1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 ppm 0.964 1.336 1.354 1.373 1.391 1.411 1.588 1.607 1.627 1.645 1.664 1.966 1.983 1.996 2.015 2.196 2.214 2.232 3.310 3.312 3.313 3.833 4.259 4.827 5.355 5.368 6.725 6.863 5.97 1.98 2.00 2.13 3.08 2.99 1.99 2.01 2.03 1.00 The Extrac+on of Capsaicin and Parallel Quan+ta+ve Analysis Nicolle S. Jackson, Andrew R. Schroeder, Dr. Christopher Nicholson, Dr. Carrie A. Simpson, Dr. Karen S. Molek Department of Chemistry, University of West Florida, 11000 University Parkway, Pensacola, FL 32514 Abstract Acknowledgments Conclusions 1 H NMR Quan;ta;ve Analysis 1 H NMR Qualita;ve Characteriza;on Introduc;on LC-MS Quan;ta;ve Analysis Several species of pepper with varying intensity of heat on the Scoville Scale were oven dried to remove excess water, and the capsaicin was extracted using a vola+le organic solvent. ERETIC was used to analyze the 1 H NMR spectra intensity to determine the concentra+on of capsaicin in the samples. Tradi+onal uses for Nuclear Magne+c Resonance (NMR), par+cularly in undergraduate seNngs, is spectral and conforma+onal analysis of compounds. The quan+ta+ve NMR (qNMR) was employed through the use of ERETIC, a program built into the Bruker NMR soRware, to determine the capsaicin content of several peppers of the capsicum family. This soRware is used in conjunc+on with data collected from the Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of the samples to accurately determine concentra+on. Capsaicin Extrac;on The 1 H NMR spectra of the extracted samples were obtained, and a calibra+on curve was generated to determine the concentra+on of capsaicin in these samples. The capsaicin standards were analyzed via the LC-MS using the photodiode array (PDA) detector. y = 0.9936x + 0.1533 R² = 0.99915 0.0000 5.0000 10.0000 15.0000 20.0000 25.0000 30.0000 35.0000 40.0000 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 qNMR Measured Capsaicin Concentra;on (mM) Calculated Capsaicin Concentra;on (mM) Capsaicin Standard qNMR Calibra;on Curve qNMR Measured Capsaicin Sample Concentra;on (mM) Samples Run 1 Run 2 Run 3 Average SD Jalapeño 1/15 7.2014 6.6108 6.6147 6.8090 0.3399 Jalapeño 2/20 19.5666 18.8677 18.8169 19.0837 0.4189 Serrano 1/24 8.7746 8.8701 8.7793 8.8080 0.0538 Serrano 2/21 28.8685 31.2117 28.8362 29.6388 1.3623 Serrano 2/24 6.834 6.8461 9.6195 7.7665 1.6047 Habanero 9/18 25.788 24.1948 24.7285 24.9038 0.8109 Habanero (1) 1/16 27.7081 30.0199 30.5114 29.4131 1.4969 Habanero (2) 1/16 21.7927 24.0978 23.6707 23.1871 1.2263 Habanero (3) 1/16 15.1118 14.4291 14.5849 14.7086 0.3578 Habanero 2/20 20.1084 20.1244 20.2132 20.1487 0.0565 Habanero 2/21 21.0403 21.5236 21.6252 21.3964 0.3125 Habanero 2/24 23.5493 23.4652 23.502 23.5055 0.0422 Calibra;on Curve qNMR Measured Capsaicin Concentra;on (mM) Standard Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Average SD 1 33.9733 34.0307 34.0124 33.9797 33.9765 34.0184 33.9985 0.0249 2 15.0062 15.0423 15.0405 15.0171 15.027 15.0556 15.0315 0.0181 3 11.5520 11.5397 11.5733 11.4394 11.5091 11.5044 11.5197 0.0471 4 8.7888 8.7634 8.8360 8.7082 8.6951 8.7305 8.7537 0.0532 5 5.5581 5.5775 5.5237 5.5332 5.5357 5.5147 5.5405 0.0233 Extrac+on Methods Tried: • wet extrac+on – excessive amount of water in samples • freeze drying – instrumental issues • oven drying – ease of prepara+on Extrac+on Solvents Tried: • methanol – extracted both chlorophyll and capsaicin • methylene chloride (DCM) – extracted only capsaicin Final Extrac+on Methodology: • pepper samples cut thin and oven dried at 120°C for several hours • ~100mL DCM was used to extract the samples for 8 hours Percent Capsaicin by Mass Sample Capsaicin by Mass (%) 1 Jalapeño 1/15 0.061871 2 Jalapeño 2/20 0.13210 3 Serrano 1/24 0.062332 4 Serrano 2/21 0.15506 5 Serrano 2/24 0.032122 6 Habanero 9/18 0.061853 7 Habanero (1) 1/16 0.039390 8 Habanero (2) 1/16 0.039425 9 Habanero (3) 1/16 0.028407 10 Habanero 2/20 0.070974 11 Habanero 2/21 0.039250 12 Habanero 2/24 0.052205 Jalapeño Habanero Serrano y = 596.34x - 8532.5 R² = 0.99561 0 50000 100000 150000 200000 250000 300000 350000 400000 0 100 200 300 400 500 600 700 Peak Area (a.u.) Capsaicin Concentra;on (ppm) Capsaicin Standard LC-MS Calibra;on Curve Percent Capsaicin by Mass Sample Capsaicin by Mass (%) 1 Jalapeño 1/15 0.040553 2 Jalapeño 2/20 0.133526 3 Serrano 1/24 0.052882 4 Serrano 2/21 0.070096 5 Serrano 2/24 0.111511 6 Habanero 9/18 0.236487 7 Habanero (1) 1/16 0.365411 8 Habanero (2) 1/16 0.335635 9 Habanero (3) 1/16 0.238993 10 Habanero 2/20 0.208945 11 Habanero 2/21 0.190766 12 Habanero 2/24 0.133855 qNMR Measured Percent by Mass Data LC-MS Measured Percent by Mass Data 1 H NMR of Chlorophyll * overlaid on 1 H NMR of Capsaicin 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 ppm 0.964 1.336 1.354 1.373 1.391 1.411 1.588 1.607 1.627 1.645 1.664 1.966 1.983 1.996 2.015 2.196 2.214 2.232 3.833 4.259 5.355 5.368 6.725 6.863 5.97 1.98 2.00 2.13 3.08 2.99 1.99 2.01 2.03 1.00 1 2 LC-MS Measured Capsaicin Sample Peak Area and Concentra;on Sample Run 1 Run 2 Run 3 Average SD Concentra;on (ppm) Peak Area (a.u.) Jalapeño 1/15 52668 29044 36621 39444 12062 80 39444 Jalapeño 2/20 273894 270684 271582 272053 1656 471 272053 Serrano 1/24 122354 122936 132868 126053 5909 226 126053 Serrano 2/21 133566 174196 133030 146931 23614 106 54834 Serrano 2/24 163688 177481 176538 172569 7706 261 146931 Habanero 9/18 301320 353761 360032 338371 32240 582 338371 Habanero (1) 1/16 315096 266757 288098 289984 24225 501 289984 Habanero (2) 1/16 218306 216903 216465 217225 962 379 217225 Habanero (3) 1/16 174206 171975 166916 171032 3735 301 171032 Habanero 2/20 215573 203961 202260 207265 7245 362 207265 Habanero 2/21 322610 313673 311400 315894 5926 544 315894 Habanero 2/24 220286 203489 209125 210967 8549 368 210967 Calibra;on Curve LC-MS Capsaicin Peak Area (a.u.) Standard Run 1 Run 2 Run 3 Average SD 1 38158 33398 36494 36017 2416 2 79011 80992 78415 79473 1349 3 127813 123294 124433 125180 2350 4 167417 158571 159803 161930 4791 5 246297 253207 248018 249174 3597 6 363777 345153 340962 349964 12145 Capsaicin We would like to thank the University of West Florida Department of Chemistry for con+nuing support. References *Org. Magn. Resonance, 1984, 22, 779-783 y = 596.34x - 8532.5 R² = 0.99561 0 50000 100000 150000 200000 250000 300000 350000 400000 0 100 200 300 400 500 600 700 Peak Area (a.u.) Capsaicin Concentra;on (ppm) LC-MS Capsaicin Calibra;on Curve and Samples Calibra+on Curve Jalapeño 1/15 Jalapeño 2/20 Serrano 1/24 Serrano 2/21 Serrano 2/24 Habanero 9/18 Habanero (1) 1/16 Habanero (2) 1/16 Habanero (3) 1/16 Habanero 2/20 Habanero 2/21 Habanero 2/24 Linear (Calibra+on Curve) y = 0.9936x + 0.1533 R² = 0.99915 0.0000 5.0000 10.0000 15.0000 20.0000 25.0000 30.0000 35.0000 40.0000 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 qNMR Measured Capsaicin Concentra;on (mM) Calculated Capsaicin Concentra;on (mM) qNMR Capsaicin Calibra;on Curve and Samples Calibra+on Curve Jalapeño 1/15 Jalapeño 2/20 Serrano 1/24 Serrano 2/21 Serrano 2/24 Habanero 9/18 Habanero (1) 1/16 Habanero (2) 1/16 Habanero (3) 1/16 Habanero 2/20 Habanero 2/21 Habanero 2/24 Linear (Calibra+on Curve) 0.000000 0.020000 0.040000 0.060000 0.080000 0.100000 0.120000 0.140000 0.160000 0.180000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Percent Capsaicin by Mass Sample Number qNMR Percent Capsaicin by Mass Jalapeño 1/15 Jalapeño 2/20 Serrano 1/24 Serrano 2/21 Serrano 2/24 Habanero 9/18 Habanero (1) 1/16 Habanero (2) 1/16 Habanero (3) 1/16 Habanero 2/20 Habanero 2/21 Habanero 2/24 0.000000 0.050000 0.100000 0.150000 0.200000 0.250000 0.300000 0.350000 0.400000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Percent Capsaicin by Mass (%) Sample Number LC-MS Percent Capsaicin by Mass Jalapeño 1/15 Jalapeño 2/20 Serrano 1/24 Serrano 2/21 Serrano 2/24 Habanero 9/18 Habanero (1) 1/16 Habanero (2) 1/16 Habanero (3) 1/16 Habanero 2/20 Habanero 2/21 Habanero 2/24