Ascorbic Acid Rapid Determination in Fresh, Frozen, or Dehydrated Fruits and Vegetables H. J. LOEFFLER AND J. D. PONTING Western Regional Research Laboratory, U. S. Department of Agriculture, Albany, Calif.
Ascorbic acid, mg. per 100 ml. of filtrate = 10.8 (LI - L) The equation for fruit and vegetable tissue becomes: Ascorbic acid, mg. per 100 grams of tissue = 10.8 (LI - La) [ml. of acid extractant (% liquid in sample) (wt. of sample)] wt. of sample The authors use a reservoir-type automatic pipet for the acid and nonreservoir types for the 1-ml. portions of filtrate and 9ml. portions of dye, so that they can achieve a rate of 20 samples per hour (two testers and a helper). Unlike Evelyn, they have found it more suitable to add the dye to the tube outside the colorimeter and agitate the tube slightly before putting it in the instrument. The automatic 9-ml. pipet must extend to near the surface of the liquid in the tube to avoid splashing and must be calibrated to drain uniformly in less than 5 seconds.
P
LANT tissues must be finely divided and uniformly mixed as a preliminary step in analysis. These objectives, formerly accomplished only by laborious hand grinding after cutting up the sample or passing it through a food chopper, may now be attained easily by use of a commercial household food-preparing device described as a liquefier or blender. This was first pointed out by Davis in 1939 ( 2 ) . Morel1 (16) has found that in 3 per cent metaphosphoric acid plant tissues may be disintegrated in these devices, such as the Waring Blendor (made by Waring Corporation, 1697 Broadway, Yew York, X. Y.), without causing any loss of ascorbic acid. Davis ( 2 ) has also used such a blender for ascorbic determination using an extraction and titration mixture of trichloroacetic and metaphosphoric acids. hlorell’s adaptation (16) of the photometric method for determining ascorbic acid by comparing photoelectric colorimeter readings on specially prepared plant extracts with a standard curve obtained with regularly increasing increments of pure ascorbic acid is satisfactory \There only one type of material is to be tested, but the research programs on dehydrated and frozen foods under way in the Xestern Regional Research Laboratory have necessitated a method equally adaptable to many kinds and varieties of fruits and vegetables that may be fresh, frozen, or dehydrated (partially or completely), and in some cases highly pigmented. This requirement is met b y combining Morell’s technique of pre. paring plant extracts with a modification of the Evelyn et al. (4) colorimetric method for determining ascorbic acid. This combination method is an extension of that used b y Loeffler (IO) for orange juice and utilizes a rate reaction in a photoelectric colorimeter. It is somewhat simpler in operation than the excellent Bessey (1) modification of the Mindlin and Butler technique (16) which also utilizes the photoelectric colorimeter.
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Standardization of Dye The dye is standardized by noting the 15-second reading with filter 520 (when the instrument is calibrated to 100 with distilled water) given by a tube containing 1 ml. of 1 per cent metaphosphoric acid and 9 ml. of the dye solution. This value is GI, from which L1 is obtained on the calibration table. This standardization is very much easier and faster than titrimetric procedures. The dye solution is prepared simply by dissolving enough of the dye in water so that a G1 reading of about ‘‘30’’is given with the Evelyn photoelectric colorimeter. The concentration of dye to give such a reading is roughly 13 mg. per liter. The reaction between ascorbic acid and stronger dye solutions is not a linear relationship, so that a calibration curve rather than a constant factor must be used with such solutions.
Volume of Acid and Extraction Calculation Because of the thorough disintegration of the sample in the blender, the ascorbic acid is distributed uniformly through the entire liquid phase present. By determining the ascorbic acid concentration in the filtrate (per milliliter of liquid phase) and by knowing the total volume of liquid, the amount of ascorbic acid in a sample can be determined from a single extraction. Only a few milliliters of filtrate are necessary. The total amount of liquid is simply the sum of the volume of metaphosphoric acid solution added plus the liquid originally present in the sample. The authors have found a ratio of seven parts of metaphosphoric acid solution to one of vegetable or fruit tissue satisfactory. Under such circumstances the liquid originally present in the sample (water plus soluble solids) may be approximated from food tables, since a variation of 7 per cent causes less than 1 per cent variation in the ascorbic acid calculation. The results are more accurate and simpler to obtain than those after three or more extractions with small volumes of extractant (usually 1 to 1 ratio). Most of the previous workers [Mack and Tressler ( 1 3 ) , Kirk and Tressler (7), McHenry and Graham ( I 2 ) , and Rolf (IC?)] have disintegrated the tissue in a mortar and then used such multiple extractions. Bessey ( I ) uses but one extraction and suggests that the volume of the solids may be neglected. He recommends a sample of only 0.5 to 2.0 grams; but a much larger sample, 25 to 50 grams, is necessary with fruit and vegetable tissues to approach uniformity. Under such circumstances the results approach the accuracy of the method, which is about 1 per cent.
Simplified Method Blend 25 or 50 grams of fresh or frozen fruit or vegetable tissue with 350 ml. of 1 per cent metaphosphoric acid in a blending machine operated for 5 minutes at high speed, If the material is of high ascorbic acid content, such as leafy vegetables, raspberries, strawberries, or asparagus, use the smaller quantity. Fifty grams are used with foods containing less ascorbic acid, such as stored potatoes, carrots, yams, peaches, plums, and apricots. If a dehydrated fruit or vegetable is being analyzed, 5 or 10 grams of sample are sufficient, depending upon this same classification. Some thoroughly dehydrated vegetables, such as sweet potatoes or carrots, may need 0.5 hour of soaking in the acid before blending. Frozen foods may be blended without preliminary thawing. Filter the extract through coarse, fluted filter paper. Extracts of starchy vegetables, such as potatoes and corn, filter better through a Buchner funnel. They can also be cleared by centrifugation. Moderate turbidities do not interfere, since the instrument is calibrated with pro er blanks. Pipet 1-ml. portions of the figrate into three matched tubes from the Evelyn photoelectric colorimeter. Add 9 ml. of distilled water to one tube and adjust the colorimeter to read 100 with this tube, using filter No. 520. To each of the other tubes add 9 ml. of the previously standardized indophenol dye solution from a calibrated rapid delivery pipet. Take a reading in the photoelectric colorimeter, using filter KO.520, 15 seconds after the start of the dye addition. This reading is GI, from which the corresponding LZvalue is obtained, from the calibration table provided with the instrument, and substituted in the following equation of Evelyn: 846
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ANALYTICAL EDITION
November 15, 1942
Type and Concentration of Extracting Acid Ascorbic acid usually is extracted by strong acid to inhibit enzymic and autoxidation and is determined by oxidation with 2,6-dichlorophenolindophenol or less specifically with iodine. Lyman, Schultze, and King (11) have shown that metaphosphoric acid prevents the autoxidation of pure ascorbic acid solutions, while Morell (16) found that 3 per cent metaphosphoric acid prevents loss of ascorbic acid during blending of vegetable tissue. Harris and Ray (6) recommend trichloroacetic acid, but Fujita and Iwatake ( 5 ) disagree. Though workers describing photoelectric methods (Mindlin and Butler (15), Bessey ( I ) , Morell (16), etc.] have noted the fading of the dye in strong acid solutions and buffer their extracts t o a pH of 3.6 before testing, some analysts still use the erratic titration with an unbuffered acid, such as the 5 per cent sulfuric acid plus 2 per cent metaphosphoric acid recommended by Mack and Tressler (13). Kreuther and Roe (9) suggest that the final pH after addition of dye should be no greater than 3.0, since at higher values the effect of interfering substances, particularly sulfhydryl compounds, was more pronounced.
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TIME IN SECONDS
FIGURE1. FiDING O F INDOPHENOL RE.4GEXT
180
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ascorbic acid per 100 grams. When 5 per cent sulfuric plus 1 per cent metaphosphoric acid was used, 30-second end point indicated 27.3 mg., a 1-minute end point indicated 41.0 mg., and a 3-minute end point indicated 372 mg. per 100 grams. Titration of the unknown and standard under identical circumstances in the same time interval is not satisfactory, since the buffering action of vegetable tissue greatly reduces the fading from that found with the standard alone. With sulfuric acid, however, fading continues even a t higher p H values. Figure 1indicates that with a 15-second end point the fading in 1 per cent metaphosphoric acid is negligible. (Because of present war conditions it is difficult to obtain metaphosphoric acid. Preliminary experiments have shown 0.4 per cent oxalic acid to be the best substitute for 1 per cent metaphosphoric acid. No loss was incurred during blending of pure solutions; vegetable filtrates lost no more ascorbic acid in oxalic than in metaphosphoric acid when stored a t room temperature in the presence of added copper; and the fading of the dye reagent was about the same in either acid of the indicated concentration.) The dye fades a t a significant rate in 3 per cent metaphosphoric acid, but only one tenth as fast after reaction with ascorbic acid. Hence the dye itself should be standardized with 1 per cent metaphosphoric acid, but the test material, if strongly buffered may be extracted and tested using 2 per cent metaphosphoric acid. One per cent metaphosphoric acid added to cabbage in a 7 to 1 ratio protects ascorbic acid during blending, as shown in Table I. Fifty grams of shredded cabbage were blended a t high speed and small samples ivere removed every 2 minutes. (Stone, 19, has shown that ascorbic acid is lost very rapidly from shredded cabbage,) Even the heating that results from the blending does not cause a significant loss. Essentially similar results were obtained with 3 per cent metaphosphoric acid plus a different cabbage sample. Filtrates from raw cabbage extractions were tested before and after 3 days’ storage at room temperature in stoppered flasks. The results, given in Table 11, show that the actual loss in ascorbic acid is no g-reater in 1 per cent metaphosphoric acid than in 3 per cent metaphosphoric or 2 per cent metaphosphoric plus 5 per cent trichloroacetic acid, under the conditions of this test. The authors’ many previous experiments are confirmed, since the actual amount of ascorbic acid obtained in two extractions is shown to check very closely with
The authors have found that the buffering can be eliminated, since the use of a large proportion of 1 per cent metaphosphoric acid yields a p H sufficiently IOK to prevent losses during blending and yet sufficiently high to prerent fading during the reaction n-ith the dye. The extracts with 1 per TABLEI. ASCORBICACID RETENTIOX DURIXG BLEWDISG OF cent metaphosphoric acid of all vegetables and fruits tested, CABBAGE including strongly buffered soy and lima beans, hare ranged (Netsphosphoric acid solution and cabbage mixed in ratio of 7 t o 1) between p H 2.4 and pH 3.1 before addition of the dye. The Blending TemperaAscorbic Acid Ascorbic Acid p H may increase as much as 0.5 during the dye addition. Time ture 1% H P O i 1% HPOI 3 % HPOa Extracts Tyith 3 per cent metaphosphoric acid have ranged .\fin. C. .Ifg./iOO cc. .filtrate M g . / l O O g . cabbage from p H 1.8 to 2.2. 2 27 3 .OO, 3 , 0 3 23.8,24.0 23.5 4 34 3.00,3.05 23.8,24.2 23.9 The extremely unstable end points in strong acid are indi6 38 3.08,3,09 24.4,24.5 24.4 cated in Figure 1, where the rate of fading of the dye is 8 42 3.03,3.06 24.0,24.2 24.4 10 45 3 .OO, 3 . 0 5 23.8,24.2 24.3 plotted in the absence of ascorbic .. 23.5,23.S 12 49 2.97,3.00 acid. It is evident that i n ‘ a mixture of 5 per cent sulfuric O X ASCORBIC ACIDCOXTEST OF CABBAGE TABLE11. EFFECTOF THREEDays’ STORAGE and 2 per cent met,aphosphoric EXTRACTS acids a high ascorbic acid conAscorbic Acid tent would be indicated when Calculated f r o m Ascorbic Acid l e t extraction After 3 none is present. Similar errors Total in 2 ex(90‘; liquid days’ n-ere observed in tests with vegeSample Extractaiit tractions a 8 sum ed 1 storage Loss J1o.i 100 g r a m s Mg./lOO grams table tissue filtrates. SDinach extracted with 1 per cent I 1% HPO3 23.3 l5,l 8.2 I -1 1% HPOi 23.1,23.3 ?3.3,23.3 9.4 13.9 nietaphosphoric acid or 1 per I1 3L’: H P 0 3 21.8 24.8,25.0 11.2 13.7 11-4 36: HPOa 21.3 21.8,21.2 7.4 14.1 c e n t metaphosphoric acid I11 2 % HPOs + 5% ClsH.4~ 25.1,23.0 11.1 13.9 plus 5 per cent trichloroacetic 111.4 2% HPOI + 5 % CbH.4c 23.6 25.4,25.4 11.9 13.5 acid tested 12 to 14 mg. of
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 14, No. 11
preliminary dilution of the clear filtrate with an equal volume CRUSHED RASPBERRIES of saturated sodium chloride solution maintains the ionic TABLE111. ASCORBICACIDIN FROZEN Ascorbic Acid strength and prevents the turbidities. Recovered Weight of Volume of 1 % (Assuming 100% Filtrates from extractions of dehydrated vegetables will Sample Sample HPOI Liquid Phase) occasionally cause fading of the indophenol dye to an a p Grama lM1. 1Mg./iOO cc. preciable extent other than by reduction with ascorbic acid, because interfering substances have been concentrated by the dehydration. Under such circumstances the approximate correction suggested by Bessey (I)-that is, to subtract from the 1.5-second reading the extent of fading that occurs b e tween a 15- and a 30-second reading-has been satisfactory. the calculation based on a few milliliters of filtrate and the Elliott, Sklar, and Acree (3) have shown that blood plasma assumption of 90 per cent liquid content. reduces the rate of reaction between ascorbic acid and the indophenol dye; hence a more careful extrapolation to zero Application to Fruits and Berries time as suggested by Evelyn may not always be desirable. The method has been applied successfully to fresh and Preliminary experiments on dehydrated vegetables treated frozen fruits and berries, particularly peaches, nectarines, with sulfur dioxide prior to drying indicated that, somewhat raspberries, loganberries, strawberries, boysenberries, and contrary to the results of Kirkpatrick (8), sulfur dioxide does youngberries. interfere with the indophenol reaction by supplementing the Color in the sample is not an interfering factor, since the reducing effect of the ascorbic acid. However, the rate of photoelectric colorimeter, when properly adjusted to allow reaction is sufficiently slow to give a rapidly drifting reading for color in the sample, measures the added red color of the with the photoelectric colorimeter. The suggestion of Mapdye in acid solution. The dye is standardized with the colorson (14), that the sulfur dioxide can be removed by evacuaimeter adjusted to 100 with distilled water alone; but for the tion or sweeping with nitrogen, was not substantiated on extract being analyzed the colorimeter is adjusted to 100 with strongly acid bisulfite solutions. However, Mapson's other a tube containing 1 ml. of the extract plus 9 ml. of distilled suggestion of binding the sulfur dioxide with an excess of water. acetone has proved successful in preventing interference in With most fruits the volume of the solids can be neglected, pure ascorbic acid solutions. Ten per cent of acetone in the since the distribution of ascorbic acid is in the liquid phase ascorbic acid solution prevented the interference of 500 present-that is, water plus soluble solids-rather than in the p. p. m. of sulfur dioxide, provided the indophenol dye was water alone. Most fruits and berries do not possess sufficient standardized with 1 per cent metaphosphoric acid solution insoluble solids to alter t h e results materially. The formula containing the same acetone concentration. then becomes: With dehydrated vegetables, the moisture content (if beMg. of ascorbic acid per 100 grams = low 5 per cent) can usually be neglected. The formula then (d- L21(volume of acid wt. of sample becomes : wt. of sample Mg. of ascorbic acid per 100 grams = of acid added If the insoluble solids can be neglected, the formula above 10.8 (1' - volume wt. of sample should hold despite wide variations in extracting volumes and ratios. Table I11 demonstrates that such conditions are Additional dilution may be necessary for some foods, such met and that very consistent results can be obtained. The as citrus concentrates, strawberries, and brussels sprouts. berries had been crushed before freezing; hence they were A small portion of the filtrate can be quickly diluted to d e uniform in character. sired volume with 1 per cent metaphosphoric acid when Table 111, together with unpublished results obtained at necessary. the Western Regional Research Laboratory, demonstrates that many berries, besides strawberries, are good sources of Summary ascorbic acid. The statement of Morgan (17)that berries Ascorbic acid can be determined quickly in fruits and are a poor source of ascorbic acid, averaging 3 to 10 mg. per vegetables, whether fresh, frozen, or dehydrated, by disints 100 grams, has not been confirmed. Fresh California berries grating the sample with dilute metaphosphoric acid in a high purchased commercially, have averaged in ascorbic acid conspeed cutter and measuring the decolorizing effect of the extent approximately as follows: raspberries 25 mg., boysentracted ascorbic acid on indophenol dye with a photoelectric berries 15 mg., loganberries 35 mg., and blackberries 15 mg. colorimeter. The ascorbic acid is distributed within the total per 100 grams. Hence, berries may provide ascorbic acid or liquid phase present, which includes the water and dissolved vitamin C for farm and other communities whose principal solids originally in the sample. Hence by using a large profruit consumption has been apples, pears, apricots, and portion of extractant and knowing the approximate amount peaches, which have relatively lower ascorbic acid contents, of liquid in the sample, the ascorbic acid can be determined averaging about 5 mg. per 100 grams. from as little as 3 ml. of filtrate after only one extraction.
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Application to Other Products Since most food products can be disintegrated with a blender, especially after a preliminary soaking, and since interfering colors or turbidities can be blanked out in the photometric method, almost any food oan be tested by these techniques. Some products may require slightly different t r e a t m e n t for example, clear filtrates from extractions of certain varieties of fresh lima beans may become turbid when the dye is added. Apparently protein fractions soluble in 1 per cent metaphosphoric acid solution are precipitated because of the reduction of ionic strength on dilution with dye solution. A
By using a large amount of 1 per cent metaphosphoric acid as the extractant the buffering step is avoided, since the p H obtained is sufficiently low to prevent losses during blending, yet sufficiently high to prevent fading of the dye reagent. The rate of fading is high with many previously recommended extraction mixtures. Rapid ascorbic acid determinations can be made easily on highly pigmented berries and tough dehydrated vegetables
Literature Cited (1) Bessey, O.,J . B i d . Chem., 126,771 (1938). (2) Davis, W.B.,News Ed. (Am. C h m . SOC.),17,752 (1939); IND. ENQ.CHEM.,34.217 (1942).
ANALYTICAL EDITION
November 15, 1942 (3)
Elliott, Sklar. and Acree, J . Research Natl. Bur. Standards, 26, 117 (1941).
(4) Evelyn, Malloy, and Rosen, J . Biol. Chem., 126, 645 (1938). (5) Fujita and Iwatake, Biochem. Z.,227, 295 (1935). (6) Harris and Ray, Biochem. J . , 27, 580 (1933). (7) Kirk and Tressler, Food Research, 6, 395 (1941). (8) Kirkpatrick, H. F., J . SOC.Chem. Ind., 60, 226 (1941). (9) Kreuther and Roe, Proc. SOC.Ezptl. Biol. Med., 47, 487 (June, 1941). (10) Loeffler, H. J., IND.ENG.CHEM.,33, 1308 (1941). (11) Lyman, Schultae, and King, J . Biol. Chem., 118, 757 (1937).
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(12) McHenry, E. W., and Graham, M., Biochem. J., 29, 2013 (1935). (13) Mack and Tressler, J . Biol. Chem., 118, 735 (1937). (14) Mapson, L., Chemistry & Industry, 60, 802 (1941). (15) Mindlin and Butler, J . Biol. Chem., 122, 673 (1937-38). (16) hforell, S., IND.ENG.CHEM.,A N ~ LED., . 13, 793 (1941). (17) Morgan, A. F., Fruit Products J., 21, 75 (1941). (18) Rolf, L. A., J . Agr. Research, 61,381 (1940). (19) Stone, William, Biochem. J., 31, 508 (1937). OUTSIDID Publication No. 3664. Bureau of Agrioultural Chemistry and Engineering, U. S. Department of Agriculture.
Agar-Agar as a Coagulant for Barium Sulfate EDGAR J. BOGAN' AND HARVEY V. RZOYER The Ohio State University, Columbus, Ohio
T
HE precipitate of barium sulfate in the determination
of sulfate usually consists of very fine particles. Standard procedures recommend digestion for several hours and preferably overnight in the hot solution before filtration. Numerous methods have been proposed to improve the character of the precipitate.
Lindsly (6) proposed the addition of a saturated solution of picric acid as a means of increasing crystal size. Krak (6) recommended digestion in an ammonium acetate solution after decanting the mother liquor through the filter. Ziegeler (8) suggested coagulation of the barium sulfate by precipitating silver chloride in the suspension and subsequent removal of the silver chloride with concentrated ammonium hydroxide. Orlow (7) precipitated a known amount of aluminum hydroxide with the barium sulfate and made a correction for the weight of aluminum oxide in the ignited precipitate. None of these methods has proved entirely satisfactory. Caldwell and Moyer (3) roposed the use of the sensitization enomenon as a means oPcoa ulating analytical precipitates. !key showed that finely divide$ zinc sulfide can be coagulated by the addition of minute quantities of gelatin.
It has been found that positively charged barium sulfate which is obtained when sulfate is precipitated by an excess of barium ions can be coagulated by the addition of a trace of 1 Present address, Department of Chemistry, University of Maine, Orono, Maine.
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MOBILITY OF PARTICLES OF BAF~IUM SULFATE IN ABRAMSON ELECTROPHORESIS CELL Measurementa on same eolutions as in Figure 1
agar-agar. Approximately 1 mg. of agar-agar will cause the flocculation of a n ordinary analytical precipitate of barium sulfate obtained in the determination of sulfate. On the other hand, negatively charged barium sulfate obtained in the determination of barium is not appreciably improved by the addition of agar-agar. However, this precipitate, as a rule, causes little difficulty in filtration. Another troublesome property of finely divided barium sulfate is its tendency to creep up the sides of the funnel or filtering crucible. Creeping seems to be completely eliminated from precipitates coagulated with agar-agar,
Procedure
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:FIGERE1. EFFECTOF AGAR-AGARON TIMEOF SETTLISG OF PRECIPITATES OF BARIUM SULFATE Each point represents separate sample
The sulfate is reci itated as usual in a hot solution containing 1 ml. of 1 N hygochiric acid by the slow addition of 5 per cent barium chloride solution. After precipitation, from 0.5 to 1 ml. of a solution of agar-agar containing 1 mg. per ml. is added a drop a t a time. After each drop the suspension is stirred rapidly for about 10 seconds. Some coagulation can be observed after the introduction of the first drop; however, the best coagulation usually appears after the addition of about 10 drops of the coagulating agent. The optimum amount of the agar-agar solution is indicated by the flocculation and rapid settling of the precipitate. After settling, the precipitate somewhat resembles coagulated silver chloride. A study was made of the effects of adding agar-agar to precipitstes of barium sulfate in order to determine the limit-
