The reagent reacts rapidly with most phosphate insecticides, requiring no digestion other than volatilization of solvent and excess reagent on a steam bath and/or a hot plate, a process which usually requires about 10 minutes. However, some phosphorus-containing materials, including aryl esters of phosphates, phosphonamides, and dithiophosphate esters such as O,O,Striethyl phosphorodithioate, required 30 minutes or more of digestion on a steam bath in a covered crucible. Triphenyl phosphine required an even longer digestion. Thiophosphate esters required no special treatment. Dithiophosphate esters, in aqueous or nonaqueous solution, were pretreated with aqueous bromine solution. The excess bromine was boiled off, reagent was added, and procedure was continued as described. This allowed determination of phosphorus without further digestion. The digestion reagent forms nonvolatile calcium phosphorus-containing salts, thus allowing removal of solvents, excess reagent, and carbonaceous materials without loss of phosphorus. In the process of volatilizing solvents and removing carbonaceous materials by combustion, the conversion of phosphorus to orthophosphate is completed.
Recovery of Phosphorus by Use of
The relatively high temperatures used in the procedure will cause loss of phosphorus-containing materials unless provision is made to produce nonvolatile salts. Table I1 shows the results of tests with phosphoric acid salts of sodium, potassium, and calcium. Only with calcium was recovery quantitative. While other bivalent metals may be as effective as calcium for retaining orthophosphate, their use was not investigated. The loss of orthophosphate when monovalent alkali metals are used is probably due to dehydration of the phosphate, and formation of pyrophosphates which do not produce molybdenum blue color. When these metals are used, the color develops slowly a t steam bath temperature, whereas with calcium, maximum color develops in a few minutes. The molybdenum blue method is a convenient, sensitive, and reproducible method for determining orthophosphate. The color intensity is dependent upon pH; more color is obtained at 1N H&04 than at either 0.5N or 1.5N. Since the residue after combustion is neutral, the required pH control is obtained by adjusting the acidity of the molybdate reagent. With the reagents Calcium.
described, maximum color is produced in about 7 minutes on a steam bath. Much longer heating periods of an hour or more did not change the color density. Once the color had developed, allowing the solution to stand a t room temperature for 2 or 3 days caused no change in absorbance. Because of dehydration by the heat treatment, silicate does not interfere. Since the procedure allows processing of several samples at once, a large number of determinations can be made per day. Results are shown in Table 111. LITERATURE CITED
(1) Barney, J. E., Bergmann, J. G., Tuskan, W. G., ANAL. CHEX. 31, 1394-6 (1959). ( 2 ) Fleischer, K. E., Southworth, B. C., Hodecker, J. H., Tuckerman, M. N., Zbid., 30, 152-4 (1958). \ - - - - I
(3) Goodwin, J. F., Thibert, R., McCann, D., Boyle, A. J., Zbid., p 1097-9. (4) Hoffman, F. F., Jones,%. C., Robbine, 0. E., Alsbert, F. F., Zbid., pp. 1334-6. (5) !o.hnson, Clarence hl., Llrich, Albert, Divlsion of Agricultural Sciences, University of California, Berkeley, Calif., Bull. 766, 1959. (6) M-ay, Roy, ANAL. CHEK 31, 30s-10 (1909). RECEIVEDfor review May 4, 1962. Resubmitted May 31, 1963. Accepted October 17, 1963.
Colorimetric Determination of Vitamin A and Its Derivatives with Trifluoroacetic Acid RICHARD E. DUGAN, NORMAN A. FRIGERIO, and JOHN M. SIEBERT Division of Biological and Medical Research, Argonne National laboratory, Argonne, Ill.,and Chemistry Department, St. Procopius College, lisle, 111.
b A colorimetric determination for vitamin A and its derivatives with trifluoroacetic acid is described. This reagent produces a spectrally identical colored species to that formed by SbCI, the Carr-Price reagent. Analysis with triftuoroacetic acid retains the advantages while eliminating some of the disadvantages of the CarrPrice determination. Reactions of vitamin A and similar polyenes with a number of Lewis acids are described also. The feasibility of these systems for analytical determinations and the similarity of the chemistry of the reactions is examined.
T
determinations of vitamin A were performed with H2S04 (6) and AsCla (IS). Treatment of vitamin A solutions with these reagents produced a transient blue color. In 1926, Carr and Price markedly improved the reliability of the HE EARLIEST COLORIMETRIC
114
ANALYTICAL CHEMISTRY
determination by reacting vitamin A solutions with SbCL in CHCL (3). Since that time a number of other reagents have been proposed for colorimetric analysis of vitamin A but use of SbCla, the Carr-Price reagent, has not been displaced as the method of choice (IO). Inherent limitations and precautions to be taken with these analyses have been discussed in reviews by Kofler and Rubin (8) and by Morton ( I I ) , and also by Moore in his treatise on vitamin A (IO). Unfortunately SbC13 is both disagreeable to handle and unstable to moisture. Reaction with even traces of water yields the insoluble SbOCl causing turbidity during analysis and forming opaque films which are very difficult to remove. Furthermore the colored species produced with SbC& is shortlived and color production is not specific for vitamin A. Carotenoids which often accompany vitamin A in the nonsaponifiable extracts of animal
products produce interfering colored species. Because of these shortcomings a reagent that would retain the sensitivity of the SbCL reaction while eliminating some of its disadvantages would improve the ease and reliability of colorimetric analysis. EXPERIMENTAL
Apparatus. A Cary Model l l M recording spectrophotometer with 1cm. quartz cells was used to measure spectra and absorbance changes as a function of time. A Bausch and Lomb Spectronic 20 spectrophotometer with 13- X 100-mm. borosilicate glass test tubes of 11.15-cm. path length was used to prepare calibration curves. This instrument was also used for the assay of samples of crystalline vitamin A and its derivatives and 8carotene and vitamin A content in saponified oils. Reagents. All crystalline vitamin A derivatives were obtained in sealed ampoules from Distillation Products
1 5 -
(zero t rnBi
:
> E
10
-
anhydrous SnCll for TFA and a saturated CHC13 solution of SbCL for TFA. SbC13 analyses for vitamin A were also performed at the usual higher ratios of SbCb reagent to vitamin A solution, 5:l. USP XVI vitamin A assays on the same samdes were made. Analyses also were carried out using TFAA in place of TFA as the color producing reagent. RESULTS
Figure 1. species
Spectra 'of TFA-vitamin A
1.5 ml. of 2 X 1 O-6M vitamin A alcohol in CHCls plus 1.5 ml. of TFA in 1-cm. cuvettes, Cary recording spectrophotometer
A number of Lewis acids were tested with vitamin A. Some examples of those that produced color are listed in Table I. In most cases, a blue color formed first with the solution absorbing maximally near 616 m p , ,A[ for these species have been reported between 616 and 620 mp from various laboratories using different instruments. For the Carr-Price reaction Morton 3 mp ( I I ) ] . The blue reports 618 decayed t o a longer lasting pink absorbing maximally a t wavelengths between 520 and 550 mp depending on the Lewis acid. The absorptivity of the pink species was less than l/3 that of the blue. The pink then faded to yellow or colorless. Spectra of the blue and pink species produced by TFA are shown in Figure 1. TKOof these Lewis acids, TFA and SnC4, produced a blue species with vitamin A identical in spectrum and equivalent in absorptivity (10) and stability to that produced by SbCl,, the Carr-Price reagent. Molar absorptivities and wavelengths of maximum absorption for the primary colored species produced by TFA with vitamin A derivatives are listed in Table 11. The Am.= for vitamin A aldehyde, like vitamin A alcohol, with TFA corresponds with that reported for SbCL (12). The Amax for vitamin A acid with TFA was the same as our results with SbC13. Equivalent results also were obtained with TFA and SbC13 for the absorptivity of each vitamin d derivative. Spectra of the primary colored species produced by reaction of TFA with vitamin il derivatives are shown in Figure 2. In the case of p-carotene, production of a highly colored species was slower and required a higher ratio of TFA than the 1 : l ratio of the TFA method. The short-lived, low-absorbing peak a t 585 mp that is sometimes used for carotene estimation with SbCl3 was produced using the TFA method. The absorption maximum, however, was at 780 mp and full color production required thirty minutes. At higher ratios of TFA to p-carotene (5:l and 1 O : l ) the absorption maximum was a t 710 mp. Maximum absorption a t this wavelength was reached in one to two minutes after mixing and the molar absorptivity exceeded 120,000. Measurement at this wavelength where
*
Industries and stored under vacuum a t 1' C. 6-Carotene and vitamin A palmitate in corn and palm oil were obtained from Hofmrtnn-LaRoche Co., Nutley, N. J., and stored similarly. Trifluoroacetic acid (TFA) and its anhydride (TFAA) were obtained from Matheson, Coleman rind Bell. Stannic chloride was Fisher Certified and antimony trichloride was Baker Analyzed. Solvents were reagent grade. Antimony trichloride reagent was a saturated (approximately 20%) solution of SbC13 in CHCL. The solution was filtered and stored in a brown bottle. Procedure. DETERMINATION OF VITAhrm A WITH TF A. Prepare solutions of saponified viitumin A reference standard in CHCIJ through the concentration range 3 X 10-6M to 1.2 X lo-W. Place 1.5 m . in a colorimeter test tube and add arb equal volume of TFA by syringe or rapid delivery pipet with agitation. Rapid delivery of TFA from a syringe with the needle below the surface of the CHC& suffices for agitation. Measure 2,bsorbance 10 seconds after delivery tit 616 mp in the Spectronic 20 or other suitable colorimeter. Prepare a calibration curve. Use the same procedure for 1.5-ml. CHC13 solutions of the vitamin A sample to be assayed. Saponificatjon and extraction of oils and other natural products is performed the same as in the USP XVI vitamin ii assay except that the ether extract is taken just to dryness with a helium or nitrogen gas stream and redissolved in CHC13instead of isopropyl alcohol. This procedure also may be used to analyze vitamin A Esters, vitamin A aldehyde, and vitaruin A acid. In each case, the initial concentrations may be determined by comparing the molar absorptivities to that of vitamin A (Table 11). Absorbance measurements are made a t the appropriate wavelengths listed in Table 11. Crystalline vitamin A and its derivatives and vitamin A ccntaining oils were analyzed by the TFA method described above. Analyses were also performed by the TFA method substituting liquid
Table 1.
Lewis Acids Producing Color with Vitamin A
LeR% acid CFaCOOH (CF&0)20 CzFbCOOH C9,COOH SnCl, SbCla HiSOi HClOi ClSOaH ClSO&H, TiC14 FeClr BFs SbClr
Initial color Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue -~.. Blue Blue-green Violet Violet ~
Secondary color Pink Pink Pink Pink Pink Pink Pink Pink Pink Pink Green Green Green Yellov
Table II. Optical Characteristics of Colored Species Produced with TFA
WaveMolar length absorptivity, maxima, liter/mole Carotenoid mp X cm. Vitamin A 616 145,000 Vitamin A acetate 616 145,000 Vitamin A palmitate 616 145,000 Vitamin A aldehyde 604 95,000 Vitamin A acid 574 53,000
little interference occurs of a species more stable and with a much greater absorptivity than the one peaking a t 585 mp should give a more :murate estimation. Both TFA and SnClr produced with vitamin A a blue species absorbing maximally a t 616 mp and conforming to Beer's law through the conceutration range 1 X 10-6X to 1 X l O - 5 M . The species produced by TFX with the vitamin A derivatives listed in Table I1
A (mpi
Figure 2.
Spectra of vitamin A alco-
hol, aldehyde, and acid ,before and after addition of TFA 1.5 ml. of CHCla solution of vltamin A derivatives plus 1.5 ml. of TFA 1 = Vitamin A alcohol 2 = Vitamin A acid 3 = Vitamin A aldehyde * = After treatment with TFA V O L 36. NO. 1, JANUARY 1964
1 15
Table 111.
Molar Absorptivity of Vitamin A Derivatives with TFA, TFAA, and SbCls by TFA Method
Vitamin
e(TFA) 145,000 145,000 145,000
Crys. vitamin A alcohol CTS. vitamin A acetate Vitamin A palmitate in corn oil
Table IV. Determination of Units of Vitamin A per Gram of Unsaponified Oils
USP Oil
Squibb cod liver Lofoten cod liver
XVI 1645 1695
TFA SbC13 (1:l) (1:l) 1050 1275
300 300
also conformed to Beer's law. Both TFA and SnC14 were completely stable in storage and miscible with the vitamin A solvents CH2C12, CHC13, and CCL. TFA was the least noxious of the two reagents with handling properties comparable to glacial acetic acid. TFA and its derivatives do not produce fluorosis, and have, in fact, been reported to be nontoxic (9). Anhydrous liquid SnC14 fumed so strongly as to be noxious and on prolonged exposure to air the surface became encrusted with solid, water soluble SnClr hydrates.
Table V.
e(TFAA) 95 ,000 95 ,000 95 ,000
Dilution of TFA and SnC1, with CHCl3 eliminated fuming and the solutions reacted with vitamin A solutions to form the blue species. After a period of days, TFA in CHC13 solutions decayed to products that did not form the blue species. The maximum value for the absorptivity of the adduct of vitamin A was obtained with TFA when an equal volume of the acid was mixed with vitamin A in CHCL. Most methods employing the Carr-Price reagent specify a t least a 4:1 ratio of a saturated solution of SbC13 in CHC13 to vitamin -4solution. Thus the dilution factor for vitamin A in the Carr-Price test is a t least 2 I / 2 times as great as with TFA so that the minimum detectable quantity of vitamin A is thereby increased by the same factor. When SbC1, was tested in the 1:l proportion used in the TFA method, the absorptivity was less than 90% of that obtained with TFA (Table 111). At the higher prescribed ratios of SbCla to vitamin A which reduce the
Determination of Units of Vitamin A per Gram
Oil Squibb cod liver Lofoten cod liver Vitmin A palmitate (in palm oil) Vitamin A palmitate (in corn oil) Peanut oil
Table VI.
t(SbCl8) 130,000 130,000 130,000
USP XVI 1645 1695 1,780 ,000 1,010,000 0
of Saponified Oils
Manufacturers specific& tions SbCls (5:l) TFA (1:l) 1700 1655 1690 2250 1865 1825 1,695,000 1,795,000 1,805,000 1,000,000 1,030,000 1,005,000 0
0
Absorbances of Carotenoid Mixtures with TFAA and TFA at 61 6 mp
Component A (1 ml.)
A acetate A aldehyde A acid
B-carotene A acetate A acetate A acetate A acetate A aldehyde A acetate
Component B (1 ml.1 CHCls CHCL CHClr CHCls A aldehyde A acid p-carotene CHCL CHCla A aldehyde
Component C (2 ml.) TFAA TFAA TFAA TFAA TFAA TFAA TFAA TFA TFA TFA
Absorbance 0.70 0.05 0.00 0 .oo
0.71 0.69 0.71 1.06 0.36 1.40
Molar concentration-of carotenoid solutions in CHCla X lo6: Vitamin A acetate, 7.5; Vitamin A aldehyde, 7.7; Vitamin A acid, 8.0; 8-carotene,3.5.
116
ANALYTICAL CHEMISTRY
Figure 3. Time dependence of TFAvitamin A absorbance at 61 6 mp 1.5 ml. of CHC13 solution of vitamin A alcohol plus 1.5 ml. of TFA
sensitivity, increasing the minimum detectable quantity, the absorptivity was equal to that obtained with TFA. Determinations of unsaponified fish oils by the TF-4 method with TFA and with SbCls were compared with each other and with the values obtained by the USP XVI analysis (Table IV). The inhibition of color by oil components ma9 much less for TFA than for SbCl3. Nevertheless, the low results as compared to those achieved by the USP XVI method indicate that saponification and extraction are necessary prior to determination. Analysis of saponified oils by the TFA method, the Carr-Price method, and the USP XVI assay are presented in Table V. Amounts of vitamin A obtained by the TFA method and SbC13 (5:l) were determined by preparing calibration curves using vitamin A reference oil. TFA proved 10% more sensitive and the species formed slightly more stable than that formed by SbCla with the saponified oils. The correspondence of the results obtained indicates that the TFA method substitutes satisfactorily for the CarrPrice method. The stability of the blue-colored species (Figure 3) was dependent on both the purity of the system and the type of cuvette used. For example, addition of crude fish oils or aliphatic peroxides such as boiled linseed oil to vitamin A solutions greatly shortened the lifetime of the blue species. Similarly, its lifetime was much longer in the narrow-necked, Teflon-stoppered, Cary cuvettes than in open test tubes. I n either case decay of the blue species to a pink one (A, 535 mp, e = 40,000 liter per mole x cm.) proceeded in still solutions from the upper surface downwards. This suggested that decay of the blue species was catalyzed by some atmospheric component. Small amounts of added water or carbon dioxide failed to accelerate decay. Therefore, it is possible that decay is promoted by absorption of oxygen perhaps with formation of aliphatic
peroxides. Such percxides are known to cause epoxidation of carotenoids (7). These epoxides in turn rearrange rapidly in the presence of acids to pink species. Large amounts of water, ethanol, ethers, unsaturated aliphatics, and similar Lewis bases interfere by tying up the added Lewis acid, but their action can be partially reversed by addition of still more Lewis acid. Our attempts invariably geJveless than 50% recovery on the readdil ion of Lewis acid. Lewis bases should never be used as solvents for production of the blue species nor be present in even moderate concentration during determination. TFAA produced a species spectrally identical to that produced by TFA and other Lewis acids, although the absorptivity was less (Table 111), probably because the weaker Lewis acid, TFAA, did not react completely. Unlike TFA, however. TFAA prodt ced a measurable blue color only with vitamin A alcohol and its esters. Faint color was produced with vitamin L4 acid and pcarotene only after several minutes. Vitamin A aldehyde pith TFAA gave a blue flash of less than 0.2 second duration. TFA-4 added to mixtures of vitamin A alcohol with vitamin A aldehyde. vitamin A acid, or /3carotene produced absorbances a t 616 mp that corresponded to vitamin A alcohol content (Table VI). Interference from p-caro,ene, vitamin A aldehyde, and vitarrtin A acid was negligible. TFAA reacted with vitamin A aldehyde to produce an adduct that did not react with TFA or other Lewis acids to produce color; however, T F h A did not destroy &carotene or vitamin A acid. With these, suhsequent addition of TF.4 gave rise to the species absorbing maximally at 780 and 574 mp. These results indicate a more specific reagent for vitamin A determination and the possibility of determining vitamin A, its derivatives, and carotene in the presence of on(: another. However, results with fish oils did not match those in Table VI for solutions of crystalline compounds. The nonsaponifiable fraction of the oils contained enough impurities to prevent full color production by the weaker Lewis acid, TFAA. Also, the decay of the color produced was too rapid for a practicable analytical method. DISCUSSION
Examination of re2gents that had previously been emplo.yed for vitamin A determination revealed that almost all were strong Lewis acids. Furthermore, the absorption maxima of the species produced were charctcteristic of the carotenoid but independent of the Lewis acid. Thus, the reacion could be re-
garded as a coupling of the empty orbitals of these Lewis acids with the ?r electrons of the conjugated polyene. This,perhaps, is followed by dismutation into two charged species, with that formed from the carotenoid absorbing at longer wavelengths because of the increased probability space available to the T electrons. There are several exceptions to the two generalizations proposed above. For example, the analytical reagent glycerol dichlorohydrin (16) is neither a strong Lewis acid nor does it produce a blue species absorbing maximally near 616 mp, as is characteristic. Rather it produces a pink species absorbing maximally a t 550 mp. However, the primary blue species formed by TFA, SbC13, and a number of other Lewis acids decay to a secondary pink species absorbing maximally from 520 and 550 mp. This decay is greatly accelerated by the introduction of Lewis bases such as alcohols. Inasmuch as glycerol dichlorohydrin is an alcohol, this may account for the almost instantaneous decay of the blue species and observation only of the pink one. I t has also been shown (1) that to be effective for vitamin A color production glycerol dichlorohydrin must be “activated” and the “activators” &re strong Lewis acids. Other methods of vitamin A determination, such as those with modified SbClr procedures (4, 14), HzS04 (W), and FeS04 (5) that depend on the formation of the pink species, likewise utilize Lewis bases such as alcohols, phenols, and water in the milieu. It seems likely that analytical methods based on color production by Lewis acid action on vitamin A are devised to adjust conditions to produce optimally one of these two species, the more sensitive blue or its Lewis base-induced decay product, the more stable pink. The purple color often observed in impure systems is the result of eye translation from a combination of the blue and the pink species. From the results of stability studies, it appears that formation of the pink species is the result of irreversible oxidative attack catalyzed by Lewis bases. It is also possible that the Lewis bases attack the colored species irreversibly and that apparent reversibility by addition of more Lewis acid is caused by reaction with hitherto unreacted vitamin A, or more likely anhydrovitamin A. It has been shown that SbC&removes water from vitamin A to form anhydrovitamin A (15) prior to formation of the blue species. The results of the examination of a number of Lewis acids for colorimetric estimation of vitamin A indicate that TFA is the best. This reagent is water soluble and does not form insoluble films or turbid solutions on contact with
water as SbCla does. It is far more stable in storage than the Carr-Price reagent. When working with microgram quantities the TFA method can detect a smaller amount of vitamin A than the Carr-Price test. With respect to other disadvantages. lack of specificity and instability of colored species, results with TFA were equal to the best achieved with the Carr- Price test and are more easily obtained in practice. A monograph on TFA listing physical and chemical properties, reactions, derivatives, and safety precautions is available from Halocarbon Products Corp. TFAA might answer the specificity problem in the colorimetric determination if a workable analytical method could be developed. This weaker Lewis acid is in the correct range to produce the colored species with the stronger Lewis base, vitamin A alcohol, but not with the weaker carotenoid hydrocarbons. LITERATURE CITED
(1) Allen, R. S., Fox, S. W., ANAL. CHEW22, 1291 (1950). (2) Biffoli, R., Boll. Lab. Chim. Provinciali (Bologna) 10,380 (1959).
(3) Carr, F. H., Price, E. A. , Biochem. J . 20, 497 (1926). (4) Cavina, G., Rend. Ist. Super. Sanitu
20,913 (1957).
(5) Craig, R. G., Bergquist, L. M., Searcy, L. R., Anal. Biochem. 1, 433
(1960). (6) Drummond, J. C. Watson, A. F., Analyst 47,341 (1922). (7) Karrer, P., Y3ostanze Naturale,’’ pp. 79-96, Accademia Nazionale et Lincei, Rome. 1961. (8) Kofler, M., Rubin, S. H., Vitamins and Hormones 18, 315 (1960). (9) Largent, E.. J., “Fluorosis” p. 68, Oh10 State Universitv Press. dolumbus,
. goc. 77,4120 (1955). ’ (13) Rosenheim, O . , Drummond, J. C., Biochem. J. 19, 753 (1925). (14) Rosenthal, E., Erdelgi, J., Biochem. J . 28,41 (1934). (15) Shantz, E. M., Cawley, J. D., Embree. N. M.. J . Am. Chem. SOC.65, 901 (1943). ’ (16) Sobel, A. E., Werbin, H., J . Biol. Chem. 159, 681 (1945). RECEIVEDfor review June 15, 1962. Resubmitted September 9, 1963. Accepted October 24, 1963. Division of Biological Chemistry, 140th Meeting, ACS, Chicago, Ill., September 1961. This work was performed in part under the auspices of the U. S.Atomic Energy Commission and supported in part by a granbin-aid from the Research Corporation Inc., t o St. Proco ius College. The work is taken, in part, Prom a dissertation submitted by J. M. Siebert t o the faculty of St. Procopius College in partial fulfillment of the requirements for the degree of Bachelor of Science. Division of Biological chemistry, 140th Meeting, ACS, Chicago, Illinois, September 1961. VOL. 36, NO. 1 JANUARY 1964
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