Chromatographic behavior of small organic compounds in low‐temperature high‐performance liquid chromatography using liquid carbon dioxide as the mobile phase

Abstract

Low-temperature high-performance liquid chromatography, in which a loop injector, column, and detection cell were refrigerated at -35ºC, using liquid carbon dioxide as the mobile phase was developed. Small organic compounds (polyaromatic hydrocarbons, alkylbenzenes, and quinones) were separated by low-temperature high-performance liquid chromatography at temperatures from -35 to -5ºC. The combination of liquid carbon dioxide mobile phase with an octadecyl-silica (C18 ) column provided reversed phase mode separation, and a bare silica-gel column resulted in normal phase mode separation. In both the cases, nonlinear behavior at approximately -15ºC was found in the relationship between the temperature and the retention factors of the analytes (van't Hoff plots). In contrast to general trends in high-performance liquid chromatography, the decrease in temperature enhanced the separation efficiency of both the columns.

Effect of Column Structure on Separation Efficiency in Low-Temperature HPLC Using Pure Liquid Carbon Dioxide as the Mobile Phase

“…For all conditions, the retention factors of each compound increased with a decrease in the temperature, and the slope of the van't Hoff plot changed at approximately -15°C (1/T = 0.0039). This behavior has been reported in our previous research [19]. As shown in Fig.…”

“…The low-temperature HPLC used in this study was almost the same arrangement as our previous research [19]. The apparatus was composed of a CO 2 gas cylinder, an automated rotary valve injector (Valve Unite FCV-20AH2 , Shimadzu, Kyoto, Japan), a backpressure regulator (KPB1L0A422P20000, Swagelok, Solone, OH, USA), two pressure gauges (KDM30, Asone, Osaka, Japan), a gas flowmeter (Mass Flow Sensor Model 3810DSII, Kofloc Kyoto, Kyoto, Japan), a UV light source (high-power UV-Vis fiber light source L10290, Hamamatsu Photonics, Shizuoka, Japan) equipped with a band pass filter (detection wavelength, 254.66 ± 4.68 nm, VPF-25C-10-12-25370, Sigma Koki, Tokyo, Japan), fiber optics (CUV-CCE, Ocean Optics, Dunedin, FL, USA), a photomultiplier (H5783-06, Hamamatsu Photonics), and a low-temperature incubator (38 L JF-NU40G-S, Haier, Qingdao, China), in which the temperature was controlled by a heater set inside of the incubator.…”

“…containing water were mainly used as the non-freezing mobile phases. In addition, low-temperature HPLC using liquefied gasses as the mobile phase has also been reported [17][18][19][20]. For example, Pirkle et al demonstrated enantioselective separations at low temperatures above -47°C using mobile phases based on liquid CO 2 [17,18].…”

“…Recently, we investigated the basic chromatographic behaviors of small organic compounds in low-temperature HPLC using a pure liquefied CO 2 mobile phase [19]. In that study, retention factors and separation efficiencies of analytes at low temperatures (-5 to -35°C) were discussed for both ODS and bare-silica columns packed with general fully porous spherical particles.…”

Ultralow-Temperature HPLC Using Low-Molecular-Weight Hydrocarbons as Mobile Phases

“…Results of low-temperature HPLC down to -65°C have been reported, including analysis of thermally labile compounds [12][13][14], preparative separation of unstable compounds [15,16], selectivity enhancement in enantiomer separation [17,18], and the use of water ice as the stationary phase [19], in which organic solvents and their mixtures were used as non-freezing mobile phases. The use of liquid carbon dioxide as an alternative mobile phase for low-temperature HPLC has also been reported [20][21][22][23]. However, freezing makes these mobile phases unsuitable for cryogenic HPLC at near liquid nitrogen temperatures.…”