Guanidine improves DEAE anion exchange‐based analytical separation of plasmid DNA

Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles...

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Bibliographic Details
Published in:Electrophoresis 2021-12, Vol.42 (24), p.2619-2625
Main Authors: Černigoj, Urh, Vidič, Jana, Ferjančič, Ana, Sinur, Urša, Božič, Klemen, Mencin, Nina, Martinčič Celjar, Anže, Gagnon, Pete, Štrancar, Aleš
Format: Article
Language:English
Subjects:
Analytical chromatography
Anion exchanging
Anions
Chromatography, Ion Exchange
Concentration gradient
DNA
Electrophoresis
Elution
Ethanolamines
Ethylenediaminetetraacetic acids
Guanidine
Guanidine hydrochloride
Guanidines
Isomers
Monoliths
pDNA isoforms
pDNA multimers
Plasmids
Process analytical technology
Quaternary ammonium salts
Selectivity
Separation
Sodium Chloride
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Summary:Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open‐circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the diethylaminoethyl‐Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than CE, and resolution approaching CE.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.202100210