Some Factors Influencing the Quantitative Determination of Amino Acids Separated by Circular Paper Chromatography K. V. GIRI, A. N. RADHAKRISHNAN, AND C. S. VAIDYANATHAN Department of Biochemistry, Indian I n s t i t u t e of Science, Bangalore, India
AX attempt to develop a simple and accurate procedure Itheforextraction the quantitative determination of amino acids, based on of the ninhydrin-stained bands of amino acids with r\;
alcohol ( I , Q), by the technique of circular paper chromatography developed in this laboratory, some factors were encountered which influence the tone and color intensity of the bands and of the alcohol extracts. These findings have an important bearing on the quantitative estimation of amino acids by paper chromatographic techniques. EXPERIMEYTAL
Influence of Copper on Tone and Intensity of Color of Alcoholic Extract of Ninhydrin-Stained Bands. Firstly it was observed that traces of copper markedly affected the tone and intensity of the color of the alcoholic extracts of the ninhydrin-stained bands. The bluish-violet color of the alcoholic eluate of the bands was changed to reddish-purple color on the addition of copper sulfate. The effect of different concentrations of copper (as copper bulfatr pentahydrate) on the alcoholic eluate of the ninhydrin color is illustrated in Figure 1. The color density curves show that the reddish-purple color obtained was mavimum at a concentration of 0.2 mg. copper sulfate in 4 ml. of alcoholic eluate and decwased very slowly with increasing concentrations of copper up to 1.2 mg. This was true for most of the amino acids tried. In the light of these experiments it is clear that the sensitivity of the colorimetric method can be greatly enhanced by the addition of the optimum concentration of copper. The calibration curves using the optimum amount of copper (0.2 mg. of copper sulfate in 1 ml. of alcoholic extract) showed direct proportionality between the color density and the concentration of amino acids in the range 2.5 to 12.5 micrograms. By this modified method asparagine at low concentration could he quantitatively determined, a. the optical density is very much increased in presence of added copper. Relation between Area of the Band and Color Density. Secondly it was observed that the intensity of the color extracted from the band obtained after reaction with ninhydrin on paper varied with the area of the band. In circular paper chromatography the area of the ninhydrin-stained bands was found to increase with the increase in the distance traveled bv the amino acid, and therefore larger surface area was exposed for rcaction nith the reagent resulting in increase in the intensity of color evtracted. The results of some of the typical expeiinients carried out on the influence of the area of the band on the color intensity are shorn in Table I. The amino acids were chromato-
Table I.
Amino acid (10.8 Y ) Threonine
Relation between Area of Band a n d Color Intensity Distance Traveled by Amino Acid, Inches 0.1 0.5 1.2
T'aline Leucine
0.5 1.1 2.4 0.8 1.46 3.0
Area of the SinhydrinStained Bands, Sq. Inch 0.12 0.19 0.23 0.27 0.33 0.71 Q.21 0.34 0.75
Color Density 78 114 120 120 142 177 137 167 215
graphed as described in (2) and allowed to travel varying distances from the origin of the spot. This can be easily achieved by drawing concentric circles on the paper and spotting the solution on the circumference of each circle in a spiral form. When the chromatogram is run to the full, it is clear that the drop3 travel varying distancep. The drop nearest to the center travels the most. The air-dried paper was then sprayed with 0.5% ninhydrin reagent, heated a t 65' C. for 30 minutes, and the color bands were extracted with 4 ml. of 75y0ethanol containing 0.2 mg. of copper sulfate. The color density was determined using a Klett-Summer-on photoelectric colorimeter with green filter (540 mp).
O
U./ 0.2 0.3 CONCN. of CuSo 4
0.4
550,
0.5
0.6
my
Figure 1. Effect of Different Concentrations of CuS04.5H20on Intensity of Ninhydrin-Stained Bands Solid circle = valine, circle = leucine, X = isoleucine, triangle = glutamic acid, and square = aspargine
The area of the bands was measured by means of a planimeter. Giri et al. ( I ) had stated in an earlier report that it is desirable to verify the value obtained from the calibration curve by running a mixture of knon n amino acids of known concentrations on the same paper and comparing the color intensitiw of the solutions v ith those obtained by running the mixture of unknown amino acids-e.g., protein hydrolyxates. It was found, however, that it is imperative to run the amino acids on the same paper to obtain reproducible results, since the intensity of the color increases with the size of the band, x-hich in circular paper chromatography, depends on the distance ti aveled by the band and the solvent front. FIN4L PROCEDURE
-1rigorous set of conditions, under which the method is highly successful, ivere selected after a thorough study of the variables. The chromatogram was run as described by Giri and Rao (2). A circle 4 cm. in diameter as drawn from the center of the filter circle. The solutions of known and unknown mixtures of amino acids were applied alternately along the circumference of the
1677
ANALYTICAL CHEMISTRY
1678 circle using an accurately calibrated capillary tube. The adjacent spots should not coalesce. The spots were dried a t room temperature, and the chromatogram was developed using Partridge's solvent (butanol-acetic acid-water, 40: 10:50). The paper was air-dried and uniformly sprayed Trith 0.5% ninhydrin in 95% acetone. After drying in a current of air the paper was k t y t a t 65" C. for 30 minutes. The zones w r e cut out and extracted with 4 ml. of 75% ethyl alcohol containing 0.2 mg. of copper sulfate, care being taken t o avoid contamination of paper from the hands or by dust. While running the chromatogram the distance of the movement of solvent was closely controlled. For purposes of drawing calibration curve?, the distance of the solvent boundary from the center of the paper (9 cm.) was always kept constant. The optical density was measured using a KlettSummerson colorimeter with green filter (540 mp). d blank detprmination was also made with uncolored area, and correction was applied in each case.
determined accurately with ease and facility. Thompson et a / . ( 3 ) drew attention to the fact that considerable losses of amino acids occurred in two-dimensional chromatograms. Circular paper chromatography eliminates errors due t o this t o a very great extent, as shown by recovery experiments. Using this method the nitrogen metabolism of leaves and leguminous seeds and the role of transamination in protein synthesis are being investigated. LITERATURE CITED
(1) Giri, K. V., Krishnamurthy, K., and T'enkatasubramanian, T. A., CzLrrent Science, 21,44 (1952). ( 2 ) Giri, K. V., and Rao, N. A. N.. ,Vatwe, 169,923 (1952); J . Indiun Inst. Sci., 34, S o . 2, 95 (1952). (3) Thompson, J. F., and Steward, F. C., Plant Phusiol., 26, 421-40
ADVANTAGES OF T H E METHOD
This quantitative procedure has proved to bc a very useful tool. I n view of the clear separation obtained by circular paper chromatography the individual amino acids in a mixture can be
(4)
(1951). Thompson, J. F., Zacharius, R. XI., and Steward, F. C., I b t d . , pp. 375-97 (1951).
RECEIVKD for reyiew April 4, 1952. Accepted .June 14, 195'2
Determination of Tracers in the Presence of Their Radioactive Daughters H. W. KIRBY Moccnd Laboratory, Miamisburg, Ohio
IXTURES of genetically related radioisotopes are frequently encountered when tracers are employed to follow a chrmical reaction. If the particles emitted by parent and daughter are of the same kind, and especially if they are of similar energies, the question arises as to what proportion of the measured radioactivity is due to each nuclide. very simple equation can be derived which is applicable to a great many tracers, since, in general, it is the percentage carried rather than the absolute amount of thp tracer which is sought. Consider the case of a radioactive tracer having only one radioactive daughter. Let = = -41 = kl = A\-l
X1
Equations 1 and 4 are now solved simultaneously for A ,
the activity of the parent a t time, tl. If this mixture undergoes a fractionation, so that unknown amounts of the parent and daughter are present in each fraction, T , it and if each fraction is then counted a t times t 2 and t f follows from Equation 5 that the activity of the tracer in the fraction a t time t? will be
+
the number of atoms of the parent a t an arbitrary time, t l the decay constant of the parent (X = 0 . 6 9 3 / T l / ~ ) the activity of the parent (A-1E.l) a constant representing counting yield ("geometry")
and let N,, A,, A*, and kl have similar connotations with respect to the daughter. The total counting rate in the mixture a t time tl will he
C1 At a later time, tl ( 11
klNlX1
+ kz'V2X2
= ki-41
+ k2A2
where C, and C, are the counting rates of the mixture a t f l and tP T , respectively. Since the total activity of the parent a t t2 will be Ale-A1(t2-Li), on dividing Equation 6 by Equation 5
+
(1)
+ T, the counting rate for the parent will be Furthermore, it frequently happens that t, - tl can be made very small compared with the half-life of the parent, in which case, as X l ( f 2 - t l ) approaches zero
and the counting rate for the daughter will he
The total counting rate for the mixture a t tl
+ T will be
Equation 8 defines the ratio of tracer in the fraction to total tracer, if aliquots of each are counted a t nearly the same time. I t is not necessary that the chemical procedure be of short duration in order that tx - tl may be kept small, since an aliquot of the original mixture can be retained for counting a t the later time. I n practice the time T , however, should be chosen equal to or greater than the shorter of the two half-lives involved. Equation 8 is applicable only to the case of a radioactive tracer having a daughter which decays to a stable isotope. Similar
