Instrumentation
Continuous Flow Analysis Although continuous flow analysis has been most extensively applied in clinical chemistry and biomedical projiling, it has achieved practical application in agricultural chemistry, enuironmental science, pharmaceutical chemistry, and metallurgy. A recently announced computer-controlled sequential multiple analyzer, SMAC, is reported t o be capable of 40simultaneous assays a t a rate of 200 sampleslhr, using less t h a n 500 p1 of sample
Morton K. Schwartz
Department of Biochemistry, Memorial Sloan-Kettering Cancer Center. New York. N.Y. 10021
Automation in chemical analysis has been developed primarily along two distinct paths. These are discrete sample analysis, in which each step of a manual assay is carried out in robot fashion by a series of machinedriven belts, pumps, and syringes, and continuous flow analysis, which is the subject ofthis review. Continuous Flow Analysis in Chemical Laboratories
Although continuous flow analysis has been most extensively applied in clinical chemistry and biomedical profiling, it has achieved practical application in agricultural chemistry, environmental science, pharmaceutical chemistry, and metallurgy. As indicated in Table I, about 90% of clinical chemistry laboratories that participate in the CDC proficiency testing program and use automation employ continuous flow techniques. The use of these procedures has permitted clinical chemistry laboratories to keep up with ever-increasing demands reflected in many institutions by two to threefold increases in workload during the past decade. Although continuous flow procedures can be just as accurate as manual techniques and in most instances more precise, research workers have
heen reluctant to adopt these techniques in their laboratories, probably because of fear of losing intimate control over the precision and accuracy of the assay. A great advantage of continuous flow and other forms of automation for the investigator is his ability to plan a study without concern for the absolute number of needed assays. Continuous flow analysis describes the techniques developed by Skeggs in 1957 ( I ) and carried out by use of a series of connected instrumental modules collectively known as the AutoAnalyzer (Technicon Instrument Co., Tarrytown, N.Y.). The original AutoAnalyzer is shown in Figure 1. This instrument was modified by the manufacturer over the years to permit more sensitive colorimetry, dialysis a t a constant temperature, and a sampler that introduced a water wash betweensamples. During the past five years this instrument has heen replaced by the AutoAnalyzer I1 and sequential multiple analyzers (SMA systems). A new continuous flow analyzer has recently heen announced. This is a computer-controlled sequential multiple analyzer, SMAC, reported to he capable of 40 simultaneous assays a t a rate of 200 samples/ hr and using less than 500 fi1 of sample.
Figure 1. Original basic AutoAnalyzer ANALYTICAL CHEMISTRY, VOL. 45. NO. 8. J U L Y 1973
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Basic Principles of Operation
ACIDS Sulfuric Acetic
Nitric Hyd roc h Ioric Perchloric Phosphoric
BASES Ammonium Hydroxide Potassium Hydroxide Sodium Hydroxide
SOLVENTS Analytical Electronic Spectrophotometric
ELECTRONIC CHEMICALS Dopants Epitaxials Acid Etches
STANDARD SOLUTIONS Volumetric Buffer Percentage
SPECIALTY REAGENT CHEMICALS Methoxides Sodium Biphenyl E.D.T.A.
CUSTOM CHEMICAL SYNTHESIS
The principles of operation of the user-tested AutoAnalyzers (AutoAnalyzers I and I1 and the SMA 12/30, SMA 6/60, and SMA 12/60) are essentially the same. In Autohnalyzer systems, samples are placed in plastic cups held in holes in a plate on a constant-speed turntable equipped with a crook which dips a catheter into the sample and then into wash water at a prechosen rate. The second module is a proportioning peristaltic pump with moving steel rods which compress a prechosen group of plastic tubes and draw sample and reagents forward through the system. The tubes include one attached to the dipping catheter on the sampler module and others inserted into reagent bottles. The volume of aspirated liquid is a function of the inner diameter of the individual plastic tubes. Thus, a tube with an inner diameter of 0.065 in. will aspirate 1.60 ml/min; one with an inner diameter of 0.100 in. will aspirate 3.40 ml/min. The plastic tube sizes are chosen so that the final concentrations of reactants are similar to those in the manual method on which the automated procedure is based. The tube sizes permit aspiration of as little as 0.015 ml/min or as much as 3.90 ml/min by a single tube. The inner diameters of the plastic tubes will change with use, and the absolute concentrations of reactants will change. This will not usually affect the assay, since standards are subjected to the same chemical environment as the unknown solutions. The continuous flowing reactants are mixed by passage through glass coils with concentric helices that allow the heavier fluids to mix into the lighter fluids as the two reverse top-to-bottom positions in their passage through the coil. The flowing reactants are interspersed with bubbles of air which serve to regulate the flow, maintain the sample as discrete portions during passage through the modules, and act as a “squeegee” to clean the glass-plastic tubing between samples. If it is necessary to heat the flowing solution, it is passed through a glass
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coil in a heating bath thermostatically maintained at the desired temperature. The time of incubation is controlled by the length of glass coil and the volume of flowing fluid. Protein or particulate matter is removed during passage through a dialysis module. The diffusible compounds pass through a semipermeable membrane into a recipient flowing reagent stream, and the remaining solution flows into waste. Dialysis is not complete; usually much less than 50% of the available dialyzable material passes across the membrane. An assumption is made that dialysis of standards and unknowns proceeds at the same rate. Divalent ions and un-ionized weak acids and bases dialyze at the slowest rate, whereas the fastest rate of dialysis is observed for univalent anions of strong acids and bases and un-ionized compounds with low molecular weight and a high degree of water solubility ( 2 ) .In one study, 2-hr dialysis from a donor serum into a recipient pool of distilled water resdlted in 6% of the serum calcium and 22% of the uric acid passing across a standard AutoAnalyzer cellophane membrane (pore size, 40-60 A; thickness, 0.0009 in.). The dialysis rate increased to 13% for calcium and 22% for uric acid when a newer, thinner membrane was used (Type C, pore size 40-60 A; thickness, 0.0005 in.) (2). After passage through the dialysis module, variable lengths of plastic tubing and construction of the manifold permit additional reagents to be added in the sequence they are needed; the mixture passes through additional heating modules, if necessary, and then through a constant flow cuvette in a module chosen to measure the end point of the reaction. In most continuous flow procedures this is a filtertype split-beam colorimeter, an ultraviolet colorimeter, or a fluorometer. For specific applications, nephelometers, pH meters, flame photometers, or various types of radioactive counting apparatus have been used. The analog voltage output of the detecting module is applied to operate the pen of a strip chart recorder. The analog output may also be fed into a computer. It is possible, by proper choice of Auto-
, Analyzer modules, to automate any manual assay. The assay is usually represented by a flow diagram which permits construction of the manifold and demonstrates the sequence of the continuous flow analysis. An example of a flow diagram for the determination of an enzyme, 5'-nucleotidase, is shown in Figure 2 (3). Simultaneous Analyses
Continuous flow techniques can also he used for simultaneous analysis for several constituents. Plastic-glass tubing manifolds can be constructed in which the sample is split and entered into several reaction mixtures and then passed through separate incubation and detecting modules. The AutoAnalyzer I1 (Figure 3) and the SMA 12/60 (Figure 4) were specifically designed for multiple analysis. In these systems suitable lengths of glass-plastic tubing in the flow circuit stagger the arrival to the detecting modules of the several streams and allow the recording on one chart of the analysis of a group of compounds. In these AutoAnalyzer systems a new sampler and pump are used, and the dialyzer, appropriate heating bath, and some mixing coils are incorporated into an analytical cartridge. In these cartridges, glass tubing is used for carrying aqueous solutions, and Kel-F for nonaqueous solutions. Air is introduced a t the steady rate of one hubhle every 2 sec through an acrylic plastic air injection block. There have been numerous changes in AutoAnalyzer modules during the past fifteen years. The original AutoAnalyzer dialyzer contained 88 in. of circular groove length. In the SMA and AMI instruments, this length is reduced to a U-shaped
Figure 4. SMA 12/60 Courtesy
01 Technicon lnsfrumenls Co
From F/C
065
160
Tube size ml/min Proportioning ourno
Waste
Three double mixers
@/DplJ To pump Colorimeter Recorder 660nm Filters 1 5 mm F/C
One channel digital printer
Figure 2. Flow diagram of AutoAnalyzer I I method for 5'-nucleotidase (3) determination
Figure 3. AutoAnalyzer I1 CDurteSyol Technicon ln51rumenls Co
groove, 6-12 in. in length. The flow cell light paths were maintained a t 15 mm, hut the inner diameter was reduced from 3 to 1.5 mm to increase the washcapahilities. For adequate 6O-sample/hr operation, about 1ml/ minis required to wash out the new flow cell, compared to 3-4 ml/min in
older systems. For this reason, the new instruments are capable of analysis with smaller flowing volumes and, therefore, less reagent consumption. Another advantage of the low fluid flow is the use of plastic tubes with small inner diameters which permit more accurate proportioning of solutions. These instruments have electronic blank correction obtained by simultaneous analysis through two channels. The SMA 12/60 provides for simultaneous analysis of 16 channels (including four blanks) a t a rate of 60 samples/hr. A major problem is the phasing of the system to permit a portion of the colorimeter output from each analytic channel to reach the single recorder without interference from the other channels. This is accomplished by adding or subtracting lengths of tuhiog to the analytical train. To help the operator in analysis of the phasing, the SMA 12/60 is equipped with an oscilloscope which produces a continuous trace of concentration vs. time for all 16 channels. Rate of Analysis The nature of continuous flow analysis utilized in the basic AutoAnalyzer and then in the AutoAnalyzer I1
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new absorbance monitor ej4r
*
Figure 5 . SMAC courtesy OfTeChn,con l"slrYrne"t5
The ISCO Model UA-5 absorba n c e monitor gives you the high sensitivity, stability, and response speed required for high speed, high pressure chromatography-plusthewide absorbance ranges and specialized flow cells required for conventional chromatography, density gradient fractionation, electrofocusing, and gel scanning. Stationary cuvettes allow recording of enzyme and othei reactions. High sensitivity. 8 full scale absorbance ranges from .01 to 2.OA, plus %T. 13 wavelengths include 254 and 280nm supplied in the basic instrument; 310nm, 340nm, and 9 other wavelengths to 660nm are available at low cost. Options in. clude a built-in lOcm recorder, a Peak Separator to automatically deposit different absorbance peaks intc different tubes, and a multiplexer. expander which allows monitorinp of two separate columns or one column at any two wavelengths. A u t o matic 4X scale expansion prevent! oversized peaks from going off scale, T h e c u r r e n t ISCO c a t a l o g d e scribes the Model UA-5 as well ar ISCO fraction collectors, metering and gradient pumps, and additiona instruments for chromatography anc other scientific research. Your cop) is waiting.
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and the SMA 12/60 limits the rates of analysis to a usual maximum of 60 specimens/hr. The problems that limit the rates of analysis are basic to continuous flow of a liquid stream in a tube, The flow is fastest at the center of the tube and slowest a t the tube walls, because the flow is retarded by friction. Therefore, there are possible mixing and contamination of the successive central core of the stream by the slower moving preceding lateral portions ( 4 ) .Thus, the faster the rate, the greater the chance of contamination because sample segments are closer together. The air segmentation in AutoAnalyzer procedures lessens this contamination, hut there are still varying degrees of sample-to-sample interaction and carryover from one analysis to the next. Carry-over increases as the rate of analysis increases, hut it is also influenced by the nature of the flowing reactants as well as the design ofthe manifoldsystem ( 4 ) . Mathematical considerations of these interferences have been presented, and it is possible to assess and define the interreactions for any determination (4-6). The speeds of analysis have been increased by some workers by using nonsteady state conditions and computers to correct for sample-sample interaction and to monitor peak heights (5, 6). SMAC
The newest entry in continuous flow instrumentation has not yet been field tested, hut it is reported by the manufacturer to perform analysis of up to 200 samples/hr with steady state analysis. The instrument is known as SMAC: sequential multiple analysis plus computer (Figure 5).
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The computer portion of the instrument is programmed to make most of the usual operator judgments needed in other AutoAnalyzer systems, including corrections for drift, noise, short samples, standardization, and linearity. The computer also stores the colorimeter output data related to a specimen and eliminates the need for the SMA 12/60 type phasing required for graphic data representation. Information related to the sample and the requested tests is operator entered into the computer via a keyboard on a video terminal. The instrument has the ability to aspirate standards (quality control serum) from a pool placed in a refrigerated reservoir on the console a t any desired frequency. The SMAC-analyzed values are computer checked against the established limits of the expected values entered on a magnetic tape cassette prepared for each lot of quality control standard serum. The SMAC system reports the data in digital as well as graphic form. Although the SMAC system components occur in the same sequence as in other continuous flow systems, there are major differences. The sampler is an open-ended linear motion system rather than the circular turntable used in previous systems. The sampler is built to directly accept centrifuge carriers. The sampler can accommodate 19 carriers with eight samplesjcarrier. hut the sampler can be loaded continuously. Sample aspiration is modified to permit better maintenance of sample integrity and wash characteristics. In the SMA systems two intersample air bubbles encompassing a water slug are used, hut in SMAC two additional air huhhles
segmenting the sample are added. In one example, the time to achieve steady state was reduced from 21 to 9 sec by the air segmentation of the sample. After aspiration the sample is prediluted as in the SMA systems, debubbled and rebubbled with four large intersample bubbles, and then pumped at a high flow rate up a riser. The solution is resampled by peristaltic pumps on either side into the analytical cartridges. This represents a major departure from older AutoAnalyzers in which pumping is in a horizontal plane. Segmenting air bubbles are introduced at precisely the same rate as the rate of tube occlusions of the peristaltic pumps, and the segmentation has been increased from 30 to 90 segments/min to optimize wash in the analytical cartridge. In the SMAC system the flow cell is included in the analytical cartridge; most tubing has been eliminated and light transmission to and from the flow cell is via fiber optics. The flow cell diameter is reduced to 0.5 mm, and the light path to 10 mm. The volume in the flow cell has been reduced from 27 pl in the SMA or AutoAnalyzer I1 to 2 pl. Another innovation in the system is that air bubbles are permitted to pass through the flow cell, and their infinite absorbance is subtracted electronically. The SMAC also differs from other systems in the introduction of a multichannel colorimeter monitoring up to 23 flow cells in the 400-700-nm range and up to 12 flow cells at 340 nm. A single light source and photomultiplier tube are used, and a scanning disk allows exposure of one channel at a time. In addition to the innovations in instrumentation, the SMAC incorporates new chemical methods. Enzymes are determined kinetically a t two or three time points, as well as by the single time point procedure of the older methods. Sodium and potassium are determined potentiome-
trically by using ion-selective electrodes placed in line with the continuously flowing fluid. A volume of 10 pl is sufficient for each analysis. The SMAC system is a major advance in laboratory automation. It makes use of miniaturized hydraulics, new optical detection systems, ionselective electrodes, an instrument dedicated computer, and new, simplified handling and loading techniques. It is truly laboratory automation in the sense that the operator’s role is merely placing samples in the instrument. Calibration, analysis, and correction of machine problems, as well as calculation of data, are all carried out by the computer. Standardization in Continuous Flow Analysis A review of continuous flow analysis would be remiss if it did not point out that a major problem is standardization and then calculation of the assayed value. Standardization is usually achieved with previously analyzed serum (secondary standards), not with primary standards. The problem is particularly acute when enzymes are analyzed. Calculation of enzyme activity is complicated by a number of factors. These include the assumption of linearity between a zero time and a single time point for the assay; changes in reaction mixture concentrations as the delivery volumes of the manifold tubes change with use; the effect of partial dialysis on the enzyme activity; variations in incubation time; and the type of standard used. It has been recommended that commercially available control serums with label values be used as standards in continuous flow assays. The advantage in the use of such serums is obvious. Since the standard is treated in the same fashion as the unknown, changes in tube size, incubation time, the effects of dialysis, and other intrinsic continuous flow variables are eliminated. The disadvan-
tages of the use of such materials are the possible lability of the constituent, the difficulty in establishing the true concentration of the “standard,” and the possibility that the material in the control serum (particularly in the case of enzymes) is from a different tissue or species than the unknown sample and acts in a different kinetic fashion. Lyophilized serum with high constituent concentrations can be used as a standard for continuous flow methods if it is analyzed by a conventional manual method and appropriate dilutions are used. These problems of standardization can all be overcome, and the precision of continuous flow automated methods is excellent. In Table I1 are listed quality control data of successive daily SMA 12/60 analyses during a one-month period. The versatility of AutoAnalyzer procedures is such that any laboratory worker can adapt a manual method to continuous flow automation without special equipment or training. Continuous flow manifolds or cartridges can be rapidly changed and permit multiple tests to be run with a single instrument. Although there have been numerous other instruments and systems described for use in the automation of the chemical laboratory, the AutoAnalyzer remains the most useful single instrument for this purpose. References (1) L. T. Skeggs,Amer. J. Clin. Pathol., 28,311 (1957).
(2) E. Seifter, K. Demetrious, and A. Chanas, Advan. Automated Analysis, p 121, Technicon International Congress, 1969, Mediad, Inc., White Plains, N.Y., 1970.
(3) V. G. Bethune, M. Fleisher, and M
K. Schwartz, Clin. Chem., 18,1524 (1972). (4) W. H. C. Walker, C. A. Pennock, and G. K. McGowan, Clin. Chim. Acta, 27, 421 (1970). (5) R. E. Thiers, J. Meyn, and R, F. Wilder,mann, Clzn. Chem., 16,832 (1970). (6) R. L. Habig, B. W. Schlein, L. Walters, and R. E. Thiers, ibid.,15, 1045 (1969).
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