Sample-independent approach to normalize two-dimensional data for orthogonality evaluation using whole separation...

Area of Science:

• Analytical Chemistry
• Chromatography
• Separation Science

Background:

• Orthogonality is critical for evaluating the separation power of two-dimensional chromatography systems.
• Current scaling methods for orthogonality assessment are sample-dependent, leading to variable results.
• Sample composition can significantly impact the perceived orthogonality by influencing the normalized retention space.

Purpose of the Study:

• To introduce a novel, sample-independent scaling approach for orthogonality evaluation in 2D chromatography.
• To develop the Whole Separation Space Scaling (WOSEL) method for a more robust assessment of chromatographic systems.
• To demonstrate the superiority of WOSEL over existing methods in terms of stability and accuracy.

Main Methods:

• Development of the Whole Separation Space Scaling (WOSEL) approach, considering the entire separation space defined by the analytical method.
• Comparison of WOSEL with two conventional sample-dependent scaling methods.
• Validation using in silico-generated chromatograms and real-world separation data from human biofluids and petroleum samples.

Main Results:

• WOSEL demonstrated superior sample-to-sample stability (3.8%) compared to existing methods (7.0% and 10.1%) on real samples.
• The WOSEL approach provides an orthogonality evaluation that is independent of the specific sample analyzed.
• Existing methods were shown to potentially yield misleading conclusions about the overall orthogonality of 2D chromatographic systems.

Conclusions:

• The Whole Separation Space Scaling (WOSEL) method offers a more reliable and stable assessment of orthogonality in two-dimensional chromatography.
• WOSEL ensures that the evaluation of separation system efficiency is not biased by sample-specific retention characteristics.
• This method enhances the accuracy of orthogonality assessment, crucial for optimizing complex separation techniques.