the isolated internal standard technique can be used to correct any observed counting rate. However, a more important application is the study of complex counting systems to separate the effects of color and chemical quenching. I n this respect, the technique offers two important advantages over the spectrophotometric method. The isolated internal standard is faster requiring only
one additional measurement. Also, expensive accessory equipment is unnecessary; an easily prepared standard assembly that is stable indefinitely is all that is required. LITERATURE CITED
(1) DeBersaques, J., Intern. J . A p p l . Radiation Isotopes 14, 173 (1963).
(2) Guinn, V. P., “Liquid Scintillation Counting,” Proceedings of Xorthwestern
University Conference on Liquid Scintillation Counting, C. G. Bell and F. N . Hayes, eds., p. 173, Pergamon, New York. 1958. (3) Ross, H: H., Yerick, R. E., ANAL. CHEM.35,794 (1963).
RESEARCH sponsored by the U. S. Atomic Energy Commission under contract with Union Carbide Corp.
Increased Sensitivity in Automatic Amino Acid Analysis Obtained with a Special Hybrid Recorder Aline U. Orten, Herbert J. Doppke, and Harry H. Spurrier, Wayne State University School of Medicine, Detroit 7, Mich.
SPECIAL
“hybrid”
recorder,
as-
A sembled a t our request, which may
be suitable for many analytical and !ndustrial applications, was found to increase the sensitivity of automatic amino acid ion exchange column chromatography to an extent that measurements of amounts of amino acids in the nanomole range may be cai-ried out with existing commercial or bench-built equipment and the conventional logtype chart paper. Hamilton ( 3 ) described the use of a variable span recorder to obtain improved sensitivity and later (2) determined amino acids at 10-nanoniole levels to nithin *5y0 using a highly refined bench assembly. Hubbard and Kremen ( 5 ) reported that sensitivities approaching 5 nanomoles with 3yo precision could be obtained with an improved photometer. Kirsten and Kirsten (6) obtained amplification by reducing column diameter and thereby determined 2 nanoinoles and below with a *2% average error. I n the present work, an increase in sensitivity was obtained at lev& of 5, 2, and 1 nanomoles with an average deviation from the mean recovery within 2, 3, and 5Y0, respectively. EXPERIMENTAL
Recorder. Two analyzers, a commercial model, Spinco 120 B, and our own bench-built analyzer constructed in 1957 employing the design of Spackman, Stein, and Moore (8) were used. I n place of the recorders used with these instruments, a modified recorder composed of two units was substituted. I t was constructed from a Speedomax G, three-channel, twelvepoint recorder (hfodel S, Series 13, Serial S o . 63-29091-1-1 printing 30 dots per minute) and a separate portable AZAR H, variable span, variable range, control console. This AZAR unit was built a t our request by the Leeds and Korthrup Co., Philadelphia, Pa. The combined units permit the use of the
rapid switching and precise control advantages of the M A R unit along with the three channels, the wide chart paper, and the stability of the Speedomax G recorder. Spans of 10, 5, 2, 0.67, and 0.35 mv. (corresponding to absorbances of m , 0.301, 0.097, 0.0302, and 0.0155, respectively) full scale were used. The 0.35-span was obtained by placing a 1000-ohm Helipot in series with the reference voltage source. Spans as low as 0.26-mv. full scale were thereby reached, but base-line noise created a practical limit of 0.35 mv. Only 1 second is needed to switch spans. Internal calibration is incorporated in this recorder, therefore no further adjustments are necessary after the initial calibration. The amplification is linear with respect to voltage and hence to light received from the photocell. Reduction in photocell voltage is proportional to the reduction of the light intensity, and is also proportional to the reduction in transmittance-Le., the fraction of light transmitted by the sample. Thus, color peaks are amplified linearly. The span settings through 0.67 mv. were found to be reproducible within 0.13% using the standard e.m.f. from a millivolt potentiometer (L&N, Yo. 8690) as a source. Zero suppression can be moved smoothly and reproducibly throughout all ranges. The hybrid recorder proved to be sensitive and highly stable-e.g., with a 40-mv. input, input variations of 1 part in 50,000 were detected on the 0.67 range; the prevailing noise level was 1 wv. A paper speed of 8 i.p.h. was used for spans 10, 5, and 2 mv. while 12 i.p.h. was helpful in the calculation of peak areas recorded with spans of 0.67 and 0.35 mv. Integration of Peaks. T h e H X JV peak integration method of Spackman, Stein, and Moore (8) was used. The usual chart paper (8) such as L and N No. 578, log type, has a 0- to m absorbance scale which, in this work, can be read directly for the 0- to 10-mv. range and span only. However, it is convenient to use this chart paper throughout because in many determinations, for the majority of peaks, the 0-
to 10-mv. range suffices, but smaller spans also may be used to advantage throughout the chromatogram. The as-read absorbance values, A s , on the smaller spans, are converted to true absorbance, A , by appropriate calculations. The conversion is accomplished by a method similar to that used by Hamilton (3) who converted linear displacement to per cent transmittance. For any span used, unity transmittance remains at the zero line (right side) of the chart paper; under amplification, transmittance a t the m line, T,, depends on the range and span used and is equal to the voltage a t the line as determined by calibration, divided by the voltage at unity transmittance (normally 10 mv.). Electrical zero for any span smaller than 10 mv. lies to the left of the m side of the chart paper; that is, zero is off the chart paper to the left. The transmittance of any point, T p , on the chart paper scale, therefore, equals the sum of two portions: (1) the transmittance from 0 to T m a known constant (linear) for any span, plus (2) a portion read from the chart paper which is the product of: (a) the antilog of ( - A , ) , the apparent transmittance of the printed point on the chart paper and a linear fraction of the total scale and (b) the quantity (1 - T,) the actual transmittance range covered in full scale deflection on any span. I n the calculation it is simpler to express all transmittance values as fractions of unity rather than as per cent transmittance. Thus : ~
Tp
=
T,
+ (1 - T,)
X antilog ( - A , )
and
A (absorbance a t the point T p )
=
- log T p The conversion of A , values in tabular entries of 0.001 absorbance unit to 6place A values is easily made by means of Absorbance Conversion Tables which we prepared for each span. Their VOL. 37,
NO. 4, APRIL 1965
623
Table 1.
Recoveries at Amino Acids in Mixtures a t Various Column Loads
Namomoles of Each Amino Acid Placed on Column 10 (3)a
5 (6)O
99.8 f 0.8 99.6 i0 . 5 99.2 f 0 . 7 100.0 f 1 . 5 98.3 f 1 . 9 100.7 f 1 . 5 98.8 f 1 . 9 96.9 f 0 . 9 9 8 1 f 0 6 9 7 5 1 1 8 9 7 0 f 0 8 97.2 i 0 . 8 99.4 f 1 . 0 98.1 f0.7 93.2 f 1 . 4 96.4 + 0.7 95.3 f 1 . 4 95,l f 1.5 97.8 f 1 . 1 6
99.6 f 1 . 2 100.1 f 1 . 6 9 7 6 f 1 9 9 8 6 i 1 3 981f27 100.5 f 1 . 4 101.2 f 1 . 5 98.4 f 1 . 7 98.8 f 2 . 1 98.7 f 3 . 8 97.2 f 1 . 5 97.0 f 1 . 5 99.5 f 1 . 0 99.1 f 2 . 6 93.3 f 1 . 4 96.9 f 1 . 0 96.8 f 1 . 9 95.8 f 2 . 2 98.2 f 1.79
2 (8)'
1(7P
Mean Recovery in Per cent with Average Deviation from the Mean Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cvstine Kline Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine Av. a
96.7 f 2 . 2 99.4 f 2 . 7 9 5 8 f 3 2 9 4 6 f 5 1 99Of56 9 6 O f 2 5 9 4 4 f 1 6 9 7 6 f 2 8 9 4 l f 3 3 9 7 6 f 3 4 983+14 9 6 7 f l 7 98Of3O 101 5 f 3 1 8 6 7 f 3 4 9 7 8 f 1 7 9 8 i f 2 0 93 7 f 3 . 5 96.4 f 2 . 9
103.8 f 4 . 2 97.0 f 4 . 3 968f 3 5 9s.7 f 4.4 (76.7 f 1 3 . 4 ) 98.3 f 2 . 1 96.3 f 3 . 5 9 3 8 f 3 4 9 5 7 i 4 2 9 7 9 f 5 0 104 4 f 2 4 955f 6 3 9 7 5 f 6 5 9 3 4 f 5 2 867f 3 1 9 7 8 f 2 7 9 4 8 f 3 3 9 2 4 f 4 9 9 6 3 f 4 0
No. of chromatograms.
use eliminates laboratories and repetitive arithmetical calculations. The 26page Tables with insructions for their use and for their exrension to other additional >pans are on deposit at the IIedical Library, Kayne State Yniversity School of Nedicine. Photocopy may be obtained by application. RESULTS
Separation of an equimolar mixture of 18 amino acids was made by the accelerated procedure of Spackman ( 7 ) in which the basic amino acids are analyzed on a 10 X 0.60 cm. column and the neutral and acidic amino acids on a 60 X 0.90 cm. column. The results obtained with spans 0.67 and 0.35 are given in Table I. The recoveries were calculated from the standard color values obtained for 0.5 hAk! (10 mv. span) with a precision of fl%. The data presented were obtained with the Spinco model. Similar results were obtained with our laboratory-construched model, the original photometer (8) of which was modified only to the extent of mounting the exhaust blower externally and connecting it to the vent
624
ANALYTICAL CHEMISTRY
with a flexible duct to reduce the mechanical vibration of the photometer; thermal effects on the photocells were reduced by changing the ventilation path. Even a t the I-nanomole level, the recoveries of all the amino acids were satisfactory with the exception of proline. ,4t the lowest level, the proline peak was too low for accurate estimation. From base-line chromatograms and those run a t 1.0- and 0.9nanomole levels it was evident that there were small base-line irregularities which occurred chiefly under methionine and isoleucine and were associated with the buffer change. Reagent-blank chromatograms run with the sample-diluting buffer, pH 2.2, in the amounts used in the study (1 or 2 ml.) do not show other visible base-line irregularities. However, if 6 ml. of the buffer is applied to the column, there are small but surprisingly reproducible elevations in the positions of aspartic acid, threonine, serine, glutamic acid, glycine, and alanine of the magnitude of 0.06 to 0.6 n J i . From six such chromatograms and with two lots of sample-diluting buffer, appropriate correction figures of
0.02 to 0.09 n M were applied to the recovery figures a t the 1 and 2 n M levels where the additive effects are discernible. A t these more sensitive levels, contamination from any source, even from the hands (4),may unwittingly introduce amino acids. A system of scrupulous cleanliness should be maintained with particular attention to the sample itself, the glassware, distilled water, air lines, column fittings, the reagents, and their components. Individual laboratories should be aware of the possibility of errors arising from their own techniques and reagents. The 1-second span switching time on this recorder makes it possible to detect in one run less than 0.05 nanomole of an amino acid in the presence of 1500 nanomoles of a neighbor amino acid. In biological work it is advantageous to use several spans for one chromatogram to avoid dual chromatograms and high column loads. The increased sensitivity, range, and flexibility of this new recorder system, particularly if coupled with the use of spherical resin, reduced column diameter (6), and more sensitive photometry ( I ) , should facilitate investigations in protein metabolism and protein structure a t low concentration levels. The present system also makes possible the detection of even minute amounts of impurities in an individual natural or unnatural amino acid that can be separated chromatographically. LITERATURE CITED
(1) Crestfield, A. M., ANAL. CHEW 35, 1762 (1963). 12) Hamilton. P. B.. Ibid.. D . 2055. (3) Hamilton: P. B., Ann: N. Y . Acad. Sci. 102 ( l ) ,55 (1962). (4) Hamilton, P. B., Xature 205, 285 (1965). (5) Hubbard, R. W.,Kremen, D., Fed. Proc. 23, 372 (1964). (6) Kirsten, E., Kirsten, R., Biochem. Zeit. 339,287 (1964). ( 7 ) Spackman, D., Fed. Proc. 22, 244 (1963). (8) Spackman, D., Stein, W. H., Moore, S., ANAL.CHEM.30, 1190 (1958).
WORK was supported b y Grant A-693, National Institutes of Heakh.
