Expanded Capability of the Coulter Counter with a New Aperture Apparatus D. A. Flinchbaugh Homer Research Laboratories, Bethlehem Steel Corporation, Bethlehem, Pa. 18016 This paper describes the development and evaluation of a new aperture apparatus for use with a commercially available Coulter Counter or similar electronic particle-sensing instrument. The new apparatus makes it possible to accurately and precisely size and count very small numbers of particles, either as an overall size distribution or as the few largest particles in a total particle population. This is accomplished by quantitatively recycling large fractions of the sample suspension through the aperture and back into the sample beaker. No mercury siphon i s used. Tests on known numbers of particles gave an estimated recovery of 100% of the number present and a standard deviation of about 10%. In addition to permitting more reliable counting of particles of analytical concern that occur in small numbers, the apparatus is useful in cases where the analyzed sample must be quantitatively recovered because of its high economic value or potentially hazardous properties.
THISPAPER DESCRIBESthe development and evaluation of a new aperture apparatus for use with a commercially available Model B-M Coulter Counter. This apparatus can also be adapted for use with other models of the Coulter Counter. A Coulter Counter determines the number and size of particles suspended in a n electrically conductive liquid. This is done by forcing the suspension to flow through a small aperture between two immersed electrodes. As the particle passes through the aperture, it causes a resistance change between the electrodes. The resistance change produces a voltage pulse, the magnitude of which is proportional to particle volume. The pulses are then electronically scaled and counted. A schematic diagram of the commercially available aperture apparatus is shown in Figure 1. When the vacuum stopcock is opened, a vacuum initiates flow from the beaker through the aperture and unbalances the mercury siphon. When the stopcock is closed, the siphoning action of the mercury continues the sample flow. The advancing mercury column makes contact with start and stop probes, which activate the electronic counter. Hence, a constant sample volume is provided for all counts. Although the above aperture apparatus design is in widespread use and is adequate for most applications, it has the following disadvantages that either seriously restrict the range of applications or unduly complicate the use of the counter for routine laboratory purposes: (a) The sample cannot be quantitatively recovered from the siphon, connecting tubing, stopcock, and trap flask after it has been drawn through the aperture. As a result, limitations are imposed on the number of data points that can be taken on any one sample and/or on the volume of suspension that can be drawn through the aperture during a given pass. This is especially true when working with large apertures and is most critical when only a few particles in the size range of interest are present in the sample. (b) When one is working with the usual quantities of large, dense particles, the bottom of the aperture tube frequently fills up with particles that do not remain in suspension without the aid of mechanical agitation. This accumulation of particles can cause orifice blocks. Also, some of the particles can pass through the orifice back into the beaker and 172
cause extraneous counts. It is therefore sometimes necessary to remove and clean the aperture tube several times during the course of a run. This practice results in the loss of valuable sample solution. (c) A less serious disadvantage is that the commercially available aperture apparatus cannot be used with samples which are appreciably reactive with mercury. (d) Finally, when the commercial apparatus is used with biologically or chemically hazardous materials, the mercury siphon system must be dismantled and thoroughly cleaned after each series of determinations. Possible solutions have been advanced (1-3) in attempts to overcome one or more of these difficulties. However, none of the suggested aperture apparatus designs have solved all of the problems nor have they become commercially available. The new aperture apparatus designed and built at Homer Research Laboratories and described in this paper is capable of circulating sample suspension through the aperture and back to the sample beaker without losing any of the sample particles and without altering the total volume of sample suspension. No mercury siphon is used. The new apparatus offers several advantages over existing equipment. First, for each data point, a large fraction of the total sample can be drawn through the aperture and counted, and a n unlimited number of data points can be determined on a given sample suspension, T o illustrate, consider a sample having about 2000 particles large enough to require the use of a 400-p aperture tube. With the commercially available Coulter Counter aperture stand, a counting aliquot of 2 ml would be used. Assuming a sample suspension of 250 ml, only 16 particles would pass through the orifice during any one counting cycle, Furthermore, only a fraction of these 16 particles would be detected at most diameter settings. Obviously it would be difficult to construct a reliable size-distribution curve under such conditions. However, as will be shown later in this paper, using the same sample with the new apparatus makes it possible to draw about 600 particles through the aperture during each counting period. In addition, a n unlimited number of data points can be obtained o n the sample. Therefore these conditions provide a much more quantitative basis for constructing meaningful size-distribution curves. Second, the design features of the apparatus which allow complete sample recovery also eliminate the problems associated with particle buildup in the bottom of the aperture tube. Specifically, this is achieved by mounting the orifice in a vertical tube of sufficient length that large particles which do not remain in suspension without mechanical agitation fall to a collection point sufficiently below the orifice to eliminate the possibility of any extra counts. Finally, since the new apparatus does not contain a mercury siphon, and since it is easily cleaned, it can be used with materials which would react
(1) C . T. Morgan and W. H. Coulter, British Patent 1,125,289 (1968). (2) J. R. Coulter, Jr., U.S. Patents 3,340,470and3,340,471 (1967). (3) C. T. Morgan and W. H. Coulter, US. Patent 3,395,343 (1968).
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IW \) Figure 1. Schematic diagram of Coulter aperture apparatus with mercury or which are biologically or chemically hazardous. With the improvements made possible by the new aperture apparatus, the Coulter Counter can now be used for several new general applications, as follows: (a) For accurately sizing and counting extremely limited numbers of very sizable particles in the presence of large numbers of smaller particles. An example of this type of application is in studies to determine the effect of macroinclusions on the mechanical properties of steel. Our use of the apparatus for this purpose will be described in detail in a forthcoming paper. (b) For measuring overall size dihtributions on extremely limited numbers of particles-especially with instruments not equipped with multichannel analyzers. (c) When working with potentially hazardous materials and those materials which chemicallyreact with mercury. The following section presents detailed descriptions of the new aperture apparatus and the procedure to be employed when a Coulter Counter or similar electronic particle-sensing device is used with the new apparatus. These descriptions are followed hy an evaluation of the effectiveness and reliability of the new instrument configuration. EXPERIMENTAL
Apparatus. Our instrument is shown in Figure 2. It consists of a Model B Coulter Counter with a Model M Volume Converter attachment as supplied by the vendor and a Coulter aperture-stand with a 12-in. plywood extension between the base and the plastic equipment support on which the new aperture apparatus is mounted. A transfer flask sits beneath the aperture apparatus on an independent plywood stand. The aperture stand sits in an electrically grounded copperscreen cage to eliminate extra counts due to air-borne electrical interference. Details of the aperture apparatus are shown in Figure 3. The sample beaker is round-bottomed and has a capacity of 250 ml. A vertical section of the beaker wall is removed to accommodate a glass tube. This tube, which contains a Coulter 400-p orifice, is secured to the beaker wall by means of epoxy resin. The orifice is at the lowest possible point, i.e.,just above the point where the curve of the beaker bottom diverges from the tube. The external electrode is fastened to the inside of the beaker just above the orifice. The upper part of the tube contains a platinum internal electrode. Attached to this vertical tube is a vacuum port and a calibrated bulb of about an 80-ml capacity. The bulb can be graduated to provide an option of taking more than one data point during a single pass through the aperture. The bulb is con-
Figure 2. Modified Coulter Counter
Figure 3. New aperture apparatus nected to the lower part of the glass tube with a piece of flexible plastic tubing. A pinch clamp is attached to the tubing. A second piece of plastic tubing with pinch clamp is attached to the bottom end of the glass tube. The glass stirrer has 4 vertical blades about in. high and in. long. The transfer vessel is an inverted 125-ml Erlenmeyer flask with the neck drawn to a diameter (about 10 mm 0.d.) appropriate for attaching tubing. A piece of plastic tubing with a pinch clamp is attached to the neck. A female standard taper joint is positioned in the top of the inverted flask. Electrolyte. The electrolyte can be any particle-free electrically conductive solution capable of maintaining sample suspension with the aid of mechanical stirring. The electrolyte used in tbis work was selected because of its ability to hold large, heavy particles in suspension. It was prepared by mixing 125 ml of a 4.8% saline solution with 125 mlof glycerine and adding 10 drops of a 4.0% solution of Triton X-100 (alkyl phenoxy polyethoxy ethanol, Rohm and Haas, Philadelphia). The solution was then filtered through a 0.45-p membrane filter. Calibration. The instrument is calibrated according to standard procedures (4) using the monosized standards available from Coulter Electronics. For our 400-p orifice we selected corn pollen, which measured 97.4 p in our electrolyte. The calibration data were taken over 4-sec intervals. Procedure. Attach the pinch clamp to the tubing at the bottom of the aperture apparatus. Disperse the sample in (4) “Instruction Manual, Coulter Counter Industrial Model B M,” Coulter Electronics Industrial Division, Hialeah, Fla.
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Dilute to 250 ml. Position the stirrer in the center of the beaker ahout 1 in. from the bottom. Faulty positioning of the stirrer can lead to erroneous counts. Fasten the pinch clamp at the bottom of the volume-measuring bulb. Apply sufficient vacuum to wet the internal electrode. Turn off the vacuum stopcock. Set the Coulter Counter controls for the type of data required. For cumulative counts the following instrument settings are recommended: upper threshold, out; lower threshold, 20; counting mode, differential; time, 128 sec; division factor, 1; reset switch, right. Restore vacuum. When the sample level hits the calibration mark in the bulb, press the start button on the Model M to activate the counters. Press the stop button when the sample level crosses the upper calibration line. Turn off the vacuum stopcock and bleed the system. (Note that the particles large enough to settle out of suspension without the aid of mechanical agitation are trapped immediately above the pinch clamp.) Drain the sample suspension into the transfer flask. (These large 174
iettle at the pinch clamp.) Lift the trnnsfcr flask just above the sample beaker. \Vhile gmtly swirling the flask to prevent particles irom adhering to the ualls, drain the sample suspension into the sample beak:r. Repeat the abov: steps, changing current and amplification settings as needed to obtain the desired size-distribution data. I.nwcr background counts at smdler dinmeters arc obtained by increasing the aperture ci.rrent and keeping the amplificationas low as possible. Calculations. 'The calculations and data-handling operations used with the modified apparatus are similar to those used with the commercially availlblc Model B-M Coultsr Counter. The only dilrerence i s that the sample aliquot is larger. EVALUATION OF THE MODIFlED INSTRUMENT
The advantages and limitations of Coulter Counter-type electronic particle-sensing instruments for determining the
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size distribution of powdered materials have been extensively and critically reviewed (5-10). Most of the limitations noted in these references have centered on electronic noise level (5), linearity of response with particle volume (5, 6), coincidence, i.e., multiple particles being counted as a single pulse (9, flocculation of sample particles (5), and surface charge distribution on sample particles (8). However, the effect of many of these inherent limitations on the accuracy of size distribution measurements on most materials can be minimized, if not essentially eliminated, through the application of properly designed laboratory procedures. The fact that proper procedures are generally successful in this regard is evidenced by the widespread acceptance and use of these instruments in research and production control (5, 9, 10). Although these inherent limitations do not present a serious obstacle, there still remains, as was noted in the introductory section, the problem of the types and quantities of materials that can be handled effectively and safely. Therefore, the tests described below were conducted primarily to test the effectiveness of the new aperture apparatus. Calibration. The capability of the modified instrument to respond linearly to changes in particle volume was demonstrated by calibrating with pecan and corn pollen-two monosized standards supplied by Coulter Electronics. The standards were suspended in the saline-glycerine electrolyte, and photomicrographs of the particles in suspension are shown in Figure 4. The actual diameters of the materials were measured from the photographs. Calibration constants, shown in Table I, were calculated from the measured diameters and the corresponding instrument settings of threshold, current, and amplification. The agreement between the two values is well within experimental error; for example, a n error of in. in the pecan pollen measurement would result in a li value of 3690, representing a change of about 10%. O n the other hand, an error of in. in the corn pollen measurement would result in a k value of 3755, or an error of about 3z. Given the much lower probable error for the corn pollen as a standard, we routinely calibrated with this standard with an assigned diameter of 97.4 p . Background Counts. The level of background counts observed with the modified Coulter Counter is illustrated in Table 11. The data were obtained by passing 78 ml of a 250-ml electrolyte solution through a 400-p aperture. Each determination takes about 2 minutes. It is assumed that all of the observed counts are due to electrical noise since the electrolyte was prefiltered through a 0.45-p membrane filter. Note the exceptionally low background counts at diameters greater than 50 p . This size range represents the most frequently used diameter range of the 400-p aperture tube and clearly demonstrates the electronic stability of the instrument. From these data we conclude that the modified instrument is capable of operating under sufficiently noise-free conditions to allow the accurate counting of very small numbers of particles. Furthermore. it should be noted that equally low counts could also be obtained in the less-than-50-p diameter range if a
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( 5 ) T. Allen in “Particle Size Analysis,” The Society for Analytical Chemistry. London, England. 1967, p 110. (6) W. B. Mercer, Rev. Sei. brstriini., 37, 1515 (1966). (7) J. Beresford, W. Carr, and G. A. Lombard in “Particle Size Analpis,” The Society for Analytical Chemistry, London, England, 1967, p 95. (8) R. R. [rani. ANAL.CHEM.. 32, 1162 (1960). (9) R. H. Berg in “Symposium on Particle Size Measurement,” ASTM Special Technical Publication No. 234, Philadelphia: Pa., 1959, p 245. (10) “Bibliography.” Coulter Electronics Industrial Division: Hialeah, Fla., 1968.
Calibration Constants Obtained on Coulter Monosized Standards Coulter Our k, diameter, diameter, calibration Standard J ! !J constant Pecan pollen 47 49.2 3282 Corn pollen 83.5 97.4 3647 Table I.
Table 11. Background Count Level Obtained with the Modified Coulter Counter Number of counts Diameter: P Run 1 Run 2 Run 3 Run 4 Average 0 ndQ nd 0 167 0 0 nd 1 0.3 0 133 0 nd 2 1.3 105 2 1 nd nd 2 84 3 4.3 7 4 nd 66 2 25 13 10 17 53 19 11 24 31 23 42 29 71 93 60 nd 35 61 139 27 111 217 90 nd 630 693 nd nd 22 568 a nd = not determined. Table 111. Ability of Modified Coulter Counter to Count Corn Pollen Particles PartiNo. Z recover) 2 Sample Particles cles per stated size No. taken pass 66p 52p 41p Average 87 91 96 665 91 1 2104 95 113 115 108 150 2 483 135 116 102 112 109 3 342 169 97 106 109 104 4 529 Average (samples 2, 3, 4) 98 110 120 I09 Average (all samples) 96 107 112 Grand average + 1 u I05 + 14 (all 48 data points) smaller aperture were ~ised. Use of a smaller aperture makes it possible to drive the aperture current and signal amplification circuits at lower levels. Counting Precision and Accuracy. The ability of the modified instrument to count smaller numbers of particles than is possible with the commercially available configuration was demonstrated by performing actual counting experiments using known numbers of particles. Corn pollen and alumina particles were counted under a microscope, suspended in electrolyte, and then counted with the modified Coulter Counter. I n all cases, the Model B-M Coulter Counter was set to count all particles equal to o r greater than the stated size. Therefore, one should ideally expect 100% recovery a t each diameter setting smaller than the smallest particle in the sample. The corn pollen experiment was performed with our prototype aperture apparatus, which had almost the same overall design as that of the final version described in the Experimental Section. The one difference was that the vertical tube that contains the orifice was positioned a t about the same location in the sample beaker as the Coulter aperture tube is in a standard sample beaker o n a Coulter aperture stand. The recycling apparatus was exactly as described in the Experimental Section. The corn pollen experiment included four samples ranging from 342 t o 2104 particles. Four data points were taken a t each of the three diameters o n all four samples. The averages of the count values are shown in Table 111. Most of the per cent recoveries fell within 15 % of the theoretical 100% value. Although the recovery data o n samples 2, 3,
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Table IV. Ability of Modified Coulter Counter to Count Corn Pollen Particles (Corrected for Background) PartiNO. recovery 2 Sample Particles cles per stated size No. taken pass 66p 52p 41p Average 1 2104 665 90 96 87 91 2 483 150 92 102 99 98 3 342 109 97 97 112 102 4 529 169 94 102 95 97 Average (samples 2, 3, 4) 94 100 99 98 Average (all samples) 93 99 97 Grand average i 1 u (all 48 data points) 96 9
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Table V. Ability of Modified Coulter Counter to Count Alumina Particles No. recovery >_ stated size Diameter, p E A C D 167 1 1 0 0 151 11 4 3 3 132 54 28 26 29 120 71 50 55 58 105 94 76 85 89 95 93 99 98 94 83 118 103 91 96 76 114 93 90 100 66 84 112 96 95 60 85 114 112 101 53 106 111 107 99 48 nda 114 nd 107 No. particles counted 1615 1895 2026 2269 No. particles expected 504 590 631 707 per pass Average recovery, 100 106 99 99 48 through 95 p Average and standard loo i 10 deviation of all values at 95 p and below Average and standard 101 Z t 11 deviation of all 59 data points at 95 p and below nd = not determined.
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and 4 showed a n increase above the expected 100% level as the diameters decrease, this divergence was largely eliminated by taking into account the electronic background counts. Table IV gives the per cent recovery data as corrected by subtracting the background counts given in Table 11: 63 microns, 5 counts; 52 microns, 17; and 41 microns, 24. A comparison of these corrected values with those in Table 111 shows that the corrected average per cent recovery for samples 2, 3, and 4 is 98 as compared with the uncorrected value of 109. Since sample 1 contained 4 to 6 times as many particles as the other samples, correction for background count is not required. The average recovery and 1 value of 96 i 9% calculated from all 48 data points is considered more than adequate for most applications. For the second experiment, alumina was selected as representative of the large, dense materials which are usually difficult to analyze with the Coulter Counter because they are difficult to keep in suspension long enough to draw through the orifice. Furthermore, once inside the aperture tube, such particles tend to accumulate in sufficient quantity to block the orifice from the inside of the tube. F o r this experiment the alumina powder was presieved to a size range of 105 to 150 p. About 2000 particles were suspended in 250 ml of salineglycerine electrolyte, and their size distribution was deter176
Table VI. Modified Coulter Counter Data on Sample C, Table V Counts Diameter, p Set 1 4 Set 2 7 Set 3 4 Set 4 t Average nd 1 191 1 1 nda 2 3 167 3 1 5 151 23 26 nd 26 30 132 151 146 162 195 164 nd 303 290 nd 297 120 494 486 448 105 403 410 95 537 625 nd nd 582 589 605 66 1 567 607 83 nd nd 547 568 76 526 713 628 662 638 669 66 664 nd nd 674 60 684 653 652 655 53 7656 66 1 nd 11 d 673 1014b 674 48 a nd = not determined. * Discarded in calculations, falls outside of 3 u limit. Table VII. Modified Coulter Counter Data on Sample E, Table V Diameter, Counts -___ J ! S ? $ Set 2 4 Set 3 t Average 191 0 0 n@ 0 167 1 2 nd 2 151 26 22 nd 24 132 226 156 228 20 3 120 502 338 40 1 414 105 756 522 602 627 95 691 660 640 664 83 698 753 590 680 76 718 733 663 705 66 644 739 644 676 60 640 795 707 714 53 623 732 739 698 48 634 850 78 I 755 a nd = not determined. Table VIII. Average Per Cent Recovery over Diameter Range 48 through 95 1.1 of Each Run of Samples C and E Per cent recovery =tI u Sample C Sample E Set 1 106 & 14 94 i 5 2 107 i 6 103 i 5 94 7 3 114 i 4 , . . 4 104 i 6 Average 106 i 9 98 9 (all data points) Grand average 102 i 10 (all 39 data points) ~~
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mined with the modified Coulter Counter. The results of four such tests are shown in Table V. The recovery data a t 95 p and below should all be 100% and, therefore, these data can be used to calculate the precision and accuracy of the alumina determinations. (Previous data, Tables 11-IV, have shown that background count corrections need not be made with particle concentrations and size ranges of this order.) As shown in Table V, the average per cent recovery with standard deviation for all values at 95 p and below is 100 i 10%. Most of the data shown in tests C, D, and E were determined by averaging replicate determinations at each diameter setting. In order to compare the precision and accuracy of the alumina particle counts with those obtained on corn pollen (Table IV), the average and standard deviation of all 59 data points taken over the 95- through 48-p diameter range was calculated. The resultant value of 101 i 11 compares well with the 96 i 9 value found in the corn pollen experiment. Therefore, it appears that the instrument is
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
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Figure 6. Size distribution of alumina(0) and alumina plus latex plus Lycopodium( *)
Figure 7. Size distribution of alumina(0) and alumina plus latex plus Lycopodium plus pecan pollen(*)
capable of counting the large, heavy particles of the type that are most difficult for the standard aperture apparatus to handle. Inspection of the per cent recovery data in Table V at any specific diameter in the range 105 through 167 p indicates that the agreement is good among samples C, D, and E but these samples do not agree with sample A. No special significance need be attached to this observation, since no attempt had been made to homogenize the sieved alumina powder prior to counting out the particles for instrumental testing. An application paper, which has been prepared for publication, contains data to demonstrate that good sample-to-sample precision is obtained in the rising portion of the number m. diameter plot when all samples come from the same population. Examples of typical raw data are given in Tables VI and VII. The arrows indicate the order in which the data were taken. Recall that each data point involves cycling the sample suspension from the sample beaker, through the orifice and volume measuring bulb, into the transfer flask, and then back to the sample beaker. Visual observation of the data shows that none of the sample is lost during the transfer operations, even when as many as 39 data points are taken on the sample. The mathematical evidence for this conclusion is given in Table VIII, which lists the average and standard deviation of recovery values obtained over the diameter range 95 through 48 p for each run of samples C and E as shown in Tables VI and VII. The fact that particle recovery does not decrease as each succeeding set of data points is obtained, demonstrates that particles are not lost during the data-taking operations. Effect of Small Particles on the Efficiency of Counting Large Particles. An experiment was designed to show that the modified instrument is able to accurately count a few large particles in the presence of millions of smaller particles. A sample of 50- to 105-p alumina particles was suspended in electrolyte and its size distribution determined. An estimated quantity of three monosized standards was then added to the original suspension in the order latex, lycopodium (fern spore), and pecan pollen. The size-distribution measurements were repeated after each addition. The quantity of each standard added to the sample was estimated as follows: A drop of the Coulter standard suspension was dispersed in fresh electrolyte and counted by standard Coulter procedures.
The number of particles per drop of original suspension was then calculated. This information was used to calculate the quantity of monosized standard to be added to the alumina sample. Figure 5 shows the data taken on the sample before and after the addition of about 6.6 million particles of 3.49-p diameter latex. The data points are superimposable. It is therefore concluded that the latex does not interfere with counting the alumina particles. Figure 6 shows that the addition of 150,000 particles of 27-p lycopodium to the aluminum-latex sample has no effect on the counting accuracy. When, as shown in Figure 7, 90,000 particles of 45 to 50-p pecan pollen were added to the alumina-latex-lycopodilrm suspension, no effect on counting rate was observed above 50 p . The pecan pollen was being detected and counted below 50 microns. The results of the experiment to test the effect of small particles show conclusively that large particles can be accurately counted in the presence of very large quantities of smaller particles. As demonstrated by the test results, our new aperture apparatus has expanded the capabilities of the standard Coulter Counter equipment. I n particular, the new apparatus makes it possible to size and accurately count very small numbers of particles because of the recycling feature; count large, dense particles, which remain in suspension as a result of the configuration of the new apparatus; and recover the full quantity of each sample after data-taking is complete. ACKNOWLEDGMENT
The author thanks a number of colleagues at the Homer Research Laboratories, in particular T. H . Lamb for assistance in fabricating the aperture apparatus, B. s. Mikofsky for assistance in the preparation of the manuscript, and R. J. Noll, B. M. Thomas, R . W. Goerlich, and Jane H. Goerlich for technical assistance. RECEIVED for review July 13, 1970. Accepted November 2 , 1970. An abbreviated form of this paper was presented at the Pittsburgh Conference o n Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 4, 1970. A patent application has been filed which covers the improvements to the Coulter Counter aperture apparatus.
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