Optimization model for the gradient elution separation of peptide mixtures by reversed-phase high-performance liquid chromatography

Stadalius, M.A.; Quarry, M. A.; Snyder, Lloyd R.

doi:10.1016/s0021-9673(01)81640-x

Cited by 96 publications

(33 citation statements)

References 19 publications

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“…For the short column, no increase in peak capacity is observed with a gradient of more than 15 min, but for the long column the peak capacity increases up to a 75-min gradient. A possible explanation can be found in the study of Stadalius et al [11, 12], who demonstrated that peak capacity will increase with gradient time, until it reaches a maximum, after which it will even decrease. The gradient time at which this maximum is obtained is greater for longer columns.…”

Section: Resultsmentioning

confidence: 99%

“…The first is increase of the gradient length; gradients of up to 10 h have been reported on single columns [10]. However, according to theory, peak capacity increases to a maximum and then decreases as the gradients become longer [11, 12]. The second approach is the use of longer columns, as the peak capacity increases linearly with the square root of the plate number and thus with the square root of the column length.…”

Section: Introductionmentioning

confidence: 99%

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Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection

Meent

Jong

2007

Anal Bioanal Chem

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Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC-UV-mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3-and 15-min gradients with the short column and 1,376 and 993 for the 15-and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection

Meent

Jong

2007

Anal Bioanal Chem

View full text Add to dashboard Cite

show abstract

“…The long conventionalpacked column could be used within the pressure limit of the instrument in proportion to the column length and plate number. Furthermore, peak capacity (n) [16], which indicates the degree of separation, was calculated using Eq. (1), where t G is gradient time and 4r is peak width that is nearly constant in linear gradient runs.…”

Section: Resultsmentioning

confidence: 99%

Performance of wide‐pore monolithic silica column in protein separation

Morisaka

Kobayashi

Kirino

et al. 2009

J of Separation Science

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A monolithic wide-pore silica column was newly prepared for protein separation. The wide distribution of the pore sizes of monolithic columns was evaluated by mercury porosimetry. This column, as well as the conventional monolithic column, shows high permeability in the chromatographic separation of low-molecular-sized substances. In higher-molecular-sized protein separation, the wide-pore monolithic silica column shows better performance than that of the conventional monolithic column. Under optimized conditions, five different proteins--ribonuclease A, albumin, aldolase, catalase, and ferritin--were baseline-separated within 3 min, which is faster than that using the particle-packed columns. In addition, the monolithic wide-pore silica column could also be prepared in fused silica capillary (600 mm long, 0.2 mm i.d.) for highly efficient protein separation. The peak capacity of the wide-pore monolithic silica capillary column is estimated to be approximately 300 in the case of protein separation, which is a characteristic performance.

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“…The four peptides eluted with an average peak width of 0.36 min when hydrophobic interactions were suppressed with 40% ACN. According to the definition of peak capacity in gradient elution by Snyder and coworkers [55], the peak capacity was calculated to be 28, which is almost the same as that of particulate-based SCX columns [3,4,7], and surpasses most polymer monolithic SCX columns [15,41].…”

Section: Separation Of Peptidesmentioning

confidence: 99%

Polymeric strong cation‐exchange monolithic column for capillary liquid chromatography of peptides and proteins

Chen

Tolley

Lee³

2009

J of Separation Science

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A strong cation-exchange (SCX) monolithic stationary phase was prepared in 75 microm id capillaries by direct in situ polymerization of sulfopropyl methacrylate and polyethylene glycol diacrylate in a ternary porogen system consisting of methanol, cyclohexanol, and water. The resulting monolith exhibited good dynamic binding capacity, fast kinetic adsorption of proteins, and high permeability. The monolith had a dynamic binding capacity of approximately 52 mg/mL of column volume for lysozyme and cytochrome C. The monolith was evaluated for SCX capillary LC of synthetic peptides, natural peptides, and protein standards. Fast separation of proteins was achieved in less than 4 min. The average peak capacity for peptides was 28 using a relatively steep gradient when hydrophobic interactions were suppressed with 40% acetonitrile.

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Optimization model for the gradient elution separation of peptide mixtures by reversed-phase high-performance liquid chromatography

Cited by 96 publications

References 19 publications

Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection

Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection

Performance of wide‐pore monolithic silica column in protein separation

Polymeric strong cation‐exchange monolithic column for capillary liquid chromatography of peptides and proteins

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