TY - JOUR
T1 - Characterization of capillary-channeled polymer fiber stationary phases for high-performance liquid chromatography protein separations
T2 - Comparative analysis with a packed-bed column
AU - Nelson, Dwella M.
AU - Marcus, R. Kenneth
PY - 2006/12/15
Y1 - 2006/12/15
N2 - Capillary-channeled polymer (C-CP) fibers are investigated as reversed-phase (RP) stationary phases for high-performance liquid chromatography of proteins. A comparative analysis of column characteristics for polypropylene and poly(ethylene terephthalate) C-CP fiber columns and a conventional packed-bed (C4-derivatized silica) column has been undertaken. Five proteins (ribonuclease A, cytochrome c, lysozyme, myoglobin, bovine serum albumin) were used to investigate the separation characteristics under typical RP gradient conditions. Column performance was compared under standard (identical) and optimized RP Chromatographic conditions. The gradient compositions utilized with the C-CP fiber columns are similar to those used with conventional columns, employing flow rates in the 1-6 mL/min range and gradient rates of ∼1%/min. The packed-bed column was operated as prescribed by the column manufacturer. The retention factor (k'), separation factor (α), resolution (Rs), asymmetry factor (As), elution order, and peak capacity values of a four protein separations performed on the C-CP fiber columns are compared to the same separation on the C4 column. One unique feature observed here is the lessening of the percentage of organic modifier necessary to elute the proteins from the fiber phases with increased linear velocity. The potential contribution of the different stationary phases to protein denaturation was evaluated through a spectrophotometric enzymatic activity assay. The repeatability of retention times under both sets of conditions for six consecutive injections of lysozyme on each C-CP fiber column is ≤1.5% RSD. The column-to-column reproducibility of retention times for three columns of each fiber type is also ≤ 1.5% RSD. The overall performance of the C-CP fiber columns was comparable to the conventional column used in these studies. Basic characteristics demonstrated here suggested further developments in the areas of ultrafast protein separations and preparative-scale protein chromatography.
AB - Capillary-channeled polymer (C-CP) fibers are investigated as reversed-phase (RP) stationary phases for high-performance liquid chromatography of proteins. A comparative analysis of column characteristics for polypropylene and poly(ethylene terephthalate) C-CP fiber columns and a conventional packed-bed (C4-derivatized silica) column has been undertaken. Five proteins (ribonuclease A, cytochrome c, lysozyme, myoglobin, bovine serum albumin) were used to investigate the separation characteristics under typical RP gradient conditions. Column performance was compared under standard (identical) and optimized RP Chromatographic conditions. The gradient compositions utilized with the C-CP fiber columns are similar to those used with conventional columns, employing flow rates in the 1-6 mL/min range and gradient rates of ∼1%/min. The packed-bed column was operated as prescribed by the column manufacturer. The retention factor (k'), separation factor (α), resolution (Rs), asymmetry factor (As), elution order, and peak capacity values of a four protein separations performed on the C-CP fiber columns are compared to the same separation on the C4 column. One unique feature observed here is the lessening of the percentage of organic modifier necessary to elute the proteins from the fiber phases with increased linear velocity. The potential contribution of the different stationary phases to protein denaturation was evaluated through a spectrophotometric enzymatic activity assay. The repeatability of retention times under both sets of conditions for six consecutive injections of lysozyme on each C-CP fiber column is ≤1.5% RSD. The column-to-column reproducibility of retention times for three columns of each fiber type is also ≤ 1.5% RSD. The overall performance of the C-CP fiber columns was comparable to the conventional column used in these studies. Basic characteristics demonstrated here suggested further developments in the areas of ultrafast protein separations and preparative-scale protein chromatography.
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U2 - 10.1021/ac061223w
DO - 10.1021/ac061223w
M3 - Article
C2 - 17165840
AN - SCOPUS:33845546540
SN - 0003-2700
VL - 78
SP - 8462
EP - 8471
JO - Analytical chemistry
JF - Analytical chemistry
IS - 24
ER -