Luisa Pereira

Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a ‘virtual’ narrow bore column☆

Narrow bore columns find advantage in HPLC applications when volumetric flow is important, For example, for detection processes that are volume limited. Yet there are significant drawbacks to narrow bore columns. Due to their small column volume relative to analytical scale columns, narrow bore columns are more affected by system dead volume. In addition the wall effect and the variation in packing density from the centre to the wall are more significant in these columns relative to larger scale analytical columns. In this study we operate a 4.6mm i.d. parallel segmented flow column in such a manner that it emulates 2.1mm i.d. and 3.0mm i.d. columns. By using a parallel segmented flow column in this way, it was possible to combine the benefits of narrow bore and analytical scale columns.

Enhancing the separation performance of the first-generation silica monolith using active flow technology: parallel segmented flow mode of operation.

Active flow technology (AFT) columns are designed to minimise inefficient flow processes associated with the column wall and radial heterogeneity of the stationary phase bed. This study is the first to investigate AFT on an analytical scale 4.6mm internal diameter first-generation silica monolith. The performance was compared to a conventional first-generation silica monolith and it was observed that the AFT monolith had an increase in efficiency values that ranged from 15 to 111%; the trend demonstrating efficiency gains increasing as the volumetric flow to the detector was decreased, but with no loss in sensitivity.

The utility of porous graphitic carbon as a stationary phase in proteomics workflows: two-dimensional chromatography of complex peptide samples.

We present the first investigation into the utility of porous graphitic carbon (PGC) as a stationary phase in proteomic workflows involving complex samples. PGC offers chemical and physical robustness and is capable of withstanding extremes of pH and higher temperatures than traditional stationary phases, without the likelihood of catastrophic failure. In addition, unlike separations driven by ion exchange mechanisms, there is no requirement for high levels of non-volatile salts such as potassium chloride in the elution buffers, which must be removed prior to LC-MS analysis. Here we present data which demonstrate that PGC affords excellent peptide separation in a complex whole cell lysate digest sample, with good orthogonality to a typical low pH reversed-phase system. As strong cation exchange (SCX) is currently the most popular first dimension for 2D peptide separations, we chose to compare the performance of a PGC and SCX separation as the first dimension in a comprehensive 2D-LC-MS/MS workflow. A significant increase, in the region of 40%, in peptide identifications is reported with off-line PGC fractionation compared to SCX. Around 14,000 unique peptides were identified at an estimated false discovery rate of 1% (n=3 replicates) from starting material constituting only 100 μg of protein extract.

Assessing the performance of curtain flow first generation silica monoliths

Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses.

Improving quantification using curtain flow chromatography columns in the analysis of labile compounds: A study on amino acids ☆

The performance of curtain flow chromatography column technology with MS detection was evaluated for the analysis of labile compounds. The curtain flow column design allows for separations that are faster and/or more sensitive than conventional columns, depending on how exactly the curtain flow column is configured. For example, when mass spectral detection is employed, the curtain flow column can yield separations that are 5-times faster than conventional columns when the curtain flow and the conventional columns have the same internal diameter. Or when the internal diameter of the conventional column is reduced in order to yield the same analytical through-put as the curtain flow column, the sensitivity on the curtain flow column can be as much as 66-fold higher than the conventional column. As a consequence of the higher analytical through-put less standardization is required in the analysis of labile compounds because less sample degradation is apparent. Consequently the sample integrity is preserved yielding data of a higher quality.