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FEATURE
The Benefits of Refractive Index Detection for UHPLC
Dan Some
Ph.D., Wyatt Technology

Introduction

Analytical high-performance liquid chromatography (HPLC) is ubiquitous throughout the world of analytical chemistry as a means of identifying and quantitating molecular species present in a sample. HPLC systems incorporate a variety of on-line detectors including UV/Vis absorption, fluorescence, evaporative light scattering (ELS) and mass spectrometry (MS). Differential refractive index detection (dRI) is also common, serving a large variety of HPLC applications ranging from small molecules to proteins, petroleum, polysaccharides and synthetic polymers.

Ultra-high performance liquid chromatography (UHPLC) is similar in concept and application to HPLC but operates with smaller columns packed tightly with smaller beads. Since its commercial introduction in 2003, UHPLC has proliferated thanks to three significant factors: cost savings in time, sample and solvent quantity, and improved resolution. While UV/Vis, fluorescence, ELS and MS detection have made the transition to UHPLC, dRI detection has been lagging.

This article presents several basic UHPLC analyses based on an on-line dRI UHPLC detector. These examples show that on-line UHPLC dRI detectors do not compromise UHPLC resolution. They also highlight the advantages of a large dynamic range and universal detection. The applications covered include column calibration, discrimination between eluting materials based on the ratio of UV:dRI, response linearity to different injected masses, detection of a non-UV- active impurity, and detection of a sample peak against the background of a solvent gradient.

Protein Fragment or Impurity

A common reagent for calibrating protein quantitation and concentration measurements is bovine serum albumin (BSA). Though the monomeric content of BSA is usually as high as 97-98%, there is a small quantity of proteinaceous material in the solution that is not BSA monomer. Most of the additional protein are irreversible aggregates of BSA i.e. dimers and trimmers. These concentration-independent oligomers are clearly detected and analysed by HPLC size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS).

High-resolution separation by means of UHPLC brings out a new feature in BSA — a small peak eluting later than the primary monomer, shown in Figure 1.

The new peak appears in the dRI chromatographic traces. The possible sources of this peak are threefold: a misfolded BSA monomer (a precursor of the irreversible aggregates usually found by HPLC), a fragment of BSA or a foreign molecule (that may have been left over from the source serum). UV or RI detection alone does not discriminate between any of the possible sources of the small peak. However, the UV:RI ratio does inform assignment of the new species: this value depends on the chemical composition and especially on the ratio of UV-active amino acids, such as tryptophan and tyrosine, to non-UV-active components. Any variants of whole BSA molecules such as aggregates or misfolded protein have the same composition ratio and therefore a constant UV:RI ratio; e.g. the BSA dimer exhibits the same ratio as the monomer. On the other hand, the UV:RI ratio of the new peak is 60-70% higher than that of the monomer, which indicates that the new species is enriched in UV-active material relative to the monomer and therefore cannot be an aggregate or misfolded BSA. It must either be a fragment or foreign.

Isomers

The interaction between a macromolecule and a size-exclusion column is primarily (though not entirely) hydrodynamic in nature. This means that the elution time of any given species is mostly governed by the size and shape of the molecule. Proteins with the same molar mass but different conformations interact a little differently with the column beads to elute at slightly different times. Standard HPLC size exclusion chromatographies, or UHPLC SEC using a short column (150mm), do not usually resolve the isomers unless they have vastly different shapes, e.g. one is denatured. However installing a long column (300 mm), greatly improves the separations of UHPLC, permitting less drastically differentiated isomers to be resolved.

Universal Detection and Column Calibration

Most detection techniques have specific sample or solvent-dependent limitations. UV or fluorescence analysis requires that the sample contain suitable UV-active chromophores; evaporative light scattering requires a volatile solvent. Mass spectrometry does not work with larger macromolecules. Refractive index detection is quite versatile and nearly universal, as long as an isocratic method is used. A partial list of samples compatible with the dRI is as follows: proteins, carbohydrates, oligonucleotides, lipids, small-molecule drug compounds and oils. Figure 2 illustrates how the online UHPLC dRI detector was used in conjunction with a reverse-phase UHPLC column developed for the separating of sugars.

UV-Inactive Impurity Detection

Quality control of a UV-active substance, such as a protein, typically includes analytical UHPLC-SEC incorporating a UV detector. However, impurities present in the sample may not always contain chromophores and so will pass through undetected. In that case, adulterated samples will slip by QC tests without raising any warning flags.

Sample Detection against a Solvent-Gradient

In general, solvent gradients are accompanied by significant changes in the refractive index of the mobile phase, overwhelming and saturating most on-line differential refractive index detectors. Whilst the preference is generally to use a different detection technology in gradient-based separations, sometimes RI may be the only option available. Figure 3 shows an example of gradient chromatography with dRI detection, taking advantage of the extended range of the detector on a standard HPLC system. Though the refractive index changes greatly across the chromatogram, the eluting lysozyme peak is clearly captured and an impurity or variant is resolved from the primary species. No other RI detector provides the combination of range and sensitivity required for this analysis.

About the Author

As Director of Marketing and Principal Scientist at Wyatt Technology Corp., Dr. Some currently investigates techniques for characterization of macromolecular interactions, developing both hardware and software for new instruments and applications. His prior record of experimental research and applied R&D encompasses semiconductor diode lasers, photoluminescence spectroscopy of semiconductors, ultrafast THz spectroscopy, ultrafast laser amplifiers, laser-coupled scanning tunneling microscopy and atomic force microscopy, and laser scattering for defect detection on patterned semiconductor wafers. His publication list includes:

  • 11 patents in the field of laser-based wafer inspection

  • 3 patents and applications in the field of macromolecular interactions

  • 7 peer-reviewed scientific publications

  • A book chapter “Characterization of protein-protein interactions via static and dynamic light scattering”

  • An entry on composition-gradient multi-angle light scattering in the Encyclopedia of Biophysics

Dr. Some obtained his B.Sc. from the Israel Institute of Technology, and his Ph.D. from Brown University, both in physics. Prior to obtaining his doctoral degree, he served as an officer of the Israel Defense Forces in the role of test and evaluation engineer. He held post-doctoral positions at Los Alamos National Laboratory and the Weizmann Institute of Science. As a Member of Technical Staff at Applied Materials' Process Diagnostics and Control Division, Dr. Some conducted experimental work and numerical simulations towards the development of laser scattering for patterned wafer defect detection, contributing key elements of PDC's highly successful wafer inspection tools.

About the Author

Based in Santa Barbara, California, Wyatt Technology is the world’s leading provider of instruments for absolute macromolecular and nanoparticle characterization. With over 40 years’ experience developing multi-angle light scattering detectors, working with customers in the biotechnology, chemical, petrochemical, pharmaceutical, academic and government arenas, Wyatt prides itself on its entrepreneurial spirit, and the uniqueness of its offerings. The Company’s groundbreaking technology and uncompromising levels of customer care make Wyatt the global hallmark in its field.

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