Table I. R, Values of Vitamins D and Some Other Unsaponifiable Compounds'
Compound Retinol Ergocalciferol Cholecalciferol Ergosterol D,L-a-Tocopherol Stigmasterol Cholesterol, campesterol, @-sitosterol Anhydroretinol a Amounts applied: 0.2-1 mg.
Table I€. Recovery of [4-~4C]cholecalciferol Added to 5 ml of Calf Plasma
Rf 0.17
[4-14C]cholecalciferol added, ,ug
0.35
Recovery,
3.0 1.25 0.625 0.15 0.0075
0.42
0.64 0.69
0.71 0.75 0.97
Q
92.5 f 2 . 1 ~ 92.0 k 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]cholecalciferol were 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 M I E L , 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
201
i;: W
T-LoB
06'
05
550
+ +
nm
610
'50
WAVE LENGTH
Figure 1. Absorption spectra of U(VI)-chromazurol S-CPB system Curve A . 5 ml 10-3 M chromazurol S at pH 4.8 diluted to 50 ml Curve B. 5 ml low3M chromazurol S 1 ml 100 pg/ml U(V1) at pH 4.8 diluted to 50 ml Curve C. 5 ml M chromazurol S 1 ml 100 pg/ml U(V1) 5 mi M CPB at pH 4.8 diluted to 50 ml
+ +
+
PH
Figure 2. Variation of absorbance with pH
+
Curve A . 2 ml 10-3 M chromazurol S 2 ml M CPB diluted to 50 ml and at varying pH, measured against water blank Curve B. 2 ml M chromazurol S 2 ml M CPB 0.5 ml 100 pg/ml U(V1) diluted to 50 ml and at varying pH, measured against water blank Curve C. Same as B measured against reagent blank
+
+
nium(V1) complex, respectively, at pH 4.8. Curve C shows the effect of the addition of CPB to the complex as represented in Curve B . The expected bathochromic shift ( I , 2 ) in the absorption maximum of the complex from 495 to 625 nm where the reagent absorbance is small facilitates analytical measurements. Optimum Conditions for Color Development. EFFECTOF pH. The effect of pH on the absorption of the complex was studied at 625 nm with solutions containing 0.5 ml of 100 pg/ml U(V1) solution, 2 ml of 10-3M chromazurol S and 2 ml of 10-*M CPB solutions. In Figure 2, Curve A and Curve B show the variation with pH of the absorbance of the reagent and the complex, measured against water as blank, while Curve C shows the absorbance variation of the complex 202
Curve A. 1 ml100 pg/ml U(V1) 5 ml M CPB at pH 4.80 and diluted to 50 ml with varying amounts of chromazurol S Curve B. 1 ml 100 fig/ml U(V1) 3 ml 10-3 M chromazurol S at pH 4.80 and diluted to 50 ml with varying amounts of CPB
measured against the reagent blank. pH 4.8 was chosen for all further work. No buffer was used in the study, the solutions being carefully adjusted with dilute hydrochloric acid to the required pH before absorbance measurements were taken and the pH values were checked again afterwards. Figure 3 shows the EFFECTOF REAGENT CONCENTRATION. effect of the variation of the concentrations of chromazurol S and CPB. EFFECTOF TIMEAND ORDEROF ADDITIONOF REAGENTS. The stability of the color was studied at an average temperature of 20 "C. Maximum absorbance was obtained in 15 minutes and the absorbance remained constant for at least 24 hours. The order of addition of reagents was unimportant. SOLVENTEXTRACTIONSTUDY. The complex was not extractable by the following solvents. Chloroform, carbon tetrachloride, ether, nitrobenzene, nitromethane, isobutyl ketone, isoamyl alcohol, benzyl alcohol. PREPARATION OF CALIBRATION GRAPH. The calibration graph was prepared by transferring from 1.0 ml to 8.0 ml of 25 pg ml-1 U(V1) standard solution and 3.0 ml of 10-3M chromazurol S into a series of beakers; 2.0 ml of 10-2M CPB was then added, and the solution was diluted to about 40 ml with distilled water. The pH of each solution was adjusted to 4.8 with dilute hydrochloric acid measured with a pH meter. The solutions were then transferred to 50-ml calibrated flasks and diluted to the mark. After 15 minutes, the absorbances were measured in 1-cm cuvettes against a reagent blank at 625 nm. BEER'SLAWAND SENSITIVITY OF REACTION.The calibration graph obeyed Beer's law in the range 0 to 100 pg of U(V1) in 50 ml of solution. The color reaction has a molar absorptivity of 9.9 X l o 4at 625 nm. REPRODUCIBILITY OF REACTION.The reproducibility of the color reaction was studied. The relative standard deviation was 3.0z [12 variates of 25 pg U(VI)]. INTERFERENCES STUDY. A foreign ion was taken to interfere if it caused an error in absorbance corresponding to twice the standard deviation of the pure solution, containing 100 pg U(V1). No interference was found for 100-fold weight excess of Ca(II), Pb(II), Mn(II), Co(II), Mg(II), Ni(II), Zn(II), W(VI), Ti(IV), La(III), Nd(III), Sm(III), Gd(III), and 10-fold weight excess of Eu(II1). A 100-fold weight excess of Al(III), Cr(VI), Be(II), Fe(III), Bi(III), Mo(VI), Yb(III), Dy(III), and Ce(II1) gave serious interferences, while a 2-fold weight excess of Th(1V) gave interference.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
The proposed reaction suffers from serious interferences from many ions, and preliminary separation of U(V1) by such techniques as ion exchange (5) is necessary before determination.
Discussion. The proposed ternary system is one of the most sensitive for U(V1) comparable to the reagent rhodamine B (3) which has a molar absorptivity of 1.02 x lo5. The rhodamine B method, however, involves extraction of the uranium-rhodamine B complex into a benzene-etherhexane solvent. Other sensitive reagents for U(V1) include chlorophosphonazo I11 ( 4 ) with a molar absorptivity of 7.86 X lo4.
RECEIVED for review May 3,1972. Accepted July 31,1972.
(4) A. A. Nemodruk, Yu. P. Novikov, A. M. Lukim, and I. D. Kalinina, Zh. Anal. Khim., 16, 180 (1961). (5) F. Tera and J. Korkisch, Anal. Chim. Acta., 25,222 (1961).
(3) H. H. Moeken and W. A. H. Van Neste, Anal. Chim. Acta, 37, 480 (1967).
Graphite Rod Atomizer in Atomic Absorption Spectrometry for Direct Determination of Iron in Serum M. T. Glenn and J. Savory' Department of Pathology, School of Medicine, Gainesuille, Fla. 32601
S. A. Fein,2 R. D. reeve^,^ C. J. Molnar, and J. D. Winefordner4 Department of Chemistry, University of Florida, Gainesuille, Fla. 32601
DURING THE PAST FEW YEARS, several types of non-flame atomizers for atomic absorption spectrometry have been fully described (1-3). Because non-flame atomization has resulted in atomic absorption absolute detection limits in the picogram region, much attention has been given recently t o the possible advantages for using non-flame atomic absorption spectrometry for the measurement of trace metals in real samples, e.g., in jet engine oils (4-7) and also in biological materials (8-10). Most recently, Kubasik, Volosin, and Murray (11) have described a method for analysis of lead in whole blood where only a dilution of the whole blood sample was required. In the present study, a method for direct analysis of iron in 1-p1 samples of serum is described, and a correlation study with a n automated spectrophotometric procedure is presented. Present address, Clinical Laboratory, North Carolina Chemistry Laboratory, University of North Carolina, Chapel Hill, N.C. 27514. * Present address, 6575 Allison Rd, Miami Beach, Fla. 33141. On leave, Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand. Author to whom reprint requests should be sent.
(1) G. F. Kirkbright, Analyst, 96, 609 (1971). (2) M. D. Amos, Amer. Lab., 33 (Aug. 1970). (3) T. S. West and X. K. Williams, Anal. Cliim. Acta, 45,27 (1969). CHEM., 43,1557 (1971). (4) K. G. Brodie and J. P. Matousek, ANAL. (5) J. F. Alder and T. S . West, Anal. Cliim. Acta, 58, 331 (1972). (6) S. Omang, ibid., 56, 470 (1971). (7) R. D. Reeves, C. J. Molnar, M. T. Glenn, and J. D. Wine-
fordner, ANAL.CHEM.,in press. (8) M. D. Amos, P. A. Bennett, K. G. Brodie, P. W. Y.Lung, and J. P. Matousek, ibid., 43,211 (1971). (9) J. P. Matousek and B. J. Stevens, Clin. Ciiem., 17, 363 (1971). ( I O ) M. Glenn, J. Savory, L. Hart T. Glenn, and J. D. Winefordner, Anal. Ciiim. Acta, 57, 263 (1971). (11) N. P. Kubasik, M. T. Volosin, and M. H. Murray, Clin. Cliem., 18, 410 (1972).
0.1
SERUM 432
SERUM 432
Y
* a
V
-
2 0.05 0. I v)
1,
0
Figure 1. Typical recorder tracings EXPERIMENTAL Apparatus. A modified graphite rod atomizer (GRA) described by Molnar et al. (12) was mounted in place of the burner in a Perkin-Elmer 303 atomic absorption spectrophotometer (wavelength 284.3 nm, slit 3, scale expansion 1, noise 1) equipped with a recorder readout unit, deuterium background corrector, and a Brown strip chart recorder. The photomultiplier output was also connected to an Autolab 6230 digital integrator with print-out facility. A high intensity Perkin-Elmer iron hollow-cathode lamp was operated at manufacturer's recommended maximum current. Graphite rods were machined from Poco FX91 graphite (Poco Graphite, Inc., Decatur, Texas 76234), and a cylindrical (12) C. J. Molnar, R. D. Reeves, J. D. Winefordner, M. T. Glenn,
J. R. Ahlstrom, and J. Savory, Appl. Spectrosc., in press.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
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