A steam distillate mas obtained from emulsion CS. Its spectrum (Figure 28) has the general spectral characteristics of an anhydride. Figure 29 shows that the nonionic emulsifier isolated from emulsion CS had the general characteristics of a Tween-type emulsifier (Figure 30), a sorbitan fatty acid polyoxyethylene derivative (8), but there are differences in the carbonyl stretching region. Figure 31 shows the spectrum of Tm-een 20 (Atlas Powder Co.) recovered from a prepared emulsion. The bands in the carbonyl stretching region have changed in the same manner as the bands in nonjonic emulsifier from emulsion CS. It is also possible to make quantitative determinations from the spectra of emulsion films. Because of surface tension effects it is not possible t o obtain quantitatively reproducible film thickness of these water emulsion polishes on silver chloride. However, quantitative or semiquantitative determinations are made possible by using the relative absorbance of the C-H deformation (or stretching bands) to compensate for variations in film thickness. A method has been developed for determining the amount of ester wax present in one of the wax emulsions. The ester wax content is measured b y the absorbance of the ester carbonyl at 1738 cm.-’, and the film thickness is corrected by the strength of the absorb-
Table 1.
Determination of Resin Content
Sample
Known
Phenolic
similar problem arises in the potassium bromide pellet technique, when the sample scatters light (6).
Infrared Detn.
LITERATURE CITED
Bellamy, L. J., “Infrared Spectra of Complex Molecules,” p. 31, Wiley, New Tork, 1954. Bhatt, H. A., Kamath, K. A., Naokarni, J. M., J . Sci. Ind.
ance of the CH, and CH, deformation bands at 1468 ern? A similar analysis of the amount of a phenolic resin in an emulsion formula has been developed. The absorption band at 1612 cm.-’ (benzene ring deformation) can be used to determine the per cent phenolic resin content of the formulation. The symmetric aliphatic C-H stretching band at 2830 cm.-l was used as a measure of film thickness. This analysis was used on samples which varied in phenolic resin content from 35 to 45%; apparently no serious error was introduced by correcting for film thickness in this manner. The results of a series of determinations on emulsions containing known amounts of this phenolic resin are given in Table I. The error calculated from the standard deviation (3) is of the order of 2%. These quantitative measurements are subject to additional error if the film scatters light and thus changes shape and intensities of absorption bands. A
Research 14B, 270-5 (1955). ( 3 ) Dean, R. B., Dixon, W. J., i l x . 4 ~ . CHEJI. 23, 636-8 (1951). (4) Hausdorff, H. H., “Analysis of Polymers by Infrared Spectros-
copy,” Perkin-Elmer Corp., Korwalk, Conn., 1951. ( 5 ) Kagarise, R. E., Weinberger, L. A., “Infrared Spectra of Plastics and Resins,” Office of Technical Services, U. S. Dept. Commerce, PB-111,438 (1954).
Kirkand, J. J., 4 ~ a CHEX ~ . 27, 1537-41 (1955).
Krimm, S
J . Chem. Phys. 22,
567-8 (1954).
McCutcheon, J. W., “Synthetic Detergents,” p. 420, MacPu‘airDorland Co., New York, 1950. Potts, W. J., Jr., Wright, Iiorman, ANAL.CHEX 28, 1255-61 (1956). Randall, H H., Fowler, R. G., Fuson, Felson, Dangl, J. R., “Infrared Determination of Organic Structures,” p. 20, Van Sostrand, Xew York, 1949. Sternglanz, H., Appl. Spectroscopy 10, 77-82 (1956).
Wasserman, K. J., Soap Chem. Specialties 32, S o . 1, 127-33, 173 (1956).
RECEIVEDfor review October 20, 1956. Accepted September 16, 1957.
Improved Method for Performing DensityGradient Electrophoresis ROBERT
1. BERG
and RICHARD G. BEELER
Departments of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Mass.
b Density-gradient electrophoresis is performed in a compact apparatus which reduces the volume of solutions needed, and by use of silver-silver chloride electrodes avoids release of gas during electrophoresis. A method is described for producing a variable-density gradient automatically. The chief advantage is better stabilization of the initial zone occupied by the sample, with a resultant greater symmetry of the individual components, thus avoiding excessively long trailing edges.
Z
is being used increasingly because a protein mixture may be simultaneously analyzed and fractionated by this method. OX’E ELECTROPHORESIS
126
ANALYTICAL CHEMISTRY
Among the most useful modifications is that of density-gradient columns, as devised by Philpot ( 7 ) and developed by Brakke (2, S), Brakke, Vatter, and Black (A$), and Sorof, Ott, and Young (9).
Electrophoresis takes place in a liquid column whose density gradient prevents the production of convection currents. Sucrose was the neutral substance used by Brakke ( 2 ) and Sorof, Ott, and Young (9). The advantages of a liquid supporting medium compared with starch are evident. A much larger number of fractions may be cut with precision and ease. No elution is required from the solid supporting material, there is no need for centrifugation, and the material is not diluted before use.
The apparatus (Macalaster-Bicknell
Co., Cambridge, Mass.) pictured in Figure 1 is considerably more compact and saving of solutions than other apparatus (10) for density-gradient electrophoresis. The use of silver-silver chloride electrodes, which do not give off gaseous products during electrophoresis, makes it possible to omit trapping devices. The electrodes were prepared by making a tight spiral of a 100-em. length of No. 17 silver wire of 0.5-em. diameter. One end, 12 cm. long, was seated in glass tubing with DeKotinsky cement. The glass tubing was capped a t the outer end with a 1-em. length of copper tubing, 0.375 inch in diameter, for easy attachment to an electrode cap. The upper and lower electrodes
Figure 1.
0
Apparatus
A , Density-gradient electrophoresis apparatus which may be filled from below. Lg,15 cm. Diameter of gradient column, 2.0 em. Volume, 47 ml. (in practice a working volume of 40 ml. has been used) B. Arrangement for generating variable-density gradient
SOLUTION
-RUBBER STOPPER
are exchanged in alternate runs. When the density gradient has been formed with the ball and socket joint closed, the joint must be opened prior t o electrophoresis. This usually results in some turbulence, and a special bypass arrangement is included, so that equilibration may be made slowly and without turbulence before the lorrer member of the joint is pulled down. In preparing the apparatus, enough saturated sodium chloride solution is put in the wells in which the silversilver chloride electrodes sit t o cover them and the chambers are filled with the appropriate solution: buffer a t the top, and the most concentrated sucrose solution in buffer in the lower chamber. About 15ml. of buffer is allowed to run int o the lower end of the gradient tube, and is later displaced upward to the upper portion of the tube surrounded b y the electrode chamber.
ER CHAMBER
COOLING JACKET
GRADIENT TUBE
GROUND GLASS BALL AND SOCKE
LECTRODE CHAMBER
CHAMBER GLASS TUBING SLIDES IN RUBBER STOPPER
ELECTRODE
MAGNET
,MAGNETIC STIRRER
B. A.
El00 0 7
a w
Various methods have been described for preparation of the density gradient. Commonly, equal aliquots of increasing densities of sucrose and buffer have been layered in retrogradely; the column is gently agitated by dipping a long stirring rod through its length and out again, and is then allowed to sit overnight for a smooth gradient to be formed. Mechanical devices (1) have been described for making gradients of any shape; such continuous gradients may be used at once without sitting. I n Figure 1,B, a simple but efficient device operates on the principle used in changing eluant concentrations in chromatography (6). The mixing chamber is filled with the least dense liquid desired, usually a n aqueous buffer solution, and connected to the outlet of the gradient tube. Liquid of the greatest density desired (in this case 55% sucrose in buffer) is placed in the reservoir, and is then allowed to flow slowly into the mixing chamber, where it is vigorously agitated, and displaces an equal volume of liquid. This displaced liquid then flows into the column, where it produces a variable-density gradient which can be calculated b y the following formula, derived b y Cherkin, Martinez, and Dunn ( 5 ) : c = e__ k-1 ca ek -
% DISTANCE FROM TOP OF COLUMN T O BOTTOM
Figure 2. Variation in concentration with distance from top of column
Density gradient at any point is proportional t o slope of tangent at that point volume of liquid in mixing chamber r = volume of liquid in filled column
% D I S T A N C E FROM TOP OF COLUMN TO BOTTOM
Figure 3.
Density gradients
---Theoretical . . . Actual Mixing chamber volume, 20 ml. Column volume, 40 ml. r = 0.50
where c
concentration of e1utan.t. in solution leaving mixing chamber co = concentration of elutant in solution in reservoir volume of eluate collected k = volume of diluent in mixing =
chamber VOL. 30, NO. 1; JANUARY 1958
127
250 r
A volume for the mixing chamber may be chosen to produce various nonlinear gradients, as indicated in Figure 2. I n Figure 3 an actual density gradient, as determined by refractometer determinations, was of the shape indicated by the dotted line. Except for an initial roundoff phase, the shape of the curve conforms well to that anticipated for r = 0.50. The p H in the apparatus has shown no variability greater than 0.05 p H unit with buffers a t pH 3, 7, and 10 during actual operation.
MIXING CHAMBER ALBUMIN- 5 GLOBULIN pH 8 6 5 HOURS 200 V , 3 - 4 rnA
>.
t v) z
105
110
115
120
125
130
135
140
145
500r
The gradient is steeper in the portion of the column with the lowest density. I n the lower part of the column, where the gradient is less steep, convection is hindered by the greater viscosity of the solution. This advantage is somewhat offset by the greater tendency t o heat production because of the greater resistance, but this is partly controlled by the cooling jacket. The gradient is steeper in the upper part of the column where the sample is introduced; this leads t o a more distinct layering of the sample.
2
145
When the gradient is prepared from a neutral s u b t a n c e which diffuses much more rapidly than the protein, the height of the stabilized protein zone, L,, is a function of the specific proteins and of the volume and concentration of the protein solution used. L, also depends on the solution density and
LAYERED I N , NOT STOOD OVERNIGHT
Figure 4. Separation of human albumin and gammaglobulin by electrophoresis
226c[ 40
95
100
105
110
115
120
125
130
135
140
T.UBE NUMBER
n
i',
DENS,TY G R A D I E I WUMAh
SERUM
VOLUMF OF EFFLUENT
Figure 5.
DENSITY GRADIENT ELECTROPHORESIS HUMAN PLASMA
ELECTROPHORESIS
(MLJ
ALB
D(2
b
SALT
VOLUME OF EFFLUENTlMLJ
Electrophoresis in density gradient of human serum and plasma compared with free electrophoresis
0.05M Tris buffer a t pH 8.6 a t 200 volts and 4 ma. for 18 hours. Tiselius electrophoresis performed in diethyl barbiturate buffer with r / 2 = 0.1 for 55 minutes
128
ANALYTICAL CHEMISTRY
density gradient at the point of pseudoequilibrium of the protein solutionthat is, a t the point where the injected protein solution floats. This is due to the instability of the initial narrow, sharply defined zone formed by introduction of the protein solution, which is caused by the following chain of events ( 1 1 ) . In the protein zone the solution has a lon-er sucrose concentration than the solution above and below. Sucrose diffuses into this zone faster than protein diffuses out. The density of the solution a t the lower (and upper) surface becomes greater than that inmediately below, and droplets begin to settle out. Brakke ( 2 ) explains in detail the means for controlling this droplet sedimentation. The protein zone becomes stable as soon as a smooth sucrose gradient is re-established, and a protein gradient is formed which is of opposite sign and of no greater magnitude than that of the sucrose gradient. The depth of this stabilized protein zone can be minimized by the use of high density columns, in which the density increment due to the protein is relatively smaller, but the high viscosity of such a column makes this undesirable, A second method is to use a column m-hich has a steep density gradient; the steeper the density gradient of the sucrose, the steeper the gradient of the protein, and thus the narrower the protein zone. This narrower protein zone, L,, niight be expected t o give greater resolution of the electrophoresis procedure, for it is a ratio of the height of t h e original protein zone t o the total height of the column which defines the resolution that can be expectedthat is, the broader the initial protein zone, the broader will be the bell-shaped curve of any component after electrophoresis. The protein gradient formed within
a poorly stabilized protein zone, of opposite sign to the sucrose gradient, may give rise to asymmetry of the peaks of the individual components. This will result in asymmetry of the type in which the leading edge is blunted and the trailing edge is extended. I n the experiment reported in Figure 4, to stabilize the layered gradients, a long stirring rod was inserted from the top to the bottom of the column, gently rotated, and removed. Fifteen milligrams each of human serum albumin and globulin were dissolved in 0.5 ml. of buffer and dialyzed overnight against 5% sucrose solution in 0.05;ll Tris buffer a t pH 8.6. This sample was inserted a t a point about 10% of the distance from the top of the gradient to the bottom, and descending electrophoresis vas used. Although the width of the individual components is not noticeably greater in the gradients formed by layering, there is a perceptibly greater asymmetry than where a nonlinear gradient has been used, (Figure 4,first curve) as produced automatically by the apparatus shown in Figure 1,A. Thus use of the steeper gradient at the point where the sample is layered in probably results in a narrower stabilized protein zone. Figure 5 presents the results of the electrophoresis of human serum, human plasma, and the same sample of human serum analyzed by the usual Tiselius analytic electrophoresis. Protein determinations were carried out b y the Buffalo Black dye method of precipitating proteins reported by Plum, Hermansen, and Petersen (8). This extremely sensitive method avoids the difficulties involved in exchanging very viscous samples in cuvettes used for ultraviolet spectrophotometry. It is well adapted to the analysis of protein, except that plasma albumin combines in a different stoichiometric proportion than the plasma globulins. Thus the
peak of albumin in these diagrams is somewhat exaggerated. The p-globulins in plasma do not exceed the a-2-globulins, as might be expected, and this may be due t o the presence of some albumin in the a-2 peak. The correspondence of these peaks with known fractions of plasma proteins has been established by paper electrophoresis. It is advantageous t o use as small a volume as possible for the sample; however, good separation can be achieved with 1-nil. samples containing from 50 to 300 mg. of protein. ACKNOWLEDGMENT
The authors are indebted to John Gregory for helpful advice. LITERATURE CITED
Bock, R. M.,Ling, X.-S., ANAL. CHEM.26, 1843 (1954). Brakke, 11.K., Arch. Biochem. and Biophys. 55, 175 (1955). Brakke, RI. K., Phytopathology 43, 467 (1953). Brakke, RI. K., Vatter, A. E., Black, L. RI. “Abnormal and Pathological Plant Growth,” pp. 137-56, Brookhaven Sational Laboratory, Upton, N. Y., 1954. Cherkin, il., Martinez, F. E., Dunn, RI. S..J . Am. Chem. SOC.75, 1244 (i953j. Mitchell, H. K., Gordon, M.,Haskins, F.’B.,J . Biol. Chenz. 180, 1071 (1949). Philpot, J. St. L., Trans. Faraday SOC.36, 38 (1940). Plum, C. L., Hermansen, L., Petersen, I., Scand. J . Clin. Lab. Invest. 7, Suppl. 18 (1954). Sorof, S., Ott, h’. G., Young, E. ?VI., Arch. Biochem. and Bzophys. 57,
140 (1955). Svensson, H., 1 . V . d . 25, 252 (1954). Svensson, H., Hagdahl, L., Lerner, K. D., Science Tools 4, 1 (1957). RECEIVED for review Sovember 1, 1985. Accepted September 6, 1955. Kork supported in part by a research grant, G-4253, from the U. S. Public Health Service.
Process Control Methods in the Chlorination of Benzene RALPH A. TROUPE and JEROLD J. GOLNER Northeastern University, Boston, Mass.
F A rapid, simple, and accurate method of analysis of reaction mixtures of chlorinated benzenes is based on density measurements. It can be used for either process control or product analysis, if a density-product distribution chart is prepared for the specific chlorination system under the conditions used in its operation. Vapor phase chromatography was used as a primary standard and was found
to be an accurate qualitative and quantitative method of analyzing mixtures of chlorobenzenes.
I
N THE AI4KUFbCTURE O f most O f the
chlorobenzenes, such as the commercially important p-dichlorobenzene, a complex reaction mixture results @), a-hich contains not only the desired product and hydrogen chloride but
also other chlorobenzenes. For example, in the manufacture of p-dichlorobenzene, monochlorobenzene, odichlorobenzene, and some trichlorobenzenes may be produced in addition to the principal product. As the economics of the process depend upon being able t o produce the maximum yield of the desired product or combination of desired products and upon knowing the concentration of the by-products, VOL. 30, N O . 1, JANUARY 1958
129
