In the Laboratory
Pungency Quantitation of Hot Pepper Sauces Using HPLC Thomas A. Betts Department of Physical Sciences, Kutztown University, Kutztown, PA 19530
Several interesting applications of high-performance liquid chromatography (HPLC) for the analysis of consumer products in undergraduate laboratories have appeared in this Journal. These include the analysis of vitamin A in infant formula (1); acetaminophen, caffeine, aspirin, salicylamide, phenacetin, and paracetamol in analgesic tablets (2–4 ); chlorophyll a, chlorophyll b, and β-carotene in collard greens (5); aspartame, caffeine, saccharin, and benzoic acid in colas and other beverages (6–9); coumarin in vanilla extracts (10, 11); theophylline, ephedrine, and phenobarbital in asthma medication (12); riboflavin in milk and cheese (13); sugars in milk, colas, and beer (14); UV absorbers in sunscreens (15), and enantiomers of beta-blockers (16 ). An additional application of HPLC that is suitable for undergraduate laboratories is the quantitation of the components of hot-pepper sauces responsible for the perceived “heat”. As an undergraduate laboratory experience, our goal was not simply to determine the heat of various hot pepper sauces. Rather, the goal was to allow students to develop an HPLC method to separate and quantify capsaicins that would precede pungency quantitation. The HPLC method presented here was developed collectively by students during a course in instrumental analysis. The compounds responsible for the pungency of hot peppers (Capsicum spp.) are a class of compounds known as capsaicinoids (Fig. 1). The two capsaicinoids responsible for over 90% of the heat in hot peppers are capsaicin (N-[(4hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide) and dihydrocapsaicin ( N-[(4-hydroxy-3-methoxyphenyl)methyl]-8-methylnonanamide) (17). Other structurally related capsaicinoids (nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin) in trace amounts contribute to the remainder of the heat. Capsaicinoids have been used in over-the-counter pain-relieving lotions, in sprays to ward off would-be attackers (such as bears), and in squirrel-
Capsaicin
Dihydrocapsaicin
resistant bird seed (18). (Birds do not have capsaicin receptors, but squirrels do.) One early method of quantifying the perceived heat from hot peppers is based on an organoleptic method known as the Scoville method (19). The Scoville method involves a series of trained pepper-tasters who evaluate pungencies of peppers based on “bite” and duration of bite, and assign the pepper a Scoville heat (pungency) value. This method provides a direct evaluation of pepper heat perception by humans but is fraught with subjectivity and irreproducibility. The Scoville heat unit, however, remains the standard in the food industry. Todd et al. (20) determined Scoville pungency levels for five individual capsaicinoids (Table 1). These organoleptic experiments determined how each of the capsaicinoids contributes to the overall oral sensation of the pepper or pepper product. For example, capsaicin, dihydrocapsaicin, and nordihydrocapsaicin produce rapid pungent sensations near the back of the palate and throat, whereas homocapsaicin and homodihydrocapsaicin tend to produce longer, low-intensity bite sensations in the midmouth and midpalate regions (20). These Scoville pungency levels for individual capsaicinoids allow us to calculate the perceived heat of a pepper product given the concentration of each of the individual capsaicinoids. Todd et al. used gas chromatography to quantify individual capsaicinoids (20); others have used visible spectrophotometry (21, 22) and HPLC (17, 23-29). Of these methods HPLC offers suitable precision and accuracy with minimal complications. The instrumental analysis lab at Kutztown University typically involves six pairs of students working on six different experiments during a given lab period. Because students performed this experiment “in rotation”, each pair of students was assigned one aspect of the method to develop (e.g., mobile phase composition). No detailed instructions were provided; however, all students were provided access to a set of papers involving HPLC analysis of capsaicinoids (23–28). Students were required to read these papers and propose an approach to address their component of the method. All procedures were approved by the instructor before they were implemented. After the students formulated and executed a systematic approach to the portion of the method they were assigned, they were required to communicate their procedures and results to the remainder of the class in the form of a lab report. Sub-
Nordihydrocapsaicin
Table 1. Threshold Pungencies of Capsaicinoids from Ref 20 Homocapsaicin
Homodihydrocapsaicin
Figure 1. Structures of natural capsaicinoids.
240
Capsaicinoid
Threshold Pungency/ Scoville Units
Capsaicin
16.1 × 106
Dihydrocapsaicin
16.1 × 106
Nordihydrocapsaicin
9.3 × 106
Homocapsaicin
6.9 × 106
Homodihydrocapsaicin
9.2 × 106
Journal of Chemical Education • Vol. 76 No. 2 February 1999 • JChemEd.chem.wisc.edu
In the Laboratory
Experimental Details
Instrumentation The liquid chromatograph included two Beckman Model 110 B pumps driven by a Beckman Model 421A controller. Sample injections were performed with a Beckman model 210A sample injection valve with a 20-µL sample loop. A 4.6-mm by 25-cm Beckman Ultrasphere ODS (C-18) column with a 5-µ m packing diameter was used. The signal from an SSI model 500 variable-wavelength UV–vis absorbance detector was collected by a computer using Waters Maxima 820 software. A Hewlett-Packard 8451A diode array UV–vis spectrophotometer and quartz cuvettes were used to obtain absorbance spectra. Reagents CAUTION: Natural capsaicin is highly toxic and is a severe irritant, especially to mucous membranes and upper respiratory tract. Wear gloves, protective clothing, and a mask. Do not breath dust. The natural capsaicin (Sigma) used for preparation of standards was a mixture of 65% capsaicin and 35% dihydrocapsaicin. It is strongly recommended that the initial capsaicin stock solution (250 ppm) be prepared by the instructor. Methanol and acetone used for extractions were reagent grade. Dehydrated ethanol was used for dilution of the extracted capsaicinoids. HPLC grade acetonitrile was used for the solidphase extraction (SPE). Mobile phases were composed of HPLCgrade methanol and distilled, deionized water, and were degassed and filtered prior to arriving at the pump inlet. Extraction of Capsaicin from Hot Peppers and Pepper Sauces Although a variety of extraction solvents could be used, our students chose to extract capsaicinoids from hot pepper sauces using acetone according to the procedure described by Weaver and Awde (27). Attuquayefio and Buckle (30) evaluated several extraction solvents and showed that acetone was one of the solvents that produced a high yield. Acetonitrile not only produced a high yield, but minimized interferences during HPLC analysis as well. Ten grams of the hot sauce was placed in a 250-mL round-bottom boiling flask with 100 mL of acetone. The mixture was refluxed for one hour. The resulting mixture was filtered by vacuum, and the volume of the supernatant was reduced to approximately 5 mL by removing acetone using a rotary evaporator. The resulting capsaicin extract was diluted to a final volume of 50 mL with dehydrated ethanol. A 10-mL aliquot of this ethanol solution was further diluted to 25 mL
with mobile phase. Several solutions contained oil droplets, which were dissolved by sonicating for 2 minutes. The final solution was filtered through a 0.45 µm filter before injection. An alternative to the acetone liquid extraction is solidphase extraction (SPE). A simple SPE of capsaicinoids has been described by Attuquayefio and Buckle (30). A Suppelco 3-mL LC-18 SPE cartridge was conditioned first with 5 mL of acetonitrile and then 5 mL of water. Acetonitrile (10 mL) was added to a 2–5-g sample of hot sauce (or 1 g of dehydrated crushed pepper) and blended for 2 minutes. A 1-mL aliquot of the acetonitrile extract was dissolved in 9 mL of water. This solution was passed through the conditioned SPE cartridge. Slight head pressure on the column was maintained using a syringe, which greatly speeded elution. The aqueous eluant was discarded. The capsaicinoids were then eluted from the column with 4 mL of acetonitrile followed by 1 mL of acetonitrile containing 1% acetic acid. The eluted capsaicinoids were injected directly into the liquid chromatograph for analysis. The initial extraction of the oil-based Pure Cap sample had to be performed differently because the oil was insoluble in the acetonitrile. Pure Cap (0.1 g) was dissolved in 2 mL of hexane, and the capsaicins were transferred to acetonitrile by performing a liquid–liquid extraction with four 5-mL portions of acetonitrile. A 1-mL aliquot of the acetonitrile extract was dissolved in 9 mL of water, and the SPE cleanup procedure was carried out on this solution as described above. Results and Discussion
Choice of Detector Wavelength To determine the best wavelength for the detection of capsaicinoids, an absorbance spectrum of the capsaicin standard dissolved in 80:20 methanol–water was obtained (Fig. 2). Students immediately chose 216 nm and 230 nm as the wavelengths that would afford the greatest sensitivity. However, the absorbance for the 65-ppm capsaicin–35 ppm dihydrocapsaicin standard was quite high (2.3) at these wavelengths, and it was likely that calibration curves would be nonlinear if higher concentration standards were used. For quantitative work absorbances should remain below 1.0 because the low intensity of light reaching the detector creates large errors. A chromatogram of Dave’s Total Insanity gourmet hot sauce extract was obtained at 216, 230 and 284 nm to de2.5
2.0
Absorbance
sequent groups needed the cumulative results and conclusions from the previous groups if progress was to be made. Each pair of students was assigned a portion of the method small enough to be investigated in one 3-hour lab period. The portions of the method were divided as follows. (i) How do you extract capsaicinoids from hot peppers or hot pepper sauces? (ii) What wavelength should be used to detect the capsaicinoids? (iii) Which mobile phase will provide adequate resolution in a reasonable analysis time? (iv) How will the capsaicinoids be quantified? The final groups of students then evaluated the capsaicinoid content of a variety of hot pepper sauces.
1.5
1.0
0.5
0.0 190
210
230
250
270
290
310
Wavelength / nm
Figure 2. Absorbance spectrum of 65 ppm capsaicin–35 ppm dihydrocapsaicin standard in 80:20 methanol–water.
JChemEd.chem.wisc.edu • Vol. 76 No. 2 February 1999 • Journal of Chemical Education
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In the Laboratory
Choice of Mobile Phase An isocratic mobile phase of methanol–water was chosen as the basis for the separation. The resolution and capacity factors for capsaicin and dihydrocapsaicin were determined as a function of mobile phase composition (Fig. 4) by evaluating chromatograms of McIlhenny’s Tabasco extract as the concentration of methanol in the mobile phase was varied from 60 to 85% (v/v). As expected, retention of capsaicinoids on the C-18 column decreased as the concentration of methanol increased. A reasonable capacity factor would lie between 1 and 10, and an acceptable resolution would be greater than 1.5 (31). A mobile phase composed of 75:25 methanol–water satisfied both criteria; however, our students chose an 80:20 methanol–water mobile phase to keep the analysis time under 5 minutes, with only a slight sacrifice in resolution.
Quantitation For the purpose of this laboratory exercise, we chose to focus on the two capsaicinoids that contribute more than 90% of the heat from hot peppers. Quantifying only two of the 5 possible capsaicinoids may slightly underestimate pungency because we are ignoring the minor capsaicinoids. Quantitation of minor capsaicinoids is possible, but it would add a degree of complexity that is not necessary to introduce students to HPLC method development. Another factor that limited the accuracy of the pungency quantitation was the nature of the standard. Natural capsaicin, as purchased from Sigma, is reported to be a mixture of ≈65% capsaicin and ≈35% dihydrocapsaicin. Pure standards of each capsaicinoid would enhance accuracy, but they are inevitably more expensive.
Table 2. Relative Standard Deviation of Peak Height and Peak Area from 5 Replicate Injections of Calibration Standard Relative Standard Deviation, %
Component
Peak Height
Peak Area
Capsaicin
2.57
0.57
Dihydrocapsaicin
2.69
1.25
In an effort to assess the capsaicin content of the natural capsaicin standard, peak areas (expressed as percentages of the combined peak areas) were determined for capsaicin and dihydrocapsaicin for all of the calibration standards. An average of (67 ± 1)% of the combined peak area was due to capsaicin and (33 ± 1)% was due to dihydrocapsaicin. Iwai et al. (23) reported that the capsaicin analogues have spectra similar to that of capsaicin, with similar absorptivities. Assuming the absorptivities are identical, the peak areas indicate that the standard contains (67 ± 1)% capsaicin and (33 ± 1)% dihydrocapsaicin. Although these values differ slightly from the values Sigma provides, they could be explained by slight differences in absorptivities. Therefore, the values of 65% capsaicin and 35% dihydrocapsaicin were used in all calculations. Figure 3 compares the chromatogram of Dave’s Total Insanity gourmet hot sauce extract with the 65 ppm capsaicin– 35 ppm dihydrocapsaicin standard. The first peak on Figure 3 at 1.6 min contains most of the polar contaminants not removed by SPE. Incidentally, this peak is much larger for the acetone liquid extraction because no attempt was made to separate the polar components from the extract before analysis. The retention times for capsaicin and dihydrocapsaicin are 3.19 and 3.96 min, respectively. The unidentified shoulder on the capsaicin peak and additional unidentified peaks could be impurities or additional capsaicinoids in low concentrations. Any shoulders on capsaicin or dihydrocapsaicin peaks were skimmed using the integration software to minimize their effect on the peak area. It was necessary to make a choice between peak height and peak area as the quantitative parameter. Five replicate injections of the 65 ppm capsaicin–35 ppm dihydrocapsaicin
50
5.34
6
5.33
5
Capsaicin
5.31 5.30
Dihydrocapsaicin
5.29 5.28
Capacity Factor
Detector Response
40 5.32
4 30 3 20 2 10
Resolution
termine if a particular wavelength proved advantageous in minimizing interference from possible co-eluting species. An unidentified shoulder on the capsaicin peak was minimized (but still present) by using 284 nm (Fig. 3). A detector wavelength of 284 nm, which provided adequate sensitivity for these analyses, was used in all subsequent determinations. Unfortunately, capsaicins do not absorb well at 254 nm (see Fig. 2). Therefore, a fixed-wavelength detector operating at 254 nm is not suitable for capsaicin analysis.
1
5.27 5.26
0
2
4
6
Time / min
Figure 3. Chromatograms of Dave’s Total Insanity Gourmet Hot Sauce extract () and 65 ppm capsaicin–35 ppm dihydrocapsaicin standard (᎑ ᎑ ᎑ ᎑). The mobile phase was 80:20 methanol– water flowing at 1.5 mL/min through a 4.6 mm × 25 cm Beckman Ultrasphere ODS (5-µm particle diameter) column. UV detection was performed at 284 nm.
242
0 60
65
70
75
80
0 85
% Methanol in Methanol/Water Mobile Phase
Figure 4. Capacity factors for capsaicin ( –䉭—䉭– ) and dihydrocapsaicin ( –䊊—䊊–) and the resolution ( –䊏—䊏–) as a function of methanol concentration in the methanol–water mobile phase. The mobile-phase flow rate was 1.5 mL/min. The column was a 4.6 mm × 25 cm Beckman Ultrasphere ODS (5-µm particle diameter). UV detection was performed at 284 nm.
Journal of Chemical Education • Vol. 76 No. 2 February 1999 • JChemEd.chem.wisc.edu
In the Laboratory
Table 3. Capsaicin and Dihydrocapsaicin Concentrations in Spicy Condiments Solid Phase Extraction
Acetone Extraction
Product
% by Weight Capsaicin
% by Weight Dihydrocapsaicin
Scoville Units
% by Weight Capsaicin
% by Weight Dihydrocapsaicin
Scoville Units –
Pure Capa
1.8 ± 0.25
1.4 ± 0.16
520,000
–
–
Endorphin Rusha
0.312 ± 0.004
0.295 ± 0.003
97,700
–
–
–
Dave's Total Insanity b
0.210 ± 0.006
0.138 ± 0.003
56,000
0.205 ± 0.005
0.142 ± 0.003
56,000
Scorned Womanc
0.272 ± 0.003
0.0478 ± 0.0008
51,500
–
–
–
Crushed Red Pepper d
0.105 ± 0.008
0.109 ± 0.005
35,000
–
–
–
Inner Beauty Real Hot Saucee
0.086 ± 0.004
0.010 ± 0.002
15,000
0.074 ± 0.004
0.009 ± 0.002
13,000
McIlhenny's Tabascof
0.012 ± 0.002
0.008 ± 0.001
3,000
0.012 ± 0.002
0.007 ± 0.002
3,000
Arizona Gunslinger g
0.009 ± 0.002
0.0066 ± 0.0007
2,500
–
–
– –
0.003 ± 0.002
0.0010 ± 0.0008
600
–
–
Frank's Original Red Hot i
0.001 ± 0.002
0.001 ± 0.001
400
0.001 ± 0.002
0.001 ± 0.001
Red Devilj
0.001 ± 0.002
0.0009 ± 0.0008
300
–
–
Thai Sweet and Sour
h
400 –
NOTE: Precision was estimated using the standard deviations of the slopes and intercepts of the calibration curves. Scoville heat units were calculated from weight-to-weight ratios of each capsaicinoid and the Scoville threshold pungency from Table 1. a Garden Row Foods, Inc., Elmwood Park, IL. b Dave’s Gourmet, Inc., 1232 Montgomery Ave., San Bruno, CA 94066. c Oak Hill Farms, P.O. Box 888302, Atlanta, GA 30356. d Echo Hill Country Store, R.D. #1 Box 1029, Fleetwood, PA 19522. e Inner Beauty, Inc., Cambridge, MA 02139. f McIlhenny Company, Avery Island, LA 70513. g Arizona Pepper Products Company, 638 W. Broadway, Ste. #302, Mesa, AZ 85210. h Thai International Foods, P.O. Box 130, 325 Bowers Road, Bowers, PA 19511. i Durkee Famous Foods, SCM Corporation, Westlake, OH 44145. j Trappey’s Fine Foods, Inc., P.O. Box 12638, New Iberia, LA 70562.
calibration standard were made. The relative standard deviations in peak height and peak area for capsaicin and dihydrocapsaicin are presented in Table 2. From the low relative standard deviation of peak area compared to peak height, it is clear that peak area offers greater precision. Peak area was used in all subsequent quantitative measurements. A series of capsaicin–dihydrocapsaicin standards was prepared by diluting aliquots of the stock solution containing 250 ppm total capsaicinoids (163 ppm capsaicin and 87 ppm dihydrocapsaicin) with 80:20 methanol–water. An 80:20 methanol–water blank was also prepared. Figure 5 shows the analytical calibration curves used to determine the capsaicin and dihydrocapsaicin concentrations in a variety of hot pepper sauce extracts.
Peak Area / (mV ⋅ s)
800 600
400 200 0 0
50
100
150
Capsaicinoid Concentration (ppm)
Figure 5. Analytical calibration curves for capsaicin ( –䉭—䉭–) and dihydrocapsaicin ( -䊉- - - 䊉-). Regression line for capsaicin calibration: y = (5.006 ± 0.032)x + (1.627 ± 2.950), R2 = .9998. Regression line for dihydrocapsaicin: y = (4.659 ± 0.027)x + (1.023 ± 1.310), R2 = .9998.
Analysis of Hot Pepper Sauces Capsaicinoids from a variety of hot pepper sauces were extracted using acetonitrile followed by C-18 SPE cleanup of the extract as described above. Twenty microliters of the extract was chromatographed on a 4.6-mm by 25-cm C-18 column using an isocratic 80:20 methanol–water mobile phase with a flow rate of 1.5 mL/min and UV absorbance detection at 284 nm. The peak areas of the resulting chromatograms were compared with the calibration curves for capsaicin and dihydrocapsaicin (Fig. 5) and the concentrations of both capsaicinoids in the original sample were determined. Table 3 shows the results of these analyses by organizing the pepper sauces from highest capsaicinoid concentration to lowest. The precision of the concentrations reported in Table 3 was estimated using the random error associated with the slopes and intercepts of the capsaicin and dihydrocapsaicin calibration curves (Fig. 5) (32). The random error in the concentrations of the capsaicinoid extracts was then propagated to ultimately arrive at the random error associated with the weight percent. Scoville values were calculated by multiplying the weight-toweight ratio of both capsaicinoids by the threshold pungency as described by Todd et al. (20) (e.g., for Endorphin Rush (0.00312 × 16.1 × 106) + (0.00295 × 16.1 × 106) = 97,700 Scoville Units). The Scoville ratings are reported with the correct number of significant figures based on the random error in the capsaicinoid weight percent. Besides the Consumer Reports-type information provided by Table 3, there are two interesting notes regarding this table. First, the Pure Cap product boasts a Scoville rating of 500,000 on the label. With the method described here a Scoville rating of 520,000 has been determined. This value is in excellent agreement with the Pure Cap label considering the single significant figure on the label. Second, the agreement between results from the acetone extraction and the SPE is good (within 14%).
JChemEd.chem.wisc.edu • Vol. 76 No. 2 February 1999 • Journal of Chemical Education
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In the Laboratory
Extraction Efficiency The efficiency of the acetonitrile extraction followed by SPE cleanup was evaluated by using a single spike in McIlhenny’s Tabasco sauce. Two McIlhenny’s samples were extracted and analyzed: one was not spiked, and the other was spiked with 2.2 mg of the capsaicin–dihydrocapsaicin standard (1.4 mg capsaicin and 0.8 mg dihydrocapsaicin). This relatively large spike was used to determine how this extraction would hold up with the hottest of hot sauces. The increase in peak areas due to the spike corresponded to 95% and 92% recoveries for capsaicin and dihydrocapsaicin, respectively. Published recoveries for total capsaicinoids using this procedure were 98.2% (30). The acceptable recoveries presented here are slightly lower than the published value possibly because of the high spike concentration. The efficiency of the acetone extraction was not determined. SPE provided several advantages over acetone extraction. (i) Sample preparation time was greatly reduced owing to elimination of the reflux period. (ii) Polar contaminants were significantly reduced. (iii) Students were exposed to the advantages of SPE. SPE provided results that were comparable to the acetone extraction with a much shorter sample preparation time. Several samples were extracted using both techniques and are compared in Table 3. Conclusions In traditional instrumental analysis laboratory exercises a question is posed and students are told exactly how to answer it. Students typically follow a prescribed instrumental method and are expected to obtain valid results. Using an approach where students must collectively develop a method provides pedagogical advantages by placing additional realistic expectations on students. Here, a set of problems was presented to the class, and students were required to collectively solve these problems using the literature and instrumental resources available. All students were expected to (i) review pertinent primary and secondary literature, (ii) design and execute an experiment, (iii) analyze data, and (iv) communicate their methodology, results, and conclusions to their peers. Evaluation of student performance was based on how well they performed with regard to the expectations outlined above. Grading this laboratory exercise was a bit subjective because groups of students were performing very different experiments. Although students were solving different problems, they were all working toward a common goal: a useful HPLC method for capsaicin analysis in hot pepper sauces. Acknowledgments We gratefully acknowledge Sean Park for initiating this project and Kimberly Bucolo of the Reading Spice Co.,
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Reading, PA, for donation of the Scorned Woman hot sauce and the Thai Sweet and Sour sauce. Literature Cited 1. Bohman, O.; Engdahl, K.; Johnson, H. J. Chem. Educ. 1982, 59, 251–252. 2. Kagel, R. A.; Farwell, S. O. J. Chem. Educ. 1983, 60, 163–166. 3. Haddad, P.; Hutchins, S.; Tuffy, M. J. Chem. Educ. 1983, 60, 166–168. 4. Beaver, R. W.; Bunch, J. E.; Jones, L. A. J. Chem. Educ. 1983, 60, 1000–1001. 5. Silveira, A., Jr.; Koehler, J. A.; Beadel, E. F.; Monroe, P. A. J. Chem. Educ. 1984, 61, 264–265. 6. DiNunzio, J. E. J. Chem. Educ. 1985, 62, 446–447. 7. Strohl, A. N. J. Chem. Educ. 1985, 62, 447–448. 8. Delaney, M. F.; Pasko, K. M.; Mauro, D. M.; Gsell, D. S.; Korologos, P. C.; Morawski, J.; Krolikowski, L. J.; Warren, F. V., Jr. J. Chem. Educ. 1985, 62, 618–620. 9. Bidlingmeyer, B. A.; Schmitz, S. J. Chem. Educ. 1991, 68, A195– A200. 10. Sparks, L.; Bleasdell, B. D. J. Chem. Educ. 1986, 63, 638–639. 11. McKone, H. T.; Chambers, T. E. J. Chem. Educ. 1988, 65, 628. 12. Mueller, B. L.; Potts, L. W. J. Chem. Educ. 1988, 65, 905–906. 13. Munari, M.; Miurin, M.; Goi, G. J. Chem. Educ. 1991, 68, 78– 79. 14. Luo, P.; Luo, M. Z.; Baldwin, R. P. J. Chem. Educ. 1993, 70, 679–681. 15. Davis, M. R.; Quigley, M. N. J. Chem. Educ. 1995, 72, 279– 281. 16. Tran, C. D.; Dotlich, M. J. Chem. Educ. 1995, 72, 71–73. 17. Yao, J.; Nair, M. G.; Chandra, A. J. Agric. Food Chem. 1994, 42, 1303–1305. 18. Rouhi, A. M. Chem. Eng. News 1996, 74(10), 30–31. 19. Official Analytical Methods of the American Spice Trade Association, 2nd ed.; ASTA: Englewood Cliffs, NJ, 1968; Method 21.0. 20. Todd, P. H., Jr.; Bensinger, M. G.; Biftu, T. J. Food Sci. 1977, 42, 660–665. 21. Suzuki, J. I.; Tausig, F.; Mores, R. E. Food Technol. 1947, 11, 100– 104. 22. Palacio, J. R. J. Assoc. Off. Anal. Chem. 1977, 60, 970–972. 23. Iwai, K.; Suzuki, T.; Fujiwake, H.; Oka, S. J. Chromatogr. 1979, 172, 303–311. 24. Hoffman, P. G.; Lego, M. C.; Galetto, W. G. J. Agric. Food Chem. 1983, 31, 1326–1330. 25. Weaver, K. M.; Luker, R. G.; Neale, M. E. J. Chromatogr. 1984, 301, 288–291. 26. Krajewska, A. M.; Powers, J. J. Chromatogr. 1986, 367, 267–270. 27. Weaver, K. M.; Awde, D. B. J. Chromatogr. 1986, 367, 438–442. 28. Cooper, T. H.; Guzinski, J. A; Fisher, C. J. Agric. Food Chem. 1991, 39, 2253–2256. 29. Woodbury, J. E. J. Assoc. Off. Anal. Chem. 1980, 63, 556–558. 30. Attuquayefio, V. K.; Buckle, K. A. J. Agric. Food Chem. 1987, 35, 777–779. 31. Hamilton, J. R. Introduction to High Performance Liquid Chromatography, 2nd ed.; Chapman and Hall: New York, 1982; Chapter 6. 32. Miller, J. C; Miller, J. N. Statistics for Analytical Chemistry; Wiley: New York 1984; Chapter 4.
Journal of Chemical Education • Vol. 76 No. 2 February 1999 • JChemEd.chem.wisc.edu
