LC Capillaries and Fittings

Importance of the Topic

Understanding and controlling extracolumn dispersion is critical for high-performance liquid chromatography. Poorly chosen or oversized capillaries and fittings introduce dead volume, cause peak broadening, reduce resolution and sensitivity, and can lead to leaks or unwanted interactions between analytes and metal surfaces.

Study Objectives and Overview

This application note aims to:

• Quantify the contribution of capillaries and fittings to system dispersion.
• Compare different capillary materials, diameters and lengths.
• Review common fitting types and their impact on performance.
• Recommend optimized connections for various Agilent LC systems.

Methodology and Instrumentation

Extracolumn volume was determined by combining theoretical calculations (Taylor–Aris equation) and experimental chromatograms using a StableBond SB-C18 or Eclipse Plus C18 column. Peak widths and resolution were measured at varying capillary inner diameters (0.17 vs. 0.12 mm) and lengths (10–150 mm). The impact of optimized versus standard tubing on UHPLC performance was demonstrated using sunscreen samples and model peptides.

Instrumentation Used

• Agilent 1100, 1200, 1260 and 1290 Infinity series HPLC/UHPLC systems with DAD/MWD or VWD detectors.
• Semimicro flow cells for reduced dead volume.
• Capillaries: stainless steel, PEEK, PEEK-coated fused silica, PEEK-lined stainless steel, titanium, MP35N.
• Fittings: Swagelok, Parker, Waters styles; Agilent InfinityLab Quick Turn and Quick Connect; UHP-FF.

Main Results and Discussion

• Experimental chromatograms show that increasing extracolumn volume from 10 µL to 50 µL leads to significant peak broadening and loss of resolution.
• Using shorter, narrower capillaries (0.12 mm id, 50 mm length) reduces extracolumn volume by ~50% compared to 0.17 mm id, improving peak width by more than twofold.
• Optimized system tubing for UHPLC (0.12 mm vs. 0.17 mm id) doubled resolution (from 0.96 to 1.90) and halved peak width (~0.019 min vs. ~0.038 min).
• Quick Connect and Quick Turn fittings with spring-loaded design eliminate dead volume, maintain performance after 200 reconnections, and provide reliable leak-free operation up to 1300 bar.

Benefits and Practical Applications

• Enhanced chromatographic resolution and peak capacity for complex mixtures.
• Greater reproducibility and robustness in routine QA/QC and method development.
• Reduced sample dilution and increased sensitivity, critical for trace analysis.
• Streamlined workflow with finger-tight, spring-loaded InfinityLab fittings, minimizing installation time and user error.

Future Trends and Possibilities

• Further miniaturization toward micro- and nano-LC with ultralow-dispersion capillaries.
• Development of advanced bio-inert materials to prevent analyte adsorption in biopharmaceutical analysis.
• Integration of automated capillary and fitting selection guided by instrument control software and AI diagnostics.
• Enhanced modularity allowing rapid reconfiguration for multidimensional separations and high-throughput applications.

Conclusion

Selecting the appropriate capillary diameter, length and fitting type is essential to minimize extracolumn dispersion and maintain high chromatographic performance. Agilent’s range of materials and spring-loaded Quick Connect/Quick Turn fittings offers robust, low-volume connections compatible with all major HPLC and UHPLC platforms. Optimizing system plumbing significantly enhances resolution, sensitivity and reproducibility across analytical workflows.