The Modern Student Laboratory The Analysis of Artificial Sweeteners Beverages by HPLC An Undergraduate Experiment Brlan A. Bldlingmeyer, Present Address: Consultant, P.O. Box 99, Hopkinton, MA 01748 and Stefan Schmitz, Present , Address: Institute for Applied Physical Chemistly University of Saarlands, S a a r b ~ c k e nGermany Waters Chromatography Divlsion of Millipore Corporation, 34 Maple St., Milford, MA 01757
High-performance liquid chromatography (HPLC) has become the primary tool used for quantitative analysis of compounds at the trace level, and,~accordingly,this technique is increasingly taught in undergraduate instrumental analysis laboratories. These laboratories have the need for experiments that are rugged, safe, and can he performed in a nhort period of time. In addition.exoeriments that deal with an issue t o which the students cak relate are more "fun" and can be an important impetus in fostering a longer term interest in science. A basic LC experiment that demonstrated the fundamentals in a vivid manner vet was safe (used no hazardous chemicals) and used relativeiy common materials (e.g., Kool Aid) was an inexpensive separation of food dyes ( I ) . This experiment used syringes and solid phase extraction devices, not an HPLC instrument. Another experiment t o which students can relate and that demonstrates the power, utility, and ease of HPLC is the analysis of additives in beverages such as soft drinks (2-4). Many soft drinks contain benzoic acid, and, historically, most cola drinks contained caffeine. In recent years, responding to popular concern about the health effects of caffeine and other drugs, soft drink manufacturers have introduced caffeine-free sodas. This adds an extra element of interest and provides a broader ranee of commerciallv available samples. Currently the most popular sweetener in dietary soft drinks is aspartame. The analysis of this sweetener has a social relevance since individuals who have phenylketonuria must avoid . phenylalanine, a metabolic product . of aspartame. Saccharin is sometimes used as a sweetkner for dietary soft drinks. Therefore, the capability to measure either sweetener is desirable. A facile HPLC separation of saccharin, sodium benzoate, and caffeine in beverages was developed by Smyly (2) who accomplished the separation in 35 min. This was the basis of an undergraduate experiment by Grayski (3).Delaney et al. (4) broadened the scope and developed an experiment that included aspartame: thus, the student could auantitate four additives in carbonated beverages in one separation. This determination was performed on a reverse-phase column using an isocratic mobile phase in a short period of time (10 min). Although this separation was effective, i t required high flow rates (4 mL/min) with the total solvent consumption of 40 mL for each analysis and the use of acetonitrile in the mobile phase. Acetonitrile is toxic, expensive, and i t creates significant disposal problems.
In this paper we describe an undergraduate laboratory experiment for saccharin, caffeine, benzoic acid, and aspartame using a methanol/acetic acid mobile phase. This experiment is of particular merit since i t illustrates the power of OHcontrol on retention. It also emphasizes the auantitati;e role of HPLC and can he used to indicate the risks in usine HPLC as a aualitative tool. These issues are important t o emphasize to the student. An HPLC is not a "blaek box" instrument and must be approached interactively with regard to the analysis of interest. A principal reason for using methanol is its significantly reduced toxicity relative to acetonitrile. In addition, we note that the price of HPLC-made methanol is about 40% of that of acetonitrile. heref fore, the use of this solvent can effect a significant savings in experimental cost. The separation can be performed in less than 10 min using 15 mL of mobile phase, making it a very effective experiment to demonstrate the power and flexibility of HPLC.
lnshvmentation The modular liquid chromatograph (WatersChromatography Diviaion of Millinare . Cornoration. Milford MA) consisted of a Model 6 0 0 0 ~Sulvrnt Delivery s ~ S I Q Ia~Model , 712 WISP autoinjector (optional for the student), and a Model 440 Absorbance Detector that was uperated st a fired detection wavelength of 254 nm. Instrument control, which is also optional to the student laboratory, was provided by a Model 84OChromatopaphy Contn,lStation (Waters) with analogue data monitored on an SE-I20 dual channel recorder Boveri, Austria). Digital data was auto-archived to a (Asea BIOA Model 860 networking chromatography station (Waters) for prothat the automated eessine~.and storaee. ,. It should be emohmized . fearurea used in this work are not rrquired for a teaching laboratory. and nsimglrr syatemconaiatingofaaolvenc delivery system, manual injector, fixed wavelength detector, and strip chart recorder can be used very effectively in this experiment. A model 811pH meter wm used (Orion, Boston, MA).
.
~
~
~
~
~~~~
~
~~~
Columns, Eluents, and Reagents A pBoudapak C18 column (3.9 X 150 mm) (Waters)was used in this work. An in-line filter kit containing a 2-pm filter assembly (Waters)was placed in line between the injector and the column and used throughout this work. Purified and filtered water was obtained (Continued on page A193 Volume 68 Number 8 August 1991
A195
The Modern Student laboratory
from a Milli-Q water purification system (MilliporeCorp., Redford, MA). HPLC-pade methanol and acetic arid were obtained from J.T. Baker IPhilliusburz. NJ). The mobile nhaae used for this work was an 80120 1M &tic~id/methanol (vlvi solution. The nH of the aqueou- phase wan adjusted with 50%NaOH using a pH meter. Caffeine (Fastman USP), sodium sarcharin tMallinkmdt. USP), benzoic and (fisher ACSI, and aspartame (Sl~maJwere used to prepare standards. All reagents were used as received. A calibration solution was prepared by dissolving 40 mg of saccharin and benzoic acid, 20 mg caffeine, and 200 mg of aspartame in 100 mL of the mobile phase to be used for the analytical separation, 80120 1 M acetic acid (pH = 4.2)lmethanol (vlv). To determine the retention time of each comnaund as a function of nH. individual solutions of each additive were required. These weie prepared hy adding the weight of the compound indicated above (e.g.,200 mg ofaspartame) to 100 mL of 8OtZO warer/methanol.
Labwatory Procedure The laboratory work to be performed by the student consists of two separate experiments. The first experiment is the determination of the optimum pH for the separationof the additives,while the second involves the qualitative and quantitative analysis of the additives in beverages. If the instructor desires not to study the pH effect, the first part of the experiment may he presented verbally ta the students before doing the actual analysis of the additives. To determine the optimum pH for the separation, the student should determine the retention time for each compound at five pH values, and prepare a plot of the retention time for each as afunctiou of the pH (refer to Fig. 1).The student should prepare 100 mL of each mobile phase to he used (80120 acetic acid, pH = 3.0, 3.5,4.0, 4.2, and 4.5lmethanol) by adding 5.26 mL (6.0 g) of acetic acid to a sufficient quantity of water toprepare 100mL of solution. The pH is adjusted by dropwise addition of a 50% solution of NsOH. (The approximate volume of NaOH will range from 1to 2 mL depending upon the final pH attained.) A pH meter should be used to monitor the addition of the base (the small volume change due to the addition of the NaOH will not have a significant effect on the separation). After the pH is properly adjusted, 80 mL of the solution is added to 20 mL of methanol. The pH of 1M acetic acid is 2.4; thus, no adjustment will be required for this solution. It is recommended that the mobile phases be prepared fresh daily and not stored. Prolonged storage of this mobile phase may result in the formation of methylacetate by an esterification reaction of the mobile phase components. If sufficient methylacetate is formed in the mobile phase this may effect the retention time of the compounds of interest. At a given pH, approximately 10 mL of the mobile phase is pumped through the column at a flow rate of 1.5 mllmin to equilibrate the column. The detector is set to 254 nm, 0.2 absorbanceunits full scale (AUFS). A 15-pL sample of a standard solution is injected and the chromatagram obtained. This procedure is followed for all standard solutions so that the retention time for each compound is obtained at each pH value. For the determination of the additives in beverages, the column is to be equilibrated with 10 mL of the 80120 1 M acetic acid (pH = 4.2Vmethanol mobile phase at a flow rate of 1.5 mL/min. The detector is set to 0.2 AUFS at 254 nm.Five different volumes of the calibration solution are injected (2, 5, 10, 15, and 20 pL) and the chromatoerams recorded. Each iniection should he reneated . at least three timrs, and a calibration cuke for earh compound should be prepared using the peak height (or peak area if an integrator is availablel. Eachcalibration plot should providea linear relationship between the absolute amount of the compound injected and peak height (and peak area if an integrator is used). The beverages (a 5-mL sample is sufficient)to he analyzed should be degassed in a sonic bath for 10 min or left open overnight and then filtered throueh a 0.8-um Millex PF filter (Millinore Corn.. Bedford MAI. he-filtration is important for m&imi& the r,'d: umn 1ifetime.A IS-rl.sampleisinjectedintothe HI'I.Cryatem,and the prak herght (or peak area) is used to determine the level of the additives in the beverage.
To maintain column performance and enhance lifetime, it is recommended that 10 mL of an 80120 waterlmethanol mixture should be pumped through the column at the conclusion of the day. The column should never be stared in a mobile phase containing 1 M acetic acid.
Results and Dlscusslon Chromatographic Separation of the Food Additives I n the development of this aeparation the variable of p H was studied in an effort to optimize the required time and the resolution. When the students perform this activity, they observe the dramatic effect of p H on the retention time of ionizable species in a reverse-phase separation. I n Figure 1 the retention time of the four compounds of interest is presented as a function of pH. It can be seen that there aretwo regions where sufficient resolution can be obtained, around p H = 3.3 and around pH = 4.2. When the higher p H is used for the experiment, the overall separation time is slightly shorter. In addition, aspartame, which is subject to acidic hydrolysis, is more stable a t the higher p H (5). At p H = 4.5, benzoic acid and caffeine cannot be separated, which points out the uncertainties that can arise when using retention times alone for verification of comoound identification. This aspect will be further reinforced-when, as we will discuss later in the text, another compound present in one soft drink we studied eluted a t the same retention time as saccharin, and a change in the mobile phase was required to indicate that the compound was not the artificial sweetener. T h e chromatogram obtained from a n injection of 15 fiL of the calibration solution is shown in Figure 2A, while that from a "sugar-free" cola is shown in Figure 2B.This separation uses a mobile phase of 80% 1M acetic acid (pH = 4.2)/ 20% methanol and takes approximately 8min, with satisfactorv resolution between each comoonent to allow for the quantitative analysis. The chromatogram from the sugarfree cola includes a number of unidentified components that eluted a t the beginning of the chromatogram. ~ h e s did e not interfer with the s e ~ a r a t i o nor auantitation of saccharin. (Continued on page A199)
pH Dependence
of Retention Time
~~~~~~~
A196
Journal of Chemlcal Education
Flgve 1. Effect of pH upon reternion times Column: ~BondapskCl8 13.9 X 150 mm) Mobile Finss: 20% MsOHI8O% 1 Macetlcacideluent,pHadlusted wim 50% ~ s o H horn 3.0 104.5.Flow Rate: 1 5 rn~/min.yenion voi~me:15 pL of indlvldual standard solutions (40 mg1100 ml for saccharin, caffeine, benzolc acid, and 200 mg/iOO mL fw aspllame).
CaNbratlon The concentrations of the calibration standards were selected so that the peak heights for the additives in typical soft drinks would lie within the calibration range for each of the compounds. We observed excellent linearity (R = 0.9999) in each calibration plot. The calihration standards are designed to provide a range of 19-190 mg/l2-oz serving (a can) for saccharin and benzoic acid, 9.5-95 mg/12 oz for caffeine and 95950 mg/12 oz for aspartame. The sample volumes and/or the concentration of the standards could be varied to adjust the calibration range. We are aware of at least one soft drink that contains caffeine at the level of approximately 200 mgIl2-oz can. If that were used as a sample, an extended calihration plot would be needed (or the sample should he diluted). We recommend that the calihration axes be peak height (and peakarea if an integrator is available for comparison purposes) on the vertical axis and the weight of the additive injected (in pg) on the horizontal axis. To obtain the concentration of a given additive in the beverage from the peak height (or area), the student determines the amount of additive present by using the calibration plot and locating the value equivalent to the peak height of the additive. That value is multiplied by the volume of the container and divided by the injection volume. All volumes must be in the same units. Getting the Student involved In the analysis of a common nondietmy soft drink we observed a peak that had a retention time identical to that of saccharin (Fig. 2C). However, the label did not indicate the presence of this additive, and the manufacturer's consumer information center indicated that the beverage did not contain that additive. If this occurs in the laboratory, the student has to ask "How can this he?" By reminding him of the earlier observations that the composition of the mobile phase has an effect on the retention time (i.e., in Figure 1at pH = 4.5 benzoic acid and caffeine co-elute), the student should conclude that an unknown compound having the same retention time as that of saccharin could he present in the chromatogram. This would explain the apparent anomaly. The typical approach to determine if the suspect compound is saccharin is to study the chromatographic behavior of a saccharin standard and the suspect compound in the sample as a function of the composition of the mobile phase. Inspection of Figure 1indicates that the effect of pH on the retention time of the saccharin peak is trivial. Therefore, varying the methanol concentration of the mobile phase should be done to determine if the observed peak moves in the same way as the saccharin peak does. When we used 1M acetic acid as the mobile phase, the chromatograms shown in Figure 3 were obtained. Because the peak for the unknown component had a different retention time than that of saccharin (Fig. 3), it is clear that saccharin was not present in the sample. Another approach to verify that the peak is not saccharin is to collect the eluting peak and compare its UV/ Vis spectrum to that of saccharin. (Note: As additional confirmation that the peak was not due to saccharin, we obtained the mass spectrum of the liquid chromatographic fraction that corresponded to the unknown compound. This likewise indicated that the peak was not due to saccharin.) In the development of this experiment, we analyzed eight different naturally sweetened colas, of which one contained a peak that interfered with the analysis of the additives.
Figure 2. Chmmatograma of calibration standards and cola beverages. Column: rBDndapak C18 (3.9 X 150 mm). Mobile Phase: 80120 1 M acetic acid. pH 4.2lMeOH. Flow Rate: 1.5 mUmln. UV detection at 254 nm, 0.2 AUFS. lniection Volume: 15 gL. (A) CalMIatlon standards: 11) Saccharin, 40 mgJ100 mL; (2) caffeine, 20 mg1100 mL: (3) benzoic acid, 40 mg/100 mL; (4) aspartame. 200 mg/100 mL. (8)Diet Colasample containing caffeine, benzoic acid, and aspartame. (6) Cola sample with a suspected saccharin peak at 1.75 mi" reternion time. See text tor details.
Figure 3. Comparison of retention timas of peaks of a saccharin standard to peaks in a cola sample. Column: pBondapak C l 8 (3.9 X 150 mm). Mobile phase: 1 M acetic acid (pH = 2.4). Flow rate: 1.5 mumin. Injectionvolume: I 5 gL. A: Saccharin standard; B: cola sample. Note that the suspected "saccharin" peak has adlfferent refentlontime than lhatof the saccharh standard. See text f a details.
(Continued on page A2W)
Volume 68 Number 8 August 1991
A199
The Modern Student laboratory However, inasmuch as cola beverages contain a fairly complex variety of natural producta, it is not unreasonable to expect that occasionally such a chromatographic dilemma will he encountered. This provides the instructor with the ability to demonstrate the concept of the power of changing the mobile phase to effect changes in the separation and to explain to the student that, while HPLC is an excellent quantitative technique, it provides only limited qualitative information. Analysis of Soft Drinks
The table provides the observed level of the additives in a number of soft drinks from cans and from a soda fountain. The results that were obtained for the common additives were similar in the various samples that were analyzed. In some cases, the levels of the sweeteners may be available from the manufacturer of the soft drink (6) and should be obtained to verify that the analytical results are valid. (It should be noted that label claims are generally upper limits for rhe concentration of the additiveaand the observed results may . vary- from the manufacturer's indicated maximum concentration levels.) Additional Experiments
The separation of these compounds in soft drinks may lead to a number of additional experiments to be performed by advanced students. For instance, the analytical data is presented in terms of the retention time of each component.
Althoueh this orovides a suitable method to identifv the compounds, the instructor might add uracil as a void volume marker to the standards and the samples so that the value of k' can be calculated (7, 8). Students can then use k' to identifv oeaks rather than relvine on the observed retention times. kiditionally, the separ&& can be run at several flow rates to observe the constant nature of k' values and to use the chromatograms to judge which flow rate is most appropriate for the analyses. A second activity could focus on a variety of other beverages including coffee, tea, and fruit juices that contain the additives described in this experiment. It mieht he instructive to have students analvze such samples w determine the concentration of caffeine in coffee and decaffeinated coffee. In our work we found that a CUD of coffee contained approximately 75 mg of caffeineIl2-oz ;up, with the level varvine fairlv widelv. The caffeine level will depend on the amount of coffee used to brew the coffee as well as the brewing process. u
Conclusion
The experiment descrihed in this paper can he performed in a twical undereraduate laboratory in instrumental analysis in; single laboratory period. It demonstrates the power of HPLC, how to develop a separation, and the limits of qualitative and quantitative usage. Furthermore, it provides the student with an analysis that has a high degree of relevance. There are a number of advantages of this method over currently existing methods for beverage additives, including safetv. ease of use. meed of analwis. and reduced solvent cons&nption. perhaps, the most beneficial is the fact that the mobile ohase contains methanol rather than acetonitrile, which reduces the exposure of students to this potentially toxic substance. Lastlv, a number of additional areas that could be explored by &interested student are suggested. Acknowledgement
The authors would like to thank P. Froehlich for helpful discussions and perspectives, C. Niederlander for laboratory work and C. Galgano for assistance in the preparation of the manuscript. Llterature Clted 1. Bidlingmeycr, B. A : Warren, F. V . J.Chem. Educ. 1384,61,716720. D.S.:Woodward,B. B.;Conrad,E. C. J Aaaar O//.Ano!. C h e m 1376.59.14-19. 3. Grayeski,M. L.; Woolf,E. J.;Sfrsub,T. S.LC 1385.3.538540. 4. Delsnoy.M.F.:Pasko,K.M.;Mauro.D.M.:GsII,D.M.:Korologas,P.C.;Mo~awki,J.; Krolikowaki, L. J.;Warren. F.V. J. Chsm. Educ 1985,62,618420. 5 . N e i w c k , W.; Nollet, L.Balg. J. Food Chsm. Biomhnol. 1988.43.83-88. 6. Consumer InfarmationBulletin entitled"YauAsked A b u t Soft Drinks born CocaCole USA'I; Conwmer Information Center. Coca~ColaUSA: P O . Drawer 1734, Atlanta, GA 30301, May 1990. 7. Sadek.P.C.:Csrr.P. W.:Bowers. L.D.LC l985,3,590-592. 3. Bid!ingmeyer,B.A.: Warren,F.V.;Weston,A.:Nugent,C.:Froehlich.P.M.,J.ChromoLopr Sci. (L991)29.275-279. 2. Smyly,
AddHlve Amounts In Saldad hverages in mg/l2 11-02 Can Saccharin Cola 1 Diet Cola 1 b m n Lime Diet Cola 2 Grape Cola 2 Diet Cola Fountain Cola Fountsin 'NF =not found.
A200
Journal of Chemical Education
NF' NF NF 161.4 NF NF 41.7 NF
Cafielne
BenroicAcid
Aspartame
