- Email: [email protected]
Thin-layer chromatography of steroids: specificity of two location reagents
NOTES
Exanz;bZes of diflerently
reactiq
273
steroids
Some examples of the use of the above location reagents are given in Table I. No& added irt proof The tetrazolium reagent is also effective on silica gel, provided that an 8 y0 (2 IV) solution of sodium hydroxide is used to ensure alkaline conditions on the adsorbent. Glaxo Laboratories Ltd., Montrose, (Great Britain)
PETER J, STEVENS
Angus
AND J. MITCXIXLL, Amd. Chenz., “-5 (1953) 1376. BROOKS, R. M. EVANS, G. I?. H. GRIZIZN, J. S. HUNT, A. G. LONG,B. MOONEY AND L. J. WYMAN, J. Chew. Sot., (1958) 4614. 3 R. I<. CALLOW, D. H. W. DICKSON, J. ELKS, R. M. EVANS, V. H. T. JAMES, A, G. LONG,J. F. OUGNTON AND J. E. PAGE, J. Cl&em?.Sot., (1955) 1966. 4 J, KUEERA, 2% PRO~HAZKA AND I<. VBREB, Collecl,ion Czectboslov. Chem. Commzms., 22 (1957) I 185. 6 J, F. NYC, J, l3. GARST, H, B. FRIEDGOOD AND D. M. MARON, Arch. Biochem., “-9 (1950) 21g. * P. J. STEVENS, Pvoc. Assoc. Clia. Biochem, P (1963) 156. 1 S. D. NOGARE
2 S. G.
Received
August 23rd, ag63 J. Chvomatog.,
Reverse phase thin-layer 2,43dinitrophenylhydrazones
chromatography
14 (1964) 269-273
of
of noalkanals and n-alkan-&ones*
URBACW~of C.S.T.R.O. (Melbourne) has recently published a comprehensive thin-layer chromatographic (TLC) study of the z,.+dinitrophenylhydrazones (DNPH’s) of the aliphatic monocarbonyls. The author briefly reviewed the pertinent literature, and presented methods for both class and homolog separations. Our purpose in this communication is to briefly describe an alternate method for the separation by TLC, of an homologous series of the DNPEI’s of aliphatic aldehydes and ketones. In URBACN’S system, chain-length separation is achieved on Kieselguhr G plates impregnated with 2-phenoxyethanol; the impregnated plates are spotted with derivatives, and developed several times with 4% diethyl’ether in light petroleum. Resolution of the DNPEI’s of C,-C,, rc-alkanals and C&Z,, ti-alkan-2-ones was achieved; the higher members of the series moved the fastest. In our method we have adapted the KLEIN AND DE JONG~paper chromatographic procedure for TLC. Glass plates 5 x 20 cm were coated with a 250 ,u layer of silica gel G using the Brinkmann apparatus. After allowing about rg min for the adsorbent to set, the plates were placed in an oven and heated at IIO’ for at least I h. The plates were then cooled to room temperature and v&y slowly immersed in petroleum ether (boiling range 30-60”) containing 10% (v/v) of Shell Ondina 27 mineral oil. After impregnation, the petroleum ether was allowed to evaporate at room temperature, and the plates were then spotted. The plates were given a single development with * This investigation was supported by Public Health Service Research Grant Division of Environmental Engineering and Food Prdtection. J. Clwontatog.,
El?-269
I
4
(I 964)
from the
“-73-z75
274
Fig. I. TLC of C,-C,, n-allranal DNPEI’s (A, and 33 are from two different runs). Developed for 7 11 with dioxane-lvnter (63: 35, v/v) using the ordinary TLC cylinder as a chromatographic chamber. The C, derivative moved the fastest, the C,, the slowest.
NOTES
Fig. a. TLC using “continuous clevelopmcnt”. Developed for 7 h with clioxanewater (65: 35, v/v) in a 1000 ml beaker. covered with Saran film; 3-4 cm of the top of the plate was exposed to the atmosphere through a slit in the film. (A) ?z-alkan-z-one DNPI-I’s. (13) and C,--C,,1, It-alkanal DNPFI’s. (C) C,--C,,
J. Chromatog.,
14 (1964)
273-275
375
NOTES
dioxane-water (65 :35, v/v). A single development took about ‘6 h at 25"; multiple development, in this particular system, did not result in further resolution. Fig. I shows a photograph, taken under U.V. light, of the TLC of the C,-Cl, rt-alkanal DNPI-I’s using the technique described. We have observed that the best separation is achieved with the middle members of the C,-C1, series; the C, and C, derivatives tend to run together, and the Cl3 and C,, derivatives are often not well separated, Although multiple development did not enhance resolution, we have used what we call “continuous development” to increase resolution. In this technique the derivatives are spotted into the impregnated plates, and the plates are developed with the diosane-water system with 3-4 cm of the top of the plates esposed to the atmosphere. This is conveniently done in a Saran*- covered IOOO ml beaker, with a slit cut in the Saran film for the plate. Using this technique the slow and medium-mobility fractions are usually well resolved. Overdevelopment, however, can cause tile “piling-up” of the fast-moving fractions at the top of the plate. Fig. 2 shows a plate that had been run with “continuous development”. De;hnrtmmt
of Food Science and Teclwzology, Oregon State University, Corvallis, Oreg. (U.S.A.)
1 C;. URBACI-I,J. Chr~owzatog., 12 (1963) 1g6. 1 F. KLEIN AND Ii. DE JONG, REC. Tvcm. Chim.,75
Received * Sr.ran
September is a trade
xzth, name
L. M. LTBBEY E. A. DAY
(x956)1zS5.
1963
for polyvinyliclene
chloride.
J. Ckvonznlog., 14 (1964) 273475
Thin-layer
chromatography
of tetra-
and pentacyclic
triterpenes
Thin-layer chromatography has occasionally been applied to the separation of triterpenes, e.g. by TSCHESCWE~~2 to those of Brederneyem floribzcndn, by TI-IO&IAS~to those of Colnn@1$ora glarzddosn and by HUNECI CQ to those of S07bz~stornzimtis. In experiments with Israeli peat,. which will be reported elsewhere, we have developed a system that proved useful in the separation of triterpenoid compounds The solvent mixture used was heptaneand permitted their easy identification. benzene-ethanol (50: 50 :0.5), applied to alumina G. This mixture has the advantage that an increase in the alcohol concentration increases and a decrease in its concentration decreases the rate of migration. For example, the RF values for betulin (No. 3) are o, 0.14~ 0.73 for o Oh, 0.5 o/0and 2 o/oalcohol, for lupeol (No. IO) 0.16, 0.37, 0.94 for the same three alcohol concentrations. A systematic study has given the following results, which will be extended by (No. 15) and e$i-lupeol (14) can be separated further investigations : e$i+Amyrin from their diastereoisomers /I-amyrin (No. 6) and lupeol (IO) ; the e$i-compounds have higher RF values. J. Cirromato,q., 14 (1964) 275479
Exanz;bZes of diflerently
reactiq
273
steroids
Some examples of the use of the above location reagents are given in Table I. No& added irt proof The tetrazolium reagent is also effective on silica gel, provided that an 8 y0 (2 IV) solution of sodium hydroxide is used to ensure alkaline conditions on the adsorbent. Glaxo Laboratories Ltd., Montrose, (Great Britain)
PETER J, STEVENS
Angus
AND J. MITCXIXLL, Amd. Chenz., “-5 (1953) 1376. BROOKS, R. M. EVANS, G. I?. H. GRIZIZN, J. S. HUNT, A. G. LONG,B. MOONEY AND L. J. WYMAN, J. Chew. Sot., (1958) 4614. 3 R. I<. CALLOW, D. H. W. DICKSON, J. ELKS, R. M. EVANS, V. H. T. JAMES, A, G. LONG,J. F. OUGNTON AND J. E. PAGE, J. Cl&em?.Sot., (1955) 1966. 4 J, KUEERA, 2% PRO~HAZKA AND I<. VBREB, Collecl,ion Czectboslov. Chem. Commzms., 22 (1957) I 185. 6 J, F. NYC, J, l3. GARST, H, B. FRIEDGOOD AND D. M. MARON, Arch. Biochem., “-9 (1950) 21g. * P. J. STEVENS, Pvoc. Assoc. Clia. Biochem, P (1963) 156. 1 S. D. NOGARE
2 S. G.
Received
August 23rd, ag63 J. Chvomatog.,
Reverse phase thin-layer 2,43dinitrophenylhydrazones
chromatography
14 (1964) 269-273
of
of noalkanals and n-alkan-&ones*
URBACW~of C.S.T.R.O. (Melbourne) has recently published a comprehensive thin-layer chromatographic (TLC) study of the z,.+dinitrophenylhydrazones (DNPH’s) of the aliphatic monocarbonyls. The author briefly reviewed the pertinent literature, and presented methods for both class and homolog separations. Our purpose in this communication is to briefly describe an alternate method for the separation by TLC, of an homologous series of the DNPEI’s of aliphatic aldehydes and ketones. In URBACN’S system, chain-length separation is achieved on Kieselguhr G plates impregnated with 2-phenoxyethanol; the impregnated plates are spotted with derivatives, and developed several times with 4% diethyl’ether in light petroleum. Resolution of the DNPEI’s of C,-C,, rc-alkanals and C&Z,, ti-alkan-2-ones was achieved; the higher members of the series moved the fastest. In our method we have adapted the KLEIN AND DE JONG~paper chromatographic procedure for TLC. Glass plates 5 x 20 cm were coated with a 250 ,u layer of silica gel G using the Brinkmann apparatus. After allowing about rg min for the adsorbent to set, the plates were placed in an oven and heated at IIO’ for at least I h. The plates were then cooled to room temperature and v&y slowly immersed in petroleum ether (boiling range 30-60”) containing 10% (v/v) of Shell Ondina 27 mineral oil. After impregnation, the petroleum ether was allowed to evaporate at room temperature, and the plates were then spotted. The plates were given a single development with * This investigation was supported by Public Health Service Research Grant Division of Environmental Engineering and Food Prdtection. J. Clwontatog.,
El?-269
I
4
(I 964)
from the
“-73-z75
274
Fig. I. TLC of C,-C,, n-allranal DNPEI’s (A, and 33 are from two different runs). Developed for 7 11 with dioxane-lvnter (63: 35, v/v) using the ordinary TLC cylinder as a chromatographic chamber. The C, derivative moved the fastest, the C,, the slowest.
NOTES
Fig. a. TLC using “continuous clevelopmcnt”. Developed for 7 h with clioxanewater (65: 35, v/v) in a 1000 ml beaker. covered with Saran film; 3-4 cm of the top of the plate was exposed to the atmosphere through a slit in the film. (A) ?z-alkan-z-one DNPI-I’s. (13) and C,--C,,1, It-alkanal DNPFI’s. (C) C,--C,,
J. Chromatog.,
14 (1964)
273-275
375
NOTES
dioxane-water (65 :35, v/v). A single development took about ‘6 h at 25"; multiple development, in this particular system, did not result in further resolution. Fig. I shows a photograph, taken under U.V. light, of the TLC of the C,-Cl, rt-alkanal DNPI-I’s using the technique described. We have observed that the best separation is achieved with the middle members of the C,-C1, series; the C, and C, derivatives tend to run together, and the Cl3 and C,, derivatives are often not well separated, Although multiple development did not enhance resolution, we have used what we call “continuous development” to increase resolution. In this technique the derivatives are spotted into the impregnated plates, and the plates are developed with the diosane-water system with 3-4 cm of the top of the plates esposed to the atmosphere. This is conveniently done in a Saran*- covered IOOO ml beaker, with a slit cut in the Saran film for the plate. Using this technique the slow and medium-mobility fractions are usually well resolved. Overdevelopment, however, can cause tile “piling-up” of the fast-moving fractions at the top of the plate. Fig. 2 shows a plate that had been run with “continuous development”. De;hnrtmmt
of Food Science and Teclwzology, Oregon State University, Corvallis, Oreg. (U.S.A.)
1 C;. URBACI-I,J. Chr~owzatog., 12 (1963) 1g6. 1 F. KLEIN AND Ii. DE JONG, REC. Tvcm. Chim.,75
Received * Sr.ran
September is a trade
xzth, name
L. M. LTBBEY E. A. DAY
(x956)1zS5.
1963
for polyvinyliclene
chloride.
J. Ckvonznlog., 14 (1964) 273475
Thin-layer
chromatography
of tetra-
and pentacyclic
triterpenes
Thin-layer chromatography has occasionally been applied to the separation of triterpenes, e.g. by TSCHESCWE~~2 to those of Brederneyem floribzcndn, by TI-IO&IAS~to those of Colnn@1$ora glarzddosn and by HUNECI CQ to those of S07bz~stornzimtis. In experiments with Israeli peat,. which will be reported elsewhere, we have developed a system that proved useful in the separation of triterpenoid compounds The solvent mixture used was heptaneand permitted their easy identification. benzene-ethanol (50: 50 :0.5), applied to alumina G. This mixture has the advantage that an increase in the alcohol concentration increases and a decrease in its concentration decreases the rate of migration. For example, the RF values for betulin (No. 3) are o, 0.14~ 0.73 for o Oh, 0.5 o/0and 2 o/oalcohol, for lupeol (No. IO) 0.16, 0.37, 0.94 for the same three alcohol concentrations. A systematic study has given the following results, which will be extended by (No. 15) and e$i-lupeol (14) can be separated further investigations : e$i+Amyrin from their diastereoisomers /I-amyrin (No. 6) and lupeol (IO) ; the e$i-compounds have higher RF values. J. Cirromato,q., 14 (1964) 275479