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.