New Baseline Correction Method Using Near-Infrared Absorption of Water in Water/Acetonitrile Gradient High-Performance Liquid Chromatography with Far-Ultraviolet Absorbance Detection

Abstract

We have previously reported the development of a far-ultraviolet (FUV) absorbance detector capable of detecting wavelengths down to 175 nm in high-performance liquid chromatography (HPLC). Although the FUV detector can detect substances with weak to non-existent ultraviolet (UV) absorption (e.g., sugars at 185 nm and peptides at 190 nm), a large baseline drift occurs in the gradient elution due to differences in the FUV absorbance properties of water and acetonitrile. To overcome the problem of baseline drift, we have proposed a new baseline correction method using the absorption of water at 1450 nm. It is well known that water has a relatively large absorption peak at 1450 nm in the near-infrared (NIR) region. By contrast, acetonitrile used in reversed-phase HPLC shows negligible absorbance compared to water at 1450 nm. Sugars and peptides also show negligible absorbance at 1450 nm. Thus, it is expected that changes in absorbance at 1450 nm only reflect the volume fraction of water in the gradient elution. The baseline correction method by a linear combination of FUV and NIR chromatograms was applied to the HPLC separation of sugars and peptides in water/acetonitrile-gradient HPLC coupled with FUV detection. The results showed that flat baselines were successfully obtained in the gradient HPLC coupled with FUV detection.