lnstrumentation Donald A. Burns
Technicon Industrial Systems 51 1 Benedict Avenue Tarrytown, NY 10591
Automated Sample Preparation Scene I: Analytical laboratory, mid-morning: Carl Chemist (scowling):“Blast! This analysis can he done in five minutes, but it takes 20 minutes to get each sample ready. I’ll he here all night!” Anabelle Analyst (equally dejected): “You think that’s had? The cleanup procedure for these samples requires nine different steps and a ton of glassware to wash. There must he a better way!” Scene II: Same laboratory, several weeks later:
This brief dialogue addresses two important problems facing the analytical chemist the labor-intensive aspect of sample cleanup prior to analysis and the inefficiency that results when sample preparation and sample analysis operate in different time frames. These two analysts, and most of their colleagues, surely have sufficient reason to squawk when they discover that many so-called fully automated methods have all hut ignored the major effort required for manual sample treatment prior to sample introduction into the system. Analysts want access to an instrument that will accept unmeasured, untreated samples at one end and provide a full report, in correct concentration units, a t the other end, requiring no operator involvement beyond keeping the reagent bottles full. Such instruments (see Figure la) do exist and have been described for gas chromatography (GC) (I),and more recently for high performance liquid chromatography (HPLC) (2). Inside these analyzers several types of operations may he performed, some examples of which are shown in Figure lb. But not all instruments are fully automated, nor should they be. It is often advisable to stop part way through the procedure and insert an additional step: temporary storage. Figure ICillustrates such a pause: a 0003-2700/81/A351-1403~1.00~0 @ I981 American Chemical Society
Carl (smiling): “Say, this off-line sample prep unit great! The instrument processed 40 samples overnight, unattended, and the analyzer can now handle them a t 12 per hour. They’ll all he finished by noon!” Anabelle (equally ecstatic): “You think that’s good? Take a look at this machine: It’s doing the entire sample cleanup by itself, with no dirty glassware, and the RSDs are lower than I used to get. Thank goodness for automation!”
Figure 1. An analytical instrument: (a) the analyst’s dream: (b) typical operations; (c) two major divisions
break in the previous listing at some appropriate point where the time frame changes. In this REPORT, I address three aspects of automated sample preparation (ASPI: the principles of the unit operations most frequently required; the instrumentation necessary to perform these unit operations; and ASP systems for typical analyses, including the upgrading of existing instruments to attain total automation. Sampling was addressed in ANALYTICAL CHEMISTRY earlier this year in terms of process analyzers (3) and plans for optimizing results ( 4 ) , hut neither paper dealt with automated sample cleanup as it is applied to an analyst’s daily work load. Here sample preparation will he emphasized, and the various means of detection, data handling, and instrument control will not be addressed. ~~~~~
Principles There are two basic approaches to automation-discrete and continuous-and both are widely used. Either approach may be employed in any given system, and sometimes both are found. As we examine various types of operations, the reader should keep in mind that total automation is simply a combination of partially automated
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Flgure 2. Sample definition for discrete analyzers: (a)with reversible stepperdriven pump; (b) with automatic diluterldispenser; (c)with looplvalve combination steps. To get from one step to the next, materials must often be transported from one place to another. In discrete or batch systems, the prime mover may be a turntable, racks of tubes, or an endless chain. Samples are contained in individual vessels such as small cups, beakers, or tuhes. In continuous systems, the prime mover is generally a fluid stream flowing through a conduit, and these liquid streams may or may not be segmented with air. Previous papers in this JOURNAL. have addressed both segmented continuous flow (5) and unsegmented continuous flow (6).In both modes of operation, samples are introduced into the flowing stream at periodic intervals and are kept separated from one another by periods of no sample. For the remainder of this section, reference to Figure l h will he helpful. Sampling and Sample Definition. Definition of a liquid sample is generally combined with the action of transporting it from the original container into the analytical system. In a discrete system this aliquoting is usually accomplished by one of three standard methods: a reversible pump, a pair of mechanized syringes, or a sample loop in a valve. These work in synchronization with a movable probe which alternates between two positions, aspirating a small volume of sample from one vessel and then dispensing the sample with a selected volume of diluent into a second vessel in which a reaction and/or incubation will take place. The peristaltic pump version is diagramed in Figure 2a. With the probe in position a, the pump operates leftto-right and dispenses a small portion of the sample from ita container (frequently on a turntable that holds many sample cups). The probe then moves to position b and the pump opera& right-to-left to dispense the 1404 A
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sample and a defined volume of diluent into a reaction vessel. The pump may be driven by a stepping motor, and the number of steps (operator-selectable) dictates the volumes moved. Typically, one might aspirate 100 pL and dilute it with 0.9 mL to achieve a 1:lO dilution. Needless to say, the sample volume is sufficiently small so as not to travel any appreciable distance from the end of the probe-certainly never back through the pump and into the diluent container. Figure 2b depicts a two-syringe version of an automatic diluter/dispenser. With the probe in the sample cup, syringe c draws in a defined volume of sample while syringed is likewise charging itself with diluent. A valve prevents syringed from affecting the sample volume. When the probe is over the reaction vessel, both syringes empty, dispensing sample and diluent t h r o y h the probe. The syringes can
Multichannel PerisMiic Pump
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Flgure 3. Sampling systL ous-flow analyzers
ANALYTICAL CHMISTRY, VOL. 53. NO. 12. OCTOBER I981
he driven electrically, pneumatically, or manually. A great many instruments are available: ANALYTICAL CHEMISTRY’S1980-81 LabGuide (7) lists 27 automatic diluters and 49 automatic pipetters, many of which are mechanized for nonmanual cvcline. Chromatographers are weli awge of injection valves with sample loops, since this is a common means of automatically introducing samples in HPLC. GC may also use such valves, albeit with smaller external or internal loops. Figure 2c shows a typical sixport valve with an external sample loop that can be filled by either pressure or aspiration. Whev the valve is rotated 60°, the precisely defined contents of the loop are introduced as a plug into the flowing stream of an analytical system. In most systems additional sample equal to two to three times the loop volume is required to flush the loop to reduce sample carryover to an acceptable level. Figure 3 shows the hydraulic arrangement for sampling and diluting simultaneously in the continuous-flow mode. The sample probe alternates between successive cups in the sampler’s turntable and the wash vessel, the relative times in each pwition defining the sample/wash ratio. The airsegmented diluent and the sample are mixed in coil m as will be described later. Aliquot dispensing in continuous flow systems is accomplished by choosing the correct combination of tube size and pumping time. Since the multichannel proportioning pump employs rollers of constant linear velocity, it follows that pump tube internal diameter (i.d.) controls the pumping rate. The sampler’s probe will aspirate sample from ita container for as long as ita adjustable cam dictates. Sampling rates are typically 10-150h. with sample-to-wash ratios varying between one and nine (but special circumstances may require values out-
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J rigure 4. Mixing coil: (a)side view: (D)
end view with liquid segment
side these ranges). Thus, a pump tube flow-rated at 1.2 mL/min aspirating 60 samplesh with a sample/wash ratio of two to one would dispense an aliquot of 0.8 mL of sample per test. Between testa the system would aspirate 0.4 mL of wash liquid. When a sample prohe moves from one cup to another, there will always he some material transferred to the next cup, thus contaminating it. To limit this sample carry-over, the prohe must be washed in some manner. Ordinarily, acceptable decontamination is achieved by a simple intermediate stop in a wash vessel through which there is a continuous flow of fresh wash liquid. SamDle Reduction. When we’re dealingwith solid samples, it may he on a weight basis (e.g., 5 g of animal feed) or a per unit basis (e.g.. a single pharmaceutical tablet). If samples must be weighed, they can either be all the same (e.g., 3.5 g of powder) or an actual value can be recorded for each individual sample. The former is easier for subsequent data handling, but requires more operator involvement than simply loading an approximate weight (perhaps by means of a scoop) and noting ita actual weight. Often these weights can be accommodated by the system’s data handler, either automatically as they are measured or via operator entry prior tQ report generation. After solid samples are weighed, it is customary for them to he treated in such a manner that they can he handled as liquids throughout the remainder of the sample preparation scheme. This usually means reducing panicle size by grinding or homogenizing with an appropriate liquid, dissolution of the analyte when poasihle, and occa. sional extraction of the active ingredient into an aqueous or organic solvent. This sample reduction may be accomplished gently or vigorously, as re. 1406I
quired, by mixing, rotation, inversion, qonic or ultrasonic vibration, or shearng. It is almost always a batch pro:em, since any continuous process would likely lead to nonhomogeneous mixtures and consequent irreproducibility. A solution, suspension, or slurry From a hatch procedure, on the other hand, can generally he made as uniForm as processing time permits. Prior to, and as part of, physical deanup, the operations of mixing and lilution may he required. Mixing is lone during sample reduction, of :ourse, and must be done again if anylhing is added. In automatic discrete systems this operation is performed by stirring motors with paddles that are mechanically lowered into the reaction vessel, turned on, and eventually raised while still spinning to fling material onto the inside walls of the container and avoid contamination of the next sample. Alternatives to this approach include rotation of the vessel itself, directing a stream of air or inert gas obliquely against the liquid surface to impart a rotary motion, activation of an external magnetic field (vibratory or rotational) to move a stirring bar or hall inside the container, or even bubbling a gas through the liquid when its viscosity is not tw high. In continuous-flow systems, three mixing principles are used diffusion, bolus flow, and gravity. For nonsegmented streams diffusion is the means of homogeneity, and ita completion may he time-limited. Segmented streams have the benefit of bolus flow, and if they contain liquids of different densities and are flowing through horizontal coils, they have the additional benefit of gravity mixing. Flow is typically through a coil whose pitch and tube i.d. have been selected to spread each segment over about one-third of the coil’s circumference (Figure 4). As each segment rises and falls during ita passage through the helical coil, the repeated inversions result in the more dense liquid being above the less dense liquid half of the time, thus letting gravity help in mixing. Bolus flow is another means of mixing within a single segment, whether or not there is a density difference. It is dependent upon friction at the fluid-wall interface, and has been described in detail previously (5). Dilution and reagent addition need little elaboration. Dispensers-single syringe versions of the diluters described earlier-are usually used in discrete systems to introduce liquids downstream from the point of sample entry. In continuous-flow instruments this is handled hy additional pumps 01 additional channels on multichannel proportioning pumps. Materials added could include reagents, diluents, immiscible solvents for partition.
ANALYTCAL CkEMISTRY, VOL. 53, NO. 12. OCTOBER 1981
ing, internal standards, or buffers for pH adjustment. Concentration of an analyte is prohably required as frequently as dilution-more often in very low level work, where “trace enrichment” is a popular expression. If the analyte is volatile, then distillation or the use of a purge-and-trap concentrator is in order. When the analyte isn’t volatile, routes to ita concentration include: volume reduction via evaporation: partitioning into some smaller volume of solvent; * liquid loss via dialysis; and sorption/desorption with an appropriate resin. Physical Cleanup. Analytical chemistry may he simply defined as the process of separating an analyte from interfering excipients, then measuring the interference-free analyte. Many of these separation steps constitute the physical cleanup grouping of Figure lb. This group includes such unit operations as: centrifugation, decantation, or filtration for removal of insoluble particles; dialysis or distillation for removal of soluble excipients; evaporation as a route to concentration or solvent exchange; and separation schemes involving chromatography or liquid-liquid parttioning. Centrifugation is seldom done in automated procedures, except as a manual pretreatment (e.g., preparing serum from whole blood in most clinical analyses). When there is a substantial density difference, particles can he permitted to settle out in discrete systems or be made to fall through one leg of an appropriate fitting in continuous-flow systems. Filtration is the last resort, and can he done by hatch with self-cleaning filters (8)or continuously hy employing a moving strip of filter paper. In hoth cases, the addition of a flocculating agent is often helpful. Dialysis is a form of molecular filtration, and can he used on a continuous basis for removal of hoth macromolecules and particulates. Solutions on each side of a membrane will approach equilibrium with the other solution whether or not they are in motion. The technique is thus applicable to both discrete and continuous analyzers, and is particularly useful in obtaining particle-free solutions for HPLC. It has also been incorporated in an automated antibiotic bioassay machine where it served the dual purpose of letting dialyzable analyte reach an inoculated growth medium while preventing contamination by dehria from the fermentation broth
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(9).
When the analyte is volatile, sepa-
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Floure 5. Cartridge for automatic sample processw ration may he achieved by distillation. Although generally regarded as a hatch process, it can be performed on a continuous basis by pumping the sample through a coil which is maintained at some temperature between the boiling points of the substances to be separated. It is commonly employed in the analysis of phenols and volatile pesticides (IO). Evaporation is necessary for concentrating certain analytes, and when a sample is taken to dryness, evaporation can lead to redissolution in a second liquid to effect solvent exchange. This is required quite often in chromatography, where the best solvent for initial solution or extraction is generally not the best solvent for injection into a GC or HPLC system. It can be included in both discrete and continuous automated systems when two requirements are satisfied Sufficient energy can he directed where it is needed, and the resulting vapor can he safely and completely removed. Separation by chromatographic means may be either high- or low-resolution. The low-resolution version is frequently encountered in ASP where one substance is separated from every thing else. It is most often done as a discrete operation, employing either multiple disposable columns or a single one that is hack-flushed and/or regenerated. Sometimes the analyte is retained for elution after interfering materiale have been washed off, and 1408A
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other times the material of interest passes through the column while excipients andlor particles are retained for removal by back-flushing or washing with the appropriate solvent. One way this can be accomplished continuously, however, is by pumping the resin as a slurry from a vessel that is being stirred, allowing the resin to absorb the interfering materials, then removing theresin by continuous filtration. Roy and Buccafuri (11)have described such a scheme for the automated analysis of the vitamin calcium pantothenate. Another very popular separation scheme involves partitioning between two immiscible liquids. Whether discrete or continuous, the procedure includes mixing followed by phase separation. In the batch mode, a vessel containing the two liquids is stirred, the phases are allowed time to separate, and one of the liquids is aspirated into a probe for transfer to the next step. Partitioning is done routinely by continuous flow in much the same fashion as addition, mixing in a coil, and debuhhling prior to reading in a flowcell. Continuous phase separation requires proper selection of wall material for the phase separators, e.g., hydrophobic when the organic phase is to be retained. It is sometimes also necessary to add an appropriate wetting agent, at the correct concentration, to avoid emulsions or to break them if they are formed during mixing. Chemical Modification. Chemical modifications of the analyte (or an interferent) may be required to complete the sample preparation procedure. This is often as simple as a pH adjustment by acid, base, or buffer addition. It is common in drug analyses where such pH adjustment permits moving acidic or basic drugs between aqueous and organic phases. Or it could be as complex as precolumn derivatization reactions to provide increased selectivity in the subsequent chromatographic separation. Although chemistry may also he performed after HPLC (post-column derivatizations), it is the precolumn activity that is more likely to be part of ASP. For these reactions to he reproducible, temperature control is often required. This is particularly true when the immune reaction is employed, as in automated radioimmunoassay (RIA) or in procedures involving enzyme-labeled antibodies (ELA); each of these has been reviewed previously (12). Internal Standarda In a fully automated system, it is generally unnecessary to use internal standards as long as an external standard (reference) is analyzed at appropriate intervals, along with the samples. However, when a system for ASP produces treated samples that may not be ana-
ANALYTICAL CHEMISTRY, VOL. 53. NO. 12, OCTOBER 1981
lyzed for some period of time, and when sample concentration could change with time (due to evaporation or degradation), the incorporation of an internal standard can improve precision. Internal standards can be added to the sample cup, if both the initial sample volume and the added volume are constant. They can also be added as a component of one of the reagent lines, if two disacivantages are acceptable: First, it will be present even when the sample isn’t, thus eliminating the distinction between a zerolevel sample and no sample, and second, the farther downstream from the sample line, the more dependent is the ratio upon the constancy of all pump tubes or dispensers adding components between these two lines. Finally, internal standards can be added via a separate probe in the same arm as the one aspirating the sample; this requires a dual row of cups in the sampler, but circumvents all of the ohjections of the other methods.
Instrumentation In the “198041 Guide to Scientific Instruments” (13)there are no listings for ASP; the nearest category is “Sampling System, Injection, Automatic,” where 13 chromatography manufacturers are listed. Their offerings are limited to injectors for handling prepared samples. Similarly, the JOURNAL’S current LabGuide (7) has a category “Samplers, general,” but none that deals with ASP. Even though ASP systems p e not generally well known, the subject has been addressed for HPLC (14) and for the field of pesticide analysis (15, 16). All of the operations described in the preceding section have been automated. In this section we will consider what automated equipment is available and how it performs the various procedures. First, let’s look at some commercially available instruments designed specifically to automate sample preparation-not necessarily designed to perform the complete analysis. Commercially Available Systems. Gel permeation chromatography (GPC), first described by Stalling et al. (17)in 1972 and presented in ANALYTICAL CHEMISTRY (18) the same year, has been automated in an instrument (19) that combines three unit operations: sample definition, transport, and separation. The apparatus accommodates up to 23 samples that are defined by 5-mL Teflon loops. These samples are typically crude extracts of animal or plant tissues, soils, feeds, or water for residue analysis. After manual loading, the samples are automatically and sequentially removed and transported by a positive displacement pump to a cleanup col-
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umn where the analyte is separated from interfering substances and collected in individual containers as “chromatographically clean” samples for subsequent analysis. Typical elution solvents are MeClz/cyclohexane and tolueneiEt0Ac. Samples can be processed in 30-60 min, including time for automatic cleaning and regeneration of the column. The automation of sample transport and various combinations of addition, mixing, etc., have been described by Allington and Hansen (20).The instrument is user-programmable and moves samples in tubes around on a rectangular platform, operates pumps for fluid addition or withdrawal, controls a digital diluting dispenser, and activates a host of external devices. Up to 975 program steps (commands) may be entered to control up to 35 racks of six tubes or four plates each. Mixing of added materials is accomplished either by spinning the tube or gently rocking the rack of tubes or plates. The apparatus bas been used for peptide synthesis and enzyme studies. In contrast to the automated GPC, which uses a single column for multiple samples, another instrument (21) employs small disposable columns in its cleanup procedure. A reversible centrifuge is combined with a clever mechanism to automatically perform several standard operations on up to 12 samples simultaneously. Before operation, samples are manually loaded into cartridges containing a sample reservoir and resin bed (Figure 5). Centrifugal force is used to drive the sample through the resin bed on which the analyte is retained. After the bed has heen thoroughly washed, the spin direction is reversed to realign the cartridge over a collection vessel. Following programmed elution of the analyte, heated air can evaporate the solvent from the collection cup (optional). The time required to automatically prepare 12 samples is 16 min, with recoveries reported to ex: ceed 90% and with RSDs of 5%. Other single-use columns are available (22) and one has been evaluated (23). Whereas the above three instruments all operated as discrete devices, this last commercially available instrument (24) is based upon continuous flow. The basic modules include a sampler, proportioning pump, sample treatment cartridge, and a fraction collector. A controller is added to maintain synchronization, and an optional evaporation-to-dryness module is available when required. The sample treatment cartridge can be configured (plumbed) to accommodate any combination of such unit operations as reagent or solvent addition, mixing, heating, cooling, dialysis, partitioning, 1410A
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Flgure 6. Continuous-flow ASP system for pharmaceutical tablets
phase separation, digestion, etc. The rate of sample preparation is dependent upon the complexity of the sample treatment cartridge, but the continuous-flow nature of the operation permits a series of samples to be in various stages of cleanup. Typically, the system might run at 3&60h, with the first sample emerging after a residence time of 5-10 min. Riviera (25)confgured the system to prepare lozenges containing hexylresorcinol for GC analysis. The bydraulic system is depicted in Figure 6. Each sample consisted of five lozenges in one of the 20 cups of the solid sampler. The tablets were first ground up with water, then chloroform containing an internal standard was dispensed by an auxiliary diluent pump and the mixing was continued to partition the analyte into the organic solvent. In this particular arrangement, no attempt was made to separate the solvent from the aqueous phase. Both phases were pumped to the tray of the fraction collector, which had been prepared by inserting vials from a GC’s automatic sampler into the normally used sample cups. At the end of the run the vials were capped and placed in the GC sampler for analysis. Any aqueous phase entering the vial presented no problem, since the probe of the GC sampler descended through the upper layer and aspirated only the heavier organic layer. Standard deviations were reported to he statistically similar to those obtained with manual sample preparation. The solid sampler is such an important module (26) in ASP that it will be described in more detail. It contains a tray of 20 sample cups (plastic or glass-lined) which can hold %log of material, depending upon sample density. Once each cycle a cup tilts to allow its contents to fall through a
ANALYTICAL CHEMISTRY, VOL. 53. NO. 12, OCTOBER 1981
large glass funnel into a homogenizing vessel, all components of which are constructed of inert materials (glass, Teflon, Kel-F, ceramic). For samples other than tablets or capsules, it is sometimes necessary to position the instrument’s wash jets so that a liquid stream is directed into the cup to quantitatively transfer the sample into the funnel. Similarly, a ring of jets around the perimeter of the funnel insures that all material is flushed into the homogenizer. The cup, funnel, and jet arrangement are diagrammed in Figure 7. The operator may select aqueous or organic solvents in the range 25-125 mL for introduction with the sample. Six different grindinghomogenizing speeds are available along with such options as a heater for the vessel and an auxiliary pump for a second liquid. At the end of the operator-selectable sample reduction time, the homogenate (or solution, or slurry, or suspension) can he quickly aspirated into an external coil from which it is removed at a lower rate for further treatment or analysis. A cleaning sequence is initiated automatically between samples. Another type of ASP device is a sample acquisition system for dissolution rate analysis. It combines controlled mixing, filtration, transport, and storage with yet another featur+ timed removal of portions of the sample. Of particular interest to pharmaceutical analysts, the sample preparation part involves dissolution vessels that are manually loaded (usually with single tablets), maintained at constant temperature and stirred with precision mixers of well-defined geometry and speed (27),and solution removal and storage modules (28).The latter group perfnits the simultaneous filtration and removal of samples from all vessels at operator-specified intervals,
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Figure 7. Solid sampler’s cup-funneljet arrangement
and storage of these samples in individual coils. The coils are then emptied serially into an analytical system for qnantitation of analyte in each vessel at the time of sample removal. Multiple sampling times provide for a dissolution profile for the contents of each veasel. A final example of a commercially available instrument for ASP is based upon the purge-and-trap concentrator first described by Bellar and Lichtenberg (29).Useful for liquids containing a volatile analyte, it performs three operations: Volatile material in a sample tube is continuously purged by an inert gas (flowing through the sample) and trapped in a resin-filled tube (e.g., Tenax); the trap is heated and a carrier gas transfers the desorbed analyte to a GC column for analysis; and a trap-cleaning cycle is completed before the next purge cycle begins. Several manufacturers (30) offer this concentrator, and one (31)has an accessory which permits the unattended loading of up to 10 samples. Hybrid Systems. The following examples of instrumentation for ASP result from users either modifying commercial devices or assembling new combinations of existing modules. Hofman et al. (32)have constructed a 1412A
two-level turntable for holding Sephadex columns above sample vials in such a manner that the columns can be used repeatedly for batch cleanup )f serum samples in a competitive proein-hinding assay for thyroxine. This sample preparation system performs the following steps automatically: definition of sample by timed pumping, addition of labeled reagent, mixing via coil, transfer to column, washing excipients from column via grabity flow, addition of thyroxine-binding globulin, elution and collection of labeled sample, and three-step regeneration of +.hecolumns. A standard sampler and xoportioning pump have been comined with a special fraction collector o prepare samples at 6 0 h with 5% RSD, and it correlates well with the nanual method (r = 0.98).Prepared ramples are subsequently placed in a :amma counter interfaced with a data iandler. A very ambitious undertaking by Hormann et al. (33) involves a method !or extraction and cleanup of triazine herbicides in soil. These workers modified a solid sampler to accommodate $0-gsamples of sieved soil, and extracted the samples with a hot acetonitrilewater mixture. Flocculation of the soil colloids was achieved by pumping in a calcium chloride solution, mixing, and permitting the sediment to settle for 5 min. A portion of the clear supernatant liquid was pumped into the helix of a digestor where the 75 OC temperature evaporated most of the solvent, leaving the herbicide in the aqueous phase. A second extraction was performed by mixing the pesticide-containing water from the helix with a hexaneether solution in a coil, then separating the two phases in a U-shaped vessel. The lighter organic phase was pumped to a fraction collector where heated air drove off the solvent and left the residue for redissolution in a known volume for later transfer to a GC. A typical experiment with the automated sample cleanup system handled 34 samples (water, soil, hay, orange peel, cherries, apple leaves) along with four standards and two fortified samples, and provided RSDs of 2.9% (as compared with about 5.4% for manually prepared samples). Whereas the manual analysis permitted a maximum of 12 soil residue samples/day to be analyzed per analyst, the automated system handled six samples/h (240/week) and kept one person m u pied half-time. Marsh and co-workers (34) have designed an apparatus to prepare soil extracts for mineral-nitrogen determinations by conventional wet chemistry. Even the sample preparation part isn’t totally automated, since operator involvement is required for part of the
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12. OCTOBER lgal
weighing and reagent addition procedure. But once the 40 100-mLplastic beakers (each containing about 20 g of soil, a proportionate weight of reagent, and magnetic stirring bar) are placed in their holders on a conveyor belt, the mechanism automatically performs these operations: stirs sample and reagent for 48 min, lets it settle for 12 min, filters, and transfers filtrate to clean sample cups for later analysis via continuous flow. The authors prepared more than 4000 samples with this equipment and reduced their labor by about 60%. A final example of automated column cleanup is offered by Ramstad et al. (35).Their system converts “dirty” samples on a turntable to clean ones in a synchronized fraction collector, all operations being controlled by 11 cam-actuated switches on a 60-pwition drum timer. Samples are defined in a 5-mL loop on a six-port valve, transported via peristaltic pumping, pushed through a silica gel column for separation of analyte and interferences, and collected in clean tubes for later GC or GC/MS analysis. With appropriate valving, the flow is reversed, and a sequence of solvents flushes the retained contaminants from the column to waste and regenerates the column. The system processes one sample every 96 min, providing 10 clean samples on an overnight run. The manual procedure required 3 h per sample. Special-Purpose Modules. A hatch extraction device designed by Siggia (36)involved a howl-shaped vessel attached to a centrifuge motor and configured to permit introduction of sample and solvent followed by sequential removal of the extract and spent solution. A porous harrier on the top of the vessel, and a means of inserting various tubes for addition or aspiration of solutions, renders the apparatus useful for extraction and phase separation or for precipitation and filtration. In his Wiley award address, Coulson (37)described a similar module for the analysis of poisons in tissue samples. It automated such operations as blending, centrifugation, phase separation, and transfer. Typically the vessel accommodated a 10-g sample, 50 mL of aqueous solvent, and 100 mL of organic solvent. An extract could be prepared in about 20 min. Although these two modules required operator attention to load each sample, they nevertheless freed the operator for more demanding tasks while they performed their assigned task ASP. Bart& et al. (38)described an automated solid-liquid extraction system that added wet milling to a spinning centrifuge howl. A module for automating continuous evaporation to dryness was first
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-virtually no waiting for a usable spectra to appear. Other options allow you to employ energy or
Easy Step-BySte Decision making for easy with the EDAX PV Keys, shown above, o trol of our system software. Each key, twelve in all, is lynamically labeled providing choices for the next .unction in the analytical process; make your :hoice, depress a key, and the next series is relax l e d for your next choice.The system software IOU by the hand and literally walks you through malysis. The flicker free color display can be Iositioned and expanded or contracted via your Jse of a special set of monitor control keys. And hat's not all.
we have been pioneers in the practical dispersive x-ray analysis. And We offer more application throughout the world than anyone; we customer reports in the times a year; we hold regular seminars and training sessions throughout the world; and no one can match our service support.
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The EDAX PV9100 is an easy-to-usesystem 'or practical multi-element analysis. t offers you a wide range of programs for 2ualitative. semi-quantitative,and qualitaive analysis on samples ranging from )ulk to thin sections. rhanks to our ECON detector you can go 3s low as carbon, which includes the xitical elements of oxygen and nitrogen. ;uesswork and complex manipulations of lata for these elements are eliminated. doreover, you get almost instant peak identifi:ation thanks to our patented Dynastatic Display'
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CIRCLE 173 ON READER SERVICE CARD
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 12. OCTOBER 1981
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Chemists are in the
1
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Dialogbrings knowledge them. F m all over the WOCICI.
described by Burns (39).Organic solvents can be evaporated up to ahout 1 mL/min, and the residue is continuously redissolved in a second solvent. The modus operandi is a loop of Teflon wire wound around two pulleys and threaded through a glass evaporator tube, through which flows airor other inert gas, heated if necessary. The evaporation-to-dryness module has been incorporated into a number of analyses, one example of which is given in the last section.
There are a lot of chemists in your field around the world. They publish a lot of information that could be helpful, if only you could get your hands on it. Turn to Dialog. It extends your reach around the world. Dialog is the worlds largest online information retrieval system. It has more databases, more abstracts of articles, more references, hy far, than any other system. In seconds, Dialog puts you in touch with the latest developments in your field, and it also retrieves information going back 10 years. It places the knowledge of the world at your fingertips. And it‘s surprisingly low in cost. A search can cost as little as $5. You can search Dialog from a computer terminal in your office. Or your librarian may already be using Dialog. Write Lockheed Information Systems, Dept. 52-80AC, 3460 Hillview Avenue, Palo Alto, CA 94304. In the US. call toll-free (800) 227-1927 or (800)227-1960. In California, (800) 982-5838.
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Fully Automated Systems Here the adverb “fully” means just ‘that: These systems accept unmeasured, untreated samples and produce a printed report without operator in-
I
I
Flgure 9. Flltration device (from Refer-
ence 41, with permlssion)
CIRCLE 126 ON READER SERVICE CARD
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
tervention. By definition, they incorporate ASP. All have some limitation to sample form or size (e.g., none will accept a head of cabbage!), but rehtively Little should be required if a system is to be dubbed fully automated. The first (40) of two discrete analyzers automatically takes and measures a sample, adds one to five reagents, and makes a measurement with one or more sensors (e.g., titrametric, colorimetric, selective ion). The heart of the system is a rotary reaction cell as diagramed in Figure 8. Liquid addition is accomplished by digitally pulsing a stepper motor to achieve the positive displacement of microliter increments. The reaction vessel, which spins at 400 rpm, can accommodate up to three sensors for multiple analyses, and the microprocessor (which controls the instrument and data output) can handle up to 200 samples without attention. The second instrnment (41) automates the GPC analysis of polymers, plastics, and resins to provide molecular weight distributions. The operator need only fill up to 16 vials with weighed samples and cold solvent. The instrument then completes sample preparation by agitating, filtering, and injecting 10-500 pL (up to nine injections from each vial). Samples are treated sequentially at a spin station, a filter station, and an injection station. The filtration device, diagramed in Figure 9, provides particle-free solutions for injection. A typical overnight run would handle 16 20-min
At last. A time saving and efficient method for routine sample preparation. J.T. Baker introduces the ‘BAKER’- Lo EXTRACTION SYSTEM--a specially designed vacuum manifold capable of simultaneously processing up to 10 samples through ‘BAKER’ DISPOSABLE EXTRACTION COLUMNS.
a
Time S a v i n g - . T h i s simple method yields a 75% time reduction over conventional extraction techniques. Efficient-m.Higher, purer and drier sample recoveries are achieved a s compared to resin columns and liquid-liquid extraction. Reproducible--Uniform phase bonding and narrow particle and pore size distribution of the sorbent insure consistent extractions.
High C a p a c i t y . -‘BAKER’ DISPOSABLE EXTRACTION COLUMNS offer a Highly Surface Active Bonded Phase which accoimmodates sample volumes ranging from a few microliters to several hundred milliliters. E c o n o m i c a l . - T h e system reducers the expense of labor, glassware, solvents and ancillary equipment used in sample prepeirat ion. S a f e - - O f f e r sa 90% volume reduction in the use of hazardous solvents.
CIRCLE 31 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL.. 53, NO. 12, OCTOBER 1981
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Finally a Tunable Dve Laser that's easG to use By design, the Model 2100 DyescanTMTunable Dye Laser from EGBG Princeton Applied Research Corporation is the most convenient tunable pulsed dye laser in existence. And, you won't believe how low the price is. The system is the first ever to incorporate nitrogen pump laser, dye laser assembly, and tuning mechanism ail under microprocessor control in one cabinet. The tunlng range of 360 to 800 nanometerscan be scanned in either wavelength (nm) or energy units (cm-0. Scan limits, repetition rate (1-100 pps) and other parameters are entered via simple front panel keystrokes. The only requirements for operation of the Model 2100 Dyescan are standard electrical power and a source of nitrogen gas. No vacuum systems or pumps are needed and since the dyes are contained in standard, low volume cuvettes, changes are fast and easy. The Model ZIW Dyescan providesoutput pulses of 1 nanosecond duration and 25 kilowatts of peak power with a iinewidth of .04 nm. .I)
owe it to yomeif to fino out more about our new laser Wrlte or call today complete SpecIiIcatlons and your copy of our Dyescan brochure X G PRlhCETON APPLiED RESEARCH, P 0 Box 2565. Princeton. NJ 08540, USA (609) 452-2111; Circle 61 for information only.
lor
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Figure 10. Continuous-flow analyzer (ASP porlion) for serum theophylline (redrawn from Reference 43, with permission) analyses in triplicate. There is no single continuous-flow analyzer; the modular approach results in almost limitless combinations configured to the needs of each individual analysis. However, for purposes of illustration, an HPLC-based clinical analyzer (42) for the drug tbeophylline will he described. The system has been evaluated by Dolan et al. (43),so only the ASP portion is addressed here and in Figure 10. Untreated serum is aspirated directly into an air-segmented stream of buffer containing an internal standard. After mixing, an organic solvent is added and the analyte is partitioned into it in an extraction cartridge. During this operation, protein is precipitated and the particulates conveniently go to waste (W) with the spent aqueous phase. The heavier organic phase is resegmented with air and pumped to an evaporation-to-dryness module where a solvent exchange occurs. The analyte, now in a solvent that is compatible with the forthcoming mobile phase, is sent to the HPLC for automatic injection and analysis. (Alternately, it could be stored in a fraction collector for later analysis). It will be apparent to the reader that appropriate combinations of modules can be added to semiautomated analyzers to upgrade them to full automation. The components of Figure 10, for example, can precede al.
most anv manufacturer’s HPLC as long as a control module maintains synchronization between the sampler and the Lc‘s injection valve actuator. Such upgrading by adding ASP to existing analyzers can greatlyexpand their usefulness and efficiency.
(3) Nichols, G. D. Anal. Chem. 1981,53 (3), 489 A. (4) Kratoebvil, B.; Taylor, J. K. Anal, Chem. 1g81,53 (a), 925 A, ( 5 ) Snyder, L.; Levine, J.; Stoy, R.;Conetta,A. Anal. Chem. 1976.48 (12),942 A. (6) Betieridge, D. Anal. Chem. 1918.50
Summary ASP can be implemented as: part of fully automated systems;, an on-line addition to semiautomated instruments to upgrade them to full automation; and as off-line stand-alone configurations to simplify one aspect of an analysis without limiting the output of the hasic instrument. Nearly every operation performed by analytical chemists has yielded to automation, and the logical combination of these unit operations can greatly simplify the analyst’s task. Acknowledgment The valued input of J. Russel Gant, group leader in chromatography, and Robert Weinherger, applications chemist, is much appreciated. References 11) Burns, D. A.;Snyder, L. H.;Adler, H.J. In “Advances in Automated Analysis”: Mediad Tarmown. N.Y.. 1973:
(8) Bums, D. A.; Hansen, G . D. Ann. N.Y.
Vol. 6.
Burns, D. A. In “Laboratory Management & Automation”:Technicon Instru. rnentaCo.. Ltd.: Bashestoke. U.K.. 1979 VOl. 9,p Bu-1.
(2)
~
, c . , 1.71,
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(7) Anal. CI%em.,1980,52(10), 100,148,
resp.
Acad. Sei. 1968,153 (21,541. (9) Burns, D. A,; Williams, P. R.;Hansen, G. D. Biotechnol. Bioeng. 1969. ZZ, 1011. (10) Ott, D. E.;Friestad, H.0. J . Assoc. Off. Anal. Chem. 1977,60,218. (11) Roy R. B.;Buccafuri, A. J . Assoc. Off. Anal. dhem. 1978.61 (3), 720. (12) Burns, D. A. In “Trace Organic Analysis: A New Frontier in Analytical Chemistry”; NBS Special Publication 519, Proceedings of the Ninth Materials Re-
search Symposium; NBS:Gaithersburg, Md., 1979; pp 587400. (13) “198041 Guide to Scientific Instruments”; Am. Assoc. Ad”. Sci.; Washington, D.C., 1981. (14) Burns, D. A,; Fernandez, J. I.; Gant, J. R.;Pietzantonio, A. L.,Am. Lab. 1979, October, p 79. (15) Burns, D. A. In “Pesticide Analytical Methodology”;ACS Symposium Series No. 136;Harvey, J.; Zweig, G.,Eds.; ACS: Washington. D.C., 1980,pp 15-30. (16) Burns, D. A. In “Analytical Methods for Pesticides and Plant Growth Regulators”; Zweig, G.;Shenna, J., E&.; AMdemic Press: New York, 1980, Part I,
Vol. 11,pp 3-53. (17) Stalling, D.L.; Tindle, R.C.; Johnson, J. L. J. Assoc. Off. Anal. Chem. 1972.55, 19
(l(ij’Tindle,R.C.; Stalling, D. L.Aml. Chem. 1972,44,1968. (19) Analytical Bio Chemistry Labs Inc., Columbia, Mo., GPC 1002.
ANALYTICAL CHEMISTRY, VOL. 53. NO. 12, OCTOBER I981
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(22) For example: Sep-Pak (Waters Associates, Milford, Mans.):Extuhe (Analvtiehem International, Lawndale, Calif.i. (23) Lantz. R. K.: Eisenberg, R.9. Cfin. Chem. 1978.24 (5j.821. (24) Technicon Industrial Svstems..Tarn. . town, N.Y.: ASP. (25) Riviera, G. M. In "Advances in Automatad Anal is"; Mediad Tanytown, N.Y., 1976; rol. 2, pp 347-9. (26) Technicon Industrial System, Tarrytown, N.Y.: SOLIDprep-II. (27) For exam le Hanson Research Corp., Northridge, h f . :Dim aph (28) Technicon Industrial?yste& Tarrytown, -.: SASDRA. (29) Bellar, T. A,; Lichtenberg, J. J. Report No. EPA-670/4-74-009 (NERC, 0 " : Cincinnati. Ohio. 1974. (30) For example: Chemical Data Svstem. Oxford, Pa.;Hewlett-Packard, Aiondale, Pa.: NuTech Corp.. Durham, N.C.; Spex Industries. Mewrhea. NJ.: Tekmar Co.. matic Sampler Model ALS. (32) Hofman, L. F.: Bouley, A. M.: Barron, E. J. Clm. Chem. 1377.23 19). 1628. (33) Hormann, W. D.; FOdC&'G.; Ramsteiner, K.:Eberle, D. 0.J.Assoc. Off, Anal. Chem. 1972,55,1031. (34) Marsh, J. A. P.; Kibble-White, R.; Stent, C. J. Analyst (London) 1379,136. (35) Ramstad, T.; Mahle. N. H.; Matalon. R.Anal. Chem. 1977,49,386. (36) Siggia, S. In "Instrumental Methods of Oreanic Functional Groun Analvsis": WileG New York, 1972;p 25. (37) Coulson, D. M. J. Assoc. Off. Anal. Phon -. ._... -I97S.SX -. __, -174 .. (38) Bartels, H.; Werder, R. D.; Schurmann. W.: Arndt. R.W. J.Automatic C h i IS%, I (I), 28. (39)Burns, D. A. Res./Deu. 1977. p 22. (40)Ionies he., Watertmvn, Mass.; DigiChem 4000 oroermmable chemical analysis syskmi (41) Waters AeaoCiates Inc., Milford, Maae.: Model 15CC liquid/gel permeation chromatograph. (42) Technicon Industrial S y s k m . Tarrytown, N.Y.: FAST-LC. (43) D o h , J. W.; van der Wal, S.; Bannister, S. J.; Snyder, L. R.Clin. Chem. 1980,26 (7), 871.
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ANALYTICAL CMMISTFIY. VOL. 53, NO. 12. OCTOBER 1981
Donald Burns received his AB in chemistry from Syracuse University, his MS in organic chemistry from the University of Puget Sound in Tacoma, Wash., and his PhD in biochemistry from Purdue University. He is currently director of R&D for Technicon Industrial Systems where his research involves automation and computerization of analytical instruments.
