INTRODUCTION
Capsaicin, the pungent ingredient of the red pepper or Capsicum annum has been widely used in food preparation. Capsicum fruits generally contain between 0.1 and 1.0 % of capsaicin. Observed toxic responses to excess capsaicin are nerve damage, gastric ulcers, and liver cirrhosis while the LD50 for Tabasco sauce is reportedly 24 mls/kg body weight.
More novel uses of capsaicin have been its utilization as a herpes treatment, a tear gas or rodent repellent, and a deterrent to dog fights and swine cannibalism. In humans, the limit for taste is approximately 10 ppm which is 70 times lower than that for piperine, the active spice in black pepper, and 1000 times lower than for zingerone, the active ingredient in ginger.
Although puzzling, the popularity of this "masochistic gustatory phenomenon" seems to be rising. In 1992, 165.5 million pounds of red chili peppers were purchased for or processed into food products in the United States. Capsaicin targets pain receptors in the mouth. The desired response of "hotness" appears to originate from the physiological placement of the pain receptors in the same location as the receptors for high temperatures; thus one feels hot when consuming this incendiary substance. Contrarily, some people appear to tolerate or "enjoy" larger amounts of hot peppers than others. Apparently capsaicin causes a desensitization of the nerve fibers containing the pain and heat receptors upon repeated ingestion.
The structure of capsaicin is shown below. The purpose of this
lab is to acquaint you with the active ingredient of hot peppers,
and the extraction, clean-up, and analysis of capsaicin as a fun
analytical exercise. Please bring a hot pepper sample to class
and the group with the "hottest" pepper as determined
by the quantitation of capsaicin will win a free lunch of the
hottest Mexican food we can find in Toronto (Las
Iguanas along Bloor).
PROCEDURE
NOTE: The extract is very noxious and will cause contact burns on exposed skin; all extraction steps should be carried out in a hood.
Extraction. From the available equipment in the laboratory (see the list below) determine the method by which you will most efficiently and completely extract all the capsaicin from the pepper material which you brought to class. Your sample must be a fresh pepper and you are to weigh exactly 10 g of it of the whole pepper for extraction ~ for competition purposes there is to be no selective isolation of parts of the pepper; it is suggested you finely "chop" your pepper prior to extraction. Divert a small proportion of the chopped sample for dry weight determination (see the TA). The extraction solvent to be used is acetonitrile. Vacuum filtration flasks with Buchner funnels are to be used to remove insoluble plant parts. The volume of your extract must be 25 ml (Volume must be measured exactly, if necessary rinse with 5 ml more ACN). Be aware of the time required for the method you choose. Transfer 1 ml of extract to a 10 ml volumetric flask and dilute to the 10 ml mark with deionized water. This diluted sample must next be cleaned up for analysis.
Extraction Equipment:
Mortar and Pestle
*Tissue Grinder (Blender)
Soxhlet Extractor (Requires at least 1-2 hrs of extraction. Followed by time for solvent reduction.)
*Sonicator Bath
Separation Flasks (Liquid-Liquid extraction)
*This equipment must be shared amongst all the groups in the lab.
Cleanup.. Condition a C-18 solid-phase extraction
cartridge with 2 column volumes of methanol, then 2 column volumes
of acetonitrile, and then 2 column volumes of deionized water.
Centrifuge out all of the water, then add your 10 mls of diluted
extract to the column. Flow through the cartridge should be
less than 5 mls per minute. Be careful not to let the cartridge
go dry during the conditioning or extract addition. Elute your
capsaicinoids from the solid-phase extraction cartridge with 4
mls of methanol followed by 1 ml of methanol containing 1% acetic
acid. Please check with the TA whether your sample needs dilution
at this point.
Analysis.. Analysis of your extract will be by reverse-phase HPLC with UV detection at 281 nm (optimizes for capsaicin) or with a diode-array detector, C-18 stationary phase, and either acetonitrile or methanol:water mobil phase. You will quantitate by calibrating the HPLC with standard solutions of capsaicin. See the TA for standard concentrations and injection volumes. Given we do not know the level of capsaicin in your sample prior to analysis you will may need to adapt your sample dilution or injection volumes. Good Luck.
LAB REPORT
In your Results & Discussion section please comment
on the extraction, clean-up, and analysis techniques you utilized
in this lab. Particular attention should be paid to the use of
solid-phase extraction cartridges. For example, explain clearly
why the particular stationary and mobil phases were chosen. Be
sure to clearly identify the source and type of your hot pepper.
Results (for purposes of determining who wins lunch)
On a separate piece of paper, determine the amount of capsaicin
in your sample on a ppb or ppm basis. Also from the dry weight
numbers for your pepper please calculate the Scoville Heat Unit
value from the "heat" coefficients found in Thomas et
al. Show all your calculations. Be sure to be descriptive as
you make these determinations.
REFERENCES
Leary, W. 1989. Peppers Helpful in Studies of Herpes. The New York Times, December 27, 1989.pp. B-7.
Monsereeunusorn, Y., S. Kongsamut, and P. Pezalla. 1982. Capsaicin, A Literature Survey. CRC Critical Reviews in Toxicology, 10:321-339.
Szolcsanyi, J. and A. Jancso-Gabor. 1975. Sensory Effects of Capsaicin Congeners. Arzneim.-Forsch. (Drug Res.), 25:1877-1881.
Willoughby, J. 1994. The Tip of Your Tongue Knows the Bitter Truth: Flavor Can Be Painful. The New York Times, April 27, 1994, pp B-1.
Attuquayefio, V.K., and K.A. Buckle. 1987. Rapid Sample Preparation Method for HPLC Analysis of Capsaicinoids in Capsicum Fruits and Oleoresins. J. Agric. Food Chem. 35:777-779.
Thomas, B.V., A.A. Schreiber, and C.P. Weisskopf. 1998.
Simple Method for Quantitation of Capsaicinoids in Peppers Using
Capillary Gas Chromatography. J. Agric. Food Chem. 46:2655-2663.
Related Problem Example:
A former colleage of mine (Dr. Linda Aston) dissertation work
involved analyzing pine needle extracts for organophosphate pesticides,
one of which is diazinon. In her method, she starts with about
100 g of fresh pine needles which she cuts up, then grinds up
in 150 ml of hexane. The ground needles are filtered to remove
the insoluble plant parts. The hexane extract is reduced by rotary
evaporation to 5 ml. Of the 5 mls of extract, 1 ml is measured
out and cleaned up by using a florisil column. This is done
by conditioning the florisil with 30 ml of hexane, (which is discarded),
adding the 1 ml of sample to the column, and eluting the column
with 40 ml of 15%diethylether:hexane. As the eluting solvent
flows through the column it is collected in a round bottom flask.
This extract, now containing the diazinon, is reduced by rotary
evaporation to near dryness, it is transferred quantitatively
to a graduated centrifuge tube, and the volume is adjusted to
2 ml. In this example, analysis of the final extract by GC (as
measured against an external standard curve,) yields 1.5 ng/µl
diazinon for a 3 µl injection. The concentration of diazinon
in the pine needles (assuming 100% recovery for the method) is
211 ppb fresh weight.
| Sample calculation for diazinon in pine needle: |
|
| Original Sample Amount: | 95 g |
| First extract volume: | 5.2 ml |
| Amount of first extract used: | 0.8 ml |
| Second extract volume: | 2 ml |
Conc. of injection 1.5 ng/µl = 1.5 µg/ml (= concentration
of final extract)
| Quantity of diazinon in second extract = | 1.5 µg/ml | * final volume of extract from which the injection was made |
| 1.5µg/ml * 2 ml = 3 µg | ||
0.8 ml ÷ 5.2 ml = 0.15 of the original 95 g needle sample.
Therefore, the second extract represented the amount of material
extracted from 14.25 g of fresh needles.
Concentration of diazinon in pine needles:
3 µg (diazinon) ÷ 14.25 g (needles fresh weight) =
0.211 µg/g = 211 ng/g = 211 ppb
Note the importance of getting accurate measurements for the
mass of material used and for each volume of extract!!