Simple Labor-Saving Calibration Curve Creation Using Autosampler Automatic Dilution Function Part 2

Significance of the Topic

High-performance liquid chromatography (HPLC) requires accurate sample and standard dilutions for reliable quantitation. Manual pipetting of small volumes is labor-intensive, prone to errors, and consumes large amounts of organic solvents. Integrating an automatic dilution function into an autosampler streamlines routine calibration and sample preparation, enhancing productivity and sustainability in analytical laboratories.

Objectives and Study Overview

This article demonstrates the use of the Nexera XR autosampler’s automatic dilution feature to:

• Create calibration curves directly from stock standards diluted by user-defined factors using organic solvents.
• Reduce manual dilution steps and solvent consumption for standard and sample preparation.
• Maintain a single method file for varying dilution ratios to minimize setup errors.

Methodology and Instrumentation

An autosampler pretreatment program was configured in LabSolutions to execute aspiration, dispensing, mixing, and injection steps:

• Pretreatment commands define dilution ratios, mixing volumes, and rinse cycles.
• Batch table variables specify stock vial location and dilution factor for each sample.
• Mixing performed in 1 mL polypropylene vials; stock and rinse solutions in septum vials.

Analytical conditions:

• Instrument: Shimadzu Nexera XR UHPLC
• Column: Shim-pack GIST-HP C18 (150 × 3.0 mm, 3 μm)
• Mobile phase: water (A) and acetonitrile (B), gradient from 50 % to 100 % B
• Flow rate: 0.8 mL/min; column temperature: 40 °C
• Injection volume: 5 μL; detection at 276 nm (coumarin) and 288 nm (cinnamaldehyde) using SPD-M40

Main Results and Discussion

• Calibration curves generated for coumarin and cinnamaldehyde over 0.1–10 mg/L exhibited excellent linearity (r² ≥ 0.999).
• Repeatability studies (n=6) at 0.2 mg/L showed retention time RSD ≤ 0.09 %, area RSD ≤ 1.8 %.
• Analysis of Cinnamomum cassia extract, after dSPE cleanup, with 100-fold automatic dilution yielded well-resolved peaks and quantitative results consistent with manual dilutions (RSD < 1.1 %).

Benefits and Practical Applications

• Eliminates manual pipetting for dilution, reducing labor and potential errors.
• Minimizes organic solvent usage by preparing dilutions on-demand.
• Facilitates method maintenance since a single protocol handles multiple dilution factors.
• Enables rapid, reproducible quantitative analysis of standards and real samples.

Future Trends and Opportunities

The integration of automatic dilution with advanced autosamplers is expected to:

• Expand to complex multi-analyte workflows with variable dilution steps.
• Interface seamlessly with laboratory information management systems (LIMS) for automated reporting.
• Incorporate AI-driven optimization of dilution protocols and gradient methods.
• Support green chemistry by further reducing solvent and consumable use.

Conclusion

Implementing the autosampler automatic dilution function on the Nexera XR platform simplifies calibration curve creation and sample analysis workflows. This approach improves throughput, data consistency, and sustainability, representing a significant advancement for routine HPLC laboratories.

Reference

Shimadzu Application News No. 01-00717
Shimadzu Application News No. 01-00233