Anal. Chem. 1990, 62, 1545-1547
Although HDITCP does not show bleaching under laser diode radiation, the flow through system is more advantageous if other, more sensitive dyes are used. Previous work with NIR laser dyes has shown the possibility of bleaching, especially in aqueous solutions (7). The flow operation of this system permits cleaning and solution replacement so that the cell need not be removed from the optical path between measurement of different solutions. Thus, optical alignment of the system is easier. The data presented in Figure 2 are indicative the multipass effect, Le., a more than 50% change is observed in the PIN diode signal at C0.06 absorption units. The highest absorbance that could be detected without compromising linearity was 0.08 AU, in good agreement with the theoretical calculations. The ( I - Ith)dependence of the sensitivity of the method was studied earlier. It was found (11)that the sensitivity of the LDIS does depend on how far the system is operated from the threshold current as predicted by eq 12. This indicates that individual laser diode parameters can influence sensitivity. Naturally, the absolute maximum rating of the recommended electrical characteristics of a particular laser diode will determine the maximum operating current and therefore the sensitivity. In conclusion, we have shown that a simple laser diode intracavity device that may be simply constructed will operate reliably and produce a linear output signal without using an external detector. Further studies will be necessary to determine the dependence of average effective optical path length on the reflectance of the front face mirror of the laser diode chip. It is expected that the best results can be obtained by using antireflective coated laser diode chips (eq 8 refers
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to losses from front facet mirror reflectance). These results may prove useful in HPLC, where NIR dyes could be used to label compounds and selectively detect them in the presence of a complex matrix. This process could be effectively used to minimize background interference. NIR dyes and laser diodes may also prove to be useful in the determination of biologically active molecules if NIR dyes are used as labels, where the inherently low interference of the NIR spectral region should improve detection because of higher detection power and higher selectivity. Higher detection power arises from the laser intracavity detection method and the high molar absorptivity of NIR dye labels. Higher selectivity is a direct result of the low interference of the NIR spectral region.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)
Harris, J. M.; Dovichi, N. J. Anal. Chem. 1980, 52,695A. Bennett, H. S.; Forman, R. A. Appl. Opt. 1977, 76, 2834. Tran, C. D. Anal. Chem. 1988, 6 0 , 182. Kawabata, Y.; Imasaka, T.; Ishibashi, N. Talanta, 1986, 33, 281. Bialkowski, S. E.; He, 2 . F. Anal. Chem. 1988, 6 0 , 2674. Harris, T.; Mitchell, J.; Shirk, J. S. Anal. Chem. 1980, 52, 1701. Sauda, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1986, 58, 2649. Imasaka, T.; Tsukamoto, A.; Ishibashi, N. Anal. Chem. 1989, 67, 2285. Svelto, 0. Principles of Lasers; Plenum Press: New York, 1989. Unger, E.; Patonay, G. Anal. Chem. 1989, 67, 1425. Hicks, J.; Patonay, G. Anal. Instrum. 1989, 18, 213. Harris, T. D.; Mitchell, J. W.Anal. Chem. 1980, 52, 1706.
RECEIVEDfor review December 13,1989. Accepted March 26, 1990. This work was supported in part by a grant from the National Science Foundation (CHE-8920456). Acknowledgment is also made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research.
Device for Subambient Temperature Control in Liquid Chromatography Lane C. Sander* and Neal E. Craft National Institute of Standards and Technology, Center for Analytical Chemistry, Gaithersburg, Maryland 20899 Although regulation of column temperature in liquid chromatography is less common than in gas chromatography, temperature remains an important separations parameter. In reversed-phase liquid chromatography, as with gas chromatography, absolute retention varies invenely with temperature (In (123 However, unlike gas chromatography, retention in LC is more conveniently adjusted by altering the mobile-phase composition. For this reason, the potential benefits of temperature control in liquid chromatography have gone largely unnoticed. Separations are commonly carried out under ambient conditions, although greater reproducibility usually results when isothermal conditions are maintained (1). In addition, the use of elevated column temperature has been reported as a way of increasing separation efficiency (2), particularly for large molecular weight solutes (3). Recently, changes in column selectivity that occur at subambient temperatures have been reported ( 4 ) . Enhanced shape recognition was observed among isomeric polycyclic aromatic hydrocarbons (PAHs) a t reduced temperatures. Changes in selectivity with temperature also have been observed for other classes of solutes, including carotenoid (5) and steroid isomers (6). The variation in column selectivity with temperature is a universal effect that is not specific to column brands or phase type (4). In general, enhanced specificity for isomeric mixtures is usually possible for separations carried out a t reduced temperatures.
l/n.
Commercial instrumentation for column temperature regulation typically consists of an insulated metal block in which elevated temperatures may be achieved by resistive heating. Other designs include forced hot air ovens and column "jackets" through which a fluid is recirculated by a controlled-temperature bath. The latter method has the potential for subambient operation and provides excellent, uniform temperature control. The disadvantages of this approach include difficulty in changing columns, need for a bulky external refrigerated bath, and hazards associated with use of solvents other than water (for temperatures below 0 "C). The thermoelectric design described below and illustrated in Figure 1 eliminates these disadvantages and permits column cooling to approximately -25 "C. In addition, the device can be used for column heating (to approximately 80 "C) by reversing the polarity of the power supply. The column cooler has added benefits of portability and reduced cost compared to refrigerated bath systems. The thermoelectric cooling plate assembly was fabricated to specification by Thermoelectrics Unlimited, Inc. (Wilmington, DE). The unit consists of water cooled thermoelectric heat exchangers attached to an aluminum top plate measuring 0.32 X 3.8 X 30.5 cm X 1.5 X 12 in.). To this plate is attached an aluminum block 2.5 X 3.8 X 30.5 cm (1 X 1.5 X 1 2 in.) machined to accept standard length (25 cm) LC columns (see Figure 1). Shorter column lengths could also
This article not subject to US. Copyright. Published 1990 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
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Lutein
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Figure 1. Sketch of column cooling device. The shaded area represents the column block assembly, which is machined to accept 0.64 X 25 cm commercial LC columns.
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Flgum 2. plot of the rate of heating and cooling for the column cooling
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be incoporated into the design by slightly modifying the block. The column block is attached to the cooling plate with silicone sealant (at each end) and silicone heat sink compound (along the length of the plate). The glued area is minimized to permit disassembly of the column block should modification become necessary. The chromatographic column is held in contact with the block by two 'I4 X 20 Allen head screws. Such contact facilitates thermal transfer between the column and the column block. An adhesive, magnetic strip was placed on the base of the heat exchanger assembly so the assembly could be placed at various locations on the liquid chromatograph. During operation, the column block assembly is covered with foam insulation to provide uniform temperature across the length of the column. Heat is removed from the heat exchanger with room-temperature ("tap") water flowing at a minimum rate of 1 L/min. The degree of cooling is dependent on the temperature and flow rate of this water. With water at 18.5 OC with a flow rate of 1.25 L/min, it is possible to achieve temperatures at the column block of -24 "C. With chilled water, it should be possible to attain even lower column block temperatures. Temperature regulation is accomplished by varying the voltage output of the power supply to the thermocouples. Temperatures within the range -25 to 80 "C were possible with the power supply, as provided with the thermoelectric heat exchanger. Power supply design is not critical; this design used an unregulated dc power supply capable of supplying 10.6 V at 7 A. Output voltage is varied with a rheostat placed in series with the power supply output. By reversing the polarity of the output, elevated temperatures were possible. The efficiency of the column cooler in the heating mode is greater than that of the cooling mode, and the manufacturer of the thermoelectric cooling module warns against using the device at temperatures exceeding 99 OC. The rate of cooling and heating is illustrated in Figure 2. Constant temperatures were usually achieved within about 45 min of operation using a constant power output setting; however, it is possible to attain a more rapid equilibrium by manually varying power supply settings.
Separation of carotenoid isomers at near ambient and subambient conditions.
Figure 3.
Although active temperature regulation through feedback was not incorporated into the design of the column cooler, column block temperature varied during operation by less than *0.2
"C. Recently, the effects of subambient temperature operation in liquid chromatography have been examined (4). In general better separations of isomers and other classes of solutes that differ primarily in overall shape are possible at reduced temperatures. This trend has been demonstrated for numerous PAH isomer mixtures (4). The same trends have been observed for planar and nonplanar geometric steroid isomers (e.g., 5a- and 5(3-androstane) (6). The observed changes in selectivity are not related to increases in retention that necessarily occur with subambient temperature operation. For example, a t ambient temperature, chrysene and benz[a]anthracene coelute on most commercial CI8 columns, regardless of mobile-phase composition or absolute retention. Under subambient temperature conditions, chrysene and benz[a]anthracene are easily resolved ( 4 ) . Another example of enhanced shape discrimination at subambient temperatures is illustrated in Figure 3. Carotenoids are long-chain conjugated compounds, often differing only in the geometric conformation a t a double bond. A t ambient temperature, the separation of 9 4 s - and trans-@carotene is difficult with most CIBcolumns. Base-line separation can be achieved, however, under reduced temperature. Although the absolute retention of these geometric isomers is increased under subambient conditions, enhanced shape discrimination does not result from increased retention. With a weaker mobile phase composition at ambient temperature, 9-cis- and trans-@carotene are not resolved. The enhanced selectivity toward solute shape observed at subambient temperatures has been attributed to ordering of individual alkyl chains of the stationary phase (4, 7). IR studies have indicated that conformational disorder such as gauche-gauche bends and gauche-trans-gauche' kinks in immobilized alkyl chains are reduced with reductions in tem-
Anal. Chem. 1990, 62, 1547-1549
perature (8),and such changes are well-known for bulk alkanes (9,lO). Ordered alkyl conformations are comparable to liquid-crystal structures, and in fact the retention behavior of liquid-crystal phases in GC is remarkably similar to ordered polymeric CISphases in LC (I 1). A review of shape discrimination effects in liquid chromatography has been published (7)
In conclusion, the column cooler provides a compact, portable means of cooling LC columns below ambient conditions. The design allows for maximum flexibility in use and permits the device to be moved between instruments with little effort, as the need arises.
ACKNOWLEDGMENT We thank Richard Christensen for assistance in machining the column block assembly. LITERATURE CITED (1) Gilpin, R. K.; Sisco, W. R. J. Chromatogr. 1980, 794, 285-295. (2) Warren, F. v,; Bidlingmeyer, B, A. Anal, Chem, lg88, 6 0 , 2021-2824.
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Snyder, L. R.; Kirkland, J. J. Introduction to AMern LiquM Chromatography, 2nd ed.; Wlley-Interscience: New York, 1979. Sander, L. C.; Wise, S. A. Anal. Chem. 1989, 67, 1749-1754. Craft, N.; Sander, L. C.; Pierson, H. F. Submitted to J. Micronoh. Anal. Olsson, M.; Sander, L. C.; Wise, S. A., unpublished research. Sander, L. C.; Wise, S. A. LC-GC 1990, 8, 378-390. Sander, L. C.; Callis, J. B.; Fieid, L. R. Anal. Chem. 1983, 55, 1068- 1075. Snyder, R. G.; Maronceiii, M.;Qi, S. P.; Strauss, H. L. Science 1881, 274, 188-190. Snyder, R. G.; Schachtschneider, J. H. Spectrochim. Acta 1963, 79, 85-116. Wise, S. A.; Sander, L. C.: Chang, H.; Markides, K. E.; Lee, M. L. Chromatographia 1988, 25, 473-480.
RECEIVED for review January 12,1990. Accepted April 1,1990. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
Effects of Ion Source Parameters on Ion Beam Energy in Mass Spectrometry Kuangnan Qian, Ani1 Shukla,* and Jean Futrell Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
INTRODUCTION Tandem mass spectrometry (MS/MS) has become one of the most powerful gaseous-ion chemistry techniques in analytical and structural chemistry (1). For MS/MS studies most commercial mass spectrometers provide good mass resolution but relatively modest energy resolution, especially when primary ion currents are increased for sensitivity reasons. To obtain a good intensity ion beam under electron impact source conditions, strong repeller fields or strong field penetration are often used. The potential field inside an ion source may also be affected by space charge a t higher pressure and electron currents. This combination of circumstances both introduces broad energy distributions and causes the kinetic energy of an ion beam not to be equal to the nominal energy defined by the voltage difference between the source block and the final exit lens. In our ongoing studies (2, 3) of the fundamental reaction dynamics of colliiion-induced dissociation (CID) of polyatomic ions we measure the absolute kinetic energy of the ion beam many times during the course of an experimental series. In these measurements we have observed that the ion energy is dependent on the ion source chamber pressure and the nature of the gas. In this paper, we report a study of the effects of ion source parameters on ion energy and rationalize our results on the basis of a combination of field penetration and surface and space charge effects. Since our experiments have utilized an unmodified (commercial) ion source, we assume that these effects are ubiquitous and should be an important consideration in other experiments for which ion kinetic energy is a significant parameter.
EXPERIMENTAL SECTION The instrument has been described in detail in an earlier publication (4). Briefly, the ion beam is produced by a VG 7070E double-focusing mass spectrometer. The 3-keV ion beam is decelerated by a group of tube and rectangular deceleration lens to the desired energy at the point where it collides with a su-
personic beam of neutrals. The kinetic energy of the parent and fragment ions is measured by a hemispherical energy analyzer. A quadrupole mass filter is used for mass analysis. AU the voltages on the lenses and energy analyzer are referenced to the ion source voltage. Recently the entrance and exit slits on the energy analyzer were reduced to 3 mm X 7 mm each for improved resolution in both energy ( M / E = 0.02) and angle. Prior to installation in our instrument the energy analyzer was calibrated for measuring absolute ion kinetic energy in several ways. First, the geometry of the device defines ion energy quite accurately except for fringing fields at the ion entrance and exit slits of the analyzer. Computer simulations of three-dimensional ion trajectories provided us with a reliable estimate for these effects and an assessment of the transmission function for different slit sizes. Calibration using an ion source having a well-defined ion energy and energy distribution provided us with experimental confirmation of the theoretical predictions and basis for a correction to the theoretical equations. Finally during each experiment the energy analyzer calibration is checked independently by measuring the absolute energies of both the parent and the daughter ion formed by unimolecular metastable decay within the collision region of the tandem mass spectrometer. The precision with which the kinetic energy can be measured is 0.1 eV, and the absolute energy is determined with an estimated accuracy of 10.1 eV.
RESULTS AND DISCUSSION Figure 1 shows the effect of ion source pressure on the kinetic energy of argon, acetone, propane, and nitromethane primary ions. Data are plotted as the mean of the energy distribution as measured by the hemispherical energy analyzer after primary ions are decelerated to 50 eV versus the pressure reading from an ion gauge connected to the ion source vacuum chamber. The actual ion source pressure depends on the gas and is about 100 times greater than the plotted value. The ordinate is the maximum of the measured energy distribution minus the nominal energy defined by the potential difference between the ion source of VG 7070E mass spectrometer and the potential of the last plate of the deceleration lens. These
0003-2700/90/0382-1547$02.50/00 1990 American Chemical Society
