Peak Shape Improvement Using the Auto- Pretreatment Function (Co-injection) of i-Series Plus Integrated Liquid Chromatograph

Importance of the Topic

The quality of chromatographic peak shape is critical for sensitive and reliable quantitation in high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC). Modern UHPLC columns use very small internal diameters (2.0–3.0 mm I.D. and sub-0.1 mm tubing) to achieve high efficiency, but this increases extra-column dispersion and makes mixing of sample and mobile phase more challenging. Poor mixing, especially when the sample solvent is stronger or significantly different in composition from the mobile phase, often leads to broadened or distorted peaks, reduced theoretical plate counts and compromised quantitation.

Objectives and Overview of the Study

This application note demonstrates how Shimadzu’s i-Series Plus (Nexera™-i MT) automatic pretreatment “Co-Injection (Simple)” function can be used to co-inject a dilution or reagent solvent together with the sample to improve peak shape. Two use cases were studied: standard reversed-phase analysis of caffeine in water/methanol and ion-pair chromatography of berberine chloride with an ion-pairing reagent in the mobile phase. The goal was to compare conventional injections versus co-injection at various volumes and assess effects on theoretical plate count and peak integrity.

Methodology and Used Instrumentation

Instrumentation and conditions for the caffeine study:

• System: Shimadzu Nexera™-i MT with automatic pretreatment autosampler
• Column: Shim-pack™ XR-ODS II, 75 mm L × 3.0 mm I.D., 2.2 μm particle size
• Mobile phase: Water/methanol (7:3, v/v)
• Flow rate: 1.0 mL/min; Column temperature: 40 °C
• Detection: UV at 272 nm; Injection volumes: 1, 2, 5, 10 μL
• Co-injection solvent: Water (15 μL aspirated for each injection)

Instrumentation and conditions for the berberine ion-pair study:

• Column and system as above
• Mobile phase: 3.4 g monobasic potassium phosphate + 1.7 g sodium lauryl sulfate per 1000 mL water/acetonitrile (1:1)
• Flow rate: 0.8 mL/min; Column temperature: 40 °C
• Detection: UV at 345 nm; Injection volume: 15 μL
• Co-injection reagent: Mobile phase containing ion-pair reagent

Main Results and Discussion

Caffeine study:

• Standard injections showed a progressive drop in theoretical plate count as injection volume increased: ~84 % retention at 2 μL and only ~13 % at 10 μL compared to the 1 μL reference.
• With co-injection of water, plate counts improved markedly: ~89.9 % at 5 μL and ~61.3 % at 10 μL, and peak shapes remained sharp enough for accurate quantitation.

Ion-pair study:

• Without co-injection, berberine peaks exhibited splitting and distortion, attributed to inadequate mixing of sample and ion-pair reagent.
• Co-injection of mobile phase containing the ion-pair reagent resulted in complete mixing in the needle, yielding single, symmetric peaks.

Benefits and Practical Applications

• Improves peak shape and efficiency in both reversed-phase and ion-pair UHPLC/HPLC without additional hardware.
• Allows larger injection volumes while maintaining quantitation performance.
• Simplifies method development by handling dilution or reagent addition in a single autosampler template.
• Enhances reproducibility and throughput in routine QA/QC and research laboratories.

Future Trends and Opportunities

As UHPLC moves toward even smaller column dimensions and higher pressures, automated in-needle mixing strategies like co-injection will become increasingly important. Potential developments include:

• Multi-reagent co-injection for on-line derivatization or cleanup.
• Integration with sample preparation workflows (e.g., SPE or dilution series).
• Advanced software control for dynamic solvent programming based on sample composition.
• Application to high-throughput screening and metabolomics where matrix effects are critical.

Conclusion

The i-Series Plus co-injection function effectively addresses peak distortion caused by mismatched sample and mobile phase solvents in UHPLC/HPLC. By aspirating a dilution or reagent solvent and injecting it together with the sample, users can restore theoretical plate counts, eliminate peak splitting in ion-pair analyses, and simplify workflow without extra hardware.