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Investigating effects of sample pretreatment on protein stability using size‐exclusion chromatography and high‐resolution continuum source atomic absorption spectrometry
Author(s) -
Rakow Tobias,
El Deeb Sami,
Hahne Thomas,
ElHady Deia Abd,
AlBishri Hassan M.,
Wätzig Hermann
Publication year - 2014
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201400189
Subject(s) - chemistry , chromatography , size exclusion chromatography , adsorption , mass spectrometry , atomic absorption spectroscopy , myoglobin , analytical chemistry (journal) , sample preparation , liquid nitrogen , organic chemistry , quantum mechanics , enzyme , physics
In this study, size‐exclusion chromatography and high‐resolution atomic absorption spectrometry methods have been developed and evaluated to test the stability of proteins during sample pretreatment. This especially includes different storage conditions but also adsorption before or even during the chromatographic process. For the development of the size exclusion method, a Biosep S3000 5 μm column was used for investigating a series of representative model proteins, namely bovine serum albumin, ovalbumin, monoclonal immunoglobulin G antibody, and myoglobin. Ambient temperature storage was found to be harmful to all model proteins, whereas short‐term storage up to 14 days could be done in an ordinary refrigerator. Freezing the protein solutions was always complicated and had to be evaluated for each protein in the corresponding solvent. To keep the proteins in their native state a gentle freezing temperature should be chosen, hence liquid nitrogen should be avoided. Furthermore, a high‐resolution continuum source atomic absorption spectrometry method was developed to observe the adsorption of proteins on container material and chromatographic columns. Adsorption to any container led to a sample loss and lowered the recovery rates. During the pretreatment and high‐performance size‐exclusion chromatography, adsorption caused sample losses of up to 33%.