Article

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides.

Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
Journal of Chromatography A (Impact Factor: 4.61). 11/2007; 1169(1-2):139-50. DOI: 10.1016/j.chroma.2007.09.005
Source: PubMed

ABSTRACT Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.

0 Bookmarks
 · 
307 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A set of tryptic peptides was analyzed in reversed-phase liquid chromatography using gradient elution with acetonitrile, methanol, or isopropanol. We used these retention data as training sets to develop retention prediction models of peptides for the three organic eluents used. The coefficients of determination, R2, between predicted and observed data were approximately 0.95 for all systems. Retention coefficient values of twenty amino acids calculated from a model were utilized to investigate differences in separation selectivity between acetonitrile, methanol, or isopropanol eluents. The experimentally observed difference in separation selectivity appears to be a complex interplay of multiple amino acids, each contributing to a different degree to overall peptide retention. While retention contribution of hydrophilic amino acids was higher in methanol than acetonitrile, peptides containing aromatic amino acids (tyrosine, phenylalanine, tryptophan) exhibit relatively lower retention in methanol compared to acetonitrile. The differences between acetonitrile and isopropanol eluents were less pronounced. We also compared the relative elution strength of the three organic eluents for peptides. The relationship between the elution strength of two solvents is not linear, rather it was best fitted by a cubic polynomial function. Three solvents can be arranged in the order of increasing elution power as methanol < acetonitrile < isopropanol. The equations for relative solvent strength conversion were proposed.
    Journal of Chromatography A 01/2014; · 4.61 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A dual labeled oligonucleotide used as TaqMan® or 5' nuclease probe for in vitro diagnostic has been purified through orthogonal ion-pairing reversed phase chromatography, using polymeric semi-preparative and preparative PRP-1 column. We studied the mechanism of separation of oligonucleotides using ion-pairing reversed phase chromatography. We found that elution profiles of dye labeled oligonucleotides can be controlled by use of specific ion-pairing reagents. Here, we report a method for purification of an oligonucleotide containing an internally positioned rhodamine dye using two orthogonal chromatographic steps, in which the primary step resolves mostly by differences in hydrophobicity by using a weak ion-pairing reagent, and a secondary step uses a strong ion-pairing reagent for separation of length variants. Purification is demonstrated for both 1 and 15μmol scale syntheses, and amenable to further scale up for commercial lot production.
    Journal of Chromatography A 01/2014; · 4.61 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Recent findings have elucidated numerous novel biological functions for oligonucleotides. Current standard methods for the study of oligonucleotides (i.e., hybridization and PCR) are not fully equipped to deal with the experimental needs arising from these new discoveries. More importantly, as the intracellular capacity of oligonucleotides is being harnessed for biomedical applications, alternative bioanalytical techniques become indispensable in order to comply with ever-increasing regulatory requirements. Owing to its ability to detect oligonucleotides independent of their sequence, LC-MS is emerging as the analytical method of choice for oligonucleotides. In this article, the current applications of LC-MS in the analysis of oligonucleotides, with an emphasis on RNA therapeutics and biomarkers, will be examined. In addition, the theoretical framework of oligonucleotide ESI is carefully inspected with the purpose of identifying the contributing factors to MS signal intensity.
    Bioanalysis 06/2014; 6(11):1525-1542. · 3.25 Impact Factor

Full-text (2 Sources)

Download
162 Downloads
Available from
May 19, 2014