Sugar Determination in Foods with a Radially Compressed High Performance Liquid Chromatography Column Martin G. Ondrus. J a n e Wenzel. and Gerald L. Zlrnmerman University of Wisconsin-Stout, Menomonie, WI 54751 Carbohydrates are present in nearly all food products. With the recent awareness of hiah sugar levels in some ioods, the interest in rapid, sensitive, &d reliable methodology for sugar analysis has increased. A recent Analytical Chemistry application review referenced over 20 HPLC sugar determination methods published in the last two years ( I ). Many commercial laboratories are dedicating automated, microprocessor controlled HPLC systems to the analysis of sugars in foods. Excellent resolution of mono- and disaccharides can he achteved with columns containing amine honded phases ('21 (31. . . and with . ., with s~ecialized"carbuhvdrate"cc~lumns (4.51, columns containing silica modified in situ with amines (6,7). In the exoeriment rewrted here. we have chosen to use a silica stationary phase treated w ~ t h trtrnethylenepentamine (TEPAj (8). The column is a 100-mm long hy 8-mm 11) ra. dially compressed polyethylene cartridge with a silica stationarv Dhase (Waters Associates "Radial Pak R"). The mw consists of an alkaline acetonitrile-water mixture bile containing TEPA. We are using the Waters Associates Radial Compression Separation System for all undergraduate HPLC experiments and strongly recommend it regardless of the make of liquid chromatography equipment available. A "radial compression module" hydraulically subjects columns to radial pressures of over 2000 psi. This ensures a uniformly packed stationary phase. Sudden pressure changes expected when the flow rate is abruptly changed will not damage the column. Column voids which can he produced by improper instrument operation or by silica dissolution a t high pH are essentially squeezed out of the radially compressed column. Columns can he changed in the radial compression module in less than one minute without disconnecting any high pressure fittings. Solvent leaks often encountered when one steel column is exchanged for another are eliminated. The polyethylene "radial pak" columns are stored dry with no special precautions. They appear to he nearly indestructible. Although the radial compression module is fairly expensive, columns can he purchased for about one-third the cost of stainless steel columns. T h e recirculation of mohile ohase described in this experiment i~ convenient and can iesult in considerable solvent savinrs. Effluent from the detector is returned to the solvent reser;oir rather than to a waste container. In this manner, a liter of elution solvent can he used for a week or more. Students choose a 2-hr block of time to run their standards and prepared samples. Because the liquid chromatograph is con~
776
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Journal of Chemical Education
tinuously operating, students need not wait for the instrument to warm up or establish a straight haseline. This experiment has been used in our food chemistry course for the analysis of such products as soft drinks, fruit juices, wine, ice milk, and breakfast cereals. lnstrumentatlon and Reagents Our equipment consists of Waters Associates components including an M6000A pump, U6K Universal Injector, RCM-100 Radial Compression Module with 8 mm ID Radial Pak B, and a Model R 401 Differential Refractometer (Water$ Associates, Milford, MA). Chromatograms are displayed on an Omniscrihe chart recorder (Houston Instruments, Austin, TX). Technical grade tetraethylenepentamine (Eastman, Rochester, NY)is used without purification. An aqueous 10% (vlv) TEPA stock solution is convenient for preparation of column pretreatment and mohile phase mixtures. Solvents are prepared with HPLC grade acetonitrile (Fisher, Pittsburgh, PA) and deionized distilled water. Acetonitrile-water mixtures are degassed by vacuum filtration through Teflon@, 0.4 fim memhrane filters (Millipore, Bedford, MA). Reagent grade carbohydrates (Eastman, Rochester, NY) are used to prepare standard solutions. The column pre-treatment solution consists of 100 ml of 70% (vlv) acetonitrile-water containinzO.l% TEPA. The DH oft his solution is adjusted to ahout 9.U kith dropwise addit& of elacial acetic acid rind filtered throueh a U.4 urn memhrane through filt;?r. T o condition the column, 50-75kl is a silica ~ a c k e dcolumn a t a flow rate of 2.0 mllmin. One liter of 750; acetonitrile-water c~nrainingO.O?~oTEPA serves as the final elution solvent. The solution is adiusted to a pH of 9.0 and filtered as with the pre-treatment kdution. Following column conditioninp, the final elution solvent (which should he constantly stirred) is pumped at 2.0 mbmin until the enttre system is flushed. The effluent tuhe is then transferred to the mohile phase supply flask and the column stabilized by recirculating the solvent overnight. The solubility of silica becomes appreciable above a pH of 8.0. It is tempting to lower the pH of the mohile phase, hut doing so significantly reduces chromatographic resolution and baseline stability (8). Recirculation of solvent produces a saturated silica solution and significantly reduces the loss of packing material from the column. Column compression compensates for any silica loss and eliminates the formation of column voids.
'
Analysis is generally carried o u t with t h e solvent flowing
at 2.0 o r 3.0 mllmin. T h e higher flow rate results in shorter analysis time along with a slight reduction in resolution. T h e refractometer attenuation is generally s e t at 16X o r 8 X . Procedure Standard solutions containing 5, 10, and 20 pg per microliter are utilized. The first is prepared by weighing 0.50 g each of fructose, glucose, sucrose, and lactose and combining them in a 1W-ml volumetric flask. Water is added to dissolve the sugars and the mixture is diluted to the mark. The other standards are prepared from 1.0 g and 2.0 g of each sugar, respectively. Depending on the nature of the food product under investigation, samole ranees from essentiallv unnecessarv to relstivelv . .oreoaration . involved. The followingbaragraphs contain suggestiansfor preparirk! samples based upon our experiences with the products.
2) Fruit juices. Apple juice and grape juice can he analyzed following membrane filtration. Citrus products and other juices with a great deal of pulp require centrifugation followed by membrane filtration of the decantate. Some products restrict the flow through the filter very rapidly, but even 5-10 drops of clear filtrate is more than sufficient for injection into the liquid chromatograph. 3) Ice milk and related dairy products. Soft serve ice milk and milk shake productsand sweetened yogurts ~rovidean interesting mixture of sugars. With a medicine dropper transfer 1.00 g of the melted product to a 25-ml Erlenmeyer flask. Add 5.00 ml distilled water and 9.00 ml isopropanol. Place on a shaker-stirrer and mix gently for at least
2
1) Soft drinks and wines. Generally these products can he injected without filtration or pre-treatment. Products which have a cloudy appearance (such as Mello Yellom, Mountain De+, and Sunkistm Orange Drink) should he filtered with a 0.45 um cellulose membrane filter in a Swinney type filter holder.
PEAK IDENTIT7 1. 2.
3. I. 5.
Solvent f r o n t Glycerol Fructose Glucose Sucrose
3
*"
5
-
%
15 "1"
..
Figure 1. Separation of Standard Carbohydrate Mixture. injectim Volume. 10 pL; Mobile Phase. 76% CH3CNH2Ov/v(0.02% TEPA, pH = 9.0); Flow Rate. 2.0 mllmin; Detectw Attenuation, 8 X .
Figure 3. Separat onof Carkhydrates m Bvgundy W ne inect on Volume. 10 #L; Mobile Phase. 7690 CH3CN-h20v l r (0 02% TEPA, pH = 9 0 1 F ow Rate. 2.0 milmin; Detector Anenuation, EX PEAK IDENTITY
Solvent
1. 2. 3. 4. 5.
sucrose Maltose
6.
Lactose
front
Fructose Glucose
PEAK IDENTITY 2.
Solvent front Fructose
3.
Glueore
4.
sucrose
1.
15 min
Fgure 2. Separation of Cartohydrates in Ccca Colam.lnlectim Volume, 2.5 pL: Mobile Phase, 76% CHSCN-H.0 vlv (0.02% TEPA, pH = 9.01; Flow Rate. 2.0 mllmin: Detector Attenuation. 8X.
Figure 4. Separation of Carbohydrates in Ice Milk. Injection Volume. 10 pL: Mobile Phase. 76% CH3CN-H20vlv (0.02% TEPA, pH = 9.0): Flow Rate. 2.0 P milmin; Detector Attenuation. 8 X .
Volume 60
Number 9
September 1983
777
Student Deterrnlned Sugar Percentages In Selected Food Products Food Products
Fr~ctose 1%)
Glucose
Sucrose
Lactose
Total
1%)
(%)
1%)
(%)
1.0 5.0 13.0 0.0
41.0 36.0 42.0 5.0
Son Drinks
Sprite* no**
coke* Wines
Chateau LaSallem Lambrusco* Rhine Juices
Apple Juice (unswtenedl ~ra~ehuit ~ u i a Imswmened) t Ice Milk Cereal
Sugar Frosted Flakesm Lucky Charms*
Sugar Smacks*
Chseriosm
2.0 0.0 1.0 0.0
an hour. Transfer the contents to a test tube and centrifuge. Filter the clear supernatant solution through a 0.45 pm filter. The filtrate is readv for HPLC analysis. 4) Breakfast cereals. Weigh 1.M) g of dry cereal in a small beaker. Add 20 ml water and stir for 15 mi". Centrifuge and filter the decantate
through a membrane filter. Some cereals produce colloidal suspensions which are difficult to filter even after thorough centrifugation. Cereals which have proven to be relatively straightforward for student analysis include Sugar Smacksm, Sugar Frosted Flakesm, Lucky Charms@,and Cheeriosm. A n injection volume of 10 rrl is recommended for standards. Complete spparation of sugars rpquirri ahout 10 min with a solvent flow rate of .3.U ml min. The sugars elute in the order: fructore, glucose. sucrose, lactose. If maltose is present, it appears between sucrose and lactose. A chromatogram obtained by injecting 10 pl of a solution containing 10 pg of each sugar per microliter is shown in Figure 1. To obtain oeak heiehts which are com~arableto those of the ~ volumes are recommended standard solutions the f o l l u w ~ ninjection for products prepared as drprrthpd earlier. Typ~ralrhnxnatograms are illustrated in Figures 2-4. 10 pl Dry Wines (Rhine, Burgundy) Sweet Wines (Chateau LaSalle, Lambrusco) 2.5 pl 2.5 pl Soft Drinks (Coke', Rondoa, Spritem) 2.5 pl Fruit Juice (Apple. Grapefruit) 5.0 p1 Sweetened Cereals 15 p1 Unsweetened Cereals Ice Milk 15 pl
Data Analysis Measure the peak height of each sugar under investigation. Using data from the standards, prepare a standard curve plotting peak height (for each sugar) as a function of microerams iniected. ~ e t e r m i n eby comparison to the standard m e the number of microerams of each suear oresent in the food sample injected. ~ k c u l a t the e percent sugar.
-
sugar) (dilution factor) (p1 injection) Liquid foods have a dilution factor of 1,while ice milk and cereal have dilution factors of 15 and 20, respectively.
%sugar =
(10)
Discussion Some interesting student-generated results are listed in Table 1. The data appear reasonable and are intended to provide the reader with a general idea of the sugar levels to be
778
Journal of Chemical Education
0.0 0.0 0.0 0.0
44.0 41.0 56.0 5.0
expected in certain foods. A recent Consumer Reports study listed Sugar Frosted Flakes@,Lucky C h a m ~ kand , Cheeriok as containing total sugar concentrations of 41,41, and 3 percent, respectively (9).Our totals of 44,41, and 5 percent are in good agreement with the report. While satisfactory data is readily obtained without the use of an internal standard, ribose has been suggested as a reliable choice because it produces a sharp, well-resolved peak (10). Using ratios of sugar to ribose peak heights may improve the experimental precision and increase the range over which the standard cur& is linear, but we have not used this procedure to date. A mobile phase composition of 75% acetonitrile/25% water, vlv, is generally satisfactory hut may require slight adjustrnents.-~esolution and retention times increase with increasing acetonitrile concentration. Impurities in injected sampl&reduce resolution (after several weeks of constant use) due to column contamination. T o a great degree this can be overcome hv. aonro~riatelv increasing the acetonitrile con.. . centration up to a maximum of about85%. The amine modified column described in this experiment can be regenerated somewhat by reversing the column and treating with 0.1% TEPA as in the initial pre-treatment step. To prolong plunger seal life, pumps should not be allowed to stand idle with the system containing the mobile phase described in this experiment. Pumps should he thoroughly flushed with water followed by methanol a t the completion of the experiment. Acknowledgment The authors are grateful for support which was provided by the National Science Foundation Instructional Scientific Equipment Program, the Stout Foundation, and the University of Wisconsin Undergraduate Teaching Improvement Program. Literature Cited 11) Solman, K. G.,Foltz.A.K.,andYerandan. J . A . . A w l . Chrm..53,242R (1981). 12) Schwartnnbaeh. R.. J. Chmmatapr.. 117.206 11976). (3) Jones. A. D.. Burns. I. W.. Sellinpa. S. G.. and Cox. J. A.. J. Chramoto8r. 144,169 11971) , .- .. ,. 14) Linden. J. C.. and Lawhead. C. L.. J. Chmmotoer.. 195.125 (19751. 15) Magher. R. B..and Purat.A.. J. Chmmalo8r.. 117.211 l19761. 161 Wheal.. 8. B..end W h k P C.. J Chromlun.. 176,421(1979). (7) Aitzefmuller. K., J. Chromologr. 156.354 11978). (6) Hendrix. D. 11981).
L.. Kee. R. E.. Beust, J. C., and James. H.,J Chromologr., 210, 45
(9) canrumera Union of the Unitad Stale%Coruumar Reports. 46.68 11981). (10) Hunt, D. C.. Jaekaon, P. A . Martloek. R. E., and Kirk, R. S.. Anoiyil!, 102. 917
119771.
