Anal. Chem. 1988, 60, 377-380 80
Electrokinetic Injection
LITERATURE CITED
Hydrostatic Injection
(1) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, Th. P. E. M. J. Chromatogr. 1979, 769, 11-20. (2) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (3) Pretorius, V.; Hopkins, 6. J.; Schieke, J. D. J. Chromatogr. 1974, 99, 23-30. (4) Tsuda, T.; Nornura. K.; Nakagawa, G. J. Chromatogr. 1983, 264, 385-392. (5) Tsuda, T.; Mizuno, T.; Akiyama, J. Anal. Chem. 1987, 59, 799-800. (6) Lukacs, K. D.; Jorgenson, J. W. HRC CC,J . H/gh Resolot. ChromatWr. 1985, 8, 407-411. (7) Hiang, X.; Pang, T.-K. J.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1987, 59, 2747-2749.
40 -
-0
4
8
12
Resistance
377
Xiaohua H u n g Manuel J. Gordon Richard N. Zare*
16
(kill
Flgure 2. Plot of K+ and Li+ peak areas as a function of sample solution resistance for both electrokinetic and hydrostatic Injection. Electrokinetic injection causes a bias linear in sample solution resistance (which is inversely proportional to electrolyte concentration).
almost linearly with sample solution resistance. This bias needs to be recognized when comparing the results from different sample solutions if it is desired to place all data on a common footing.
Department of Chemistry Stanford University Stanford, California 94305
RECEIVED for review August 10, 1987. Accepted November 2,1987. Support for this work by Beckman Instruments, Inc., is gratefully acknowledged.
TECHNICAL NOTES Temperature Control and Local Heating Effects in Laser- Illuminated Samples Cooled by Closed-Cycle Helium Refrigerators J. W.Hofstraat,*’ A. J. Schenkeveld, C. Gooijer, a n d N. H. Velthorst Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Here we report on the applicability of a closed-cycle helium refrigerator for cooling of samples in laser-excited Shpol’skii and fluorescence line-narrowing (FLN) spectroscopy. In both techniques low-temperature solid samples are employed to obtain spectra that show vibrational resolution, and hence have similar selectivity as an infrared spectrum, but with the inherent sensitivity of fluorescence spectroscopy (1-3). The closed-cycle helium refrigerator contains gaseous helium as refrigerant; the gas is cyclically pumped through a closed system so that no helium is consumed (4),as in the generally applied helium-bath cryostats. However, in the latter instrument cooling is more direct as the sample is in contact with the refrigerant; the closed-cycle apparatus applies conductance cooling. In studying the applicability of a closed-cycle cooling system in low-temperature spectroscopy it is appropriate to discern several aspects. Firstly, it has been shown that in Shpol’skii spectroscopy fast freezing of samples is required to obtain narrow-line spectra of compounds that do not fit well into the n-alkane matrix (5). A study in our laboratory has shown that such fast freezing can also be realized in a closed-cycle system provided that a suitable construction of the sample holder is employed to ensure good thermal contacts (5). ‘Present address: Ministr of Traffic and Public Works, Department of Public Works, Ti& Water Division, Nijverheidsstraat 2, 2288 BB Rijswijk, The Netherlands.
Secondly, particularly in FLN sufficiently low temperatures, i.e. below 30-50 K depending on the mode of excitation and the nature of the system studied, are required in order to get useful spectra. Namely, as the temperature increases an intensity shift occurs from the narrow lines to broad spectral features (“phonon wings”) that arise from the coupling of the electronic transitions of the guest molecule with vibrational transitions of the host lattice (1). In addition, temperature rise results in a broadening and shift of the narrow lines (I). Recent experiments in our laboratory have shown that temperatures down to 10 K, readily attainable with closed-cycle refrigerators, suffice for application of low-temperature high-resolution fluorescence techniques (6). From the above it is evident that effective temperature control and stability are crucial for the reproducible application of low-temperature high-resolution fluorescence techniques. Especially when highly intense laser excitation is employed, one has to be alert for (local) heating of the conductively cooled sample. In this paper, first the effects of inadequate cooling will be discussed as related to the FLN spectrum of tetracene in a polyethylene film. Secondly, optimal conditions for experiments with closed-cycle systems will be sketched. Under such optimal conditions the performance of the system with respect to temperature control, stability, and homogeneity across the sample will be investigated. As an internal temperature probe the phenalenyl radical is used, a compound with temperature-dependent spectral
0003-2700/88/0360-0377$01.50/00 1988 American Chemical Society
378
ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988
Figure 1. Effect of temperature on the 0-314 cm-' vibronic FLN band of tetracene in polyethylene film. A mixture of glyceroVwater was added to the sample to improve the thermal contact. The sample was excited with 2 mW of the 476.5-nm laser line.
features (7,8). This compound permits reasonably accurate temperature measurement in the 10-70 K region. Another compound with temperature-dependent spectral features is xanthone (9). The phosphorescence spectrum of xanthone in n-pentane shows emissions from three dynamically coupled states enabling temperature determination up to about 15 K. EXPERIMENTAL SECTION Methods. Samples were contained in a sample holder consisting of two sapphire plates separated by a 0.4-mm-thick indium ring and held in a gilted-copper mount made in one piece. The mount was attached to the cold tip of a CTI-Cryogenics Cryodyne Model 21 closed-cycle helium refrigerator equipped with a homemade gilted-copper radiation shield. The temperature could be varied with a homemade thermostat between 10 and 300 K. A calibrated DT-500 silicon diode from Lake Shore Cryotronics was attached to the cold tip to measure the temperature. For excitation the 476.5- and 514.5-nm lines of a Coherent CR 10 argon-ion laser were used. A spot of about 3 mm2 was irradiated. The laser intensity was determined with a Model 404 power meter from Spectra Physics. The emitted light was separated by a 2 X 0.425 m Jeol JRS 400D double monochromator. The signal was processed by an EM1 95586 photomultiplier cooled to -20 "C and a Jeol photon counting system. Materials. The phenalenyl radical was obtained by photolysis at 40 K (for about 2 h with the 254-nm line of a 100-W highpressure mercury arc) of a M solution of 6b,7a-dihydro-7Hcycloprop[a]acenaphthylene in n-pentane or n-hexane (7).
This compound was prepared according to the method described by Hunadi and Helmkamp (IO). Following photolysis, the nhexane solution was annealed to 80 K to simplify the spectrum. The total annealing cycle took about 15 min. The tetracene in polyethylene film sample was prepared by soaking the polymer sheet for 1 day in a saturated solution of tetracene in ethanol at 80 "C. Tetracene was obtained from Eastman Kodak. Solvents used were n-pentane, n-hexane, and ethanol, all Baker analyzed reagents, glycerol (99+ % ) from Janssen Chimica and demineralized, distilled water from our laboratory. RESULTS AND DISCUSSION It is very important to maintain the sample at a constant temperature, in particular in FLN, as these spectra are much more sensitive to temperature fluctuations than the Shpol'skii spectra (cf. Figure 1). Moreover, in FLN laser excitation is always employed; when high laser energies are used, one has to be aware of possible local heating effects, as many of the excited molecules lose their energy nonradiatively, provided that the fluorescence quantum yield is not 100%. When a bath cryostat is employed, no problems are expected, because the sample is directly in contact with the cryogenic substance, which has a constant temperature of 4.2 K. The closed-cycle
refrigerator relies on conductance for cooling, so that the heat conduction properties of the matrix materials and sample-cell construction become important. In this section we will first show that an inadequate thermal contact can be determined from the spectral data and subsequently try to define optimal experimental circumstances. Next, under these circumstances, the local temperature in the sample will be determined by means of an internal probe with a strongly temperature-dependent spectrum. As the organic solvents that are generally used in highresolution fluorescence spectroscopy are very poor heat conductors, one has to reckon with possible local heating of the sample. Heating may be invoked by bad contacts between cold stage and sample holder or between sample holder and sample. Especially when solid substrates as polymer sheets or TLC plates are employed as samples, one has to be aware of the latter effects. In our experimental setup we have done the utmost to avoid the first type of bad thermal contact by using (1) thin indium sheet between cold stage and sample holder, (2) a sample holder constructed from one piece of copper, (3) sapphire in stead of quartz windows in the sample holder, (4) indium wire between the optical windows and the sample holder and in between the optical windows as spacer, and (5) a gilted-copper sample holder and a gilted-copper radiation shield enclosing the sample to reduce the increase in heat load due to ambient radiation. Despite all of the above modifications, in some measurements with the closed-cycle helium refrigerator marked local heating effects were observed, which fortunately can be easily assessed. One example is shown in Figure 2 for the fluorescence of tetracene in polyethylene film after excitation by the 476.5-nm argon-ion laser line. Figure 2a shows the rapid decay that is observed for the 0-314 cm-l vibronic fluorescence line of tetracene when 100 mW of laser light is focused on the sample. The decay appears to have a half-life of about 3 s; however, when laser irradiation is ceased and the sample is kept in the dark for approximately 45 s the original fluorescence intensity has been completely restored. The reversibility of the decay implies that it cannot be the result of a hole burning process. The attribution to local heating is sustained by Figure 2b where a scan of the steady-state 0-314 cm-l vibronic fluorescence band under 476.5 nm excitation with increasing laser intensities is depicted. It appears that up to 2 mW of laser intensity the general shape of the band, e.g. the relative intensities of ZPL and PW, remain unchanged. Moreover, the intensity of the ZPL increases linearly with the laser intensity. At the same time no fast and reversible decay similar to that shown in Figure 2a is observed. When higher excitation intensities, e.g. 10 mW, are employed, the ZPL is found to be strongly reduced in intensity as compared to its PW and has ceased to vary linearly with the laser photon flux. Now a similar, though less sharp, reversible decrease in intensity of the ZPL as shown in Figure 2a is observed. These results can be easily explained on the basis of local heating; up to laser powers of about 2 mW the cooling capacity of the closed-cycle refrigerator is sufficient to maintain the low temperature (in this case 10 K) of the sample. At higher laser intensities the cooling capacity appears insufficient so that heating occurs at the place of excitation. Consequently the spectra reflect an effectively higher temperature than indicated by the silicon diode that is attached to the cold tip of the cryogenic. However, we have been able to reduce the local heating effects in polyethylene film to a great extent by adding some glycerol/water solution to the polymer sample. This mixture is highly polar so that tetracene (and most other PAHs) hardly dissolves and easily forms a glass of good optical quality at low temperatures.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 4, FEBRUARY 15, 1988 a
30s
c3
379
Table I. Temperature Stability in Closed-Cycle Helium Cooler laser irradiation power, W 0.015 0.045 0.14
temp: K
laser irradiation power, W
temp: K
24.5-25.5 26-27 27.5-28.5
0.42 0.83 1.35
28-30 30-33 30-33
"Temperature was set to 25 K; measurements were made in npentane with the phenalenyl radical as the internal temperature Drobe.
b
Flgure 2. (a) Decays of the 0-314 cm-' vibronic fluorescence ZPL of tetracene in potyethyhe film. The sample was Illuminated with 100 mW of laser light. In between the decays the sample was kept in the dark for 45 s. (b) Scans of the steady-state 0-314 cm-' fluorescence band of tetracene in polyethylene at several laser intensities. The 476.5-nm laser ilne was used for excitation both in a and b. The temperature of the cryogenic cold tip was 10 K.
When no precautions are taken, severe local heating can also be observed for molecules on thin-layer chromatography (TLC) plates. For these kind of samples thermal contact can be improved by either adding glycerol/water solution or by replacing the sapphire window a t the back of the sample by a copper disk covered with indium sheet. The latter procedure is to be prefered as addition of solvent may lead to blurring of the spots on the TLC plate. Having improved the obviously bad thermal contacts the question remains how well the low temperature is maintained in the sample when a conventional organic solvent is employed as the matrix. As such solvents themselves are poor heat conductors here also local heating effects or even temperature gradients across the sample may occur, especially when high laser powers are appljed. Unfortunately, the silicon diode used to register the temperature cannot give information on the actual, local, temperature in the sample as it is mounted on the cold stage of the cryogenic. An elegant way to measure the temperature in the sample is to use as a probe a system that shows temperature-dependent spectral features. An example is the phenalenyl radical in n-pentane and n-hexane Shpol'skii matrices; it can be excited by the 514.5-nm argon-ion laser line as its fluorescence spectrum starts around 535 nm. Its spectrum shows a number of bands whose intensities are temperature dependent (8). In particular, the intensities of the three
dynamically coupled sites in the 0-0 transition region can be used for temperature estimations. In n-pentane the site at 535.36 nm, which is the strongest transition at low temperature, loses intensity upon temperature increase. The two close-lying higher energy sites, at 534.67 and 534.87 nm, at the same time gain intensity. In n-hexane, two sets of three dynamically coupled sites are observed: one at about 536 nm, the other in the 539 nm region. The temperature dependence of the site intensities was examined quantitatively by using relatively low intensities of the 514.5 nm line for excitation, in order to avoid possible local heating of the matrix. We will not go into the mechanism behind the temperature dependency of the phenalenyl fluorescence here, but merely mention that the intensities reflect a normal Boltzmann distribution law, varying as exp(-l/7') with the temperature. The temperature dependencies of the spectra that are obtained in n-pentane and n-hexane are slightly different. In n-pentane the energy differences evaluated from the Boltzmann law amount to 35 h 5 cm-' for the two higher energy sites with respect to the one with the lowest energy. In practice, this implies that temperatures down to 15 and 25 K, respectively, can be measured with sufficient accuracy. Phenalenyl in n-hexane shows a much more complicated 0-0 site structure. The intensities of the components of the main site, around 536 nm, appear to be separated by only 20 f 5 cm-' according to the Boltzmann law so that temperatures down to 10 K can be determined from their intensity ratio. Using the phenalenyl radical as internal temperature probe, we have attempted two kinds of experiments. First, we have measured the effective temperature at different places in the sample to get an impression of its thermal homogeneity. Secondly, we have determined the effective temperature as a function of laser intensity. It appears that in our experimental setup the thermal homogeneity is surprisingly good. Even when relatively high laser intensities (0.5 W) are employed the temperature at any place in the sample is the same within 1-2 K. At lower laser intensities even better results are obtained. The measurements have been done at 20 K, maintaining the temperature with our homemade thermostat. Replicate measurements at one position show that the temperature is constant in the time within 1 K. In the second type of experiments the thermostat has been set to 25 K and the laser intensity varied from 15 mW to 1.35 W. Up to about 50 mW no significant increase in effective temperature is measured (it varied from 24 to 26 K). However, when the laser intensity is increased further, a gradual increase in temperature is observed (see Table I). Illumination of the sample with 1.35 W of laser light finally leads to a temperature rise of 5-8 K. Still, at such high excitation intensities no striking temperature variations over the sample are measured so that the cooling efficiency at any place in the sample must be about the same. Thus, for each laser power a thermal equilibrium is reached depending on the intensity used. The equilibrium temperature of course increases as the intensity
380
Anal. Chem. 1988, 60,380-383
grows. Furthermore it is interesting to note that at higher intensities the sensor a t the cold tip also indicates a temperature increase, but less than the internal probe. Though the experiments described above are exemplary, they show clearly that when conductance cooling in a closed-cycle helium refrigerator is employed quite good temperature stability can be realized even with relatively high laser powers. One always has to be aware, however, of the possibility of a slight rise in effective temperature at high excitation intensities. This temperature increase is also observed when precautions have been taken to ensure good thermal contacts between sample and cold tip, as in our experimental setup. When thermal contacts are bad, like for tetracene in the polyethylene measurements where no attention was paid to the contact of the polymer and the sample holder, increased laser intensities give rise to poignant temperature effects which are fortunately easily recognizable.
LITERATURE CITED Hofstraat, J. W.; Gooijer, C.; Velthorst, N. H. I n Molecular Luminescence Spectroscopy: Methods and App//caflons,Part 2 ; Schulman, S . G., Ed.: Wiley: New York, to be published. Wehry, E. L.; Mamantov, G. I n Molecular Ruorescence Spectroscopy: Wehry. E. L., Ed.; Plenum: New York, 1981; Vol. 4, p 193. De Lima, C. G. CRC Crit. Rev. Anal. Chem. 1986, 76, 177. Vibrational Spectroscopy of Trapped Species: Hallam, H. E., Ed.: Wiley: New York, 1973. Hofstraat, J. W.; Freriks, I.L.; De Vreeze, M.; Gooijer, C.; Velthorst, N. H., submitted to the J. Phys. Chem. Hofstraat, J. W.; Schenkeveld. A. J.; Gooijer, C.; Velthorst, N. H., submitted for publication in Spectrochim. Acta. Cofino, W. P.; Van Dam, S. M.; Karnming, D. A.; Hoornweg, G. Ph.; Gooijer, C.; MacLean, C.; Velthorst, N. H. Mol. Phys. 1984, 57, 537. Hofstraat, J. W.; Schenkeveld, A. J.; Gooijer, C.; Velthorst, N. H., submitted for publication in J . Mol. Struct. Griesser, H. J.; Bramley, R. Chem. Phys. Lett. 1982, 88, 27. Hunadi, R. J.: Helmkamp, G. K. J . Org. Chem. 1978, 43. 1586.
RECEIVED for review May 29,1987. Accepted September 22, 1987.
Determlnation of Aluminum in Dialysate Concentrates by L'vov Platform Graphite Furnace Atomic Absorption Spectrometry Johanna Smeyers-Verbeke* Farmaceutisch Instituut, Laboratory for Pharmaceutical and Biomedical Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium Dierik Verbeelen Academisch Ziekenhuis, Renal Unit of the Department of Medicine, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium The determination of aluminum in biological materials by means of graphite furnace atomic absorption spectrometry has been the subject of many publications (1-5). Most attention has been given to the analysis of serum since serum A1 values can be used for diagnosing and for monitoring dialysis patients a t risk of aluminum intoxication (6, 7). In patients with renal failure treated by means of dialysis it is well established that, besides the ingestion of aluminum containing phosphate binders, aluminum contaminated water and dialysis fluids can cause aluminum toxicity. It is evident that proper control of A1 concentrations in water and dialysis fluids reduces the risk of an aluminum accumulation in these patients. Although the water used for the dilution of the dialysate concentrates seems to be the main source of the aluminum present in the dialysis fluids, a contamination of the concentrates themselves remains possible. Most work on dialysis solutions has been done on diluted which are prepared before use from hemodialysis fluids (8,9) dialysate concentrates by a dilution of about 35 times with water. The problem with the analysis of the concentrates is the very high salt content of these solutions. Typically they contain about 400 g/L of sodium, potassium, calcium, and magnesium chloride and sodium acetate. Here we report on the determination of aluminum in dialysate concentrates by means of graphite furnace atomic absorption spectrometry using the L'vov platform and ammonium nitrate as matrix modifier. The difficulties encountered during the development of the procedure are discussed. MATERIALS A N D METHODS Equipment. A Zeeman 3030 atomic absorption spectrometer equipped with an AS-60 autosampler and a PR-100 printer (Perkin-Elmer Corp., Norwalk, CT) was used for the measure-
ments. Pyrolytic graphite tubes (part no. B010-9322) with pyrolytic platforms (part no. B012-1091) were used. The instrumental conditions were as follows; drying at 160 "C for 15 s in ramp and 15 s in hold mode; charring at 600 "C for 30 s in ramp mode and 10 s in hold mode and 1600 "C for 40 s in ramp and 10 s in hold mode; atomization at 2500 "C, 0 s ramp (maximum power) 5 s hold; clean out at 2700 "C for 4 s. Argon gas flow was 300 mL/min except for the atomization, which was done in gas stop condition. An aluminum hollow cathode lamp was used at a wavelength of 309.3 nm. Background correction was used for all measurements. Contamination Control. Precautions were taken to avoid contamination as described elsewhere ( I ) . Composition of the Concentrate. The composition of the concentrate used in this investigation was as follows: NaCll87.0,
[email protected],KC19.5, CaC12.2Hz08.8, and MgC12.6H203.3 g/L. For use in the dialysis unit this solution was diluted 32 times with reverse osmosis purified water. Reagents. Standard Al solutions were prepared from Titrisol standard solutions containing 1 g/L of aluminum (Merck, Darmstadt, FRG). The water used to prepare all solutions was doubly distilled in a quartz device just before use. It contained no detectable Al. Nitric acid was of Suprapur grade and ammonium nitrate of "pro analysis" grade (Merck). A 100 g/L (10%)solution of ammonium nitrate in water was prepared in a plastic container and used as matrix modifier. Spiking of Concentrates with Al. The addition of A1 to concentrate solutions was performed by pipetting 20,50, and 100 pL of a 1 mg/L A1 solution to 1mL of concentrate. This results in samples with,respectively, 19.6,47.6, and 90.9 pg/L of Al added. For comparison, standard solutions were prepared in the same way by replacing 1 mL of concentrate by 1 mL of HN03 0.2%. Recommended Analytical Procedure. Standards of 0, 10, 20, 40 and 60 pg/L of aluminum in "OB 0.2% (v/v) were prepared in quartz volumetric flasks. These were used during
0003-2700/88/0360-0380$01.50/00 1988 American Chemical Society
