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Hans Veening, Department of Chemistry, Bucknell University,. Lewisburg, PA 7 7837. DETECTORS. There have been several noteworthy de- velopments in the...
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Chemical Instrumentation Edited by GALEN W. EWING, Seton Hall University, So. Orange, N. J. 07079

These articles are intended to serve the reoders o j ~ mJOURNAL s by calling attention lo new developmals in the themy, design, or availability of chemical laboralory inslrumenlalia, 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, modern inslrumenlatia a n d instrumenlal techniques. The editor invites correspondence from prospeclive catributors.

LXXII. Recent Developments in Instrumentation for Liquid Chromatography (continued) Hans Veening, Department of Chemistry, Bucknell University, Lewisburg, PA 7 7837

DETECTORS There have been several noteworthy developments in the area of LC detectors since the subject was last reviewed in this journal in 1970 (I). LC detectors have also been reviewed more recently by Munk (9), Byrne ( 1 0 , Perry, et al. (13), Brown (15) and Polesuk and Howery (16). These reviews deal specifically with theory, performance, criteria, detectability, sensitivity and applications of various detectors. Detectors based on UV or visible light absorption and refractive index account for the majority of devices which are "tilized a t the present time. Fluorescence detectors are also rapidly gaining importance for certain specific applications. LC detectors hased on flame ionization and canductance are utilized in certain instruments; the heat of adsorption detector, however, has become less popular. Palarographic (23) and radiometric detectors (24) have been reported and evaluated by Huber, e t al., and offer wide ranges of patential application. Other detectors which have been proposed or utilized recently in-

dude argon ionization (25), scintillation (26), and coulometry (27). Special emphasis will be placed in the present discussion on new or improved detectors which have been reported recently. A unique concept which will be covered deals with the emergence of "reaction" detectors, i.e., mixing the column effluent with a reagent stream prior to the detector for the purpose of developing a chromophor or fluorophor. Harmon and Folt have reported the application of multidetectors (refractometry, infrared, and W) to the analysis of polymeric materials by steric exclusion (28). Takata has evaluated the application of a coulometric detector to rapid ion exchange chromatography of copper, zinc, nickel, lead, cobalt and cadmium (27). A constant potential flow coulometer was designed and utilized. Lyons has reported a liquid chromatographic detector used as a pollution monitor and a total organic carbon analyzer (29). A new type of detector hased on the volume change accompanying the sorption and desorptian of ions an a strip of ion exchange membrane has been reported by Gilbert and Dobhs (30). A DuPont Model 941 Thermomechanical Analyzer was used in conjunction with a Model 900 Thermal Analyzer to measure changes in linear dimension of the membrane. The device was evaluated for the detection of alkali metals and inorganic anions separated by ion exchange chromatography.

UV detector far monitoring carbohydrates in body fluids has been developed by Katz and Thacker (31). This system depends upon the rapid production of ehramophores with absorption maxima in the 290-310 nm region by dynamic mixing of column eluent with sulfuric acid. The unit was used with the "Carbohydrate Analyzer" (20). In order to provide better discrimination from other constituents in body fluids which absorb strongly below 300 nm, this UV photometer was provided with an analytical channel at 306 nm rather than the 296 nm optimum wavelength, due to the availability of a suitable phosphor. The 254 nm channel was retained. The analytical channel consisted of a phosphor-rod light source (LKB, 839002). a quartz flow cell with a 0.27 cm optical path, a Corning 7-54 filter, a Sylvania phosphor type 2382 and a photoconductor. The phosphor rod is activated by 254 nm energy from a mercury lamp and emits the broad spectrum shown in Figure 21. The UV portion passes through the filter and is absorbed by the Sylvania phosphor with the efficiency also shown in Figure 21. A maximum at 306 nm is thus produced. The dual wavelength UV photometer is shown in Figure 22. This detector was found to be far more sensitive for monitoring carbohydrates than the earlier detector which operated a t 254 nm only. A new oxidative-fluorescence LC detectian system hased on the production of cerium(II1) fluorescence has been reported by Katz and Pitt (32) and has been evaluated for monitoring aromatic acids by Katz, Pitt and Jones (33). In this method the column effluent is mixed with M Ce(1V) dissolved in 1M sulfuric acid. The mixed stream is then passed through a heated reaction bath and then to the fluorometer where the fluorescence of cerium(ll1) is monitored. The flow system is shown in Figure 23. An "annular mixer" was designed and used effectively to meter the two streams. Column effluent comes in through the inner channel while reagent enters through the larger of the two concentric tubes in the mixer as shown in Figure 24. Efficient metering was achieved with this design. This reaction detector is very versatile and responds to any materials which are oxidizable by Ce(IV) under the given conditions. The method has been

Reaction Detectors

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Figure 21. Combination of Emission and Absorption Spectra Utilized in UV Detection System. Reprinted from ( 3 1 ) . with permission of the Journal of Chromatography.

The concept of forming a reaction product immediately after column elution and prior to the detector has been used effectively in a number of specific biochemical LC applications. This particular technique can oecasionslly he troublesome because of the flow fluctuations produced when mixed streams are involved. A simple and sensitive dual wavelength

Figure 22. Two Wavelength Ultraviolet Photomster Head. Reprinted from ( 3 1 ) . with permission of the Journalof Chromatography.

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Chemical Instrumentation

Figure 23. Flow System for Cerate Oxidative Fluorescence Detection System. Reprinted from (32).with permission of Analytical Letters.

reported to he 103 times more sensitive for aromatic acids than previously reported methods, and was successfully used far monitoring aromatic acids in urine (33). A new reagent known as "fluorescamine" (or "Fluram") has been reported by Udenfriend, et al. for the fluorescence monitoring of amines and amino acids separated by ion exchange chromatagraphy (34). Fluram is non-fluorescent, but reacts very rapidly with primary amines to farm highly fluorescent fluoraphors (390 nm excitation, 475 nm emission) s t pH 9, while excess reagent is destroyed. An aeetone solution of fluorescamine is mixed with the aqueous column effluent prior t o the fluorescence detector and the resulting fluorophors are then monitored with a flow fluorimeter. This method of detection promises to he far more sensitive than the well-known ninhydrin detection technique, and has been incorporated into Aminco's Aminalyzer (see later section an LC units). A description of new and/or improved commercially available detectors now follows.

(shown in Figure 25). It is a single beam unit adjustable from 210 to 700 nm. The detector has deuterium and tungsten sources and uses a grating monochromatar; it is also supplied with low volume flow cells. I t is thus possible to plot a spectrum for a component when the flow is stopped. Chromatee also manufactures a two module unitized UV detector which operates a t 254 and 280 nm (singly or simultaneously). The cell for this unit is 10 mm in path length and 8 rrl in volume. Output is in ahsorbanee units, and the detector is easy to calibrate. An external control enables one to place a known absorbance in the optical path. The refractometer in the Chromatec LC units is manufactured by LDC.

Chromatronix UV Detectors The Model 220 UV Detector is a dual wavelength detector with a single channel recorder output. The Model 230 is a dual wavelength deteetor with dual channel recorder outputs for simultaneous monitaring a t different wavelengths. The two models are otherwise identical. A unique "mixed wavelength" system of optics allaws 254, 280, 254-280 and 280-254 nm operation. This is done by having the cells themselves acting as apertures which separate the 254 and 280 nm radiation, directine each to different ohotoeells. The ~ o d e i 2 3 is 0 shown in Figure 26.

Aminco Fluoro-Microphotometer This instrument can serve both as a filter fluorometer and as a calorimeter. It is a compact, integrated, solid state unit with three ranges of blank subtraction, allowing suppression of a light signal up to ten times full scale so that small changes in signal can he measured with increased precision. The voltage supplied to the photomultiplier is fixed s t 700 V and has two stages of regulation: 1) a continuous sensitivity adjustment, and 2) seven fixed steps of 100, 30, 10, 3, 1, 0.3 and 0.1. Three readout scales are provided: relative intensity of 0-10090 and 0-32% and absorbance, 0-2 AU. A recorder outlet for a 50 mV recorder is provided. Aminco offers a complete line of flaw cells for using the instrument as a fluorescence and colorimetric detector.

Chromatec UV Detectors Chromatec has recently come out with its new tunable Model 800 UV detector

Figure 25. Chromatec Modal 800 Tunable UV Detector. Figure 24. Annular Mixer.

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Figure 26. Chromatronix Model 230 Dual Channel UV Detector.

Chromatronix also offers the Model 200 UV detector which is sold separately and is an optional possibility for all their chromatographs. The Model 200 is a single wavelength (254 nm) instrument employing a chopped beam for high stability.

DuPont Multiwavelength Detector The Model 835 is a filter photometer, which is an outgrowth of the DuPont 400 process stream analyzer, the 410 photometer and the newer single wavelength detector. This detector does not utilize phosphors to generate additional UV wavelengths. The unit provides energy a t 254 nm with a low pressure mercury source and also a t 280, 313, 334, and 365 nm with a medium pressure mercury source. Selected wavelengths between 380 and 650 nm are available with a quartz-iodine source. Absorbance ranges between 0.01 and 2.56 AU are provided. Short term noise is +5 X A U with the low pressure source, and +1 X lo-* A U with the other source lamps. The cell is 8 rrl in volume, 1 mm in diameter with a path length of 8 mm. A 24 pl cell is standard with the medium pressure mercury and quartz-iodine lamps. The DuPont multiwavelength detector is shown in Figure 21. DuPant also has its single wavelength UV and refractometric detectors available. These detectors have been described previously ( I ) .

Gilson Recording UV Spectrophotometer This is a simplified UV recording spectmphatameter, which uses a deuterium light source and a precisian grating monochromator to provide continuous wavelength adjustment from 215 to 310 nm (other optional ranges between 200 and 400 nm are also available). Two types of cuvets are available: one is a blown

Figure 27. DuPont Multiwavelength Detector

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Chemical Instrumentation quartz-cuvet (150 pl, 10 mm path) and the other is Kel-F with quartz windows (32 pl, 10 mm path). The main cahinet also cantains a 200 mm wide servo recorder, an event marker, a chart drive, a power supply and an amplifier. A gel scanning attachment for this UV monitor is also available from Gilson. The Gilson spectrophotometric UV detector is shown in Figure 28.

Gow-Mac Christiansen Effect Detector (CED). This unique detector was recently announced by Gow-Mac a t the 1973 Pittsburgh Conference. The CED utilizes the developments described in 1884 by Christiansen in his work with crystal filters (35).A sample cell, provided with an inlet and outlet connection, is packed with a solid having the same refractive index as the moving phase eluting from the column. Visible light is transmitted through the cell as long as the refractive index of the solid and liquid remain the same. When a sample is eluted from the column and carried through the cell, the refractive index changes and no longer matches that of the solid. This is indicated by a change in the amount of light transmitted and is measured by the photodetectors. The optical schematic is shown in Figure 29. If ~ o l ~ c h r o m a tenergy ic is employed, the central part of the transmitted beam will exhibit the color corresponding to the wavelength a t which both substances have the same refractive index. The remaining wavelengths will be refracted in other directions causing a "halo" effect. When a sample appears in the liquid stream, its refractive index changes and the wavelength in the center of the beam shifts, causing a change which can he measured by photodeteetors.

Figure 29. Gow-Mac Christiansen Effect Detector (optical schematic)

Gow-Mac has developed a wide range of solids (1.3 to 1.6 RI) to match the various solvents used in LC. Minimum detectable RI difference is 10-6 RI units. The deteetor should have a wide range of applications in isoeratic LC. The detector optics are shown in Figure 30 and the CED modules in Figure 31.

ISCO UA-5 Absorbance Monitor ISCO has recently made its new UA-5 absorbance monitor available. This is a high performance detector whieh is a great deal more sensitive than the previous Model UA-4. This dual beam detector offers 254-280 nm operation (with other wavelengths available), eight absorbance ranges from 0.01 to 2.0 full scale, and a typical noise level of +0.00005. This unit can be supplied with a built-in 10 cm recorder which has 8 chart speeds. Flow cells are pressure-resistant to 2000 psi with 19 pl volume per em of optical path. The UA-5 monitor is shown in Figure 32.

JEOL Photometric Detector The JEOL Amino Acid Analyzer comes equipped with a high sensitivity visible detector consisting of two sets of double beam optical systems a t 440 and 570 nm. Detection sensitivity for amino acids is listed a t 1 X male (10 mm cell) and better than 5 x mole (2 mm cell). Measurement ranges are 0-100 %T ( X I ) ; 70-100 %T (X3); and 90-100 %T (X10).

Laboratory Data Control (LDC) Detectors LDC presently markets the Model 122 dual wavelength UV detector. The duoMonitor is a high sensitivity microflow cell detector for simultaneously and differentially monitoring one or two streams a t 254 and 280 nm. The detector incorporates a low pressure mercury lamp which illuminates a screen partially coated with a phosphor capable of converting 254 nm radiation to 280 nm. The screen thus becomes a composite light source radiating a t both wavelengths in different areas. The dual flow cell has two slot shaped absorption chambers sealed by lens windows. Each of the two wavelengths are separated into two beams (one for each cell chamber), and by means of a unique optical system,

Figure 28. Gilson Specfrophotometric UV Detector.

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Figure 30. Gow-Mac Christiansen Effect Detector Optical Componentr.

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Figure 33. LDC duoMonitar.

Figure 31. Gow-Mac Christiansen Effect Detector.

the two completely separated beams are then sensed by dual photodetectors. The unique feature of this detector is that while the two wavelengths are completely separated after having passed through the flow cell, they are, nonetheless, " m i x e d within the flow eell chambers. As b result, measurements a t the two wavelengths are truly simultaneous-in the same cell a t the same time. An additional optical system is provided with this detector, enabling the operator to view a magnified image of the flow cell to inspect it for dirt, bubbles, etc., while the unit is operating. The dual flow cell has a 3 mm path and a volume of 25 @I. Readout is in absorbance a t 254 and 280 nm in six binary ranges (0.02 to 0.64 AU) plus a non-linear range to 1.28 AU. Minimum detectable absorbance is 0.0002 AU. A photograph of the duoMonitar is shown in Fieure 33. L ~ also C offers a low cost Model 1520 LuvManitor, which contains a built-in, 5 inch, single chart speed recorder. It monitors a t 254 or 280 nm, readout is in absorbance units, and the eell is 25 in volume with a 3 mm path length. The unit has been expressly designed for gel permeation, column electrophoresis, and preparative work. The LDC Model 701 conductoMonitor is a canductance detector which can be used for monitoring solution conductance in a differential or absolute mode. The instrument provides direct read-out in specific conductance units and can measure differences as small as 0.01% (in the differential mode) between the carrier and carrier plus solute. The linear dynamic range in the absolute mode varies from 0.01 to 100,000 @mha/cm and the instrument will function with samples varying from distilled water to concentrated salt solutions without having to change cells. The cell volume is 2.5 pl and the cell constant is 20 cm-1 (nominal). Automatic thermistor temperature compensation is also provided. The

Figure 32. ISCO Model UA-5 Absorbance Monitor.

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instrument, shown in Figure 34, is.packaged in two separate modules: the electrical control unit and the measuring cell assembly. The conductoMonitor is especially useful in monitoring ion exchange columns and measuring buffer gradient profiles, in the detection of non-UV absorbing ionic surfactants using HzO-methanol gradients, and as a chromatographic detector for metal ions. LDC also supplies its well known uvMonitor (280, 350, 440 and 550 nm), refractohlonitor, and fluorohlanitor. These units have been described previously (1).

LKB Uvicord Ill In addition to the Uvieords I and 11, described previously (I), LKB now offers a new Uvicord I11 with dual light sources, providing simultaneous sample and reference measurements a t two different wavelengths. Filters select the required wavelengths from each beam and a continuously rotating mirror system causes light from each source to be alternately directed to sample and reference cells. One measuring cycle therefore consists of four measurements and takes four seconds. Another unique feature is automatic scale expansion, which ensures that the recorder will not go off scale, even when the output signal from the Uvicord varies greatly. A wide choice of UV and visible wavelengths are available. Several 0.1 ml cells with optical paths of 0.5 to 5 mm are available. Mieracells which are 10 in volume with a 10 mm optical path are also offered. The Uvieard I11 is shown in Figure 35.

Micromeritics UV Detector Micromeritics manufactures its own high pressure, high sensitivity UV detector and offers a refractive index detector for the Model 7000 LC. The UV detector, which operates a t 254 nm, is a dual beam unit which continuously calculates the log of the intensity of the reference beam and subtracts this from the log of the intensity of the sample heam. Flow cells are constructed of stainless steel with sapphire windows. The cells are 8 @Iin volume with

Figure 34. LDC conductoMonitor.

transformation system in contrast to the previous pyrolysis method. The linear range of the detector is better than three orders of concentration, with a detection limit for many organic compounds of hetter than 1 pg/ml. The detector responds to all organic compounds which can be burned to COz. A special advantage is that the response of the detector is independent of the solvent used because it is evaporated prior to sample combustion. This detector should therefore be particularly useful for gradient elution. The detector module is shown in Figure 37. Figure35. LKB Uvicord I l l .

a light path of 10 mm; they are pressure resistant to 2500 psi. Drift is 0.0004 AU; noise is 0.0002 AU.

Packard Detectors Two high performance detectors may be installed and operated simultaneously in the Packard Model 8200 LC. The refractometer provides a sensitivity of greater than lo-' RI units with a wide linear dynamic range and a cell volume of 8 pl. The UV detector offers dual wavelength (254 and 280 nm) operation and absorbance ranges of 0.01 to 2.56, typical peak to peak noise levels of 0.0002 AU and an optical path of 10 mm.

Schoeffel Yodel 770 Spectrotlow Monitor This' is a new low-cost UV-VIS cantinuously variable wavelength LC detector which was recently introduced by Sehoeffel. The unit has two light sources: deute-

rium fur the wavelength range 200 to 400 nm and tungsten for 350 to 630 nm. The UV-VIS monochramstor features a digital wavelength selector and provides a band width of 5 nm. The instrument is calibrated in absorbance units over 8 full ranges from 0.01 to 2.0 AU and has a typical sta(Continued onpage A490)

Perkin-Elmer Detectors Perkin-Elmer provides a UV and RI detector for its liquid chromatographs. These are the former Nester/Faust detectors and have been described previously (1). The Model 250 W operates a t 254, 366, 480, 546, and 600 nm. Cells are 12 p1 in volume with a 10 mm path length. The measuring range extends from 0.01 to 0.50 AU. The RI detector (LDC) is capable of detecting differences as small as 2 x 10-7 RI units. Double prisms are utilized to monitor the entire RI range from 1.320 to 1.520.

Pye Unicam Phase Transformation Detector The Pye LCM2 liquid chromatograph incorporates the phase transformation (moving wire) detector. The transport principle of the detector is well known and is illustrated in Figure 36. This, however, is an improved version of the moving wire system (36) in that the sample which is coated on the wire is no longer pyrolyzed hut is burned in excess air to form carbon dioxide. A molecular entrainer, using a hydrogen-nitrogen mixture draws COz from the oxidizer oven to be mired with hydrogen and passes it over a nickel catalyst which reduces COz to CHn. The methane is then detected by a flame ionization detector. Far greater sensitivity can be realized with the described phase

Figure 36. Pye Unicam Transport wire) Principle.

(moving

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Figure 40. Varian Stainless Steel Flaw Cell.

est, such as the solvent. Carr (37) has reported the advantages of multiwavelength detection using the Varian system, and Munk (38) has evaluated the performance and characteristics of the Techtron 635 fitted with high pressure, thermostatted cells (volume 8 pl, path length 10 mm). T h e cells are made of stainless steel, fused silica a n d FEP Teflon. A eut-away view of the cell showing the liquid passage through it is shown in Figure 40. Liquid enters the cell through the left channel, flushes the left window and passes along the 1 m m i.d., 1 cm length bore, flushes the right window and exits on the right. The internal geometry is the same as the Varian 254 nm single wavelength detector described previously ( I ) . T h e cells are also water jacketted as shown in Figure 41 in order to reduce their sensitivity to flow. T h e reason for this decreased flaw sensitivity is temperature equilibration of the incoming solvent with t h e cell. The Techtron 635 multiwavelength detector has four linear ranges in absorbance with sensitivities of 0.1, 0.5, 1.0 and 2.0 AU full scale on a 10 mV recorder. The high transmission dual chopper optics, dark current correction a n d matched sample and reference beams of the Varian Techtron 635 suit it well for use with the type of high performance microflow cells which are standard in high pressure LC. Varian also nates that it ie possible to stop the flow through the column when a component is in the cell, thus enabling the operator to record an absorption spectrum. T h e Varian Techtron 635 sample compartment and microflow cell assembly are shown in Figure 42. Detailed descriptions of the Varian refractive index, and single wavelength UV detectors have been reported previously (I).

Waters R I and UV Detectors Waters manufactures its R-400 Series differential refractometers (I). They are based on a unique, optical deflection de-

Figure 41. Varian Flow Cell Block with Water Jacket Cover Removed.

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~ i g m 43. Waters R-401 D ~ l f e r e n t i a l Refractometer.

uid chromatographs is the LDC uvMonitor described previously ( I ) . Figure 42. Varian Techtron 635 Microflow Cell Assembly.

sign rather than light reflection. The optical deflection configuration permits the use of one sample cell throughout the entire refractive index range from 1.00 to 1.75. Higher sensitivity coupled with greater stability in the presence of dirt or air bubbles are inherent in the deflection design in comparison to the light reflection design. Additional advantages of the deflection design include a wider linear dynamic range for analytical and preparative scale work. Stability a t flow rates as high as 20 ml/rnin and the ability to monitor refractive index changes in intensely colored solutions are also prnvided by the R-400differential refractometer. Waters provides the R-401 (optimized for high-speed LC), the R-403 (optimized for gravity feed columns), and the R-404 (far preparative LC). The R-401 is shown in Figure 43. The UV detector utilized in Waters' liq-

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REFERENCES 1231 Kcen. J . G.. H u k r . .I. F. K.. Popoe, H. and don Boef.G..J Chrnmotoe. S r i . 4,192l19701. (241 van Urk-Schoen. A. M. and Huher. J. F. K.. A n d Chim. A d a . 52,519l19701. 1251 Maggs, R. J.. Chromalogrophio, I . 4 3 I l S W . (261 M c G u i n n e ~ .E. T. and Cullen. M. C.. J. Chrm. Edur 47. A9 119701. (271 Takats, Y., Paper No. 272. Piftshurgh Conference. Cleudand. Ohio, March 1973. 1281 H8rmon.D. J.andFolt, Y. L.. PaperNo.202, ibid. (291 Lyons. J. G.. Eighth International SymposiumAdvances in Chmmatogsphy. Tomnto. April,

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m i a l h & T. W.. and Dahbs. R. A.,Anohtical Chem.. 45. 1- 119731. 1311 ~ a f z .s.. and Thaeker. L. H.. J Chromofoe.. 64, 247 (19721. 1321 Katz, S. and Pitt, W. W.. JI., A n d Left. 5, 177 ,>WV

(331 Katz. S.. Pitt. W. W.. .Jr., and Jones, G.. Jr.. Clinical ?hem. 19.817 119731. (341 Udcnfriend. S., o t al.. Science 178,871 119721. I351 Chrirfianwn, C . . A n n P h y ~Cham., 23.298 118841. I361 Scott. R. P. W, and Lawrence. J . G.. J. Chromotop. Sci 8. 65119701. (371 c s T r , . k . D.. Paper No. 270. Pittsburgh Conference. Cleveland. Ohio. March. 1973. 1381 Munk, M. N., Paper No.271. ibid