Extraction of Ascorbic Acid

from Plant Tissues

WARD B. DAVIS
Agricultural Chemical Research Division,
Bureau of Agricultural Chemistry and Engineering,
U. S. Department of Agriculture,
Los Angeles, Calif.
HE extraction of ascorbic acid by a new procedure which
utilizes the Waring Blendor (2) is briefly described.
T This procedure will avoid the laborious grinding neces-
sary for plant tissue hard to triturate, as in the method of
Thornton (6) and others. Plant material was extracted by
covering the cutting knives of the Blendor with about 150
ml. of extractant, adding a sample of 10 to 20 grams, and
running the motor at high speed for 5 minutes. The Blendor
and reagents were kept in a room maintained near 5" C.
Sufficient Super-Cel filter aid was added to the finely divided
material suspended in the extractant to give quick filtration
by suction on a Biichner funnel. Convenient aliquots of
the clear, almost colorless filtrate which was made to a volume
of 150 ml. were titrated with 2,6-dichlorophenolindophenol.
The dye was standardized by the method of Menaker and
TABLE I. COMPARISON OF METHODS FOR EXTRACTION OF As-
CORBIC ACID (MQ. ASCORB~C ACID PER GRAM FRESH WEIQHT)
-Hand Method- -Blendor Method-
a b a b
Parsley
First extract 1.93 2.20 2.00 2.20
Second extract 0.07 0.06 0.03 0.03
Third extract 0.01 0.02 0.01 0.01
0.07 0.12 0.02 0.02 Residue
Total 2. 08 2. 40 2.06 2.26
First ext 7 of total 92.78 91.67 97.09 97.38
- - - -
Extract& &e, min. 24 20 10 10
Guerrant (4). Except where 2 per cent metaphosphoric
acid was used, the extractant was a mixture of 3 per cent
trichloroacetic acid and 2 per cent metaphosphoric acid.
After extraction, 8 per cent acetic acid was used for diluting
the mixture to volume. Ballentine's iodate method (1)
was used on some of the tissues. The end point is sharper
and results are higher than by the dye titration.
The Blendor method of extracting ascorbic acid was com-
pared with the usual method of grinding by hand. Five
grams of the plant tissue were ground 5 minutes with 25
ml. of extractant and 3 ml. of sand to a thin paste, which wm
poured into a 50-ml. conical centrifuge tube. Samples
ground in duplicate were centrifuged at the same time for
3 minutes. The fairly clear liquid was decanted, and then
10 ml. of fresh extractant, used to rinse the mortar, were
stirred with the residue in each tube. These were centrifuged
and decanted, and a final extraction was made with 5 ml.
The total liquid decanted was made to 50 ml. This was the
first extract. An aliquot of the first extract was made to
about 50 ml. and titrated. Two additional extractions were
made by grinding the residue in the mortar again for 2 min-
utes, each time with 25 ml. of extractant, and centrifuging.
The residue of tissue fragments and sand was diluted to 50
or 100 ml. and titrated. The end point, ordinarily obscured
by suspended particles, was clearly seen after centrifuging
a small quantity of the suspension in an angle centrifuge for
Cabbage 1 minute.
First extract 0.32 0.30 0.30 0.23 Twenty-gram samples of the same tissues extracted by the
0.00 0.00 0.01 0.01 hand grinding were reduced in the Blendor, in the manner
Residue 0.01 0.01
already described, for 5 minutes. Two additional extractions
Extraction time, min. 19 19 12 13 Only 5 grams of grapefruit peel and 10 grams of fresh peas
were used in the Blendor. The extractant for these two tis-
0.01 0.01 sues was 2 per cent metaphosphoric acid (3). Samples of the
Third extract 0 " : : ; ::ti fresh peas from the same l ot were weighed, frozen, and kept
Total 0.25 0.22 0.22 0.23 frozen until extracted to avoid loss of ascorbic acid on stand-
First ext %, of total 88.00 86.36 86.30 78.80 ing. Centrifuging the fresh pea tissue after extraction gave
Extractizn time, min. 18 18 10 11
the same results as filtration with acid-washed filter aid.
Some tissues foam in the Blendor, making the transfer to
First ext., % of total 75.54 79.83 the centrifuge tubes bothersome. A few drops of alcohol aid
Mustard greens in causing the foam to subside.
As shown in Table I, the new procedure appears not only
to be just as efficient but also to require less time and effort
Potato
First extract 0.09 0.07 on the part of the worker than the older, grinding method.
88.49 Even in cabbage tissue, which contains ascorbic acid oxidase,
First ext., of total
Grapefruit peela the new procedure seems satisfactory. The titration figures
Av. of 4 samples 1.75 1.92
for the tissue residues, although only approximate in nature,
indicates the completeness of the extractions.
Av. of 6 samples 0. 07 0.11
Av. of 6 samples, acid-washed
Higher results were obtained from samples in which acid-
Super-Cel ... 0.16
Av. of 6 samples, centrifuged ... 0.16 washed in comparison with unwashed Super-Cel was used.
a Extractant, 2% metaphosphoric acid. To test the recovery of added ascorbic acid, 20 mg. of pure
ascorbic acid were added to the titrated residues from 20
0.01 0.01 0.01 0.01
0.34 0.32 0.33 0.26
Second extract
Third extract
First ext., % of total
94.12 93.76 90.90 88.47 of 2 minutes each were made on each sample in duplicate,
Total
Frozen peas
0.22 0.19 First extract
Second extract
Residue
- - - -
Chard
First extract 0.40 0.42
First extract 1.11 1.07
First ext., 70 of total 90.16 85.88
86.79
Fresh peas5
217
218 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 34, No. 2
grams of extracted tissues mixed with filter aid and fragments no plant tissue and the motor was run at high speed for
of filter paper. After the ascorbic acid was stirred into the 10 minutes.
residue, the extractant was added and the suspension was Much time and effort may be saved by this procedure, be-
placed in the Blendor. The procedure was then the same as cause a large number of workers have been making the
described for the extraction of fresh material. The degree ascorbic acid assay by the chemical method.
Literature Cited
of completeness of extraction of added ascorbic acid was
similar to that of the first extraction of naturally occurring
ascorbic acid.
90 to 101 per cent.
The total percentage recovered varied from
Boiled cabbage tissue residue gave about
(1) Ballentine, R.8 1x0. ENG. CHEM. 9 ANAL. ED., 13, 89 (1941).
(2) Davis, W. B., IND. ENG. CHEM., NEWS ED., 17, 752 (1939).
(3) King, C. G., IND. ENG. CHEX., ANAL. ED., 13, 228 (1941).
(4) Menaker, M. H., and Guerrant, N. B., Ibid., 10, 25 (1938).
( 5) Thornton, N. C., Contrib. Boyce Thompson Inst , 9, 273 (1938).
CovTRiBuTroN 37 from the Division of Agricultural Chomioal Research.
the same percentage recovery of added ascorbic acid as the
unboiled residue. A recovery of 99 per cent was made
when ascorbic acid was added to the extractant containing
Effect of Reinforcing Pigments
on Rubber Hydrocarbon
F. S. THORNHILL AND W. R. SMITH
Godfrey L. Cabot, Iiic., Boston, Mass.
The unsaturation values and amount of combined
sulfur at various states of vulcanization for a num-
ber of rubber compounds have been studied.
While the anticipated loss in unsaturation of the
rubber hydrocarbon was noted in stocks containing
nonreinforcing fillers, no such loss in unsaturation
could be detected in rubber compounds containing
reinforcing channel blacks. It is suggested that
this alteration in the mechanism of sulfur vul-
canization may be mostly responsible for the
physical characteristics of reinforced rubber stocks.
It has not been possible to detect any effect of
carbon black on the unsaturation value of natural
rubber. Calculation indicates that, while such an
effect would not be detected in the present case
with the analytical method employed, such an
effect, if present, should be detectable with carbon
blacks of greater surface area than those employed
in the present investigation.
EVERAL investigators (20) have established that sulfur
vulcanization of rubber involves chemical combination
S of sulfur and rubber hydrocarbon. A definite decrease
in unsaturation of the rubber hydrocarbon as vulcanization
progresses has been generally noted (S, 10, 18).
While it has often been concluded that a double bond is
saturated for each atomic equivalent of combined sulfur (10,
18), recent work by Brown and Hauser (3, 7 ) demonstrates
that this conclusion cannot be applied in all cases. I n certain
compounds they found the loss in unsaturation with extent
of vulcanization to be considerably less than anticipated on
the above basis. Their results indicated that stocks reaching
optimum cure with the least loss of unsaturation possessed
the greatest tensile strength.
Although a considerable amount of work has been done on
this problem, wehave not found a published account of simi-
lar investigations performed on stocks containing significant
loadings of reinforcing fillers. Since, as pointed out below,
the nature of the bonding between such fillers and the rubber
molecule has not been clearly defined, one is not justified in
applying previous results obtained on stocks containing no re-
inforcing fillers to those bearing appreciable loadings of such
substances. Accordingly one portion of the present investi-
gation was concerned with determining the effect of various.
fillers on the course of sulfur vulcanization, as judged from
combined sulfur and unsaturatioii values.
Aapointed out by Gehman and Field (S), it is undoubtedly
true that the black particle in a carbon-black-reinforced
rubber stock is firmly attached to the rubber molecule. The
nature of the bonding between the black and rubber has not
been clearly defined. Some investigators (4, 8, 14, 17) main-
tain that the association is physical and involves dcfinitc
forces of adhesion or adsorption; othcrs ( I S ) have suggested
formation of primary valence linkages with the rubber hydro-
carbon. The opinion of the present autho& is that if such
linkages are formed, the ethylenic bonds of the rubber mole-
cule would probably beinvolved. If this latter view is correct,
then a specific loss in unsaturation of the rubber hydrocarbon,
due to the reinforcing filler, should occur. Thus the second
objective of the present study was to determine whether it
was possible by chemical means to detect such a linkage. If
measurable, this effect, together with the surface area deter-
minations reported previously (15), would be particularly
valuable in estimating the reinforcing value of various fillers.
Experimental Procedure
The unsaturation of the rubber stocks was determined by
addition of iodine chloride. The procedure followed was es-
sentially Kemps technique (9, 11) as modified by Blake and
Bruce ( 9) :
A 0.1-gramsample of stock was dissolved in boiling p-dichloro-
benzene. Solution be-
camemore difficult with well cured compounds. This was over -
This usually required from 2 to 3 hours.