automated reaction-rate methods of analysis - ACS Publications

AUTOMATED REACTION-RATE METHODS OF ANALYSIS HOWARD V. MALMSTADT EMIL A. CORDOS COLLENE J. DELANEY School of Chemical Sciences Roger Adams Laboratory University of Illinois Urbana, Ill. 61801

Reaction-rate methods of analysis often offer advantages in selectivity and accuracy. New automated instrumentation encourages the development of specific procedures and the likely adoption of these methods

'"THE

FIRST " A U T O M A T E D " reaction-

rate (kinetic) methods and in strumentation for the rapid selective determination of glucose a n d other constituents were reported over a decade ago (1-6). T h r o u g h the 1960's, analytical methods based on the measurement of initial rates of chemical reactions grew in impor tance as m a n y new sensitive and selective procedures were reported, and the instruments were improved. T h e frequent journal review articles and recent books a t t e s t t o the in terest in kinetic methods (7-12). It seems now t h a t it will be t h e middle of the 1970's when reaction-rate methods become generally accepted and widely used on a routine basis. I t is certainly not coincidence t h a t t h e conversion from research in terest to general routine use of reac tion-rate methods corresponds to the development of a new generation of elegant, completely a u t o m a t e d , and computer-controlled chemical in strumentation. T h e r a t h e r recent concepts a n d advances in analytical instrumentation make it just as easy to obtain sensitive q u a n t i t a t i v e chemical results with reaction-rate methods as with conventional stoi chiometric (equilibrium or endpoint) methods. By elimination of t h e barriers imposed by difficult labora tory rate techniques, it now becomes worthwhile to consider the inherent advantages of reaction-rate meth ods. I n this report the inherent ad vantages and possible limitations of reaction-rate methods as compared to equilibrium methods are re viewed, and the general concepts of

26 A · ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

encoding reaction-rate information are presented. However, the major discussion is focused on the a u t o mated systems t h a t make it possible to perform hundreds of accurate, sensitive, and selective q u a n t i t a t i v e determinations per hour via r a t e d a t a and to develop new methods more rapidly.

ADVANTAGES AND LIMITATIONS OF REACTION-RATE METHODS

W i t h rate methods it is often possible to measure, immediately after mixing the reactants, the r a t e of change of some parameter Ρ of the reactant whose concentration is to be determined, or other reactant or product of the reaction, and n o t wait for the reaction to go to com pletion (equilibrium). This is il lustrated in Figure 1. T h e saving in time may or m a y not be sig nificant depending on the specific reaction, b u t there are good ex amples (1-12) of obtaining q u a n t i t a tive r a t e results in seconds for selective reactions t h a t would h a v e required many minutes or hours t o go to completion. This is especially t r u e for many of the highly selective enzymatic reactions. Because it is possible to obtain q u a n t i t a t i v e rate d a t a shortly after the reagents are mixed, the measure ment m a y be completed before interfering side reactions begin. This can be a distinct advantage in providing higher accuracy for some determinations. One of the most i m p o r t a n t char acteristics of the reaction-rate method is t h a t it involves a relative

REPORT FOR ANALYTICAL CHEMISTS measurement. T h e absolute value of t h e parameter (i.e., absorbance, cell potential, fluorescence) chosen to monitor t h e reaction does n o t have t o be measured accurately, as shown in Figure 1. I t is only nec essary t o measure t h e parameter's time rate of change with high pre cision a n d accuracy. Hence, even for extremely rapid reactions, t h e reaction-rate method can offer free dom from those interferences which contribute t o the absolute value of the p a r a m e t e r (turbidity, dirty cells, junction potentials, a n d other fluorescing materials) b u t do n o t enter t h e chemical reaction a n d do not contribute t o t h e r a t e of change of t h e parameter with time. Of course, there are those applica tions where it is t h e rate per se, rather t h a n the absolute concentra tion of a specific species, which is t h e i m p o r t a n t q u a n t i t y t o be deter mined. M o s t noteworthy in this category is t h e determination of enzyme activity. Another possible a d v a n t a g e of kinetic methods is t h a t they some times provide a means of determin ing t h e concentration of two or three constituents of closely related chemical properties without physi cal separation. As a rule of t h u m b , t h e successful development of differ ential r a t e methods requires t h a t the first-order rate constants of t h e individual components differ b y a t least a factor of 10. F o r example, silicate a n d phosphate in mixtures h a v e been determined b y a differ ential rate procedure based on t h e formation reactions of t h e heteropolymolybdate a n d t h e reduced heteropoly blues (13). There are some limitations in t h e general application of reaction-rate methods. T h e most i m p o r t a n t is t h a t imposed b y t h e reaction r a t e itself. T h e half-time of t h e reac tion must be greater t h a n the mixing time of t h e i n s t r u m e n t a l system available. Considering t h e other extreme, very slow reactions with half-times greater t h a n a few hours are n o t t o o practical for routine analyses. Also, t h e accuracy a n d precision of t h e measurement d e pend upon good reproducibility (although n o t necessarily good a c curacy) for all experimental con ditions such as t e m p e r a t u r e , p H , ionic strength, size, a n d shape of reaction vessels.

GENERAL CONSIDERATIONS IN ENCODING CHEMICAL REACTION-RATE INFORMATION

Determination of Glucose. A few practical examples of reactionr a t e methods are presented here to illustrate t h e general consider ations involved i n t h e encoding of rate information. T h e first ex ample is t h e q u a n t i t a t i v e deter mination of glucose b y use of t h e well-known selective oxidation of glucose in t h e presence of t h e en zyme, glucose oxidase, as illustrated by E q u a t i o n 1. Glucose oxidase

Glucose + 0 2 >Gluconic acid + H 2 0 2

(1)

Encoding Indirectly Via Coupling Reaction. I n o u r own laboratory we first became heavily committed to t h e development of rate methods more t h a n a dozen years ago b y devising a n a u t o m a t e d r a t e mea surement system for the determina tion of glucose in blood serum (1). For expediency we first chose t o determine t h e r a t e of change of glucose b y indirectly following t h e rate of formation of H 2 0 2 . Since the H 2 0 2 did n o t h a v e a physical parameter t h a t could be easily a n d directly measured, t h e relatively fast coupling reaction of H 2 0 2 with an organic d y e w a s used. This was conventional practice a t t h e time for t h e end-point methods

because t h e colored reaction prod uct, as illustrated in E q u a t i o n 2, provided high sensitivity b y photo metric measurements. .

Peroxidase

Organic dye A + H 2 0 2 — °

>•

Enzyme

Colored product Β (2) Unfortunately, the peroxidase en zyme t h a t catalyzed t h e coupling fast reaction was rather unstable and expensive, a n d t h e a u t o m a t e d two-point, reciprocal time, ratemeasuring system was a t first rela tively crude. B u t from this ex ample, i t became a p p a r e n t t h a t sensitive a n d precise q u a n t i t a t i v e measurements could be realized with the aforementioned advantages over end-point methods if suitable r e actions could be selected a n d con trolled; sensitive devices for con verting (transducing or encoding) t h e concentration change for one of t h e reaction species t o a mea surable electrical signal could b e developed; a n d reliable electronic systems for t h e r a t e measurement could be developed. T h e glucose reaction t h e n b e came a test system for demonstrat ing new encoding systems a n d r a t e measurement systems. By use of a different fast coupling reaction, as illustrated in E q u a t i o n 3, i t was possible t o determine t h e r a t e of change of H 2 O 2

Figure 1. Initial rates of reaction propor tional to concentration of sought-for species

ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972 · 27 A

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I , " + 2 0 H - (3) by following t h e I 3 _ with potentiometric (2), spectrophotometric (3), and amperometric (14) encoding (transducer) systems, a n d t o eliminate t h e troublesome peroxidase enzyme. I n E q u a t i o n 1 t h e rate of formation of gluconic acid could be used to obtain t h e initial rate information. Therefore, a sensitive digital p H - s t a t system (15) was devised wherein t h e p H was held constant by adding small increments of N a O H during t h e glucose reaction. T h e number of increments of base added during a short fixed period of time proved t o be directly proportional t o t h e glucose concentration. However, t h e system is n o t as sensitive as t h e methods based on t h e H 2 0 2 coupling reactions. Encoding Directly Via Primary Reaction. T h e obvious desire t o eliminate a n y t y p e of secondary or coupling reaction also led t o t h e investigation of direct methods for following t h e rate of change of 0 2 . Voltammetric encoding systems t h a t could measure 0 2 directly (16-18) were developed, although again the method was n o t as sensitive as t h e indirect H 2 0 2 color methods. T h e conversion of glucose or gluconic acid rate information t o a directly measured physical parameter has n o t been reported. Determination of Phosphate. Encoding Indirectly Via Secondary Reaction. T h e determination of phosphate b y a rate method illustrates similar considerations as those for t h e glucose determination. First, t h e classical molybdenum blue procedure could be developed into a sensitive rate method involving t h e primary reaction of phosphate with M o (VI) t o form 12-molybdophosphoric acid (12-MPA) a n d i t s subsequent reduction t o form the heteropolyphospho molybdenum blue, P M B (19), as shown in E q u a tions 4 a n d ô . H3P04 + 6Mo(VI),—>

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12-MPA + 9 H +

(4)

12-MPA + wiled —*• P M B + «Ox (5)

CIRCLE 25 ON READER SERVICE CARD 28 A · ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972


RAW SAMPLE

REAGENTS

SAMPLE PREPARATION

PREPARATION

tively large choice of methods is now available (1-21). The method of choice for encoding the rate information from either the primary or coupling reactions depends, of course, on several factors including sensitivity, freedom from interferences, simplicity, and dependability.

REAGENT

ALIQUOTING 8 MIXING OF REACTANTS

DATA DOMAIN

INSTRUMENTATION SYSTEMS FOR REACTION-RATE METHODS CONTROL SYSTEM

CONVERTER (TRANSDUCER)

RATE

Figure 2. Block diagram of complete instrumentation system for reaction-rate methods

MEASUREMENT

AND DATA MANIPULATION

OSCILLOSCOPE

RECORDER

OUTPUT DATA DISPLAY

DIGITAL

In this case, the measured reaction rate depends on the nature of the reductant, order of adding reagents, as well as acidity, etc. (19). Encoding Directly Via Primary Reaction. I t was again obvious that it would be advantageous to eliminate the reduction reaction (Equation 5) and convert one of the species in the primary reaction to a physical parameter that could be readily measured. Fortunately, the 12-JVIPA has a relatively high absorptivity at readily available wavelengths. However, for the first time in our development of quantitative reaction-rate procedures, there was a confrontation with a reaction whose half-life was so short that it was necessary to make rate measurements in milliseconds rather than seconds. A rather long development program was thus required to develop the new automated equipment that would provide rapid mixing and readout of rate information. After the automated system was developed, it was then possible to determine

TELETYPE

phosphate directly {20), utilizing the primary reaction shown in Equation 4. It is feasible to make as many as 3000 determinations of phosphate concentration per hour with this type of system. General Conclusions. It is seen from these examples for glucose and phosphate that a sequence of chemical reactions is often used to obtain a chemical species that is related to the desired rate information and readily converted to a sensitive measurable signal, or to obtain a readily measured chemical species whose rate of formation is slow enough to be measured by available equipment. Although the methods utilizing multiple sequential reactions can be dependable under carefully controlled conditions, almost inevitably they will be less reliable than methods using measurements on one primary reaction. Much research effort has been invested in providing more sensitive and selective encoding systems and high-speed automated ratemeasuring instruments, and a rela-

The work on automating reaction-rate methods in the past decade primarily provided improved rate measurement devices (as described in this month's Instrumentation feature, page 79 A) and sensitive data domain transducers for converting the chemical rate information to measurable electrical signals. Today, the attention is being focused on complete systems that start with the raw samples and reagents and end with a formatted printed readout in the desired quantitative units. A general system for obtaining quantitative data is illustrated by the block diagram in Figure 2. By preliminary treatments (e.g., dissolution, dilution, nitration, ion exchange) the sample and reagent solutions are prepared as required for the specific procedures. Predetermined volumes of sample and reagents are then introduced, mixed, and transported to a vessel which serves as the reaction cell. The chemical reaction is monitored by a suitable transducer which converts the rate information about a specific species inherent in the reaction to a measurable signal in the electrical domain. Frequently, several interdomain conversions are required to obtain data in the preferred form. The control and rate measurement systems can be hardwired for specific applications, or they can be incorporated in a minicomputerinterfaced system that can provide through software much versatility in control of the measurement sequence and the processing and readout of data. Readout is visually displayed with digital lights or printed out on serial teletype or a high-speed parallel printer. When desired, a servo recorder or storage oscilloscope can display the parameter vs. time and rate curves.



MOTOR DRIVEN STIRRER UNIT Β INJECTION PIPE Τ FOR COMPOSITE REAGENT

TO WASHOUT DILUENT

TO VACUUM

INJEC TION PIPE Τ FOR DEPR0TE1NIZED SAMPLE

TO STOPCOCK DRIVER

ASPIRATOR PUMP

TRANSDUCER

CONTROL

INTERFACE

SYSTEM

DIGITAL

CALIBRATED^ PI PET TIP \

CELL

READOUT

[|3l7l6|2l| STEP I MOVABLE PLA TFORM MAGNETIC STIRRER

Figure 3a. Block diagram of automatic potentiometric reactionrate analyzer (5) T h e weakest links in reactionr a t e instrumentation systems in t h e recent past have been the; sam ple and reagent preparation and aliquoting and mixing systems. I n fact, it becomes apparent t h a t the greatest differences in completely automated reaction-rate instru ments will probably be the methods of sample and reagent prepara tion, aliquoting, and mixing. These operations are not only tedious, repetitious, and time de manding when done, manually b u t are subject to h u m a n error and bias. I t is not unusual t h a t the sample-handling procedures re quire much more time t h a n the r a t e measurement itself. This, of course, is not a problem exclusively associated with the development of reaction-rate methods; rather, it is an i m p o r t a n t consideration in the development of all a u t o m a t e d analytical methods. One of the first reaction-rate instruments t h a t incorporated auto m a t e d pipetting of sample and reagents and provided rapid deproteinization and filtration of serum samples was presented by M a l m s t a d t and P a r d u e (5). T h e instrument was designed specifically for glucose in serum determina tions. A potentiometric concen t r a t i o n cell, Figure 3a, was designed as the transducer to encode the initial rate information. One auto mated injection pipet {22) was used to deliver a 1-ml aliquot of composite reagent to the sample compartment. A second auto mated pipet delivered a measured

STEP 2

STEP 3

STEP

4

Figure 3b. Manipulations for filtering, measuring, and deliver ing deproteinized serum or plasma samples. Step 1: picking up polyethylene sleeve. Step 2: picking up filter paper. Step 3: filtering deproteinated sample. Step 4: delivering sample into reaction cell

aliquot of diluent to wash out the calibrated delivery tip t h a t con tained an accurate aliquot of de proteinized serum. T h e rate in formation for glucose was auto matically measured within about 30 sec. T h e reaction mixture was t h e n removed through a n aspira tion tube, and the cycle repeated. T h e deproteinization, nitration, and pipetting of an accurate aliquot of sample into the reaction cell is schematically represented in Figure 3b. T h e serum sample and deproteinizing reagents are added to a small cuvette. Another sam pling pipet immediately draws an accurate aliquot of the deprotein ized sample solution through a glass-fiber filter which retains the precipitate. When t h e calibrated tip is filled with sample filtrate, the filter is knocked off, and the threeway valve is turned to connect the diluent injection pipet to wash out the sample and diluent into the reaction vessel. All of t h e oper ations illustrated by Figure 3b can be performed automatically. Although it is, of course, pref erable to develop a procedure t h a t does not require filtration, t h e method does illustrate how classical deproteinization, filtration, and pipetting operations can be auto m a t e d in discrete steps. M a n y of the newest commercial instru ments operate in similar discrete steps. Considerable research, de sign, and engineering activities are currently being directed to auto mation of aliquoting and mixing of reactants, and many commercial


models have only recently appeared on the market.

Automated Stopped-Flow Re action-Rate System. The stopped-flow a p p a r a t u s is widely used for kinetic studies {23, 24) of fast reactions, and it has been modi fied {20, 31) for rapid a u t o m a t e d re action-rate methods, as illustrated in Figure 4. ΛI any sample and reagent han dling systems are only suitable for utilizing relatively slow reactionrate systems. As pointed out ear lier, there are advantages in select ing a fast reaction for analytical purposes. T h e limitation in making r a t e measurements on a fast re action is t h a t the time taken for mixing and observation must be shorter t h a n the half-life; of the reaction. Today, observation time can be reduced to the millisecond range by use of fast-responding detector-readout systems {25, 26) so t h a t the real limitation becomes the time required for physical mix ing of reactants and initiation of reaction. To minimize this time, mechanical systems are used which drive reactants rapidly enough to promote; turbulent flow through the system and thereby insure rapid mixing and uniformity of solution composition in the observation cell. Although a number of flow meth ods have been used for studying fast reactions, t h ç stopped-flow method has been most widely a p plied for analytical purposes. This technique consists of rapidly mixing reactants by forcing the solutions through a mixing chamber and into


as a control system for a number of operations. At the end of a measurement cycle, the logic cir cuit provides a signal which acti vates the syringe drive circuit, thereby injecting sample and re agent automatically. The number of rate measurements per injec tion can be preset on the logic unit. Likewise, the number of injections per sample cup is programmed. A preselected number of injections are used to insure complete flushing

an observation cell. The flow of solution is abruptly stopped, creat ing a back pressure which completes the mixing, and the rate measure ment is rapidly made. Once ex perimental parameters have been selected, and samples and com posite reagent have been loaded on the sample tray, the modified stopped-flow measurement system (81) operates automatically and con tinuously until reagents are ex hausted. A logic circuit is used STOP BAR

TO

STOPPING SYRINGE

DRAIN CHECK VALVE

MANUAL' VALVE ,

LIGHT

MONO-

SOURCE

CHROMATOR

OBSERVATION

I TO V AMPLIFIER

PM TUBE

MIXING

SAMPLE CELLS

CELL

SAMPLE Dl Ρ TUBE

Automated System with Pre packaged Reagents. A novel

DIGITAL RATE METER A CONTROL SYSTEM

CHAMBER

DOUBLE 3-WAY STOPCOCK

Figure 4. Schematic diagram of automatic stopped-flow system (20)

STOPCOCK . MOTOR

vM^

SAMPLE PICKUP/ SYSTEM^

REAGENTS

2-ML

SYRINGES

TURNTABLE AND SAMPLE DIP_ TUBE MOTOR £

TRIGGER CIRCUIT CAM

M,

SYRINGE. DRIVE MOTOR

of the flow system of previous re acted sample. During this flush ing interval, the readout system is locked by the control circuit. After the preset number of injec tions and measurements have been made, the logic circuit activates the sample tray, and the next sam ple cup is brought into position. A teletype is used to log the data and to generate a paper-punched tape. The paper tape is sub sequently used to input the data to a small computer (Digital Equip ment Corp. PDP-8L). Results for standards and samples are com puter averaged and corrected for blank if necessary. A least-squares routine is used to provide printout of concentration of the samples.

MICROSWITCH

method for automation of reactionrate and equilibrium techniques minimizes the problem of laboratory reagent preparation and contami nation'by prepackaging all required reagents in stable form in a dispos able plastic pack. The same pack also serves as the reaction chamber and observation cell. This approach has been initiated by Du Pont ( E . I . du Pont de Xemours & Co., Wil mington, Del.) in the development of the automatic chemical analyzer (aca) system. As shown in Figure 5, the re agents are contained in plastic bubbles near the top of the pack. The pack header contains the name of the test and a binary code which is interpreted by a small, ROM

CHROMATOGRAPHIC COLUMN PACK HEADER

TEST NAME BINARY CODE

TO OUTLET VALVE

NEEDLE CARRIAGE RAIL POSITION Β DRAIN

TUBING O F ' Y E F L O N "

POSITION A SAMPLE INTAKE CUVETTE

INPUT TRAY

AREA

NEEDLE CARRIAGE TEMPORARY SEALS

PERMANENT SEAL

REAGENT

SAMPLE CUP EXIT TRAY

COMPARTMENTS

POSITION D SAMPLE & DILUENT INJECTION

POSITION C SAMPLE & ELUANT INJECTION

Figure 5. Analytical pack for Du Pont automatic clinical analyzer

Figure 6. Filling station for Du Pont automatic clinical analyzer

Courtesy of E. I. du Pont de Nemours & Co., Wilmington, Del.

Courtesy of E. I. du Pont de Nemours & Co., Wilmington, Del.



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(read-only-memory) computer. T h e code specifies experimental conditions such as sample volume, reagent dilution, and processing cycle in addition to providing for automatic setting of specified in strumental parameters and for manipulation of the d a t a so t h a t the final printout is in desired units. Since the code is embossed on the t o p of the header, the packs may be loaded in any order to provide t h e desired sequence of analyses for a given sample. An additional fea t u r e is t h e inclusion of a gel filtra tion or ion-exchange column in those packs for determinations which require the elimination of inter fering components. Initially, t h e sample is placed either manually or by automatic sampler into a plastic cup to which an identification card is clipped. T h e cup assembly and test packs, after being manually loaded on the input t r a y of the analyzer, are automatically transported into the filling station shown in Figure 6. Here, t h e binary code on the in dividual test packs is decoded by t h e R O M computer, and the filling needle moves sequentially to per form the operations of flushing the delivery needle (position Β in Figure 6), withdrawing the specified vol umes of diluent (or elution diluent if a chromatographic column is used) and sample (position A in Figure 6), and injection of sample and diluent into the pack (at posi tion D or C in Figure 6). T h e fluid metering system con sists of a series of reagent reser voirs which are connected to a piston p u m p through a series of precision micro valves. T h e p u m p is driven by a stepping motor. T h e volume of liquid drawn and delivered by the p u m p is proportional to the number of impulses applied to the stepping motor by t h e built-in com puter. T h e operation of the microvalves is also computer-controlled. At t h e conclusion of the filling cycle, each pack is automatically moved into the main processing section of the analyzer. As the pack proceeds on a transport chain, it is heated to and maintained at a constant temperature, and reagents are released and mixed at two breaker-mixer stations by r u p t u r e of the reagent bubbles and oscilla tion of t h e pack.

CIRCLE 153 ON READER SERVICE CARD


Upon completion of this cycle, the pack advances to the photom eter station where a set of mechan ical jaws close around the pack generating hydraulic pressure within t h e pack to form a precision cuvette between quartz windows. T h e ini tial rate is measured by use of the fixed-time technique, a n d t h e com puter automatically supplies an appropriate conversion factor so t h a t concentration or activity is printed out on a report sheet along with t h e sample identification n u m ber. T h e R O M computer is also used for surveillance of all opera tions required for processing the sample. Should a malfunction be detected, the system also prints out a code on the report sheet in dicating the source of error.

Parallel Vs. Sequential Mixing of Samples and Reagents. In the sample and reagent handling approaches described in t h e above methods, the reactants are aliquoted and dispensed into t h e mixing cham ber, and t h e process repeats se quentially for a series of samples. I n most cases, the reagents are directly expelled from a pipet into the mixing chamber where the mix ing operation is immediately car ried out. An alternate approach can be t a k e n by using a time separa tion of these two operations and then t h e simultaneous (parallel) mixing of reactants for a group of samples. All reactants are premeasured and introduced into rea c t a n t wells. T h e mixing operation takes place a t a later time. This principle of parallel mixing is applied in the unique G e M S A E C analyzer developed b y Anderson {27) and Coleman et al. {28) a t Oak Ridge National Laboratory (Oak Ridge, Tenn.). T h e latest sys t e m allows parallel mixing of re actants for u p to 42 samples. Cen trifugal force is used to transfer reactants into separate mixing chambers which also serve as the observation cells. Samples and reagents are manu ally or automatically pipetted into individual holding c o m p a r t m e n t s of t h e transfer disk as shown in. Figure 7. T h e transfer disk is then manually locked into place in the rotor which also insures proper indexing of the samples. T h e hold ing compartments are arranged in two or more concentric circles in the CIRCLE 38 ON READER SERVICE CARD

>•


SAMPLE BEFORE TRANSFER AXIS OF ROTATION

REAGENT BEFORE TRANSFER LIGHT TO PHOTOMULTIPLIER TUBE

QUARTZ WINDOWS SPACER REMOVABLE TRANSFER DISK REACTION MIXTURE AFTER TRANSFERS

LIGHT FROM MONOCHROMATOR

Figure 7. Sections of the GeMSAEC fast analyzer. Upper left: loading of transfer disk. Upper right: transfer disk and centrifugal analyzer section (27, 28) Courtesy of International Scientific Communications, Inc., Green Farms, Conn.

transfer disk, with each circle containing t h e same number of comp a r t m e n t s . T h e compartments are aligned radially and are angularly arranged so t h a t samples a n d reagents are dumped through a t r a n s fer cavity into t h e corresponding reaction chamber (which also serves as a photometric cuvette) when t h e rotor reaches a sufficient speed (typically 400 r p m ) . T o prevent lateral splashing of t h e solutions in t h e transfer disk, t h e rotor is gradually accelerated, a n d t h e n during a breaking interval, a pulsed v a c u u m is applied which draws a stream of small air bubbles through each cuvette. T h e resultant turbulence produces complete mixing of t h e reactants. T h e air bubbles are removed by rapid acceleration followed by deceleration to the normal operating speed of about 500 rpm. I n present designs this entire process requires only a couple of seconds. T h e cuvettes r o t a t e past a stationary light b e a m completely interrupting it between cuvettes.

Figure 8. Oscilloscope traces from photometer output of 15cuvette GeMSAEC analyzer. Upper: signal for a single cuvette. Lower: display for water blank and duplicate sets of protein standards (28) Courtesy of International Scientific Communications, Inc., Green Farms, Conn.

T h e signal from the photomultiplier is continuously displayed on an oscilloscope which is synchronized with the rotor to provide an immediate visual monitor of t h e signals for all of t h e cuvettes. T h e photomultiplier signal is also passed t h r o u g h a buffer amplifier, directed to an analog-to-digital converter, and subsequently to a P D P 8/1 computer. During each rotation of t h e rotor, t h e d a r k current value is obtained between each peak, and a blank peak value equivalent to 100% transmittance, in . addition to a series of s t a n d a r d and experimental peak values, is determined, as shown in Figure 8 for a 15-place rotor. After the spectrophotometric rate or stoichiometric measurements are completed, t h e samples are dumped through drainage siphons located in each cell by a p plication of an air stream to t h e center cavity of t h e rotor which extends radially t o each cell. T h e rotor is t h e n stopped, and wash water is added from a wash bottle to both t h e sample a n d reagent


compartments of the transfer disk. T h e rotor operational sequence is repeated, allowing the wash water to be transferred to t h e cuvettes which are drained as described above. T o increase sample t h r o u g h p u t rate, research is being conducted to provide automation of sample and reagent loading and of cleaning and drying of the rotor between runs. T o extend t h e utility of the analyzer to more complex methodology, two additional research objectives reported (27) are incorporation of ion-exchange and gel filtration columns into the syst e m and provision for posttransfer self-decanting of supernatant from a precipitate. Also, throwaway disks already loaded with reagents are being considered. W h e n an end-point determination is made, t h e readout is obtained by averaging t h e results for a n u m ber of rotor passes. F o r a reaction-rate procedure, a fixed-time technique is used. Two or more sets of digitized absorbance readings


for each cuvette are stored si multaneously, and the difference is calculated and expressed as Δ Α / min. The commercial versions of G e M S A E C are the Centrifichem (Union Carbide, Tarrytown, N.Y.), the Rotochem (American Instru ment Co., Silver Spring, M d . ) , and the G e M S A E C (ElectroNucleonics, Fairfield, N.J.). ELLA. The use of a small compu ter with a spectrophotometric rate system can be extended to mecha nistic investigations by programmed decision-making. Automation of such investigative experiments en hances the collection of d a t a required for the development of new, reliable reaction-rate methods. This ap proach to automated instrumenta tion has been incorporated into a system called E L L A (29). T h e hardware used for E L L A (Experimental L I N C Laboratory Analytical System), excluding the computer I / O devices, is shown in Figure 9. T h e reactants are placed on the sample tray in the order of desired addition to the system. For example, for an en zymatic experiment the order is buffer, enzyme, buffer, substrate, buffer. T h e initial volume of en zyme to be aliquoted and incuba tion time required prior to rate measurement are specified via a teletype by the experimenter in response to the system's pro grammed request. T h e thermostated digital pipet sequentially draws reactants through a dip tube

from the turntable and into a hold ing coil. T h e volumes of solutions to be picked up and delivered are determined by the number of clock pulses received from the accumu lator buffer of the computer ( D E C P D P - 1 2 ) . These impulses activate a stepping motor which allows delivery of 5 μΐ from the pipet per pulse. T h e reactants are expelled from the pipetting system into a thermostated mixing chamber, and the reaction mixture is transferred by vacuum into the flow cell where t h e spectrophotometric measure ment is made. T h e software used for E L L A permits performance of a kinetic experiment to a desired end point, making all necessary decisions and controlling all instrumentation via a special-purpose time-sharing system. This software consists of four sub programs called the initialization routine, the on-line operating sys tem, the updating system, and the cleanup routine. T h e initializa tion routine requests the experi menter to enter the volume of en zyme and the incubation time to be used, sets up a blank reading tube and the first series of reaction tubes containing varying amounts of substrate, calibrates the reactionrate reading system, and activates the sample-reagent introduction sys t e m automatically. T h e on-line operating system takes readings of the rate of reaction, controls the reaction-rate interface, and performs a least-squares fit to the data. When a sufficient

COMPUTER

ACCUMULATOR BUFFER

SEQUENCE TIMER

RATE METER

WASH SPECTRO PHOTOMETER

TURNTABLE

DIGITAL PIPET

THERMOSTATED CELL SOLENOID VALVES

THERMOSTATED PLUNGER DELAY C O I L HEATING BATH MIXING CHAMBER

VACUUM PUMP MAGNETIC STIRRER

WASTE FLASK

Figure 9. Block diagram of hardware used in ELLA (29) 38 A · ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972

number of data points have been obtained which exhibit an error of estimation below the level pro grammed by the experimenter, a flag is set indicating the experiment is completed. T h e updating program runs after each tube to process information obtained from the previous t u b e and to set up and activate the hard ware for the next run. T h e pro gram performs bookkeeping tasks and calculates the rate. T h e cal culated initial rate is corrected for blank and printed along with other desired information. If the experi ment is not completed, control is returned t o the on-line operating system. If the experiment is completed, the cleanup routine is called which stores the data in the file on mag netic tape for successive experi ments. T h e system then prints out this material upon command. M u l t i m o d e Instrumental S y s t e m . Recently, Doming and P a r due (SO) have developed a computercontrolled instrumental system for characterization of chemical reac tions. T h e system consists of four distinct elements: a program mable digital computer (controller), a spectrophotometer used to con vert the chemical information into electrical information, an analogto-digital converter, and a device for initiation and control of the experiment. The initiation and con trol device is composed of a num ber of electrical and mechanical components which enable the sys tem to introduce into the spectro photometer cell the proper volume of reagents and diluents. Four stepping motors are used to drive four calibrated syringes which pull the reagents from a stock reservoir and dispense them. T h e volume of delivered solution is controlled by applying a corresponding num ber of pulses to the stepping motors. T h e system is also provided with the ability to remove the contents of the cell, rinse the cell, and mix the reagents. The automated instrumental sys tem has been evaluated for three types of operations : routine opera tions, experimental design, and d a t a interpretation. For routine operations, the ex periments are preprogrammed into t h e information base, and data


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are plotted on the storage scope and printed out on the teletype. In the experimental design opera tion, the concentration of reagents and the limits over which they must be varied are introduced into the computer. Then the instru ment plots the reaction rate vs. concentration of the species. Data interpretation type of oper ation is accomplished by using the results of precision experiments. These results are interpreted, and new experiments are automatically designed to complete the characteri zation. The automated instrumental system allows the time required for the operations involved in the experiment preparation to be de creased. The efficiency of the ex perimenter is greatly increased by the rapid availability of the results of a characterization and by more free time to accomplish other tasks that are more difficult to auto mate. CONCLUSIONS

A new generation of automated and computer-controlled instru mentation makes it possible to per form reaction-rate methods of anal ysis at times per sample that are equivalent to the fastest equilib rium methods. The rate methods often have advantages in selectiv ity and accuracy that will encour age their adoption as the new auto mated instrumentation becomes available in laboratories, and more methods are consequently de veloped. The present interest in automating basic chemical reac tion-rate studies should make it possible to develop specific new procedures many times faster than in the recent past. Because of space, no attempt was made to review all of the new auto mated methods or the well-known Technicon continuous-flow analy zers. The methods presented illus trate that the major differences in chemical reaction-rate analyzers are in the sample and reagenthandling and mixing procedures. The functional units of the instru ments are similar, and with suit able modular designs, it should be possible to easily modify analyzers for various general approaches to either reaction-rate or equilibrium analytical methods.


of Chemistry at the University of Illi nois in Urbana. After receiving a BS degree in chemistry from the Univer sity of Wisconsin (194-3) and serving as a radar officer in the Pacific, he re turned to Wisconsin and earned his MS and PhD degrees in 1948 and 1950. He joined the faculty at the

University of Illinois in 1951 follow ing a postdoctoral year at Wisconsin. He is the author or coauthor of more than 90 technical publications in cluding the well-known and widely used books: "Electronics for Scien tists," "Digital Electronics for Scien tists," and "Computer Logic." He was a Gugenheim Fellow in 1960, the 1963 ACS Chemical Instrumen tation award winner, and recipient of the 1970 Eckman aivard in edutation (Instrument Society of Amer ica). Dr. Malmstadt was chairman of the ACS Analytical Division in 1964 and served on Analytical Chem istry's Advisory Board (1961-63). His major areas of research are in new spectroscopy methods and in strumentation; short-time phenomena in laser plumes, flames, and spark discharges; reaction-rate methods; and instrumentation automation.

Emil A. C o r d o s is an assistant pro fessor in the Analytical Division of the University of Cluj, Romania, and is also a member of the Directing Council of the Center for Research

in Analytical Chemistry, Cluj. Dr. Cordos received his BS and PhD degrees in chemistry from the Univer sity of Cluj in 1959 and 1969. In 1967-68, he studied at the University of Illinois under H. V. Malmstadt as an exchange visitor and returned to Illinois in 1970 as a two-year post doctoral research associate. Dr. Cordos' research interests include the development of rapid methods of analysis, electrolysis in molten salts, inorganic ion exchangers, gas chro matography of inorganic gases, auto mated atomic absorption and fluo rescence spectroscopy, and automated reaction-rate methods. He has pub lished more than 20 papers on the above topics.

C o l l e n e J. D e l a n e y is a postdoc toral trainee in clinical chemistry at University Hospital, University of

Washington, Seattle, Wash. She re ceived her BS in chemistry from Pur due University in 1965 and earned her MS and PhD degrees from the University of Illinois in 1967 and 1972. Her research interests include reaction-rate methods of analysis; flame emission, absorption, and fluo rescence techniques; and automation in clinical chemistry. She is espe cially interested in developing in-ser vice training and continuing education programs for clinical laboratory per sonnel. Dr. Delaney is coauthor of six papers on the above topics. She is a member of Iota Sigma Pi, the ACS,andAAAS.

At present, only t h e stoppedflow analyzer has been applied for rapid quantitative procedures based on measurement of initial reaction rates for extremely fast reactions. REFERENCES

(1) H. V. Malmstadt and G. P . Hicks, Anal. Chem., 32, 394 (1960). (2) H. V. Malmstadt and H. L. Pardue, ibid., 33, 1040(1961). (3) H. V. Malmstadt and S. I. Hadjiioannou, ibid., 34, 452 (1962). (4) H. V. Malmstadt and T. P . Hadjiioannou, ibid., ρ 455. (5) Η. V. Malmstadt and H. L. Pardue, Clin. Chem.., 8, 606 (1962). (6) W. J. Blaedel and G. P . Hicks, Anal. Chem., 34, 388(1962). (7) G. A. Rechnitz, ibid., 38, 513R (1966); 40, 455R(1968). (8) G. G. Guilbault, ibid., 42, 344R (1970). (9) Κ. Β. Yatsimerskii, "Kinetic Methods of Analysis," Pergamon Press, New York, N.Y., 1966. (10) H. Mark and G. Rechnitz, "Kinetics in Analytical Chemistry," Wiley, New York, N.Y., 1968. (11) G. G. Guilbault, "Enzymatic Meth ods of Analysis," Pergamon Press, Ox ford, England, 1970. (12) H. L. Pardue, Rec. Chem. Progr., 27, 151 (1966). (13) J. D. Ingle, Jr., and S. R. Crouch, Anal. Chem., 43, 7 (1971).· (14) H. L. Pardue, ibid., 35, 1240 (1963). (15) H. V. Malmstadt and Ε. Η. Piepmeier, ibid., 37, 34 (1965). (16) H. V. Malmstadt and A. C. Javier, unpublished internal report, 1965. (17) T. Kajihara and B. Ilagihara, Rinsho Byori, 14, 322 (1966). (18) Kadish, E. Litle, and J. Sternberg, Clin. Chem., 14, 116 (1968). (19) S. R. Crouch and H. V. Malmstadt, Anal. Chem., 39, 1084, 1090 (1967). (20) A. C. Javier, S. R. Crouch, and II. V. Malmstadt, ibid., 4 1 , 239 (1969). (21) H. V. Malmstadt, C. Delaney, and E. Cordos, CRC, Critical Rev. Anal. Chem., 2,559(1972). (22) H. V. Malmstadt and G. P. Hicks, Anal. Chem., 32, 445 (1960). (23) B. Chance, J. Franklin Inst., 229, 455,613,737(1940). (24) Q. H. Gibson and L. Milnes, Biochem. J., 91,161(1964). (25) E. Cordos, S. R. Crouch, and H. V. Malmstadt, Anal. Chem., 40, 1812 (1968). (26) H. V. Malmstadt, C. J. Delaney, and E. Cordos, ibid., 44 (12), 79A (1972). (27) N. G. Anderson, Amer. J. Clin. Pathol., S3, 778(1970). (28) R. L. Coleman, W. D. Shults, M. T. Kelly, and J. A. Dean, Amer. Lab 3, 7,26(1971). (29) A. A. Eggert, G. P. Hicks, and J. K. Davis, Anal. Chem., 43, 736 (1971 ). (30) S. Deming and H. Pardue, ibid., ρ 192. (31) C. J. Delaney and H. V. Malmstadt, unpublished report, 1971; or C. J. De laney, PhD thesis, University of Illinois, Urbana, 111., 1972.

Howard V. Malmstadt is Professor


automated reaction-rate methods of analysis - ACS Publications

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