1. 1.01.52.02.53.03.54.04.55.05.56.06.57.0 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
1H NMR Quan;ta;ve Analysis 1H 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 1H 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 1H 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
1H NMR of Chlorophyll* overlaid on 1H NMR of Capsaicin
1.01.52.02.53.03.54.04.55.05.56.06.57.0 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
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