The characterization of aggregates or fragments in a biomolecular sample for applications such as bioanalytical chemistry and clinical diagnostics has traditionally been carried out using size exclusion chromatography (SEC). Conventional SEC relies on reference standards to calibrate column elution time as a function of molar mass. However, these standards often do not accurately represent the conformation of the sample, and in some cases the sample may demonstrate column interactions that change its elution properties relative to the standard, producing erroneous results. The combination of size exclusion chromatography and a multi-angle light scattering detector (SEC-MALS) with conventional HPLC is commonly utilized to determine molar masses independent of reference standards and elution time.
This article demonstrates how a new set of MALS and concentration detectors have been engineered for coupling to ultrahigh-performance liquid chromatography (UHPLC) to enable absolute quantification of molar mass in conjunction with UHP-SEC.
MALS for UHPLC applications
UHPLC enables fast, efficient separation of biomolecular samples. Compared to standard SEC, UHP-SEC provides improved resolution, higher throughput, less solvent, and smaller sample consumption. Light scattering detectors designed for HPLC have generally been incompatible with UHPLC. Multi-angle light scattering detectors and refractive index (RI) detectors specifically engineered for UHPLC applications can now be used in combination to measure the absolute molar mass and size of eluting species in UHPLC, which was previously not possible.
For the first time, the narrow peak widths typical of UHPLC can be maintained within the MALS detector while also providing even more information than standard SEC-MALS due to the high-quality resolution obtained with UHPLC columns. This capability has meant that researchers can now transition from existing HPLC methods to UHPLC while maintaining the same accurate, high-quality molecular weight data.
Measuring absolute molar mass
In this study, UHPLC was performed with bovine serum albumin (Sigma, St. Louis, MO) using an ACQUITY® UPLC pump, autosampler, and UV detector (Waters, Milford, MA). This instrumentation was coupled to the μDAWN™ multi-angle light scattering (MALS) detector and Optilab® UT-rEX™ refractive index (RI) detector (Wyatt Technology, Santa Barbara, CA), designed specifically for UHPLC applications. This combination of instrumentation enabled the measurement of absolute molecular weight (or molar mass) and size eluting species in UHPLC.
For a given protein, the entire UHPLC analysis can be completed in less than 5 min (Figure 1, red), compared to 20 min or more per sample for traditional HPLC (Figure 1, blue). For both traditional HPLC and UHPLC, the combination of light scattering intensity and concentration measured by RI is used to determine the molar mass for each peak. Figure 1 overlays the chromatograms with the monomer, dimer, and aggregate molar masses for standard SEC-MALS (determined by the miniDAWN TREOS and Optilab T-rEX) and for UHP-SEC-MALS (determined by the μDAWN and Optilab UT-rEX). The chromatograms in Figure 1 (top) are rescaled as a function of column volume (bottom) for easier comparison. The perfect agreement in molar mass for each peak means that it is now even easier for researchers to translate existing HPLC methods to UHPLC while maintaining the same accurate, high-quality molecular weight data.
Figure 1 – Light scattering data and measured molar mass for a protein separated by UHPLC and detected using the μDAWN and UT-rEX (red) overlaid with the separation by standard HPLC and detected with the miniDAWN (Wyatt) TREOS and Optilab T-rEX (blue). The top plot shows the chromatograms as a function of elution time. The same data are rescaled in the bottom plot as a function of column volume.
Not only does UHP-SEC deliver faster analysis time, the small particle size of the UHPLC stationary phase means improved resolution of aggregate and fragment peaks. Dispersion caused by the large internal volume of standard columns and detectors broadens out these small peaks, leading to the “vanishing” of certain poorly resolved species, such as the fragment shown in Figure 2 (blue traces). The μDAWN is the only MALS instrument optimized for minimal internal volume in order to preserve the resolution in a UHPLC elution profile; this means that previously unknown species can now be detected and analyzed with MALS coupled to UHP-SEC.
Figure 2 – Light scattering data and measured molar mass for a protein separated by UHPLC (red) overlaid with the separation by standard HPLC (blue). The box indicates a fragment peak that was separable only by UHPLC. The figure at the right is zoomed in to show the fragment and its molar mass as measured by the μDAWN.
Because of the optimized internal volume of these detectors, the fragment peak is preserved as it travels between detectors (Figure 2, red traces). Thus, the molar mass of the fragment peak can now be quantified. Since the refractive index increment (dn/dc) is nearly constant for all proteins, there is no need to know the extinction coefficient of the peak (and thus the identity of the fragment) in order to determine the eluting concentration and molar mass. The combination of UV and RI detection may even be used to determine the extinction coefficient of each species and aid in their identification.
In addition to quantifying absolute molecular weight, the μDAWN offers all the in-depth analyses available with the miniDAWN TREOS for UHPLC. As with standard HPLC-MALS, changes in molar mass across the UHPLC-MALS peak quantify polydispersity of a biopolymer or assess reversible protein oligomerization. Triple detection with UV, MALS, and RI enables the application of protein conjugate analysis to identify the amount of post-translational modification, associated detergent, drug-conjugate, or other additions to a polypeptide background.
In order to continue to raise standards in molecular analysis carried out using UHPLC, researchers need a technique that can deliver efficient and repeatable characterization of biomolecular samples. This study has demonstrated that with a MALS and RI detector specifically engineered for UHPLC, users can enjoy the efficiency of UHPLC without sacrificing the ability to measure absolute molar mass with MALS. By successfully overcoming the limitations of conventional detectors, this capability will enable unprecedented levels of in-depth analysis to be achieved for UHPLC applications.
Sophia Kenrick, Ph.D., and Aym Berges, Ph.D., are both Application Scientists at Wyatt Technology Corp., 6300 Hollister Ave., Goleta, CA 93117, U.S.A.; tel.: 805-681-9009; e-mail: firstname.lastname@example.org; www.wyatt.com