silanols. Rapid analysis of a variety of proteins of industrial, pharmaceutical, clinical, and general research interest is possible using commercial liquid chromatographic equipment.
LITERATURE CITED E. Heftmann, "Chromatography", Reinhold Publishing Corporation, New York, 1967, pp 405-428. W. Haller, "Material and Method for Performing Steric Separations", U.S. Patent 3,549,524. C. W. Hiatte, A. Shelokov, E. J. Rosenthal, and J. M. Galimore, J. Chromatogr. 56,362 (1971). F. E. Regnier and R. Noel, J. Chromatogr. Sci., in press. E. Von Rudloff, Can. J. Chem., 43, 2260 (1965). J. D. Roberts and M. C. Ccserio. "Basic Principles of Organic Chemistry", W. A. Benjamin, New York, 1964, p 762. S.H. Chang, K. M. Gooding and F. E. Regnier, submitted to J. Chromatogr. Sci. P. D. Henry, R. Roberts, and B. E. Sonel, Clin. Chem. ( Winston-Salem, N.C.), 21,884 (1975).
H. J. Kentel, K. Okabe, H. K. Jacobs, F. Ziter, L. Maland, and S. A. Kuby, Arch. Biochem., 150,648 (1972). S.B. Rosalki, J. Lab. Clin. Med., 69,696 (1967). "Worthington Enzyme Manual", Worthington Biochemical Corporation, 1972. Howard Purnell, "Gas Chromatography", Wiley, New York, 1962, p 240. E. C. Horning, E. A. Moscatelli, and C. C. Sweeley, Chem. lnd. (London), 751 (1959). J. C.Giddings, "Gynamics of Chromatography Part K", Marcel Dekker, New York, 1965, p 58. Y. Birk, A. Gertler, and S. Khalef, Blochem. J., 87,281 (1973). A. Skeson and H. Theorell, Arch. Biochem. Biophys., 91,319 (1960). S.H. Chang and F. E. Regnier, in preparation. D. A. Nealson and A. R . Henderson, Clin. Chem. ( Winston-Salem, N.C.), 21, 392 (1975).
RECEIVEDfor review April 12,1976. Accepted July 26,1976. Parts of this work were presented at the 26th and 27th Pittsburgh Conferences on Analytical Chemistry and Applied Spectroscopy. The work was supported in part by a grant from Corning Glass Works, Corning, N.Y.
Universal Detector for Liquid Chromatography Based upon Dielectric Constant Leon N. Klatt Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830
A liquid chromatography detector based upon dielectric constant is described. The detector employs a modular phaselocked-loop circuit with its operating frequency determined by the dielectric constant of the column effluent. Readout is the shift in frequency resulting from changes in solution dlelectric constant as a solute band elutes from the column. Response is independent of intrinsic solvent conductivity and dielectric loses associated with dipole-dipole relaxation. The detector has excellent stability and reproducibility, negligible sensitivity tto variations in solvent flow rate, and the capability of operating without any restrictions on solvent dielectric constant. Separations employing low and high dielectric constant eluants were monitored. An average detection limit of 0.0005 A€ was measured in low dielectric constant media. Comparison with the uv and refractive index monitors indicates that the dielectric constant detector should find numerous applications in liquid chromatography.
Liquid chromatography detectors are classified either as universal or specific; the universal detector responds to a change in some bulk property of the mobile phase as the solute elutes from the column while the specific detector responds to some unique property of the solute. Since dielectric constant is an electrical property possessed by all materials, a liquid chromatography monitor based upon dielectric constant is classified as a universal detector. The first use of dielectric constant as the basis for detection in liquid chromatography was probably made by Troitskii ( I ) . The response consisted of a change in an audio tone upon location of an absorption zone on an alumina column. Monaghan and co-workers ( 2 ) described the use of a heterodyne chemical oscillometer for chromatographic detection. The capacitor plates were separated from the solution by the insulating glass walls of the detector cell. This same instrumental concept was used by Baumann and Blaedel ( 3 ) and
Johansson et al. ( 4 ) to monitor a change in solution conductivity as a solute band passed a detector head attached to the chromatographic column. Based upon the equivalent circuit for these systems (5),one senses a combination of capacitance and conductance with the relative magnitude of each dependent upon the operating frequency of the oscillometer and the conductivity of the media. Vespalec and Hana (6) reported a capacitance detector for liquid chromatography that used a detector cell with direct contact between the capacitor plates and the column effluent. The instrument operated at 18 MHz, and the cell formed the capacitor in the tuned network that determined the oscillator operating frequency. Readout was obtained via a heterodyne technique. The detector cell had to be altered to permit operation with mobile phases of different dielectric constants (7). Use of the detector cell in the resonant network of the oscillator precludes operation with high dielectric constant solvents because the energy losses to the solution from dielectric relaxation quench the oscillations. Erbelding (8)described a very simple circuit based upon a triangular wave generator whose output was the input to a difference-differentiator containing a reference capacitor and the capacitance detector cell. Under ideal conditions, the amplitude of the square wave output from the differencedifferentiator is proportional to the capacitance difference between the detector cell and the reference capacitor. This circuit also was unable to operate with high dielectric constant solvents, although sufficient energy to compensate for dielectric losses was available. Because solution conductivity and dipole relaxation appear as a resistive component in parallel with the capacitance, the transfer function of the difference-differentiatoris dependent upon solvent dielectric constant producing an output waveform that is not uniquely related to capacitance and results in the detector's failure to operate with high dielectric constant solvents. The instrument described herein overcomes many of the difficulties inherent in previously described dielectric constant detectors and possesses sufficient sensitivity for use in modern liquid chromatography.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
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properties of the solution, respectively. The parallel R,C, model of a cell containing a fluid media is well documented and accounts for solution conductivity (5) and dielectric losses (9). It can be shown that the circuit impedance is a maximum when
SA
wc,
I
Figure 1. Tuned
I
Frequency Shift Readout
Phase Detector
T
(3)
Voltage Contrailed Osciiiator
c D.C.Gain Amplifier
Figure 2.
Low - Pass Filter
Block diagram of dielectric constant detector
DESCRIPTION OF INSTRUMENT The determination of dielectric constant involves measuring the capacitance of a cell in which the material of interest constitutes the dielectric medium separating the capacitor plates. The observed capacitance is related to the dielectric constant by C = kc
(1)
with k being determined by the particular cell geometry. Capacitance, in turn, is determined by measuring the resonant frequency of a tuned network, either in a constant or variable frequency mode, and calculating the capacitance from the appropriate resonant condition equation. The constant frequency approach requires use of a calibrated reference element or elements whose impedances must be varied to satisfy the resonant requirements of the tuned network. The use of a Wein bridge to measure capacitance is an example of this technique. The variable frequency mode involves observing some property of the tuned network, such as the total current in a series LC network, and varying the frequency of the applied signal until resonance is achieved. This approach requires only a single standard to calibrate the system. Because the variable frequency mode is easier to implement, this mode was selected for the dielectric constant detector. Consider the circuit shown in Figure 1. R , and C, are the solution resistance and the capacitance due to the dielectric
Figure 3.
1846
(2)
and it is the sum of the resistances R and R,. w is the angular frequency in radiansls. Since conductivity can change without a significant change in dielectric constant (10), measurement of the voltage, current, or power in the resonant network does not provide the unique information necessary to determine whether the resonant frequency, WO, is applied to the network. However, the phase angle between the voltage a t points V R and V,, as given by Equation 3
network of dielectric constant detector Power Amplifier
- l/wL = 0
is a monotonic function of the frequency, and is zero a t the resonant frequency independent of the resistive elements in the network. This is the basis for the operation of the detector. The instrument consists of four basic elements, and is shown in block diagram form in Figure 2. The first element is a voltage-controlled-oscillator (VCO)/power amplifier combination. The output from this unit drives the parallel inductive-capacitive network containing the detector cell as the capacitor. The third element consists of the phase detector, low pass filter, and dc gain amplifier. these units complete the feedback loop to the VCO. These modules operate as a phase-locked-loop. The final element contains the frequency shift readout circuitry that provides the analytically useful information. Figure 3 is a functional block diagram of the first three elements of the instrument. For the particular detector cell geometry and frequency shift readout circuitry, the inductor L was chosen such that the upper operating frequency was 5 5 MHz. A digital phase detector was used because its output is independent of input amplitude variations that result from dielectric loses and/or solution conductivity. Since the two signals derived from the cell network are sinusoidal waveforms, a dual high speed comparator was used to convert them into the logic levels required as inputs to the phase detector. In order to maintain the phase integrity of these signals during the conversion, an ultra high speed emitter-coupled-logic comparator (MECL 111, Motorola Inc., Phoenix, A r k ) was used. The output from the phase detector appears as two pulse trains whose duty cycle depends upon the phase difference between the input signals. These are filtered with dual 2-pole active low-pass filters, subtracted, and amplified to produce the control voltage applied to the voltage-variable capacitance
Functional block diagram of phase-locked-loop portion of the detector
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
Overrange Code
Latch 1
BCD upldown
7 BCD Digits
Counters
Latch 2
5*
Y
DAC Analog Readout
A -A = BDigital
Digital Readout
Comparators A>B
4
Zero
Cont ro I
I
Clock 0
I
I
I
Flgure 4. Functional block diagram of frequency shift readout circuitry
diode. A two-position coarse tuning switch allows operation from 0.6 to 5 MHz. The other active components provide the necessary impedance matching between the various circuit elements, and were constructed from discrete components. The frequency shift data are obtained via digital counting techniques with T T L logic elements. Data are presented in digital and analog formats. Figure 4 contains a block diagram of the readout circuitry. The time base is derived from a 1-MHz crystal controlled oscillator with the output frequency of the dividers switch selectable. The readout operates as follows. With eluant passing through the detector cell, a baseline is set by pressing a momentary contact push button switch. This interrupts the current counting sequency, clears the counters, and enables them in the up-count mode. During the next clock period, a digital count directly related to the VCO operating frequency is determined. At the end of the clock period, this is stored in Latch 1.The control logic then enables the decade counteers in the down-count mode and loads them with the baseline data from Latch 1.During the next clock period, the counters count down toward zero. If F , has shifted to lower frequencies, the counters will not reach zero at the end of the clock period and the counter output is the frequency shift. However, if F , has shifted to higher frequencies, the counters will reach zero before the clock period ends. When the counter outputs attain zero, the digital comparators, through the control logic, enable the counters in the up-count mode for the remainder of the clock period. Again the counter output indicates the frequency shift at the end of the clock period. This is loaded into Latch 2 via the control logic. Termination of the load to Latch 2 reloads the counters from Latch 1 and enables them in the down-count mode. This sequence is repeated on subsequent clock periods. T o establish a new baseline, either as the result of a readout range or eluant change, the baseline switch is depressed and the entire operation is repeated. It should be noted that this mode of measuring the frequency shift results in positive and negative shifts to appear at the output as unipolar signals. Thus, the observed output for solutes that increase or decrease the dielectric constant of the mobile phase all appear as positive elution peaks. An overrange indication is also included in the readout. This is accomplished by using the A > B output of the digital comparator connected to the fifth digit of the decade counters. If this A > B output is true when Latch 2 is loaded, the control logic selects word Y of the multiplexer input for clocking to the readout. If during some subsequent counting interval the overrange condition no longer exists, the A > B comparator output is false when Latch 2 is loaded, and the control logic
: B N C CONNECTOR
CONTACT TO INNER CAPACITOR P L A T E
TOP ASSEMBLY PLATE
DETECTOR BODY SERVI AS OUTER CAPACITOR
TEFLON INSULATOR
INNER CAPACITOR P L A T E
SOLUTION O U T L E T
SOLUTION I N L E T
LIQUID S E A L
TEFLON INSULATOR
BOTTOM ASSEMBLY PLATE
hGT-
Figure 5. Cross sectional diagram of detector cell
selects the output of Latch 2, word X, for clocking to the readout. The frequency shift information is converted to an analog voltage with a 4-BCD digit DAC and recorded with a strip chart recorder. A digital display of the frequency shift data is also incorporated into the completed instrument. The switch selectable time base provides full scale frequency shifts from 5 KHz to 1MHz in a 1-2-5 sequence. A second digital readout provides a continuous four-digit display of the VCO frequency. Complete schematics for the instrument are available from the author upon request. The capacitance detector cell is constructed from 316 stainless steel and Teflon, and employs concentric cylinders as the capacitor geometry. A cross sectional diagram is shown in Figure 5. The outer and inner cylinder diameters are 1.000 cm and 0.990 cm, respectively. Each cylinder has an active , the calculated length of 1.000 cm. Cell volume is 16 ~ 1and capacitance with air as the dielectric media is 55 pF. Contact to the inner cylinder is made via a gold plated pin soldered to the conductor contact of a BNC connector and press fitted into a hole in the inner cylinder upon attachment of the BNC connector to the detector cell. Contact of the completed cell
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
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7.50 I
Table I. Operating Frequency and Noise of Detector Sclvent Wgter Acetonitrile Methanol Ethanol Acetone Isopropanol Methyl ethyl ketone o -Dichlorobenzene Methylene chloride Tetrahydrofuran Chloroform Benzene Cyclohexane Hexane
Dielectric constant at 25 O C fo, MHz RMS noise, Hz 78.54 0.827 20 22 36.70 1.221 34 32.63 1.275 59 24.30 1.481 20.70 1.604 23 57 19.92 1.661 32 18.50 1.718 9.93
2.297
39
8.895
2.448
61
7.58
2.658
74
4.798 2.274 2.015 1.882
3.248 4.538 4.775 4.876
72 61 46 68
assembly to the resonant circuit is made via the appropriate mating BNC connector mounted on the etched circuit board. Solution inlet and outlet lines are attached with conventional low pressure Teflon chromatographic fittings. The detector cell has two turns of 0.635-cm i.d. copper tubing brazed unto the body, and a coolant fluid at some selected temperature is circulated through the coil to control the temperature of the detector cell. EXPERIMENTAL Reagents. All chemicals were reagent grade quality and used without further purification,except that all solvents were filtered (5 pm, Type LS, Millipore Corporation, Bedford, Mass.) prior to use as chromatographic eluants. Apparatus. A reciprocating pump (Model 396, Milton Roy Company, LDC Division, Riviera Beach, Fla.) was used for all studies reported herein. No effort was made to dampen the pump pulses. Conventional Teflon tubing, 0.79-mm id., and connectors were used for assembly of the chromatographicsystem. A rotary sample injection valve with interchangeable sample loops was used for sample injection. An ultraviolet absorption monitor (254 nm, Model 1200,Milton Roy Company, LDC Division, Riviera Beach, Fla.) was used in selected comparison studies. Chromatograms were recorded on a strip chart recorder. Initial studies employed a 6-mm i.d. X 14-cm glass column packed with glass beads to simulate chromatographic conditions as well as providing a small back pressure to ensure constant solution delivery by the pump. Prepacked columns, 4.6 mm X 25 cm, (Reeve Angel, Whatman, Inc., Clifton, N.J.) containing either silica (Partisil 10) or permanently bonded ODS (Partisil ODs) were used in the chromatographic studies. Procedure. All chromatograms were obtained with the columns at ambient laboratory temperature. Just prior to entering the detector cell, the column effluent was equilibrated to the detector cell temperature through use of a coil heat exchanger. The capacitance de1848
I
I
I
I
*
6.50 0
20 40 60 DIELECTRIC CONSTANT, E
80
Figure 6. Comparison of the observed frequency dependence upon dielectric constant with the parallel capacitor model. Points are data from Table I and the solid curve is calculated from the equation f,, = 1/2av'1.14 X 10-e(4.105X lO-"c: 4- 1.5 X lo-")
tector was maintained a t 25 f 0.03 "C by circulating constant temperature water through the cooling coils attached to the outside of the
detector cell. Samples were diluted with the same batch of solvent being used as the eluant for a particular separation. All other procedures were conventional techniques practiced in liquid chromatography. RESULTS A N D DISCUSSION
Table 11. Dependence of Frequency Shift upon Flow Rate Flow rate, ml/min Frequency shift, AHza 0.61 315 f 41 1.19 210 f 24 1.79 178 f 26 2.38 161 f 26 111 f 26 3.00 a The error estimates are the 95% confidence interval for five measurements.
I
1
General Detector Characteristics. Table I contains operating frequency and noise data as a function of dielectric constant. The noise figures were obtained by pumping the specified solvent through the chromatographic system containing the glass bead column until a stable baseline was observed. Then a 12.5-min segment was recorded at the highest readout sensitivity from which 31 frequency shift values at equally spaced intervals were taken and analyzed according to a least-squares fit to a first-degree polynomial. This method was used with the objective of decoupling drift, i.e., long term noise, from the short term noise. The square root of the variance of this least-squares fit was taken as the RMS noise level. These values agreed quite closely with the RMS noise calculated from the peak-to-peak envelope of the same data set, and that calculated from the standard deviation of the mean frequency shift. Although, the RMS noise data appear to increase with operating frequency, a statistically significant correlation does not exist. The average RMS noise of 48 Hz is larger than the typical noise specification of the VCO ( I I ) ,and probably is determined by the temperature changes within the detector cell, because the cyclic noise pattern was in phase with the on-off cycling of the temperature controller. For the readout system employed in the instrument, this noise level is 1% of full scale a t the highest sensitivity. According to Equations 1and 2, the product of fo.\/; should be independent of dielectric constant. This is true for frequencies below 2.5 MHz, i.e., > 8. The cell constant calculated from this low frequency data is 41 pF. Above 2.5 MHz, fo.\/; decreases regularly indicating an apparent increase in the proportionality constant in Equation 1 as the dielectric constant decreases. This change info.\/; a t high frequency is not due to the electronic system, because the analogous constant in which silvered mica capacitors replaced the detector cell is constant over the entire operating range of the instrument. Also, deviations of the dielectric constant from its static value cannot account for this effect because dipole relaxation times are -lo6 times shorter than the period of the alternating electric field applied to the cell (10). This result can be explained, however, if one assumes that the detector cell has a stray capacitance component, independent of the solution dielectric constant, which is in parallel with the capacitance determined by the solution dielectric constant. Figure 6 shows the excellent agreement between the experimental data from Table I and the parallel capacitor model.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
T
I
I
0 T
6 6
S
10
12
14 16 T I M E , minutes
18
20
22
24
._-i:i'i 0.5KHz
Figure 7. Separation of o-dichlorobenzene and nitrobenzene on a 4.6-mm X 25-cm Partisil 10 column with n-heptane as eluant Flow rate 0.56 ml/min. ( A ) 250 pl of o-dichlorobenzene and 75 pl nitrobenzene diluted to 10 ml with n-heptane. 50 pl injected. (B)40:l dilution of above sample. 50 pl injected
The long term drift was determined by recording the baseline for approximately 1h with hexane flowing through the detector. These data were treated in the same manner as the noise data. The drift is 3.5 f 1.8 AHz/min. Qualitative observations with the other solvents indicate that the drift is frequency independent. Although the response of a dielectric constant detector should be flow rate independent, a small shift in frequency is noted as the flow rate changes. Table I1 summarizes the dependence observed with hexane. It is worthy to note, that in this study the baseline was established at the highest flow rate and, because of the particular design for the readout circuitry, the data would have had the opposite trend if the baseline initially had been established a t the lowest flow rate. The dependence upon flow rate is quite small and, when considered in terms of the precision of modern liquid chromatographic pumps, it is an insignificant factor. Chromatographic Applications. Initial tests of the detector in actual chromatographic applications involved low dielectric constant solvents. Figure 7 shows the separation of o-dichlorobenzene from nitrobenzene on silica with n-heptane as the eluant and monitored with the dielectric constant detector. The shift in elution peaks with dilution is characteristic of heavy column loads. The step nature of curve B results from the digital readout circuit, which at the higher sensitivities requires longer counting times between each update of the output. The elution times of the two components were verified by injection of the individual components and with the uv monitor. As expected with this solute-solvent system, the uv monitor has a much higher sensitivity. Figure 8 shows the same separation with benzene as eluant. The retention times of both components are shortened because benzene possesses stronger solvent properties than n-heptane (12).Comparing Figure 8 to Figure 7, one observes that the response of the dielectric constant detector is essentially constant as the eluant is changed from n-heptane to benzene. The uv monitor cannot be used with benzene as the eluant. This example illustrates one capability of the dielectric constant detector, i.e., the ability to monitor separations requiring the use of uv absorbing eluants. Detector linearity in low dielectric constant media was ascertained with naphthalene benzene solutions. The data are summarized in Table 111. Excellent precision and linearity are indicated.
4
6
S
10
12
T I M E , minutes
Figure 8. Separation of o-dichlorobenzene and nitrobenzene on a 4.6-mm X 25-cm Partisil 10 column with benzene as eluant Flow rate 0.55 ml/min. ( A ) 250 pl of o-dichlorobenzene and 75 pI nitrobenzene diluted to 10 ml with benzene. 50 wl injected. (8)40:ldilution of above sample. 50 pI injected
Table 111. Linearity and Detection Limit in Low Dielectric Constant Solvents a A€
x 10-3
b
Frequency shift, A KHzC
0.47 0.38 f 0.10 0.94 1.09 f 0.07 2.06 2.62 f 0.04 5.56 & 0.19 5.02 7.06 7.85 f 0.25 11.75 f 0.22 9.92 20.02 23.22 f 0.10 Glass bead column and the data were taken from the steady-state response observed when -2 ml of sample was injected into the flow stream. Flow rate 1.48 ml/min. Regression line calculated from data is: Af = [(1.164 f 0.014) X lo6] A t - (70 f 80). Errors expressed as the 95% confidence interval. €benzene = 2.274, tnaphthalene = 2.54. Ac calculated assuming additivity on a volume fraction basis. Four replicate measurements.
Because the dipole-dipole interactions between solvent and solute with low dielectric constants are relatively small, the dielectric constants of the individual components are additive on a volume fraction basis (13),and one can, therefore, express a detection limit applicable to these systems in terms of a change in dielectric constant. Analysis of the data in Table I11 according to the method of Hubaux and Vos ( 1 4 ) yields a detection limit at the 95% confidence level of 0.0008Ac. Using three times the RMS noise level to define the detection limit (15),0.0002 Ac is obtained. Chromatograms B in Figures 7 and 8 are representative of elution bands, which at the peak produce a change in dielectric constant equal to 0.0005Ac. This is the mean of the above detection limit estimates. Previous designs of dielectric constant detectors have been unable to operate with high dielectric constant solvents (6,8). Curve A , Figure 9 is a separation of chloroform from l-chlorobutane with permanently bonded ODS using 40-60% (v/v) isopropanol-water mixture as eluant. The solvent peak cor-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER
1976 * 1849
~
Table IV. Reproducibility and Linearity of Detector in Isopropanol-Water Solvents a Retention time, min Peak height, AF KHz
I
0
~
II
10
~
20
I
,
30
TIME, minutes
Figure 9. Separation of chloroform and 1-chlorobutane on a 4.6-mm X 25-cm Partisil ODs column with 40-60% isopropanol-water mixture
as eluant Flow rate 0.36 ml/min. ( A ) 100 pl chloroform and 100 pl I-chlorobutane diluted to 10 ml with isopropanol-water eluant. 50 pl injected. Dielectric constant monitor. ( B ) 150 pl chloroform and 150 pI I-chlorobutane diluted to 10 ml with isopropanol-water eluant. 50 p1 injected. UV monitor with air n reference beam
responds to a negative frequency shift, and is the result of water being displaced from the stationary phase by the less polar solutes. The solute peaks are positive frequency shifts. Curve B , Figure 9 is the chromatogram obtained with the uv monitor a t 254 nm. It should be noted that the solute concentrations used to obtain chromatogram B is 50% larger than that used in A . This higher concentration represents the solubility limit of 1-chlorobutane in the isopropanol-water solvent at 25 OC. Clearly, the present instrument can be used with high dielectric constant solvents, and in specific applications represents the optimum detector. Linearity and reproducibility of the complete chromatographic-detector system was ascertained by injection of a series of chloroform and 1-chlorobutane mixtures. Table IV summarizes the data. Peak shape was independent of the quantity of solute injected. The excellent precision of the retention time data indicates that the discrete nature of the digital readout does not degrade the precision of the total chromatographic system. Peak height is linearly related to the volume of solute injected, with the scatter comparable to the noise level of the detector. Analysis of the peak height vs. solute volume data yields detection limits of 0.04 gl chloroform and 0.05 ~1 1-chlorobutane. Due to the large dipole-dipole interaction in high dielectric constant solvents, the volume fraction additivity of the individual dielectric constants no longer holds, and the above detection limits, expressed as a quantity of solute, cannot be converted to a change in dielectric constant.
Volume of solute 1-Chlorobu1-Chlorobuinjected, pl CHCl3 tane CHC13 tane 0.500 15.5 20.7 3.025 2.475 0.375 15.4 20.3 2.325 1.945 0.250 15.3 19.9 1.505 1.305 0.125 15.4 19.7 0.725 0.650 0.125 15.4 19.7 J 0.680 0.670 0.100 15.3 19.5 0.505 0.485 0.100 15.3 19.5 0.580 0.530 0.050 15.4 19.5 G.275 0.100 0.050 15.5 19.5 0.305 0.265 a Column is 4.6 mm X 25 cm packed with permanently bonded ODS (Partisil ODs). Fifty ~1 total sample injected which contained the indicated volume of solute. Elutant was 40-60% isopropanol-water mixture. Flow rate was 0.36 ml/min.
lating the two properties, E = q2, where q is the refractive index. Based upon this relationship, measurement of unit change in dielectric constant woidd be required to achieve the same detection limit with the dielectric constant detector as reported for the refractive index monitor (16). This has not been achieved with the present instrument. However, this does not necessarily mean that a dielectric constant detector has an inherently lower sensitivity, because Maxwell's relationship is only valid for substances without a permanent dipole moment and when E and 7 are measured with electromagnetic radiation in the optical frequency range. Because of distortional and orientational polarization, relatively few solvents obey Maxwell's equation (IO),and thus, an a priori comparison of the two detector sensitivities is not useful from a practical viewpoint. Literature reports indicate a detection limit for the refractive index monitor of 4.3% (163,suggesting that, although very small changes in the refractive index can be measured, this does not translate into the corresponding analytical sensitivity. The detection limits determined from the data of Table IV are -0.1%, which indicates that the dielectric constant monitor described herein is a competitive detector and should be useful to the practicing chromatographer.
LITERATURE CITED (1) G. V. Troitskii, Biokhimiya, 5, 375 (1940). (2) P.H. Monaghan, P. B. Moseiey, T . S. Burkhalter, and 0. A. Nance, Anal. Chem., 24,-193 (1952). (3) F. Baumann and W. J. Blaedel, Anal. Chem., 28, 2 (1956). (4) G. Johansson, K. J. Karrman, and A. Norman, Anal. Chem., 30, 1397 11958) ---, (5)C. N. Reilley and W. H. McCurdy, Jr., Anal. Chem., 25, 86 (1953). (6) R . Vespalec and K. Hana, J. Chromatogr., 65, 53 (1972). (7) R. Vespalec, J. Chromatogr., 108, 243 (1975). ( 8 ) W. F. Erbelding, Anal. Chem., 47, 1983 (1975). (9) S. Haderka, J. Chrornatogr., 54, 357 (1971). (10) R . A. Robinson and R. H. Stokes, "Electrolyte Solutions", 2nd ed., Butterworths, London, 1959, Chap. 1. (11) Semiconductor Data Library, MECL Integrated Circuits", Vol. 4, Series A, Motorola Semiconductor Products, Inc., Phoenix, Ariz., 1974, p 4-3. (12) Lloyd R. Snyder, "Principles of Adsorption Chromatography", Marcel Dekker, New York, N.Y., 1968, Chap. 8. (13) 6.W. Thomas and R. Pertei, "Treatise on Analytical Chemistry", Part 1, Vol. 4, Wiley-Interscience, New York, N.Y., 1963, pp 2631-2672. (14) A. Hubaux and G. Vos, Anal. Chem., 42,849 (1970). (15) H. Kaiser, Anal. Chern., 42 (4), 26A (1970). (16) E. S. Watson, Am. Lab., September 1969. \
CONCLUSION The main focus of this research effort was to design, test, and evaluate a practical dielectric constant detector suitable for use in liquid chromatography. The results presented above indicate that this objective was achieved. This is the first known report of a dielectric constant detector that is capable of operating without any restrictions on solvent dielectric constant. Because the detector responds to changes in bulk properties, direct use with solvent gradients is not possible. However, the availability of reproducible gradient generators and inexpensive microcomputer systems suggest that a readout mode can be designed to permit operation with gradient elution. Dielectric constant detectors are often compared to refractive index monitors bbecause of Maxwell's equation re-
1850
RECEIVEDfor review April 14,1976. Accepted July 29,1976. ORNL is operated by the Union Carbide Corp. for the U.S. Energy Research and Development Administration.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 13, NOVEMBER 1976
