m
6 5 + 3 M M . O.D.
‘
I
I
r 0 E A D
WALL
1
2MM.
, 102 f 3 M M O D
1
500 M L
Figure 1.
has been proved to be generally Mfe, precautions such as the use of gloves, shields, etc., should be taken. LITERATURE CITED
(1) Chem. Eng. News 26,883 (1948). (2) Cheng, F. W., Smullin, C. F., Microchem. J . 4, 213 (1960).
Borosilicate flasks for oxygen combustion procedures
(3) Committee on MicrochemicalAppara-
tus, Division of Analytical Chemistry, ACS, ANAL. 26, 1186 (1954); 28, 112, 1993 (1956); 30, 1702 (1958); 32, 1045 (1960). (4) Committee for Standardization of Microchemical Apparatus, Division of Analytical Chemistry, ACS, Ibid., 21, 1283, 1555 (1949); 22, 1228 (1950); 23, 523, 1689 (1951).
(5) Juvet, R. S., Chiu, J., I&id., 32, 130 (1960). (6) A. J*, Deveraux, Ibd., 31, 1932 (1959). (7) SchCiniger, W,, MikTochim. Acta 1954, 74; 1955, 123; 1956,869. (8) Steyermark, A., “Quantitative Organic Microanalysis,” 2nd ed., p. 292, Academic Press, New York 1961. RECEIVED for review August 9, 1961. Accepted August 9, 1961.
Estimation of Microgram Amounts of Protein Using a Modified Ring Oven SIR: The ring-oven technique described by m7eisz (34) offers a rapid, simple means for concentrating and separating materials in small samples, so that conventional reactions may be employed for identification and quantitation (4, 6). The method has gained recognition for analyses of inorganic ions (6, 7 ) but does not seem to have been seriously applied to organic or biological materials. A modified apparatus and technique extend the ringoven concept to such materials. Fundamentally, the modified apparatus differs from the Weisz ring oven in that a strip rather than a circle of 1790
*
ANALYTICAL CHEMISTRY
filter paper is used, and the sample migrates in one direction along this strip to a pair of rectangular electrically heated aluminum blocks. Materials contained in the sample are thus deposited on the paper a t the heated edge of the blocks in a single, narrow straight line across the width of the strip. The apparatus might be termed a “line oven.” This modification improves the ability to concentrate materials from the sample. The ring oven concentrates a sample by causing it to migrate from a diffuse spot in the center of a circular filter paper to a thin, sharp ring a t the
heated evaporation zone. An aqueous sample of 2 5 4 . volume will make a diffuse spot about 21 111111. in diameter with an area of approximately 350 sq. mm. The dissolved materials in such a sample can quantitatively migrate to the evaporation zone and be deposited in a thin ring approximately 22 mm. in diameter and usually less than 0.5 mm. wide. Thus, the area on the paper occupied by the materials from the sample has been reduced from about 350 to approximately 35 sq. mm., a tenfold concentration. Ideal circwnstances may yield a ring of such width thac the sample is concentrated fifty-
fold. A similar sample placed upon a filter paper strip 2.5 cm. wide and caused to migrate in one direction to an evaporation zone will produce a single straight line 2.5 cm. long and usually less than 0.5 mm. wide. The area occupied by the sample is thus reduced to approximately 13 sq. mm., nearly a thirtyfold concentration. Ideal circumstances may allow the sample to concentrate more than a hundredfold. The apparatus is illustrated in Figure 1. It is 7 inches long, 4 inches wide, and 3l/n inches high. The base plate and blocks are aluminum for high heat capacity. Heating is accomplished by a 30-watt, 110-volt, capsule-type heater controlled by a thermoswitch. Heater, thermoswitch, and a miniature dial thermometer are all inserted in the lower block in close tolerance wells (not shown). The thermoswitch adjustment and a neon pilot light are housed in the projecting case on the end of the lower block. The three upper blocks are hinged to the lower block and are heated by conduction. The long solvent reservoir is a plastic “parts box.” In use, the temperature of the block is adjusted to approximately 130” C., and distilled water is placed in the solvent reservoir. A 2.5 X 10 cm. strip of Whatman No. 1 chromatography paper is placed between the upper and lower blocks, so that a 4-cm. length extends out to the front, with the end about midway above the solvent reservoir. This extended end of the strip is then immersed in the solvent, and sample is applied to the strip from a microliter pipet along the advancing solvent front. The solvent is allowed to flow up the strip to the evaporating zone for 5 minutes. The reservoir is then removed and a dry zone 1 to 2 mm. wide is allowed to develop a t the edge of the block, so that the sample line will remain intact. The paper is removed and dried in an oven or, better, in the warm air stream from a heat gun. The dry strip is ready for appropriate treatment to identify or quantitate the materials concentrated from the sample. This procedure has been satisfactory for analysis of dilute protein solutions containing large amounts of both organic and inorganic impurities. The samples were essentially solutions of human blood serum in the concentration range 0 to 100 p.p.m. of protein. The impurities were inorganic salts, amino acids and protein fragments, biological pigments, and unidentified materials. The preponderance of interfering materials as well as the low concentration of protein in these samples made existing techniques such as the use of phenol reagent, ninhydrin, ultraviolet spectra, etc., useless (2). A specific reaction which seemed easily adaptable to this problem was the protein binding of bromophenol blue. A standard method for staining paper
electrophoresis strips using an alcoholic solution of the dye (1) was chosen as rapid and reproducible. Several experiments were undertaken to determine the validity of the line-oven method.
-
The effect of differences in dye-binding capacity of different serum proteins was checked by preparing three series of standard dilutions, one from human serum, one from human serum albumin, and one from human 7-globulin. The materials used were commercially avail-
F i g u r e 1. apparatus
M o d i f i e d ring-oven
Table 1.
protein per 100 ml. was diluted 1 part in 700 with isotonic saline. This solution was then added to an equal volume of each ultrafiltrate, and aliquots were analyzed. Finally, the serum protein solution was added to an equal volume of each of the original samples, and aliquots were analyzed. Results of these experiments are reported in Table I. Similar experiments have repeatedly yielded comparable data. The maximum disagreement between calculated quantities of protein and amounts actually found in the samples after addition of diluted human serum was j=8Y0. The mean error for the values reported was +O.l%, which does not seem to indicate a large systematic error. This method has been successful for nearly two years for estimating the protein content of various biological solutions. Three aliquots of a sample may be concentrated on strips in less than 10 minutes. Staining requires about 2 hours. The method is sensitive to as little as 10 pg. of protein per ml. and requires only 0.2 ml. of sample. Bacterial growth in the samples can cause interference when the organisms become numerous enough to stain visibly.
Specificity of Method
A Unknown Ultrafiltrate Ultrafiltrate and 1: 700 serum s o h , equal volumes Unknown sample and 1: 700 serum s o h , equal volumes
able samples of high purity and known protein content. Each series of dilutions ranged from 1 to 10 pg. of protein per 100 pl. a t 2-pg. intervals. Corresponding strips prepared by carrying the same concentration of each of the three standards through the entire process were visually indistinguishable; the incremental increases, however, were easily recognized, and the strips were adequate for quantitation by the method of Weisz (6). The question of specificity rtated in the following manner.
was
Aliquots of five unknown protein solutions were analyzed by the lineoven method. A portion of each sample was ultrafiltered through a collodion membrane, and an aliquot of each ultrafiltrate was analyzed. Human blood serum known to contain 7.0 grams of
5 Calcd. Found Calcd. Found
50 51 53 51
Sample B C D Protein, pg./Ml. 10 51 37 None detectable 60 50 50 48 54 51 69 55 75 71 57 69
E 80 50 46
90 91
LITERATURE CITED
(1) Beckman/Spinco, Palo Alto, Calif.,
Manual RIM-5,1957.
(2) Kunkel, H. G., Ward, S. M., J. BWZ. Chem. 182,597-604 (1950). (3) Weisz, H., Mikrochim. Acta 1954, 140-7. (4) Ibid., pp. 376-87.
(5) Weisa, H., “Micro-analysis by the Ring Oven Technique,” Pergamon Presa, New York, 1961. (6) West, P. w., ANAL. CHEW 28, 757 (1956). (7) West, P. W., Mukherji, A. K., Ibid., 31,947 (1959). ANDREWF. FARR ALBERTL. CHANEY Rancho Los Amigos Hospital Downey, Calif. Albert L. Chaney Chemical Laboratory Glendale 6, Calif. RECEIVED for review June 28, 1961. .4ccepted August 28, 1961. VOL 33, NO. 12, NOVEMBER 1961
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