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IS 16039 : 2012
ISO 4052 : 1983
Hkkjrh; ekud
dkWiQh oSQiQhu dh ek=kk Kkr djuk (lanHkZ ifr)
Indian Standard
COFFEE DETERMINATION OF CAFFEINE
CONTENT (REFERENCE METHOD)
ICS 67.140.10
BIS 2012
BUREAU OF INDIAN STANDARDS
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002
NATIONAL FOREWORD
This Indian Standard which is identical with ISO 4052 : 1983 Coffee Determination of caffeine
content (Reference method) issued by the International Organization for Standardization (ISO) was
adopted by the Bureau of Indian Standards on the recommendation of the Stimulant Foods Sectional
Committee and approval of the Food and Agriculture Division Council.
The text of ISO Standard has been approved as suitable for publication as an Indian Standard without
deviations. Certain conventions are, however, not identical to those used in Indian Standards. Attention
is particularly drawn to the following:
a) Wherever the words International Standard appear referring to this standard, they should be
read as Indian Standard.
b) Comma (,) has been used as a decimal marker while in Indian Standards, the current practice
is to use a point (.) as the decimal marker.
In this adopted standard, reference appears to certain International Standards for which Indian
Standards also exist. The corresponding Indian Standards which are to be substituted in their respective
places are listed below along with their degree of equivalence for the editions indicated:
International Standard Corresponding Indian Standard Degree of Equivalence
ISO 1447 Green coffee IS 3581 : 1982 Green coffee Technically Equivalent
Determination of moisture content Specification
(Routine method)
ISO 3726 : 1983 Instant coffee IS 16092 : 2012 Instant coffee Identical
Determination of loss in mass at Determination of loss in mass at
70C under reduced pressure 70C under reduced pressure
ISO 4072 : 1982 Green coffee in bags IS 10814 : 1984 Method of sampling
Sampling green coffee in bags Technically Equivalent
ISO 6670 : 2002 Instant Coffee IS 16035 : 2012 Instant Coffee
Sampling method for bulk units with Sampling method for bulk units with Identical
liners liners
ISO 6673 : 2003 Green coffee IS 16034 : 2012 Green coffee
Determination of loss in mass at Determination of loss in mass at do
105C 105C
In reporting the result of a test or analysis made in accordance with this standard, if the final value,
observed or calculated is to be rounded off, it shall be done in accordance with IS 2 : 1960 Rules for
rounding off numerical values (revised).
IS 16039 : 2012
ISO 4052 : 1983
INTERNATIONAL STANDARD ISO 4052-1983 (E)
Indian Standard
COFFEE DETERMINATION OF CAFFEINE
CONTENT (REFERENCE METHOD)
Coffee - Determination of caffeine content
(Reference method)
2 References
4.4 Ammonia, 70 g/l Solution (1 volume of concentrated
ISO 1447, Green coffee - Determination of moisture content ammonia Solution, e20 = 0,9 g/ml, + 2 volumes of water).
(Rau tine methodl.
4.5 Diethyl ether, pure, or repurified (sec 7.5) by
ISO 3726, lnstant coffee - Determination of Ioss in mass at chromatography as follows, and saturated with water.
70 OC under reduced pressure.
Pass 800 ml of diethyl ether through a column containing 100 g
ISO 4072, Green coffee in bags - Sampling. of basic aluminium Oxide of activity grade 1. The diethyl ether,
thus repurified, shall be kept in dark bottles until used.
ISO 6670, lnstant coffee in cases with liners - Sampling. 1)
(Alternatively, diethyl ether, recently distilled and free of perox-
ISO 6673, Green coffee - Determination of Ioss in mass at ides, tan be used instead of diethyl ether repurified by
105 OC.) chromatography.)
1
IS 16039 : 2012
ISO 4052-1983 (EI
ISO 4052 : 1983
5.1 Chromatographie columns (see figure 1), 250 mm Calculate the dry matter content after determining the moisture
long, 21 mm in internal diameter (column 1) and 17 mm in inter- content on part of the test Sample (7.1) in accordance with the
nal diameter (column II), with stopcocks preferably of PTFE. method specified in the relevant International Standard?)
5.2 Ultraviolet sp ectrometer, accurate to within 0,004 ab- 7.3 Test Portion
sorbance unit within the range used.
7.3.1 Green coffee and roasted coffee
5.4.4 One-mark pipettes, of capacities 2 and 5 ml. Proceed in accordance with 7.3.1, but using a test Portion of
0,5 g, and an aliquot Portion of the turbid Solution of 2 ml, and
3 g of the diatomaceous earth.
5.4.5 Analytical balance.
Sample in accordance with the method specif ied in the relevant 7.3.5 Dried decaffeinated coffee extract
International Standard.1)
Proceed in accordance with 7.3.4, but using a test Portion of
015 g*
7 Procedure
7.3.6 Liquid decaffeinated coffee extract
7.1 Preparation of test Sample
Proceed in accordance with 7.3.4, but using a test Portion
If necessary, mill the Sample, using the apparatus specified in between 1 and 2,5 g corresponding to approximately 0,5 g of
5.5 or 5.6, as appropriate, until it Passes the sieve (5.7). coffee solids, and 7 to 8 g of the diatomaceous earth.
1) For the sampling of green coffee in bags, see ISO 4072; for the sampling of instant coffee in cases with liners, see ISO 6670. Methods for other
types of coffee and coffee products have not Yet been elaborated.
2) For the determination of the moisture content of green coffee, see ISO 1447, and, for the loss in mass at 105 OC of green coffee, see ISO 6673;
for the determination of the loss in mass at 70 OC under reduced pressure of instant coffee, see ISO 3726. Methods for other types of coffee and
coffee products have not yet been elaborated.
2
2
IS 16039 : 2012
ISO 40524983 (E)
ISO 4052 : 1983
7.4 Determination stopcock carries only a faint smell of diethyl ether (see the
warning below). Elute column II with 45 to 50 ml of chloroform
(4.7). Collect the eluate in a 50 ml one-mark volumetric flask
7.4.1 Filling of columns
I5.4.3), dilute to the mark with the chloroform (4.7) and mix
carefully.
7.4.1.1 Column I (alkaline l
column)
The flow rate of the diethyl ether and the chloroform under
conditions of natura1 flow should be between 1,5 and
7.4.1.1.1 Layer A
3 ml/min. If this rate is exceeded, channelling should be
suspected and the determination recommenced.
Mix carefully, by kneading with a flexible spatula blade, 3 g of
the diatomaceous earth (4.3) and 2 ml of the sodium hydroxide WARNING - The additions of diethyl ether and chloro-
Solution (4.2), until homogeneous (sec the note). A slightly wet form should be carried out in a weil-ventilated fume-
powder is obtained. Transfer this powder, in portions of cupboard to prevent both the possibility of inhalation of
approximately 2 g, into the 21 mm diameter chromatographic solvent vapours and the possibility of an explosion.
column (5.1), the lower part of which is packed with a wad of
cotton wool or glass wool. Tamp down the mixture after each
7.4.3 Spectrometric measurement (sec figure 2)
addition, without excessive forte, using a glass rod one end of
which is flattened to the diameter of the column, until a per-
fectly homogeneous and compact layer is obtained. A small 7.4.3.1 Measurement of the test Solution
wad of cotton wool or glass wool may be placed on the top of
Avoiding error from evaporation of chloroform, measure the
layer A.
absorbance of the Solution of caffeine in chloroform (7.4.2), us-
NOTE - Column packing material may be prepared in bulk in advance ing the silica cells (5.3), against chloroform (4.7) at the
and stored in closed Containers. A mass of 5,16 g is required for each wavelength of maximum absorbance obtained on the spec-
alkaline column. trometer used (about 276 nm), and at wavelengths 30 nm
above and below this wavelength in Order to verify the purity of
the caffeine obtained.
7.4.1.1.2 Layer B
If the maximum absorbance exceeds the limit of accurate
Transfer the mixture of the diatomaceous earth and the test
measurement of the instrument used, repeat the measurement
Portion (7.3) into the column on top of layer A. Dry the beaker
on a diluted aliquot Portion of the Solution of caffeine in
twice with portions of about 1 g of the diatomaceous earth
chloroform (7.4.2). In this case, take the dilution into account
(4.3), transferring this into the column. Tamp down to obtain a
in the calculation; the appropriate factors of the formulae in
homogeneous layer and place a wad of cotton wool or glass
8.1.1, 8.1.2 and 8.1.3 will have to be adjusted accordingly. If
wool on the top of this layer 9.
the maximum absorbance measured is lower than 0,2, repeat
the determination using a test portion of greater mass.
7.4.1.2 Column II (acid column)
3
3
IS 16039 : 2012
ISO
ISO 4052-1983 (El
4052 : 1983
c is the concentration, in grams per millilitre, of caffeine in Take as the result the arithmetic mean of the values obtained,
the reference Solution (7.4.3.2); provided that the requirement for repeatability (see 8.2) is
satisfied.
Al is the corrected absorbance of the purified extract
(7.4.2) obtained in 7.4.3.1, i.e.
Repeatability Reproducibility*
Amount of caffeine
Sample g caffeine/ g caffeine/
g/lOO g coffee 100 g coffee
100 g coffee
green coffee beans about 2 0,12 0,38
about 1 0,08 0,3l
decaffeinated < 0,l < 0,Ol 0,Ol
roasted coffee beans about 2 0,lO 0,32
about 1 0,04 0,19
decaffeinated < 0,l < 0,Ol 0,Ol
[ subecoffee
1decati;E;i<
o,3
j g 1 ;;z;
* The values of repeatability and reproducibility are critical differentes at the 95 % probabili-
ty level.
4
4
ISO 4052-1983
IS 16039 (El
: 2012
ISO 4052 : 1983
@21 mm
Column I
(alkaline)
250 mm long
Mixture of diatomaceous
earth and test Portion
Q3&y
Gotton wool or glass wool
Stopcock,
preferably PTFE
@17mm,
Column II
(acid)
250 mm long
Glass wool
Glass wool
- a,y i;
4 - LI
* 3
.I
Stopcock,
preferably PTFE
4
55
ISO 40524983
IS 16039 (El
: 2012
ISO 4052 : 1983
Absorbance at 276 nm
12 -
LO /
09
Absorbance at 246 nm
02 -
Absorbance at 306 nm /
w
1 w
220 240 260 280 300 320 340
Wavelength, nm
6
Bureau of Indian Standards
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Enquiries relating to copyright be addressed to the Director (Publications), BIS.
Amendments are issued to standards as the need arises on the basis of comments. Standards are also
reviewed periodically; a standard along with amendments is reaffirmed when such review indicates that
no changes are needed; if the review indicates that changes are needed, it is taken up for revision. Users
of Indian Standards should ascertain that they are in possession of the latest amendments or edition by
referring to the latest issue of BIS Catalogue and Standards: Monthly Additions.
This Indian Standard has been developed from Doc No.: FAD 6 (2018).