Instrumentation Continuous Flow Analysis Although continuous flow analysis has been most ex tensively applied in clinical chemistry and biomedical profiling, it has achieved practical application in ag ricultural chemistry, envi ronmental science, pharma ceutical chemistry, and metallurgy. A recently an nounced computer-con trolled sequential multiple analyzer, SMAC, is reported to be capable of 40 simulta neous assays at a rate of 200 samples/hr, using less than 500 μΐ of sample
M o r t o n K. S c h w a r t z 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 of this review. Continuous Flow Analysis in C h e m i c a l L a b o r a t o r i e s
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, pharmaceuti cal chemistry, and metallurgy. As in dicated in Table I, about 90% of clin ical chemistry laboratories that par ticipate in the CDC proficiency test ing program and use automation em ploy continuous flow techniques. The use of these procedures has permitted clinical chemistry laboratories to keep up with ever-increasing de mands reflected in many institutions by two to threefold increases in work load during the past decade. Al though continuous flow procedures can be just as accurate as manual techniques and in most instances more precise, research workers have
been reluctant to adopt these tech niques in their laboratories, probably because of fear of losing intimate con trol 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 con cern for the absolute number of need ed assays. Continuous flow analysis describes the techniques developed by Skeggs in 1957 (1) 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 per mit more sensitive colorimetry, dialy sis at a constant temperature, and a sampler that introduced a water wash between samples. During the past five years this instrument has been replaced by the AutoAnalyzer II and sequential multiple analyzers (SMA systems). A new continuous flow ana lyzer has recently been announced. This is a computer-controlled sequen tial multiple analyzer, SMAC, re ported to be capable of 40 simulta neous assays at a rate of 200 samples/ hr and using less than 500 μΐ of sam ple.
Figure 1 . Original basic A u t o A n a l y z e r ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973 · 739 A
T a b l e I. U s e of A u t o m a t e d T e c h n i q u e s in Clinical Chemistry Laboratories
Analysis Glucose Creatinine Urea nitrogen Uric acid
Total labs. no. 362 294 324 265
Basic Principles of Operation
ACIDS Sulfuric
Nitric Hydrochloric
Acetic
Perchloric Phosphoric
BASES Ammonium Hydroxide Potassium H y d r o x i d e Sodium Hydroxide
SOLVENTS Analytical Electronic Spectrophotometric
ELECTRONIC CHEMICALS Dopants Epitaxials A c i d Etches
STANDARD SOLUTIONS Volumetric Buffer Percentage
SPECIALTY REAGENT CHEMICALS Methoxides Sodium Biphenyl
E.D.T.A. CUSTOM CHEMICAL SYNTHESIS
CORCO CHEMICAL
CORPORATION
Manufacturers Reagent and Electronic
of Chemicals
Tyburn Road & Cedar Lane, Fairless Hills, Pa. 19030 'Phone: ( 2 1 5 ) 2 9 5 - 5 0 0 6
The principles of operation of the user-tested AutoAnalyzers (AutoAnalyzers I and II and the SMA 12/30, SMA 6/60, and SMA 12/60) are essentially the same. In AutoAnalyzer 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 prechosenrate. 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
CIRCLE 41 ON READER SERVICE CARD 740 A · ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY
1973
Automated labs. no. 229 173 216 161
Automated labs using con tinuous flow techniques No.
%
200 165 201 153
87.3 95.3 93.0 94.9
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 resulted in 6% of the serum calcium and 22% of the uric acid passing across a standard AutoAnalyzer cellophane membrane (pore size, 40-60 Â; 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, t o a u t o m a t e any m a n u a l assay. T h e assay is usually represented by a flow d i a g r a m which p e r m i t s construction of t h e manifold a n d d e m o n s t r a t e s t h e sequence of t h e continuous flow analysis. An e x a m p l e of a flow d i a g r a m for t h e d e t e r m i n a tion of a n e n z y m e , 5'-nucleotidase, is shown in Figure 2 (3).
__
Waste
Sample
375°C
Ό30 .056 .030 030 .056 .040 .040
032 1 20 0.32 0.32 1.20 0.60 0.60
Sample H ? 0 .065
1.60
Simultaneous Analyses
C o n t i n u o u s flow t e c h n i q u e s c a n also be used for s i m u l t a n e o u s analysis for several c o n s t i t u e n t s . P l a s t i c - g l a s s t u b i n g manifolds can be c o n s t r u c t e d in which t h e s a m p l e is split a n d en tered into several reaction m i x t u r e s a n d t h e n p a s s e d t h r o u g h s e p a r a t e in cubation and detecting modules. The AutoAnalyzer II (Figure 3) a n d the S M A 12/60 (Figure 4) were specifi cally designed for m u l t i p l e analysis. In these s y s t e m s s u i t a b l e lengths of glass-plastic t u b i n g in the flow cir cuit stagger t h e arrival t o t h e detect ing modules of t h e several s t r e a m s a n d allow t h e recording on one c h a r t of t h e analysis of a group of com p o u n d s . In t h e s e AutoAnalyzer sys t e m s a new s a m p l e r a n d p u m p are used, a n d t h e dialyzer, a p p r o p r i a t e h e a t i n g b a t h , a n d some mixing coils are incorporated i n t o a n analytical car tridge. In t h e s e cartridges, glass t u b i n g is used for carrying aqueous solutions, a n d K e l - F for nonaqueous solutions. Air is introduced a t t h e s t e a d y rate of one b u b b l e every 2 sec t h r o u g h a n acrylic plastic air injection block. T h e r e have been n u m e r o u s changes in AutoAnalyzer modules during the p a s t fifteen years. T h e original AutoAnalyzer dialyzer contained 88 in. of circular groove length. In the S M A a n d AAII i n s t r u m e n t s , this l e n g t h is reduced t o a U - s h a p e d
IV Sampler Rate: 60 per 2T hour
06S 1.60 Tube size ml/rrtir
Colorimeter Recorder 660 nm Filters 15 mm F/C
One channel digital printer
Figure 2 . F l o w d i a g r a m of A u t o A n a l y z e r II m e t h o d for 5 ' - n u c l e o t i d a s e (3) d e termination
t
'Λ
•j.l.-iÎWiÎ'.iÎ.Îirf
Figure 3. A u t o A n a l y z e r II Courtesy of Technicon Instruments Co.
groove, 6-12 in. in length. T h e flow cell light p a t h s were m a i n t a i n e d a t 15 m m , b u t t h e inner d i a m e t e r was red u c e d from 3 t o 1.5 m m to increase the w a s h capabilities. For a d e q u a t e 6 0 - s a m p l e / h r operation, a b o u t 1 m l / m i n is required t o w a s h out t h e new flow cell, c o m p a r e d to 3-4 m l / m i n in
older systems. For this reason, the new i n s t r u m e n t s are capable of analysis with smaller flowing volumes a n d , therefore, less reagent c o n s u m p tion. Another a d v a n t a g e of the low fluid flow is t h e use of plastic t u b e s with small inner d i a m e t e r s which p e r m i t more a c c u r a t e proportioning of solutions. T h e s e i n s t r u m e n t s have electronic b l a n k correction o b t a i n e d by s i m u l t a n e o u s analysis t h r o u g h two channels. T h e S M A 12/60 provides for simult a n e o u s analysis of 16 channels (including four blanks) a t a r a t e of 60 s a m p l e s / h r . A major problem is t h e p h a s i n g of t h e system t o p e r m i t a portion of t h e colorimeter o u t p u t from each analytic c h a n n e l t o r e a c h t h e single recorder without interference from t h e other c h a n n e l s . T h i s is accomplished by a d d i n g or s u b t r a c t i n g lengths of t u b i n g t o t h e analytical t r a i n . T o help t h e operator in analysis of t h e phasing, t h e S M A 12/60 is e q u i p p e d with a n oscilloscope which produces a continuous trace of concent r a t i o n vs. t i m e for all 16 c h a n n e l s . R a t e of A n a l y s i s
Figure 4. S M A 1 2 / 6 0 Courtesy of Technicon I nstruments Co.
T h e n a t u r e of continuous flow analysis utilized in t h e basic AutoAnalyzer a n d t h e n in t h e AutoAnalyzer II ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973 · 741 A
new absorbance monitor utrftir
mi
Figure 5. SMAC Courtesy of Technicon Instruments Co.
The ISCO M o d e l UA-5 absorbance monitor gives you the high sensitivity, stability, and response speed required for high speed, high pressure chromatography —plus t h e w i d e absorbance ranges and specialized f l o w cells required for conventional chromatography, density gradient fractionation, electrofocusing, and gel scanning. Stationary cuvettes all o w recording of enzyme and other reactions. High sensitivity. 8 full scale absorbance ranges f r o m .01 to 2.0A, plus % T . 13 wavelengths include 254 and 2 8 0 n m supplied in the basic instrument; 3 1 0 n m , 3 4 0 n m , and 9 other wavelengths to 6 6 0 n m are available at l o w cost. Options inc l u d e a built-in 10cm recorder, a Peak Separator to automatically deposit different absorbance peaks into different tubes, and a multiplexerexpander w h i c h allows m o n i t o r i n g of t w o separate c o l u m n s or one c o l u m n at any t w o wavelengths. A u t o matic 4X scale expansion prevents oversized peaks f r o m going off scale. The current ISCO catalog describes the M o d e l U A - 5 as w e l l as ISCO fraction collectors, metering and gradient p u m p s , and additional instruments for chromatography and other scientific research. Your copy is w a i t i n g .
BOX 5347 LINCOLN, NEBRASKA 68505 PHONE (402) 434-0231 TELEX 48-6453 CIRCLE 102 O N READER SERVICE CARD
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 at 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, but 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, but it is also influenced by the nature of the flowing reactants as well as the design of the manifold system (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, but 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).
742 A · ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
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 contEol serum) from a pool placed in a refrigerated reservoir on the console at 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 samples/carrier, but 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, but in SMAC two additional air bubbles
T a b l e I I . S M A 1 2 / 6 0 Quality Control Control 1. π - 2β
Ca+ + , mg/dl Pi, m g / d l BUN, mg/dl UA, m g / d l TP, g m / d l Alb., g m / d l Bili, m g / d l Alk, U / L LDH,U/L GOT, units
Control 3. η = 24
Control 2, π = 28
Mean
SD. ±
CV,
Mean
SD. =
CV. %
Mean
SD, m
CV, %
6.7 8.0 30 8.1 4.6 2.3 4.12 7 63 10
0.12 0.12 0.61 0.11 0.08 0.063 0.09 1 2 1
1.8 1.5 2.0 1.4 1.7 2.7 2.2 14.2
8.4 5.9 49 9.2 5.9 2.8 3.48 209 390 162
0.13 0.09 0.74 0.14 0.06 0.063 0.07 9 6 5
1.5 1.5 1.5 1.5 1.0 2.3 2.0 4.3 1.5 3.1
9.5 3.8 12 6.4 6.9 2.1 0.57 39 167 49
0.11 0.06 0.38 0.06 0.07 0.05 0.034 1.6 2.5 1.33
1.2 1.6 3? 0.94 1.0 2.4 6.0 4.1 1.7 2.7
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 peristal tic pumps on either side into the ana lytical cartridges. This represents a major departure from older AutoAnalyzers in which pumping is in a hori zontal plane. Segmenting air bubbles are intro duced at precisely the same rate as the rate of tube occlusions of the peristaltic pumps, and the segmenta tion has been increased from 30 to 90 segments/min to optimize wash in the analytical cartridge. In the SMAC system the flow cell is includ ed 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 di ameter is reduced to 0.5 mm, and the light path to 10 mm. The volume in the flow cell has been reduced from 27 μΐ in the SMA or AutoAnalyzer II to 2 Ail. Another innovation in the system is that air bubbles are permit ted to pass through the flow cell, and their infinite absorbance is subtract ed electronically. The SMAC also dif fers from other systems in the intro duction 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 incorpo rates new chemical methods. En zymes are determined kinetically at two or three time points, as well as by the single time point procedure of the older methods. Sodium and po tassium are determined potentiome-
3.2 10.0
trically by using ion-selective elec trodes placed in line with the contin uously flowing fluid. A volume of 10 μΐ is sufficient for each analysis. The SMAC system is a major ad vance in laboratory automation. It makes use of miniaturized hydraulics, new optical detection systems, ionselective electrodes, an instrument dedicated computer, and new, simpli fied handling and loading techniques. It is truly laboratory automation in the sense that the operator's role is merely placing samples in the instru ment. Calibration, analysis, and cor rection of machine problems, as well as calculation of data, are all carried out by the computer. S t a n d a r d i z a t i o n in Continuous Flow Analysis
A review of continuous flow analy sis would be remiss if it did not point out that a major problem is standard ization and then calculation of the assayed value. Standardization is usually achieved with previously ana lyzed 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 mix ture 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 se rums with label values be used as standards in continuous flow assays. The advantage in the use of such se rums is obvious. Since the standard is treated in the same fashion as the un known, changes in tube size, incuba tion time, the effects of dialysis, and other intrinsic continuous flow vari ables are eliminated. The disadvan
tages of the use of such materials are the possible lability of the constitu ent, 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 differ ent tissue or species than the un known sample and acts in a different kinetic fashion. Lyophilized serum with high constituent concentrations can be used as a standard for contin uous 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 preci sion of continuous flow automated methods is excellent. In Table II are listed quality control data of succes sive 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 equip ment or training. Continuous flow manifolds or cartridges can be rapidly changed and permit multiple tests to be run with a single instrument. Al though there have been numerous other instruments and systems de scribed for use in the automation of the chemical laboratory, the AutoAn alyzer 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, ρ 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. Wildermann, Clin. Chem., 16, 832 (1970). (6) R. L. Habig, B. W. Schlein, L. Wal ters, and R. E. Thiers, ibid., 15, 1045 (1969).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973 · 743 A
