Talanta 78 (2009) 781–789 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Estimation of uncertainty in size-exclusion chromatography with a double detection system (light-scattering and refractive index) Alexis Oliva ∗ , Matías Llabrés, José B. Fari˜ na Dpto. Ingeniería Química y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de La Laguna, Avda. Fco. Sanchez, s/n, La Laguna, 38200, La Laguna, Tenerife, Spain article info Article history: Received 16 June 2008 Received in revised form 16 December 2008 Accepted 18 December 2008 Available online 30 December 2008 Keywords: Light-scattering Uncertainty Peptides and proteins Validation Method reproducibility abstract Size-exclusion chromatography (SEC) coupled with online laser light-scattering (LS) and refractive index (RI) detection provides an excellent approach to determine the molecular weights (Mw) of proteins by the “two-detector” approach. Mw is determined only at the maximum of a peak, using either peak heights or area ratio from the two detectors. However, proper calibration of the SEC/LS/RI system is critical to obtain high precision. Today, an essential part of any analysis is to evaluate the uncertainty associated with the method. Basically, it is possible to distinguish between factors related to signal nature, precision and those due to signal processing. Given the signal of interest is the peak height or area ratio from two detectors, the signal ratio uncertainty was calculated using the random propagation of error formula. In this case, the effect of signal correlation was evaluated to avoid the uncertainty overestimation. In the second case, the sources of uncertainty affecting analytical measurement were estimated with the information from the precision assessment. For this, two designs with two-factor fully nested were followed for each method. Finally, the contributions from various uncertainty sources related with calibration are also analysed in detail. There are in fact only three main sources of measurement uncertainty: intermediate precision, calibration and repeatability. Of these, method precision is always the greatest, regardless of approach. For all proteins and peptides studied, the Mw calculated using both methods are close to the theoretical results, independently of the design, but the contributions of individual terms to combined uncertainty depend on both the design and method used. For example, the combined uncertainty varied between 223 and 813.2 Da for carbonic anhydrase, although higher values were found for human insulin and ovalbumin dimer. Other considerations that can have a significant impact on the results are discussed. The reproducibility of the two methods versus that based on ASTRA software used as reference method was performed using the concordance correlation coefficient. The methods’ reproducibility depends on the permitted losses in precision and accuracy. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Recombinant proteins intended for clinical use must be exten- sively characterized with regard to their molecular and biological properties and monitored for structural and biological integrity during manufacturing and storage. Various analyses are used to characterize the biomolecules. Molecular weight, conformation, size and shape, and state and extent of aggregation are a few of the physicochemical properties studied. Recent technological advances have significantly increased the speed of characterizing proteins. It is now feasible to measure the LS, RI and intrinsic viscosity (IV), along with UV-absorption characteristics of protein component peaks separated on a SEC column in real time by coupling four detectors online with HPLC equipment. A combination of two or ∗ Corresponding author. Tel.: +34 922 318 452; fax: +34 922 318 514. E-mail address: amoliva@ull.es (A. Oliva). more of these four detectors online with chromatographic systems has been used in several laboratories for determination of protein Mw, hydrodynamic radius, protein aggregation, and protein glyco- sylation [1–5]. Size-exclusion chromatography (SEC) coupled with online laser light-scattering (LS) and refractive index (RI) detection provides an excellent approach to determine the molecular weights (Mw) of proteins by the “two-detector” approach [1,3]. Mw is determined only at the maximum of a peak when using peak heights, or for a selected portion of the eluting peak, if using peak areas ratio from the two detectors. This approach circumvents the need to know the specific refractive index for the protein, a parameter that is oth- erwise needed to determine Mw using the commercial available software for LS data analysis e.g. ASTRA (Wyatt Corp., Santa Barbara, CA). However, proper calibration of the SEC/LS/RI system is cru- cial to obtain high precision since the experimentally determined Mw depends on the precision of the instrument calibration con- stant, and Mw of the calibration standard [1–5]. To obtain precise 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.12.039