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Real World Project - HPLCJames Bergenti
Jessica MelhornKimberly Duncan
http://www.youtube.com/watch?v=1tRq8ExAHzk
Paper Review“Determination of the Scoville Heat Value for
Hot Sauces and Chilies: An HPLC ExperimentJames D. Batchelor, Bradley T. JonesTaken from the Journal of Chemical
Education
BackgroundCapsaicinoids are a group of compounds that give
various peppers their characteristic taste.Capsaicin is comprised of seven capsacinoids, or
closely related alkanoids.Upon contact with capsaicin, neurons in the
nociceptors in the palate become excited, emitting a perception of pain, and release local inflammatory mediators.
SHV = 16.1 million
Dihydrocapsaicin and NordihydrocapsaicinDihydrocapsaicin: accounts for 22% of total capsaicinoid
mixture. The same pungency as capsaicin. SHV = 16.1 million
Nordihydrocapsaicin: Accounts for 7% of the total capsaicinoid mixture. Half as pungent as capsaicin. SHV= 9.3 million
Scoville Heat ValueFirst brought to light by in 1912 by Wilbur
Scoville.The Scoville heat value is relative to the final
concentration of capsaicin in a compound, expressed in grams of compound per gram of sample.
The value is obtained by multiplying the literature established SHV of the pure compound by the final concentration.
Comparative Scoville Values
Purpose of ExperimentTo quantify and determine the Scoville Heat
Value of various peppers through High Performance Liquid Chromatography.
Three alkaloids were tested: Capsaicin, Dihydrocapsaicin, and Nordihydrocapsaicin.
SHV was calculated from integrated absorbance data.
MaterialsChemicals:
Ethanol (95%) used as an extraction solventSynthetic Capsaicin (97%) used to prepare
working standards.Instruments:
HPLC with a UV/Vis absorption detectorReference Instrument: UV/Vis
A 50ppm sample was run through UV/Vis in order to find the wavelength of the capsaicin peak .
Approx. 224 nm
UnknownsTabasco Pepper Sauce- Red Pepper Homemade Hot Sauce – JalapenoEl Yucateco Hot Sauce – Habanero Dried Red Pepper
ProcedurePreparation of samples:
1000 ppm stock was made by dissolving 25 mg in 25 mL of 95% ethanol.
Serial Dilutions: (1000 ppm)(V1) = (50)(10) = .5 mL 1000 ppm stock (1000 ppm)(V1) = (40)(10) = .4 mL 1000 ppm stock (1000 ppm)(V1) = (30)(10) = .3 mL 1000 ppm stock (1000 ppm)(V1) = (20)(10) = .2 mL 1000 ppm stock (1000 ppm)(V1) = (10)(10) = .1 mL 1000 ppm stock (1000 ppm)(V1) = (5)(10) = .05 mL 1000 ppm stock (1000 ppm)(V1) = (1)(10) = .01 mL 1000 ppm stock
Preparation of Hot SauceGround Red Pepper: 2.9846g dissolved in 50mL of
ethanol. Stirred continuously, held at a slow boil for 30 minutes.
El Yucateco: 15.0218g dissolved in 50mL of ethanol. Stirred continuously, held at a slow boil for 30 minutes.
Homeade: 14.9652g dissolved in 50mL of ethanol. Stirred continuously, held at a slow boil for 30 minutes.
Tobasco – 14.9841g dissolved in 50mL of ethanol. Stirred continuously, held at a slow boil for 30 minutes.
MethodSolutions were filtered via vacuum filtration,
brought up to volume in a 100mL Erlenmeyer flask.
Approximatley 10 uL was injected onto the column for each sample.
All standards and samples were run three times.
Day 1
Concentration (ppm) Time (min) Area50 2.68 55184440 2.56 50374630 2.55 44419720 3.39 19666610 3.37 1532685 3.44 1456621 3.37 123038
Calibration Curve for CapsaicinArea vs Concentration
0 20 40 600
200000
400000
600000
R2= 0.9323y = 9844x + 83249
Concentration (ppm)
Are
a
Day 2
Concentration (ppm) Time (min) Area50 1.15 24814740 1.17 22809130 1.18 21594520 1.18 16218510 1.18 1236365 1.18 1078431 1.17 90089
Calibration Curve for CapsaicinArea vs. Concentration
0 20 40 600
100000
200000
300000
R2 = 0.9704y = 3373x + 92828
Concentration (ppm)
Are
a
Day 3
Concentration (ppm) Time (min) Area50 1.19 19224640 1.19 13212230 1.18 10713320 1.19 5520810 1.18 273375 1.20 184071 1.18 8670
Calibration Curve for CapsaicinArea vs Concentration
20 40 60-50000
0
50000
100000
150000
200000
250000R2= 0.9787y = 3674x - 4580
Concentration (ppm)
Are
a
Sample Calculation
HabaneroLiterature Value: 100,000-300,00 SHV
Calculated Value: 77,195 SHV
CayenneGround Red Pepper:
Literature Value: 30,000-50,000 SHVCalculated Value: 65,912 SHV
Tabasco SauceLiterature Value: 30,000-50,000 SHVCalculated Value: 71,635
JalapenoLiterature Value: 2,500-8,000 SHV
Calculated Value: 5,121 SHV
Taste TestScale of 1-101 = not hot. 10 = unbearableJalapeno – 2Tabasco – 6Red Pepper – 5Habanero - 8
Discussion Calculated values were not extremely accurate, however the
pattern remained constant. Errors:
Low baseline resolution Voltage range was not constant throughout experiment Peaks were truncated
Heat of peppers could have varied based upon soil they were grown in and time of year they were harvested.
Standards and calibration curves could have been made of the Dihydrocapsaicin and Nordihydrocapsaicin, however due to high cost and limited availability, this was not practical.
Only 50mg of synthetic capsaicin was available, so only one stock solution was able to be made.
Future ExperimentsStock solution would be remade each time
standards were made and run.Dihydrocapsaicin and Nordihydrocapsaicin
calibration curves could be made.Use of a wider variety of peppers.