t
L
E a
5
o.g
-
1
44
0
--
x
1,6
L
3
E
N
0
L 0
' 2 :
1
3
'
I
6
3
6
9 9
12 12
'
dL*/(?M
2M
2,3
-u 1
15
15
Figure 3.
tailed badly on all components (Figure 2). This seems characteristic of this column with this class of compounds, since it operates satisfactorily with a paraffin mixture in the same boiling range. To achieve a separation without peak tailing, a number of substrates operable in this range (150' to 200' F.) were investigated. The Ucon 2000 200-foot X 0.01-inch column separated the 1-ethyl- and 2-ethylnaphthalenes without tailing and in a reasonable elution time (Figure 3). Retention data for these 15 components are tabulated in Table 11. I n investigating the quantitative aspects of this system, it was found that
18 18
21 21
24
~
21
30
33
36
39
42
- .-
45
EL U 1 I O N 1 IM E - Mi n ut es Separation of naphthalenes with Ucon 2000
these high boiling components could not be quantitatively split unless the temperature of the splitter tee was kept above the normal boiling point of the components being analyzed.
other complex compound mixtures, which are similar in boiling point and structure. LITERATURE CITED
&., Preprints, Division of Analytical Chemistry, 142nd Meeting ACS, Atlantic City, N. J., September 1962. (2) Fredrick, D. H., Cooke, W. D., Preprints, International Gas Chromatography Symposium, Michigan State University, East Lansing, Mich., 1961. D. 21. (3) King, R. W., Fabrizio, F. A,, Donnell, A. R., Zbid., p. 101. RECEIVEDfor review May 13, 1963. Accepted September 9, 1963. Southwest Regional Meeting, ACS, Dallas, Texas, December 1962.
(1) Ahlberg,. D. L., Walker, J. DISCUSSION
It nas possible to achieve qualitative analytical results for the naphthalenes boiling through 270' C. by correlating the spectra obtained from the m-bis(mphenoxyphenosy)benzene column and the Ucon 2000 column, since neither column would separate all the points of interest. This method (with appropriate liquid phases) may be applied to
Column Partition Chromatography of Vitamins A and D on Fluoropak 80 PHILIP S. CHEN, Jr., A. RAYMOND TEREPKA, and NANCY REMSEN Department of Radiation Biology, University of Rochester, Rochester, N . Y .
A reversed-phase column chromatographic system allowing the separation of vitamin D from vitamin A i s described. The stationary phase consisted of Fluoropak 80 impregnated with iso-octane and the mobile phase was aqueous methanol. Advantages include the use of easily volatile nonu.v.-absorbing solvents which facilitate spectrophotometry and liquid scintillation counting of eluted fractions. Examples illustrating the use of this column are presented. I.T€IOUGH
A
LARGE:
VARII, 1Y
01
techniques are available for purtition chromatography of polar conipounds, relatively few systems have 2030
ANALYTICAL CHEMISTRY
been described for such separation of nonpolar materials-e.$., vitamins A and D (4). Most methods for the latter substances rely on the theoretically less desirable adsorption systems (f ). This report introduces the u>e of Fluoropak 80 impregnated u ith isooctane as a stationary phase for the column partition chrornatographv of vitamin D. Use of this support eliminates a serious drawback of available partition methods, namely the difficulty of obtaining separated material free of the solvents used in chromatography. The mobile phase employed in moat of these studies ha. been '30% methanol. The column has been used mostly for
evaluating the purity of and for purifying radioactivity-labeled vitamin DS which may then be used for physiological or biochemical studies. In the latter studies, the Fluoropak column has been used to separate radioactive vitamin D3 from vitamin A in tissue extracts. EXPERIMENTAL
Apparatus. A 3 - f O O t glass column (12 mm. i d . ) with fritted glass disk and Teflon stopcock was used with the G M E Model V 152 fraction collector. Ultraviolet absorption was measured either on the collected fractions with the Ueckman DK-2 recording spectrophotometer or between the column and
fraction collector with the GME Model U.V. 265 I.F. recording attachment coupled to a Texas Instruments recorder. Radioactivity m s counted in tlie TMC Model LP-1. liquid scintillation counter. Reagents. Flouropak 80 (full range particle size) was purchased from tlie Fluorocarbon Co., 1206 East Ash Ave., Fullerton, Calif. Sieving revealed that practically all the particles were betvi-een 20 to 40 mesh, and material between these sizes was used. Isooctane (2,2,4-trinietl:ylpentane) T ~ obtained from Phillip:; Petroleum Co., Bartlesville, Okla. The scintillator solution added to each sample for rounting was 10 ml. oi' toluene containing 0.4% diphenylosa zole (DPO) and O.Olyo 1,4-di [2-(5-phenylosazoly1)]bcmenc (POPOP). Procedure. Equal volumes of isooctane and 90yo methanol were equilibrated. Some of the stationary liquid phase (iso-octane) was mixed with dry Fluoropak 80 to imp]-egnate the solid support. The column was filled with iso-octane and the impregnated Fluoropak 80 added ;o a height of 80 cm. The solid sur'port was then gently tamped with a glass rod until the column was 7 5 to 77 em. The isooctane level was lowered to the t'op of the Fluoropak and a t least 100 ml. of mobile phase (90% methanol) was s l o d y run through and the sample applied in about 1 nil. of mobile phase. Several hundred fig. of unlabeled vitamin D3 marker may be added to facilitate location to the vitamin D3 zone when separating radiolictive vitamin D3 from tissue extracts. Elution was then begun and fractions oi' 5 or 10 ml. were collected. For radiowtivity measurement, the test tube contents were transferred to a 10-dram glass vial, dried with air,, dissolved in scintillator solution, and counted. RESULTS AND D SCUSSION
Figure 1 shows a typical separation of itamin AI and D3 standards. Toluidine 131ur 0, 13 hich partitions exclusively into the methanol phase, was added simply to illustrate the solvent front, peak of which was at b O ml. With 90% methanol, vitamin -4 emerged very soon after the solvent front, while the peak of vitamin D3 occurred a t 220 ml. As evident from the figLre, separation of these tr\ o vitamins W Es excellent. Stability of T. itamin D on the Fluoropak 80 column was checked by running paper chromatograms of pooled column eluates in the syste ns described by Ihdicek ( 2 ) . The paper chromatograms were heated to 80° C. in an oven after dipping through 25% antimony trichloride/CHCl3 ( 3 ) . KO colored or fluorescent breakdown products were observed. Recoveries of vitan in D3 (as well as vitamin A) Lvere esseni ially quantitative a t 4' C. (column set up in walk-in refrigerator), as shown in Table I. A t summer room temperatures, re\
a MOBILE PHASE 9 90% 0% METHANOL STATIONARY STATIONARY PHASE PHASE ISG-OCTANE ISG-OCTANE - FLUGROPAK FLUGROPAK 80
Tot" din8 Blue 0
o*
2 m W W
i
z2
ea
10.50
a a
04-
9.
O4\
A
,F.l
034
02-
S
01-
P 5 IO FRACTION NUMBER
Figure 1.
Table 1.
15
20
25
30
35
40
25-30" c'.
60
55
Separation of vitamins A and D:%on Fluoropak 80
Added,
c.
50
Recoveries of Vitamins A and D from Fluoropak 80 Column
Temperature 4O
45
Sample Vitamin Dt Vitamin D3 Vitamin AI Vitamin D3 Vitamin D3 Vitamin Ds Vitamin A1 Vitamin D2
coreries of the less stable vitamin A were not so good as a t the lower temperature, but vitamin DS recoveries always exceeded 92%. A primary application of the Fluoropak column has been to purify radioactively labeled vitamin D3 for physiological or biochemical studies. An example is illustrated in Figure 2 where 1 mg. of vitamin Dr7-H3 was run. The radiochemical purity of the vitamin D3 exceeded 97y0 and the nonvitamin D3radioactivity was present in u.v.-absorbing material more polar than the vitamin and eluted earlier from the column.
fig.
Recovered, P&
Recovered, %
300 3000 420
295 2985 412
98.3 99.6 98.2
1077 1255 940 2950 400
1007 1155 896 2150 3S8
93 5 92.0 96.4 73.0 97.2
The radioactive contaminant present with the original labeled vitamin D3-i-H3could have arisen from breakdown of the vitamin D3 itself. A variety of experimental conditions can cause destruction of vitamin D. While it is not the purpose of the present paper to present extensive data on this point, it is necessary to indicate t,hat the Fluoropak column effectively separates vitamin D from its more polar breakdown products produced by ultraviolet irradiation, exposure to oxygen, and dispersion into water. An illustration of this type of separation is shown in Figure 3, where the last mentioned
01
Img. VIT D,-7-H3
A TEST TUBE FRACTION (IOrnl.) Figure 2.
Vitamin D3-7-H3 VOL. 35, NO. 13, DECEMBER 1963
2031
3500
H,O EFFECT ON VIT D3-4-CI4
LYOPHILIZED RAT KIDNEY
0.6
00 m p
3000
0.5
2500
0.4
P
2000
0.3
&
Q i,
8
I5O0
3 8 2P
0.2
1000
0. I 500 0
0
5
IO
15 20 25 30 35 40 45 50
Y
Figure 4. Lyophilized rat kidney with vitamin D3 marker
400
pg. added
Effect of water on vitamin D8-4-CI4
procedure was employed. Five fig. of vitamin Da4-C1* in 10 pl. of methanol was rapidly squirted into 25 ml. of water, the dispersion allowed to stand for 24 hours, dried under vacuum, and applied to the Fluoropak column. With concentrations of vitamin D large enough to be followed by U.V. absorption-e.g., 10 to 20 p g . per m1.-this breakdown could be observed as a loss in absorbance and shift in peak to longer wavelengths. Further details on studies of vitamin D stability will be reported elsewhere. The Fluoropak column may also find application to physiological studies in the chromatography of tissue extracts. In preliminary experiments lyophilized or saponified rat tissues, extracted with chloroform (or dichloromethane) and ethyl acetate were used. Chromatographic eluates from liver extract prepared in the above manner exhibited an early peak of relatively polar u.v.absorbing material running with and near vitamin A, as in Figure 4. Most of the absorbance was due to this vitamin or its breakdown products. In the region where vitamin D was eluted (130 to 250 ml.), practically no ultraviolet absorbance was evident with extracts of normal liver or other soft tissues. This indicated the futility of demonstrating normal tissue vitamin D levels by ultraviolet absorption alone
2032
IO
FRACTION NUMBER
15
FRACTION NUMBER (IOml.) Figure 3.
5
ANALYTICAL CHEMISTRY
but gave hope that more sensitive quantitation methods might be developed in the future for chemically estimating tissue vitamin D levels following a separation of this type. Exhaustive surveys of either the stationary liquid phase or the mobile phase were not made, and perhaps other combinations of solvents can be developed for other purposes. The distribution ratio of vitamin D between 90% MeOH and iso-octane was about 0.22, rising to 0.5 a t a concentration of 95% MeOH. Thus elution chsracteristics may be easily varied by altering the alcohol concentration of the mobile phase. The liquid stationary phase was less amenable to changes. Without isooctane, there must be some adsorption effects, for if methanol in concentrations of 70% or above was run over Fluoropak 80 powder, the sterols all remained in the alcohol. However, a t 65% methanol separations between vitamins A and D were possible, although there was tailing and the solubilities of sterols in this concentration of methanol were poor. The major advantages of the reversedphase system described above are: the inert stationary support which does not decompose or otherwise contribute impurities to the eluates; use of solvents which are available in pure form a t low
cost and which are transparent in the visible and U.V. regions allowing direct absorbance measurements without evaporation or further purification; and use of solvents which are completely and readily volatile, facilitating the application of liquid scintillation counting or other analytical techniques to column eluates, A disadvantage which may arise in attempting to chromatograph large amounts of crude extract is the necessity of dissolving the material in 90% methanol. Some extracts are rather poorly soluble in this solvent and may require preliminary purifications before running on Fluoropak 80. LITERATURE CITED
(1) Heftmann, E., “Chromatography,”
Reinhold, New York, 1961.
( 2 ) Kodicek, E., Ashby, D. R., Biochem. J . 57, xii (1954). (3) Yield, C. H., Russell, W. C., Zimmerli, A.. J . Biol. Chem. 136.73 (1940). (4) U.S.P. XVI, 910 (1960):
RECEIVEDfor review April 25, 1963.
Accepted August 12, 1963. Pittsburgh Conference on Analytical Chemistry and
Applied Spectroscopy, Pittsburgh, Pa., March 1963. Paper is based on work performed under contract with the United States Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, N. Y.
