Automated Preparation of Calibration Curves Using the Autosampler's Dilution Function

Importance of the Topic

Automating sample dilution and calibration curve preparation in high-performance liquid chromatography streamlines workflows, reduces manual labor and potential errors, and enhances throughput. This is critical for laboratories handling large sample volumes or conducting routine quality control analyses of organic acids, where reliability and efficiency are paramount.

Objectives and Article Overview

This application note demonstrates how the autosampler’s built-in auto-dilution function on the Nexera X3 system can fully automate calibration curve generation and sample quantification. The goals are to eliminate manual pipetting steps, minimize human error, and show performance in organic acid analysis, including a real-world sample test on a bath salt product.

Methodology and Instrumentation

Sample pretreatment is controlled by a programmable pretreatment script in LabSolutions. Users specify vial positions for undiluted standards and target dilution factors in the batch table. The autosampler aspirates a calculated volume of standard solution and diluent (ultrapure water) into a mixing vial, mixes, then injects the diluted aliquot onto the column. Key instrument components:

• Liquid chromatograph: Nexera X3 high-performance system with conductive detection for organic acids
• Autosampler: Equipped with auto-dilution and mixing functions
• Column: Shim-pack SCR-102H analytical column (300×8 mm, 7 µm) with guard
• Software: LabSolutions with SIL pretreatment variables batch add-in
• Reagents: p-toluenesulfonic acid buffer, Bis-Tris, EDTA; ultrapure water

Main Results and Discussion

Using a 1000 mg/L mixed standard of ten organic acids, 100-fold dilutions were prepared automatically to generate calibration curves at five levels (10–1000 mg/L).

• Repeatability: Retention times RSD ≤0.04%, peak areas RSD ≤2.83% (n=6)
• Linearity: Coefficients of determination (r²) >0.9999 for all acids
• Accuracy: Automated dilution peak areas within ±1.4% of manual dilution reference at 50 mg/L

Application to bath salt samples (1 g in 10 mL water) showed clear detection of malic acid (344.6 mg/L, RSD 1.53%) and formic acid (421.2 mg/L, RSD 1.84%) after 50-fold auto-dilution. Other acids were below detection.

Benefits and Practical Applications

By centralizing dilution settings in the batch table and using a single method file, users avoid creating multiple methods for different concentration ranges. The process:

1. Prepares undiluted standards and samples once
2. Automatically generates calibration points and analyzes samples
3. Reports concentrations back-calculated to the original solution

This reduces labor, standardizes procedures, and minimizes errors, making it ideal for high-throughput environmental, food, beverage, and pharmaceutical testing.

Future Trends and Potential Applications

Further developments may include integration with advanced LIMS systems, expansion to isotopic dilution workflows, and real-time process monitoring in manufacturing. The auto-dilution concept can extend to other analyte classes (e.g., pesticides, pharmaceuticals) and detection modes (MS, UV), driving fully unattended analysis platforms.

Conclusion

The Nexera X3 autosampler auto-dilution feature successfully automates calibration curve creation and sample preparation for organic acid analysis. Demonstrated performance meets rigorous repeatability, linearity, and accuracy criteria, while significantly reducing hands-on time. Adoption of this approach can elevate laboratory productivity and data quality.

References

No external literature references cited in this application note.