1490 COPPOLA ET AL.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 61, NO.
6, 1978)
FRUITS AND FRUIT PRODUCTS
High Pressure Liquid Chromatographic Determination
of Major Organic Acids in Cranberry Juice
ELIA D. COPPOLA, EDWARD C. CONRAD, 1 and RICHARD COTTER 1
Ocean Spray Cranberries, Inc., Bridge St, Middleboro, MA 02346
A reverse phase high pressure liquid chro-
matographic method is presented for the simul-
taneous separation and determination of quinic,
malic, and citric acids in single strength, un-
diluted cranberry juice. After a 1:10 dilution
and cleanup through a disposable column,
major organic acids in cranberry juice are sepa-
rated on a Bondapak/Cig column and quanti-
tated by using a differential refractometer.
Twenty-seven samples of different single strength
cranberry juice were analyzed using this
method; the mean content of quinic, malic,
and citric acids were 1.32 (std dev. 0.150),
0.92 (std dev. 0.079), and 1.08% (std dev.
0.111), respectively. Mean percent recoveries of
each acid were quinic 95.4 (std dev. 6.8),
malic 96.6 (std dev. 5.8), and citric 94.0%
(std dev. 4.8).
Because they occur in foods and comprise a
significant part of plant foods, organic acids are
becoming increasingly important. The official
AOAC methods (1, 2) are quite lengthy and
usually exist only for a few acids. Recently,
Palmer and List (3) used high pressure liquid
chromatography (HPLC) for a number of food
acids, using a porous resin ion exchange column
and a refractive index detector. Other ion ex-
change methods for organic acids in grape and
wine must have been described by Rapp and
Ziegler (4) and by Kaiser (5) for various
mono-, di-, hydroxy-, and ketocarboxylic acids.
All these methods specify ion exchange resins
which must be eluted at elevated temperature
with critical pH control.
We have developed an ambient temperature,
reverse phase method for determining major
organic acids in cranberry juice.
METHOD
Apparatus and Reagents
(a) Liquid chromatograph.—Waters Associates
Model ALC/GPC 244, or equivalent, with Model
6000A solvent delivery system and TJ6K injector
(Waters Associates, Inc., Milford, MA 01757).
0004-5756/78/6106-1490$00.90
© Association of Official Analytical Chemists, Inc.
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Operating conditions.—Flow rate 0.50 ml/min,
chart speed 2.0 cm/mo, and differential refrac-
tometer at attenuation SX and room temperature
to separate quinic, malic and citric acids from
each other and from aqueous matrices.
(b) Detector.—Model 401 differential refractom-
eter (Waters Associates, Inc.), or equivalent.
(c) Recorder.—Single pen (Houston Instrument,
1 Houston Square, Austin, TX 78753).
(d) HPLC column.—ISO cm X 4 mm Bondapak/
Ci 8 (Waters Associates, Inc., No. 27324).
(e) Mobile phase.—2.0% potassium dihydrogen
phosphate (KH 2 P0 4 ) adjusted to pH 2.4 with
phosphoric acid.
(f) Organic acid standards.—Quinic and malic
acids ACS reagent grade (Aldrich Chemical Co.,
904 W St. Paul Ave, Milwaukee, WI 53223).
Prepare separate 1.00% aqueous stock solutions of
quinic and malic acids. Prepare 1.00% citric acid
solution, by dissolving 1 5306 g ACS reagent grade
sodium citrate dihydrate in 100 ml water.
Preparation of Sample
Dilute each sample of single strength cranberry
juice 1:10 and filter through qualitative paper. In-
sert small amount of silanized wool in bottom of
Pasteur pipet and tap down with stirring rod.
Pour % to 1 in. Bondapik Ci 8 /Porasil B (Waters
No. 27324), 37-75 /im, into pipet and tap gently
to settle particles. Rinse with 5-10 ml acetonitrile-
water (50+50) to elute organic impurities and at
same time condition minicolumn. Place 8-10 ml
filtered cranberry juice on column and discard
first 8-10 ml eluate. Add 2 ml more of sample
and collect this portion. Percolation of sample
may be facilitated by connecting piece of Tygon
tubing to air or dry nitrogen supply and applying
low pressure to pipet. Inject 10 /A into chro-
matograph.
Results and Discussion
Aqueous standard solutions containing all 3
acids, quinic, malic, and citric, in the range
0.0800-0.180%, were prepared and analyzed
1
Present address: Waters Associates, Inc., Food/Agricul-
tural Laboratories, 34 Maple St, Milford, MA 01757.
 COPPOLA ET Ah.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 61, NO. 6, 1978) 1491
Quinic
Malic

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FIG. 1—Chromatogram showing separation of 10 ml standard solution of 0.100% each quinic, maiic, and citric
acids at attenuation 8 x .

under the above conditions. A typical chromato-
gram of a 0.100% standard solution of quinic,
malic, and citric acids is shown in Fig. 1 and
a typical chromatogram of a 10% cranberry
juice sample is shown in Fig. 2. The total analy-
sis time from the time of injection is less than
12 min with quinic acid eluting at 6.11, malic
acid at 7.1, and citric acid at 11.0 min. The
acids are eluted according to increasing polarity
with quinic acid (less polar) first and citric
acid last. The peak after the solvent front is
thought to be from the naturally occurring
sugars. Benzoic acid is also naturally present at
low levels but is not identified and quantitated
by the present procedure. Unpublished data
(Coppola, E. D., Ocean Spray Cranberries,
1977) using the same HPLC column but a dif-
ferent procedure show that benzoic acid is
naturally present in cranberry juice at levels
below 0.0200%. The amount of each acid pres-
ent in the sample was calculated by measuring
the peak height of each acid and by compari-
son with a standard curve.
Peak heights of each standard acid solution
were measured at operating conditions described
previously and were directly proportional to the
concentration of each acid in the range 0.080-
0.180%. The practical slope of peak heights vs.
percentages of each acid was 3.76 cm/0.10%
citric acid.
Twenty-seven samples of different single
strength undiluted cranberry juices were ana-
lyzed for individual acid content. The quinic
acid content ranged from 1.11 to 1.62%, with
a mean content of 1.32% and a standard devia-
tion of 0.150. The malic acid content ranged
from 0.75 to 1.14%, with a mean content of
0.92% and a standard deviation of 0.079. The
citric acid content ranged from 0.94 to 1.30%,
with a mean value of 1.08% and a standard de-
viation of 0.111.
Recoveries of organic acids added prior to
the cleanup step were determined. The amounts
added to 4 different 10:1 diluted cranberry
juice samples ranged from 0.10 to 0.20% quinic,
malic, and citric acids, respectively. For 4 de-
terminations, the amount of quinic acid recov-
ered ranged from 89.7 to 105.0%, with a mean
value of 95.4% and a standard deviation of
6.8; malic acid recovered ranged from 92.0 to
104.0%, with a mean value of 96.6% and a
standard deviation of 5.8; citric acid recovered
ranged from 90.0 to 101.0%, with a mean of
94.0% and a standard deviation of 4.8%. The
first 8-10 ml sample passing through the clean-
up column is discarded in order to eliminate
an interfering substance that has the same re-
tention time as citric acid.
T I M E (min)
FIG. 2—Chromatogram showing separation of quinic,
malic, and citric acids present in 10 ml sample of
unsweetened cranberry juice at attenuation 8X-
1492 COPPOLA ET AL.: J. ASSOC. OFF. ANAL. CHEM. (VOL. 61, NO. 6, 1978)
Preliminary work with apple juice and wines
shows that the present procedure can be suc-
cessfully applied to these and similar products
for the separation and determination of organic
acids.
Acknowledgment
Elia D. Coppola is grateful to Martin S. Starr
of Ocean Spray for bringing the problem to his
attention and for useful suggestions.
v S ^ O CT^g,
REFERENCES
(1) Official Methods of Analysis (1975) 12th Ed.,
AOAC, Washington, DC, sec. 11.041
(2) Official Methods oj Analysis (1975) 12th Ed.,
AOAC, Washington, DC, sec. 20.001
(3) Palmer, J. K., & List, D. M. (1973) / . Agric.
Food Chem. 21, 903-906
(4) Rapp, A., & Ziegler, A. (1976) Chromato-
graphia 9, 148-150
(5) Kaiser, U. J. (1973) Chromatographia 6, 387-
389
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Received April 4, 1978. Accepted June 23, 1978.