title: Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography.
abstract: Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension.
authors.affiliation: Life Sciences Chemistry R&D, Waters Corporation, Milford, MA 01757, USA. Martin_Gilar@waters.com
authors.forename: Martin
authors.initials: M
authors.lastname: Gilar
authors.forename: Amy E
authors.initials: AE
authors.lastname: Daly
authors.forename: Marianna
authors.initials: M
authors.lastname: Kele
authors.forename: Uwe D
authors.initials: UD
authors.lastname: Neue
authors.forename: John C
authors.initials: JC
authors.lastname: Gebler
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008962:Models, Theoretical; D010455:Peptides / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010455:Peptides