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Automated determination of ascorbic acid in orange and grapefruit juice
ANALYTICAL
38, 3ij-39
BIOCHEMISTRY
Automated
(1970)
Determination
of Ascorbic
and M. HOFFMAN, Department
of Horticulture,
Grapefruit
in Orange
Juice1
A. BAR-AKIVA, The Volcani Bet Dugan,
Acid
AND
Institute Israel
LEA TANHUM of Agricultural
Research,
AND
Y. BERKENSTADT “Levant”
X-Rav Received
Co., February
Tel
Aviv,
Israel
10, 1970
Until recently, determinations of ascorbic acid in citrus fruit juice were carried out in this laboratory by titrating the juice with indophenol solution in the presence of oxalic acid (2). This report discusses the conversion of the oxidation reduction titration to an automated procedure with a Technicon AutoAnalyzer. METHODS
The procedure carried out by a Technicon AutoAnalyzer is based on a modification of the Nesset et al. method (3) proposed for ascorbic acid determination in rat ovaries, and Gang and Owen’s method (1) proposed for determining vitamin C content in blood. The orange juice is centrifuged for 5 min at 3000 rpm, decanted into sampling tubes, and placed in the sampler (model II). The juice is drawn at the rate of 40 samples per hour, diluted with water, segmented, mixed, and passed through one side of the dialyzer. A recipient stream of segmented dye solution is passed through the opposite side of the dialyzer and mixed in a single mixing coil. The distance and time required for the sample to reach the flow cell in the calorimeter after leaving the mixing coil have been kept to a minimum to offset the fading color of the dye. Absorption of the solution is determined with a Technicon 520-18-28 filter. Standard solutions of ascorbic acid are prepared in 3% metaphosphoric acid and should be reprepared when results are not reproducible from day to day. ‘Contribution from The Volcani Israel, 1970 Series, No. 1669-E.
Institute 35
of Agricultural
Research,
Bet
Dagan,
36
HOFFMAN
ET AL.
Since sampler II draws in water between samples, there is no need to introduce wash tubes after every sample tube. A minimal baseline noise and peak reproducibility can be achieved by eliminating the sponge effect of the tubing reacting with the dye and replacing it with glass tubing (Fig. 1). Tween-80 (4 ml/liter) should be added to the dilution and wash water and the dye solution. The dye solution is prepared (as suggested by Nesset et al. (3)) by adding 50 mg 2,6-indophenol to 200 ml water, heating to 95”C, diluting to 1 liter, and adding to it 0.5 liter sodium acetate solution (22.65 gm sodium acetate is dissolved in 500 ml and adjusted to pH 7.0 using a 6% acetic acid solution). RESULTS AND DISCUSSION Results were reproducible (Figs. l-3) and in good agreement with the standard method used. Data on the determination of ascorbic acid in orange and grapefruit juice are as follows: Sample
1. Mean titration
Mean Sample 2. Mean Mean Sample S. Mean Mean It is evident that magnitude of 270.
value AutoAnalyzer titration value AutoAnalyzer titration value AutoAnalyzer
391.5, S.E. 2.5. value 400, S.E. 0. 323.7, S.E. 6.0. value 318.5, SE. 5.0. 316.0, S.E. 6.0. value 321.0, S.E. 8.0.
the difference between the two methods
Proportional w-w
Type C Membrane I H.
Colorimeter 15 mm Flowcell 520 rnp Filter FIIG.
acid
(all
1. Flow diagram pumping lines
utilizing are Tygon
1
Tube
-h* w 2
Size
is in the
_‘+-..
Sampler ..
U. 02” 0.061 ‘I 0.065” H20 0.090” Dye Solution 0.056” Air 0.065” H20 To sampler II wxh Receptacle - ---.. -.
Recorder
Technicon tubing).
modules
for
determinations
of ascorbic
ASCORBIC
Steady State at 500 mgll
ACID
Sample
IN
ORANGE
no. 2 Sample
no.1’
4
FIG.
2. Typical also shows
standard steadiness
Vitamin a8 CD
0
chart
37
JUICE
C Standards mg/l
0
and sample run of ascorbic of the baseline and the steady
acid in orange juice. state at 506 mg/liter.
The
Data on the ascorbic acid content of diluted orange juice, and the recovery from ascorbic acid added to it, are presented in Table 1. There is a good agreement in vitamin C content in orange juice as determined by the two methods. Upon dilution, the values obtained with the AutoAnalyzer were in relatively close agreement with the calculated values which should have been found in the juice. (Values found were within 2.5% of the calculated mean.) The results indicate that this method is applicable for the determination of ascorbic acid content in orange and grapefruit juice. (In parallel Ascorbic
Acid
(A.A.)
TABLE 1 in Diluted Orange Juice and Recovery Acid Added to the Juice
Detn.
Determination
by AutoAnalyzer
of Ascorbic
(mg/liter)
-.J
Sample number 1 2 3
titration” (mg/liter)
Whole juice
Diluted 1: 1 with Hz0
Mixed 1: 1 with 600 ppm A.A.
Mixed 1: 1 with 800 ppm A.A.
524.0 550.2 576.4
527.5 540.0 565.0
260 (264)b 270 (270) 280 (282)
560 (564)a 580 (565) 600 (582)
654 (664)b 670 (670) 680 (682)
= Determination in both methods b Values in parentheses calculated
repeated four times for 100% recovery.
on each sample,
38
HOFFMAN
ET AL.
FIQ. 3. Standard calibration curve for determination of ascorbic acid run at the rate of 40 samples per hour. The reaction obeys the Beer-Lambert law within a relatively wide concentration range.
trials, we found that the method is not applicable for the determination of vitamin C in lemon juice because of its high total acid content.) The results of this method were reproducible and in good agreement with the standard method used; per cent recovery was about 100%. The method differs from other similar methods (1, 3) by introducing dialysis to overcome the turbidity of the juice and absorption of the ascorbic acid through the membrane directly into the dye solution, thus eliminating unnecessary reagents. SUMMARY
An automated method for the determination of ascorbic acid in orange and grapefruit juice, based on the reduction of 2,6-dichloroindophenal, was developed by modifying the methods of Nesset et al. (1) and Garry and Owen (3). The method is accurate and reproducible with standard ascorbic acid solution and orange and grapefruit juice. REFERENCES 1. GARRY,
P. H., AND OWEN,
Technicon
Symp.
Pub.
G. M., in “Automation (1967).
1, 507
in Analytical
Chemistry,”
ASCORBIC
ACID
IN
ORANGE
JUICE
39
W., ed., “Official Methods of Analysis.” Association of Official Agricultural Chemists, Washington, D. C., 832 pp., 1960. 3. NESSET, B. L., WINDSOR, B. Ii., HUMPHREY, R. R., AND COLLATINE, M. R., Ad. Biochem. 19, 89 (1967).
2. HORWITZ,
38, 3ij-39
BIOCHEMISTRY
Automated
(1970)
Determination
of Ascorbic
and M. HOFFMAN, Department
of Horticulture,
Grapefruit
in Orange
Juice1
A. BAR-AKIVA, The Volcani Bet Dugan,
Acid
AND
Institute Israel
LEA TANHUM of Agricultural
Research,
AND
Y. BERKENSTADT “Levant”
X-Rav Received
Co., February
Tel
Aviv,
Israel
10, 1970
Until recently, determinations of ascorbic acid in citrus fruit juice were carried out in this laboratory by titrating the juice with indophenol solution in the presence of oxalic acid (2). This report discusses the conversion of the oxidation reduction titration to an automated procedure with a Technicon AutoAnalyzer. METHODS
The procedure carried out by a Technicon AutoAnalyzer is based on a modification of the Nesset et al. method (3) proposed for ascorbic acid determination in rat ovaries, and Gang and Owen’s method (1) proposed for determining vitamin C content in blood. The orange juice is centrifuged for 5 min at 3000 rpm, decanted into sampling tubes, and placed in the sampler (model II). The juice is drawn at the rate of 40 samples per hour, diluted with water, segmented, mixed, and passed through one side of the dialyzer. A recipient stream of segmented dye solution is passed through the opposite side of the dialyzer and mixed in a single mixing coil. The distance and time required for the sample to reach the flow cell in the calorimeter after leaving the mixing coil have been kept to a minimum to offset the fading color of the dye. Absorption of the solution is determined with a Technicon 520-18-28 filter. Standard solutions of ascorbic acid are prepared in 3% metaphosphoric acid and should be reprepared when results are not reproducible from day to day. ‘Contribution from The Volcani Israel, 1970 Series, No. 1669-E.
Institute 35
of Agricultural
Research,
Bet
Dagan,
36
HOFFMAN
ET AL.
Since sampler II draws in water between samples, there is no need to introduce wash tubes after every sample tube. A minimal baseline noise and peak reproducibility can be achieved by eliminating the sponge effect of the tubing reacting with the dye and replacing it with glass tubing (Fig. 1). Tween-80 (4 ml/liter) should be added to the dilution and wash water and the dye solution. The dye solution is prepared (as suggested by Nesset et al. (3)) by adding 50 mg 2,6-indophenol to 200 ml water, heating to 95”C, diluting to 1 liter, and adding to it 0.5 liter sodium acetate solution (22.65 gm sodium acetate is dissolved in 500 ml and adjusted to pH 7.0 using a 6% acetic acid solution). RESULTS AND DISCUSSION Results were reproducible (Figs. l-3) and in good agreement with the standard method used. Data on the determination of ascorbic acid in orange and grapefruit juice are as follows: Sample
1. Mean titration
Mean Sample 2. Mean Mean Sample S. Mean Mean It is evident that magnitude of 270.
value AutoAnalyzer titration value AutoAnalyzer titration value AutoAnalyzer
391.5, S.E. 2.5. value 400, S.E. 0. 323.7, S.E. 6.0. value 318.5, SE. 5.0. 316.0, S.E. 6.0. value 321.0, S.E. 8.0.
the difference between the two methods
Proportional w-w
Type C Membrane I H.
Colorimeter 15 mm Flowcell 520 rnp Filter FIIG.
acid
(all
1. Flow diagram pumping lines
utilizing are Tygon
1
Tube
-h* w 2
Size
is in the
_‘+-..
Sampler ..
U. 02” 0.061 ‘I 0.065” H20 0.090” Dye Solution 0.056” Air 0.065” H20 To sampler II wxh Receptacle - ---.. -.
Recorder
Technicon tubing).
modules
for
determinations
of ascorbic
ASCORBIC
Steady State at 500 mgll
ACID
Sample
IN
ORANGE
no. 2 Sample
no.1’
4
FIG.
2. Typical also shows
standard steadiness
Vitamin a8 CD
0
chart
37
JUICE
C Standards mg/l
0
and sample run of ascorbic of the baseline and the steady
acid in orange juice. state at 506 mg/liter.
The
Data on the ascorbic acid content of diluted orange juice, and the recovery from ascorbic acid added to it, are presented in Table 1. There is a good agreement in vitamin C content in orange juice as determined by the two methods. Upon dilution, the values obtained with the AutoAnalyzer were in relatively close agreement with the calculated values which should have been found in the juice. (Values found were within 2.5% of the calculated mean.) The results indicate that this method is applicable for the determination of ascorbic acid content in orange and grapefruit juice. (In parallel Ascorbic
Acid
(A.A.)
TABLE 1 in Diluted Orange Juice and Recovery Acid Added to the Juice
Detn.
Determination
by AutoAnalyzer
of Ascorbic
(mg/liter)
-.J
Sample number 1 2 3
titration” (mg/liter)
Whole juice
Diluted 1: 1 with Hz0
Mixed 1: 1 with 600 ppm A.A.
Mixed 1: 1 with 800 ppm A.A.
524.0 550.2 576.4
527.5 540.0 565.0
260 (264)b 270 (270) 280 (282)
560 (564)a 580 (565) 600 (582)
654 (664)b 670 (670) 680 (682)
= Determination in both methods b Values in parentheses calculated
repeated four times for 100% recovery.
on each sample,
38
HOFFMAN
ET AL.
FIQ. 3. Standard calibration curve for determination of ascorbic acid run at the rate of 40 samples per hour. The reaction obeys the Beer-Lambert law within a relatively wide concentration range.
trials, we found that the method is not applicable for the determination of vitamin C in lemon juice because of its high total acid content.) The results of this method were reproducible and in good agreement with the standard method used; per cent recovery was about 100%. The method differs from other similar methods (1, 3) by introducing dialysis to overcome the turbidity of the juice and absorption of the ascorbic acid through the membrane directly into the dye solution, thus eliminating unnecessary reagents. SUMMARY
An automated method for the determination of ascorbic acid in orange and grapefruit juice, based on the reduction of 2,6-dichloroindophenal, was developed by modifying the methods of Nesset et al. (1) and Garry and Owen (3). The method is accurate and reproducible with standard ascorbic acid solution and orange and grapefruit juice. REFERENCES 1. GARRY,
P. H., AND OWEN,
Technicon
Symp.
Pub.
G. M., in “Automation (1967).
1, 507
in Analytical
Chemistry,”
ASCORBIC
ACID
IN
ORANGE
JUICE
39
W., ed., “Official Methods of Analysis.” Association of Official Agricultural Chemists, Washington, D. C., 832 pp., 1960. 3. NESSET, B. L., WINDSOR, B. Ii., HUMPHREY, R. R., AND COLLATINE, M. R., Ad. Biochem. 19, 89 (1967).
2. HORWITZ,