with the PN-MIP-AAS system. The value obtained was 9%. The glasa frit nebulizer uses a lower argon radiation making less flow turbulence and longer sample residence time in the plasma than the pneumatic nebulizer; it nebulizes finer and lesser amounts of aerosol, thus enhancing the atomization efficiency compared to the PN. The glass frit nebulizer is thus superior for the present system in terms of extended torch lifetime and analytical figures of merit. However, some pneumatic nebulizers operate at an argon flow rate similar to that for the glass frit nebulizer; w e of these nebulizers will then make the comparison proceed under similar operating conditions. The present torch provides the hollow cathode lamp radiation with a absorption path length of approximately 2 cm in the MIP, which is several times longer than the path length in the conventional ICP. The hollow cathode beam fills the entire discharge tube. The discharge tube also has a geometry and viewing mode similar to those of the graphite tube employed in electrotheral atomic absorption spectrometry. Therefore, this plasma torch has a potential for sensitive atomic absorption measurement, and the present analytical figures of merit should be improvable.
ACKNOWLEDGMENT K.C.N. is grateful to California State University a t Fresno for the 1987-1988 assigned time for research. The assistance of Dave Zellmer in drawing Figure 1 is acknowledged. Registry No. Mn, 7439-96-5; Cu, 7440-50-8; Ca, 7440-70-2. LITERATURE CITED Wendt, R. H.; Faasel, V. A. Anal. Chem. 1988, 38, 337. Greenwdd, S.; Smith. P. B.; Breeze, A. E.; Chllton, N. M. D. Anal. chkn.Acta 1988, 41, 385. Veilon, C.; Margoshes, M. Spectrochkn. Acta, Part 8 1988, 238, 503. Morrison, 0. H.; Talma, Y. Anal. Chem. 1970, 42, 809.
(5) Mermet, J. M.; Trassy, C. Appl. Spectrosc. 1977, 31, 237. (8) Bordonall, C.; Planclflorl. M. A. Roc. X I V CSI 1908, 1153. (7) Bordonall, C.; Plancltiori, M. A. US. Patent 3884884, August 15, 1972. (8) Huang, X.; Lanauze, J.; Wlnefordner, J. D. Appl. Spemsc. 1981, 38. 1042. (9) Greenfield. S. 1984 Winter Conference on Plasma Spectrochemistry, Sen Diego. CA, Paper 54. (10) Magyer, B.; Aeschbach, F. Spsctwchh. Acta, Part 8 1980. 358, 839. (11) Downey, S. W.; Nogar, N. S. Appl. Specbosc. 1984, 38, 878. (12) Gillson. G.; Horllck, G. Spectrochlm. Acta, Part 6 1988, 418, 431. (13) Umemoto, M.; Kubota, M. Spectrochm. Acta, Part 8 1987, 428, 1053. (14) Llang, D. C.; Blades, M. W. Anal. Chem. 1988, 60, 27. (15) Ng, K. C.; Shen, W. Anal. Chem. 1988. 5 8 , 2084. (16) Haas, D. L.; C a m , J. A. Anal. C h m . 1985, 57, 846. (17) Deutsch, R. D.; Keilsohn, J. P.; Hleftje. G. M. Appl. Specbosc. 1985, 38. 531. (18) Urh J. J.; Carnahan, J. W. Anal. Chem. 1985, 57, 1253. (19) Ng, K. C.; Brechmann, M. J. Specboscopy(Spr/ngW, oreg.)1987, 2 , 23. (20) Beenakker. C. I. M. Spectwchlm. Acta, (Part 8 ) 1978, 318, 485. (21) Long, 0.L.; Perklns, L. D. Appl. Specfrosc. 1987 41, 980. (22) Nlsemaneepong, W.; Haas, D. L.; Caruso, J. A. Spectrochlm. Acta, Part 8 1985, 408, 3. 'Present address: BSK 8 Associates, 1414 Stanlalaus St., Fresno, CA 93708.
Kin C. Ng* Rader S . Jensen Michael J. Brechmann' William C. Santos Department of Chemistry California State Univeristy-Fresno Fresno, California 93740 RECEIVED for review December 22,1987. Resubmitted July 18,1988.Accepted September 7,1988. Portions of this paper were presented a t the 1987 Pacific Conference on Chemistry and Spectroscopy, October 28-30, Irvine, CA.
Influence of Temperature on Column Efficiency in Reversed-Phase Liquid Chromatography Sir: The increasing commercial availability of temperature-control systems has provided chromatographers with better access to the utilization of temperature in liquid chromatography (LC). Commonly, the column is heated to a few degrees above ambient temperature for purposes of improving the precision of chromatographic data. In reversed-phase LC, changes in chromatographic resolution may be expected due to the influence of temperature on thermodynamic and/or kinetic contributions (1). In some cases, a change in the column temperature has led to improvements in chromatographic selectivity that are not readily achieved through the manipulation of other variables (2-4). In other cases, the primary effect is upon retention (5). In addition to a possible influence on chromatographic selectivity,an increase in column temperature is generally said to improve column efficiency (6),due to a reduction of eluent viscosity and an increase in solute diffusivity. While this generalization appears to hold true for ion exchange separations (7,8), the demonstration of the anticipated influence of temperature on reversed-phase LC is the subject of surprisingly few literature reports. The results of such investigations are mixed and include reports that increased temperature has a beneficial effect (*12), no effect (13),or even a negative effect (14-16)on column efficiency. In one report 0003-2700/8S/0360-2821$01.50/0
(In,
efficiency improved with temperature for a 10-pmCI8 packing but worsened for a 5-pm packing under identical experimental conditions. The reasons for the conflicting results of these studies are difficult to judge for several reasons. In most reports, a full plot of the height equivalent to a theoretical plate (H) w linear velocity (u) is not determined as a function of temperature; rather, H is measured at several temperatures for one or two flow rates. Furthermore, sample loading is not generally addressed, even though this variable can intluence peak shape and hence the experimental value of H. In addition, the apparatus used to control temperature has varied widely. This is significant,since an improperly designed apparatus can lead to peak broadening or even peak splitting (181, thus influencing the observed value of H significantly. As a result of previous work in our laboratq (I)concerning the parameters that influence column efficiency in LC, we chose to investigate the influence of temperature on column efficiency across a wide range of eluent flow rates. The data are used to construct H,u plots and to determine the A, B, and C constants of the van Deemter equation H = A ( B / u ) + Cu (1)
+
where H (millimeters) is the height equivalent to a theoretical 0 1988 American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
SET TEMPERATURE FROM KEYBOARD
i RAM
I
$’
4-m PROPORTIONkL
TEMPERATURE DlFFERENCEi IDENTITY
INTEGRAL
DIFFERENCE
ACTUAL -TEMPERATURE
-
-
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0
SOLID STATE RELAY
-
COLUMN HEATER
Flguro 1. Control system diagram for the temperature control model.
plate. Conventionally, u is the eluent linear velocity, determined as the column length (millimeters) divided by the void time (seconds). Since the proper method for the determination of the void time is a matter of some controversy in the LC community (19))particularly when a range of temperatures is to be used, we have substituted flow rate (milliliters per minute) for linear velocity in the H,u plots presented in this report. The determination of void time, and hence linear velocity, has been considered in some detail in our laboratory and will be discussed in a separate report. The collected efficiency data for this reversed-phase system is compared to theoretical predictions of the influence of temperature on H,u curves. The anticipated decrease of H with increasing temperature is not observed in practice. Rather, H is higher at all flow rates as temperature is increase, both for a steel column and for a rapidly compressed cartridge containing the same reversed-phase packing.
EXPERIMENTAL SECTION The modular chromatographic instrument included a Model M6000A solvent delivery system, a WISP Model 710B automated injector, a Model M441 absorbance detedor, and an SE-120dual pen chart recorder, all from Waters Chromatography Division of Millipore Corp., Milford, MA. Data collection and processing as well as system control were provided by a model 840 Data and Chromatography Control Station (Waters). Temperature control was provided by a TCM temperature control module (Waters) connected to a steel column heater (Waters) or an RCM-100/ column heater (Waters). While the thermostating medium is air in both heaters, preheating of the eluent stream prior to entrance into the column occurs through a length of tubing (approximately 21 in. in the steel column heater and approximately 48 in. in the RCM heater) which is in intimate contact with a metal heating block. In the steel column heater, the column is contained in a metal block and in the RCM-100 heater the entire RCM-100 module is placed into the heating chamber. The schematicof the oven is shown in Figure 1. A programmed microprocessor serves as the control element in the TCM. The program provides a coupled data feedback control system in which the difference between the temperature entered into the memory
and the actual measured temperaturedeterminesthe rate at which the heater reaches the desired temperature. This feedback system results in rapid warmup and stabilization of the system while minimizingthe possibility of a temperature overshoot (20).The microprocessor control also contains a feed-forward compensation to accommodate fluctuations in the power line voltage. A single steel column (15 X 0.39 cm) and a single cartridge (10 X 0.8 cm) both containing Nova-Pak C18 packing material from production batch 1036 were used for all studies. Nova-Pak C18 packing consists of chemically modified 4-pm spherical particles with an average pore size of 70 8, (21). The eluent was 50/50 acetonitrile/water (v/v). To improve the precision of eluent preparation, a laboratory balance was used to measure out the 776.6 g of acetonitrile and 1000.0 g of water that were used to prepare each 2-L batch of eluent. To prepare the test mixture, acenaphthene (0.049 g) dissolved in acetone (600 pL) plus dibutyl phthalate (125 pL) was dissolved in 50 mL of eluent. A study of peak asymmetry as a function of amount injected was done in order to determine the dilution of the test mixture that should be injected for the efficiency checks. Based upon this study, a 1:9 dilution was selected. This dilution provides a suitably strong detector response with a minimum of peak asymmetry. At higher injected amounts, the peaks for dibutyl phthalate were slightly tailed. For the steel column, a 1GpL injection contained 0.98 pg of dibutyl phthalate. For the radially Compressedcartridge, a 26-pL injection was used to maintain the same injected mass per gram of packing. Duplicate chromatogramswere run at every temperature and flow rate. Pressure and flow rate were monitored throughout the study as a check for stable performance of the pumping system. Pressure fluctuations less than about 50 psi were considered acceptable. If pressure fluctuated more than 125 psi during a run, the data were not accepted. All chromatographic runs at a given temperature were repeated if the pressure record revealed unexpected pressure spikes or if the average pressure was not constant. A 5-min equilibration period followed every change of flow rate, in order to allow the pressure trace to stabilize. At each temperature, chromatogramswere run in duplicate at a series of flow rates, starting with the highest flow rate and descending in order to the lowest. For the Radial-Pak cartridge, the flow rates were 3.5,3.0, 2.5, 2.0, 1.5, 1.0, and 0.75 mL/min. For the steel column, chromatogramswere determined at 2.0,1.5, 1.0, 0.75, 0.5, 0.3, and 0.1 mL/min. Upon completion of the experimentsat each temperature, a new temperature was set and the system was allowed one or more hours to reach thermal equilibrium with eluent flowing. Temperatures were changed in the order 35,45,55, and finally 65 “C. Runs at “ambient” temperature (temperature controller switched off) were added for comparison. On several occasions, a chromatogram was rerun at 2.0 or 2.5 mL/min after the completion of the flow rate sequence. Such repeat plate counts were found to agree to within &3% with the plate counts originally measured at the same flow rate. Plate counts were calculated by the area/height method (1). The Scanner utility of the Expert software included with the Waters 840 system was used to determine peak areas and heights with base lines set interactivelyby the user. The Rs/1 Integrated Data Analysis System (BBN, Cambridge, MA) was used for graphics, curve fitting, and data analysis. Specific routines were written within RS-1 for the preparation of H,u curves.
RESULTS AND DISCUSSION Figure 2 presents a typical chromatogram from the study. The experimental conditions were selected to emphasize the column’s kinetic efficiency (1). Neutral solutes and a simple, low-viscosity eluent were selected (6) to ensure rapid diffusion and easy access to the pore structure and to minimize the possibility of undesirable thermodynamic influences on peak symmetry. The previously described loading study ensured that a sufficiently dilute sample was used to minimize the effect of sample amount on peak symmetry. The degree of retention was long enough to keep the influence of extracolumn band broadening a t an insignificant level (6),since k’ values were greater than 7 for acenaphthene and 12.5 for
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988 E0
in Figure 3. Here, the curves have been estimated by using Englehardt's approximate van Deemter equation (22)
+ (2Dm)/u + (0.047.dp2/Dm)u
H = 3.dp
I 10
5
15
20
Minutes
Figure 2. Typical chromatogram, showing separation of the test solutes acetone (11, acenaphthene (2), and dibutyl phthalate (3) using the radially compressed Nova-Pak C18 cartrldge. For this example, the flow rate was 3.0 mL/min and the temperature was 55 OC. 0 OsOOT
0
osa-
D 0,So-
2823
~
I1 I I
-2% 3%
......... 4 %
-- 55c
Llnear Velocity
lmm/secl
Flgue 3. Predicted H,u curves for acenaphthene based on eq 2. (See text for details.)
dibutyl phthalate at all temperatures. Band-broadening theory predicts that the H,u curve should respond to an increase in temperature in the manner indicated
(2) with the particle diameter (dp) set to 4 Mm and the solute diffiwivity (Dm) calculated for acenaphthene according to the WilkeChang equation (23) with viscosity data for the eluent obtained from the literature (13). The linear velocities presented are typical of those that would be measured for the steel column used in this study. According to theory, H is insensitive to solute retentivity for k' of 5 or greater (22). Thus, it was not necessary to consider the influence of temperature on k' in the calculation of the curves in Figure 3. According to Figure 3, two major effects on the H,u curve result from an increase in temperature: (1)the optimal linear velocity is shifted to higher values, and (2) the slope of the right-hand portion of the curve decreases. Note that the A term of eq 2 in independent of Dm, and hence of temperature, leaving the minimum H value unchanged for the curves in Figure 3. From a practical point of view, Figure 3 indicates that H improves with temperature at high linear velocities but worsens at low linear velocities. Since chromatographers frequently work at higher than optimal linear velocities, the general expectation is for column efficiency to improve with increasing temperature (6). Our results for two column types are presented in Figure 4 based on acenaphthene as the test solute. The plots for dibutyl phthalate were very similar to Figure 4. Contrary to expectations based on theory, an increase in column temperature leads to higher H values regardless of eluent flow rate. There is an overall upward shift in the H,u curves for both column types, reflecting an unanticipated effect of temperature on the A term of the van Deemter equation. The effect of temperature on the H,u curve is more dramatic for the radially compressed cartridge. Across the entire flow rate range, the plate count for the cartridge is 26-30% lower at 65 "C than a t ambient temperature. For the steel column, the largest difference in plate count is 20% at 0.3-0.5 ml/min. In addition, the minimum H value for the cartridge is observed to be lower than that of the steel column. This is consistent with a previous report (24) that indicated a substantial re-
>
0.040 0.03a 0.036
0.034 0.032
0.021
I
0.010--
0 .Oi4-0.012--
t
0.0
I
0.1
i.0
i.S
FLOW RATE (rnl/rnin)
f
2.0
a H w
0.021
a
0.020
\
\-.
+
L
0.018 0.014
o.oi2 0.5
FLOW RATE
(rnl/rnin)
Figure 4. Experimental H,u curves for acenaphthene: (A) steel column; (9) Radial-Pak cartridge. Curve symbols: A, ambient; 3, 35 O C ; 5, 55 OC; 6, 65 OC. Curve line types are identical in A and B for a given temperature.
OC;
4, 45
2824
Anal. Chem. 1988, 60, 2024-2027
duction of the A term due to radial compression. However, the steel column used in this study was not specifically selected on the basis of its efficiency, and other steel columns containing the same packing are expected to have lower minimum H values. Some authors have previously reported a negative impact of increased temperature on column efficiency of bonded phases (14-17),although we are not aware of another report that presents the full H,u curve across a range of temperatures for modern (4-10 fim) packings. Poppe et al. (14, 15) and others (16) attribute the negative impact of temperature on H to radial thermal gradients that may exist for thermostated columns if the eluent temperature is not precisely equal to the temperature of the outer walls of the column. Such an effect may explain the trend observed in Figure 4. Indeed, this may be a “fact of life” for commercially available column heaters. Axial thermal gradients (W,26)may also occur due to resistive heating in the column, which causes the outlet end of the column to be slightly higher in temperature than the inlet. Additional studies to test the possibility of thermal gradient effects are ongoing in our laboratories. On the basis of the resulta summarized in Figure 4, we find that improvements in column efficiency for reversed-phase LC systems are not a guaranteed consequence of increased column temperature when temperature-control systems are used. The results presented here do not support the conventional wisdom that efficiency will rise with column temperature in reversed-phaseLC. As mentioned above, it is not possible to rationalize the disagreement between our findings and previous results due to differences in heating devices, eluents, solutes, and packings, as well as the limited data presented in most previous reports. Thus, the decision to use column temperature control in reversed-phasesystems should not be dictated solely by a requirement for increased efficiency, but by other goals such as the improvement of retention time precision or the control of selectivity.
ACKNOWLEDGMENT The authors wish to acknowledge our colleagues, J. Chinen, H. Hirasawa, and T. Sakai, at Nihon Waters (Japan) for bringing to our attention their observation of decreased column efficiency with a reversed-phase column at elevated temperatures. We also thank C. Nugent and A. Weston for assistance with data collection, and U.Neue, T. Dourdeville,
and W. Carson for helpful discussions. We acknowledgethe technical assistance provided by A. Pomfret and R. Fernandes, and we thank J. Newman for assistance in preparing the manuscript.
LITERATURE CITED Bidlingmeye‘, 8. A.; Warren, F. V., Jr. Anal. Chem.1984, 56, 1583A. Terweijgroen, C. P.; Kraak, J. C. J . Chromatog. 1977, 738. 245. Kraak, J. C.; Huber, J. F. K. J . Chromatogr. 1974, 702, 333. Snyder, L. R. J . Chromatogr. 1979, 779. 167. Snyder, L. R.; Kirkland, J. J. Introdxblon to Modem Liquid Chromatography; Wiley: New Ywk. 1979; Chapters 7, 10, and 11. Poole, C. F.; Schuette, S. A. Contenporaty h d c e of Chromatography; Elsevier: Amsterdam, 1984; Chapter 4. Horvath, C. G.;Prelss, B. A.; Lipsky, S. R. Anal. Chem. 1967. 3 9 , 1422. Brown, P. R. J . Chromatog. 1970, 5 2 , 257. MaJors, R. E. In Bonded Statbnafypheses h Chrometography;Bushka, E., Ed.; Ann Arbor Science Publishers: Ann Arbor, MI, 1974. Tsuji, K.; Goetz. J. F. J . chrometog. 1978, 757, 165. Herbut, G.;Kowalczyk, J. S. HRC CC.J . H&h Rem/ut.Chromatogr. Chromatogr. Commun. 1981, 4 , 27. Schmidt, J. A.; Henry, R. A.; Williams, R. C.; Dleckman. J. F. J . Chromatog. Scl. 1971, 9 , 645. Czajkowski, T.; MledzColin, H.; Dlez-Masa, J. Carlos; Quiochon, 0.; iak, I . J . Chromatog. 1978, 767, 41. Poppe, H.; Kraak, J. C.; Huber. J. F. K.; van den Berg. J. H. M. Chromatographia 1981, 14, 515. Poppe, H.; Kraak, J. C. J . Chromatog. 1983, 282, 399. McCown. S. M.; Southern, D.; Morrison, B. E.; Gartelz, D. J . Chromatogr. 1986, 352, 483. 749. W. A.; Jadamec, J. R.; Sager, R. w. Anal. Chem. 1978, 50, Saner, Perchalski, R. J.; Wilder, B. J. Anal. Chem. 1979, 57, 774. Smith, R. J.; Nleass, C. S.; Wainwright, M. S. J . Llq. Chromatogr. 1986, 9 , 1387. Temperature Control System Operator’s Manual, No. 38003, Rev. E, Oct 1986. Warren, F. V., Jr.; Bidlingmeyer, B. A. Anal. Chem. 1984. 5 6 , 950. Engelhardt, H. H&h Performance Llquki Chromatography; SprlngerVertag: Berlin, 1979; p 22. Chang, P.; Wllke, C. R. J . 191ys.Chem. 1955, 59, 592. Fallick, 0. J.; Rausch, C. W. Am. Lab. (FaMleki, Conn.) 1979, 7 7 , 87. BMlingmeyer, B. A.; Hooker, R. P.; Lochmilller, C. H.; Rogers, L. B. sep. Sci. 1989, 4,439. Halasz, I . ; Endele, R.; Asshauer, J. J . Chromatogr. 1975, 712, 37.
F. Vincent Warren, Jr. Brian A. Bidiingmeyer* Waters Chromatography Division of Millipore Corporation 34 Maple Street Milford, Massachusetts 01757 RECEIVED for review November 4, 1987. Accepted June 6, 1988.
Quantitative Infrared Emission Spectroscopy Using Multivariate Calibration Sir: In a recent report on process analytical chemistry, noninvasive, remote methods of analysis were identified as highly desirable (1). Infrared emission spectroscopy (IES) is obviously such a technique but its use has been limited due to (a) lack of suitable instrumentation of requisite sensitivity and ruggedness and (b) difficulty in deriving quantitative information from the observed spectra ( 2 , 3 ) . The advent of process-qualified Fourier transform infrared spectrometers has eliminated the instrumentation problem, but quantitation remains a challenge. Spectral interferences/anomalies due to blackbody emission, temperature gradients, self-reabsorption, and internal reflections all contribute to the complications inherent in infrared emission spectroscopy (2, 4 ) . Some of these problems can be overcome by using optically thin samples (2,5).In fact, IES methods have been used with moderate 0003-2700/88/0360-2624$01.50/0
success for quantitative analysis of gaseous samples-for example, atmospheric studies, stack emissions, physical studies of gases in cells (6-8)-and very thin samples-such as polymer and organic films, molten glasses and salts, and fine powders (4, 5, 9-11). Most approaches to this technique have tried to minimize complicationsby empirical methods. Griffiths has suggested, as have others, that the emission spectrum be ratioed to a blackbody at the same temperature (2). Hvistendahl has proposed ratioing the emission spectrum to an optically thick sample for best results (4). Chase has commented on the problem of multiple passing of reflected radiation through the spectrometer (12). He uses the method of Kember et al. involving four measurementsand interferogram manipulations to remove background contributions(13). Emission intensities 0 1988 American Chemical Society
