Simplified Procedure for Extraction and Determination of Vitamin A in

ISOMERIZATION OF VITAMIN A IN VIVO. T. K. Murray , D. W. Stainer , J. A. Campbell. Canadian Journal of Biochemistry and Physiology 1959 37 (12), 1469-...
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ANALYTICAL CHEMISTRY

a definite yellow. Six milliliters of 6 N nitric acid were added, followed by the addition of 25 ml. of 0.lOOON silver nitrate, No precipitate was observed. On titration of the resulting solution with 0.1000N potassium thiocyanate, the titration value corresponded exactly to that obtained when 25 ml. of the silver nitrate were titrated directly with potapeium thiocyanate. The same results were obtained in the use of both propionaldehyde and acetaldehyde, indicating that the reactions with sodium cyanide were complete a t the pH of the indicator. Propionaldehyde was used to test the procedure for the determination of chloride in the presence of cyanide as follows: Ten milliliters of a 0.l O O O S standard sodium chloride solution were added to 10 ml. of approximately 0.2N sodium cyanide solution and 1.5 ml. of technical grade propionaldehyde (calculated to he an excess). Indicator, 0.1N nitric acid, 6 N nitric acid, and 25 ml. of 0.LV silver nitrate (an excess) were added according to the procedure already described. The silver chloride was filtered and washed. The filtrate was titrated in the usual way with standard potassium thiocyanate. The original sodium chloride solution was also analyzed by the Volhard method. Each determination was run in triplicate. The results for the 0.lOOON standard sodium chloride solution were 0.0999N and 0.100021' in the absence and presence of cyanide, respectively. T h w , thc procedure is suitable foi the quantitative determination

of chloride in the presence of cyanide. It may be suitable for other halides as well. The quantitative removal of acetaldehyde and propionaldehyde using hydrogen cyanide solutions has been described (1). The m e of sodium cyanide solutions would be preferable because of the smaller hydrogen cyanide losses and the rapidity of the reaction in basic solution. S o specific experiments were performed to validate a procedure using sodium cyanide solutions, but from the above experimental work, a suitable procedure is readily suggested. One would prepare a standard solution of sodium cyanide, add a sample of aldehyde solution to an excess of the Etandard sodium cyanide solution, adjust the p H using bromocresol purple and then determine the unreacted cyanide by the procedure in (1), LITERATURE CITED

(1) Svirbely, W. J . , and Roth, J. F., J . A m . Chem. Soc., 75, 3106 (1953). RECEIVED

for review March 30,1954

Accepted &May21, 1964

Simplified Procedure for Extraction and Determination of Vitamin A in liver STANLEY

R. AMES, HUGH A. RISLEY, and PHILIP L. HARRIS

Research Laboratories, Distillation Products Industries, Division of Eastman Kodak Co., Rochester,

C

URRENTLY wed methods for determining vitamin A in liver tissue were critically evaluated during the course of developing a modified (1) liver-storage type vitamin A bioassay procedure (4, 7 , 11). None of the published methods proved to combine the accuracy and precision desired with the speed arid ease of performance needed for routine analyses. Consequently, a simplified procedure was developed which involved drying thc liver by grinding with anhydrous sodium sulfate, adding ethyl ether, and determining thc vitamin A content of an aliquot by means of the standard Carr-Price (antimony trichloride) reaction. This new method eliminntrs the need for saponification (e),;L step which leads to low results unless carefully conducted Ethyl ether was used, since it has been shown ( 8 ) to extract vitamin .A more completely from biological samples than other solvents. The use of anhydrous sodium sulfate to dry liver tissue has been reported (6, 14), but these methods differ either in tlitl type of solvent or in thr details of extraction of the liver lipides. ANALYTICAL PROCEDURE

A 3- to &gram portion of liver tissue, or an ent'ire rat liver, is ground in a mortar with three to five times its weight of anh;.drous sodium sulfate until completely dry. If the grinding it* started while the livers are in the frozen state, less sodium sulfat'c. is required and the drying is more rapid. Too much sodium sulfate is undesirable, because it results in a fine, slow-settling suspension in the ether layer during the subsequent extraction. The dry powdered liver is transferred quantitatively to a 250-ml. Perma-red glass-stoppered Erlenmeyer flask. Exactly 100 ml. of peroxide-free anhydrous ethyl et,her are added and the flask is shaken for 2 minutes. It is then eit,her set aside until the solids have separated from the ether layer (ea. 1 hour) or the suspended solids are removed by centrifugation. An aliquot of the ether layer is transferred quantitatively to an Evelyn colorimeter tube and the solvent is removed by evaporation under nitrogen. The residue is dissolved in 1 ml. of chloroform, the tube is positioned in the colorimeter, and 2 drops of acetic anhydride are added. Ten milliliters of a saturated solution of antimony trichloride in chloroform (ca. 20 to 25%) are then added and the intensity of the resulting blue color is determined using a 620 mp filter. The

N. Y.

amount of vitamin A present is determined by reference to a standard curve prepared using the United States Pharmacopoeia vitamin %. reference standard. Liveis containing less than 0.2 y of vitamin A per milligram of liver lipide (5 to 10 y per gram of liver tissue) cannot be satisfactorily analyzed by this procedure. Ether-soluble materials in the liver extract result in turbidity on addition of the antimonytrichloride reagent. .%bout 20 mg. of total ether-extractable material in the colorimeter tube are sufirient to induce this disturbing turbidity. This is not solely a triglyceride effect, since 60 mg. of either cottonseed oil or lard do not give turbidity under rimilar conditioris. The method, however, i.; very satisfactory for use in conjunction with the modified rat liver-storage bioassay procedure ( I ) , since livers from the bioassay contain between 100 and 1500 y of vitamin A per liver. RECOVERY EXPERI,,IERTS

.Assays of livers from vitamin A-deficient rats by the antimony trichloride-blue color procedurr indicated no perceptible blue color potency. Results of recovery experiments using livers from vitamin A-deficient animals are given in Table I. dliquots of an ethyl ether solution of vitamin .4 acetate [?ere evaporated onto the whole liver before grinding it with anhydrous sodium sulfate. The fortified livers were analyzed by the simplified procedure. Excellent recovery was realized a t all three levels added (30, 150, and 300 y). The over-all average recovery of 98.6 + 0.56% standard error showed that there was no significant loss of vitamin A during the analysis. Also, there was no indication of extraction of materials from the liver which interfere with the vitamin A determination. CONFIRMATION O F IDENTITY

The possibility of the presence of nonvitamin A substances yielding a blue color with antimony trichloride was considered.

V O L U M E 2 6 , NO. 8, A U G U S T 1 9 5 4 Table I. Animal So.

Recovery of Vitamin A Added to Livers from Vitamin A-Depleted Rats

Vitamin A Added, Recovered, Average blue color units blue color units Recovery, yo Recovery, % Controls, S o Vitamin .4 Added

Lon Le\el Added, 0.10 RIl of ca. 300 ?/RIl. 406

,570 ,546 361 547 240 542

105 105 105 107 107 107 107

108 105 103 105 104 105 107

103 100 98 98 97 98 100

99.1

COMPARISOh- WITH OTHER METHODS

Intermediate Level Added, 0.50 MI. of ca. 300 ?/MI. 97.5

High Level .4dded, 1.00 hI1. of ca. 300 y/Ml.

,564 ,545 $44 388 396 3R 5

1053 1053 1053 1053 1075 1075

1035 1087 1079 1035 1028 1050

Over-all average recovery = 98.6 a

98 103 102 98 96 98

+ 0.56%

S.E. (r

99.2

= 2.4%)

Table 11. Test of Identity of Vitamin A in Rat Liver Lipides (Composite ethyl ,ether extract from animals receiving 300 or 600 y of vitamin A a s alcohol, acetate, or palmitate) Liver Lipides, Unsaponifiable Fraction Analysis Units per Gram of Liver Lipides Units per Gram' 2970 SbCls-blue color 2860 4035 3330 Ens (uncorr.) X 1900 EW (corr.) X 1900 (ESP XIV) 2480 Rlorton-Stubbs correction ... 74.4% factora Dehydrationb 3018 90% Vitamin A a s esterc

...

h C

Comparison of methods for the determination of vitamin A in liver tissue was complicated hy the difficulty of obtaining samples of uniform composition. Aliquots of chopped or homogenized liver tissue were analyzed by the various procedures according to a Latin square design and the results were subjected to analysis of variance. By this procedure the variation between samples was isolated and a statistical comparison of methods was made. Five procedures for the determination of the vitamin A content of liver tissue were compared. Similar sized samples (3 to 5 grams) were used in all cases and the vitamin A content of the extract was determined colorimetrically. The procedures are summarized briefly as follows:

No blue color.

Ether extracts from a nunibcr of animals on a routine liver $torage bioassay (each of which had received 300 or 600 y of vitamin A as alcohol, acetate, or palmitate) were pooled and the solvent was removed under nitrogen. This composite sample of rat liver lipide was saponified and both the unsaponifiable fraction and the original lipide extract were analyzed as indicated in Table 11.

a

spoiidence of the potency, 3015 unitv per gram, by dehydration with that obtained by the other procedures is further indication that under these conditions the blue-color potency is a reliable measure of the vitamin .4 present. The vitamin A in the liver lipide fraction was 90% in the ester form. This corresponds well with the value of 86 to 86.5% previously reported from these laboratories (8). I n this sample of rabliver fat any blue-color yielding substances were formed from vitamin A, since nearly pure vitamin A or its esters had been fed. The approximate agreement of the various procedures for estimating vitamin A shows that the bluecolor method is satisfactory for the determination of vitamin A in rat livers under the conditions of the modified liver-storage bioassay.

- AT

Correction factor x 100 ( 1 9 ) . La Determined a s described b y Shantz Cawley a n d Embree (IS). Determined a s described b y Kaschdr a n d B l x t e r (9).

The vitamin A content of the liver lipides was 2970 units per gram by conventional blue-color procedures compared with a blue-color potency of 2860 units per gram in the unsaponifiable fraction. Spectrophotometric analyses indicated an ES2S uncorrected potency of 3330 units per gram. The Eans corrected potency (U.S.P. XIV procedure) resulted in a value of 2480 units per gram with a Morton-Stubbs correction factor (12) of 74.4%. The ratio of blue-color potency to U.S.P. XIV potency of 1 15 is identical with the mean ratio of 1.15 considered ( 3 ) characteristic of good quality vitamin A-bearing liver oils. As a further proof of identity the unsaponifiable fraction was dehydrated with acid and the anhydro vitamin A formed was analyzed spectrophotometrically as described by Shantz, Cawley, and Embree ( I S ) . Since this procedure is specific for vitamin A , the corre-

Metbod A. Simplified procedure as described in this paper. Method B [Glover, Goodwin, and Morton ( 6 ) ] . Sand and anhydrous sodium sulfate were added to the liver and the mixture was ground until dry and homogeneous. The dried powder was extracted once with 150-ml. and then twice with 75-ml. portions of anhydrous et,hyl ether. Extraction was facilitated by stirring for 2 minutes on a warm water bath. The extracts were separated from the residue by decantation and filtration and the residue was washed. The combined extracts and washings were evaporated to dryness under nitrogen and vitamin A was determined. Method C (Yoxhlet). The mixture obtained by grinding the liver n-ith anhydrous sodium sulfate was placed in a thimble in a Soxhlet apparatus and was continuously extracted with anhydrous ethyl ether for 3 hours. The extract was evaporated to dryness under nitrogen and vitamin A was determined. Method D [(Benham @)I. In a flask, 4 ml. of 5% alcoholic potassium hydroxide were added for each gram of liver. The flask was heated on a st'eam bath with occasional shaking until saponification was complete. An equal volume of water wafi added and the mixture was extracted with one 100-ml. and two 50-ml. portions of anhydrous ethyl ether. The combined ether extracts were washed with one 100-ml. and two 10-ml. portionP of water. The washed extracts were diluted to a known volume, and dried with anhydrous sodium sulfate, and the vitamin A content of an aliquot was determined. Method E [Week and Sevigne (Id)]. The mixture obtained by grinding n-ith anhydrous sodium sulfate was extracted four times with 50-ml. portions of petroleum ether (boiling point, 40" to 60" C.). The extracts were filtered, the residue was washed, the combined extracts and washings were evaporated to dryness under nitrogen, and vitamin A was determined. Uniformity of distribution of vitamin A in samples and reduction of sampling errors to the minimum were prime objects in these comparison experiments. Pork and beef livers were used because of the quantity of sample needed. I n a preliminary experiment, samples of minced fresh pig's liver were analyzed in duplicate by each of the five methods on each of 5 days. The analysis of variance showed that the difference between samples within days (error variance) was large and the difference between average units per sample determined on different days (days variance) was likewise large. Even with the great variation between samples, t,he variance due to difference in methods was significant due primarily to low values obtained by Method E. This inferior method was, therefore, omitted from further comparisons.

ANALYTICAL CHEMISTRY

1380 I n view of the large sampling variation in the first experiment, a refined sampling technique was devised for the second comparison test. Fresh calf liver was thoroughly homogenized in a Waring blender and the homogenate was passed through cheesecloth under slight pressure to remove large particles and fibrous tissue. The filtered homogenate was thoroughly mixed and poured into damp Visking cellulose sausage casings (23/32 inch in diameter). The casing was tied off in sections containing ample amounts for a 1-day analysis. The sections were wrapped carefully in foil, frozen as quickly as possible, and stored at -2OOC.

Table 111. Comparison of Methods Using Homogenized Calf Liver Latin Square Used to Randomize Sampling Errors (For details of methods see text) Order of Samples Selerted 1st 2nd 3rd

Day

Simplified procedure. Glover et al. Soxhlet. D . Benham.

Cnits A/Gram _ of Vitamin _ _ ~ 1st day 2nd day 3rd day 4th day

_

Method Simplified procedure

718 661

702 666 687 670 670 632 617 628 693 635 677 648 623 603

646

B.

Gloreretal.

651 675 658

641

C.

611 690 652

Soxhlet

649 D.

633 603 596 586

Benham

Average units/ gram 0

564

613 604

639.6

G50.0

= 19.6

Source of Variation Treatment Method Day Interaction Error Totals

or 3.0%

671

623

602 585 570 586

629 636 661 663

607.3

633.1

670.0

647.9

668 651

662 660 633 632

645 641 654

S.E.

Average I-nits ~ ~ Per Gram

679 683 692 669 673 675 693 631 712 688 677 669

631

671 6

631.3

661.6

= 2.44 or 0.38%

Analysis of Variance Sum Squares dj' 38,597 13,032 3763 18,394 -~ 73,788

-

Comparison of hrethod A (simplified procedure) and Method C (Soxhlet). Mean of Nethod A = 671.7 (15 observations). Mean of Method C 658.2 (15 observations.) Error 14 982.9 70 2 Sample 14 13,035 931 0 13 3 & Method 1 1,360 1,360 0 19 40 Total 29

a b

C.

Table IV. Comparison of Methods Using Homogenized Calf Liver

A.

[Analysis of variance of adjacent samples (paired observations)] F Source of Variation df Sum Sciiiarrs Mean Square Comparison of Method 4 (simplified procedure) and Method B (Glover et 0 1 . ) . RIean of Method A = 671.6 (15 observations). Mean of Method B = 662.6 ( 1 5 observations). Error 14 3.089 7 220.7 Sample 14 20,663 1.475 9 R.69 Nethod 1 2,707 .5 2,707.5 12.270 Total 29

Comparison of Method B (Glover et a l l and Method D (Benham). Mean of .\lethod B = 050.0 (15 observations). Mean of Method D = 607 5 (15 observations). Error 14 4,762.8 340 2 Sample 14 14,044 1,003 2 4.3 Method 1 19,611 13,611 40 O b Total 29

4th

.4. B.

Methods. Methods. Methods. Methods.

Table V. Comparison of Jlethods Using Homogenized Calf Liver

Mean Square

3 3 9 48 __.

12.800 43-14 418

383

F 33 59b 11 34" 1 09

Significant. Highly significant.

of sampling, using homogenized liver, was much superior to that previously used. By reduction of the variance due to sampling, the variance due to method proved to he highly significant a8 evidenced by an F value of 33.6. I t remained to determine which of the four methods was the best. I n the design of the Latin square used to aspign consecutive samples to each method, methods A and B, . I and C, and B and D, but not .4 and D, \yere always awigned to adjacent Famplesi.e., 15 times out of 16 samples. The comparison of adjacent samples by analysis of variance for paired observations and statistical analyses for these pairs of methods are given in Table V. This method was used in preference to Student's t-test, since an estimate of the sample variance is obtained in addition to the method variance. It is evident that Method I), involving saponificat,ion, is distinctly inferior to the other three methods. Method h (simplified procedure) givw significantly higher recoveries of vitamin A than either lfethod B or llethod C. Thus. drying with anhydrous sodium sulfate followed by addition of ethyl ether and analysis of an aliquot yields somewhat higher results than either estraction three t,imes with hot ethyl ether or Soxhlet estraction.

...

63

Significant. b Highly significant.

a

At the time of analysis a section of the cylindrical casing containing the homogenized liver was cut into 3- t o &gram samples, the casing was removed, and the still-frozen homogenate was weighed. The assignment of the samples t o the four methods compared was randomized by following the Latin square schedule given in Table 111. The initial row of the Latin square was altered on each of 4 days. This procedure was followed to minimize not only the error variance (within methods variation on a given day) but also the variance between days. The order of sampling was quadruplicated each day so t h a t four samples by each of four methods on 4 days or a total of 64 determinations were run. The results of these determinations and their treatment by analysis of variance are given in Table IV. The data showed excellent agreement within methods and within days as evidenced by an error mean square which was about one fourth that obtained in the preliminary experiment. I n addition, the day-to-day variance was greatly reduced, from an F value of 128 to 11.3 These results showed that the method

PRECISION OF hIETIIOD

An estimate of the precision of the simplified procedure can be made from the recovery experiments included in Table I. The standard deviation of a single determination was zk2.4% with a standard error of 0.56% (19 values). Similarly, the data of Table IV indicated that the four methods have a comparable precision. Based on the total error mean square which includes both variation between eamples and the true method variation, a standard deviation of 3.0% with a standard error of 0.38% (64 values) was obtained. The liver storage bioassay for vitamin .I\ ( 1 ) specifies 10 analyses per dose level and would yield a standard deviation (owing to method) of about 2.?5% and a standard error of about 0.6%. DISCUSSIOh

One of the inferior procedures involves hot saponification of the liver followed by a single extraction with petroleum ether (Skellysolve F) ( 5 ) . A recovery from the liver of only 25 t o 35% of orally administered vitamin A was obtained by this method in contrast to the 60 to 70% recovery observed with the simplified

V O L U M E 26, N O . 8, A U G U S T 1 9 5 4 procedure. This ie ronfirniation of Renham’s conclusions ( 2 ) t h a t an e\cesi of alkali and the uce of petroleum ether both result in lon-er and variable results. The ney, simplified prorrdure used in conjunction with the modified liver-storage hioessay ( I ) yields data from which a docieresponse curve is ohtained which is linear and passes through the point, of zei’o dose-zri,o iesponse (origin). The use of less accurate analytic~alterhniques usually leads to considerable destruction of vitamin A i\-hich is proportionatelygreaterat thelower concentrations of vitmnin A in the liver. Such data results in a dose-response relationship \vith a positive y-intercept a t zero response-i.e., a dose of 1 to 20 ;’ of vitamin -1must he fed before storage is observed. I-wuspected complications of this type led Koch and Kaplan (10) to the prroneous conclusion that t,he threshold dose of storage (y-intercept of dose-response curve) was proportional to the potency of the material fed. The nexv, simplified proc.edure thus result5 in significant,ly higher recovery of the vit;Lmin A from liver, compared with currently used procedures. This i,esults from an improved extraction technique with feiver manipulations and involving less destruction by avoiding both hezit and prolonged manipulation of the extract. I t comhines both speed and precision which recommends it for use as a routine analysis. It also serves as an essential p u t of a liver-storage type bioassay which was developed

1381 for the accurate and economical determination of vitamin A in oils and pharmaceuticals ( 1 ) . LITERATURE C I T E D

Ames, S.R., and Harris, P. L., to be published. Benham, G. H., C a n . J . Resrarch, 22, 21B (1944). Fox, Sereck, Chairman, Informal Committee on U.S.1’. Assay Vitamin A, personal communication. Foy, J. R., and llorgareidge, K., .Ss.u.. CHEY.,20,304 (1948). Gallup, 14’. D., and Hoefer, J. d..IXD.ENG.CHEM..;is.i~.ED.,18, 288 (1946). Glover, J.. Goodxin. T. W,, and llorton, R. A, Riochem. J . , 41, 97 (1947). Guggenheim, K., and Koch. W., Ihid.,38,256 (1944). Harris, P. L., Ames, S.K.. and Brinkman, J. H., J . Ani. Chen. Soc., 73, 1252 (1951). Kascher, H. AI., and Baxter. J. G., ISD. Eso. C H E x . , . ~ A L . ED.,17,499 (1945). Koch, W.,and Kaplan, D., 2. Vitaminforsch,, 22, 15 (1950). Lemley, J. AI., Brown. R. -4., Bird, 0. D., and Emmett, A. D.. J . S t d r i f t o n ,33, 53 (1947). Morton, Ifay 8, 1954. Paper X I 1 a series entitled +,BiochemicalStudies on Vitamin *,,, Communication 183 from the ~~~~~~~hLaboratories, Distillation products Industries, ~ i ~of i Eastman Iiodak Co., Rochester, N.Y .

Spectrophotometric Determination of Arsenic, Phosphorus, and Silicon in the Presence of Each Other M I C H A E L A. DeSESA’ and L O C K H A R T B. R O G E R S Department of Chemistry and Laboratory for Nuclear Science, Massachusetts Institute of Technology, Cambridge 39, Mass.

D

U R I S G the investigation leading to the spectrophotometric determination of traces of silica in magnesium oside and

magnesium carbonate ( 1 as the yellow molybdosilicic nc.id, various methods for removal of the interference from phosphorus and arsenic were investigated. Selective extraction of the heteropoly acids of arsenic and phosphorus was one of the methods considered. The literature on estraction of heteropoly acids has recently been summarized by Wadelin and Mellon (3),and the technique was used for the determination of phosphorus. Hure and Ortis (2) hiid used ethyl acetate to extract molyhtlophosphoric acid from a mixture of the heteropoly acids of silicon and phosphorus This technique was briefly investigated and shown to be feasible but more time-consuming than simply using tartaric acid to bleach selectively molybd’oarsenic and niolybdophoPphoric acids. However, while investigating the solvent estraction technique, a method \vas developed for the separation and determination of arsenic, phosphorus, and silicon, as the yellow heteropoly acids formed with molybdate. This note reports the preliminary investigation%leading to this method and the brief tests conducted on the procedure. h s the authors do not intend to continue this investigation further, the results have been reported so that interested persons may use the proposed or a modified procedure for the analysis of traces of arsenic, phosphorus, and silica in one sample. APPARATUS 4 R D R E 4 G E N T S

Absorbance measurements were made with a Beckman Ilodel DC quartz spectrophotometer using matched 1.00-em silica cells. Stock solutions of 10 mg. per ml. of soluble silica, arsenic(S’), ~

1

Present address, American Cyanamid Co., Winchester, Mass

and phosphorus(V) were prepared by dissolving 4.7 grams of sodium metasilirate (SarSiOa.SHjO), 1.2 grams of disodium arsenate (Sa?H..\sOd.iH?O), and 12 grams of trisodium phosphate (?;arP04. 12H20), respectively, in distilled water and diluting the resultant solutions to 100 ml. These solutions were standardized gravimetrically, and standard solutions of 100 */ per nil. of desired constituents were prepared by dilution. EXPERI3IEZIT.4 L

Preliminary Studies. The standard procedure previouslv developed for development and measurement of the color of the heteropoly acids mas employed (1). An aliquot, of the desired standard solution \vas pipetted into a 100-ml. volumetric flask. Five milliliters of a solution containing 40 nig. per ml. of m o l j h denum(V1) oxide were added and the solution mixed. Two milliliters of 60% perchloric acid were added, and the solution was made u p to 100 ml., and thoroughly mixed. Finally the absorbance was measured at 332 rng against a blank rontitining n l l the reagents used in developing the color. Solutions prepared by the method described above had a p H of 0.7. Hure and Ortis ( 2 ) reported that ethyl acrtate would extract molybdoarsenic and molybdophosphoric, acids a t p H 1. I n order to check the possible quantitative aspwts of an extraction technique, solutions of 5 p.p.m. of eilica as silicate, 10 p.p.m. of phosphate, and 50 p.p.m. of arsenate were made up according to the standard procedure, and 20-ml. aliquots of each of the three heteropoly acids were taken. I n one study, they were extracted once with 20 ml. of ethyl acetxte; in a second, extracted twice. The absorbance of the aqueous phase was measured a t 332 mp against an extracted and an unextracted blank. The results of this study are shown in Table T. The ethyl acetate extracts the molybdophosphoric and molybdoarsenic acids t o some degree. However, if a single or double extraction was used to remove arsenic and phosphorus before measuring the remaining molybdosilicic acid, appreciable errors

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