Life Science
Measuring Absolute Molar Mass Using UHPLC SEC-MALS
istry and clinical diagnostics has traditionally been carried out using size exclusion chro- matography (SEC). Conventional SEC relies on reference standards to calibrate column elution time as a function of molar mass. How- ever, 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, produc- ing 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 refer- ence standards and elution time.
T
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 detec- tors 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 in- formation than standard SEC-MALS due to the high-quality resolution obtained with UHPLC
he characterization of aggregates or fragments in a biomolecular sample for applications such as bioanalytical chem-
columns. This capability has meant that re- searchers 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 instrumen- tation 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 spe- cifically 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 concentra- tion 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 (deter- mined 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.
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 stan- dard columns and detectors broadens out these small peaks, leading to the “vanishing”
AMERICAN LABORATORY • 12 • JUNE/JULY 2014
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.
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 pre- serve 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.
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 refrac- tive 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
by Sophia Kenrick and Aym Berges
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64