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ANALYST, MARCH 1984, VOL. 109 263

Chlorogenic Acid Composition of Instant Coffees

Luiz C. Trugo and Robert Macrae
Department of Food Science, University of Reading, London Road, Reading, RG7 5AQ, UK

A method, based on high-performance liquid chromatography, is described for the determination of

chlorogenic acid isomers in instant coffee. Identification of the individual components was assisted by the
preparation of isomeric mixtures by the isomerisation of available compounds and quantification based on
published ultraviolet molar absorptivities. A number of extraction and clearing techniques were studied to
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determine the optimum conditions for the recovery of all the isomers present. The developed method was
applied to a wide range of commercially available instant coffees.
Keywords: Chlorogenic acids; high-performance liquid chromatography; coffee

The chlorogenic acid composition of coffee products is
extremely complex with at least five major groups of
compounds present: caffeoylquinic acids (CQA), dicaffeoyl-
quinic acids (diCQA), feruloylquinic acids (FQA) ,p-coumar-
oylquinic acids (CoQA) and caffeoylferuloylquinic acids
(CFQA).1 The general structure of chlorogenic acids is shown
in Fig. 1.
There are also a wide range of more minor chlorogenic acid
components that have received little attention. Several
attempts have been made to correlate (inversely) the levels of
chlorogenic acids with beverage quality and to correlate
specific sensory attributes. such as astringency, to the pre-
sence of specific chlorogenic isomers. There is little evidence
in the literature to support these ideas but it is generally
accepted that Arabica coffee, with a lower chlorogenic acid
content, is superior to Robusta coffee.'
A significant loss of chlorogenic acids, in some instances up
to 90Y0,~takes place on roasting. Additionally, the levels
found in instant coffees will depend on the extraction
conditions. These factors, coupled with a large difference
between varieties, means that commercial instant coffees
would be expected to vary widely in terms of chlorogenic acid
composition. A number of methods have been described in
the literature for the determination of chlorogenic acids in
green, roasted and instant coffees. The simplest methods for
analysis are based on ultraviolet absorption of alcoholic
extracts3 or colorimetry.4 The former lacks specificity and the
latter. even using differential colorimetric techniques. is
subject to interferences and individual isomers cannot be
determined. 1 Chromatographic techniques have greatly
improved the precision of analytical data and, even though
Extraction solvents, aqueous methanol (40 and 70% VIV),
aqueous propan-2-01 (70% VIV) and aqueous ethanol (70%
ViV.
Chromatography solvents, tripotassium citrate (K3C6-
H 5 0 7 . H 2 0 ) ,0.01 M . Adjusted to pH 2.5 with dilute hydro-
chloric acid and HPLC-grade methanol.
Chlorogenic acid solutions. The IUPAC nomenclature8 is
used to name chlorogenic acid isomers. 5-Caffeoylquinic acid
(commerically termed chlorogenic acid) was obtained from
Sigma, UK.
5-Feruloylquinic acid solution, 1 mg ml- I . Obtained from
Dr. M. Clifford (University of Surrey).
Dicaffeoylquinic acid mixture (commercially termed iso-
chlorogenic acid). Obtained from Roth, F.R.G.
Apparatus and Chromatographic Conditions
An Applied Chromatography Systems liquid chromatograph
(two pumps, Model 750103, and a programmer, Model 750136)
was used. Injection was achieved by means of a fixed-loop
Rheodyne injection valve, Model 7120 (20 pl). A pre-column
(50 x 5 mm i.d.) dry packed with coarse silica (70-150 pm) was
placed in-line before the injection valve in order to saturate
the mobile phase. A Spherisorb 5-ODS column (Phase
Separations Ltd., UK, 2.50 x 5 mm i.d.) was used for the
chromatographic separations. The final conditions used were
a gradient of methanol (see Fig. 3) in 0.01 M tripotassium
citrate solution (pH 2..5), increasing from 20 to 70% with
5-min linear segments of 0 , 1 , 1 , 2 , 1 , 1 , 2 , 2and 0% min-1 and
a flow-rate of 1 ml min-1. Peak integration was achieved by
means of a Hewlett-Packard 3390-A integrator.

im:H
gas chromatography provides excellent resolution ,5 high-
performance liquid chromatography (HPLC) is preferred, as H
it avoids the derivation step in the analysis.6.7 I HO I
This paper presents an HPLC method for the determination
of the nine main chlorogenic acids in a range of commercial HO-C
instant coffees. Particular attention has been paid to extrac-
tion efficiency and the optimisation of chromatographic OH HO
resolution, factors that have received only brief consideration
in some published methods.
0
Experimental
Reagents
All reagents and solvents were of analytical-reagent grade
unless otherwise specified.
Carrez solutions (clearing agent). Solution (I) was pre- ' OH
pared by dissolving 21.9 g of crystallised zinc acetate,
Zn(C2H302)2.2H20, and 3 ml of glacial acetic acid in distilled (b)
water and diluting to 100 ml. Solution (11) consisted of 10.6 g
of potassium hexacyanoferrate(I1) in 100 ml of distilled water. Fig. 1. Structure of chlorogenic acids. ( a ) D-(-)-Quinic acid. ( b )
R = H, 5-p-coumaroylquinic acid; R = OH, 5-caffeoylquinic acid;
Ammonia solution, approximately 4 M . and R = OCH,, 5-feruloylquinic acid. Esterification is also possible at
Hydrochloric acid, approximately 4 M . carbon atoms 3 and 4 of the quinic acid
 View Article Online

264 ANALYST, MARCH 1984, VOL. 109

Procedure before and after precipitation of the chlorogenic acids, is

Extraction
Finely ground instant coffee (0.5 g) was dissolved in 80 ml of
an aqueous methanol solution (40%) and transferred into a
100-ml calibrated flask. Carrez solution (2 ml of each
component) was added and the mixture was allowed to stand
for 10 min after making up to volume. The precipitate was
removed by filtration under gravity (using Whatman No. 1
filter-paper) and the filtrate was used directly for chromato-
graphy. For testing extraction efficiencies 70% V/V aqueous
solutions of propan-2-01, ethanol and methanol were also
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adequate for quality control purposes. However, where the
determination of individual components is required, HPLC
provides the simplest method. With reference to the published
HPLC methods6.7 there appear to be three areas where
improvements could be made (unambiguous peak assignment,
greater chromatographic resolution and substantiation of the
methods in terms of recovery and precision) and these aspects
are addressed in this paper.
The problem of peak assignment is mainly due to the
non-availability of chromatographic standards. 5-Caffeoyl-
quinic acid and a mixture containing the three dicaffeoylquinic

used. acids are available. This range of standards can be extended by

isomerising the 5-caffeoylquinic acid to yield an equilibrium
Isomerisation of chlorogenic acids mixture of the 3-, 4- and 5-isomers. The normal isomerisation
conditions consist of heating the chlorogenic acid, either in a
5-Caffeoylquinic acid (200 mg) was dissolved in distilled water
phosphate buffer of pH 7-7.211 or in saturated sodium
(20 ml) and the pH was adjusted to 8 with dilute ammonia
hydrogen carbonate solution.12 However, a much cleaner
solution. This solution was heated for 30 min in a boiling
reaction can be realised simply by heating the acid in dilute
water-bath. The pH was then adjusted to 2.5-3.0 with dilute
ammonia solution at pH 8. The rate of isomerisation can be
hydrochloric acid, after cooling to room temperature. For the
readily followed by HPLC, the reaction being stopped by
preparation of feruloylquinic acid isomers, 20 1-11 of the
acidification after various intervals (Fig. 2). The graphs show
standard solution of 5-feruloylquinic acid were added to 5 ml
of water, adjusted to pH 8 with dilute ammonia solution and clearly that one isomer is initially formed at a greater rate than
heated for 15 min in a boiling water-bath before being the other and that the final concentrations are approximately
evaporated to dryness under reduced pressure and the residue equal. This is consistent with the isomerisation taking place via
an intramolecular mechanism with the 4-isomer being formed
dissolved in 0.1 ml of dilute hydrochloric acid. These solutions
were then used directly for chromatography. first and subsequently isomerising to yield the 3-isomer. On
this basis the isomer initially formed at the greater rate was
assumed to be 4-CQA. This assignment is also supported by a
Quantification
consideration of the polarity of the structures (see Fig. 1). The
Quantification was achieved by peak-area measurement and 3-isomer contains two equatorial and one axial hydroxyl
by comparison with a 5-caffeoylquinic acid standard. It was groups and the 4-isomer contains one equatorial and two axial
possible to quantify the individual isomers using their molar hydroxyl groups, conferring greater polarity to the former. 13
absorptivities, according to the equation
RFX X M,, X A
C=
E2 x M,,
where C is the concentration of the isomer in milligrams per
millitre; RF is the response factor of 5-caffeoylquinic acid
standard (concentration in milligrams per millilitre per unit
area); c1 is the molar absorptivity of 5-caffeoylquinic acid; e2 is
the molar absorptivity of the isomer in question; Mr, is the
relative molecular mass of 5-caffeoylquinic acid; Mr, is the
relative molecular mass of the isomer in question; and A is the K
.-+-0
area of the peak corresponding to the isomer in question.
F
Molar absorptivities ( x lo4) are as follows: at A,,,.330 nm, c
C
Q,
5-CQA = 1.95,4-CQA = 1.80,3-CQA = 1.84,3,4-diCQA = C
3.18, 3,5-diCQA = 3.16 and 4,5-diCQA = 3.32; and at A,, 0
325 nm, 5-FQA = 1.93, 4-FQA = 1.95 and 3-FQA = 1.90 10
1 mol-1 cm-1.9
Calibration graphs were plottcd using the CQA and diCQA 0 2 10 20 30
isomer mixtures diluted at five different concentrations. These Ti rnelrn in
concentrations varied from 10 to 100 pg ml-1 in the CQA
group and from 1 to 15 1-18 ml-1 in the diCQA group. Fig. 2. Isomerisation of (A) chlorogenic acid (5-CQA) yielding (B)
3-CQA and (C) 4-CQA. Conditions as described in the text
Recoveries were checked by the method of standard addi-
tions. The levels of addition to the samples (in grams of isomer
per 100 g of sample) were 3-CQA, 0.3-1.6; 4-CQA, 0.3-1.5;
5-CQA, 0.3-1.7; 3,4-diCQA, 0.1-0.8; 3,5-diCQA, 0.2-1.9;
and 4,5-diCQA, 0.2-1.7. The samples (0.2 g) were dissolved
Table 1. Recovery of chlorogenic acid isomers using HPLC. Average
in aqueous methanol (40%), spiked with the isomer solutions, of five levels of addition for each isomer (values are in grams of isomer
cleared with Carrez reagent, made up to volume (100 ml) and per 100 g of sample for wet matter)
filtered. The filtrates were used directly for chromatography.
Calibration and recovery data were not obtained for Range of addition, Average Standard
5-feruloylquinic acid as only trace amounts of the material Isomer g per 100 g recovery, YO deviation. 06
were available. 3-CQA . . .. 0.3-1.6 101.7 4.9
4-CQA . . .. 0.3-1.5 99.3 4.4
5-CQA . . .. 0.3-1.7 104.2 4.9
Results and Discussion 3,4-diCQA .. 0.1-0.8 99.9 3.6
When only a total chlorogenic acid value is required the 3,5-diCQA .. 0.2-1.9 97.3 4.4
4,5-diCQA .. 0.2-1.7 97.8 5.6
AOAC method,l0 based on differential ultraviolet absorption
 View Article Online

ANALYST, MARCH 1984, VOL. 109 265

, 4
I (b' 1 3
A
1
I\
70%
/
/
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/
/

/
4 /
/ 0 30
Timeimin

Fig. 4. Chromatograms of ( a ) FQA isomers mixture and ( b )+CQA

isomers mixture. Peaks as described in Fig. 3 and cord'itions as
described in the text

0 30
Timeimin Table 2. Coefficient of variation of chlorogenic acid isomers in an
instant coffee by HPLC. Results obtained from six different extrac-
Fig. 3. Chromatograms of ( a ) chlorogenic acid standard, ( b ) CQA tions from the same sample (values are in grams of isomer per 100 g of
isomers mixture and (c) instant coffee using Spherisorb 5-ODS and sample for wet matter)
gradient elution. (1) 3-CQA; (2) 3-FQA; (3) 4-CQA; (4) 5-CQA; (5)
4-FQA; (6) 5-FQA; (7) 3,4-diCQA; (8) 3,5-diCQA; and (9) 4,5- Isomer Mean, g per 100 g Coefficient of variation, '/o
diCQA. Detection at 325 nm; flow-rate. 1 ml min-1; and solvent and
gradient conditions as described in the text 3-CQA . . .. 1.49 0.8
4-CQA . . .. 1.67 1.2
5-CQA . . .. 2.12 1 .o
3-FQA . . .. 0.27 2.5
The same procedure was adopted with a sample of 5-feruloyl- 4-FQA . . .. 0.37 3.0
quinic acid to allow the assignment of the peaks due to the 4- 5-FQA . . .. 0.52 3.0
and 5-isomers. In each instance the coffee extracts were spiked 3,4-diCQA .. 0.17 5.4
with standards, or isomerised mixtures, to aid peak assign- 3,5-diCQA .. 0.12 4.0
ment. 4,5-diCQA .. 0.24 2.2
Quantification of the isomers in the coffee samples was
achieved by comparison of peak areas with a standard of
5-caffeoylquinic acid, allowing for differences in molar
absorptivities between the isomer in question and the stan-
dard. Pure reference compounds were not available and thus Linearity of the detection system was established over the
the molar absorptivities could not be determined experimen- concentration range expected from the samples, showing good
tally. Consequently literature values were used,9 which were correlation for all isomers studied (correlation coefficients:
recorded at the ,A values for each component. However, 5-CQA, 0.999 92; 4-CQA, 0.999 94; 3-CQA, 0.999 8; 3,4-
the ,A values only varied from 325 to 330 nm and so by using diCQA, 0.999 5; 3,5-diCQA, 0.999 995; and 4,5-diCQA,
a common detection wavelength (325 nm) only a very small 0.999 90). The major problem encountered with the chromato-
error was introduced. From a study of the absorption graphic resolution was between the peaks due to 4-caffeoyl-
spectrum of one isomer (5-CQA) this error was calculated to quinic acid and 3-feruloylquinic acid, although with careful
be about 1-1.5%, which is acceptable in terms of the over-all selection of the gradient and solvent conditions adequate
experimental accuracy. resolution can be achieved. Chromatograms of standards,
The major source of error in HPLC methods such as these is isomer mixtures and of instant coffee samples are shown in
not in the chromatographic stages but in the preceding Figs. 3 and 4.
extraction and clean-up procedures. A number of extraction All determinations were carried out in duplicate and one
systems were evaluated and of these, the following three sample was analysed six times to obtain an idea of the
produced good recoveries: (a) 70% propan-2-01; (b) hot water precision for each isomer and the results are shown in Table 2.
(80 "C) followed by clearing with Carrez solution; and (c) 40% The established method was then applied to the determination
methanol followed by clearing with Carrez solution. System of chlorogenic acid isomers in 13 commercial instant coffees,
(a) produced a clean extract but the presence of propan-2-01 in with the results shown in Table 3. The major isomer in all the
the sample produced severe distortion of the chromatogram. samples was 5-caffeoylquinic acid accounting for about 30%
With this system it is essential to evaporate the solvent and of the total, whereas the sum of the caffeoylquinic isomers
redissolve the residue in water or buffer solution prior to accounts for approximately 70%. Similarly, the feruloylquinic
injection, a process that complicates the analytical scheme and acid group and the dicaffeoylquinic acid group represent
may cause losses by oxidation. System (b) produced chromat- about 20 and lo%, respectively. However, it has been
ograms with interfering peaks and showed losses of dicaffeoyl- suggested that these low levels of dicaffeoylquinic acids are
quinic isomers, compared with systems (a) and (c). Conse- nonetheless very important for sensory qualities of coffee.14
quently, system (c) was chosen for this study. The efficiency of The highest total level found was 10.7% from a "mild" coffee
this solvent system based on the recoveries of added amounts and the lowest (3.6%) was from a decaffeinated coffee,
of CQA and diCQA isomers from one coffee sample is shown suggesting that there are considerable losses of chlorogenic
in Table 1. acid during processing.
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266 ANALYST, MARCH 1984, VOL. 109

Table 3. Chlorogenic acid isomer contents in instant coffees. Average of duplicate determinations for dry matter

Samples, grams of isomer per 100 g of sample

Isomer C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-I2 C-13
3-CQA .. .. .. . . 1.38 1.35 0.90 0.92 1.89 1.44 0.75 1.40 1.00 1.43 1.08 0.72 0.70
4-CQA .. .. .. . . 1.65 1.55 1.04 1.03 2.25 1.62 0.87 1.70 1.16 1.56 1.25 0.84 0.81
5-CQA .. .. .. . . 2.25 2.15 1.38 1.36 3.50 1.89 1.02 2.28 1.69 1.93 1.74 1.07 1.04
TotalCQA . . . . . . . . 5.28 5.05 3.32 3.31 7.64 4.95 2.64 5.38 3.85 4.92 4.07 2.63 2.55
3-FQA .. .. .. . . 0.25 0.25 0.17 0.18 0.35 0.25 0.15 0.23 0.21 0.22 0.20 0.17 0.15
4-FQA , . , . .. . . 0.59 0.34 0.44 0.37 0.76 0.29 0.32 0.54 0.49 0.26 0.37 0.43 0.40
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5-FQA .. .. .. . . 0.58 0.45 0.32 0.32 0.82 0.36 0.27 0.52 0.41 0.28 0.41 0.29 0.30
TotalFQA . . . . .. . . 1.42 1.04 0.93 0.87 1.93 0.90 0.74 1.29 1.11 0.76 0.98 0.89 0.85
3,4-diCQA.. .. .. ..0.21 0.20 0.10 0.10 0.39 (1.16 0.09 0.22 0.12 0.14 0.13 0.08 0.07
3S-diCQA . . , . .. . . 0.16 0.16 0.07 0.07 0.29 0.11 0.07 0.17 0.08 0.10 0.08 0.06 0.06
4,5-diCQA . . . . .. . . 0.03 0.02 0.14 0.14 0.48 0.17 0.07 0.19 0.10 0.14 0.13 0.10 0.09
TotaldiCQA .. .. . . 0.40 0.38 0.31 0.31 0.16 0.43 0.23 0.58 0.30 0.38 0.35 0.24 0.22
Totalchlorogenicacids . . . . 7.10 6.47 4.56 4.49 10.73 6.29 3.61 7.25 5.26 6.06 5.45 3.76 3.62

The authors thank Conselho Nacional de Desenvolvimento
Cientifico Tecnologico, Universidade Federal do Rio de
Janeiro, Brazil, the Committee of Vice-Chancellors and
Principals of the Universities of the UK for sponsorship and
Dr. M. Clifford (University of Surrey) for providing the FQA
fraction.
References
1. Clifford, M . N.. and Wight, J . , J . Sci. Food Agric., 1976,27,73.
2. Clifford, M. N . , Process Biochem., 1975. May, 13.
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5 . Konig, W. A . and Sturm, R., “Tenth International Scientific
Colloquium on Coffee,” Association Scientifique Internation-
ale du Cafe, (ASIC), Paris, in the press.
6. Rees, D . I., and Theaker, P. D., “Eighth International
Scientific Colloquium on Coffee,” ASI(:, Paris, 1979, p. 79.
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International Scientific Colloquium on Coffee ,” ASIC, Paris,
1980, p. 107.
8. IUPAC Commission on the Nomenclature of Organic
Chemistry and IUPAC - IUB Commission on Biochemical
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9. Rubach, K., Dusertation, Technical University of Berlin, 1969.
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Paper A311 15
Received April 25th, 1983
Accepted June 21st, 1983