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Separation of basic amino acids by paper electrophoresis
ANALYTICAL
223, 5467554 (1968)
BIOCEFXISTRY
Separation of Basic Amino Acids by Paper Electrophoresis J. L. FRAHN Division
of Nutritional Research
Biochemistry, Organization,
AND J. A. MILLS Commonwealth Scientific and Industrial Adelaide 6000, Australia
Received August 21, 1967
Alkaline aqueous electrolytes have not been used extensively for the paper electrophoresis of amino acids, but Jirgl (1) has described the separation of several basic amino acids in an electrolyte at pH 12. A striking feature of Jirgl’s paper is the apparent absence of the carbamates (N-carboxy derivatives) of the amino acids under his conditions. Other workers (2-4) have also separated amino acids in alkaline electrolytes without mentioning the formation of carbamates. Several aspects of the formation of carbamates from amines and amino acids of various types have been investigated by paper electrophoresis (5). Most primary and secondary amines in which the amino group is not protonated (which includes a-amino acids in alkaline solution) readily combine with carbon dioxide, giving carbamates, and the carbamate ions are fairly stable at low temperatures in sufficiently alkaline solution, although they quickly decompose when the solution is heated. The carbamate ion (B) from an a-amino acid has two negative charges in alkaline solution and is easily separated by electrophoresis from the amino acid anion (A). It was found (5) that, in general, paper electroR-CH-COSIi?&+
OH- R-CH-CO,&Ha (-9
Ok- co2 R-CH-COz+ AH-COs@I
phoresis in alkali of a compound containing n reactive amino groups gave n + 1 spots if some exposure to carbon dioxide had occurred, representing the successive stages in formation of carbamate ions from all amino groups. It was very diflicult to exclude carbon dioxide rigorously enough to prevent some formation of carbamates in alkaline solutions of amines at room temperature. Solutions of sodium carbonate were sticiently alkaline to permit formation of some carbamate from an amine. Nearly all n-amino acids gave two spots after electrophoresis in 540
SEPARATION
OF BASIC
AMINO
ACIDS
547
strongly alkaline solution, and diamino acids such as ornithine gave three. Since the carbamates usually decomposed slowly during a run, streaking back from the carbamate spots also occurred. The multiplicity of spots and streaks obtained would normally make paper electrophoresis in alkaline solution unsuitable for separations of mixtures of amino acids, although the method can give valuable information about the structure of a single compound (5). It seemed that the freedom from complications in Jirgl’s separation at pH 12 (1) must be the result of either a procedure that prevented the formation of carbamates or, more likely, a rise in temperature of the paper strip sufficient to decompose any carbamates that were present. Investigation has confirmed that spots for carbamates are always found after paper electrophoresis of amino acids by the ordinary procedure in Jirgl’s electrolyte at pH 12, if the temperature in the paper strip is between 0” and about 30”. If the electrophoresis is conducted at 50” no carbamates are present and each amino acid gives a single spot. Jirgl (6) alw described the separation of a mixture of arginine, citrulline, histidine, ornithine, and lysine by paper electrophoresis in sodium hydrogen carbonate at pH 7.5. It is now shown that results with this electrolyte are unsatisfactory on paper strips held at 20”, but satisfactory at 50”. A better separation of the five basic amino acids is obtained by using a buffer of sodium carbonate plus hydrogen carbonate at pH 9.2, and working at 50”. Other results potentially useful in separations of amino acids in alkaline media are also described. MATERIALS
1. Amino acids. Commercial samples of nn-histidine monohydrochloride, DL-ornithine monohydrobromide, and nn-citrulline (British Drug Houses), L-arginine monohydrochloride (L. Light & Co.), and Llysine monohydrochloride (Merck, Darmstadt) each gave a single spot on paper electrophoresis at 50’. Solutions used were 0.01 M; citrulline was dissolved in water, the other amino acids in 0.01 M sodium hydroxide. 2. Heavy&metal salts. All salts of metals used for impregnating paper strips were of reagent grade. 3. Electrolytes. (a) A solution of 0.05 M sodium tetraborate with pH 12.0 was obtained by dissolving 9.54 gm Na2B,0,.10Hz0 in 500 ml water, adding 430 ml 0.2 M sodium hydroxide, and diluting the mixture to 1 liter. Jirgl (1) had used 9.75 gm sodium tetraborate. (b) A mixture of sodium carbonate and sodium hydrogen carbonate showed pH 9.2 when it contained 1.06 gm NazCOs anhydrous (0.02 N) and 6.72 gm
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NaHC03 (0.08 N) in 1 liter water. (c) The elect,rolyte was prepared as in (b) and 8-hydroxyquinoline (0.1% w/v) was added. (d) A solution of 0.08M sodium hydrogen carbonate was prepared, but it was found to have pH 8.4, instead of pH 7.4-7.5, as stated by Jirgl (6; cf. refs. 5, 7). Part of it was adjusted to pH 7.5 by added carbon dioxide, and both solutions were examined 4. Spray reagent. Ninhydrin (0.2 gm) was dissolved in 100 ml nbutanol previously saturated with water, and 4 ml glacial acetic acid was added to the solution. PROCEDURE
,.The enclosed-strip electrophoresis apparatus has been described in detail- (5, 8). A strip of Whatman No. 4 paper, 13.5 X 61 cm, was enclosed in an envelope:of polyethylene film and pressed firmly against a cooling plate by means of a rectangular rubber bag, which was inflated with water to a pressure of approximately 0.4 atm. About 45 cm length of the strip was uniformly compressed and cooled. In order to protect the electrolyte on the paper strip during application of samples, the strip was covered, inside the envelop, by a separate thin mask of polyethylene film containing small holes through which samples were applied by means of a platinum loop for qualitative runs. The potential difference applied gave a gradient of about 20 V cm-’ over the working area of the paper; the current was about 40 mA. The temperature on the paper strip was measured with a thermocouple made of thin (0.2 mm diameter) wires of iron and Constantan. The junction was enclosed in a thin polyethylene envelop and inserted between the paper strip and the mask, and the output was displayed on a recorder (Yellow Springs Instrument Co., model 80) that had been calibrated with the junction of the thermocouple at 0” and 50’. The temperature on the paper during electrophoresis was about 20” when water at 18.5’ was circulated through the cooling plate at 2.5 liters/ min. When water at 48” was circulated the temperature of the paper was 50”. The paper strip was wetted with electrolyte by dipping, blotted lightly, and inserted in the apparatus, and 30 min was allowed for equilibration under pressure at the chosen temperature. The envelop was then opened and samples of about 0.5 ~1 were applied by means of the loop through the holes in the mask. The apparatus was then closed without delay, pressure was restored, and electrophoresis was carried out for 30 to 60 min. The paper strip was dried at lOO”, sprayed with ninhydrin, and heated at 100” to develop the color. Any carbamates present were decomposed during drying and development, leaving the
SEPARATION
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BASIC
AMINO
ACIDS
549
amino acids, which gave spots of the same color’ as the free. amino acids (5) In some experiments cooling was interrupted and the temperature of the paper strip was allowed to rise unchecked; in others, the temperature initially was raised to 50” for a short time, then lowered to 20” for the electrophoresis. The effect of heavy metals on electrophoresis of amino acids under these conditions was determined by pretreatment of the paper strips with dilute solutions (usually 10d3M) of calcium, magnesium, lead, copper, zinc, cobalt, nickel, or iron (III). The paper was dipped in the solution of the appropriate metal, dried in warm air in a horizontal position, then dipped in the alkaline electrolyte in the usual way. RESULTS AND DISCUSSION Effect
of pH ad
Temperature
Experiments with the electrolyte at pH 12 gave results similar to those obtained (5) for various amino acids with 0.1 N sodium hydroxide. Pherograms run at room temperature, under conditions in which some exposure of alkaline solutions of the amino acids to atmospheric carbon dioxide had occurred, always showed additional spots for carbamates. Figure la shows the pattern of spots obtained for arginine, histidine, and ornithine; the unaltered amino acid is the spot farthest from the anode in each case, and the other spots (two for ornithine) represent carbamates. The connecting streaks result from slow breakdown of carbamates during migration. A comparable result was obtained when 0” water was circulated through the cooling plate, but because carbamates are more stable near O”, the streaks were barely detectable. The single carbamate spot for arginine and for histidine presumably represents formation of carbamate at the primary amino group only for each acid. It was not expected that nitrogen atoms of the guanidino group of arginine wouId be sufficiently nucleophilic to combine with carbon dioxide, and this seems also to be true of the imidazolyl group of histidine. When the paper was maintained at 50” throughout the run there were no streaks or multiple spots (Fig. lb). Any carbamates initially present must have decomposed before they could separate from the unchanged amino acids. If the run was started at 209 and the cooling water was then turned off, the temperature in the paper strip rose steadily and reached 35” after 30 min. Most of the carbamate had decomposed by the end of such a run, leaving a small residual spot of carbamate connected by a heavy streak to the spot of free amino acid. Other amino acids behaved similarly in this electrolyte, and in general multiple spots
550
FRAHN
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may be expected at 20” in highly alkaline electrolytes,. single spots. at 50°, and serious streaking at intermediate temperatures. Carbamates can also form and be detected by electrophoresis in less alkaline solutions (5). Runs at 20” in the carbonate-hydrogen carbonate mixture of pH 9.2 gave markedly elongated spots for many amino acids, showing that carbamates had been initially present, but, had rapidly decomposed during the run. Carbamates are more stable at 2” in this electrolyte. As expected (5), carbamates of the amino acids were much less stable in sodium hydrogen carbonate solutions at pH 7.5 or 8.4, but present, and survived long enough, to sufficient carbamate was initially
FIG. 1. Effect of temperature and pH of electrolyte on electrophoretic separation of arginine, histidine, citrulline, ornithine, and lytie during 30 min at 20V cm-l on Whatman No. 4 paper. The line of application is shown.
cause considerable elongation of spots from runs in these electrolytes- at 20”. When runs at 50” were performed in any of these carbonate-containing electrolytes the spots were sharp and nearly circular. A very satisfactory separation of arginine, citrulline, hi&dine, ornithine, an& lysine Was richieved at, pH 9.2 and 50’ (Fig. lc), rather better than that 6btained at 50” with electrolytes of pH 7.5 or 8.4. It had been found (5) that carbamates could be formed when solutions of amines in 0.1 iV aqueous ammonia were exposed to carbon dioxide at room temperature, but were rather unstable. This was confirmed by
SJWARATION
OF
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AMINO
ACIDS
551
attempts to repeat the published separation (4) of arginine; histidine, ornithine, and lysine by electrophoresis in 1 N ammonia (pH 11.7); in runs at 15’ all four acids showed extensive streaking. It seems that the present results could be reconciled with those described by Jirgl and others (l-4, 6) if the temperature during the run had not been adequately controlled in the earlier work. From descriptions available (1,3, 4, 6, 9) it seems that the apparatus used could have been of a simple, open-strip type, which would not give the positive control of temperature possible with the enclosed, pressurised apparatus. Passage of current through the alkaline electrolytes with a gradient of 20V cm-l generates heat of the order of 1 cal cm-2 min-I, and the temperature rises rapidly unless cooling is efficient. If the temperature of the paper strip reaches 40-50” during most of a run the migration of part of the amino acid as the carbamate ion would be prevented. The temperature in the paper strip is an important detail that should be included in descriptions of paper electrophoresis. If uncontrolled rise of temperature is accompanied by evaporation from an unenclosed strip of paper, results may become unreproducible for several reasons. The concentration and pH of the electrolyte may change through loss of volatile constituents. The relative mobilities of compounds may change because their dissociation constants or the extent of solvation of the ions respond differently to changes in temperature. The basic amino acids provide examples of differential effects of changes in temperature. Jirgl (1) illustrated a clean separation of histidine from ornithine in the tetraborate electrolyte at pH 12 and some unmeasured, but probably fairly high, temperature; Figure 1 shows that the two acids were not well separated in this electrolyte at either 50” or 20°, but they could be separated at 2”. Arginine and lysine, and also histidine and citrulline, were not properly separated in the sodium hydrogen carbonate electrolytes of pH 7.5 or 8.4 at 20°, but at 50” these pairs could be separated in either electrolyte. Alkaline electrolytes seem generally to have been avoided in the paper electrophoresis of amino acids (cf. refs. 10, ll), but they offer advantages if the formation of spots or streaks from carbamates is prevented by positive control of the temperature near 50’. The pH value of 9.2 for the carbonate-hydrogen carbonate electrolyte was chosen as being ,most likely to favor the separation at 50” of ornithine from histidine;:for which the calculated values (12) of pK, at 56’ are 9.93 and 8:47, respectively. However, the composition of an electrolyte, as well- as its pH value, can be important with amino acids; arginine and lysine were well separated in the carbonate-hydrogen carbonate electrolyte at pH
552
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9.2 and 50”, but not in 0.05 iV sodium tetraborate (pH 9.2) at 50”, although histidine, citrulline, and ornithine were easily separated from each other and from arginine or lysine in borate at pH 9.2. Eflect of Heavy Metals The strong, nearly circular spot for an amino acid obtained after paper electrophoresis in the alkaline electrolytes at 50” was often accompanied by a faint but sharply defined shadow on the trailing edge. A similar shadow seems to be present at the spots for arginine and citrulline in Figure 1 of Jirgl’s communication (6). The defect resembled the “lagging shadows” observed in paper chromatography of organic phosphates (13) and seemed, likewise, to be caused by inorganic impurities in the paper. It was eliminated by incorporating 8-hydroxyquinoline (0.1% w/v) in the carbonate-hydrogen carbonate electrolyte, which did not otherwise affect the electrophoretic separation or the detection of the amino acids. Equally satisfactory results were obtained from electrophoresis on paper that had been treated with 8-hydroxyquinoline and thoroughly washed with water, as recommended by Hanes and Isherwood (13). Some shadows were still obtained with paper that had been extracted with 2N .acetic acid or 5% ammonia and washed with water. Attempts were made to intensify the shadows, and so determine the cause, by incorporating- additional metal cations in- the paper. Strips that had been treated with various salts were used for separations of the five basic amino acids in the carbonate electrolyte of pH 9.2 at 50”, but for no single metal was the shadow observed with all five acids. The of the presence of added Ca 2+, Fe3+, or Pb” caused some intensification shadows behind lysine and arginine. The shadows were very prominent when citrulline or histidine was run on papers impregnated with a mixture containing Ca2+, Fe3+, Cu*+, and Pb2+. It seems likely that traces of more than one metal in the paper may be responsible for formation of the shadows, and that several different cations may simultaneously interact with some of the amino acids. The strong adsorption of metal ions on paper under alkaline conditions would promote interference with migration of the acids if complexes were formed. Histidine was sensitive to the widest range of added cations,. and formed shadows .or streaks in the presence of Ca2+; Mg2+, ,FeS+, -h2t; .or Zn2*. The effect of Co2+ or Ni2+ was more -marked, and if either’ was incorporated in the paper histidine disappeared from its normal position. It seemed to be largely adsorbed near the site of application and reThe effect could provide useful sponded only weakly to ninhydrin. evidence for the presence of histidine in a mixture of amino acids.
SEPARATION
OF
BASIC
AMINO
ACIDS
553
No defects resulted from the presence of added heavy metals when the electrolyte contained S-hydroxyquinoline. Recommended
Procedure
All separations of amino acids by paper electrophoresis in alkaline electrolytes should be carried out with the temperature of the paper strip controlled near 50” to prevent interference from carbamates, and enclosed to prevent changes in composition of electrolyte. The above five basic amino acids are best separated in the mixture of sodium carbonate and sodium hydrogen carbonate of pH 9.2, to which 0.1% w/v S-hydroxyquinoline has been added. Application of 2OV cm-l for 30 min gives a satisfactory resolution. Preliminary
Destruction of Carbamates
Some basic compounds might not survive prolonged heating in alkaline solution at 50’. Since the tight seal of the paper strip in the present apparatus prevents access of atmospheric carbon dioxide after closure of the system, carbamates initially present can be destroyed by brief heating and none can be re-formed when the paper strip is cooled. Satisfactory results were obtained with the sodium tetraborate electrolyte of pH 12 when samples of amino acids were applied in the usual way, if the paper was held at 50’ for 5 min after closure of the apparatus, the current was then switched on for 2 min only, and then switched off for a further 5 min while heating at 50” was continued. When the paper was cooled to 20” or below after this preliminary treatment, electrophoresis could be conducted at the lower temperature without any spots for carbamate being detected. The brief electrophoresis at 50” ensures uniform pH through the spot and aids decomposition of the last traces of carbamate during the subsequent heating. It should be noted that this procedure may fail with electrolytes containing carbonates unless they are highly alkaline. Solutions of carbonates below about pH 11 contain sufficient free carbon dioxide to permit the slow formation of carbamates at low temperatures (5). SUMMARY
Alkaline solutions of amino acids absorb carbon dioxide, giving carbamates, which may appear as additional spots or streaks after paper electrophoresis in alkaline electrolytes. Interference from carbamates is prevented by carrying out electrophoresis at 50”. Mixtures of arginine, histidine, citrulline, ornithine, and lysine are resolved by paper electrophoresis at 50” and pH 9.2 in an electrolyte containing sodium carbonate
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and sodium hydrogen carbonate. Addition of Shydroxyquinoline interference from impurities in the paper.
prevents
ACKNOWLEDGMENT We are grateful to Dr. D. D. Perrin, Australian National calculating dissociation constants of amino acids at 50".
University,
for
REFERENCES 1. JIROL, V., Anal. B&hem. 8, 519 (1964). 2. GERLAXHE, S., AND RENARD, M., Ind. Chim. Beige 20, Spec. No., 509 (1955); Chem. Abstr. 50, 7013 (1950). 3; REXOVA-BENKOVA, L., AND MARKO&, O., Collection Czech. Chem. Commun: 24, 1728 (1959). 4. BISERTE, G., DAXITREVAUX, M., AND BOULANQER, P., Bull. Sot. Chim. France 1963, 2954. 5. FRAHN, J. L., AND MILLS, J. A., Australian J. Chem. 17, 256 (1964). 6. JIRQL, V., Anal. Biochem. 13, 381 (1965). 7. “Handbook of Chemistry and Physics,” 45th ed, (R. C. We&, ed.), p. D-73. Chemical Rubber Co., Cleveland, Ohio, 1964-1965. 8. FRAHN, J. L., AND MILLS, J. A., Australian J. Chem. 12, 65 (1959). 9. JIRGL, V., AND SOCHMAN, J., Clin. Chim. Acta 7, 388 (1962). lb. BLACKBURN, S., Methods Biochem. Anal. 13, 1 (1965). 11. EDWARD, J. T., AND WALDRON-EDWARD, D., J. Chromatog. 24, 125 (1966); see in particular p. 130. 12. PERRIN, D. D., personal communication. 13. IXANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107 (1949).
223, 5467554 (1968)
BIOCEFXISTRY
Separation of Basic Amino Acids by Paper Electrophoresis J. L. FRAHN Division
of Nutritional Research
Biochemistry, Organization,
AND J. A. MILLS Commonwealth Scientific and Industrial Adelaide 6000, Australia
Received August 21, 1967
Alkaline aqueous electrolytes have not been used extensively for the paper electrophoresis of amino acids, but Jirgl (1) has described the separation of several basic amino acids in an electrolyte at pH 12. A striking feature of Jirgl’s paper is the apparent absence of the carbamates (N-carboxy derivatives) of the amino acids under his conditions. Other workers (2-4) have also separated amino acids in alkaline electrolytes without mentioning the formation of carbamates. Several aspects of the formation of carbamates from amines and amino acids of various types have been investigated by paper electrophoresis (5). Most primary and secondary amines in which the amino group is not protonated (which includes a-amino acids in alkaline solution) readily combine with carbon dioxide, giving carbamates, and the carbamate ions are fairly stable at low temperatures in sufficiently alkaline solution, although they quickly decompose when the solution is heated. The carbamate ion (B) from an a-amino acid has two negative charges in alkaline solution and is easily separated by electrophoresis from the amino acid anion (A). It was found (5) that, in general, paper electroR-CH-COSIi?&+
OH- R-CH-CO,&Ha (-9
Ok- co2 R-CH-COz+ AH-COs@I
phoresis in alkali of a compound containing n reactive amino groups gave n + 1 spots if some exposure to carbon dioxide had occurred, representing the successive stages in formation of carbamate ions from all amino groups. It was very diflicult to exclude carbon dioxide rigorously enough to prevent some formation of carbamates in alkaline solutions of amines at room temperature. Solutions of sodium carbonate were sticiently alkaline to permit formation of some carbamate from an amine. Nearly all n-amino acids gave two spots after electrophoresis in 540
SEPARATION
OF BASIC
AMINO
ACIDS
547
strongly alkaline solution, and diamino acids such as ornithine gave three. Since the carbamates usually decomposed slowly during a run, streaking back from the carbamate spots also occurred. The multiplicity of spots and streaks obtained would normally make paper electrophoresis in alkaline solution unsuitable for separations of mixtures of amino acids, although the method can give valuable information about the structure of a single compound (5). It seemed that the freedom from complications in Jirgl’s separation at pH 12 (1) must be the result of either a procedure that prevented the formation of carbamates or, more likely, a rise in temperature of the paper strip sufficient to decompose any carbamates that were present. Investigation has confirmed that spots for carbamates are always found after paper electrophoresis of amino acids by the ordinary procedure in Jirgl’s electrolyte at pH 12, if the temperature in the paper strip is between 0” and about 30”. If the electrophoresis is conducted at 50” no carbamates are present and each amino acid gives a single spot. Jirgl (6) alw described the separation of a mixture of arginine, citrulline, histidine, ornithine, and lysine by paper electrophoresis in sodium hydrogen carbonate at pH 7.5. It is now shown that results with this electrolyte are unsatisfactory on paper strips held at 20”, but satisfactory at 50”. A better separation of the five basic amino acids is obtained by using a buffer of sodium carbonate plus hydrogen carbonate at pH 9.2, and working at 50”. Other results potentially useful in separations of amino acids in alkaline media are also described. MATERIALS
1. Amino acids. Commercial samples of nn-histidine monohydrochloride, DL-ornithine monohydrobromide, and nn-citrulline (British Drug Houses), L-arginine monohydrochloride (L. Light & Co.), and Llysine monohydrochloride (Merck, Darmstadt) each gave a single spot on paper electrophoresis at 50’. Solutions used were 0.01 M; citrulline was dissolved in water, the other amino acids in 0.01 M sodium hydroxide. 2. Heavy&metal salts. All salts of metals used for impregnating paper strips were of reagent grade. 3. Electrolytes. (a) A solution of 0.05 M sodium tetraborate with pH 12.0 was obtained by dissolving 9.54 gm Na2B,0,.10Hz0 in 500 ml water, adding 430 ml 0.2 M sodium hydroxide, and diluting the mixture to 1 liter. Jirgl (1) had used 9.75 gm sodium tetraborate. (b) A mixture of sodium carbonate and sodium hydrogen carbonate showed pH 9.2 when it contained 1.06 gm NazCOs anhydrous (0.02 N) and 6.72 gm
548
FRAHN
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NaHC03 (0.08 N) in 1 liter water. (c) The elect,rolyte was prepared as in (b) and 8-hydroxyquinoline (0.1% w/v) was added. (d) A solution of 0.08M sodium hydrogen carbonate was prepared, but it was found to have pH 8.4, instead of pH 7.4-7.5, as stated by Jirgl (6; cf. refs. 5, 7). Part of it was adjusted to pH 7.5 by added carbon dioxide, and both solutions were examined 4. Spray reagent. Ninhydrin (0.2 gm) was dissolved in 100 ml nbutanol previously saturated with water, and 4 ml glacial acetic acid was added to the solution. PROCEDURE
,.The enclosed-strip electrophoresis apparatus has been described in detail- (5, 8). A strip of Whatman No. 4 paper, 13.5 X 61 cm, was enclosed in an envelope:of polyethylene film and pressed firmly against a cooling plate by means of a rectangular rubber bag, which was inflated with water to a pressure of approximately 0.4 atm. About 45 cm length of the strip was uniformly compressed and cooled. In order to protect the electrolyte on the paper strip during application of samples, the strip was covered, inside the envelop, by a separate thin mask of polyethylene film containing small holes through which samples were applied by means of a platinum loop for qualitative runs. The potential difference applied gave a gradient of about 20 V cm-’ over the working area of the paper; the current was about 40 mA. The temperature on the paper strip was measured with a thermocouple made of thin (0.2 mm diameter) wires of iron and Constantan. The junction was enclosed in a thin polyethylene envelop and inserted between the paper strip and the mask, and the output was displayed on a recorder (Yellow Springs Instrument Co., model 80) that had been calibrated with the junction of the thermocouple at 0” and 50’. The temperature on the paper during electrophoresis was about 20” when water at 18.5’ was circulated through the cooling plate at 2.5 liters/ min. When water at 48” was circulated the temperature of the paper was 50”. The paper strip was wetted with electrolyte by dipping, blotted lightly, and inserted in the apparatus, and 30 min was allowed for equilibration under pressure at the chosen temperature. The envelop was then opened and samples of about 0.5 ~1 were applied by means of the loop through the holes in the mask. The apparatus was then closed without delay, pressure was restored, and electrophoresis was carried out for 30 to 60 min. The paper strip was dried at lOO”, sprayed with ninhydrin, and heated at 100” to develop the color. Any carbamates present were decomposed during drying and development, leaving the
SEPARATION
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AMINO
ACIDS
549
amino acids, which gave spots of the same color’ as the free. amino acids (5) In some experiments cooling was interrupted and the temperature of the paper strip was allowed to rise unchecked; in others, the temperature initially was raised to 50” for a short time, then lowered to 20” for the electrophoresis. The effect of heavy metals on electrophoresis of amino acids under these conditions was determined by pretreatment of the paper strips with dilute solutions (usually 10d3M) of calcium, magnesium, lead, copper, zinc, cobalt, nickel, or iron (III). The paper was dipped in the solution of the appropriate metal, dried in warm air in a horizontal position, then dipped in the alkaline electrolyte in the usual way. RESULTS AND DISCUSSION Effect
of pH ad
Temperature
Experiments with the electrolyte at pH 12 gave results similar to those obtained (5) for various amino acids with 0.1 N sodium hydroxide. Pherograms run at room temperature, under conditions in which some exposure of alkaline solutions of the amino acids to atmospheric carbon dioxide had occurred, always showed additional spots for carbamates. Figure la shows the pattern of spots obtained for arginine, histidine, and ornithine; the unaltered amino acid is the spot farthest from the anode in each case, and the other spots (two for ornithine) represent carbamates. The connecting streaks result from slow breakdown of carbamates during migration. A comparable result was obtained when 0” water was circulated through the cooling plate, but because carbamates are more stable near O”, the streaks were barely detectable. The single carbamate spot for arginine and for histidine presumably represents formation of carbamate at the primary amino group only for each acid. It was not expected that nitrogen atoms of the guanidino group of arginine wouId be sufficiently nucleophilic to combine with carbon dioxide, and this seems also to be true of the imidazolyl group of histidine. When the paper was maintained at 50” throughout the run there were no streaks or multiple spots (Fig. lb). Any carbamates initially present must have decomposed before they could separate from the unchanged amino acids. If the run was started at 209 and the cooling water was then turned off, the temperature in the paper strip rose steadily and reached 35” after 30 min. Most of the carbamate had decomposed by the end of such a run, leaving a small residual spot of carbamate connected by a heavy streak to the spot of free amino acid. Other amino acids behaved similarly in this electrolyte, and in general multiple spots
550
FRAHN
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may be expected at 20” in highly alkaline electrolytes,. single spots. at 50°, and serious streaking at intermediate temperatures. Carbamates can also form and be detected by electrophoresis in less alkaline solutions (5). Runs at 20” in the carbonate-hydrogen carbonate mixture of pH 9.2 gave markedly elongated spots for many amino acids, showing that carbamates had been initially present, but, had rapidly decomposed during the run. Carbamates are more stable at 2” in this electrolyte. As expected (5), carbamates of the amino acids were much less stable in sodium hydrogen carbonate solutions at pH 7.5 or 8.4, but present, and survived long enough, to sufficient carbamate was initially
FIG. 1. Effect of temperature and pH of electrolyte on electrophoretic separation of arginine, histidine, citrulline, ornithine, and lytie during 30 min at 20V cm-l on Whatman No. 4 paper. The line of application is shown.
cause considerable elongation of spots from runs in these electrolytes- at 20”. When runs at 50” were performed in any of these carbonate-containing electrolytes the spots were sharp and nearly circular. A very satisfactory separation of arginine, citrulline, hi&dine, ornithine, an& lysine Was richieved at, pH 9.2 and 50’ (Fig. lc), rather better than that 6btained at 50” with electrolytes of pH 7.5 or 8.4. It had been found (5) that carbamates could be formed when solutions of amines in 0.1 iV aqueous ammonia were exposed to carbon dioxide at room temperature, but were rather unstable. This was confirmed by
SJWARATION
OF
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AMINO
ACIDS
551
attempts to repeat the published separation (4) of arginine; histidine, ornithine, and lysine by electrophoresis in 1 N ammonia (pH 11.7); in runs at 15’ all four acids showed extensive streaking. It seems that the present results could be reconciled with those described by Jirgl and others (l-4, 6) if the temperature during the run had not been adequately controlled in the earlier work. From descriptions available (1,3, 4, 6, 9) it seems that the apparatus used could have been of a simple, open-strip type, which would not give the positive control of temperature possible with the enclosed, pressurised apparatus. Passage of current through the alkaline electrolytes with a gradient of 20V cm-l generates heat of the order of 1 cal cm-2 min-I, and the temperature rises rapidly unless cooling is efficient. If the temperature of the paper strip reaches 40-50” during most of a run the migration of part of the amino acid as the carbamate ion would be prevented. The temperature in the paper strip is an important detail that should be included in descriptions of paper electrophoresis. If uncontrolled rise of temperature is accompanied by evaporation from an unenclosed strip of paper, results may become unreproducible for several reasons. The concentration and pH of the electrolyte may change through loss of volatile constituents. The relative mobilities of compounds may change because their dissociation constants or the extent of solvation of the ions respond differently to changes in temperature. The basic amino acids provide examples of differential effects of changes in temperature. Jirgl (1) illustrated a clean separation of histidine from ornithine in the tetraborate electrolyte at pH 12 and some unmeasured, but probably fairly high, temperature; Figure 1 shows that the two acids were not well separated in this electrolyte at either 50” or 20°, but they could be separated at 2”. Arginine and lysine, and also histidine and citrulline, were not properly separated in the sodium hydrogen carbonate electrolytes of pH 7.5 or 8.4 at 20°, but at 50” these pairs could be separated in either electrolyte. Alkaline electrolytes seem generally to have been avoided in the paper electrophoresis of amino acids (cf. refs. 10, ll), but they offer advantages if the formation of spots or streaks from carbamates is prevented by positive control of the temperature near 50’. The pH value of 9.2 for the carbonate-hydrogen carbonate electrolyte was chosen as being ,most likely to favor the separation at 50” of ornithine from histidine;:for which the calculated values (12) of pK, at 56’ are 9.93 and 8:47, respectively. However, the composition of an electrolyte, as well- as its pH value, can be important with amino acids; arginine and lysine were well separated in the carbonate-hydrogen carbonate electrolyte at pH
552
FRAHN
AND
MILLS
9.2 and 50”, but not in 0.05 iV sodium tetraborate (pH 9.2) at 50”, although histidine, citrulline, and ornithine were easily separated from each other and from arginine or lysine in borate at pH 9.2. Eflect of Heavy Metals The strong, nearly circular spot for an amino acid obtained after paper electrophoresis in the alkaline electrolytes at 50” was often accompanied by a faint but sharply defined shadow on the trailing edge. A similar shadow seems to be present at the spots for arginine and citrulline in Figure 1 of Jirgl’s communication (6). The defect resembled the “lagging shadows” observed in paper chromatography of organic phosphates (13) and seemed, likewise, to be caused by inorganic impurities in the paper. It was eliminated by incorporating 8-hydroxyquinoline (0.1% w/v) in the carbonate-hydrogen carbonate electrolyte, which did not otherwise affect the electrophoretic separation or the detection of the amino acids. Equally satisfactory results were obtained from electrophoresis on paper that had been treated with 8-hydroxyquinoline and thoroughly washed with water, as recommended by Hanes and Isherwood (13). Some shadows were still obtained with paper that had been extracted with 2N .acetic acid or 5% ammonia and washed with water. Attempts were made to intensify the shadows, and so determine the cause, by incorporating- additional metal cations in- the paper. Strips that had been treated with various salts were used for separations of the five basic amino acids in the carbonate electrolyte of pH 9.2 at 50”, but for no single metal was the shadow observed with all five acids. The of the presence of added Ca 2+, Fe3+, or Pb” caused some intensification shadows behind lysine and arginine. The shadows were very prominent when citrulline or histidine was run on papers impregnated with a mixture containing Ca2+, Fe3+, Cu*+, and Pb2+. It seems likely that traces of more than one metal in the paper may be responsible for formation of the shadows, and that several different cations may simultaneously interact with some of the amino acids. The strong adsorption of metal ions on paper under alkaline conditions would promote interference with migration of the acids if complexes were formed. Histidine was sensitive to the widest range of added cations,. and formed shadows .or streaks in the presence of Ca2+; Mg2+, ,FeS+, -h2t; .or Zn2*. The effect of Co2+ or Ni2+ was more -marked, and if either’ was incorporated in the paper histidine disappeared from its normal position. It seemed to be largely adsorbed near the site of application and reThe effect could provide useful sponded only weakly to ninhydrin. evidence for the presence of histidine in a mixture of amino acids.
SEPARATION
OF
BASIC
AMINO
ACIDS
553
No defects resulted from the presence of added heavy metals when the electrolyte contained S-hydroxyquinoline. Recommended
Procedure
All separations of amino acids by paper electrophoresis in alkaline electrolytes should be carried out with the temperature of the paper strip controlled near 50” to prevent interference from carbamates, and enclosed to prevent changes in composition of electrolyte. The above five basic amino acids are best separated in the mixture of sodium carbonate and sodium hydrogen carbonate of pH 9.2, to which 0.1% w/v S-hydroxyquinoline has been added. Application of 2OV cm-l for 30 min gives a satisfactory resolution. Preliminary
Destruction of Carbamates
Some basic compounds might not survive prolonged heating in alkaline solution at 50’. Since the tight seal of the paper strip in the present apparatus prevents access of atmospheric carbon dioxide after closure of the system, carbamates initially present can be destroyed by brief heating and none can be re-formed when the paper strip is cooled. Satisfactory results were obtained with the sodium tetraborate electrolyte of pH 12 when samples of amino acids were applied in the usual way, if the paper was held at 50’ for 5 min after closure of the apparatus, the current was then switched on for 2 min only, and then switched off for a further 5 min while heating at 50” was continued. When the paper was cooled to 20” or below after this preliminary treatment, electrophoresis could be conducted at the lower temperature without any spots for carbamate being detected. The brief electrophoresis at 50” ensures uniform pH through the spot and aids decomposition of the last traces of carbamate during the subsequent heating. It should be noted that this procedure may fail with electrolytes containing carbonates unless they are highly alkaline. Solutions of carbonates below about pH 11 contain sufficient free carbon dioxide to permit the slow formation of carbamates at low temperatures (5). SUMMARY
Alkaline solutions of amino acids absorb carbon dioxide, giving carbamates, which may appear as additional spots or streaks after paper electrophoresis in alkaline electrolytes. Interference from carbamates is prevented by carrying out electrophoresis at 50”. Mixtures of arginine, histidine, citrulline, ornithine, and lysine are resolved by paper electrophoresis at 50” and pH 9.2 in an electrolyte containing sodium carbonate
554
FRAHN
AND
MILLS
and sodium hydrogen carbonate. Addition of Shydroxyquinoline interference from impurities in the paper.
prevents
ACKNOWLEDGMENT We are grateful to Dr. D. D. Perrin, Australian National calculating dissociation constants of amino acids at 50".
University,
for
REFERENCES 1. JIROL, V., Anal. B&hem. 8, 519 (1964). 2. GERLAXHE, S., AND RENARD, M., Ind. Chim. Beige 20, Spec. No., 509 (1955); Chem. Abstr. 50, 7013 (1950). 3; REXOVA-BENKOVA, L., AND MARKO&, O., Collection Czech. Chem. Commun: 24, 1728 (1959). 4. BISERTE, G., DAXITREVAUX, M., AND BOULANQER, P., Bull. Sot. Chim. France 1963, 2954. 5. FRAHN, J. L., AND MILLS, J. A., Australian J. Chem. 17, 256 (1964). 6. JIRQL, V., Anal. Biochem. 13, 381 (1965). 7. “Handbook of Chemistry and Physics,” 45th ed, (R. C. We&, ed.), p. D-73. Chemical Rubber Co., Cleveland, Ohio, 1964-1965. 8. FRAHN, J. L., AND MILLS, J. A., Australian J. Chem. 12, 65 (1959). 9. JIRGL, V., AND SOCHMAN, J., Clin. Chim. Acta 7, 388 (1962). lb. BLACKBURN, S., Methods Biochem. Anal. 13, 1 (1965). 11. EDWARD, J. T., AND WALDRON-EDWARD, D., J. Chromatog. 24, 125 (1966); see in particular p. 130. 12. PERRIN, D. D., personal communication. 13. IXANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107 (1949).