Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography

Gilár, Martin; Daly, Amy E.; Kele, Marianna; Neue, Uwe D.; Gebler, John C.

doi:10.1016/j.chroma.2004.10.092

Cited by 157 publications

(127 citation statements)

References 34 publications

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“…The isocratic retention of a solute (ln k') is assumed to be a linear function of the volume fraction of the organic modifier (ϕ): (12) where S is the slope of ln k' versus ϕ and ln k' w is the extrapolated solute retention in pure water. Using LSST theory, the retention time (t R ) in linear gradient elution is given as: 24 (13) where t 0 is the column dead time, t D is the system dwell time, k' 0 is the isocratic retention factor in the initial eluent of the gradient.…”

Section: Prediction Of Retention Timesmentioning

confidence: 99%

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

et al. 2006

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The optimization of peak capacity in gradient elution RPLC is essential for the separation of multicomponent samples such as those encountered in proteomic research. In this work we study the effect of gradient time (t G ), flow rate (F), temperature (T) and final eluent strength (ϕ final ) on the peak capacity of separations of peptides that are representative of the range in peptides found in a tryptic digest. We find that there are very strong interactions between the individual variables (e.g. flow rate and gradient time) which make the optimization quite complicated. On a given column, one should first set the gradient time to the longest tolerable and then set the temperature to the highest achievable with the instrument. Next, the flow rate should be optimized using a reasonable but arbitrary value of ϕ final . Last, the final eluent strength should be adjusted so that the last solute elutes as close as possible to the gradient time. We also develop an easily implemented, highly efficient and effective Monte Carlo search strategy to simultaneously optimize all the variables. We find that gradient steepness is an important parameter that influences peak capacity and an optimum range of gradient steepness exists in which the peak capacity is maximized.

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Section: Prediction Of Retention Timesmentioning

confidence: 99%

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

et al. 2006

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“…In gradient mode, several authors have also demonstrated studies where decreasing particle size was used for improving peak capacity and thus chromatographic resolution [52,53]. Particle size reduction has more effect than column length, gradient time, or flow-rate to improve peak capacity in gradient mode.…”

Section: Interest Of Small Particlesmentioning

confidence: 99%

Fast analysis in liquid chromatography using small particle size and high pressure

Nguyen

Guillarme

Rudaz

et al. 2006

J of Separation Science

300

164

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ReviewFast analysis in liquid chromatography using small particle size and high pressureIn order to enhance chromatographic performances in terms of efficiency and rapidity, LC has recently evolved in the development of short columns packed with small particles (sub-2 lm) working at high pressures (A400 bar). This approach has been described 30 years ago according to the fundamental chromatographic equations. However, systems and columns compatible with such high pressures have been introduced in the market in 2004 only. Advantages of small particles working at high pressure will be discussed in terms of sensitivity, efficiency, resolution, and analysis time. Potential problems encountered with high pressure in terms of frictional heating and solvent compressibility will also be discussed even if systems working at a maximum pressure of 1000 bar are not influenced by these parameters and give reliable and reproducible results. Several applications will highlight the potential and interest of this new technology.

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“…It has been predicted that a peak capacity of 15000 can be obtained in 8 h by using a high-efficiency 2nd-D column and gradient elution at both dimensions in 2D-HPLC. 76 Reversedphase mode 2D-HPLC is easy to carry out, as shown in section 6·2. The development of various stationary phases bonded to monolithic silica columns of higher performance will make 2D-HPLC a separation tool of practical utility.…”

Section: Discussionmentioning

confidence: 99%

Properties of Monolithic Silica Columns for HPLC

et al. 2006

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Monolithic silica columns and their use in high peak-capacity HPLC separations are reviewed. Monolithic silica columns can potentially provide higher overall performance than particle-packed columns based on the variable external porosity and variable through-pore size/skeleton size ratios. The high permeability of monolithic silica columns resulting from the high porosity is shown to be advantageous to generate large numbers of theoretical plates with long capillary columns. High permeability together with the high stability of the network structures of silica allows their use in high-speed separations required for a second-dimension column in two dimensional HPLC. Disadvantages of monolithic silica columns are also described.

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Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography

Cited by 157 publications

References 34 publications

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

Peak Capacity Optimization of Peptide Separations in Reversed-Phase Gradient Elution Chromatography: Fixed Column Format

Fast analysis in liquid chromatography using small particle size and high pressure

Properties of Monolithic Silica Columns for HPLC

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