slow gas-phase reaction (9) and the reasons stated above, the decrease of the m-xylenol peak was attributed exclusively to the liquid phase reaction. Ave was computed according to Equation 34. By an iterative computer program, kl was determined according to Equations 19 and 37. Figure 4 shows the experimental results obtained. The agreement with measurements performed using a classical method, a stirred micro-reactor where the reaction took place in Squalane a t 80, 90, and 100 OC, is fair. The observed difference could be explained tentatively by the contribution of the gas-phase reaction to the overall conversion.
ACKNOWLEDGMENT Helpful discussions with Helmut Ludescher of the Section of Mathematics of the Academy and with Zoltan Sza-
baday of the Centre of Chemistry, Timisoara, are acknowledged. LITERATURE CITED (1) E. Gil-Av and Y. Herzberg-Minzly, Proc. Chem. SOC.,London, 316 (1961). (2)E. Gii-Av. J. Chromatogr., 13, l(1964). (3) C. E. Doring, W. Pehle, and G. Schmid, Gaschromatographie 1968,Vortrage des VI. Symposiums uber Gaschromatographie in Berlin, May 1968. Preprints, p 143. (4)V. G. Berezkin, V. S. Kruglikova, and V. E. Shiryaeva, Teor. Eksp. Khlm., 3, 553 (1967). (5)J. C. Giddings and K. L. Maiiik, lnd. Eng. Chem., 59, 19 (1967). (6)R. L. Martin, Anal. Chem., 33, 347 (1961). (7)R. L. Martin, Anal. Chem., 35, 116 (1963). (8) R. L. Pecsok, A. de Yliana, and A. Abdul-Karim, Anal. Chem., 36, 452 (1964). (9)E. A. Moelwyn-Hughes, "Physical Chemistry," 2nd ed., Pergamon Press, Oxford, New York, London, Paris, 1961,p 1249.
RECEIVEDfor review February 3, 1975. Accepted May 20, 1975.
Dielectric Constant Detector for Liquid Chromatography William F. Erbelding lndiana University-Purdue University at Fort Wayne, 2 10 1 Coliseum Blvd. East,
A device which senses changes In dielectric constant of the effluent from a liquid chromatographlc column has been used successfully to record chromatograms. The detector cell is a capacitor whose capacltance depends on the dielectric constant of the liquid between Its electrodes. A simple electronic circuit uslng five operational amplifiers converts the dlelectric constant changes to direct current signals for recordlng the chromatogram. Although not appropriate for use In hlgh performance liquld chromatography because of the large holdup volume of the cell and Its lack of provision for temperature stabilization, it is able to detect solutes in the range of 1 mglml. It Is demonstrated uslng several different chromatographic systems and Its behavior is compared with the expected behavior of the refractlve index and the UV absorption detectors.
Little information is available in chemical literature about the use of dielectric properties of eluates as a detection system for liquid chromatography. Laskowski and Putscher ( I ) have shown that the dielectric constants of certain petroleum fractions increase with oxygenation and such changes can be monitored with a device which is essentially a capacitor in a tuned amplifier circuit. Their instrument did not record a chromatogram. In 1958, Grant ( 2 ) , using a cell as a capacitor through which column effluent passed, electronically followed and recorded the change in dielectric constant as amino acids were eluted with n-propanol-water eluent from a column of powdered cellulose. Vespalec and Hana ( 3 ) ,using a specially designed capacitor cell in an electronic circuit which contained two oscillators and frequency meter, established that the sensitivity of this detection system can exceed the sensitivity of the refractive index detector if operating conditions are carefully controlled. These authors did not use their detector in conjunction with a chromatographic column. Many books and authors briefly mention dielectric constant as a basis for a feasible detection system (4, 5 ) , without citing references to published information. A conclusion drawn by some authors (6) is that a dielectric constant
Fort Wayne, Ind. 46805
detector would not be expected to have any advantages over the refractometric detector currently in wide use. A good dielectric constant detector is fundamentally related to a refractive index detector. For a substance without a permanent dipole moment, the dielectric constant t is related to n, the refractive index, through the Maxwell relationship, = n2, where n is measured in long wavelength (for example, infrared) light. For molecules with permanent dipoles, the dielectric constant is significantly greater than the square of the refractive index. It follows that if a change in 6 can be measured with the same precision that a change in n can be, then a dielectric constant detector can be more sensitive than a refractive index detector. Commercially available differential refractive index detectors are capable of sensing changes in refractive index down to 10-7 unit corresponding to a detection limit for many substances of 1 pg/ml (7). If the relative error in determining dielectric constant can be as low as as has been reported ( B ) , a reasonable estimate of sensitivity of a dielectric constant detector would also be in the range of 1 pg/ml. To realize these detection limits, however, careful thermostating is required for both systems. Because dielectric constant is an electrical property, changes in this property can be detected by appropriate electronic circuitry. Such circuits have been described ( I , 2). Almost always, a t least part of the circuit is an electronic oscillator. The capacitor cell, in which the eluate from the column is the medium separating the plates, may or may not be part of this oscillator circuit.
EXPERIMENTAL Reagents. Benzene and a commercial mixture of hexanes were used as single component eluents. Prior to use, these were passed through activated silica gel. Dichloromethane when used with commercial hexane as part of a binary eluent was not pretreated in any way. Chromatographic adsorbents were Davison Code 12 silica gel, 28-200 mesh, activated, and J. T. Baker aluminum oxide, neutral (for chromatography). The aluminum oxide was heated a t 400 'C for 16 hours and deactivated by adding appropriate amounts of water. Columns were packed under solvent in a buret of about 25-ml capacity. No attempt was made to equilibrate the water content of the eluent with the water content of the adsorbent.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
1983
IO K
10 K
Figure 2. Schematic diagram of function generator (lower part including A-1, A-2, and A-3) and difference-differentiator (upper part including A-4 and A-5) Operational amplifiers: A-1, A-2: Burr-Brown 3501: A-3: Burr-Brown 3503 FET input: A-4, A-5: Teledyne 2740 FET input
Figure 1. Diagram of capacitor cell (1) Glass capillary, (2) Wire leads from electrodes, (3) Teflon spacer, (4) Outer brass cylinder electrode, (5)Inner brass cylinder electrode, (6) Glass jacket
Apparatus. A diagram of the detector cell appears in Figure 1. Two concentric brass cylinders about 4.8 cm long formed the plates of the capacitor cell. The outer was 5.6-mm and the inner 4.5-mm diameter. The inner one surrounded and was cemented to a glass capillary tube which fitted into the Teflon stopcock of the buret holding the column. The outer brass cylinder was closed off by soldering a cap over the end. The bottom of the glass capillary was flared slightly by softening in a flame and pressing against a piece of asbestos. This slight bulge in the glass served to space the electrodes a t the bottom of the cell. A fine strand of Teflon encircling the inner electrode but not meeting a t its ends served as a spacer a t the top. One wire lead from each electrode connected the cell to the circuit. The column effluent passed down the capillary, filled the volume between the brass cylinders, and spilled over and down the outer cylinder. The entire cell assembly was placed inside a glass tube drawn out to a small opening a t the bottom end. No attempt was made to maintain the cell a t constant temperature. Holdup volume, Le., the volume inside the capillary and between the brass cylinders, was about 0.7 ml. Cell capacitance with hexane was about 25 pF. A function generator modeled after the circuit described in Reference (9) was assembled using three operational amplifiers. A diagram of it appears in Figure 2. The output of amplifier A-3 is a triangle wave. For these studies, frequencies between 1 and 5 KHz (adjustable a t P I ) and peak-to-peak amplitudes up to 20 volts (adjustable a t Pz) were used. The triangle wave is fed to the difference-differentiator A-4 of which the detector cell is a part. The output of A-4 is a square wave whose amplitude is zero when the capacitances associated with the cell (including stray capacitance) and balancing capacitor c b are matched. When cell capacitance changes, the amplitude of the square wave increases from zero. The output of A-4 is amplified by A-5. The bridge rectifier and voltage divider process the signal from A-5 for presentation to the recorder. Because the output of the bridge rectifier is nonlinear when its input is less than 1 volt peak-to-peak, cb as a matter of routine was not exactly balanced against the cell but was adjusted so that its capacitance was always less than that of the cell. In this way, any increase in cell capacitance resulted in a proportional increase in the amplitude of the square wave output of A-5, which was adjusted initially to be more than one volt. This necessitated the provision for additional external offset a t the recorder input so that it could be zeroed. A very conventional fl5-volt zener-regulated power supply was used to power the five operational amplifiers. 1984
Procedure. Because the main purpose of this study was to demonstrate the feasibility of the dielectric constant detector, greater emphasis was placed on this aspect of the system than on chromatographic separations. Chromatograms were obtained by placing samples of known composition a t the top of a column which had been packed under solvent. Elution was downward. Eluent from a reservoir passed by gravity through the column. Flow rates depended on eluent density and viscosity, particle size of column adsorbent, and height of the eluent reservoir. These generally varied between 1and 2 ml per minute but remained essentially constant during the recording of a chromatogram.
RESULTS AND DISCUSSION Flow Rate Sensitivity. The apparent capacitance of this detector cell depends on flow rate and increases rapidly as flow rate increases from zero to about 0.5 ml/min but increases less rapidly at higher flow rates. Figure 3 shows the change in recorded signal as the flow velocity of hexane through the cell changes. I t is important that flow rates do not change substantially during the recording of a chromatogram. Obviously, a constant flow rate is essential in gas or liquid chromatography with any kind of detector. Single Component Eluents. Preliminary experiments, in which operating conditions such as triangle wave amplitude and frequency were not yet optimized, yielded chromatograms showing the separation of benzene from methoxybenzene and p - dimethoxybenzene. The latter two components were not separated from each other by elution from a 3% HzO-Al203 column with hexane. The chromatogram is shown in Figure 4. Approximately equal amounts by weight of each component made up the sample. The area of the benzene peak is less than half the area of the two-component peak. This is due to the fact that the dielectric constant of benzene is less than that of either methoxybenzene or p-dimethoxybenzene. In Figure 5 is a chromatogram showing the separation of o-nitrophenol and diphenyl ether on a silica gel column. The eluent is benzene. On the game chromatogram is shown a plot of absorbance of 2-41 fractions of the eluate measured at 276 nm on a Beckman DU Spectrophotometer. Sensitivity of the photomultiplier had to be set very high because of strong adsorption by the benzene eluent. Beyond 40 ml, absorbance became erratic and never returned
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
0
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Figure 5. Separation of diphenyl ether and o-nitrophenol on silica gel with eluent benzene. For comparison, absorbance is plotted on the same chromatogram
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Figure 4. Chromatogram showing partial separation of benzene, methoxybenzene, and pdimethoxybenzene on 3 % H20-AI203 with hexane eluent to the base line. This may be the result of the large surface area of the outer brass cylinder over which the cell effluent spilled. Figure 6 shows chromatograms of a mixture of methoxybenzene, ethyl benzoate, and acetophenone separated on 2.3% HZO-Al203 using benzene as the eluent. An estimate of the sensitivity of the detector can be made. Obviously, sensitivity varies for each component eluted. This is true in all forms of chromatography and with all detectors. In the chromatogram of Figure 6, the dielectric constant detector shows itself clearly to be more sensitive to acetophenone than to eit.her 6f the other components in the sample. The greater dielectric constant of acetophenone accounts for this greater sensitivity. The refractive index detector would not be expected to show these differences in the same way. For example, the refractive index (20 O C ) of benzene is 1.5011 and that of acetophenone is 1.5372. The dielectric constants (25 "C) are, respectively, 2.284 and 17.39. The dielectric constant detector would therefore be expected to be more sensitive than the refractive index detector when benzene is the eluent and acetophenone is the eluate. Similarly, ethyl benzoate has a refractive index of 1.5007 and a dielectric constant of 6.02. With the refractive index detector, ethyl benzoate would therefore give a small
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Figure 6. Separation of approximately equal amounts of methoxybenzene, ethyl benzoate, and acetophenone on 2.3 % H20-AI2O3 with eluent benzene negative peak but, with the dielectric constant detector, it gives a fairly large positive peak. It will usually be true in adsorption chromatography that the longer the retention time of a compound, the greater will be its polarity. An increase in polarity generally goes along with an increase in dielectric constant but not always an increase in refractive index. Longer retention times, which result in broader, flatter peaks, are thus partly compensated for by increases in the sensitivity of the dielectric constant detector toward the more polar compounds. The same effect would generally not be observed with a refractive index detector. Holdup Volume. The volume inside the capillary and
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
1985
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Figure 7. Detector linearity: peak area vs. mass of eluted compound between the brass cylinders was 0.7 ml. By reason of this large volume, the geometry of this cell makes it unsuitable for high performance liquid chromatography (HPLC). Under the worst possible conditions of turbulent flow between the electrodes of the cell containing a given concentration of solute, three or four milliliters of pure solvent must pass through the cell to reduce the solute concentration by 99%. This effect contributes to the significant broadening of peaks for compounds having small retention volumes. Any of a large number of other cell geometries could have been chosen which would have resulted in a substantially smaller holdup volume. For example the carefully machined 11.7-kl cell of Vespalec and Hana ( 3 ) could better serve the requirements of HPLC. There is no theoretical lower limit to the size of the dielectric constant detector cell, although the smaller it is, the greater will be the need for shielding, thermostating, and other forms of stabilization in the electronic circuit. Detector Linearity. If the peak area on a chromatogram for a given compound is proportional to the mass of that compound responsible for the peak, the detector is linear. It is assumed, of course, that all operating parameters, such as flow rate, eluent composition, temperature, and recorder chart speed, are constant during the recording of the chromatogram. To determine whether the detection system used for these studies was linear, samples of methoxybenzene varying in size from 4.4 to 44 mg were eluted with hexane through a column of sand 1-cm diameter X 25-cm long. Peak areas were measured by triangulation. The plots in Figure 7 show that the detector response was linear within the range of sample sizes measured. Temperature Dependence. Dielectric constants of liquids and gases generally decrease with increasing temperature. The work of Huckel and Wenzke (IO), for example, indicates that an increase of about 0.007 in mole fraction of a 2-mole percent solution of benzyl alcohol in benzene is equal in dielectric constant change to a drop in temperature from 30 to 20 OC. Thus, a 1 "C temperature decrease is approximately equivalent to a 1 mg/ml concentration increase. An experiment with the detection system used in these studies showed a similar effect of roughly the same order of 1986
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Figure 8. Elution of benzene from 2.3% H20-AI2O3 with binary eluent l % CH2CI2-99% hexane. Absorbance change is shown for comparison. magnitude. A drop in the temperature of hexane flowing through the detector cell from 29 to 27 OC brought about the same increase in signal as 4-mg sample of methoxybenzene eluted with hexane a t the same flow rate from a 1-cm diameter X 25-cm long column of sand. Width of the peak produced was about 2.6 ml, and the height was equivalent to 2 "C temperature decrease. Thus, it is estimated that an increase in concentration of about 1.5 mg of methoxybenzene per ml is equivalent in dielectric constant change to a 1 O C drop in temperature. If these can be considered typical examples, ambient temperature changes and temperature changes brought about by adsorption and desorption of compounds on the column contribute to a decrease in signal-to-noise ratio. For maximum sensitivity, therefore, thermostating of the column, detector cell, and balancing capacitor are essential. On the other hand, when required sensitivities are in the order of a few mg/ml, as is true when liquid column chromatography is used as a preparative tool by synthetic chemists, thermostating is not required, unless large ambient temperature fluctuations are expected. Amphiprotic Solvents. The behavior of this dielectric constant detector was not studied with amphiprotic solvents because the problem associated with this class of solvents, most of which have relatively large dielectric constants, is conductivity. The same geometric parameters of cell design which work to increase cell capacitance also increase cell conductance. The equivalent circuit representing a cell which conducts is a capacitor and resistor in parallel. When the cell is filled with a dry aprotic solvent such as hexane or benzene, the resistive component of its impedance is much larger than the capacitative component at audiofrequencies. However, when the cell is filled with water or propanol, the resistive component may become much less than the capacitative component. Even moist benzene shows appreciable conductivity. For this reason, it is doubtful that Grant (2) was observing only a change in dielectric constant when he used n-propanol-water mixtures to elute amino acids. Binary Eluents. Binary eluents show some unusual effects. A sample component is, in general, more strongly adsorbed on the column than any component in the eluent. If the eluent contains two components whose polarities differ,
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
a sample entering the column causes desorption of the more polar component so that, in the zone preceding the solute front, the ratio of the more polar component to the less polar component will be greater than it is in the eluent which is entering the column behind the sample. As a result, a zone of higher dielectric constant will travel through the column, not only because of elution of a sample component, but also because of a change in eluent composition. Of course, following the peaks, the reverse will occur: the more polar component will be readsorbed from the eluent creating a region of lower dielectric constant than that of the entering eluent. Therefore, each peak is preceded by a hump and followed by a depression. This same problem of "false peaks and depressions" would occur with the refractive index and microadsorption detectors for the same reasons. Figure 8 shows a chromatogram of benzene eluted with 1%CHzC12-99% hexane. Absorbance measured a t 246 nm vs. volume is plotted on the same chromatogram. As predicted, the dielectric constant maximum slightly precedes the absorption peak. The dielectric constant depression is clearly seen. Advantages and Disadvantages. The dielectric constant (DC) detector is, in general, not as sensitive as the ultraviolet absorption (UV) detector except in those instances where the solute does not absorb in the ultraviolet or where the solvent does. Like the refractive index (RI) detector, the DC detector in its simplest form cannot be used with gradient elution. Both the RI and the DC detectors require careful thermostating to realize their maximum sensitivities, whereas the UV detector is rather insensitive to temperature changes. The DC detector is sensitive to flow rate. The UV and IR detectors, in general, are not. The DC detector described here could not be used with amphiprotic solvents and other solvents which show appre-
ciable conductivity without adding some compensating components to the electronic circuitry. With such modifications, it would become a hybrid conductivity-dielectric constant detector. The biggest advantage of the DC detector is its simplicity. Although for greatest stability, it must be held a t constant temperature and be shielded from the effects of stray capacitances and external electrical fields, unlike the UV and RI systems, it requires no light sources, monochromators, light detectors, or other optical components. The DC detector response is linear over the concentration ranges of interest in liquid chromatography. There is certainly a need for a great number of better, more generally applicable detection systems for liquid chromatography and it is the opinion of this author that the dielectric constant detector is one that deserves further study.
LITERATURE CITED D. E. Laskowski and R. E. Putscher, Anal. Chem., 24, 965-969 (1952). R. A. Grant, J. Appl. Chem. (London), 8, Pt. 2, 136-140 (1958). R. Vespalec and K. Hana, J. Chromatogr. 85, 53-69 (1972). 0 . W. Ewing, "Instrumental Methods of Chemical Analysis", 3rd ed., McGraw-Hill, New York, N.Y., 1969, p 478. (5)J. J. Kirkland, "Modern Practice of Liquid Chromatography", Wiley-lnterscience, New York. N.Y., 1971, p 122. (6)S.G. Perry, R. Amos, and P. I. Brewer, "Practical Liquid Chromatography", Plenum Press, New York, N.Y., 1972, p 204. (7) P. R. Brown, "High Pressure Liquid Chromatography", Academic Press, New York, N.Y. 1973, p 28. (8) C. Barbeau, L. Ricard. J. Turcotte, Can. J. Chem., 48, 1698-1702 (1970). (9) "Handbook and Catalog of Operational Amplifiers", Burr-Brown Research Corporation, International Airport Industrial Park, Tuscon, Ariz., 1969, p 44. (10) W. Hijckel and U. Wenzke, 2.Phys. Chem., A&. B. 51, 144 (1942).
(1) (2) (3) (4)
RECEIVEDfor review February 3, 1975. Accepted June 9, 1975.
High-pressure Liquid Chromatographic Analysis of GlycerideBased Lubricants J. A. Sinsel, B. M. LaRue, and L. D. McGraw National Steel Corporation, Research and Development Department, Weirton, W. Va. 26062
Liquid chromatographic techniques for the classwise separatlon and determination of methyl esters, triglycerides, diglycerides, and monoglycerides are described. Such separations are performed via a Ilquld/solid adsorption mechanism employlng a three-step gradlent eiutlon scheme using various mixtures of diethyl ether and n-hexane as the mobile phase. Species thus separated are detected using a Dlfferentiai Refractometer Detector. Quantltation was achieved by constructing calibration curves using working curve slope factors for mlxtures of methyl esters and giycerides of known compositions In conjunction with gas chromatographically obtained composltlonal data concerning fatty acid composltions of the mixtures to be analyzed. By accounting for the different total detector responses to dlffering fatty acid composltlons of separated glyceride species, the callbration method Is completely general for any mixture containing these species. The precisions of measurement for methyl esters, triglycerides, 1,2-dlgiycer-
ides, 1,3-diglycerides, and monoglycerides were found to be f 1 . 4 3 % , f 2 . 4 0 % , f 7 . 9 2 % , f10.50% and f6.00%, respectively, at the concentration levels measured in this study.
Methods and techniques for the separation and analysis of glyceride mixtures utilizing various chromatographic mechanisms are quite abundant in the literature. Several recent reviews have been published which discuss the applications of paper, thin-layer, column, and even gas-liquid chromatographic techniques for the separation and elucidation of glyceride structures in such mixtures (1-9). References concerning the application of high-pressure liquid chromatography (HPLC) to the problem of glyceride analysis are, however, far less abundant and it appears that only gel permeation (GPC) chromatographic mechanisms have been applied extensively to such separations. These methods have been reviewed by Bombaugh (10). Most of
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