A High-PerformanceUltraviolet Photometric Detector for Use with Efficient Liquid Chromatographic Columns J. J. Kirkland Industrial & Biochemicals Department, E. I . du Pont de Nemours & Co., Inc., Experimental Station, Wilmington, Del.
A high-performance ultraviolet absorption detector has been developed to monitor efficient liquid chromatographic columns separating UV-absorbing compounds. The detector consists of a modified commercial photometric analyzer fitted with 1-cm path length, flowthrough absorption cells with internal volumes as small as 7.5 pl, With solvent flowing, the detector is capable of operating at 254 mp with a full-scale sensitivity of 0.01 absorbance unit; short-term noise is less than 3t0.0002 absorbance. Maximum drift over a 12-hour period is 0.001 absorbance without control of ambient temperature changes. The detector can be adapted for use at other wavelengths. While the sensitivity of the detector i s dependent on sample type, chromatographic peaks containing nanogram amounts of compounds of moderate absorptivity can easily be observed. The range of linearity of the detector is comparable to a thermal conductivity detector for gas chromatography, the upper limit of linearity being 1.2-2.0 absorbance, depending on the dimensions of the micro cell employed. The high sensitivity and low dead volume of the detector permits the use of efficient liquid chromatographic columns with small internal diameters.
IN RECENT liquid chromatographic studies it has been demonstrated that ultraviolet absorption provides a convenient and sensitive means of continuously monitoring UV-absorbing compounds eluting from columns (I-6). This paper describes the application of a modified commercial process-type photometric analyzer as a high-performance detector for liquid chromatography. The key advantages of this device are its unusually high sensitivity, stability, and wide range of linearity. The detector incorporates a low-volume sample cell which permits its use with efficient, small-bore analytical columns. Chromatographic peaks containing nanogram amounts of moderate-absorptivity compounds can readily be observed with the detector, and its range of linearity is roughly comparable with thermal conductivity detectors for gas chromatography. Hamilton has used another version of the photometer herein discussed to monitor amino acids eluting from ion exchange columns after these compounds are converted to colored components by reaction with ninhydrin (7). The stability, linearity, and sensitivity of the ultraviolet detector herein described closely approximates that of the Hamilton device which was used for measurements in the visible light region.
(1) F. Alderweireldt, J . Chromatog., 5,98 (1961). (2) S. Lovett and W. C. Wright, Chem. Ind. (London), Sept. 9, 1433 (1961). (3) E. Gordy, P. Hasenpusch, and G . Sieber, Anal. Biochem., 11, 377 (1965). (4) C . G. Horvath and S. R. Lipsky, Nature, 211, 748 (1966). (5) S. P. Keleman and E. T. Degens, Ibid., 211,857 (1966). (6) R. E. Jentoft and T. H. Guow, 19th Annual ACS Summer Symposium on Analytical Chemistry, Edmonton, Canada, June 23, 1966. (7) P. B. Hamilton, Reu. Sci. Znstr., 38, 1301 (1967).
EXPERIMENTAL
Ultraviolet Photometric Detector. The detector used throughout this study incorporated a modified Du Pont 400 Double-Beam Photometric Analyzer, based on the design of Glasser, Kanzler, and Troy (8). The device is represented schematically by the diagram in Figure 1. Radiation from a low-pressure mercury tube source is collimated by a lens and passes through a filter which only transmits radiation from the 254-mp mercury line. The energy passing through the filter is divided into two beams ( A ) and ( B ) by a semitransparent mirror set at a 45" angle to the direction of radiation. Ninety per cent of the energy passes through the mirror to form the sample beam ( A ) . Ten per cent of the energy is reflected off the mirror to form beam (B). This beam is reflected off another 45" mirror to form the reference beam (C). Reference beam (C) passes through a reference cell containing the carrier phase. The sample beam ( A ) is focused by a lens onto the micro flow-through cell containing the sample. As the energy emerges from the sample cell, it is focused by another lens. The radiation transmitted through each cell passes through additional optical filters to minimize stray light and then strikes phototube ( D ) or (E). The light intensity reaching phototube ( E ) is normally constant because it is passed through a solvent reference solution. Light intensity changes reaching phototube ( D ) creates a voltage output change which relates to the absorbance changes in the sample cell. Voltage output differences are obtained in the following manner: Each phototube develops a current output proportional to the light intensity striking the phototube. The logarithmic amplifier associated with each phototube produces a voltage proportional to the negative logarithm of the phototube current. The signal output from the control station is a portion of the difference of the voltages produced by the logarithmic amplifiers. The difference in logarithms is directly proportional to the sample concentration changes occurring in the sample cell. The use of the logarithmic amplifier provides an output voltage which varies linearly with sample concentration. A significant amount of energy in the sample beam is vignetted by the small aperture of the sample cell; consequently, the larger fraction of the available energy is directed through the sample section of the photometer. This arrangement provides maximum amount of energy for the sample beam while still allowing sufficient energy for the reference circuit to work properly. Quartz focusing lenses are used in the sample beam to ensure that the maximum amount of energy passes through the small sample cell opening. Detector Cells. Two sample cells based on the same design but of different internal diameters and volumes were used during the study. Both were constructed by drilling out a Teflon (Du Pont) block for the necessary flow channels and fitting the end of the block with quartz windows held in place by flanges bolted onto the outside. A schematic of the cell design is shown in Figure 2. The dimensions of the cavities for the two cells were 1.5-mm i.d. for 10-mm and
(8) L. G. Glasser, R. J. Kanzler, and D. J. Troy, Ibid., 33, 1062 (1962). VOL. 40, NO. 2, FEBRUARY 1968
391
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Figure 1. Schematic of the dual-beam UV photometric detector Figure 2. 0.79-mm i.d. by 10 mm. Total volume of these cells, including a 10-cm length of 0.02-inch inlet tubing, were 20 and 7.5 pl, respectively. A reference cell of similar design was constructed with a 5-mm i.d. by IO-cm cavity. This reference cell can be operated in a static or flowing mode, depending on requirements. Chromatographic System. A schematic of the apparatus developed for liquid chromatography is shown in Figure 3. The equipment consists of a mobile-phase reservoir [l], an infrared lamp [2], and Teflon stirring bar driven by a magnetic stirrer [3]. The carrier passes into a pump [4], which produces the flow desired for the chromatographic separation. The output pressure of the pump is monitored by means of a diaphragm-type pressure gauge [ 5 ] . The carrier from the pump passes through a 5-micron sintered metal filter [6] and is then delivered into a 5-meter length of 0.02-inch i.d. stainless steel capillary tubing [7]. This restriction, when coupled with the volume in the diaphragm gauge, effectively forms a surge-damping network to reduce the pulsating output of the pump. The flow then passes through a precolumn [8] containing the same packing as the chromatographic column. This precolumn ensures proper equilibration of the moving phase with the chromatographic stationary liquid phase. The pressure at the input of the chromatographic column [9] is monitored by means of a strain gauge-type transducer [lo]. The sample is introduced into the top of the column by means of a low-volume inlet [ll]. The sample is forced through the column by the pressure of the carrier solvent, the column and the precolumn both being thermostated by means of water jackets [12] to ensure constant temperature control. Chromatographic peaks leaving the column pass through the UV detector [13] and are presented on the recorder [14]. To provide maximum versatility, the liquid chromatographic system is designed to operate at pressures up to 1000 psi. Fittings and pump packings incorporated in the apparatus permit the satisfactory use of low-viscosity organic solvents such as chloroform, acetonitrile, hexane, etc. Carrier solvent and sample are exposed only to glass, Teflon, stainless steel, and a small area of synthetic rubber diaphragm in the sample inlet. A more detailed description of some of the more important items of the apparatus follows. CHROMATOGRAPHIC COLUMNS.Columns were constructed of 1.1-cm 0.d. glass pipe made from borosilicate glass "Trubore" precision-bore tubing of approximately 3 mm i.d. End connections were made from Corning pipe flanges grooved on the ends for Teflon O-rings. 392
ANALYTICAL CHEMISTRY
Micro flow-through sample cell
Figure 3. Schematic of liquid chromatography apparatus Inlet and exit inserts for the glass columns were fabricated from Teflon and designed with very low dead volumes to ensure that samples can be introduced and eluted with minimum mixing effects. Columns were provided with removable glass jackets so that the temperature can be maintained as desired by circulating water supplied from a constanttemperature bath. Liquid-liquid chromatographic columns were packed using techniques not greatly unlike those employed for the preparation of high-quality gas chromatographic columns. The lower end of the glass column was fitted with a small disk of porous Teflon (Fluoro-Plastics, Inc., Philadelphia, Pa.) (9). Small portions of the dry packing were placed in the tube and the bottom of the column tapped on the bench between additions, The side of the column was also gently rapped with the wooden handle of a spatula during the packing process. After filling the column, the top was then plugged with a small wad of quartz wool. The column packing was prepared by wetting the support with a solution of p,p'-oxydipropionitrile in dichloromethane. The solvent was removed by evaporation while gently stirring the mixture under a stream of dry nitrogen. After preparing the column, the carrier liquid was introduced and air from the packing was displaced by the carrier. To assure equilibrium between the carrier and the stationary liquid, a (9) P. B. Hamilton, Alfred I. Du Pont Institute, Wilmington, Del., personal communication, 1966.
CARRIER FLOW-0.26 cc / min.
Figure 4. Stability of UV detector Sensitivity4.01 absorbance, full scale 0001 As
f precolumn containing the same material was placed ahead of of the chromatographic column. An elastomer septum inlet has proved to be useful for many of the chromatographic systems tested. With this device samples can be injected through the septum directly into the top of the column packing. The system is designed so that the needle is guided into the center of the column in a manner similar to that recently described by Scott, Blackburn, and Wilkins (IO). The septum inlet normally is preferred for use at lower column input pressures, although satisfactory injections have been carried out at input pressures up to 900 psi. An alternate sampling system was designed for continuous use at higher pressures. With this inlet it is necessary to interrupt the carrier flow and disconnect a metal cap on the top of the inlet system connected to the column so that the sample can be injected. When using both sampling inlets, it is desirable to inject the sample directly into the top of the column packing for minimum band spreading. SOLVENT RESERVOIR.This apparatus consists of a heated 500-ml glass reservoir attached to a three-bulb Allihn condenser. A glass tube is inserted through the condenser down into the reservoir so that a blanket of dry nitrogen can be maintained over the solvent contained in the reservoir. The solvent in the reservoir is heated with a 150-watt heat lamp, and a magnetic stirrer and stirring bar are used to agitate the solvent slowly. A Model T-350 Proportional Temperature Controller (T&T Controls Co., Media, Pa.) was used to control the temperature of the solvent reservoir. The controller is actuated by a sensor placed in a well which extends into the solvent reservoir. The temperature of the solvent reservoir was monitored by means of a “Sim-Ply-Trol Pyrometer,” 0”-500O C (Assembly Products Co., Chesterland, (%io). This pyrometer was actuated by means of an ironconstantan thermocouple placed in the solvent reservoir well. Spectrograde solvents obtained from Matheson, Coleman, and Bell were employed throughout in this work. CHROMATOGRAPHIC PUMP. A Model 196-32 Simplex Instrument Minipump (Milton Roy Co., St. Petersburg, Fla.), driven by a 0.35-horsepower, 32.2-rpm synchronous motor, was used to supply carrier solvent to the chromatographic column. This pump is capable of delivering liquids up to 160 cc/hour at a discharge pressure of 1000 psi, maximum. The pump flow rate can be varied by means of a micrometer pump stroke adjustment, which permits the discharge of liquid at variable known rates. PRESSURE READOUT.A Model UC-3 Transducing Cell and Model UR-8 Analog Readout, 0-1000 psi (Statham Instruments Co., Berkeley, Calif.), was installed parallel with the column inlet to measure the pressure on the column inlet. RECORDER.The UV detector output was monitored by means of an Esterline-Angus “Speed-Servo,” 10-inch recording potentiometer having a full-scale response of 0.2 second and a maximum full-scale sensitivity of 1 mV.
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are desirable for the detection of UV-absorbing materials eluting from liquid chromatographic columns. The detector is capable of operating at a full-scale sensitivity of 0.01 absorbance unit (at 254 mF) with a short-term noise of about 3=0.00015 absorbance. During a 12-hour stability test run with methanol flowing through the 1.5-mm i.d. cell at a rate of 0.26 cc/minute, drift was k0.0005 absorbance, as shown in Figure 4. Drift in any 1-hour period was less than the detector short-term noise. No attempt to control ambient temperature was made during this test.
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RESULTS AND DISCUSSION
Detector Characteristics. The photometer used in this study was originally designed for continuous, sensitive, highstability process analyses (8). The same qualities that make this instrument useful for such demanding applications also (10) R.P.W.Scott, D. W. J. Blackburn, and T. Wilkins, “Advances in Gas Chromatography, 1967,” A. Zlatkis, Ed.,Preston Technical Abstracts Co., Evanston, Ill., 1967, p. 161.
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Figure 5. High sensitivity detection Sample-100 fil of 0.1 pg/ml diuron [3-(3,4-dichlorophenyl)-l,l-dimethylurea] in n-butyl ether; sensitivity-4.01 absorbance, full scale; carrier-n-butyl ether; flow r a t d . 2 6 cc/min VOL. 40, NO. 2, FEBRUARY 1968
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Figure 6. Linearity of UV detector Same conditions as given for Figure 5
The UV photometric detector herein described is capable of detecting extremely small amounts of materials which absorb at 254 mp even though these components may have their maximum absorption at much higher or lower wavelengths. While operation at 254 mp has proved to be the most generally useful, the detector can be operated at other wavelengths by changing to appropriate sources and filters. Chromatographic peaks containing nanogram amounts of compounds of moderate absorptivity can easily be observed with this UV detector. Figure 5 shows the chromatogram obtained with a 100-pl aliquot of a lOO-ng/ml solution of diuron [3-(3,4-dichlorophenyl)-l,l-dimethylurea1 when chromatographed on a 100-cm column of 4x p,p’-oxydipropionitrile on 230-270-mesh “Gas Chrom” P (Applied Science Laboratories, State College, Pa.), operating with a dibutyl ether carrier flow rate of 0.26 cc/minute. Ten nanograms of diuron (absorptivity = 88 liter/gram-cm at 254 mp) is readily detected, with the detector operating at a full-scale sensitivity of 0.01 absorbance. The large peak preceding diuron is a solvent impurity in the 100-111sample aliquot injected into the chromatographic column. The slight up-scale shift of the base line during the chromatogram is the result of the chromatographic system not being fully equilibrated before the chromatogram was begun. The level of short-term noise normally exhibited by the detector (approximately f0.00015 ) was about halved in the chromatogram in Figure 5 by applying a 500-microfarad capacitor across the input terminals of the high-speed recorder. This reduction in noise was accomplished with only a slight decrease in the speed of response of the photometric detection system. The chromatogram in Figure 5 illustrates the ability of the chromatographic system to accept relatively large sample aliquots with a minimum of detector base line disturbance. The potential of using this detector for trace analysis is obvious, assuming the desired separation from possible interferences can be accomplished. The range of linearity of the UV photometric detector is comparable to that of a thermal conductivity detector for gas chromatography, as demonstrated in Figure 6. To obtain this plot, various amounts of diuron [3-(3,4-dichlorophenyl)1,l-dimethylurea] were chromatographed in the manner used to obtain the data for Figure 5 . Peak height (in absorbance units) and peak areas (in square centimeter, calculated by peak height times the width at 1/2 peak height) were plotted against the micrograms of diuron chromatographed. The 394
ANALYTICAL CHEMISTRY
Figure 7. Peak variance study
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peak height plot begins to deviate from linearity at about 10 pg of diuron. However, peak areas are linear up to at least 20 pg, which corresponds to an absorbance of about 1.8. The detector shows only slight nonlinearity at an absorbance of 2. Based on the noise level of the detector and the upper limit of linearity indicated in Figure 6, it can be calculated that the range of linearity for this device is about 104. This compares favorably with that reported for gas chromatography thermal conductivity detectors (11). Because a somewhat smaller amount of energy emerges from the 0.79-mm i.d. cell, linear measurements up to an absorbance of about 1.2 can be made with this unit. Extra-Column Effects. A quantitative measure of extracolumn effects can be determined by measuring the variance of peaks which are formed in the chromatographic system. Peak variances can be calculated from the following equation (12):
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