- Email: [email protected]
The separation of 1-dimethylaminonaphthalene-5-sulphonamido acids (DNS-amino acids) by thin-layer chromatography
616
NOTES
The separation (DMS-amino
of l-dimethylaminonaphthalene-50sulphonamido
acids) by thin-layer
acids
chromatography
I-Dimethylaminonaphthalene-5-sulphonyl chloride, recently introduced by for the determination on a submicro scale of’ the N-terminal residues of peptides and proteins, has already aroused. a great deal of interest. These authors also described a separation of the corresponding amino-acid derivatives by high-voltage electrophoresis on paper at pH 4.4 that permits the identification of the majority of the derivatives when they are present singly. A most useful feature of this procedure is that it clearly differentiates DNS-leucine and DNSisoleucine. The derivatives of alanine, serine, proline and glycine, which run close to the I-dimethylaminonaphthalene-g-sulphonic acid produced by the hydrolysis of excess reagent, cannot always be identified unequivocally, and GRAY AND HARTLEY resorted to a second electrophoretic run at pW 12.7 to accomplish this. The separation of DNS-amino acids by paper chromatography has also been described”. The 2dimensional, thin-layer chromatographic’method that we describe here forms a convenient complement to the electrophoretic separation at pW 4.4, and permits the separation of the DNS-derivatives of all the common amino acids except those of leucine and isoleucine, which can, however, be separated by development with another solvent in the second dimension. The DNS-derivatives of glutamine and asparagine are not separetcd. As the present work was being concluded, another 2-dimensional, GRAY AND HARTLEY~~~ as a reagent
. I, Two-dimensional separation of DNS-amino acids. (I) DNS-amide; (2) DNS-isoleuc’i‘nc: (3) DNS-leucinc; (4) DNS-valine; (3) DNS-prolinc; (6) DNS-phenylalanine; (7) DNS-methiogine; (8) DNS-methionine sulphoxide (produced by oxidation of DNS-methionine after development in solvent A) : (9) DNS-alanine; (IO) di-DNS-tyrosine; (I I) di-DNS-lysine; (12) N(a)-DNS-tyrosine; (I 3) DNS-tryptophan : (14) DNS-glycine ; (15) DNS-acid (produced by decomposition of di-DNShistidine after development in solvent A) ; (I 6) di-DNS-histidine ; (I 7) N(c+DNS-histidine (produced by decomposition of di-DNS-histidine after development in solvent A) ; (IS) DNS-threonine; (19) DNS-serine: (20) DNS-glutamic acid: (21) DNS-methionine sulphone; (22) DNS-cysteine (blue) I (23) di-DNS-cystine; (24) DNS-glutamine, DNS-asparagine; (25) DNS-aspartic acid; (26) DNS-acid (blue) : (27) 0-DNS-tyrosine (deep yellow) ; (28) N(+DNS-lysinc; (29) N(Im)DNS-histidine (orange) ; (30) DNS-arginine; (31) N(a)-DNS-lysine; (32) N(a)-DNS-histidine; (33) ’ DNS-cysteic acid. Fig.
J. Clwomatog.,
20
(1965)
G16-618
.*
617
NOTES
thin-layer described The (A)
chromatographic method for the separation of DNS-amino acids was by SELLERAND WLECHMANN~. solvents used were: benzene-pyridine-acetic acid (40 : IO : I, v/v)G, (IS) Nt-butanol saturated with 0.2 N sodium hydroxide, (C) +butanol-chloroform (3 : 97, v/v). Plates (20 x 20 cm) were spread with a suspension of Silica Gel G (Merck; 30 g/60 ml water). The layer (250 p thickness) was allowed to set for ~5 min and then the plates were dried in a draught oven at IIOO for 30 min and stored in a desiccator cabinet until used. The load (O.OOI-o.oog pmole of each derivative) was applied in r-2 ~1 of ethanol-water (3 : I, v/v). If necessary, volu.mes up to IO ~1 could be applied portionwise with intermittent drying. The tanks used (Shandon “Chromatank”) were lined with filter paper and solvent was allowed to remain in the tanks permanently. No ‘deterioration of the solvents over periods of a few days was apparent. Development in solvent A was carried out for I ,5 h, which is longer than the time required for the first solvent front to reach the top of the plate. The plate was dried in a stream of air at 60” for 30 min, and then in an oven at IZO' for 10 min. (Effective TABLE I RELATIVE
MIGRATION
DNS-devivalivc
RATES
of
Isolcucinc Leucinc Valine Prolinc Phcnylslanine Mcthionine Alaninc Tyrosine (di-DNS-) Lysine (di-DNS-) Tryptophan z;;z;e (N(a) -DNS-) Histidine (di-DNS-) Thrconine Glutamic acid Serine Methionine sulphone Aspartic acicl Cysteine Asparaginc Glutamine Cystine Cysteic acid Histidine (N(a)-DNS-) Lysine (N(a+DNS-) Arginine Histidinc (N (Im)-DNS-) Lysine (N(E)-DNS-) Tyrosine (0-DNS-)
OP DNS-AMINO
ACIDS
R value referred to DNS-amide Solvent A
Solvent
0.87 0.83 o-77 0.70 0.59 o-47 0.4G 0.40 0.34 0.32 0.32 0.24 0.18 0.16
0.65 0.63 0.54 0.24 0.51 o-43 0.37 0.48 0.54 0.45 0.53 0.32 0.32 0.28 0.05
0.15 0.09
0.25 o.ao
0.06 0.04 0.03 0.03
0.04 0.40 o.rg 0.15 0.35. 0.04 0.08
0.52
0.02 0.0 0.0 0.0 0.0 0.0 0.0 0.0
B
Solvent C 0.19
0.10 0.13 0.07 0.07
0.12
0.16 0.20
0.26 o-33
* ,Tails.
J. Clrramalog.,
20
(xgG5)
61$-6x8
628
NOTES
removal of solvent A is necessary because residual solvent quenches the fluorescence of the derivatives.) The plate was developed with solvent 13 in a direction at right angles to the first. Full development required about 2.5 h, but for simple mixtures of derivatives, I h or less may be adequate. The plate was dried at IIOO for, 30 min. If separation of DNS-leucine and DNS-isoleucine only is required, the plate is developed in the second dimension with solvent C instead of solvent 53. If necessary, development with solvents I3 and C may be carried out successively, but this procedure is rather lengthy. A diagram of the separation obtained with solvents A and I3 is shown in Fig. z. The different mono-DNS-derivatives of tyrosine, histidine and lysine were identified by spraying the plates with specific reagents. The rates of movement of the derivatives referred to DNS-amide are given in Table 1. We wish to thank Mr. R. J. WWEWELL for assistance with the experimental work, and Dr. B. S. HARTLEY and Dr. W. R. GRAY for generous and helpful advice. Wool Indzcstries Research Association, Torridort, Leeds (Great Britain)
J.
M. COLE FLETCHER A. ROBSON*
C.
I W. Ii. GRAY AND B. S. HARTLEY, Bioclicm.J., 89 (1963) 6oP. W. R. GRAY AND B. S. HARTLEY, 1Jiocltcm. J., 89 (1963) 379. 3 A. A. BOULTON AND I. IL BUSH, Biockem. J., 92 (1964) III?. ,+N. SEILER AND J. WIECHMANN, E.vpeviclzlia, 20 (1964) 559. 5 M. BRENNER, A. NIEDERWIESER AND G. PATAKI, Expdrienlia,17 (1961) 145. 2
Received
March Srst, 1965
* Present Britain.
.nclclress: Department
of Textile
Industries,
University
of Leeds,
Leeds,
Great
J. Cltromalog.,20 (1965) 616-618
Trennung
van Monoterpenen
mittels
Silbernitrat-Kiesel’geldiinnschichten
Die Trennung von Monoterpenkohlenwasserstoffen mittels Kieselgeldiinnschichten bereitet Schwierigkeiten, da die Stoffe wegen ihres apolaren Charakters mit den verschiedenen Laufmitteln immer in der N%he der Laufmittelfront wandern. Im allgemeinen werden Hexan, Cyclohexan, Methylcyclohexan ,oder 2,2-Dimethylbutan verwendetl. Die Unterschiede der Xp-Werte sind dabei klein, und viele Monoterpene sind dadurch nicht zu trennen. Methode Vor x3 Jahren ist von NICIXOLS~ eine saulenchromatographische beschrieben worden, bei der dem Silicagel Silbernitrat zugesetzt ist, urn eine bessere Trennung von Fettsaureestern zu erreichen. DE VRIES~ hat diese Methode weiter gefiihrt, und BARRETT, DALLAS UND PADLEY~ sowie MORRIS" haben daraus eine cltinnschicht-chromatographische Trennungsmethode von Pettsaureglyceriden entwickelt. J. Clcvomatog.,20 (1965) 618-620
NOTES
The separation (DMS-amino
of l-dimethylaminonaphthalene-50sulphonamido
acids) by thin-layer
acids
chromatography
I-Dimethylaminonaphthalene-5-sulphonyl chloride, recently introduced by for the determination on a submicro scale of’ the N-terminal residues of peptides and proteins, has already aroused. a great deal of interest. These authors also described a separation of the corresponding amino-acid derivatives by high-voltage electrophoresis on paper at pH 4.4 that permits the identification of the majority of the derivatives when they are present singly. A most useful feature of this procedure is that it clearly differentiates DNS-leucine and DNSisoleucine. The derivatives of alanine, serine, proline and glycine, which run close to the I-dimethylaminonaphthalene-g-sulphonic acid produced by the hydrolysis of excess reagent, cannot always be identified unequivocally, and GRAY AND HARTLEY resorted to a second electrophoretic run at pW 12.7 to accomplish this. The separation of DNS-amino acids by paper chromatography has also been described”. The 2dimensional, thin-layer chromatographic’method that we describe here forms a convenient complement to the electrophoretic separation at pW 4.4, and permits the separation of the DNS-derivatives of all the common amino acids except those of leucine and isoleucine, which can, however, be separated by development with another solvent in the second dimension. The DNS-derivatives of glutamine and asparagine are not separetcd. As the present work was being concluded, another 2-dimensional, GRAY AND HARTLEY~~~ as a reagent
. I, Two-dimensional separation of DNS-amino acids. (I) DNS-amide; (2) DNS-isoleuc’i‘nc: (3) DNS-leucinc; (4) DNS-valine; (3) DNS-prolinc; (6) DNS-phenylalanine; (7) DNS-methiogine; (8) DNS-methionine sulphoxide (produced by oxidation of DNS-methionine after development in solvent A) : (9) DNS-alanine; (IO) di-DNS-tyrosine; (I I) di-DNS-lysine; (12) N(a)-DNS-tyrosine; (I 3) DNS-tryptophan : (14) DNS-glycine ; (15) DNS-acid (produced by decomposition of di-DNShistidine after development in solvent A) ; (I 6) di-DNS-histidine ; (I 7) N(c+DNS-histidine (produced by decomposition of di-DNS-histidine after development in solvent A) ; (IS) DNS-threonine; (19) DNS-serine: (20) DNS-glutamic acid: (21) DNS-methionine sulphone; (22) DNS-cysteine (blue) I (23) di-DNS-cystine; (24) DNS-glutamine, DNS-asparagine; (25) DNS-aspartic acid; (26) DNS-acid (blue) : (27) 0-DNS-tyrosine (deep yellow) ; (28) N(+DNS-lysinc; (29) N(Im)DNS-histidine (orange) ; (30) DNS-arginine; (31) N(a)-DNS-lysine; (32) N(a)-DNS-histidine; (33) ’ DNS-cysteic acid. Fig.
J. Clwomatog.,
20
(1965)
G16-618
.*
617
NOTES
thin-layer described The (A)
chromatographic method for the separation of DNS-amino acids was by SELLERAND WLECHMANN~. solvents used were: benzene-pyridine-acetic acid (40 : IO : I, v/v)G, (IS) Nt-butanol saturated with 0.2 N sodium hydroxide, (C) +butanol-chloroform (3 : 97, v/v). Plates (20 x 20 cm) were spread with a suspension of Silica Gel G (Merck; 30 g/60 ml water). The layer (250 p thickness) was allowed to set for ~5 min and then the plates were dried in a draught oven at IIOO for 30 min and stored in a desiccator cabinet until used. The load (O.OOI-o.oog pmole of each derivative) was applied in r-2 ~1 of ethanol-water (3 : I, v/v). If necessary, volu.mes up to IO ~1 could be applied portionwise with intermittent drying. The tanks used (Shandon “Chromatank”) were lined with filter paper and solvent was allowed to remain in the tanks permanently. No ‘deterioration of the solvents over periods of a few days was apparent. Development in solvent A was carried out for I ,5 h, which is longer than the time required for the first solvent front to reach the top of the plate. The plate was dried in a stream of air at 60” for 30 min, and then in an oven at IZO' for 10 min. (Effective TABLE I RELATIVE
MIGRATION
DNS-devivalivc
RATES
of
Isolcucinc Leucinc Valine Prolinc Phcnylslanine Mcthionine Alaninc Tyrosine (di-DNS-) Lysine (di-DNS-) Tryptophan z;;z;e (N(a) -DNS-) Histidine (di-DNS-) Thrconine Glutamic acid Serine Methionine sulphone Aspartic acicl Cysteine Asparaginc Glutamine Cystine Cysteic acid Histidine (N(a)-DNS-) Lysine (N(a+DNS-) Arginine Histidinc (N (Im)-DNS-) Lysine (N(E)-DNS-) Tyrosine (0-DNS-)
OP DNS-AMINO
ACIDS
R value referred to DNS-amide Solvent A
Solvent
0.87 0.83 o-77 0.70 0.59 o-47 0.4G 0.40 0.34 0.32 0.32 0.24 0.18 0.16
0.65 0.63 0.54 0.24 0.51 o-43 0.37 0.48 0.54 0.45 0.53 0.32 0.32 0.28 0.05
0.15 0.09
0.25 o.ao
0.06 0.04 0.03 0.03
0.04 0.40 o.rg 0.15 0.35. 0.04 0.08
0.52
0.02 0.0 0.0 0.0 0.0 0.0 0.0 0.0
B
Solvent C 0.19
0.10 0.13 0.07 0.07
0.12
0.16 0.20
0.26 o-33
* ,Tails.
J. Clrramalog.,
20
(xgG5)
61$-6x8
628
NOTES
removal of solvent A is necessary because residual solvent quenches the fluorescence of the derivatives.) The plate was developed with solvent 13 in a direction at right angles to the first. Full development required about 2.5 h, but for simple mixtures of derivatives, I h or less may be adequate. The plate was dried at IIOO for, 30 min. If separation of DNS-leucine and DNS-isoleucine only is required, the plate is developed in the second dimension with solvent C instead of solvent 53. If necessary, development with solvents I3 and C may be carried out successively, but this procedure is rather lengthy. A diagram of the separation obtained with solvents A and I3 is shown in Fig. z. The different mono-DNS-derivatives of tyrosine, histidine and lysine were identified by spraying the plates with specific reagents. The rates of movement of the derivatives referred to DNS-amide are given in Table 1. We wish to thank Mr. R. J. WWEWELL for assistance with the experimental work, and Dr. B. S. HARTLEY and Dr. W. R. GRAY for generous and helpful advice. Wool Indzcstries Research Association, Torridort, Leeds (Great Britain)
J.
M. COLE FLETCHER A. ROBSON*
C.
I W. Ii. GRAY AND B. S. HARTLEY, Bioclicm.J., 89 (1963) 6oP. W. R. GRAY AND B. S. HARTLEY, 1Jiocltcm. J., 89 (1963) 379. 3 A. A. BOULTON AND I. IL BUSH, Biockem. J., 92 (1964) III?. ,+N. SEILER AND J. WIECHMANN, E.vpeviclzlia, 20 (1964) 559. 5 M. BRENNER, A. NIEDERWIESER AND G. PATAKI, Expdrienlia,17 (1961) 145. 2
Received
March Srst, 1965
* Present Britain.
.nclclress: Department
of Textile
Industries,
University
of Leeds,
Leeds,
Great
J. Cltromalog.,20 (1965) 616-618
Trennung
van Monoterpenen
mittels
Silbernitrat-Kiesel’geldiinnschichten
Die Trennung von Monoterpenkohlenwasserstoffen mittels Kieselgeldiinnschichten bereitet Schwierigkeiten, da die Stoffe wegen ihres apolaren Charakters mit den verschiedenen Laufmitteln immer in der N%he der Laufmittelfront wandern. Im allgemeinen werden Hexan, Cyclohexan, Methylcyclohexan ,oder 2,2-Dimethylbutan verwendetl. Die Unterschiede der Xp-Werte sind dabei klein, und viele Monoterpene sind dadurch nicht zu trennen. Methode Vor x3 Jahren ist von NICIXOLS~ eine saulenchromatographische beschrieben worden, bei der dem Silicagel Silbernitrat zugesetzt ist, urn eine bessere Trennung von Fettsaureestern zu erreichen. DE VRIES~ hat diese Methode weiter gefiihrt, und BARRETT, DALLAS UND PADLEY~ sowie MORRIS" haben daraus eine cltinnschicht-chromatographische Trennungsmethode von Pettsaureglyceriden entwickelt. J. Clcvomatog.,20 (1965) 618-620