Introduction
Supercritical fluid chromatography (SFC) is considered to have a separation mode similar to normal-phase chromatography in HPLC. It has gained attention in recent years due to its ability to accelerate analyses while maintaining high separation efficiency, attributed to the unique properties of supercritical CO2 as the main mobile phase solvent (low viscosity and high diffusivity). However, unlike the well-established C18 columns for reversed-phase analysis in HPLC, no column has been firmly established as the first choice for use in SFC. Therefore, when searching for a column suitable for separating target components, it is effective to employ a screening measurement technique using multiple columns of different types.
In this application note, we conducted method scouting measurements using SFC with six types of columns, each having different silica gel as the base material and functional groups for the analysis of low-molecular-weight compounds with acidic, neutral, and basic properties. We compared the separation behaviors among these columns and will introduce the results.*
*All the data presented in this application note were obtained in collaboration with GL Sciences Inc.
Experimental
Instruments
CO2 pump: PU-4380
Modifier pump: PU-4180*
Autosampler: AS-4350
Column oven: CO-4065*
PDA detector: MD-4010*
BP regulator: BP-4340
* with option units
SFC Conditions
Column: Silica-based columns (4.6 mmI.D. x 250 mmL, 5 µm, Details of columns are described later.)
Eluent A: Carbon dioxide
Eluent B: 0.1 % (w/v) ammonium acetate in methanol
Gradient: A/B = 95/5 (0.00 min) → 60/40 (10.00 min) → 95/5 (10.05 min), 1 cycle; 15 min
Flow rate: 3.0 mL/min
Column temp.: 40 ºC
Wavelength: 230 nm
Back pressure: 10 MPa
Inj. volume: 5 µL
Sample: Mixture of 5 low-molecular-weight compounds (200 µg/mL each in methanol)
Structures
Schematic Diagram
Keywords
SFC, Method scouting, separation behavior, Inertsil, InertSustain
Results
Using dedicated software, we generated comprehensive measurement conditions for the combination of one modifier solvent and six columns. The method scouting SFC system automatically switched columns and performed continuous measurements. Then we confirmed the separation behavior of five low-molecular-weight compounds on each column. Table 1 provides details of the six columns used in this method scouting measurement. Additionally, Figure 1 shows chromatograms obtained from measurements conducted with each column.
Table 1 Types and characteristics of columns used in this study
Column | Column characteristics | End-capping treatment |
Inertsil ODS-EP | A polar group-containing type (embedded type) ODS column in which an octadecyl group (ODS group) is chemically bonded to silica gel via a polar group. | without |
Inertsil SIL-100A | A column filled with silica gel with a pore size of 100 Å and extremely low metal content. | without |
InertSustain NH2 | A column with amino-propyl groups chemically bonded to silica gel. | without |
Inertsil ODS-HL | An ODS column with high planar recognition ability, with ODS groups bonded to silica gel at high density. | with |
Inertsil ODS-P | An ODS column with high planar recognition ability, which is polymerically bonded ODS groups to silica gel. This column has a higher density of ODS groups bonded compared to Inertsil ODS-HL. | without |
InertSustain AX-C18 | A mixed-mode column with reverse-phase and anion-exchange properties, has ODS and alkylamine groups chemically bonded to silica gel. | with |
Fig. 1 Chromatograms of low-molecular-weight compounds measured with each column
1: Caffeine, 2: Loxoprofen, 3: Amitriptyline, 4: Carbamazepine, 5: Salicylic acid
Inertsil ODS-EP, with a polar group introduced at the base of the ODS group, was found to retain highly polar acidic or basic compounds adequately.
Inertsil SIL-100A is an unmodified silica gel column. Similar separation behavior to Inertsil ODS-EP were observed, but overall retention was stronger with Inertsil SIL-100A. Amitriptyline did not elute under the conditions in this study because basic compounds are strongly adsorbed to the silanol group.
InertSustain NH2 is used as a normal-phase column in HPLC, as is Inertsil SIL-100A, an unmodified silica gel column. However, due to the modification with basic functional groups, it tends to strongly retain acidic compounds. In the current measurements, it was observed that the elution of the acidic compound, Loxoprofen, was significantly delayed compared to using Inertsil SIL-100A, and salicylic acid did not elute at all. On the other hand, the basic compound amitriptyline did elute.
Inertsil ODS-HL has a high carbon content due to the densely bonded ODS group and is treated with end-capping, so there is almost no residual silanol group. Due to the absence of polar groups on the packing material surface, low-molecular-weight compounds, including acidic and basic ones measured in this study, showed overall weak retention and eluted rapidly.
Inertsil ODS-P has ODS groups bonded more densely than Inertsil ODS-HL but is not treated with end-capping. The presence of residual silanol groups on the packing material surface gives it the almost retention strength as Inertsil ODS-EP, but the overall peak shapes tend to be broader. The potential cause for the deterioration in peak shapes could be attributed to the extremely densely bonded ODS groups in this column.
InertSustain AX-C18 is modified with two functional groups, ODS and tertiary amine. It shows a similar separation behavior to InertSustain NH2, which is also modified with basic functional groups. However, there is a reversal in the elution order of carbamazepine and amitriptyline, and the overall retention strength is significantly different.
Conclusion
By conducting method scouting measurements using six columns with different silica gel packing materials and modified functional groups for acidic, neutral, and basic low-molecular-weight compounds in SFC, we were able to identify distinctive retention and separation behaviors for each column. In SFC, the use of a screening measurement technique with multiple columns of different types proves effective in rapidly finding optimal analysis conditions for separating target components.