Analyst, October, 1967, Vol. 92, p p . 627-633 627 A Simple Quantitative Method for the Determination of Small Amounts of Amino-acids BY J. G. HEATHCOTE AND K. J. WASHINGTON (The University of Salford, Salford 5, Lancashire) h simple method for the quantitative determination of amino-acids has been described. The method depends upon the paper-chromatographic separation of the amino-acids, followed by staining to form a coloured complex with cadmium acetate and ninhydrin. The coloured spots are eluted from the paper with methanol and determined colorimetrically. Amounts within the range 0.1 to 40 pg (0.1 to 25 ,umoles-2) can be determined by this method to within an over-all accuracy of +10 per cent. FEW simple convenient methods are available for the separation and quantitative determina- tion of amino-acids in amounts of 0.1 to 40 pg (0.1 to 25 pmoles-2).Atfield and Morris1 used high voltage electrophoresis to separate mixtures of amino-acids, which were then determined by the cadmium - ninhydrin method of Heilmann, Barollier and Watzke.2 The precision claimed was comparable with that obtained by means of the ion-exchange method of Moore and Stein, and 250 pg of protein were sufficient for a complete analysis. We have developed a similar method for the determination of amino-acids in biological fluids, involving separation of the amino-acids by paper chromatography and dispensing with the need for expensive high voltage electrophoresis equipment. The method is capable of being carried out with simple apparatus such as a chromatographic tank and a colorimeter.It consists in developing chromatograms, uni- or bi-dimensional, in suitable, preferably volatile, solvents and then drawing each of the dried sheets through a standard volume of the staining reagent. In order to reduce the background colour as far as possible, a lower concentration of ninhydrin was present in the reagent than was used by Atfield and Morris.1 Furthermore , to remove the necessity for shaking equipment and subsequent filtration, the coloured complexes were eluted from the paper with methanol. In its present form the method is applicable to the estimation of amino-acids that are present in low concentration in biological fluids, the amino-acids being first separated by paper chromatography. The method has been applied to protein-free filtrates of human gastric juice obtained from normal subjects and from pernicious anaemia patients3; samples of de-salted urine (D.M. Davies, unpublished work); and bacterial culture fluids (M. F. Dutton, unpublished work). EXPERIMENTAL REAGENTS AND APPARATUS- All solvents and the cadmium acetate were of analytical-reagent grade. The concen- trated, reagent grade, ammonium hydroxide was kept in the refrigerator. Chromatographic solvent systems-Although any suitable solvent systems can be used, the most convenient pair for the general separation of the common amino-acids is the propan- 2-01 - water - ammonium hydroxide (7 + 2 + 1 v/v) mixture of Heathcote and Washington,4 and the butanol -acetic acid - water (12 + 3 + 5 v/v) of Smith.5 These are referred to in the text as solvent systems 1 and 2, respectively.For the separation of the leucine - isoleucine mixture the solvent system of Heathcote and Jones6 is recommended. This consists of propan-2-01- formic acid - water (20 + 1 + 5 v/v) as the first solvent, and t-butyl alcohol - ethyl methyl ketone - ammonia solution - water (50 + 30 + 10 + 10 v/v) as the second solvent. Amino-acid standard solutions-Stock solutions of amino-acids, 0.05 M, were made in aqueous propan-2-01, 10 per cent. v/v, with the minimum amount of dilute hydrochloric acid added to effect solution, if necessary. The solutions were kept in the refrigerator and diluted to 0-01 M as required.628 HEATHCOTE AHD WASHINGTON : A SIMPLE QUANTITATIVE METHOD [Analyst, Vol. 92 Amino-acid stainiag reagent-The staining reagent for the separated amino-acids was essentially the same as that used by Atfield and Morris,l but a lower concentration of ninhydrin was used for the following reasons.Opienska-Blauth, Kowalska and Pietru~iewicz~ recom- mended that the concentration of ninhydrin in the cadmium acetate - ninhydrin mixture should be less than 0-5 per cent. w/v in order to ensure negligible values for the blank. Atfield and Morris1 used a concentration of 1 per cent. w/v and although this increases the final intensity of the colour it tends to produce a much higher blank. The reagent was made by dissolving 0.5 g of cadmium acetate in 50 ml of water to which 10 ml of glacial acetic acid had been added. Acetone was then added until the total volume was 500 ml.Portions of this solution were taken before use and sufficient solid ninhydrin was added until the final concentration was 0.2 per cent. w/v. Chromatographic apparatus-Smith's universal apparatus with a 10-inch aluminium frame was chosen, and pre-punched square sheets of Whatman No. 1 chromatographic-paper were used. A polythene dip-tray was used for staining the sheets of paper. Micro-Pipettes-A series of heavy-wall capillary pipettes and precision capillary pipettes were made and each was calibrated to deliver a known volume of liquid. The series of pipettes covered the range 1 to 20 p1. An Agla syringe was found convenient for transferring a series of volumes of the same liquid, such as 2.5, 5, 10 and 20 p1. Miscellaneous apparatus-The papers were handled on glass plates in order to prevent any contamination of the paper surface and supported on glass rods after staining.Volumetric and other apparatus-Beakers of 5 and 10-ml capacity were used for collecting the eluates, and the volume of each was made up to 10 ml in a B grade graduated flask. Elution apparatus-This was based on the publication of Kemble and MacPherson.8 The apparatus used is shown in Figs. 1 and 2. A piece of Whatman No. 1 filter-paper was cut, Fold \ Glass Glass trough- Cork support\ Beaker /Glass I L Fig. 1. Elution apparatus, end view Glass cover / trough, 1-1 Glass plate Fig. 2. Elution apparatus, side viewOctober, 19671 FOR THE DETERMINATION OF SMALL AMOUNTS OF AMINO-ACIDS 629 as shown in Fig. 3, to the shape of a rectangle 30 cm x I cm where I is the length of the trough. The length of trough, 17, 26, or 40 cm, was chosen according to the number of spots to be eluted.The paper was folded along the centre and in an outward direction along a line 3 cm on each side of the central fold. Projections, 10 cm long and 1.5 cm wide, were left with a gap of 1 cm between each projection along each edge. The size of the projection was always slightly larger than the size of the spot, or band, to be eluted, in order to obtain quantitative elution. Fig. 3. Plan of paper cut for elution (not to scale) One or two troughs, semi-circular in cross-section, were accommodated under a rect- angular glass cover. This maintained a solvent-saturated atmosphere in the apparatus. A 5-ml beaker was placed under each 10-cm projection, which was adjusted so that when hanging vertically, it reached one-third of the way down the beaker.METHOD The amino-acids are first resolved into groups by solvent system 1. The groups are then resolved into individual components, if necessary, by the application of solvent system 2 or other suitable systems. APPLICATION OF AMINO-ACIDS AND DEVELOPMENT- For one-dimensional paper chromatograms the papers were marked with the names and concentrations of the amino-acids used. The spacing between each pair of amino-acid spots was at least 3 cm to avoid overlap. The solution of amino-acids to be examined was applied by means of one of the calibrated pipettes, or the Agla syringe, to give a spot of diameter not greater than 0.5 cm. A volume of from 1 to 10 pl of each particular standard solution (0.01 M) of amino-acid was applied.If the volume of solution to be applied was greater than 4 pl, the application was performed in stages with intermittent drying by means of an air blast. The solution was applied as a narrow band along the base-line for one-dimensional paper chromatograms. Development was then carried out with solvent system 1 or solvent system 2, or both, as previously described. The solvent (200 ml) was placed in the aluminium tray. The aluminium frame holding five individual papers was placed in position and the heavy ground-glass lid replaced. Air-tight conditions were ensured by greasing the edges of the lid. The tank was placed on a horizontal surface to ensure a level solvent front and uniform development.The solvent was allowed to run for 11 hours at a temperature of about 21" C. Under these conditions optimum resolution was achieved, the solvent front travelling about 20 cm. The solvent front was best located as a bright line under ultraviolet light. REMOVAL OF SOLVENT- papers in a current of air. any residual smell of ammonia in about 1 hour. When development was complete, the solvent was removed by placing the frame and If solvent system 1 was used, the papers were dry and free from If solvent system 2 was used, even when630 HEATHCOTE AND WASHINGTON : A SIMPLE QUANTITATIVE METHOD [Analyst, Vol. 92 there was no detectable smell of solvent, it was found necessary to continue blowing air for at least 5 minutes over each paper, otherwise any residual acetic acid retarded the development of the colour produced by the staining reagent.Although similar results were obtained whichever solvent system was used first in a two-dimensional chromatogram, it was more convenient to develop first with solvent system 1 because of the speed with which the solvent could be removed from the paper. STAINING- The dry chromatogram was drawn slowly through 15 to 20ml of the staining reagent contained in a polythene dip-tray. After use, the reagent was discarded, the tray dried and fresh reagent poured out for the next sheet; thus concentration of the reagent by evaporation was avoided and consistent results were obtained. Accidental fingerprints were avoided by the use of forceps and by handling above the solvent front. The paper, after hanging until visibly dry, was then carefully transferred to a dark, ammonia-free atmosphere in which the papers were kept overnight. This allowed the amino-acid complexes to attain maximum intensity with minimum development of background colour.The best container was a large desiccator containing concentrated sulphuric acid. In such a container the background of small stained strips remained colourless for many weeks. A good substitute for the desiccator was provided by placing the papers on glass supports inside a well fitting cupboard containing a small beaker of concentrated sulphuric acid. This kept the background colourless for about 3 days, although the papers were best examined 24 hours after staining. The papers developed a pink background colour on standing for several days and this made the location of trace amounts of 1 to 2 pg of amino-acids difficult because of the lack of contrast.Surprisingly enough, the colour of the stained spots of amino-acids remained constant within experimental error. EFFECT OF HEAT ON COLOURED COMPLEXES- Heating caused a deepening in the colour of the complex but tended to render it insoluble in any eluting solvent. Thus heating to 80" C for 1 minute rendered the complex completely insoluble in methanol. Consequently, heating was not used to develop the colour; instead, the papers were allowed to stand in the manner described until the maximum colour had developed. ELUTION OF COLOURED COMPLEXES- The cadmium - ninhydrin complex was eluted from the paper by means of dry, acid-free methanol.Each stained spot was cut out with forceps and stainless-steel scissors and then sewn on to a paper tongue, with a glover's needle (Nos. 3 to 7) and white cotton. The spot and paper strip were held together by cotton, as metal staples cannot be used because these react with the cadmium - ninhydrin c~mplex.~ The spots were sewn on to tongues of paper, 10 cm x 1.5 cm, and the paper placed for elution as shown in Figs. 1 and 2. About 150 ml of methanol were placed in the trough, the cover replaced and the eluates collected in the beakers. It was possible, by using this apparatus, to elute as many as thirty spots at any one time, with a trough of 40-cm length. It was observed that the coloured complexes formed by most of the amino-acids were eluted within 2 hours, only one or two of relatively large amounts, 15 to 25pg, requiring a longer time.It was found convenient to allow the elution to continue overnight, during which time the strips were completely eluted. When the cover was removed, each strip was brought clear of the liquid surface to prevent it from drying out in the atmosphere and thus re-absorbing the eluate. COLORIMETRIC DETERMINATION- The eluate from each spot, which corresponded to a known amount of amino-acid, was made up to a volume of 10ml in a graduated flask. The optical density of the eluate was determined against a blank of pure methanol in a 4-cm glass cell at the position of maximum absorption on a Hilger Uvispek. The maximum wavelength of the red complex was 500 mp and that of the yellow complex of the amino-acids, proline and hydroxyproline, 352 mp.The background reading from the paper was nearly always less than 0.02 in optical density and was therefore negligible. The paper was cut as in Fig. 3.October, 19671 FOR THE DETERMINATION OF SMALL AMOUNTS OF AMINO-ACIDS 631 Graphs of optical density against pmoles-2 of amino-acid were plotted for each amino- acid. Each graph was linear over the range of amounts of amino-acids applied, 0.1 to 25 pmoles-2, but the slope was found to be different for each amino-acid. This was largely to be expected in view of the results of Heilman, Barollier and Watzke,2 who showed that the colour yield of the complex from each amino-acid was different in each case. APPLICATION AND RECOVERY EXPERIMENTS- Various amounts of standard amino-acids between 5 and 50 pmo1es-2 (4.4 to 44.8pg) were applied to sheets of chromatographic paper and the recoveries were determined after chromatography. A known weight, 1.2 mg, of alanylglycine was hydrolysed under nitrogen with 0.30 ml of 5-9 N hydrochloric acid for 20 hours at 105" C in a sealed tube. After removal of excess of acid, the contents were made up to 5 ml and duplicate samples (A and B) of 39.2 p1 each were examined by chromatography.To sample (B) 10 pmoless2 each of alanine and glycine were added and the recoveries ascertained. These conditions of hydrolysis were used for a peptide found in normal human gastric juice, but many biological samples were examined without hydrolysis for the presence of free amino-acids.These included protein-free filtrates of gastric juice shown in Table I and de-salted samples of normal urine and urine from patients with pernicious anaemia, as shown in Table 11. TABLE I FREE AMINO-ACIDS OF NORMAL GASTRIC JUICE Optical Amount of amino-acid, Concentration of amino-acid, Amino-acid density pmoles-2 mg per cent. w/v Alanine . . . . . . 1-455 Aspartic acid . . . . 0,115 Arginine . . . . . . 0.650 Glutamic acid .,. . . 0.280 Glycine . . . I . . 0.225 Leucine . . .. . . 0.74 Valine . . .. . . 0.415 19.1 3-0 7.5 2.7 8-0 9-1 5-1 Amino-acid Alanine . . Aspartic acid Glutamic acid Histidine . . Phen ylalanine Taurine . . Tyrosine . . TABLE I1 FREE AMINO-ACIDS OF URINE Concentration of amino-acids in mg per cent. w/v 1.7 0.4 1.3 0.4 0.6 1.2 0-6 Pernicious anaemia -n r Paper chromatography ion exchange .. 4.7 5.7 . . 0.66 0.93 . . 19.9 16.7 . . 11.4 15.9 . . 1.06 1.73 .. 21.5 19.8 .. 4.3 5.0 Normal Technicon rw ion exchange ion exchange 5.5 1.4 to 4-7 0.48 < 0.6 2-66 0-53 to 2-66 10.5 7.5 to 21-3 3.5 0.6 to 2.06 8-0 5.7 to 19.6 0.99 1.0 to 3.2 RESULTS AND DISCUSSION The median values of optical density that were obtained for varying amounts of 8 control amino-acids are given in Table 111. Table IV illustrates the experimental values obtained for lysine and glycine. These have been chosen because they represent the extreme values of colour yield for a given amount of amino-acid applied to the paper. The method was found to be reproducible if certain variable factors were controlled. The main sources of error arose in the addition by pipette of the standard solutions, and in the control of the amount of reagent coming in contact with each amino-acid; other errors include the sensitivity of the staining system to pH, acid preventing the development of the colour, and the presence, or absence, of background colour.The error from the last can be caused by an increase in intensity due to the presence of ammonia vapour in the atmosphere.632 HEATHCOTE AND WASHINGTON : A SIMPLE QUANTITATIVE METHOD [Analyst, VOl. 92 TABLE I11 TABLE OF MEDIAN VALUES OF OPTICAL DENSITY FOR SEVERAL AMINO-ACIDS Optical density A r pmoles-2 0.1 0.5 1-0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 10.0 12.0 13-0 15.0 17-0 20.0 22.0 25.0 Alanine 0.005 0.027 0.105 0.210 0-257 0.360 0.430 0-504 0-575 0.647 0.711 0.790 0-843 1-092 1.365 1.730 1.745 1-882 Aspartic acid 0.018 0.031 0.035 0-067 0.078 0-152 0.205 0.235 0.260 0.315 0.407 0-485 0.525 0.645 0-695 0-817 0.900 1.065 Arginine 0.015 0.049 0.100 0.188 0.280 0.360 0.450 0.530 0-644 0-653 0.950 1.044 1.215 1.285 1.476 1.796 1.870 1.925 Glutamic acid 0.014 0.052 0.180 0.284 0.300 0.380 0.470 0-543 0-620 0.670 0.860 0.990 1.100 1-242 1.387 1.665 1.860 2.090 G1 ycine 0.002 0.071 0.078 0.110 0-13e 0.234 0.190 0.200 0.205 0.230 0.235 0.267 0.325 0.340 0.390 0.430 0.480 0.540 Leucine 0.012 0.035 0-208 0.300 0.400 0.464 0.500 0-568 0.645 0.680 0.835 0.900 0.990 1-140 1-240 1.420 1-495 1.740 Lysine 0.023 0.048 0.105 0.190 0.268 0.350 0.385 0.51 1 0.635 0.700 0.900 1.050 1-151 1-27 1 1.450 1-745 1.950 2.115 Valine 0.028 0.057 0.105 0.184 0.23 1 0.323 0.41 1 0.453 0.548 0.691 0.738 0.839 0.950 1.100 1.240 1.410 1.582 1.840 The method has given consistent results as can be seen from Table IV.The percentage variation in the results was within +lo per cent., a variation similar to that obtained by Atfield and M0rris.l The method was applicable to the determination of smaller amounts of amino-acids, from 20 pg down to less than 2 pg in certain favourable cases, than the 40 to 10 pg used by Atfield and M0rris.l TABLE IV VARIATIONS I N READINGS OF OPTICAL DENSITY FOR GIVEN AMOUNTS OF GLYCINE AND LYSINE Optical density I 3 Glycine Lysine pmoles-2 & - 1.0 0.080 0.072 0.078 0.108 0.105 0.107 5-0 0.190 0.197 0.190 0.383 0.385 0.400 10.0 0.230 0.220 0.240 0.900 0.910 0.890 20.0 0.405 0.430 0.440 1.750 1.740 1.745 25-0 0.537 0-540 0.540 2.115 2.065 2.185 TABLE V RECOVERY OF VARIOUS AMINO-ACIDS AT DIFFERENT STAGES OF ANALYSIS Amount applied to paper (-Ap, Recovery, Amino-acid pmoles-2 CLg per cent.Procedure Aspartic acid . . 5.0 6.6 97 One-dimensional chromatography Alanine . . .. 5.0 4.4 97 One-dimensional chromatography 10.0 13.3 96 10.0 8.9 93 15.0 13-4 98.5 20.0 17.8 99.5 Alanine . . . . 50.0 44.8 100 Direct staining 50.0 44.8 99.7 Two-dimensional chromatography Arginine . . . . 5.0 8-7 98 One-dimensional chromatography 10.0 17.4 98 15.0 26.1 98-5 20.0 34.5 95October, 19671 FOR THE DETERMINATION OF SMALL AMOUNTS OF AMINO-ACIDS another. be determined at 1 pg (see Table 111, 1 pmole-2 == 1 pg). experiments, were shown by staining to be well defined and compact.97 per cent. or more, of individual amino-acids was obtained (see Table V). 633 The lower limit of quantitative determination varied slightly from one amino-acid to For example, glycine was difficult to determine below 3 pg, but leucine could easily The amino-acid spots, after development in the solvent system used in the present Further, good recovery, Results are given in Table VI for the analysis of the model peptide, alanylglycine. TABLE VI RECOVERY OF AMINO-ACIDS FROM A MODEL PEPTIDE (ALANYLGLYCINE) AFTER HYDROLYSIS Alanine, pmoles-2 Glycine, pmoles-2 - - Sample (A) (*) (A) (B) Theoretical amount . . 6.43 16.43 6.43 16.43 Optical density . . .. 0.500 1.280 0.182 0.280 Experimental amount . . 6.3 16.6 6-1 16.7 Percentage recovery . . 98 101 94 101.5 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Atfield, G. K., and Morris, C. J . 0. R., Biochem. J . , 1961, 81, 606. Heilman, J., Barollier, J., and Watzke, E., Hoppe-Seyler’s 2. physiol. Chem., 1957, 309, 219. Heathcote, J. G., and Washington, R. J., Nature, 1965, 207, 941. -- Chem. & Ind., 1963, 909. Smith, I., Editor, ‘‘Chromatographic and Electrophoretic Techniques,” Second Edition, W. Heine- Heathcote, J. G., and Jones, K., Biochem. J., 1965, 97, 15P. Opienska-Blauth, J ., Kowalska, H., and Pjetrusiewicz, M., Annis Univ. Mariae Curie-Sklodowska, Kemble, A. R., and MacPherson, H. T., Biochern. J . , 1954, 56, 548. Wieland, T., Fortschr. chem. Forsch., 1949, 1, 211. Stein, W. H., J . Biol. Chem., 1953, 201, 45. mann and Co., London, Volume 1, 1960. 1957, Section D . l l , 175. Received March 18th, 1966