High-Resolution Sampling 2D-LC with the Agilent 1290 Infinity II 2D-LC Solution

Significance of the Topic

The reliable quantification of coeluting analytes in complex samples remains a critical challenge in analytical chemistry. Two-dimensional liquid chromatography (2D-LC) with high-resolution sampling extends the selectivity and sensitivity of conventional 1D-LC by enabling accurate separation and measurement of overlapping peaks. This capability is particularly relevant for quality control in food, pharmaceuticals, and natural product analysis, where precise quantitation of bioactive compounds underpins regulatory compliance and product standardization.

Objectives and Study Overview

This work demonstrates the implementation of high-resolution sampling 2D-LC using the Agilent 1290 Infinity II 2D-LC solution. The primary goal was to achieve reliable quantification of caffeine and catechin derivatives in a green tea extract. Specific aims included:

• Separating (+)-catechin and (–)-epicatechin in the first dimension (1D).
• Resolving coeluting caffeine and (–)-epigallocatechin gallate via multiple small fractions transferred to the second dimension (2D).
• Assessing linearity, repeatability, and practical quantitation workflows within a single run.

Methodology

Aqueous green tea extract was prepared and analyzed using time-based high-resolution sampling. Six 2.8-second cuts spanning the coeluting caffeine and (–)-epigallocatechin gallate peak were collected in 40 µL loops and cycled through the 2D column. Both dimensions employed reversed-phase RRHD columns at 20 °C with tailored gradients and flow rates (0.2 mL/min for 1D; 1 mL/min for 2D). Sample injection volumes were 1 µL, with DAD detection at 280 nm and data acquisition rates of 40 Hz (1D) and 80 Hz (2D). Calibration curves were generated from standard solutions (5–220 µg/mL) using summed 2D peak areas for the coeluting pair.

Used Instrumentation

• Agilent 1290 Infinity II High Speed Pumps (G7120A)
• Agilent 1290 Infinity II Multisampler with cooler (G7167B)
• Agilent 1290 Infinity II Multicolumn Thermostats (G7116B)
• Agilent 1290 Infinity II Diode Array Detectors (G7117B)
• Agilent 1290 Infinity Valve Drive and multiple heart-cutting valve modules (G1170A, G4242-64000)
• Agilent OpenLAB CDS ChemStation Edition software with 2D-LC acquisition module

Main Results and Discussion

In the 1D chromatogram, (+)-catechin and (–)-epicatechin were baseline separated and quantified directly. Caffeine and (–)-epigallocatechin gallate coeluted and required high-resolution sampling. Overlaying six consecutive 2D chromatograms revealed distinct peaks for each compound, enabling summation of individual cut areas. Calibration exhibited excellent linearity (R² > 0.999) across the full concentration range. Repeatability tests yielded relative standard deviations below 1.2 % for both target analytes.

Benefits and Practical Applications

• Enables quantification of analytes otherwise unresolved by 1D-LC without extending analysis time.
• Reduces risk of sample loss by maintaining full recovery across multiple fractions.
• Facilitates streamlined QA/QC workflows in food and pharmaceutical laboratories.
• Integrates seamlessly into existing ChemStation data analysis for automated calibration and reporting.

Future Trends and Applications

Advances in multidimensional chromatography are likely to focus on:

• Automation of cut selection via real-time peak recognition algorithms.
• Integration with mass spectrometry for enhanced structural confirmation.
• Miniaturization of sampling loops to improve throughput and solvent economy.
• Application to broader compound classes, including polar metabolites and small peptides.

Conclusion

High-resolution sampling 2D-LC on the Agilent 1290 Infinity II platform effectively separates and quantifies coeluting substances within a single analytical run. By combining precise fractionation with robust 2D separation, this approach overcomes limitations of traditional heart-cutting methods, delivering high linearity, reproducibility, and applicability to complex real-world samples.

References

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2. Lee C. et al. Application of Heart-Cutting 2D-LC for Determination of Peak Purity. Chromatographia 2013, 76(1–2), 5–11.
3. Buckenmaier S. Agilent 1290 Infinity 2D-LC Solution for Multiple Heart-Cutting. Agilent Tech. Overview, 2015.
4. Stoll D. R. et al. Selective Comprehensive 2D-LC-MS. Anal. Chem. 2015, 87(16), 8307–8315.
5. Jankana B.; Wandee Y. Catechins and Caffeine in Green Tea. J. Pharm. Biomed. Sci. 2012, 22(22), 1–7.