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Acetonitrile-based solvents: Their application in thin-layer chromatography of cyclic nucleotides, nucleosides, purine, and pyrimidines
ANSLYTICAL
BIOCHEMISTRY
60, 589-595
Acetonitrile-Based Layer
Solvents:
Institute
Their
Chromatography Nucleosides,
JOSEPH
(1974)
of Purine,
Application
Cyclic
and
Nucleotides,
Pyrimidines’
F. TOMASHEFSKI, *JR., ROBERTO AND OSCAR SUDILOVSKY” of Pathology, Hospitals Received
Case Western of Cleveland, November
Reserve Cleveland,
12, 1973;
accepted
in Thin-
J. BARRIOS,’
University and Ohio 44lO6 March
University
1, 1974
Acetonitrile-based solvent mixtures have been applied to the separation of nucleotides, nucleosides, purines, pyrimidines, and cyclic nucleotides by thinlayer chromatography. The R,‘s of over 35 compounds are presented. Development times with some of the systems were as low as 16 min. The use of acetonitrile-containing solvents in adenyl cyclase and cyclic phosphodiesterase assays and in the nucleotides and nucleic acid fields is discussed.
Acetonitrile-based solvents were first employed in paper cchromatography of purines and pyrimidines by Hedrick and colleagues (1,2). Shortly thereafter, Gabriel (3) utilized them for the separation of nucleic acid derivatives, also by paper chromatography. In the course of studies involving the determination of 3’,5’-cyclic AMP4 produced by the enzyme, adenyl cyclase, we have examined the application of similar systems to thin-layer chromatography on silica gel and on microcrystalline cellulose. While these experiments were in progress the separation of nucleoside trialcohols on cellulose thin layers by two-dimensional chromatography, using acetonitrile-containing solvents, was reported (4,5). In this publication we describe data derived from thin-layer chromatography of more than 35 purine and pyrimidine bases, nucleosides, nucleotides, and cyclic nucleotides, using a range of acetonitrile-based solvent 1 This work was supported in part by Smerican Cancer Society Grant IN-57-L by Case Western Reserve University Student Fellowship (to J.F.T.). * Present address: Tecoyatitla 281, Mexico 20, D. F. Mexico. a To whom reprint requests should be made. ’ Abbreviations : 3’,5’-cyclic AMP = adenosine 3’,5’-monophosphate ; Di-Bu-3’,5’cyclic AMP = N”-2’-0-Dibutyryl cyclic 3’,5’-adenosine monophosphate ; 3’,5’-cyrlir GMP = guanosine 3’,5’-monophosphate. Abbreviations for nucleotides are as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (Biochim. Biophys. Acta 108: (1965) I).
and
Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
589 Inc. reserved.
590
TOMASHEFSKI,
BARRIOS
AND
SUDILOVSKY
systems. As with paper chromatography (1,3), we have found that some buffered acetonitrile mixtures have a very rapid solvent-front migration and that a variety of intermediaries of nucleic acid metabolism and cyclic nucleotides can be resolved. EXPERIMENTAL
Solvent and Standards
Acetonitrile (Fisher certified, 99 mole $% pure) was used from recently opened bottles without further purification; isopropanol and n-butanol were Fisher spectranalized. Adenosine 5’-triphosphate, guanosine 5’-phosphate, cytidine 5’-triphosphate, and uridine 5’-triphosphate were purchased from P-L Biochemicals (Milwaukee, Wis.) . All other nucleotides, nucleosides, bases, and cyclic nucleotides were obtained from Sigma Chemical Company (St. Louis, MO.). Developing Unit
Covered Desaga rectangular developing tanks (approx internal dimensions 21 cm wide X 10 cm deep X 21 cm high) were used. The sides of the tanks were lined with filter paper wetted with solvent. Tanks were used 2&30 min after the addition of 100 ml of solvent. Adsorbents
Microcrystalline cellulose (Avicel F) precoated glass plates [Analtech), 20 X 20 cm, 250 pm layer. Silica gel GF precoated glass plates (Analtech), 20 X 20 cm, 250 @ layer. The plates were placed on a stripping and scoring device (6). Strips of adsorbent, 4 mm wide and 1.5 cm apart, were rapidly and completely removed from the glass plate by means of a suction-scraper (7) with a modified blade. The width of the dividing strips insured that cross contamination between adjacent lanes of adsorbent did not occur. One chromatogram per tank was developed by ascending chromatography. Solvents
Solvent A: Solvent B: Solvent C:
Acetonitrile-n-butanol-0.1 M ammonium monium hydroxide (10 : 60 : 20 : 10) Acetonitrile-n-butanol-0.1 M ammonium monium hydroxide (30 : 40 : 20 : 10) Acetonitrile-n-butanol-0.1 M ammonium monium hydroxide (50 : 20 : 20 : 10)
acetate-28%
am-
acetate-28yo
am-
acetate-28%
am-
ACETONITRILE-BASED
SOLVENTS
IN
TLC
Solvent D : Acetonitrile-n-butanol-0.1 1~zammonium acetate-280/o monium hydroxide (60 : 10 : 20 : 10) Solvent E: Acetonitrile-0.1 M ammonium acetate 280/o-ammonium droxide (70 : 20 : 10) Solvent F: Acetonitrile-isopropanol-0.1 &I ammonium acetate-28% monium hydroxide (60 : 10 : 20 : IO) Solvent systems were prepared fresh immediat.ely before use. Chromatographic
591 amhyam-
Procedures
Glass plates were heated for 60 min at 90°C and dessicated until use. Utilizing Levy-Lang micropipettes, 1 ,uliter of each sample, containing l-10 nmoles of compound was applied, with intermediate drying in a stream of cool air. The spots, approximately 3-4 mm in diameter, were placed in the middle of the prescored lanes of adsorbent at 2 cm from the bottom edge. Cyclic 3’,5’-AMP was used as the reference standard for each plate. In all instances the reference standard came within Rf + 0.04 of the average. The running distance was 10 cm. Development was at a constant temperature of 23-24°C. Plates were dried with a stream of cold air in a fume hood. Developed spots were visualized under short-wave ultraviolet light (Mineralight, Ultra-Violet Products, San Gabriel, Calif.‘). RESULTS AND DISCUSSION Preparation of the solvent mixtures is fast and simple; since acetonitrile used as described in Methods is transparent even at 244 nm, it requires no further purification. Chromatographic data for microcrystalline cellulose and silica gel-GF plates are presented in Tables 1 and 2. The R, values, which are the average of several determinations in many cases, were calculated on the basis of the primary front (a secondary front was observed with several systems5). Because ribonucleotides and deoxyribonucleotides have an extremely slow migration rate, only the range of Rf’s for each group are given. The behavior of compounds in the other categories was variable. On microcrystalline cellulose for example, adenine and xanthine moved slower with higher concentrations of acetonitrile while thiouracil, thymidine and Di-Bu-3’,5’-cyclic AMP showed increased mobility. Hypoxanthine, deoxyadenosine and 3’,5’-cyclic AMP, on the other hand, showed relatively little change in their Rf’s. Resolution of sample spots appeared to be somewhat better on silica ‘We have not observederratic results which, theoretically, could be caused by the formation of two fronts.
0.43 0.54 N.D.5 N.D. N.D. N.D. N.D. N.D. 0.60 0.53 N.D. 0.66 0.41 0.68 0.64 0.48 0.81 0.33 0.01-0.11 0.02-0.12
0.52 0.39 N.D. 0.50 0.24 0.58 0.49
0.37 0.66 0.12
0.01-O. 1oc 0.03-0.11
B
0.43 0.43 0.05 0.28 0.57 0.54 0.43 0.17
A
o-0.09 o-o. 05
0.45 0.87 0.20
0.50 0.53 N.D. 0.72 0.44 0.59 0.59
0.31 0.54 0.12 0.29 0.70 0.65 0.53 0.16
C
0.02-0.09 0.03-0.07
0.48 0.91 N.D.
0.57 0.60 N.D. 0.78 0.49 0.66 0.70
0.30 0.58 N.D. N.D. N.D. N.D. N.D. N.D.
E
o-0.06 0.01-0.04
0.32 0.90 0.13
0.44 0.45 0.20 0.59 0.37 0.53 0.56
0.24 0.46 0.08 0.21 0.55 0.55 0.42 0.11
F
Cellulose Coated Glass Plates
AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP,
0.01-0.05 0.02-0.04
0.44 0.86 0.18
0.57 0.51 0.20 0.72 0.42 0.66 0.61
0.27 0.57 0.08 0.23 0.63 0.69 0.48 0.13
D
(Avicel-F)
Solvent system
TABLE 1 on Microcrystalline
0 Not determined. b The following ribonucleotides and deoxyribonucleotides were chromatographed: GTP, TMP, TTP, UMP, UDP, UTP, dAMP, dCMP, dCTP, and dGTP. c Denote minimum and maximum R/s values within the group.
Bases Adenine Cytosine Guanine Hypoxanthine Thymine Thiouracif Uracil Xanthine Nucleosides Adenosine Cytidine Guanosine Thymidine Uridine Deoxyadenosine Deoxycytidine Cyclic nudeotdes 3’,5’-Cyclic AMP Di-Bu 3’,5’-cyclic AMP 3’,5’-Cyclic GMP Nuckotde$ Ribonucleotides Deoxyribonucleotides
Compound
R, Values of Compounds Separated by Chromatography
ACETONITRILE-BASED
R,
SOLVENTS
IN
593
TLC
TABLE 2 Values of Compounds Separated by Chromatography on Silica Gel-GF Coated Glass Plates Solvent system
Compound
A
B
C
13
E
0.55 0.28 0.44 0.57 0.60 0.46 0.37
0.60 0.54 N.D.a N.D. N.D. N.D. ND.
0.61 0.54 0.68 0.71 0.85 0.68 0.65
0.68 0.54 0.68 0.68 0.76 0.68 0.65
0.65 0.54 N.D. N.D. N.D. N.D. N.D.
0.35 0.23 0.20 0.36 0.15 0.47 0.29
0.61 0.43 0.45 0.59 0.40 0.64 0.53
0.66 0.45 0.47 0.60 0.45 0.66 0.58
0.63 0.48 N.D. 0.66 0.47 0.65 0.61
0.65 0.41 0.44 0.65 0.40 0.78 0.53
0.31 0.47 0.16
0.55 0.61 0.41
0.60 0.69 0.50
0.62 0.69 0.50
0.59 0.62 0.45
__--
Bases
Adenine Cytosine Hypoxanthine Thymine Thiouracil Uracil Xanthine Nucleosides
Adenosine Cytidine Guanosine Thymidine Uridine Deoxyadenosine Deoxycytidine Cyclic
nucleotides
3’,5’-Cyclic AMP Di-Bu 3’,5’-cyclic AMP 3’,5’-Cyclic GMP Nucleotidesb
Kibonucleotides Deoxyribonucleotides
o-o. OS o-o. 08
-.
o-o. 06 o-0.06
a Not determined. * The following ribonucleotides and deoxyribonucleotides were chromatographed: AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, TMP, TTP, UMP, UDP, UTP, dAMP, dCMP, dCTP, and dGTP. c Minimum and maximum R,‘s values within the group.
gel than with microcrystalline cellulose plates, although resolution was also satisfactory in the latter case. The velocity of migration of solvent fronts in the different chromatographic systems, as well as their relative mobilities and development times are shown in Table 3. The running times were longer for silica gel-GF t.han for cellulose coated plates, but the difference disappeared at the highest concentration of acetonitrile. The velocity of the solvent front was related to the proportion of acetonitrile in the mixtures. This is not surprising since acetonitrile, which is miscible with water, exceeds it in properties required for rapid front migration (during partition chromatography) (1). The correlat’ion, however, is not entirely linear, indicating a role for other factors.
594
TOMASHEFSKI,
Solvent Mobilities
BARRIOS
AND
SUDILOVSKT
TABLE 3 on Microcrystalline Cellulose and Silica Gel of Glass Platesa Solvent system
Adsorbent Microcrystalline
B
C
D
E
F
0.17 30 59
0.28 50 36
0.42 75 24
0.50 89 20
0.56 100 18
0.63 112 16
0.12 21 81
0.19 34 53
0.34 61 30
0.42 75 24
0.56 100 18
-
cellulose
Solvent mobility (cm/min) Relative mobility Development time (min) Silica
A
Gel GF
Solvent mobility (cm/min) Relative mobility Development time (min)
a Values are the average of at least five separate 10 cm runs.
Reagents in the solvent system appear to influence the velocity of development; thus, when isopropanol was substituted for n-butanol as in solvent F (compare with solvent D), running time was only 16 min. The mobility of the front varies also with the methods of preparation and characteristics of the adsorbent coating. In additional experiments, microcrystalline cellulose on plastic support (Bakerflex) was examined. Although good separation of the compounds tested was obtained with solvent F, the development time (40 min) was more than doubled when compared with that of glass coated plates, under similar conditions. Presumably, the difference is due to the incorporation of “hardeners” into the layers, in order to increase their durability. Decomposition of the samples was not apparent during developing; this was verified by two-dimensional chromatography in the same solvent mixture or in two of the solvent systems described. Drying of the plates after development takes only a few minutes, so that multiple or two-dimensional chromatography as well as quantitation by densitometry or by removal and elution of the spots (7) can be performed with minimal delay. Recovery of samples is very good ; in unpublished experiments, 93-95% of purified 3H-3’,5’-cyclic AMP added was usually eluted with 50% ethanol. The rapidity of migration of the high acetonitrile-containing solvents constitutes a definite advantage over other solvent mixtures for the chromatography of certain nucleosides and bases and, in particular, when used for the separation of cyclic nucleotides. In preliminary experiments, several nucleotides and 3’,5’-cyclic AMP were chromatographed under similar conditions as those described above using isopropanol-28% ammonium hydroxide-glass distilled water (70: 15: 15) or ethanol-28%
ACETONITRILE-BASED
SOLVENTS
IN
TLC
595
ammonium hydroxide-glass distilled water (75 : 15: 15) and microcrystalline cellulose (Analtech). Development times of 100 min and 55 min for 10 cm runs, respectively, were repeatedly obtained. Solvent F, on the other hand, afforded good separation and resolution of 3’,5’-cyclic AMP in only 16 min. In the solvents described, nucleotides remain close to or at the origin and are therefore easily separated from nucleosides, bases and cyclic nucleotides. In particular, adenine, adenosine and hypoxanthine have migration rates that differ clearly from that of cyclic AMP in most systems. This property permits their use in adenyl cyclase assays which employ radioactive ATP and in cyclic phosphodiesterase determinations with radioactive 3’,5’-cyclic AMP as the substrate. Because nucleosides and bases are also resolved satisfactorily, the applications of acetonitrile in the isolation and identification of precursors or degradation products of nucleotides and in the elucidation of base composition of polynucleotides, RNA’s and DNA’s are obvious. The large number of compounds with useful migration rates and the individual variation of their behavior in different mixtures are desirable properties for the separation of a given substance or group of substances. The substitution or addition of components in the solvent. mixtures-a possibilty suggested by our studies-could even further increase the versatility of acetonitrile-based solvents. ACKNOWLEDGMENTS We thank Mrs. Pansi Warren and Miss Karen Nagy for excellent secretarial sistance. Dr. Robert R. Kohn kindly criticized the manuscript. REFERENCES B. A., AND HEDRICK, C. E. (1966) Anal. Biochem. 16,260. E. E., AND KOEPPEL, T. A. (1967) Anal. Biochem. 19,411. GABRIEL, T. F. (1968) J. Chromatogr. 36, 518. RANDEIUTH, K. (1970) Anal. Biochem. 34, 188. RANDERATH, K., AND RANDERATH, E. (1973) J. Chromatogr. 82, 59. SC.DIM)VSKY, O., AND KOVACH, A. J. Chromatogr., submitted. SUDILOVSKY, O., AND HINDERAKER, P. H. (1972) Anal. Biochem. 45, 525.
1. BERCIER, 2. HEDRICK,
3. 4.
5. 6. 7.
as-
BIOCHEMISTRY
60, 589-595
Acetonitrile-Based Layer
Solvents:
Institute
Their
Chromatography Nucleosides,
JOSEPH
(1974)
of Purine,
Application
Cyclic
and
Nucleotides,
Pyrimidines’
F. TOMASHEFSKI, *JR., ROBERTO AND OSCAR SUDILOVSKY” of Pathology, Hospitals Received
Case Western of Cleveland, November
Reserve Cleveland,
12, 1973;
accepted
in Thin-
J. BARRIOS,’
University and Ohio 44lO6 March
University
1, 1974
Acetonitrile-based solvent mixtures have been applied to the separation of nucleotides, nucleosides, purines, pyrimidines, and cyclic nucleotides by thinlayer chromatography. The R,‘s of over 35 compounds are presented. Development times with some of the systems were as low as 16 min. The use of acetonitrile-containing solvents in adenyl cyclase and cyclic phosphodiesterase assays and in the nucleotides and nucleic acid fields is discussed.
Acetonitrile-based solvents were first employed in paper cchromatography of purines and pyrimidines by Hedrick and colleagues (1,2). Shortly thereafter, Gabriel (3) utilized them for the separation of nucleic acid derivatives, also by paper chromatography. In the course of studies involving the determination of 3’,5’-cyclic AMP4 produced by the enzyme, adenyl cyclase, we have examined the application of similar systems to thin-layer chromatography on silica gel and on microcrystalline cellulose. While these experiments were in progress the separation of nucleoside trialcohols on cellulose thin layers by two-dimensional chromatography, using acetonitrile-containing solvents, was reported (4,5). In this publication we describe data derived from thin-layer chromatography of more than 35 purine and pyrimidine bases, nucleosides, nucleotides, and cyclic nucleotides, using a range of acetonitrile-based solvent 1 This work was supported in part by Smerican Cancer Society Grant IN-57-L by Case Western Reserve University Student Fellowship (to J.F.T.). * Present address: Tecoyatitla 281, Mexico 20, D. F. Mexico. a To whom reprint requests should be made. ’ Abbreviations : 3’,5’-cyclic AMP = adenosine 3’,5’-monophosphate ; Di-Bu-3’,5’cyclic AMP = N”-2’-0-Dibutyryl cyclic 3’,5’-adenosine monophosphate ; 3’,5’-cyrlir GMP = guanosine 3’,5’-monophosphate. Abbreviations for nucleotides are as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (Biochim. Biophys. Acta 108: (1965) I).
and
Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
589 Inc. reserved.
590
TOMASHEFSKI,
BARRIOS
AND
SUDILOVSKY
systems. As with paper chromatography (1,3), we have found that some buffered acetonitrile mixtures have a very rapid solvent-front migration and that a variety of intermediaries of nucleic acid metabolism and cyclic nucleotides can be resolved. EXPERIMENTAL
Solvent and Standards
Acetonitrile (Fisher certified, 99 mole $% pure) was used from recently opened bottles without further purification; isopropanol and n-butanol were Fisher spectranalized. Adenosine 5’-triphosphate, guanosine 5’-phosphate, cytidine 5’-triphosphate, and uridine 5’-triphosphate were purchased from P-L Biochemicals (Milwaukee, Wis.) . All other nucleotides, nucleosides, bases, and cyclic nucleotides were obtained from Sigma Chemical Company (St. Louis, MO.). Developing Unit
Covered Desaga rectangular developing tanks (approx internal dimensions 21 cm wide X 10 cm deep X 21 cm high) were used. The sides of the tanks were lined with filter paper wetted with solvent. Tanks were used 2&30 min after the addition of 100 ml of solvent. Adsorbents
Microcrystalline cellulose (Avicel F) precoated glass plates [Analtech), 20 X 20 cm, 250 pm layer. Silica gel GF precoated glass plates (Analtech), 20 X 20 cm, 250 @ layer. The plates were placed on a stripping and scoring device (6). Strips of adsorbent, 4 mm wide and 1.5 cm apart, were rapidly and completely removed from the glass plate by means of a suction-scraper (7) with a modified blade. The width of the dividing strips insured that cross contamination between adjacent lanes of adsorbent did not occur. One chromatogram per tank was developed by ascending chromatography. Solvents
Solvent A: Solvent B: Solvent C:
Acetonitrile-n-butanol-0.1 M ammonium monium hydroxide (10 : 60 : 20 : 10) Acetonitrile-n-butanol-0.1 M ammonium monium hydroxide (30 : 40 : 20 : 10) Acetonitrile-n-butanol-0.1 M ammonium monium hydroxide (50 : 20 : 20 : 10)
acetate-28%
am-
acetate-28yo
am-
acetate-28%
am-
ACETONITRILE-BASED
SOLVENTS
IN
TLC
Solvent D : Acetonitrile-n-butanol-0.1 1~zammonium acetate-280/o monium hydroxide (60 : 10 : 20 : 10) Solvent E: Acetonitrile-0.1 M ammonium acetate 280/o-ammonium droxide (70 : 20 : 10) Solvent F: Acetonitrile-isopropanol-0.1 &I ammonium acetate-28% monium hydroxide (60 : 10 : 20 : IO) Solvent systems were prepared fresh immediat.ely before use. Chromatographic
591 amhyam-
Procedures
Glass plates were heated for 60 min at 90°C and dessicated until use. Utilizing Levy-Lang micropipettes, 1 ,uliter of each sample, containing l-10 nmoles of compound was applied, with intermediate drying in a stream of cool air. The spots, approximately 3-4 mm in diameter, were placed in the middle of the prescored lanes of adsorbent at 2 cm from the bottom edge. Cyclic 3’,5’-AMP was used as the reference standard for each plate. In all instances the reference standard came within Rf + 0.04 of the average. The running distance was 10 cm. Development was at a constant temperature of 23-24°C. Plates were dried with a stream of cold air in a fume hood. Developed spots were visualized under short-wave ultraviolet light (Mineralight, Ultra-Violet Products, San Gabriel, Calif.‘). RESULTS AND DISCUSSION Preparation of the solvent mixtures is fast and simple; since acetonitrile used as described in Methods is transparent even at 244 nm, it requires no further purification. Chromatographic data for microcrystalline cellulose and silica gel-GF plates are presented in Tables 1 and 2. The R, values, which are the average of several determinations in many cases, were calculated on the basis of the primary front (a secondary front was observed with several systems5). Because ribonucleotides and deoxyribonucleotides have an extremely slow migration rate, only the range of Rf’s for each group are given. The behavior of compounds in the other categories was variable. On microcrystalline cellulose for example, adenine and xanthine moved slower with higher concentrations of acetonitrile while thiouracil, thymidine and Di-Bu-3’,5’-cyclic AMP showed increased mobility. Hypoxanthine, deoxyadenosine and 3’,5’-cyclic AMP, on the other hand, showed relatively little change in their Rf’s. Resolution of sample spots appeared to be somewhat better on silica ‘We have not observederratic results which, theoretically, could be caused by the formation of two fronts.
0.43 0.54 N.D.5 N.D. N.D. N.D. N.D. N.D. 0.60 0.53 N.D. 0.66 0.41 0.68 0.64 0.48 0.81 0.33 0.01-0.11 0.02-0.12
0.52 0.39 N.D. 0.50 0.24 0.58 0.49
0.37 0.66 0.12
0.01-O. 1oc 0.03-0.11
B
0.43 0.43 0.05 0.28 0.57 0.54 0.43 0.17
A
o-0.09 o-o. 05
0.45 0.87 0.20
0.50 0.53 N.D. 0.72 0.44 0.59 0.59
0.31 0.54 0.12 0.29 0.70 0.65 0.53 0.16
C
0.02-0.09 0.03-0.07
0.48 0.91 N.D.
0.57 0.60 N.D. 0.78 0.49 0.66 0.70
0.30 0.58 N.D. N.D. N.D. N.D. N.D. N.D.
E
o-0.06 0.01-0.04
0.32 0.90 0.13
0.44 0.45 0.20 0.59 0.37 0.53 0.56
0.24 0.46 0.08 0.21 0.55 0.55 0.42 0.11
F
Cellulose Coated Glass Plates
AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP,
0.01-0.05 0.02-0.04
0.44 0.86 0.18
0.57 0.51 0.20 0.72 0.42 0.66 0.61
0.27 0.57 0.08 0.23 0.63 0.69 0.48 0.13
D
(Avicel-F)
Solvent system
TABLE 1 on Microcrystalline
0 Not determined. b The following ribonucleotides and deoxyribonucleotides were chromatographed: GTP, TMP, TTP, UMP, UDP, UTP, dAMP, dCMP, dCTP, and dGTP. c Denote minimum and maximum R/s values within the group.
Bases Adenine Cytosine Guanine Hypoxanthine Thymine Thiouracif Uracil Xanthine Nucleosides Adenosine Cytidine Guanosine Thymidine Uridine Deoxyadenosine Deoxycytidine Cyclic nudeotdes 3’,5’-Cyclic AMP Di-Bu 3’,5’-cyclic AMP 3’,5’-Cyclic GMP Nuckotde$ Ribonucleotides Deoxyribonucleotides
Compound
R, Values of Compounds Separated by Chromatography
ACETONITRILE-BASED
R,
SOLVENTS
IN
593
TLC
TABLE 2 Values of Compounds Separated by Chromatography on Silica Gel-GF Coated Glass Plates Solvent system
Compound
A
B
C
13
E
0.55 0.28 0.44 0.57 0.60 0.46 0.37
0.60 0.54 N.D.a N.D. N.D. N.D. ND.
0.61 0.54 0.68 0.71 0.85 0.68 0.65
0.68 0.54 0.68 0.68 0.76 0.68 0.65
0.65 0.54 N.D. N.D. N.D. N.D. N.D.
0.35 0.23 0.20 0.36 0.15 0.47 0.29
0.61 0.43 0.45 0.59 0.40 0.64 0.53
0.66 0.45 0.47 0.60 0.45 0.66 0.58
0.63 0.48 N.D. 0.66 0.47 0.65 0.61
0.65 0.41 0.44 0.65 0.40 0.78 0.53
0.31 0.47 0.16
0.55 0.61 0.41
0.60 0.69 0.50
0.62 0.69 0.50
0.59 0.62 0.45
__--
Bases
Adenine Cytosine Hypoxanthine Thymine Thiouracil Uracil Xanthine Nucleosides
Adenosine Cytidine Guanosine Thymidine Uridine Deoxyadenosine Deoxycytidine Cyclic
nucleotides
3’,5’-Cyclic AMP Di-Bu 3’,5’-cyclic AMP 3’,5’-Cyclic GMP Nucleotidesb
Kibonucleotides Deoxyribonucleotides
o-o. OS o-o. 08
-.
o-o. 06 o-0.06
a Not determined. * The following ribonucleotides and deoxyribonucleotides were chromatographed: AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, TMP, TTP, UMP, UDP, UTP, dAMP, dCMP, dCTP, and dGTP. c Minimum and maximum R,‘s values within the group.
gel than with microcrystalline cellulose plates, although resolution was also satisfactory in the latter case. The velocity of migration of solvent fronts in the different chromatographic systems, as well as their relative mobilities and development times are shown in Table 3. The running times were longer for silica gel-GF t.han for cellulose coated plates, but the difference disappeared at the highest concentration of acetonitrile. The velocity of the solvent front was related to the proportion of acetonitrile in the mixtures. This is not surprising since acetonitrile, which is miscible with water, exceeds it in properties required for rapid front migration (during partition chromatography) (1). The correlat’ion, however, is not entirely linear, indicating a role for other factors.
594
TOMASHEFSKI,
Solvent Mobilities
BARRIOS
AND
SUDILOVSKT
TABLE 3 on Microcrystalline Cellulose and Silica Gel of Glass Platesa Solvent system
Adsorbent Microcrystalline
B
C
D
E
F
0.17 30 59
0.28 50 36
0.42 75 24
0.50 89 20
0.56 100 18
0.63 112 16
0.12 21 81
0.19 34 53
0.34 61 30
0.42 75 24
0.56 100 18
-
cellulose
Solvent mobility (cm/min) Relative mobility Development time (min) Silica
A
Gel GF
Solvent mobility (cm/min) Relative mobility Development time (min)
a Values are the average of at least five separate 10 cm runs.
Reagents in the solvent system appear to influence the velocity of development; thus, when isopropanol was substituted for n-butanol as in solvent F (compare with solvent D), running time was only 16 min. The mobility of the front varies also with the methods of preparation and characteristics of the adsorbent coating. In additional experiments, microcrystalline cellulose on plastic support (Bakerflex) was examined. Although good separation of the compounds tested was obtained with solvent F, the development time (40 min) was more than doubled when compared with that of glass coated plates, under similar conditions. Presumably, the difference is due to the incorporation of “hardeners” into the layers, in order to increase their durability. Decomposition of the samples was not apparent during developing; this was verified by two-dimensional chromatography in the same solvent mixture or in two of the solvent systems described. Drying of the plates after development takes only a few minutes, so that multiple or two-dimensional chromatography as well as quantitation by densitometry or by removal and elution of the spots (7) can be performed with minimal delay. Recovery of samples is very good ; in unpublished experiments, 93-95% of purified 3H-3’,5’-cyclic AMP added was usually eluted with 50% ethanol. The rapidity of migration of the high acetonitrile-containing solvents constitutes a definite advantage over other solvent mixtures for the chromatography of certain nucleosides and bases and, in particular, when used for the separation of cyclic nucleotides. In preliminary experiments, several nucleotides and 3’,5’-cyclic AMP were chromatographed under similar conditions as those described above using isopropanol-28% ammonium hydroxide-glass distilled water (70: 15: 15) or ethanol-28%
ACETONITRILE-BASED
SOLVENTS
IN
TLC
595
ammonium hydroxide-glass distilled water (75 : 15: 15) and microcrystalline cellulose (Analtech). Development times of 100 min and 55 min for 10 cm runs, respectively, were repeatedly obtained. Solvent F, on the other hand, afforded good separation and resolution of 3’,5’-cyclic AMP in only 16 min. In the solvents described, nucleotides remain close to or at the origin and are therefore easily separated from nucleosides, bases and cyclic nucleotides. In particular, adenine, adenosine and hypoxanthine have migration rates that differ clearly from that of cyclic AMP in most systems. This property permits their use in adenyl cyclase assays which employ radioactive ATP and in cyclic phosphodiesterase determinations with radioactive 3’,5’-cyclic AMP as the substrate. Because nucleosides and bases are also resolved satisfactorily, the applications of acetonitrile in the isolation and identification of precursors or degradation products of nucleotides and in the elucidation of base composition of polynucleotides, RNA’s and DNA’s are obvious. The large number of compounds with useful migration rates and the individual variation of their behavior in different mixtures are desirable properties for the separation of a given substance or group of substances. The substitution or addition of components in the solvent. mixtures-a possibilty suggested by our studies-could even further increase the versatility of acetonitrile-based solvents. ACKNOWLEDGMENTS We thank Mrs. Pansi Warren and Miss Karen Nagy for excellent secretarial sistance. Dr. Robert R. Kohn kindly criticized the manuscript. REFERENCES B. A., AND HEDRICK, C. E. (1966) Anal. Biochem. 16,260. E. E., AND KOEPPEL, T. A. (1967) Anal. Biochem. 19,411. GABRIEL, T. F. (1968) J. Chromatogr. 36, 518. RANDEIUTH, K. (1970) Anal. Biochem. 34, 188. RANDERATH, K., AND RANDERATH, E. (1973) J. Chromatogr. 82, 59. SC.DIM)VSKY, O., AND KOVACH, A. J. Chromatogr., submitted. SUDILOVSKY, O., AND HINDERAKER, P. H. (1972) Anal. Biochem. 45, 525.
1. BERCIER, 2. HEDRICK,
3. 4.
5. 6. 7.
as-