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Real World Project - HPLC

Real World Project - HPLC. James Bergenti Jessica Melhorn Kimberly Duncan. http://www.youtube.com/watch?v=1tRq8ExAHzk. Paper Review. “Determination of the Scoville Heat Value for Hot Sauces and Chilies: An HPLC Experiment James D. Batchelor , Bradley T. Jones

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Real World Project - HPLC

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  1. Real World Project - HPLC James Bergenti Jessica Melhorn Kimberly Duncan

  2. http://www.youtube.com/watch?v=1tRq8ExAHzk

  3. Paper Review • “Determination of the Scoville Heat Value for Hot Sauces and Chilies: An HPLC Experiment • James D. Batchelor, Bradley T. Jones • Taken from the Journal of Chemical Education

  4. Background • Capsaicinoids 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

  5. Dihydrocapsaicin and Nordihydrocapsaicin • Dihydrocapsaicin: 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

  6. Scoville Heat Value • First 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.

  7. Comparative Scoville Values

  8. Purpose of Experiment • To 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.

  9. Materials • Chemicals: • Ethanol (95%) used as an extraction solvent • Synthetic Capsaicin (97%) used to prepare working standards. • Instruments: • HPLC with a UV/Vis absorption detector • Reference Instrument: UV/Vis • A 50ppm sample was run through UV/Vis in order to find the wavelength of the capsaicin peak . • Approx. 224 nm

  10. Unknowns • Tabasco Pepper Sauce- Red Pepper • Homemade Hot Sauce – Jalapeno • El Yucateco Hot Sauce – Habanero • Dried Red Pepper

  11. Procedure • Preparation 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

  12. Preparation of Hot Sauce • Ground 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.

  13. Method • Solutions 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.

  14. Day 1

  15. Day 2

  16. Day 3

  17. Sample Calculation

  18. Habanero Literature Value: 100,000-300,00 SHV Calculated Value: 77,195 SHV

  19. Cayenne • Ground Red Pepper: • Literature Value: 30,000-50,000 SHV • Calculated Value: 65,912 SHV • Tabasco Sauce • Literature Value: 30,000-50,000 SHV • Calculated Value: 71,635

  20. Jalapeno • Literature Value: 2,500-8,000 SHV • Calculated Value: 5,121 SHV

  21. Taste Test • Scale of 1-10 • 1 = not hot. 10 = unbearable • Jalapeno – 2 • Tabasco – 6 • Red Pepper – 5 • Habanero - 8

  22. 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.

  23. Future Experiments • Stock 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.

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