Supercritical Fluid Chromatography with Flexible Injection Volumes at Highest Precision

Significance of the topic

Accurate and flexible sample introduction is crucial for reproducible and sensitive supercritical fluid chromatography (SFC). Fixed-loop injection avoids CO2 loss but restricts volume flexibility and wastes sample. A precise variable-volume injection enhances method adaptability in pharmaceutical, natural product, and quality control applications.

Aims and study overview

This work evaluates the performance of the Agilent 1260 Infinity II SFC Multisampler implementing a feed-injection principle. The sampler draws the sample at ambient pressure, pressurizes it to system pressure, and injects it via an ultrafast syringing process. Assessments cover isocratic and gradient separations across micro to large injection volumes.

Methodology and Instrumentation

• System components: Control Module G4301A, Binary Pump G4782A, Multisampler G4767A, Diode Array Detector G7115A, Multicolumn Thermostat G7116A
• Column: ZORBAX Rx-Sil 4.6 × 150 mm, 5 µm
• Mobile phase: CO2 (A) and methanol (B); isocratic (12 %B) and gradient (5 to 35 %B over 4 min)
• Injection volumes: 0.1 to 10 µL (standard) and 10 to 80 µL (large)
• Analytes: caffeine and theobromine
• Detection: DAD at 272 nm, data rate 10 Hz

Main results and discussion

• Linearity: R2 > 0.9995 for 0.1–10 µL (isocratic) and > 0.9999 (gradient); R2 = 0.9999 for 10–80 µL
• Precision: area RSD < 2.5 % at 0.1 µL; < 0.3 % for ≥ 0.5 µL and for 10–80 µL volumes
• Peak shape: widths at half height increase moderately from 0.036 to 0.096 min over 10–80 µL, maintaining symmetry
• Carryover: negligible at 14 ppm after a 5 µL injection of 5 g/L caffeine

Benefits and practical applications

Variable-volume feed injection delivers high precision and extensive dynamic range without sample loss. It reduces waste compared to fixed loops and maintains reproducibility across micro and large volumes. This flexibility benefits trace analysis, high-throughput screening, and complex matrix separations.

Future trends and opportunities

• Coupling with mass spectrometry for enhanced sensitivity and selectivity
• Automation in routine QA/QC for pharmaceuticals and natural products
• Expansion to environmental and bioanalytical matrices requiring variable volumes
• Refinement of injection parameters for submicroliter accuracy

Conclusion

The Agilent 1260 Infinity II SFC Multisampler achieves precise, linear, and reproducible injections from 0.1 to 80 µL with minimal carryover, matching fixed-loop performance while offering greater flexibility. This capability supports advanced SFC methods in research and quality control workflows.

Used Instrumentation

• Agilent 1260 Infinity II SFC Control Module G4301A
• Agilent 1260 Infinity II SFC Binary Pump G4782A
• Agilent 1260 Infinity II SFC Multisampler G4767A
• Agilent 1260 Infinity II DAD G7115A
• Agilent 1260 Infinity II Multicolumn Thermostat G7116A
• ZORBAX Rx-Sil 4.6 × 150 mm 5 µm column

References

1. Naegele E. Feed Speed and Over-Feed Volume – New Parameters for SFC Injection. Agilent Technologies Technical Overview publication number 5991-7626EN 2017.