Scoville Heat Values of Hot Sauces and Topical Ointments

via High Performance Liquid Chromatography.

Object

To use analytical HPLC and solid-phase extraction (SPE) to determine the

capsaicinoid content of hot sauces, peppers, or arthritis pain relief creams.

Analytical applications of high performance liquid chromatography

Field of application. Separation and quantitative determination of components in

mixtures of compounds. Organic and inorganic species, including ions, can be
separated. Method of choice for highly polar, thermal unstable, or low vapor
pressure compounds that are not amenable to gas-liquid chromatography.

State of sample. Solids or liquids soluble in appropriate solvent or solvent

mixture.

Qualitative use. With the use of standards and knowledge of sample source,
analyte identification can be made by retention times. With a scanning UV,
electrochemical, or mass spectroscopic detector analytes can be identified by
spectral interpretation.

Quantitative use. Through calibration with standards, detector response can be

converted to absolute concentrations. For relative amounts, the ratio of a single
analyte signal to total signal due to all analytes (relative peak areas) can be
used.

Sensitivity. Major and trace species can be determined. Sensitivity varies with
type of detector used and analyte assayed. Detection limits range from micro- to
subnano- grams.

Recovery. Technique is nondestructive. Entire sample may be recovered after

analysis. With larger capacity (preparatory scale) columns, technique can be
used to separate components of a mixture on a preparative scale.

Introduction

Capsaicinoids are the group of compounds that give chili peppers their
characteristic, pungent taste. More novel uses of capsaicinoids include herpes
treatment, tear gas (pepper spray), squirrel-resistant bird seed (birds do not have
capsaicin receptors, but squirrels do), and as over-the-counter arthritis creams.
The compounds responsible for the pungent taste of the red pepper, Capsicum
annuum, are listed below. Capsaicin is N-[(4-hydroxy-3-methoxyphenyl)methyl]-
8-methyl-6-nonenamide.
2 HPLC

Walter Scoville first quantified the pungency of foods in 1912 using an

organoleptic method. In this test, a panel of subjects tasted serial dilutions of a
sample until it was no longer detected. The resulting dilution factor of the sample
was called the Scoville heat value (SHV). In humans, the lower limit of detection
for capsaicin is 10 ppm, roughly 70 times lower than that of piperine, the active
spice in black pepper, and 1000 times lower than that of zingerone, the active
component in ginger.

Capsaicinoid Threshold Pungency /

Scoville Heat Units
capsaicin 16.1x106
dihydrocapsaicin 16.1x106
nordihydrocapsaicin 9.3x106
homocapsaicin 6.9x106
homodihydrocapsaicin 9.2x106

High performance liquid chromatography (HPLC) is a technique similar to

gas chromatography (GC)
but offers advantages not
found in GC. In HPLC,
the analytes do not have
to be separated as gases
as they do in GC but
rather as liquids. This
means HPLC can be used
to separate and determine
analytes such as salts or
other low vapor pressure
compounds that in GC
would be impossible to
vaporize without
decomposing. In HPLC a
range of analyte
solute/liquid mobile phase
interactions is available
since, unlike in GC, the
mobile phase can have a
specific chemical or
solubility interaction with
the analyte solute. For
this experiment HPLC
provides a convenient
method for analyzing the
capsaicinoid content of various samples.
Chemistry 112, Spring 2014
HPLC 3

In HPLC an organic solvent or aqueous solution is used as the eluting

solvent (also called the eluant or mobile phase). A non-pulsing solvent pump
must be used since liquids are incompressible. In GC, gas pressures of about 2
atm (30 lb/in2) are sufficient for useful flow rates. In HPLC, pressures up to 350
bar (5200 lb/in2, 350 atm) are needed to force the mobile phase solvent through
the tightly packed columns at useful flow rates. The original meaning of the
acronym HPLC was high pressure liquid chromatography to distinguish it from
the older, gravity-fed, low-pressure column chromatography experiment. The
development of low-pulse, high-pressure pumps and fittings provided the
stimulus for a virtual revolution in chromatography since the middle of the 1970's.
Today HPLC is known as high performance liquid chromatography.

Components of an HPLC instrument

Injectors. Considering the pressures mentioned above, a rubber septum such as

used in a GC would fail. The HPLC injector system consists of a loop into which
the sample is injected under low pressure. By suitable valves, the injected
volume is swept out of the sample loop by the high pressure liquid mobile phase
and carried into the column as "plug" of sample volume.

Columns. Both HPLC and GC columns are similar, although HPLC columns are
packed more densely and are difficult to manufacture whereas packed GC
columns are easily homemade. Both have stationary liquid phases coated or
bonded onto a solid support that should be immiscible with the mobile phase.
HPLC columns are not usually in thermostated ovens for temperature
programming. Temperature programming in HPLC is not common because of
the difficulty of heating or cooling liquids rapidly plus the fact that liquid-liquid
distribution coefficients are not highly temperature dependent. Columns might
be thermostated isothermally however to reduce any chance temperature effects
on the separation of interest. The lack of temperature control to affect
separations is more than adequately compensated by the range of distribution
coefficients available using a variety of stationary phases. In this experiment,
you will use a C18 reversed-phase column. The stationary phase is
octadecylsilane (C18SiH2) bonded to a 5 m spherical silica gel support. The C18
column is a non-polar column, and, in a polar mobile phase, polar analytes will
tend to elute faster than non-polar substances. The descriptor of reversed
phase originates from the fact that most columns are made with polar stationary
phases which means polar materials elute after the non-polar. Here in HPLC as
in GC, the basis the separation is based on the magnitude of the distribution
coefficient describing the partitioning of the analyte between the mobile and
stationary phase.

Detectors. Once the column has provided a separation, the analyte still must be
detected. The three types of detectors most often used are the index of refraction
(RI) detector, the UV/VIS detector, and the fluorescence detector. The first is
non-specific in that the method is based on the observation that the refractive
Chemistry 112, Spring 2014
4 HPLC

index of the mobile phase is changed when an analyte is dissolved in it. The RI
detector is as general as the thermal conductivity detector is in GC. A UV/VIS
detector can be a simple single-beam, single wavelength photometer or a
sophisticated spectrophotometer. In fact, practically any other instrument can be
connected in-line with the HPLC and used as a detector. A difficulty is, however,
that the mobile phase usually has to be removed. Modern, hyphenated (tandem)
techniques include LC-MS and LC-NMR.

The HP 1090 can monitor several wavelengths simultaneously, and

obtaining complete spectra every 16 ms. The presence of the rapid scanning
detector allows the purity of the eluting peak to be estimated. Since a spectrum
of the eluting analyte can be recorded in msec, a spectrum can be obtained on
the leading edge of the peak as well as the tail of the peak. If the peak
represents a pure analyte, the two spectra should be identical. Any differences
noted provide the basis for a purity estimation.

Microprocessor control. Modern HPLC instruments control the injector and the
solvent pump with a computer. Software is used to program the computer. If the
mobile phase has a constant composition, it is called isocratic or of constant
composition. Most HPLC instruments allow gradients in the mobile phase to be
generated as functions of time so that the elution can be made with any solvent
combination desired (at least up to mixtures of three solvents). The HP 1090 can
program changes, that is, gradients, in the composition of the mobile phase
during elution. Gradient elution offers to HPLC analyses the advantages that
temperature programming offers to GC analyses.

Methods

You will need to modify methods and sequences to conduct the

experiments outlined here. The method to begin with is one called scoville.M. All
methods have file names ending in .M. All sequences have file names ending
in .S. A method is a set of instructions for one sample. A sequence is a method
or set of methods automatically applied to several samples sequentially.
Sequences are possible with the HP 1090 because of its auto injector system.

Every method consists of four sections:

Method information
Instrument control
Data analysis
Run time check list

Method information. This section is used to provide descriptive information about

the method or the samples to be analyzed by the method.

Instrument control. Here the parameters of the instrument, mobile phase

composition, flow rate, injection volume, detector wavelength, and run time are
set.
Chemistry 112, Spring 2014
HPLC 5

Data analysis. Here the parameters to control data processing are set in the
subsections: Signal Details; Integration Events; Peak Identification; Peak
Quantification; Calibration; and Report.

Run time check list. The check list provides which and in which order the
instructions of the method will execute. The check list can: acquire, store and
process data for a report; execute only a portion of the method; acquire data
without analysis; reanalyze existing data; invoke user written macros for data
analysis.

These four sections are edited by specific commands or pull-down dialog

boxes.

The condensed instructions given below will take you through the software
procedures necessary to start collecting data. The procedures necessary to
construct calibration tables and curves or determine peak purities are outlined
below also but details will found in the printed How to and Help files you will
find in the binder by the instrument.

Apparatus/chemicals

The HP 1090 will be found on a cart in Keck 2334. Glassware for solution
making will be available by the instrument. Syringe filters for use in filling the
sampling vials will be available. Sampling vials are heavy walled glass
containers about 2 dram in size and capable of being capped with a metal/rubber
serum cap. Vials and the special capping tool will be located with the other
glassware.

Experiment

To establish a suitable analysis method you will need to consider the

following parameters:

Column choice (actually you will not have choice here but, in practice,
yes.)
Column temperature
Flow rate
Injection amount
Detection wavelength: fixed or variable with analyte peak
Solvent composition: isocratic or gradient elution
Maximum and minimum pressure
Stop and post-analysis times

Locate a printed copy of the method analges.M appended to this experiment and
discuss it with your instructor.
Chemistry 112, Spring 2014
6 HPLC

Your development work should, of course, involve authentic samples. Working

with known mixtures is efficient.

Please remember to pass each solution through a 0.2 micron syringe filter
when filling the sample vial with that solution.

WARNING: Capsaicinoids are severe irritants, especially to mucus membranes

and the upper respiratory tract. In high concentration they irritate the skin. Wear
gloves when working with the powder and solutions. Do not breath the dust.

All methods and data files should be stored in the c112 directory.

Method Optimization: A standard containing a mixture of capsaicin and

dihydrocapsaicin in methanol is available. Starting with the method start.M,
optimize the separation of the two components in the standard and determine its
composition, based upon the assumption that the molar absorptivities of the two
componds are identical. (This is a fair assumption, given the similar nature of the
species and the extreme expense associated with obtaining them in pure form.)
In general, if two species co-elute, you will want to increase the water content of
the mobile phase slightly to force the less hydrophilic component to partition more
strongly into the stationary phase, thus increasing its retention time. Do not
exceed 250 bar in pressure. In practice, do not exceed flow rates of 0.5 mL/min
with the microcolumn. When you have optimized your separation, write the
method to a new file, using your initials in the file name.

Sample Preparation: Condition a Supelco solid phase extraction (SPE) cartridge

with 5 mL of methanol, then 5 mL of water. You may speed this process by placing
a syringe needle on the end of the SPE cartridge, then sticking the needle through
a septum mounted on a small side-arm Erlenmyer flask attached to an aspirator.
Set the cartridge aside.

Wear latex gloves for handling all samples! Add 2-5 g of accurately-
weighed hot sauce, 2 g of arthritis cream, or 1 g of dehydrated, crushed pepper to
exactly 10 mL of methanol and mix thoroughly for two min. Allow the sample to
settle, then remove exactly 1mL of the methanol and add to approximately 9 mL of
water.

Clean up the sample by performing an extraction: Slowly pass your

solution through the SPE cartridge. Discard the aqueous eluant in the non-
halogenated waste solvent container. Obtain a small, clean and dry side-arm
Erlenmeyer flask in which to collect the capsaicinoids. The capsaicinoids are
eluted from the SPE with exactly 5 mL of methanol. Filter the methanol solution
through a 0.2 m syringe filter into an HPLC sample vial. Cap the vial. Do not
discard the SPE cartridge! Condition the cartridge with 5 mL of methanol, then 5
mL of water.

Chemistry 112, Spring 2014

HPLC 7

Devise a series of experiments to determine the capsaicinoid content of

your sample. You will have available a standard solution of capsaicin in methanol.

Disposal and cleanup

Aqueous solutions may be poured down the sink with water rinse.
Organic solutions should be disposed of in the appropriate waste container.
Glassware can be washed with soap and water and air dried. Capped vials
should be uncapped, solutions disposed of, and empty vials discarded in a waste
glass bin. Used metal-rubber septum caps can go in normal trash cans. SPE
cartridges should be returned to the counter.

Data treatment/calculations

Report the composition of the capsaicin/dihydrocapsaicin sample. Report

calibration data and your response factors for your sample, making certain to
include all dilutions in sampling. Show your calibration curve. Provide a uv-vis
spectrum of capsaicin and dihydrocapsaicin (from your diode array detector).
Speculate as to whether the capsaicin in your sample is of natural or synthetic
origin.

References

Hewlett-Packard Part No. G2070-90104 Understanding Your Chemstation, 3rd Ed

(11/95); Chapters 2 and 8.

Hewlett-Packard Installation and Maintenance Instructions, Waldrom Analytical

Division, Waldrom, FDR, 1984

Rubinson, K.A.; Rubinson, J.F. Contemporary Instrumental Analysis, Prentice-

Hall: Upper Saddle River, 2000, Chapter 14, especially Sections 14.3, 14.7, 14.9-
10.

Skoog, D.A.; Holler, F.J.; Nieman, T.A. Principles of Instrumental Analysis, 5th Ed.;
Harcourt Brace & Company: Philadelphia, 1998; Chapter 28.

Betts, Thomas A. Pungency Quantitation of Hot Pepper Sauces Using HPLC J.

Chem. Educ., 1999, 76, 240-244.

Batchelor, James D.; Jones, Bradley T. Determination of the Scoville Heat Value
for Hot Sauces and Chilies: An HPLC Experiment. J. Chem. Educ., 2000, 77,
266-267.

Huang, Jiping; Mabury, Scott A.; Sagebiel, John C. Hot Chili Peppers: Extraction,
Cleanup, and Measurement. J. Chem. Educ., 2000, 77, 1630-1631.

Chemistry 112, Spring 2014

8 HPLC

Addenda

The next several pages are either brief instructions for using methods or copies of
various outputs from the Chemstation software. In order of appearance:

Appendix A HP 1090 Condensed Operation Instructions.

A partial printout of the method analges.M.

Chemistry 112, Spring 2014

HPLC 9

Appendix A HP 1090 Condensed Operating Instructions

Note: These instructions assume that you are running a preprogrammed METHOD from
disk.

Startup

Turn on the helium cylinder to 50 psi. Turn on the nitrogen cylinder to 65 psi. Open
the valve on the back-left of the HP-1090 to purge the solutions with He; after
purging for 5 min close the valve on the back of the HP-1090.

Turn on the main power to the HP-1090. The switch is underneath the helium purge
valve on the back left of the instrument. Turn on the front panel power by pressing
the ON button located at the bottom right front of the instrument.

Turn on the HP-5 laser printer. The switch in on the lower right front. Turn on the HP-
Vectra computer. The switch is on the center front. Turn on the monitor. The
switch is on the lower center front.

After Windows 95 starts, double-click on the HP-1090 HPLC icon to launch the
acquisition and analysis software.

To run a single sample:

- From the VIEW menu, select METHODS AND RUN CONTROL.
- From the METHODS menu, select LOAD METHOD. Find the method
scoville.M, in the directory c:\hpchem\1\methods\c112
- From the INSTRUMENT menu, select SYSTEM ON
- Place your sample vial in the swing arm (vial #100).
- Double-click on the single sample vial icon. Check to see that the vial# is set to
100. Assign a file name for the data to be collected. The can be any MS-
DOS 8 character name but with the ext .D Click ENTER.
- Click on START.

To see your chromatogram and the spectra of the eluted peaks

- Select DATA ANALYSIS from the VIEW menu.
- Your previously named data file (= signal file) should be in the menu box. If you
wish to view a different signal file, use LOAD SIGNAL under the FILE
menu. A chromatogram should appear at this point.
- Select SELECT SPECTRUM from SPECTRA menu.
- Click on a peak to see the spectrum of that peak. What you should see is the
chromatogram, a panel with just the apex spectrum, and a panel with the
three sampled spectra which if the separation is good should be close to
identical.
- To print this screen of windows, go to FILE, select PRINT, select ALL
WINDOWS and click ENTER.
- To view a 3D plot of absorbance versus wavelength and retention time, select
3D PLOT from the SPECTRA menu. This can be printed.
- To print a calibration table with the calibration curves for each analyte, go to
FILE, select PRINT, select CALIB TABLE AND CURVES.

Chemistry 112, Spring 2014

10 HPLC

- To run another single sample, return to the METHOD & RUN CONTROL menu
under VIEW, and click on START.

To prepare a level 2 calibration table:

- Prepare first a level 1 calibration table by obtaining a chromatogram of a known
mixture of analytes. Go to the DATA ANALYSIS menu and select LOAD
SIGNAL. Then select CALIBRATION and NEW CALIBRATION in
succession. Fill in the table entries.
- Prepare a second sample with know concentrations of analytes whose
concentrations are likely, with the first sample, to span the expected
concentration range of the analyte. Obtain the chromatogram for this
second sample.
- Go to DATA ANALYSIS, LOAD SIGNAL, select CALIBRATION, but now select
ADD LEVEL. Fill in the menu which appears, selecting 1 as the level to
add. Type OK.
- Fill in the augumented calibration table with the amounts appropriate to the new
sample. Select OK and then print out the new CALIB TABLE AND
CURVES. The calibration curves should now should 2 data points and a
linear line joining them. This is a level 2 calibration curve. To go to a level
3 calibration, repeat the steps above.

To obtain a purity analysis:

- Go to the DATA ANALYSIS menu. If the desired signal (chromatogram) is not
already loaded, LOAD SIGNAL. Go to SPECTRA menu and select PEAK
PURITY. Accept the message that peak controlled spectra are not good
for purity assessments by selecting yes and continuing.

To run multiple samples:

- From the VIEW menu, select METHODS AND RUN CONTROL
- From the SEQUENCE menu, select LOAD SEQUENCE. Find the sequence
AFC.S in the directory c:\hpchem\1\sequence. (The sequence program will
probably need revision.)
- Load your vials into the sample tray. Vial #0 is at the lower right corner of the
tray; vials number 1 through 9 continue away from you. Vial #10 is at the
front of the second column from the right.
- From the SEQUENCE menu, select SEQUENCE PARAMETERS. Fill in the
operator name. Make certain the subdirectory is set to C112. Click OK.
- From the SEQUENCE menu, select SEQUENCE TABLE. Click on INSERT
VIAL RANGE. Fill in the entries for FROM VIAL and TO VIAL. Select 1
injection/vial.
- Click on RUN SEQUENCE.

Shut-down

From the INSTRUMENT menu, select SYSTEM OFF. From the Windows 95 START
button, select SHUT DOWN, then SHUT DOWN THE COMPTUER. Turn off the
main power on the back pannel of the HPLC. Turn off the gas cylinders.

Turn off the computer, monitor, and laser printer.

Chemistry 112, Spring 2014

HPLC 11

Method Analges.M

Below is a partial printout of analges.M, a method for the separation of analgesics in water/methanol
mixtures on a 100 x 2.1 mm Hypersil ODS 5 microM column with single wavelength monitoring at 225 nm;
spectra acquired from 200-400 nm at 2 nm resolution. Method first written by H. Van Ryswyk, 31 Dec 96,
modified 31 Mar 200 by G.R. Van Hecke
LC 1090
Pump (DR5):
Stop time 4.00 min
Post time 3.00 min
Flow 0.500 mL/min
Store Flow time Yes
Min. Pressure 0 bar
Max. Pressure 350 bar
Store Pressure Curve Yes
Oven Temperature Off
Store Temperature Curve Yes
Solvent A 78.0% (bottle A) water
Store Solvent A Curve Yes
Solvent B 22.0% (bottle B) methanol
Store Solvent B Curve Yes
Solvent C Off (bottle C) 20/80 water/methanol
Store Solvent C Curve Yes
Injector:
Injection Volume 4.0 L
Draw Speed 83.8 L/min
Contacts:
Contact 1 Off
Contact 2 Off
Contact 3 Off
Contact 4 Off
Time Table: empty

DIODE ARRAY DETECTOR

Settings:
Stop Time as Pump: 4.00 min
Post Time Off
Peakwidth 0.100 min
Sampling Interval 0.640 sec
Autobalance On
Signals:
Sample, Bw Reference, Bw [nm]
A: 225 80 550 80
B: Off
C: Off
D: Off
E: Off
F: Off
G: Off
H: Off
B: Off
Spectrum:
Store Peak controlled
From 200 nm
To 400 nm
Step 2 nm
Treshold 1.0 Mau

Chemistry 112, Spring 2014