Chromatographic evidence of silyl ether formation (SEF) in supercritical fluid chromatography.

In this article, we propose that silyl ether formation (SEF) is a major contribution to retention and selectivity variation over time for supercritical fluid chromatography (SFC). In the past, the variations were attributed to instrumentation, but high performance SFC systems have shed new light on the source of variation. As silyl ethers form on the particle surface, the hydrophilicity is decreased, significantly altering the retention and selectivity observed.

Characterization and kinetic performance of 2.1 × 100 mm production columns packed with new 1.6 μm superficially porous particles.

The overall kinetic performance of three production columns (2.1 mm × 100 mm format) packed with 1.6 μm superficially porous CORTECS-C18 + particles was assessed on a low-dispersive I-class ACQUITY instrument.

Evaluation of the kinetic performance of new prototype 2.1mm×100mm narrow-bore columns packed with 1.6μm superficially porous particles.

The mass transfer mechanism in three prototype narrow-bore columns (2.1mm×100mm format) packed with 1.6μm superficially porous particles was investigated using different instruments.

Retention mechanism divergence of a mixed mode stationary phase for high performance liquid chromatography.

Mixed mode stationary phases utilize secondary retention mechanisms to add a dimensionality to the surface of high performance liquid chromatography (HPLC) adsorbents. This approach was used by several authors to improve the separation performance of single dimension separations. We explored the magnitude of these secondary interactions by performing an off-line two-dimensional (2D)-HPLC separation with a Scherzo SM-C18 column of a β-lactoglobulin tryptic digest with a mobile phase pH of 7 in the first dimension and 2 in the second.

Black box linearization for greater linear dynamic range: the effect of power transforms on the representation of data.

Power transformations are commonly used in image processing techniques to manipulate image contrast. Many analytical results, including chromatograms, are essentially presented as images, often to convey qualitative information. Power transformations have remarkable effects on the appearance of the image, in chromatography, for example, increasing apparent resolution between peaks by the factor √n and apparent column efficiency (plate counts) by a factor of n for an nth-power transform.

Two-dimensional liquid chromatography/mass spectrometry/mass spectrometry separation of water-soluble metabolites.

Off-line two-dimensional liquid chromatography with tandem mass spectrometry detection (2D-LC/MS-MS) was used to separate a set of metabolomic species. Water-soluble metabolites were extracted from Escherichia coli and Saccharomyces cerevisae cultures and were immediately analyzed using strong cation exchange (SCX)-hydrophilic interaction chromatography (HILIC). Metabolite mixtures are well-suited for multidimensional chromatography as the range of components varies widely with respect to polarity and chemical makeup.

Martin-Synge algorithm for the solution of equilibrium-dispersive model of liquid chromatography.

An alternative method, called the Martin-Synge algorithm, is introduced to calculate numerical solutions of the equilibrium-dispersive (ED) model. The developed algorithm is based on the earlier work of Friday and Levan and on the continuous plate model of Martin and Synge. The column is divided evenly into a series of virtual vessels in which a simplified mass balance equation is solved accurately by the Runge-Kutta-Fehlberg method and the elution profile is given by the numerical solution for the last vessel.

On the optimization of the shell thickness of superficially porous particles.

The thickness of the porous shells of superficially porous particles influences the separation power of columns packed with these packing materials. Models of the mass transfer kinetics across porous adsorbents permit the prediction of the HETP curves of columns packed with particles having shells of different thicknesses, for molecules of different sizes. Decreasing the thickness of the porous layer potentially results in lower values of the "C-term" of the HETP curve and of the minimum of these curves.

Correlation between peak capacity and protein sequence coverage in proteomics analysis by liquid chromatography-mass spectrometry/mass spectrometry.

Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) allows the acquisition of a vast amount of analytical data in the aim of identifying peptides and proteins. Difficulties arise when attempting to identify proteins from the results of analyses of their peptide digests. We investigated possible quantitative correlations between the peak capacity achieved in chromatographic analyses and the protein sequence coverage.

Detection issues in two-dimensional on-line chromatography.

The dilution of analytes during their migration through two-dimensional liquid chromatographic systems was investigated. Simplified equations for the calculation of the dilution factors in the first and second dimension columns were derived considering the variance of the injected sample, the flow rates, the split ratio, the volume of the intermediate sample loop and the sampling time of the first-dimension effluent. The net dilution factor is the product of the dilution factors in the two single dimensions.

Theoretical advantages and drawbacks of on-line, multidimensional liquid chromatography using multiple columns operated in parallel.

The theoretical advantages and drawbacks of using a multiple-, parallel column approach in on-line multidimensional liquid chromatography systems were investigated. Much time or peak capacity can be gained with the use of multiple parallel columns at the second-dimension while the aggregate time of separation increases only by the increment of the gradient time of the second-dimension. Multidimensional chromatographic systems are now used to perform many tasks ranging from routine, fast analyses to specialized, arduous separations.

Generation and limitations of peak capacity in online two-dimensional liquid chromatography.

The different operating conditions of an online two-dimensional liquid chromatographic separation (2D-LC), such as the length of the column, the linear velocity and the composition of the mobile phase used in the second dimension, its initial organic content if this separation is carried out in gradient elution, the number of fractions of the first column eluent collected, and the analysis time of the first dimension all affect the achievable separation power of 2D-LC online systems. The influences of these factors on the separation performance were investigated, and an equation was derived for the calculation of the achievable peak capacity in online 2D-LC assuming (1) that the option of undersampling the first-dimension separation is acceptable, (2) that the solutes follow linear-solvent-strength behavior, and (3) that all the separations are made in gradient elution. This theoretical discussion shows that (1) highly efficient separations made with online 2D-LC require the second-dimension peaks to be very narrow, (2) the separation power of 2D-LC systems is maximum for an optimum number of fractions collected in the first dimension, (3) higher peak capacities can be achieved by using shorter second-dimension columns and collecting a relatively large number of fractions, (4) the achievable 2D peak capacity is maximum for a certain eluent flow rate and column length of the second-dimension column, and (5) the maximum achievable peak capacity increases with decreasing velocity and initial organic content of the second-dimension eluent.

Approaches to comprehensive multidimensional liquid chromatography systems.

This work compares the performance of the three different schemes implementing two-dimensional liquid chromatography (2D-LC) in terms of the peak capacity that they can generate and of the time that they need to complete a two-dimensional analysis. We discuss in detail how time is spent in these two-dimensional liquid chromatography x liquid chromatography (LC x LC) schemes and how to compare them. Keeping constant the characteristics of the first-dimension separation, we systematically varied those of the second-dimension separation and of its coupling to the first-dimension.

Comprehensive off-line, two-dimensional liquid chromatography. Application to the separation of peptide digests.

The separation of the peptide digests of myoglobin and bovine serum albumin was performed with an off-line combination of two commercial, conventional HPLC columns. The first column was packed with a strong ion exchanger and eluted with a KCl gradient. The second column was packed with particles of C18-bonded silica and eluted with an acetonitrile gradient.