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Chemical Instrumentation Edited by GALEN W. W I N G , Seton Hall University, So. O r a n g e , N. J. 07079
These articles are intended to serve the readers o f ~ m JOURNAL s by calling attention lo new developments in the theory, design, or availability of chemical laboratory instrumentation, or by presenting useful insights a n d ezplanalions of topics that are of practical importance to those who use, or teach the use. of, modem instrumentation and instrumental techniques. The editor inwiles correspondence from prospeclive contributms.
LXXII. Recent Developments in Instrumentation for Liquid Chromatography (concluded) H a n s Veening, D e p a r t m e n t of Chemistry. Bucknell University. Lewisburg, PA 17837 LIQUID C H R O M A T O G R A P H S F O R CLINICAL A N D BIOCHEMICAL APPLICATIONS
is required, since the mode of operation (ion exchange) is also aqueous.
Liquid chromatographic systems are being used routinely for the separation of complex biological mixtures such as physiological fluids and water pollutants. A comprehensive paper on this subject was recently published by Scott, Chilcote, Katz and Pitt (39). T h e majority of the instruments that have been developed for this work, are high pressure ion exchange LC units which can be used for the separation and determination of amino acids, carbohydrates, organic acids, indoles, and other components in physiological fluids as well as in polluted water. T h e advantage of usine LC instead of GC in this case is t h a t t h .ample: ~ in question nrc nqueoui a n d l h c r r i ~ r en.8 .prc~alu m p l e preparation
Development of high-resolution systems for analyzing physiological fluids has been under way for a number of years. The "UV-Analyzer," developed a t the Oak Ridge National Laboratory (40) is being used routinely in medical research facilities far the separation and determination of UV-absorbing constituents in physiological fluids. This system has been further developed to increase sample handling capacity by use of parallel columns (41, 42). The flow diagram for this type of operation is shown in Figure 44. A high pressure, multiple-column sample injection valve was designed for this instrument as shown in Figure 45. A further development
Figure 44. UV Analyzer Utilizing Parallel Columns. Reprinted from (42) with permission of the Journal of Chromatographic Science.
UV Analyzer
Figure 45. High pressure, 12-port sample injection valve for the simultaneous introduction of two samples. Reprinted from ( 4 2 ) with permission of the Journal of Chromatographic Science.
has been to operate the detectors in a differential mode to allow differential eomparison between the separated constituents of two samples (42). Differences between the urine of a normal subject, versus that of a pathological subject are notable as shown in Figure 46; furthermore the effects of drug therapy can be studied by comparison of samples before and after treatment. In a new modified version of the UV Analyzer, a UV detector is placed in series with the previously described cerate oxidative monitor, thus providing greater detection capability (33). Carbohydrate Analyzer An improved, automated, high resolution anion exchange analyzer has been developed for separating and determining carbohydrates in body fluids (20). This unit is known as the "Mark I11 Carbohydrate Analyzer" and has evolved from earlier models. The carbohydrates are separated on a n anion exchange column as their barate complexes using a barate/boric acid buffer ( p H 8.5) gradient. The compounds are detected photometrically by utilizing
Figure 46. Differential chromatograms of the UV-absorbing molecular constituents in biochemical mixtures. Reprinted from (42) with permission at the Journal of Chromatographic Science.
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Figure 51. tograph.
Figure 47. Carbohydrate Analyzer Flow System. Reprinted from (20) with permission a1 Ciioicai Chemistry.
the color developed when the column effluent is mixed with phenol and sulfuric acid. Alternatively the carbohydrates can be detected by utilizing the simplified UV ~hotometric detection system described previously (31). The flow system for the Carbohydrate Analyzer is shown in Figure 47.
lndole Analyzer An LC separation system has also been designed at Oak Ridge by Chilcote and Mrachek (43) for the separation of nanogram amounts of indoles in physialogical fluids. A coupled column configuration is used in which an anion exchange column is directly connected to a cation exchange column. The indole compounds are eluted with an ammonium acetate-acetic acid buffer which is 4F in ammonia and 5.8Fin total acetate. The indoles can be detected fluorometrically using an excitation wavelength of 292 nm and an emission wavelength of 330 nm. Coupling the anion exchange column with the cation exchange column results in a much more effective separation in the front end of the chromatopram. The "indole analyzer" is now used routinely far the determination of indole compounds in urine, blood serum, and whole blood.
separated, and are then chemically converted to chramophors or fluarophors which can be detected. Descriptions of these instruments are included in the next sectionof this review.
COMMERCIAL LIOUID CHROMATOGRAPHS Aminco Aminalyzer A new instrument far picomole peptide, amino acid and protein separations utilizing fluammetric detection was recently introduced by Aminco. This LC system is specifically designed for the optimum utilization of the new fluoraphor-producing reagent Fluram (fluoreseamine) reported previously in the section on Detectors and described in the literature (34). The Aminal~zersystem, shown in Figure 48, consists of a counter-top ion exchange chromatograph utilizing a high pressure thermastatted, stainless steel column and a new ratio filter fluorometer as a detector. Fully adjustable buffer programming with up to four buffers, high pressure (3000 psi) operation, loop valve sample introduction, pressure detection and overpressure protection are provided.
Beckman Amino Acid Analyzers
Chromatec LC-5200 ilquld chroma-
121M provides completely automatic twocolumn operation using micrabore columns for rapid, high-sensitivity analyses of sample volumes as small as 20 rl. The analyzer has a 72-sample capacity and can complete up to 16 hydrolysate analyses per day. The 121M is shown in Figure 49. The Model 121 has the same automatic features as the Model 121M, except that it uses conventional columns and can complete up to 12 hydrolysates or two physiological fluid analyses per day. The Model 119 ~rovidesautomatic single-column operation a t moderate cost and can handle up to six hydrolysates or two physiological fluids per day. The Model 118 is a semiautomatic version of the Model 119, without an automatic sample injector. It has a capability of two hydrolysates or one physiological fluid per day.
Chromatec LC Units Chromatec emphasizes its modular approach to LC, as described by Bombaugh (44). For example, two pumps and two detectors can provide three operating modes: one pump can feed one detector and the other pump the other detector; or one pump can feed both detectors; or one pump can feed the second for gradient elution. Two examples of the several LC units offered by Chromatec are the LC-2200 (shown in Figure 50) and the LC-5200 (shown in Figure 511. The LC-2200 utilizes
There are four Beckman Amino Acid Analyzers. The recently introduced Model
Amino Acid Analyzers A number of manufacturers supply liquid chromatographs (amino acid analyzers) specificslly designed for performing ion exchange separations and quantification of amino acids in physiological fluids. In these units, the amino acids are first
Figure 48.
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Flgule 50. tograph.
Chromatec LC~2200q u t d chroma-
Figure 52. Chromafronix Model 3100 liquid chromatograph with UV and Ri detectors.
Figure 55. tograph.
Figure 53. Chromatronix Model 3520 liquid chromatograph.
a solvent system, a pump, an injection system, a column oven compartment and two detectors (UV and RI) connected in series. The LC-5200 includes a solvent reservoir, a Model 5000 pump, an injection system, a column mounting rack, and UV and RI detectors.
Chromatronix Models 31 00 and 3500 The Model 3100 LC is a low-cost instrument containing a 3000 psi gas-pressurized pump, a 500 X 2.1 mm unpacked column, the Model 220 W detector, and a sensitive differential RI detector. The instrument is capable of recording two wavelengths simultaneously. Several aecessaries and two choices of recorders are also available. The Model 3100 is shown in Figure 52. The Series 3500 research chramatographs are total capability instruments for analytical, preparative, i6ocratic and gradient elution. The unique advantage of these units is the ability to do preparative work (unlimited solvent volume) for both isocratic and gradient elution, yet produce high sensitivity analytical work due to low noise pumping. The Model 3500 incorporates the new Model 740 pulse free high pressure pump (previously described), a syringe or sample loop injector system, a thermostatted column compartment, a choice of UV or RI detectors, a pressure monitor, and gradient elution can also be provided via the Model 744 (690) solvent programmer. The Chromatronix Model 3520 is shown in Figure 53.
DuPont Model 840 q u l d chrama-
forced air oven option (40-140°C). The DuPont gradient accessory, multiwavelength UV detector, and RI detector can be incorporated into the instrument, and are included in the photograph of the Model 830 shown in Figure 54. DuPont also has a low-cost high performance Model 840 unit. The previously described pressurized coil pumping system is included in the instrument. Also, the basic 840 has a low dead volume injection port, a UV detector and a column mounting accessory. The interlocked valve design and integral solvent reservoir provide tor safety, simplicity and convenience. Optional accessories include a thermal jacket and a fraction collection valve. This instrument, shown in Figure 55, should be very suitable for "dedicated" analysis where cost is a major consideration.
Durrum Amino Acid Analyzer The Durrum Amina Acid Analyzer Model D-500 operates with a single column and the Moore-Stein ion exchange chromatographic technique. Operating a t buffer solution pressures up to 3000 psi, the unit can provide peptide hydrolysate analysis in 60 minutes or less; biological fluid samples can be analyzed using lithium buffers in less than 4 hours. A dedicated DEC PDP-8 digital computer controls and monitors instrument operation, in addition to integrating areas, identifying residues, and calculating output data. Unique alternating-wavelength (590/690 nm and 440/690 nm-optional) photometers provide maximal sensitivity with ahsorhanee ranges of 0.1 to 2.0. Up to 80 sample cartridges can be loaded in the instrument which operates without further operator attention. The cost per analysis is reason-
Du Pont Models 830 and 840 The Model 830 DuPont LC features a purnp (pressure 100 to 3000 psi), a reservoir of 1.3 liters, a manual fraction collector, and a thermostatically controlled
Figure 54. tograph.
DuPant Model 8 3 0 ,quid chroma-
Figure 56. Analyzer.
Durrum Model D ~ 5 0 0Amino Acid
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Figure 57 Hewlelt~Packard (Hupe+Busch) Model lOlOB liquid chromatograph.
able because buffer and ninhydrin solution flow rates are less than 10 ml/h. A fraction collector accessory is available for use in scintillation counting of radioactive residues. Data output is in the form of strip chart recording together with a teletype printout. The instrument is shown in Figure 56.
Hewlett-Packard Model 10108 The Model lOlOB high speed liquid chromatograph is based an the Hupe+ Busch UFC1000. This is a versatile unit enabling all separation modes. Dual column operation, flow and gradient programming and preparative capability can be added as direct attachments to the basic unit. The Model lOl0B is housed in three cabinets as shown in Figure 57. The left unit contains the solvent preparation and pumping system with appropriate
controls and gauges. The column compartment is located in the center. The right hand cabinet contains the recorder and electronic modules for pump control, column compartment heating, flow and solvent gradient programming and other optional accessories. Hewlett-Packard does not incorporate any given detector permanently in the 1010B; it is felt that greater versatility is attained by not limiting detector choice. Any detector chosen by the user can be mounted in the open space below the column compartment. Safety features include overpressure protection, solvent and column overheat protection, and interlock for loss of column eompartment purge gas. The sampling system of the lOl0B is a unique septumless syringe injection block, as shown in Figure 58. It allows sample introduction a t full column pressure without interrupting solvent flow. All parts are stainless steel and Teflon. A syringe is inserted to an adjustable stop and a knurled knob is tightened, causing the Teflon cylinder to form a pressure-tight sliding seal around the needle. The transverse seal is then opened, the syringe pushed forward and the injection made.
ISCO Model 1440 ISCO combines the modules (described in previous sections) into its Model 1440 LC. This is a very versatile, high performance, modular research liquid chromatograph a t a reasonably low cost. A six-port sample injection valve can operate a t 2000 psi and accepts interchangeable sample loops from 7 to 50 &I. One or two stainless
Figure 58. Hewlett-Packard septumless syringe injection block.
steel columns are easily accommodated with minimal plumbing. Columns are available from 30 to 100 em in length and from 1.5 to 3.0 mm i.d. The complete Model 1440 includes the Model 314 pump the UA-5 UV detector, an unpacked column, a sample injection valve and loop, and a 10 cm recorder. The Model 1440 is shown in Figure 59.
JEOL Amino Acid Analyzer The operation of the JLC-GAH is fully automated from sample loading, through buffer exchange, column exchange, to recording. It is possible to analyze 12 samples successfully without operator attention, and automated operation is possible to 48 hours. An IC matrix programmer combined with an auto-sampler can be included with the unit which allows 36 samples to be analyzed successively, providing
Figure 59. graph.
iSCO Model 1440 liquid chromato-
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automated operation to 600 hours. The unit incorporates the previously described buffer pump and photometric detector (UV optional). A detection reagent pump is provided for pumping the stream-split sample with ninhydrin reagent. The instrument contains two independent flow systems, each of which consists of an eight-step buffer selector and sampler. Two channel digital integrators (JLC-DK) can be added as optional accessories. The JLC-6AH is shown in Figure 60.
LKB Model 3201 Automatic Amino Acid Analyzer The LKB 3201 is a bench level instrument supplied with a wheeled table. Fluid and electronic assemblies are mounted in a steel cabinet. The unit is capable of eight complete protein hydrolwnte runs
and three physiological fluid runs in 24 hours. Septum (1 to 100 PI) or sample loop injection (1 and 0.5 ml) are provided. Five temperatures from 0 to 100°C can be selected. High pressure Milton Roy piston pumps provide for buffer and ninhydrin flow (15-145 ml/h up to 70 a t m ) Ahsorhanees of the colored reaction products between amino acids and ninhydrin are measured alternately a t 440 and 570 nm in a 15 mm cell utilizing the instrument's visible light detector. Programming of all functions of the amino acid analysis is eontmlled by a patch-board system. The instrument comes equipped with resin and a two channel pen recorder.
Micromeritics Model 7000 The Model 7000 contains the modules described previously and is shown in Figure 61. The flow system is illustrated in Figure 62. A solvent compartment for 5
Figure 60. JEOL JLC-6AH Amlno Acid Ana-
lyzer.
one-liter reservoirs is located in the top part toward the rear. The column oven compartment is toward the frant on top. The UV and RI detector modules are in the right frant portion of the instrument; F a dient elution and flow rate controls, pressure limit controls, and a digital meter providing pressure, flow rate, percent high concentration, and temperature read-out are all located on the front panel. The 5 one-liter reservoirs are individually eannected to the two solvent select valves which are located on the left front panel, and a convenient and versatile high pressure septumless injector valve allows sample plug-injections of 1, 2, 4, or 8 PI (up to 4000 psi) hy the turn of a handle. A crosssection of the Mircamerities multi-injector valve is shown in Figure 63.
Molecular Separations Model 8-500 The MSI liquid chromatograph is s lawcast, multipurpose unit specifically designed for teaching in the university and college laboratory along with dedicated quality control use. The instrument utilizes a gas-actuated, constant flow 2WO psi pump with a 230 ml solvent volume and a 59 seeand refill time. Column mounts accommodate 4 one-meter columns, and a dual cell UV detector (254 nm) is ineluded. Other detectors can be easily adaoted into the unit. Septum, stop-flaw or valve injectors are available for the unit. The chromatomaph . . is s h a m in Fieure 64. The frant panel contains all the necessary controls for gas pressure, solvent flow and detector operation.
Packard Model 8200 The hlodel 8200 emphasizes high performance, a maximum ease of operation. complete accessibility and modular construction. The various components of the instrument have been described in previous sections of this review. Two 5-liter solvent reservoirs are located a t the top of the instrument. Direct injection a t pressures up to 250 atm is accomplished without stop-flaw. Two detectors (RI and UV) may be installed and operated simultaneously. An advanced design column oven is included, and is capable of precise, digitally-set temperature control from 0 to
Figure 61. Mioomeritics Moael 7000 liquid Chromatograph.
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Figure 6 2 . system.
Micromeritics Model 7000 LC flow
99'C (+0.05"C for 24 hours). A wide choice of column and multicolumn arrangements can be accommodated. A digital gradient elution option can also be added. A huiltin p~eparativefacility is provided; a dropflap at the front of the chromatograph provides access to a selector valve used to connect vessels or automatic fraction collectors. A full 360" access is provided by a unique, swivel base. The Model 8200 is shown in Figure 65.
Perkin-Elmer Models 1220 and 1250 The Model 1220 modular liquid chromatograph utilizes dual Perkin-Elmer digital pumps (7000 psi). This instrument is expandable from a basic isocratic ambient temperature instrument to a total capability system with a controlled temperature oven (35 to 150°C + 0.lSC), a digital gradient device, and dual chromatographic operation. A 1500 psi on-column septum injector and an optional 2000 psi stainless steel loop valve, as well as optional UV or RI detectors are available. The instrument, shown in Figure 66, can also be used for gel permeation, ion exchange, and reverse phase chromatography. The Model 1250 is a self-contained bench-top instrument. A 2-liter solvent reservoir, a positive displacement pump (1000 psi), a column compartment (not heated), and UV detector are included. Optional accessories include a loop injector, a gradient assembly, and an RI detector.
Perkin-Elmer KLA-5 Amino Acid Analyzer The KLA-5, shown in Figure 67, is a fully automated amino acid analyzer. Either the Moore-Stein ion exchange or lig-
Figure 63. Micrameritics multi-injector valve (C,OSS-SeCtonl
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