V O L U M E 1 9 , N O . 5, M A Y 1 9 4 7
355
Table IV. Analysis of Commercial Copper-Base l l l o y s by Colorimetric and Volumetric RIethods Type of Alloy
Antimony Found Tolumetrically ( 1 ) Colorimetrically
7c Heat Heat Heat Heat Heat Heat
6413, 6416, 6418, 6426, 6427, 6434,
SS-5-5-5 85-5-5-5 81-3-8-8 80-9-11 85-5-5-5 78.2 5.7 5
0
n
0 0 0 0
os3
050 071 085 104 330
% 0 0 0 0 0
n
080 054 068 070 102 32s
The method described is rapid, accurate, and less subject to manipulative errors than the volumetric method. 4CKNOW LEDGMEhT
The author wishes to express his sincere appreciation to C L. Luke of the Bell Telephone Laboratories, Murray Hill, N. J., for many helpful criticisms in the preparation of this manuscript and to E. B. Sandell, University of Minnesota, for reviewing it. LITER4TURE CITED
A number of Bureau of Standards standard samples were analyzed for antimony by the procedure given here (Table 111). Good agreement was obtained between the values of the antimony present and the antimony found. The results obtained from running a number of commercial copper-base alloys are shown in Table I V . Good agreement was obtained on the antimony values between the volumetric ( 1 ) and the colorimetric method described.
(1) I m . Soc Testing Material-, “-4.S.T.M. Methods of Chemical Analysis of J l e t n l s ” , p. 163, 1939. (21 Am. Sac. Testing Materials, “A.S.T.M. Methods of Chemical Analysis of Metals”, p 197 (1946). modified a s to iron and silicon content. (3) Ibid., p. 316. (4) F a u c h o n , M .L., J . pharm. chim., 25, 537 (1937). ( 5 ) McChesney. E. W., ISD.EXG.CHEX..ANAL.ED.. 18, 146 (1946). (6) Sandell, E. B., “Colorimetric Determination of Traces of Metals”, p. 131. New York, I n t e r s r i e n r e Publishers, 1944.
Capillary Absorption Cells in Spectrophotometry P. L. KIRK’, R. S. ROSENFELS, AND D. J. HANAHAK, University of Chicago, Chicago, I l l . Capillary absorption cells are described which yield a sensitivity of colorimetric analysis about a thousandfold greater than volumetric microgram analysis methods and a millionfold greater than standard microanalysis. -4limiting sample size for quantitative analysis is of the order of a few millimicrograms.
I
N CONIVECTIOS with various small-scale colorimetric analytical methods which were needed for control procedures in the operation of the Hanford Engineer Works plant, it was necessary to determine extremely minute quantities of a number of constituents. The spectrophotometer, which is one of the most useful instruments for such analyses, has a limiting sensitivity of a few micrograms. Certain samples requiring analysis were expected to have small fractions of microgram quantities present. . In most colorimetric methods only a small portion of the colored solution which is prepared is placed in an instrument for measurement, thereby wasting a t times the greater portion of the colored constituent. It is evident therefore that, if the color is developed in a volume which is merely sufficient t o fill an absorption cell, all of the colored constituent could be used in the instrument and would amount effectively to a corresponding increase in sensitivity. Moreover, in photometers and spectrophot.ometei-s it is customary to use an absorption cell which has a considerably greater diameter than the beam of light which passes through it. Thus the colored component through which light is not passed is also effectively wasted, even though i t is placed in the instrument. If a cell Rvere designed so as to be not larger than the beam of light passed through the instrument and if t,he total volume of solution were only sufficient to fill this cell, maximum sensitivity of the method would be achieved. With instruments such as the Beckman spectrophotomet’er, which contain photoemission rlements, the current of which is amplified, it is possible to increase the sensitivity still further by diminishing the cross section of the cell to even less than the cross section of the light beam and use the amplification to increase the measured sensitivity. When this is done, the cell length may be increased correspondingly, which will increase the light absorption exponentially with length according to the Bouguer-Beer’s law relationship. As a practical matter the cross section of the cell will be limited to some minimum value to which the amplification system of the in1 Present address, Division of Biochemistry. I-nirerslty of Califorma Medical School. Berkeley. Calif.
strument can be adjusted and the initial volume of solution will be limited by available equipment for measurement and dilution of sample and reagents. Several authors have described capillary cells for use with various types of colorimeters and photometers. More common has been the use of capillary tubes in which colored solutions were compared visually against’standards in similar tubes (6, IS). Evelyn and Gibson ( 4 ) have described a plunger-type cell for the filter microphotometer, capable of use with as little as 0.15 ml. of solution. The cell length was, however only 1 mm., which is too short to realize a large increase ’in sensitivity. Somogyi (18) utilized capillary cups in a special type of wedge colorimeter. The capillaries were 20 mm. long and contained about 10-microliter volume. Compensation of color was obtained by varying the standard wedge. Kul’berg (9) has reviewed the subject of microcolorimetry and micronephelometry. I n the authors’ original laboratory some years ago a Duboscq colorimeter was modified to carry plungers of about 1-mm. diameter which operated in conjunction with cups of about 2mm. diameter and had a depth comparable with that of the standard instrument. It. was found that the light passed was sufficient to fill the visual field of the instrument and that if the plungers and cup could be constructed accurately, t,he accuracy of the measurement was comparable to that of the standard Duboscq. Its sensitivity was, however, increased by several hundredfold. Practical difficulties in the construction of these small plungers and cups prevented any great use of this modified instrument and the description was consequently never published. Chapman ( 3 ) has described a similar ,instrument modification which required someTThat greater volumes of solution. The use of capillary cells for increasing the sensit,ivity of the Beckman spectrophotometer was developed on the basis outlined above and found to yield sensitivities higher than have previously brcn described for colorimetric procedures. \$‘ith some constit,uents it \va.s possible to analyze quantitatively as little as 0.001 microgram of material. Of the various materials test,ed, all could be analyzed in amounts as low as 0.01 microgram. This development is considered particularly significant in ultramicroanalysis because it, increases the possible range of analysis by approviniately a thousandfold over previously described ultramirrotitrimetric. methods ( 7 , 8, 10, 1;) and also extends the prac-
356
ANALYTICAL CHEMISTRY
tical working range of the spectrophotometer by a large factor. I n addition, the very large number of materials, both inorganic and organic, which can be determined by colorimetric procedures makes the method of the greatest interest to the biochemical as well as the chemical analyst. EXPERIMENTAL
The length of absorption cells for the Beckman spectrophotometer is practically limited to 10 em. with any standard cell holder so far available. Because of certain technical considerations which were encountered it was deemed wise to limit capillary cells to a 5-em. light path. A 5-em. cell with an internal diameter of 4 mm. would contain less than 1 ml., which would then by the desirable volume in which t o develop the colored constituent. If the internal diameter of the cell is reduced to 2 mm., the capacity is about 160 X 10-Bliter, which would then make a 0.2-ml. volumetric flask the proper container in which t o develop color. Since these are available commercially (Microchemical Specialties Co., Berkeley, Calif.), this would appear to be a practical procedure, provided cells as small as this cas be used with the instrument.
L
U
Figure 1. Cross Section of Teflon Capillary Absorption Cell
Tests showed that 2-mm. cells were entirely practical and even smaller ones down to 0.5 mm. may be used under certain conditions. Cells smaller than 2 mm. lead to technical difficulties in the addition of reagents and dilution in the volumetric flask and consequently were not studied. The two sizes chosen for routine use had internal diameters of 2 and 4 mm. The construction material of the cell and its manufacture became the two most critical points to be solved. Clear glass cells made from thick-walled capillary tubing with fused end windows were constructed by the American Instrument Company and tested. Their performance was excellent when the conditions were properly arranged. However, light from the instrument's light source could pass through the glass walls of the cell and register on the photoelectric cell. This light had no relationship to the absorption of the solution and produced both erratic and incorrect results. It was found that such cells required a set of three metallic masks, one between the light source and the entrance of the cell, another a t the exit of the cell, and a third with a larger orifice in front of the photosensitive element. The arrangement of these masks was technically difficult because their alignment, as well as the alignment of the cell, had to be nearly perfect and, in order to achieve this, a high degree of adjustability was required. The use of such cells was shortly abandoned because of these technical difficulties. Similar cells constructed of colored glasses which absorbed the wave lengths used were found to be much more satisfactory. They were difficult to make because of differences of the coefficient of expansion between the clear end windows and the colored glass tubing. Moreover, the external diameter of such tubing was variable and the bore of the available tubing was frequently not concentric with the periphery. Thus each cell required individual adjustment in the instrument and led to much technical difficulty. Cells of various plastics and of stainless steel were also tested. The stainless steel cell, while easily constructed and indestructible, was found to corrode sufficiently to disturb analyses for iron, chromium, and nickel. Such corrosion was not noticeable to the eye but in the range of analysis was extremely significant.
Plastics suffer inherently from a general tendency to dissolve in organic solvents, thus eliminating many of them from use with this type of system. Several plastics such as methacrylate resin, polystyrene, and polyethylene were suitable for certain aqueous systems, and polyethylene plastics showed a very considerable resistance to solution by both aqueous and certain nonaqueous solvents. The construction of cells from these materials was simple and effective, inasmuch as they could be machined and the external diameters made uniform. The best plastic tried was Teflon (E. I. du Pont de Nemours & Co., Inc.), a fluorinated hydrocarbon which was found to be completely inert to all dilute and most concentrated aqueous reagents as well as to organic solvents. It was opaque and served as its own mask. Clear windows must be installed in such cells. These may be made of a clear plastic in cases of solutions containing fluoride ion or other ions which are corrosive to glass, or they may be made of glass in order to use organic solvents or materials which would normally affect plastic. A design which allowed ready interchange and replacement of windows is shown in Figure 1. The external diameter of these cells was 1.6 em., which is almost the same as the standard 7-ml. glass absorption cell marketed by the American Instrument Company, and therefore the same cell holder could be used interchangeably. The end windows were held in by plugs which screwed into the main body of the material and contained channels for light passage, Por>s for filling were provided inside the windows a t both ends. Liquid was inserted in one port by means of a pipet, while the cell was held in a slightly slanting position, allowing the air to escape a t the upper port. The location of these ports was rather critical. Two-millimeter cells required them to be contiguous to the end window to prevent trapping of a bubble of air a t the upper end during filling. With 4-mm. cells it was preferable t o place the windows about 1 mm. inside the end window. Some difficulty was encountered at times with trapping of small air bubbles in the channels of these cells because the surface of this material was not readily wettable by water. I t was necessary therefore t o examine the cell visually after filling to make certain that this had not occurred. Less difficulty was encountered with organic solvents because of their greater wetting capacity for the material. The spectrophotometer requires special holders for cells of greater than 1-cm. light path. Such holders can be obtained from the manufacturers but are unsatisfactory for capillary cells because they do not contain easy adjustment to center the cell in the light path. For this reason a different type of holder was designed (Figure 2). These cell carriers were adjustable vertically in a slight arc and had adjustable stops for lateral movement. Thus the cell on one side was centered with respect to the light beam, the carriage was moved, and the cell on the other side centered, after which the cells, which were of standard size, could be used interchangeably without difficulty.
Figure 2.
Absorption Cell Holder, End View
Holds capillary cells or 7-ml.. 5-om. Aminco absorDtion cells
V O L U M E 19, NO. 5, M A Y 1 9 4 7
357
The Beckman spectrophotometer gives a light beam about 9 mm. in height and 2.5 mm. in width. At times a little light would pass above or below the cell, owing to the considerable height of the beam, but a simple mask of cardboard or other material could be inserted to prevent this effect. Otherwise, the cells were self-masking and all light passing through them had passed through the colored solution. The Coleman Model 11 spectrophotometer uses a beam which is approximately circular and about 6 mm. in diameter. It uses barrier layer photocells without amplification and absorption cells much smaller than the diameter of the light beam would not give sufficient sensitivity with this instrument. Of the spectrophotometers tested, only the Beckman instrument was found satisfactory for use with this type of absorption cell. RESULTS
The method was tested in the analysis of iron by the o-phenanthroline method (5, 16), chromium by the diphenylcarbazide method (12, 14), manganese measured as permanganate (IS?), and phosphate (1) and silicon (a), both measured as molybdenum blue. The sample size corresponding to 90% transmission, which is still within the quantitatively determinable range of these procedures, is shown in Table I. Not all the procedures were tested with the 2-mm. capillary cells, so that experimental values are not completely available. A comparison of the calibration curves of the various methods in which the smallest cells were used is shown in Figure 3. Similar curves were obtained in determining the other constituents listed. Little if
100
80
Table I.
Capillary Absorption Cell Sensitivities Amount Giving 90% Transmission 0.2-ml. 10-ml. 1.0-ml. volume volume volume
Constituent
Method
Iron Chromium Manganese Phosphate Silicon
o-Phenanthroline Diphenylcarbaaide Permanganate hlolybdenum blue Molybdenum blue
Y
Y
Y
0.44 0.14 3.5 0.38
0.044 0.014 0.350 0.038 0.011
0.0087 0.0028
0.11
.... ....
....
any sacrifice in precision was necessary over standard spectrophotometric techniques, though the chance of error in manipulating small volumes is increased somewhat as compared with standard procedures. The effect on the sensitivity curve is very evident and it is clear that considerable increase in sensitivity over all previously described accurate methods has been achieved. Values listed in Table I should not be confused with sensitivity as designated by Sandell (15),who lists the sensitivity as that amount needed to give 99.8% transmission in a 1-em. column of solution. Thus, his sensitivities represent the limit of detection of color, whereas values listed here represent limiting sample size for quantitative determination. The accuracy of any colorimetric method diminishes very rapidly as the per cent transmittance on which the determination is based approaches 100. Much further work remains to be done in the development of capillary spectrophotom?try, for use particularly in biological systems. The applications, for example, are not available for any organic materials nor for that matter with most inorganic materials, and the difficulties encountered with biological systems may be expected to be considerably greater than is true of most inorganic solutions. It is believed that the above listed analyses represent by a considerable margin the smallest scale analyses ever performed with th-is degree of accuracy. NOTE
60
Since the preparation of this manuscript describing work done in 1944, Lowry and Bessey (11) have described a similar technique. The sensitivities obtained in their work are not clearly defined, but apparently are similar to the authors’. The technique is considerably different in that small quartz cells are used and the mode of application to different analyses is somewhat less completely described.
D
40
5
‘a i 2
LITERATURE CITED
L
(1) Berenblum, I., and Chain, E., Biochem. J., 32,286,295 (1938).
C
s 2
(2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)
20
0
0.4 0.8 Micrograms of Iron
Figure 3.
1.2
Iron Calibration Curves
1. Volume 10 ml., I-cm. absorption cell. 2. Volume 10 ml 5-em. Aminco cell. 3. Volume 1 ml., 5-cm. capillary cell. 4. Volzme 0.2 ml., 5-om. capillary c e l l
unpublished modification to silicate determination. Chapman, G. W., Analyst, 55,443 (1930). Evelyn, K. A., and Gibson, J. G., J.B i d . Chem., 122,391 (1938). Fortune, W. B., and Mellon, M. G., Ibid., 10, GO (1938). Hshn, F. L., Ber., 57, 1394 (1924). Kirk, P. L., Mikrochemie, 14, 1 (1933). Kirk, P. L., and Bentley, G. T., Ibid., 21, 250 (1936). Kul’berg, L. M., Zavodskaya Lab., 9 , 3 7 2 (1940). Lindner, R., and Kirk, P. L., Ibid., 22,300 (1937). Lowry, 0. H., and Bessey, 0. A,, J . Bid. Chem., 163, 633 (1946). Moulin, A., Bull. SOC. chim., 31, 295 (1904) Richards, A. N., Bordley, J., 3rd, and Walker, A. &J. I.B ,i d . Ibzd.,
Chem., 101, 179 (1933). (14) Rowland, G. P., Jr., IND.ENQ.CHEM.,ANAL.ED., 11, 442 (1939). (15) Sandell, E. B., “Colorimetric Determination of Traces of Metals’’, New York, Interscience Publishers, 1944. (16) Saywell, L. G., and Cunningham, B. B., IND. ENQ. CHEM., ANAL.ED.,9, 67 (1937). (17) Sisco, R. C., Cunningham, E. B., and Kirk, P. L., J . Bid. Chem., 139, l ( 1 9 4 1 ) . (18) Somogyi, J. C., Nature, 138,763 (1936). (19) Willard, H. H., and Greathouse, L. H., J . Am. Chem. SOC.,39, 2366 (1917).
