Mikrochimica Acta [Wien] 1979 I, 81--86

9 by Springer-Verlag 1979

Department of Vegetable Crops, Punjab

Agricultural University, Ludhiana, India

Colorimetric Determination of Capsaicin in Capsicum

Fruits With the Folin-Ciocalteu Reagent
By
K. L. Bajaj and Gurdeep Kaur
(Received February 14, 1978)

Scvcral colorimctric methods 1-12 for determination of capsaicin,

the pungent principle in capsicum fruits, are based on various ex-
tractions and use of chromogenic reagents which involve the phenolic
group of capsaicin. Most of the colorimetric methods suffer from
instability of the colour developed and from turbidity formation 13.
The pigments and other interfering substances present in capsicum
fruits should be removed in all colorimetric methods 11, but in some
this is not done 1,4,s,lz and in others elaborate procedures are used
which make them inconvenient for routine analysis~, 3. Methods in-
volving diazonium salts as reagents require the preparation of these
in situ 5, v on account of their instability. Some of the methods which
involve the measurement of spot size or visual comparison of colour
are only semi-quantitative 7,1~ Recently a method using VOC13
has been reported 12 in which the colour formed was stable for only
1--1.5 min, which is rather a short for a colorimetric method. The
present communication describes a colorimetric method in which
intcrfering substances are removed on a basic alumina column and
capsaicin is determined by means of the Folin-Ciocalteu reagent
which forms a highly stable blue colour.

Experimental
Reagents
Folin-Ciocalteu reagent. To 700 ml of water add 100 g of sodium
tungstate (Na2WO4 "2H20), 25 g of sodium molybdate (Na2MoO4 92H20),
50 ml of 85% phosphoric acid and 100 ml of conc. hydrochloric acid.
6 M i k r o c h i m . Acta 1979 I/1~2

0026-3672/79/7901/0081/$ 01.20
82 K.L. Bajaj and G. Kaur:

Table I.

Total interfering
Extraction procedure (Reference) substances +
capsaicin,
%

Aqueous methanol, 50% . . . . . . . . . . . . . . . . (10) 1.76

Acetone (refluxing for 1 hr) . . . . . . . . . . . . . (13) 2.00
Acetone (refluxing for 1 hr)* . . . . . . . . . . . . (9) 1.02
Acetone (shaking for 10 min)** . . . . . . . . . . . (1, 4, 8) 0.68
Aqueous ethanol, 70% . . . . . . . . . . . . . . . . . . (5, 7) 1.23
Ethyl acetate** . . . . . . . . . . . . . . . . . . . . . . . . . (12) 0.70

* Further purification by Norit treatment (Ref. 9)

** Recommended procedures.

Reflux for 10 hr. Add 150 g of lithium sulphate (Li2SOa'H20), 50 ml of

water and 3 drops of bromine. Boil off the excess of bromine. Cool and
dilute to 1 litre with water. Filter and store in a brown bottle.
Saturated sodium carbonate solution. To 100 ml of water add 35 g of
anhydrous sodium carbonate, dissolve at 70--80 o C and allow to cool
overnight. Seed the supersaturated solution with crystals of Na2COa" 10H20
and after the crystallisation is complete, filter the mother liquor through
glass wool.
Standard capsaicin solution. Dissolve 10 mg in 100 ml of methanol.
Solvents. Dried and distilled before use.
Calibration curve. Take 0--1.5 ml (in appropriate steps) of standard
capsaicin solution in small beakers. Evaporate to less than 0.5 ml, if neces-
sary, at a temperature below 650 C (methanol interferes if more than
0.5 ml is present).
Add 0.5 ml of Folin-Ciocalteu reagent and 6.5 ml of distilled water,
let stand for 3 min, add 1.0 ml of saturated sodium carbonate solution and
mix well. Transfer quantitatively to 10-ml standard flasks, and dilute to
the mark with water. Measure the absorbance at 760 nm after 1 hr at
room temperature.

Method

E x t r a c t 0.5 g of dried, p o w d e r e d ( 3 0 - 4 0 mesh) c a p s i c u m fruits

with 25 ml of a c e t o n e or 25 ml of ethyl acetate. If a c e t o n e is used,
shake the m i x t u r e for 10 rain, let stand for 2 hr and then filter
t h r o u g h a g l a s s - w o o l plug in a s h o r t - s t e m m e d funnel. If ethyl acetate
 Colorimetric Determination of Capsaicin in Capsicum Fruits 83

Determination of Capsaicin

Capsaicin, %
Interfering
By present method By other (Re- substances,
methods ference) %
Min. Max. Average

Not applicable 0.55 (6) 1.21

0.63 0.67 0.65 0.50 (7) 1.50
0.46 0.52 0.50 0.51 (7) 0.52
0.51 0.53 0.52 0.51 (8) 0.16
Not applicable 0.55 (5) 0.78
0.52 0.54 0.53 0.51 (12) 0.17

is used, keep the extract at r o o m temperature for 24 hr, then filter

as for acetone extracts. Make up to 25 ml. Use 2 g of material for low
capsaicin content (less than 0.1%). Pass 2 ml of the extract through
a basic alumina column (Brockmann grade I, 80--200 mesh), 1.5 g
in a 10.0 • 0.9 cm column, washed with 5 ml of acetone. Wash the
column with three 5-ml portions of acetone after loading. Elute pure
capsaicin with methanol-acetone-water mixture (75 : 25 : 1) 13, col-
lecting 50 ml. Evaporate 10 ml (or other suitable volume) to dryness
(temperature ~<65 ~ C) and develop the colour as for the calibra-
tion curve.

Calculation

If the absorbance of 10 ml of eluate from the column corresponds to x

mg of capsaicin, then 0.5 g of capsicum fruit contains 62.5 x mg of cap-
saicin, i. e. 1.25 x %.

Results and Discussion

Capsaicin was extracted by various methods reported in the

literature, and its content was determined by the present and earlier
methods. The results are given in Table I (averages of six replicates).
The sum of capsaicin + interfering substances was determined as
capsaicin prior to column chromatography, and the pure capsaicin
content was determined after the chromatography. The data
show that m a x i m u m extraction of interfering substances was
6*
84 K.L. Bajaj and G. Kaur:

achieved by refluxing for 1 hr with acetone, and the minimum by

using shaking with acetone for 10 rain or extracting with ethyl
acetate. The results also show that some of the methods which do
not involve prior removal of interfering substances do not give the
true value of capsaicin content. The main disadvantage with the
VOCI3 method 12 seemed to be instability of the colour and suscept-
ibility towards moisture, making it difficult to get reproducible
results.
The results show that chromatography of the acetone or ethyl
acetate extracts on a basic alumina column gives correct and con-
sistent results. Extracts containing large quantities of interfering
substances are not suitable for this method, however, since they
give high results, probably because of elution of some other pig-
ments along with capsaicin. Correct capsaicin values were obtained
when these pigments were partially removed by treatment with
Norit 9. However, this method is not recommended since the amount
of Norit to be added for clarification of the extract has been found
to be very critical. The recovery of capsaicin from the alumina
column was found to be 100 _+0.6%, in close agreement with that
reported earlier for purification of capsaicin prior to its GLC deter-
ruination la. If 50% aqueous methanol or 70% aqueous ethanol is
used for extraction, the capsaicin cannot be purified in this way,
owing to inactivation of the alumina. It is concluded that only the
acetone or ethyl acetate extract can be purified by using a basic
alumina column 9,13. With phosphomolybdic acid or Folin-Denis
reagent, formation of a turbidity was the main problem, causing
irreproducible results. Reproducible results were obtained with the
Folin-Ciocalteu reagent, which gave a blue colour stable for 48 hr,
without turbidity formation. The method is quite sensitive, i. e.
0.5 #g/ml can be detected. The coefficient of variation found was
1.7%. Analysis of variance of the capsaicin content found by the
present method and other methods indicated no significant differ-
ence between the results.
It is recommended that capsaicin should be extracted from
capsicum fruits at room temperature by shaking with acetone for
10 min or standing with ethyl acetate for 24 hr, and purified on a
basic alumina column; the Folin-Ciocalteu reagent should be used
for colour development.

Acknowledgement
This work was done as part of the All India Coordinated Project
for Improvement of Vegetable Crops, financed by the Indian Council
of Agricultural Research, New Delhi, India.
 Colorimetric Determination of Capsaicin in Capsicum Fruits 85

Summary
Colorimetric Determination of Capsaicin in Capsicum Fruits With
the Folin-Ciocalteu Reagent

A colorimetric method for determination of capsaicin in capsicum

fruits is described. T h e acetone or ethyl acetate extract (prepared
at r o o m temperature) is purified on a basic alumina (Brockmann
Grade I) column and pure capsaicin is eluted with methanol-acetone-
water (75 : 25 : 1); the capsaicin content is determined after colour
development with Folin-Ciocalteu reagent, the absorbance being
measured at 760 nm. T h e m e t h o d is reproducible and results are in
good agreement with those obtained by existing methods.

Zusammenfassung
Eine kolorimetrische Methode zur Bestimmung yon Capsaicin in Paprika-
friichten wurde beschrieben. Der bei Zimmertemperatur gewonnene Ace-
ton- oder Essigesterextrakt wird in einer Sfiule aus basischem Aluminium-
oxid (Brockmann I) gereinigt und das reine Capsaicin durch Farbreaktion
mit Folin-Ciocalteu-Reagens und Messung bei 760 nm durchgefiihrt. Die
reproduzierbaren Ergebnisse stimmen mit denen anderer Methoden gut
iiberein.

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Correspondence and reprints: Dr. K. L. Bajaj and Mrs. Gurdeep Kaur,

Department of Vegetable Crops, Punjab Agricultural University, Lud-
hiana-141004, India.