q~1990,503,227-235.
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