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High performance liquid chromatography at -196ºC Tomohiro Motono, Shinya Kitagawa, and Hajime Ohtani Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01417 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016
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Analytical Chemistry
High performance liquid chromatography at -196ºC Tomohiro Motono, Shinya Kitagawa*, Hajime Ohtani Department of Materials Science and Engineering, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466–8555, Japan Fax: +81-52-735-5368 E-mail
[email protected] ABSTRACT: Ultra-low temperature high-performance liquid chromatography (HPLC) was developed using a liquefied gas as the mobile phase. HPLC separation of low molecular weight alkanes at -196ºC with liquid nitrogen mobile phase was successfully achieved, whereas their GC separation at -196ºC using helium gas mobile phase failed to elute the analytes due to strong adsorption. Prior to the further study of HPLC at -196°C, the effect of column temperature on the chromatographic behavior was investigated, and it was found that the retention of analytes was drastically increased when the column temperature was over the boiling point of the mobile phase. As the study of retention control in HPLC at -196°C, the mobile phases of nitrogen and methane mixtures were investigated. The addition of methane to the nitrogen mobile phase suppressed the retention of the analytes (tetradeuterated methane, ethane, and propane), i.e., the retention on HPLC at ultra-low temperature could be controlled by the mobile phase composition, akin to the typical retention in HPLC. The selectivity toward the n- and iso-alkane in HPLC at -196°C was altered compared with that in GC separation at room temperature. A significant enhancement of retention between silica and alkenes compared with alkanes were observed in HPLC at -196ºC.
INTRODUCTION SECTION High performance liquid chromatography (HPLC) is one of the most widely used analytical methods in various research fields, such as biochemistry, pharmacology, and environmental science. In order to separate analytes with a wide variety of properties, various modes, such as normal-phase mode, reversed-phase (RP) mode, ion-exchange, and size exclusion have been developed for HPLC, for which nearly all separations are performed at approximately room temperature. On the other hand, HPLC separations at both high and low temperature ranges have been investigated, as the temperature is an important parameter in chromatographic separation.1 In RP-HPLC, the use of high-temperature water from 100 to 200ºC has been reported as the mobile phase of low environmental loading, and acceleration of the mass transfer rate at high temperatures often resulted in the enhancement of the separation efficiency.2-4 On the other hand, HPLC at low temperatures has been used for the analysis of thermally labile molecules in the early days.5-7 Preparative separations have also been used for unstable synthetic and natural compounds, such as small organic compounds, complexes, and proteins.8,9 Furthermore, HPLC at low temperature has sometimes been applied for enantiomer separations, for which the selectivity was successfully enhanced at low temperatures.10,11 In another approach to HPLC at low temperature, Okada et al. proposed HPLC using ice as the stationary phase, or ice chromatography, at a low temperature of approximately -12 to -15ºC.12 At the low HPLC temperature range between 0 and -65ºC, organic solvents can be used as the mobile phases. However, HPLC separations at lower temperatures than that have not been reported to date. Since the separation factor (α) depends on temperature, the chromatography at ultra-low temperature has a potential to
separate the analytes of less enthalpy difference in van’t Hoff equation. At ultra-low temperature, unstable species, such as radicals, often become stable. So comprehensive analysis of highly reactive and unstable species in various systems may be achieved using chromatography at ultra-low temperature. Moreover, the molecular vibration is limited at ultra-low temperature, and this limitation may result in an enhancement of the separation based on molecular shape recognition. Furthermore, chromatography at ultra-low temperature will be an effective tool to investigate the interaction in a cryogenic condition. However, the study of chromatography at ultra-low temperature is currently insufficient. In the case of gas chromatography (GC), separation at ultralow temperatures has been reported, i.e., GC at liquid nitrogen temperature (-196ºC), or cryogenic GC.13-16 The separation of isotopes and nuclear spin isomers of hydrogen molecules has been reported with cryogenic GC. That is, chromatography at ultra-low temperatures has the potential to achieve separations that are difficult at ambient temperature. However, the analytes for cryogenic GC are limited to only a few compounds, because the vast majority of compounds do not exist in the gas state at -196ºC. Moreover, even for the compounds that have the ability to remain in the gas state at that temperature, their strong adsorption on the stationary phase suppresses their separation (elution) at ultra-low temperatures. In general, HPLC has the potential to transport analytes toward the column outlet when the analytes have solubility in the mobile phase. In addition, the interaction between the analytes and the stationary phase in HPLC can be controlled by the composition of the mobile phase. Therefore, HPLC has less limitations than GC as a separation method at ultra-low temperatures. However, solvents, such as water, methanol, acetonitrile, hexane, and dichloromethane, freeze at ultra-low temperatures such as -196ºC. Here, we aimed to employ a
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liquefied gas as the mobile phase for ultra-low temperature HPLC, owing to its sufficiently low freezing (melting) point. In our previous study, we reported the development of low temperature HPLC using liquefied CO2 as the mobile phase with the basic chromatographic behavior of low molecular weight compounds.17 Similar studies using CO2 as the mobile phase for low temperature HPLC have been reported by other researchers.18,19 However, CO2 is not suitable as a mobile phase at ultra-low temperatures such as -196ºC. In this study, as the first step of development of ultra-low temperature HPLC, we proposed HPLC at -196ºC using liquid nitrogen based mobile phase (critical point: -147°C, 3.40 MPa; triple point, -210°C, 0.0125 MPa; the phase diagram of nitrogen was shown in Figure S1, Supporting Information). The effect of methane addition to the nitrogen mobile phase was also attempted to control the analyte retention. The basic chromatographic behavior in ultra-low temperature HPLC was investigated using low molecular alkanes as prove analytes. The chromatographic behavior for both alkenes and structural isomers of alkanes at -196°C were also studied. EXPERIMENTAL SECTION Apparatus for ultra-low temperature HPLC. The apparatus used in this study (see Figure S2, Supporting Information) was composed of N2 and CH4 gas cylinders, two solenoid valves (VX210AGA, SMC), an injector with a 0.5 µL sample loop (Model 7520, RHEODYNE), a column (Chemcobond 5-Si 1.0 × 150 mm, Chemco) surrounded by a laboratory-made heater equipped with a thermocouple, a capillary tube as a back pressure generator (i.d. 30 µm × 4 cm), a capillary tube for sprit injection (i.d. 30 µm × 40 cm), a gas flowmeter (Mass Flow Sensor Model 3810DS, Kofloc Kyoto), and a quadrupole mass spectrometer equipped with an electron ionization source (GC-MS QP5050, Shimadzu). The column was refrigerated by immersion in a liquid nitrogen bath. The column temperature was controlled by the application of a constant voltage that ranged 0 to 12.5 V to the heater, so as to keep temperatures between -196 and -148ºC. The mobile phase composition, or mixing ratio of N2 and CH4, was controlled by an open/close sequence of the two solenoid valves. In this study, a constant pressure mode of 1.0 MPa was used to supply both the gaseous and the liquefied mobile phases to the column, except where stated otherwise. The pressure at the column outlet end was kept over 0.7 MPa, except some cases, due to the resistance tube connected at the outlet end. Therefore, the mobile phases could keep liquid state in the column. All analytes were injected in their gas state via the valve injector at approximately 0.5 MPa. In this study, we could not find an ideal void (t0) marker without retention to the stationary phase. Therefore, argon was deemed to the void marker, because argon was the analyte with the lowest retention in HPLC at ultra-low temperature. Chemicals and reagents. Nitrogen (>99.99995%), methane (>99.999%), helium (standard grade), and argon (standard grade) gases were purchased from Taiyo Nippon Sanso. Standard ethane (99.5%), propane (99.5%), ethylene (99.5%), and propylene (99.5%) gases were purchased from GL scienc es, and n-butane (>98.0%) and iso-butane (>98.0%) were obtained from Sumitomo Seika. Tetra-deuterated methane, CD4, (>99.5%) was purchased from WATARI Co., Ltd. Chemical
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properties (boiling, melting, critical, and triple points) of the analytes used in this study were summarized in Table S1 (Supporting Information) except CD4. RESULTS AND DISCUSSION HPLC and GC separation at ultra-low temperature. Figure 1A shows the typical chromatograms of low molecular weight hydrocarbons (methane, ethane, and propane) in HPLC at -196ºC using a bare-silica column and a pure liquid nitrogen mobile phase (pure nitrogen exists as liquid state at -196ºC and 1.0 MPa, see Figure S1). Since MS was used for the detection, selected-mass detection was adopted to enhance the sensitivity. The mass numbers used for monitoring are noted in each chromatogram (e.g. m/z = 40 is for Ar as the t0 marker).
Figure 1. Separations of alkanes with (A) HPLC and (B) GC at ultra-low temperature using a bare-silica column. (A) Mobile phase: liquid nitrogen, temperature: constant at -196ºC, MS conditions: SIM mode at 16, 30, 40, and 44 m/z for methane, ethane, argon, and propane, respectively. The elution time (peak end time) of ethane and propane employed in this study were marked with arrow. (B) Mobile phase: helium gas, temperature: gradient from -196 to 21.6ºC (dotted line), MS conditions: scan mode from 15 to 100 m/z, and then extraction at 16, 30, 40, and 44 m/z. In both the separations, the mobile phases were supplied to the column at a constant pressure mode of 1.0 MPa. The other conditions are given in the text.
In this study, the elution time of each analyte was defined by the following procedure. The different amount of the analyte
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was injected and analyzed in the same chromatographic condition. When the peak top time was not affected by the injection amount, this value was employed as the elution time as similar to general HPLC (e.g. argon and methane in Figure 1). Alternatively, when the peak top time depends on the injection amount and the peak end time was approximately constant (Figure S3, Supporting Information), the peak end was employed as the elution time of the analytes. In this case, the elution time of the peak was marked with arrow in Figures (e.g. ethane and propane in Figure 1). As clearly shown in Figure 1A, the analytes were eluted from the column at -196ºC with different retention times, whereas the propane peak was significantly broadening. That is, HPLC separation at -196ºC was successfully achieved. To the best of our knowledge, the chromatograms shown in Figure 1A are the lowest temperature HPLC separation ever reported in the literature. The retention factor (k) increased with the increasing carbon number in the analytes, i.e., k values of 0.06, 3.2, and 35 were obtained for methane, ethane, and propane, respectively. The relationship between carbon number in alkane and ln k values for these alkanes was almost in linear (r2 = 0.98, shown in Figure S4, Supporting Information). The methylene selectivity in HPLC at -196°C was the similar behavior of that in conventional HPLC. GC separation was performed at -196ºC with a helium mobile phase, since, due to its low boiling point, this element exists in the gas state at that temperature. Figure 1B shows both the chromatograms and the column temperature (right axis). The analytes injected in the column were not eluted over 120 min when the column temperature was kept at -196ºC. When the column temperature increased after evaporation of liquid nitrogen in the bath, all analytes eluted from the column. That is, the adsorption of the analytes on the stationary phase at -196ºC is too strong and impedes the GC separation. Figure 1 demonstrates that GC is completely inapplicable for the separation of analytes at -196°C, while HPLC at this temperature seems to be applicable a little. Effect of temperature on the retention of analytes. As shown in Figure 1A, the alkanes were successfully separated in HPLC at -196°C. However, the elution time of propane was too long and the peak was broadening. Here, we investigated the possibility of the retention control by temperature akin to GC. The effect of temperature on the chromatographic retention of analytes at ultra-low temperature was investigated. Figure 2A and B show the chromatograms of ethane and methane, respectively, at various temperatures with liquid nitrogen mobile phase of an inlet pressure of 1.0 MPa. In both cases, the elution time of the argon (assumed as void marker) decreased with the increasing temperature. This behavior indicates that the flow velocity increases with the increasing temperature, due to the decrease in the viscosity of the liquid nitrogen (note that the mobile phase was supplied to the column at a constant pressure mode). The retention factor of ethane at -166ºC (k = 1.9) is smaller than at -196ºC (k = 3.1). The reduction in the retention factor at high temperatures is a consistent behavior for HPLC. However, at -161ºC, ethane was not eluted within 60 min as shown in Figure 2A. Moreover, the ethane was not also eluted in the HPLC at -165°C. In the case of methane (Figure 2B), the retention factor at -196, -168, and 153ºC was 0.06, 0.07, and 2.0, respectively. In contrast to typical chromatographic behavior, the retention factor of methane
increased with the increasing temperature. Moreover, the retention factor of methane drastically increased at -153ºC.
Figure 2. Effect of temperature on the separation of (A) ethane and (B) methane. Detection: (A) SIM mode at 30 and 40 m/z, (B) SIM mode at 16 and 40 m/z. The other conditions are the same as in Figure 1A.
The relationship between 1/T and ln k (van’t Hoff plot) for ethane and methane is shown in Figure 3A and B, respectively. As shown in Figure 3A, the retention factor of ethane decreases with the increasing temperature (the decreasing 1/T). In the low temperature range (0.0104 to 0.0130 in 1/T, or -196 to -177ºC in T), a linear relation was obtained, which agrees with the van’t Hoff equation. However, in the high temperature range (0.00936 to 0.0104 in 1/T, or -177 to -166ºC in T), the reduction in the retention is suppressed. In addition, at temperatures above -165ºC (
