Table I. Reactivity of B C h , B O s , and ZrOZ.Si02 Zr 0 2 . Compounds ZrC14 ZrO: Si02 Direct reaction with L Borax fusion (Alizarin test) + Reaction of borax-fused sample
+
with L
Conversion to ZrCh with cc14 Conversion to ZrClr after boraxfusion with CCla
-
+
+
+
-
__ -
+ +
+
ores and therefore the hafnium base peak, l*oHfL3+,is observable 90 mass units above the zirconium base peak at about one hundredth the intensity of the latter peak. The theoretical relative abundance at the cluster of ZrL3+ which was calculated considering the stable isotopes of zirconium, carbon ( l ZC and C ) and oxygen (l 60 and IsO) is also given. 2H and 1 7 0 were neglected in this calculation because of their low abundances. The observed and theoretical mass ratios between the first and second peak in the ZrL3+clusters are also shown in Figure 1 for the sake of rapid, accurate identification of mass number without the need of a standard which can often complicate the analysis of very small samples. Rapid identification of a peak is particularly important in trace analysis using the stable isotope dilution method (IO, 11).
Table I shows a summary of the reactivities of zirconium chloride, zirconium oxide, and zircon. This method for preparing the zirconium P-diketonates has the following advantages. First, it is applicable to most compounds containing zirconium. Second, an almost completely anhydrous state can be obtained during reactions because Small amounts of water present in the reagents, which may cause hydrolysis of ZrC14 and decomposition of the formed j3-diketonates, are converted into COCh and HCI. These gaseous products are easily removed from the reaction system and do not interfere in later chelating reactions. Third, a sealed glass capillary is suitable for the preparation of the chelate samples for trace analysis of zirconium by mass spectrometry or gas chromatography. This procedure is also applicable to the synthesis of j3-diketonates of many other metals. Throughout this work, parallel synthesis of the corresponding hafnium compounds has been carried out, with further work on the trace analysis of zirconium and hafnium in rocks and ores currently in progress. ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of David Rosenthal for his cooperation and for the use of the facilities of the Research Triangle Center for mass spectrometry, which is supported by the Biotechnology Resources Branch of the NIH under Grant Number PR-330.
~~
(IO) J. K. Terlouw and J. J. De Ridder, Z . Anal. Cheni., 250, 166 (1970). (11) N. M. Frew, J. J. Leafy, and T. L. Isenhour, ANAL.CHEM., 44, 665 (1972).
RECEIVED for review May 12, 1972. Accepted August 11, 1972. Work supported by Materials Research Center, University of North Carolina, under Contract Number DAHCIS67-(2-0223 with the Advanced Research Projects Agency.
Simple Separation of Vitamins D from Sterols and Retino1by Argentation Thin-Layer Chromatography David Sklan and Pierre Budowski Faculty o j Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel THE SEPARATION OF CALCIFEROLS from accompanying materials prior to their physicochemical determination is of paramount importance in the determination of vitamins D in pharmaceutical preparations, enriched food and feeds, and biological materials. The main compounds to be removed are sterols and retinol which accompany vitamins D in tissues and many enriched products, and interfere especially with the antimony trichloride color reaction. Retinol is readily retained by Florex columns, and this separation technique is being used in combination with column partition chromatography ( I ) or digitonin precipitation of sterols ( 2 , 3). Conversion of vitamins D to jsotachysterol (4-6)
(1) United States Pharmacopeia, 17th Rev., Mack Publishing Co., Easton, Pa., 1965. p 891. (2) P. P. Nair, C. Buchana. S. De Leon, and D. A. Turner, ANAL. CHEM., 37, 63 1 (1965). (3) J. Eisses and H. De Vriess. J . Ass. Ofic.Agr. Chem., 52, 1189 (1 969).
200
provides additional possibilities of removal of interfering substances prior to gas-liquid chromatography (GLC) of isotachysterol esters. An adsorption chromatographic procedure on thin layers of silica gel G has been described, which however, does not separate vitamins D from retinol (7). This is achieved by partition thin-layer chromatography (TLC) (8), but the calciferols are not well separated from each other. The problems of efficient separation of vitamins D becomes particularly acute in low potency materials such as foods and (4) T. K. Murray, K. C. Day, and E. Kodicek, Biochem. J . , 98, 293 (1966). (5) A. J. Sheppard, D. E. La Croix, and A. R. Prosser, J . Ass. Ofic. Agr. Chem., 51, 833 (1968). (6) P. P. Nair and S. De Leon, Arch. Biochem. Biophys., 128, 663 (1968). (7) H. Janecke and I. Maas-Goebels, Freselzius’ Z . A d . Chem., 178, 161 (1960). (8) H . R. Bolliger, in “Thin-Layer Chromatography,” E. Stahl, Ed., Springer, Berlin, 1965, p 223.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1973
Table I. R, Values of Vitamins D and Some Other Unsaponifiable Compounds' Compound Rf Retinol 0.17 Ergocalciferol 0.35 Cholecalciferol 0.42 Ergosterol 0.64 0.69 D,L-a-Tocopherol Stigmasterol 0.71 Cholesterol, campesterol, @-sitosterol 0.75 Anhydroretinol 0.97 a Amounts applied: 0.2-1 mg.
Table I€. Recovery of [4-~4C]cholecalciferol Added to 5 ml of Calf Plasma [4-14C]cholecalciferol added, ,ug Recovery, 3.0 1.25 0.625 0.15 0.0075 Q
92.5 f 2 . 1 ~ 92.0 31 1.8. 92.3* 93.3 f 2.3" 93.1 f 1 . 9
Mean of 3 determinations f standard deviation.
* Mean of 2 determinations.
~~~
animal tissues where the ratio of unsaponifiables to vitamins D is very large. We describe a simple TLC technique for the separation of ergocalciferol and cholecalciferol from each other and from the usual accompanying unsaponifiable compounds. All solvents used were analytical grade and were redistilled before use, except chloroform which was washed with water and shaken with active alumina. All compounds tested were previously found to yield single spots upon TLC under the conditions described. [4-~4C]cholecalciferol (32.3 mCi/ mmole) was from the Radiochemical Center, Amersham, England. Conventional glass TLC plates, 20 cm x 20 cm, were prepared, coated with 0.4-mni thick layers of silica gel G (Merck AG, Darmstadt) impregnated with 5 (w/w) silver nitrate. After drying the plates at room temperature for 30 min and at 105 "C for 1 hour, test compounds were applied in chloroform solution. The plates were immediately transferred to glass tanks and developed with chloroform-acetone 9: 1 (v/v). The developed plates were kept in developing tanks under a stream of nitrogen before spraying with an 0.05 ethanolic solution of fluorescein and visualization under short-wave UV light. In recovery experiments, cholecalciferol areas were localized with the help of a reference standard and the marked areas were removed and extracted with acetone as quickly as possible to minimize oxidation.
z
Table I shows the approximate R f values obtained. Retinol is relatively immobile, whereas sterols and a-tocopherol move far ahead, permitting good separation of the calciferols. The latter are readily separable from each other. Chromatographic losses of cholecalciferol were about 4 %, as determined by radioactivity of [4-~4C]cholecalciferol,UV absorption at 265 nm, and the area of the double peak obtained upon GLC (9) before and after TLC. A series of recovery experiments were undertaken in which small amounts of [4-14C]cholecalciferolwere added to calf plasma samples which were carried through the saponificationextraction procedure of Nair et a/. ( 2 ) , followed by TLC as described above. Overall recovery was constant and close to 93 over a 400-fold range of concentrations of vitamin Da, even when the amount added was below the physiological range (Table 11). The present technique is simple and appears to overcome the problems of separation from retinol and sterols and losses of vitamin D during the separation. It should be useful in the colorimetric or GLC determination of vitamins D. RECEIVED for review July 14, 1972. Accepted August 28, 1972. (9) H. Ziffer, W. J. A. Vandenheuvel, E. 0. A. Hahti, and E. C. Horning, J . Amer. Clrem. Soc., 82,6411 (1960).
Spectrophotometric Determination of Uranium(V1) with Chromazurol S and Cetylpyridinium Bromide C. L. Leong National Institute for Scientific and Industrial Research, Lot IO & 12, Phase 111 Kawasan MIEL, Shah Alam, Selangor, Malaysia CONTINU~NG THE SERIES of investigations on the use of ternary complexes ( I , 2) in spectrophotometric trace determination of metals, it was found that UOpz+formed a soluble blue complex with chromazurol S and cetylpyridinium bromide (CPB). This paper reports the results of the study of the color reaction with a view to developing it into a suitable spectrophotometric method for U(V1). --__
(1) C. L. Leong, Ancrlyst (Loridon),95,1018 (1970). (2) C. L. Leong, Tolurita, 18, 845 (1971).
___
The reagents used were: lo00 ppm uranium(V1) (as u012-t)solution. Dissolve 2.1094 grams of uranyl nitrate hexahydrate in distilled water and make up to 1 liter. 10-3M Chromazurol S. Dissolve 0.5389 gram of chromazurol S in 1 liter of distilled water. 10-2M Cetylpyridinium Bromide. Dissolve 3.8445 grams of cetylpyridinium bromide in liter of 20zaqueous methanol.
,
RESULTS AND DISCUSSION Spectral Characteristics. In Figure 1, curves A and B show the absorption spectra of chromazurol S and its uraANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
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