Introduction

Supercritical fluid chromatography (SFC) uses carbon dioxide as the main solvent in the mobile phase and is characterized by its ability to maintain high separation efficiency even when performing analyses at high flow rates (linear velocity). Among the sample components that are separated and fractionated by taking advantage of this feature are chiral compounds. To investigate the optimal separation conditions for chiral compounds, a screening method is used to comprehensively analyze individual substances using solvents and columns. Recently, chiral columns with a 3 µm particle size have been developed for SFC, and are expected to provide faster and more efficient chiral-compound separation than conventional SFC using 5 µm particle-size columns.

In this application note, we used a JASCO SFC system with the Method Scouting Support Program, an add-in software for ChromNAV Ver. 2. Fast screening of hydroxyzine, an anti-allergic palliative tranquilizer, was performed using three modifier solvents and ten chiral columns with a 3 µm particle size, which are expected to provide high speed and high efficiency. Since hydroxyzine is a basic compound, it was examined under conditions in which diethylamine was added to the modifier to improve separation and peak shape.

The circular dichroism (CD) detector used is capable of outputting UV chromatograms, CD chromatograms, and the g-factor (CD/UV ratio), in real time. In the CD chromatogram, different optical isomers produce peaks with opposite signs (positive or negative).

SFC method scouting system

Experimental

Instruments
CO2 pump:  PU-4380
Modifier pump:  PU-4185*
Autosampler:  AS-4350
Column oven:  CO-4065*
PDA detector:  MD-4010*
CD detector:  CD-4095*
BP regulator:  BP-4340
* with option units

SFC Conditions
Column:   CHIRALPAK IA/SFC, IB-N/SFC,  IC/SFC, ID/SFC, IE/SFC, IF/SFC, IG/SFC, IH/SFC, IJ/SFC, IK/SFC  (3.0 mmI.D. ´ 50 mmL, 3 µm)※
Eluent :   Carbon dioxide/modifier (75/25)
Modifier:  A; Methanol/diethylamine (100/0.4)  B; Acetonitrile/ethanol/diethylamine       (80/20/0.4)  C; t-Butyl methyl ether/ethanol/diethylamine        (80/20/0.4)
Flow  rate:  1.2 mL/min
Column temp.:   40 ºC
Wavelength:   230 nm (MD-4010)  230 nm (CD-4095)
Back pressure:  15 MPa
Inj. volume:   1 µL
Sample:   0.5 mg/mL hydroxyzine in methanol
* CHIRALPAK is a trademark or registered mark of Daicel Corporation.

Structure

Schematic Diagram

Keywords

Hydroxyzine, SFC, screening, method scouting, chiral separation, CD detector, CHIRALPAK, 3 µm

Results

The column stabilization time during screening was set to 10 min only for the first column after solvent changeover and 3 min for the other columns, taking into account solvent substitution in the system. The analysis time was set to 3.0 min. The total required time for this screening was 3.4 hours per compound.

Figure 1-1 shows the screening results obtained using a photodiode array (PDA) detector at a wavelength of 230 nm, and Figure 1-2 shows those obtained using a CD detector at a wavelength of 230 nm. Each row of chromatograms represent a different modifier solvent, and each column of chromatograms represents a different chiral column. In the CD chromatograms, different optical isomers produce signals with opposite signs.

Fig. 1-1 Chromatograms of hydroxyzine obtained using method scouting system  (PDA detection wavelength: 230 nm)

Fig. 1-2   Chromatograms of hydroxyzine obtained using method scouting system (CD detection wavelength: 230 nm)

     Table 1 shows the degree of separation of hydroxyzines under each condition. Results with a resolution of 1 or less are marked as incomplete separation (I.S.). When methanol containing 0.4% diethylamine was used as the modifier, a resolution of 2 or greater was obtained for IF and IJ. Among the conditions examined in this study, good separation with a short analysis time and favorable peak shape was achieved using CHIRALPAK IJ/SFC with t-butyl methyl ether/ethanol (80/20) containing 0.4% diethylamine.

However, complete separation was also obtained under certain conditions for the IA and IF columns, and under other conditions, partial separation (indicated as I.S.) was observed for other columns. When scaling up, it is necessary to carefully select the conditions, as factors such as sample loading may adversely affect the peak shape.

Table 1   Comparison of resolution

Conclusion

In this study, the use of a size-reduced chiral column with 3 µm particle size in SFC enabled rapid method scouting, allowing the selection of separation conditions to be completed within just 3.4 hours. Conditions that resulted in only peak apex separation have the potential to be further optimized to achieve baseline separation by adjusting the column dimensions and modifier.

In chiral separation of basic compounds by SFC, the use of additives such as diethylamine is necessary to improve peak shape and resolution.

The SFC method scouting system can be effectively used to explore separation conditions for chiral compounds because SFC allows separation studies in a shorter time compared to HPLC.