A Comparison of Separation Techniques:
Analysis of Vanilla for Coumarin Contamination Laurie S p a r k s and Bob D. Bleasdell Southern Oregon State College. Ashland, OR 97520 Coumarin (2H-l-henzopyran-2-one) comes from t h e tonka bean, which grows mainly in South America. Because its taste, smell, and appearance are similar t o vanilla, coumarin was used a s a flavoring agent in chocolates, candies, confections, and baked goods. However, coumarin is known t o cause liver damaae in laboratory animals and has been banned for useas a-food or food additive in t h e u n i t e d States since 1954. Currentlv. coumarin finds limited use in the United States a s a n additive in perfumes and cosmetics.' In some parts of the world, however, coumarin is still being added t o real andlor artificial vanilla t h a t is then labeled as ~ these coumaringenuine vanilla e x t ~ a c t .Occasionally, tainted flavorings find their way into food co-ops and kitchens in the United States. This experiment involves the analysis of vanilla samples for coumarin using TLC, GC, and HPLC. A comparison of results from these techniaues will demonstrate the aualitative and quantitative nature, a s well a s t h e relative aEcuracy and sensitivitv of each tvue of chromatoerauhv. Students in a n instrumental analy& course enjoy t h i s t i p e of experiment a s a n advanced project because they are able toanalyze "real" samples using techniques a n d procedures they have been introduced t o in other courses. Procedure Several vanilla samples, at least one of which is suspected to contain coumarin, are collected by the student. (It is not uncommon that vanilla nurchased abroad for oersonal use will contain some in solid form &e also needed. Coumarin, ethyl coumarin). ~iandards vanillin, and vanillin have been included in this study.
TLC The following procedure is used by the FDA to screen suspected sample^.^ The standards are dissolved in methanol. The TLC solvent is 3%methanol in benzene, and the stationary phase is an 8- X 8-in. sheet of commercially prepared silica gel with fluorescent indi-
'
cator.Thestandards andundiluted samples are spotted on the silica gel sheet and allowed tomigrateabout 6 in. (thisrequiresless than 1 h). An aqueous solution of 10%NaOH is sprayed on the sheet and allowed to dry. The sheet is then placed under a fluorescent light to h a t e the spots. Rr values are calculated and recorded.
GC A gas chromatograph with a flame ionization detector is recommended. We used an HP 5890A Gas Chromatograph with a Series 530 micro fused capillary column and an HP 339211 Integrator for the readout. Temperature programming may he necessary depending on the column and separation conditions employed. Using nitrogen as the carrier gas (3 psi), we found the following temperature program to be effective: Hold at an initial temperature of 85 OC for 1 min, then increase the temperature at a rate of 5 "Clmin. An equimolar solution of the three standards is prepared. Asuitahle solvent is 50%methanol (vlv) in water. Most vanilla samoles will have to be diluted 125 with the solvent before injection.'~he samples and standards decompose after sitting for 2-3 d at room temperature. Therefore, solutions should be prepared the day of the analysis or refrigerated if they are to be used at a later date. The standard solution along with the diluted samples are run. The values from several injections should be averaged to obtain the best results. The retention times and relative percentages of the main peaks (eoumarin, ethyl vanillin, and vanillin) are then recorded for comparison. HPLC An HPLC equipped with a suitable column and a UV detector is required. We used a Waters Associates 441 HPLC, a radial pack C18column, a 254-nm UV detector, andan HP 3392A 1ntegrator.The mobile phase is 50% methanol (vlv) in water with a flow rate of 2 mL1min. The same diluted samples and standards can be used here as were used in the GC procedure. Again, the values for several injections should be averaged and the retention times and relative percentages recorded. Conclusion Organization of t h e data a s shown in the table is helpful for comparison of the three techniques. T h e student should he asked to comment on the qualitative and quantitative nature of the three techniaues and the relative sensitivitv of each. As outlined in this experiment, t h e T L C is used only a s a screening technique a n d may not be sufficiently sensitive to detect trace concentrations of a compo-
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Meulv. W. C. In "Kirk-Othmer Encvclo~edlaof Chemical Technology"; G&onj M Ed.: ~ l l e yNew . &k.'1979vol 7, p 196. Hopkins. H. FDA Consumer 1983, 17. (8) 11. Da las Oisrrlcl Lab Board FDA. personal communication. 1984.
Separatlon Results Coumsrin TLC
GC
HPLC
SAMPLE
R1
RT/Rel% (mi")
RTIRel% (mi")
coumarin ethyl vanillin vanillin
0.57
5.86
#3=
638
0.56
5.83123.0
Ethyl Vanillin GC HPLC RT/Rel% (mi") RTlRel% (min)
Vanlllm TLC R+
GG
HPLC
RT/Rel% (min)
RT/Rel% (min)
0.32 0.32 0.32 0.30
5.40 5.37101.5 5.341100.0 5.33173.3
1.17 1.17195.0 1.16/100.0 1.16174.4
2.87
#Is
#za
nc
2.00124.0
Journal of Chemical Education
0.43
6.58
1.59
ND~
6.56183
1.5015.0
NDd
6.5213.7
1.5811.6
nent in a given sample. If the GC and HPLC quantitative data do not agree, the student should be asked to offer a plausible explanation (e.g., insufficient data, relative detector response, injection technique). Approximately six hours are required for this experiment.
Acknowledgment
The authors would like to thank DebraLaBianco for valuable suggestions and information in the preparation of this article.
Volume 63
Number 7
July 1966
639
