A systematic investigation of sample diluents in modern supercritical fluid chromatography.

This paper focuses on the possibility to inject large volumes (up to 10µL) in ultra-high performance supercritical fluid chromatography (UHPSFC) under generic gradient conditions. Several injection and method parameters have been individually evaluated (i.e. analyte concentration, injection volume, initial percentage of co-solvent in the gradient, nature of the weak needle wash solvent, nature of the sample diluent, nature of the column and of the analyte). The most critical parameters were further investigated using in a multivariate approach. The overall results suggested that several aprotic solvents including methyl tert-butyl ether (MTBE), dichloromethane, acetonitrile or cyclopentyl methyl ether (CPME) were well adapted for the injection of large volume in UHPSFC, while MeOH was generally the worst alternative. However, the nature of the stationary phase also had a strong impact and some of these diluents did not perform equally on each column. This was due to the existence of a competition in the adsorption of the analyte and the diluent on the stationary phase. This observation introduced the idea that the sample diluent should not only be chosen according to the analyte but also to the column chemistry to limit the interactions between the diluent and the ligands. Other important characteristics of the "ideal" SFC sample diluent were finally highlighted. Aprotic solvents with low viscosity are preferable to avoid strong solvent effects and viscous fingering, respectively. In the end, the authors suggest that the choice of the sample diluent should be part of the method development, as a function of the analyte and the selected stationary phase.

Using the fundamentals of adsorption to understand peak distortion due to strong solvent effect in hydrophilic interaction chromatography.

The peak distortion observed in hydrophilic interaction chromatography (HILIC) may be caused by the sample diluent to mobile phase mismatch. The United States Pharmacopeia (USP) method for organic impurities in cetirizine HCl tablets calls for such a mismatch, having a higher concentration of strong solvent in the sample diluent than in the mobile phase. A significant peak deformation is reported for cetirizine (a second-generation antihistamine) when it is purified on a Ethylene Bridged Hybrid (BEH) HILIC column (4.6mm×100mm, 2.5µm particles) using an acetonitrile-water eluent mixture and a sample diluent containing 7% and 9% water (in volume), respectively. The mechanism and physical origin of such peak distortion are related to (1) the diluent-to-eluent excess of water that propagates along the column at a velocity similar to that of the analyte, (2) the significant drop of the Henry's constant of the analyte upon increasing water concentration in the eluent, (3) the sample volume injected, and (4) to the pre-column sample dilution factor that depends on the characteristics of the LC instrument used. This proposed mechanism is validated from the calculation of the concentration profiles of cetirizine and water by using the equilibrium-dispersive (ED) model of chromatography. The observed distortion of cetirizine peaks is successfully predicted from the measurement of (1) the excess adsorption isotherm of water from acetonitrile onto the BEH HILIC adsorbent, (2) the retention factor of cetirizine as a function of the volume fraction (7, 8, and 9%) of water in the mobile phase, and (3) of the pre-column sample dispersion related to the instrument used (HPLC or UHPLC). The results of the calculations enables the user to anticipate the impacts of the diluent-to-eluent mismatch in water content, the injection volume, the analyte retention under infinite dilution, and of the pre-column sample dispersion on the amplitude of peak distortion in HILIC. Appropriate and permitted alterations of the USP method are then suggested based on a sound physico-chemical approach.

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. SEF is expected to occur with any chromatographic particle containing silanols but is slowed on hybrid inorganic/organic particles. The SEF reaction is between alcohols on the particle surface and in the mobile phase solvent. We have found that storage conditions of a column are paramount, which can either prevent or accelerate the process. Because SEF exists as an equilibrium between the liquid phase and the particle surface, the process is also reversible. The silanols can be hydroxylated (regenerated) to their original state upon exposure to water. The next generation of stationary phases will either advantageously utilize SEF or effectively mitigate its effects. Mitigation of SEF would be a significant improvement in SFC that has the potential to vault their performance to levels of similar reproducibility and reliability observed for high performance liquid chromatography (HPLC). Further research in SEF may lead to a better understanding of the mechanism of interaction between the solutes and chromatographic surface.

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. The values of their minimum intrinsic reduced plate heights (h(min) = 1.42, 1.57, and 1.75) were measured at room temperature (295 K) for a small molecule (naphthalene) with an acetonitrile/water eluent mixture (75:25, v/v). These narrow-bore columns provide an average intrinsic efficiency of 395,000 plates per meter. The gradient separation of 14 small molecules shows that these columns have a peak capacity about 25% larger than similar ones packed with fully porous BEH-C18 particles (1.7 µm) or shorter (50 mm) columns packed with smaller core-shell particles (1.3 µm) operated under very high pressure (>1000 bar) for steep gradient elution (analysis time 80 s). In contrast, because their permeabilities are lower than those of columns packed with larger core-shell particles, their peak capacities are 25% smaller than those of narrow-bore columns packed with standard 2.7 µm core-shell particles.

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. The heights equivalent to a theoretical plate of three small molecules were measured using a mixture of acetonitrile and water as the eluent. The values reported include the contributions of longitudinal diffusion, eddy dispersion, and the solid-liquid mass transfer resistance. The bulk diffusion coefficients of the analytes were measured using the capillary method, calibrated with thiourea in pure water. The reduced longitudinal diffusion coefficient was determined from the results of a series of peak parking experiments. The solid-liquid mass transfer resistance coefficient and the intra-particle diffusivities of the analytes in the porous region of the particles were estimated from Garnett-Torquato's model of effective diffusion in dense beds packed with core-shell particles. The eddy dispersion term, mostly due to trans-column and border effects, was obtained by subtracting the longitudinal diffusion and the solid-liquid mass transfer resistance terms from the total HETP obtained from the first and second central peak moments calculated by numerical integration (Simpson's approach) after baseline correction and systematic left and right cuts of the peak profiles. The results show that the eddy dispersion controls at least 66% of the overall column HETP for small molecules beyond the optimum velocity. This work illustrates how important it is to use ultra-low dispersive very high pressure liquid chromatography (vHPLC) systems to properly measure and to practically use the high efficiencies of narrow-bore columns packed with 1.6µm core-shell particles since these columns provide intrinsic efficiencies higher than 400,000 plates per meter.

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 ß-lactoglobulin tryptic digest with a mobile phase pH of 7 in the first dimension and 2 in the second. Mechanism divergence was determined using the peak capacity and a geometric approach to factor analysis, to measure the correlation. This separation was repeated with a C18 stationary phase as a control. It was found that the C18 column had a correlation coefficient of 0.784, smaller than the mixed mode column, 0.884. This indicated that the retention mechanisms of the C18 column were more divergent under these two pH environments than the mixed mode column. However, the SM-C18 still provided alternative selectivity of the peptides to that of the C18 and could be considered as a good alternative for further 2D-HPLC separations.

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. The dispersion of the compound during the elution process is controlled by adjusting the number of virtual vessels into which the column is divided. Solving the ED model under linear conditions with this method gives exactly the same profile as the analytical solution of the Martin-Synge plate model. The Martin-Synge method gives better results than the Rouchon method (1) when the isotherms involved are sigmoidal or anti-Langmuir; and, more importantly, (2) in the case of multi-component problems. Finally, the Martin-Synge method proves to be more robust and faster than the OCFE method that, until now, was considered to be one of the most robust and accurate algorithms. The developed algorithm was used for the calculation of the coefficients of the isotherm of butyl benzoate by the inverse method, using a simplex optimization algorithm.

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. Some currently used methods employ two different separations for the detection of positively and negatively ionized metabolites by mass spectrometry. Here we developed a single set of chromatographic conditions for both ionization modes and were able to detect a total of 141 extracted metabolite species, with an overall peak capacity of ca. 2500. We show that a single two-dimensional separation method is sufficient and practical when a pair or more of unidimensional separations are used in metabolomics.

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. The profile of a Gaussian peak is not qualitatively changed, but the peak becomes narrower, whereas for an exponentially tailing peak, asymmetry at the 10% peak height level changes markedly. Using several examples we show that power transforms also increase signal-to-noise ratio and make it easier to discern an event of detection. However, they may not improve the limit of detection. Power responses are intrinsic to some detection schemes, and in others they are imbedded in instrument firmware to increase apparent linear range that the casual user may not be aware of. The consequences are demonstrated and discussed.

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. The Poppe plots calculated under isocratic and gradient conditions show that the separation power of columns packed with superficially porous particles increases significantly with decreasing thickness of the porous layer but this increase is more important for larger than for smaller molecules. The resolution between pairs of compounds increases at constant values of their retention factors when the strength of the eluent must be reduced to compensate for the decrease of their retention that is caused by the reduction of the surface area of the stationary phase. Thus, the separation power of columns packed with superficially porous particles increases with decreasing shell thickness. In contrast, if analysts do not compensate for the retention decrease, the resolution between small molecular weight compounds becomes worse with thin than with thick superficially porous particles. Finally, the importance of using instruments providing low extra-column band broadening contributions is stressed.

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. For this purpose, we used high performance liquid chromatography (HPLC) columns packed with fully and superficially porous (shell) particles of average sizes ranging from 2.6 to 15 microm in diameter. We found that the sequence coverage of bovine serum albumin obtained with a 10-min gradient elution run on a column packed with 2.6 microm shell particles is greater than that provided by 40-min gradients run on columns packed with 15 or 10 microm particles and equal to those yielded by the same 40-min gradient run on columns packed with 3 and 5 microm particles.

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. It is between 200 and 300 in typical two-dimensional liquid chromatographic (2D-LC) separations. These values are less pessimistic than those reported previously [1]. It was shown that the fraction of organic modifier or stronger eluent component in the injected sample has a significant effect on the widths and heights of the eluted peaks. Analytes with high retention factors and high molecular weights are less sensitive toward this effect than those having small retention factors. These results suggest that the optimization of the experimental conditions of two dimension separations must be made from the points of view of both the retention factor and the detector response factor. The frequency of sampling of the eluent of the first-dimension separation does not have a significant effect on the detection limit of a 2D system if the split ratio is adjusted in the ratio of the frequency of sample collection.

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. In this work, we focus on the advantages of a multiple, parallel columns approach to on-line multidimensional liquid chromatography systems. Calculations of the achievable peak capacities were made as functions of the number of columns operated in parallel. Increasing the number of second-dimension columns from one to two or three causes the largest increase in peak capacity with only a slight increase of aggregate time. We also present some practical aspects to consider when attempting multidimensional separations with multiple columns operated in parallel.

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. As a consequence, due to the time restriction of the second-dimension gradient time, online 2D-LC schemes cannot realistically afford peak capacities exceeding 10,000, even if they are implemented with exceptionally efficient columns and if long analysis times are accepted.

Optimization strategies for off-line two-dimensional liquid chromatography.

A step by step strategy of optimization of comprehensive off-line two-dimensional liquid chromatography (2D-LC) separations is proposed. The goal of an optimization process in the separation sciences is either to achieve a given resolution (a target peak capacity in 2D-LC) within as short a time as possible or to reach the highest possible resolution in a given analysis time. The proposed method takes into account the characteristics of the columns used in the first and the second dimension and the number of fractions of the first dimension eluent that should be collected. The effect of the time spent during the analysis on the second dimension column to carry out necessary tasks that are not the separation itself (called the additional time) on the maximum peak capacity that is achievable was carefully investigated. It was shown that (1) an increase in the peak capacity of the first dimension column combined with the collection of larger volume fractions permits a significant reduction of the time needed to achieve the desired peak capacity; and (2) there is an optimum fraction collection ratio (or number of collected fractions per peak) which yields the target peak capacity in the minimum time. The proposed strategy was used for the optimization of the separation of samples of BSA tryptic digest by an off-line 2D-LC using an SCXmultiply sign in circleRP-HPLC method. As a result of this optimization, a peak capacity of 4000 could be achieved in about 5h with the two columns available. The time needed for the optimized analysis was less than two thirds of the analysis time that would have been needed had the conventional rule of thumb of sample collection in comprehensive on-line 2D-LC (4 samples/peak) been followed.

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. In the process, five systems were created, based on the principles of the three known implementations of comprehensive 2D-LC. This work demonstrates an original method for the selection of the best comprehensive 2D-LC approach, depending on the desired peak capacity and on time constraints. The decision to use a 2D-LC method arises from the need to achieve a given resolution (i.e., a target peak capacity) within as short a time as possible or to reach the highest possible resolution in a given analysis time. Using the most appropriate schemes, we suggest how it is realistically possible to generate peak capacities ranging from 266 in just over 20 min or about 2800 in 2.3 h. When the time available for a two-dimensional separation is very short and the desired peak capacity cannot be achieved in 1D-LC, an on-line 2D-LC approach is unquestionably best. However, if a longer analysis time is acceptable, a 10-fold increase in the peak capacity can be obtained at the cost of a mere 7-fold increase in total analysis time.

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. The conditional peak capacities of the 2D separations achieved exceed 7000 under the experimental conditions investigated. This performance is achieved at the cost of an analysis time of the order of 28 hours. Possible improvements to the separation method described here are discussed.

Investigation of the stability of Chiralpak AD chiral stationary phase under various solvent conditions and development of a method to identify stationary phase-derived polymer contamination.

The stability of Chiralpak AD chiral stationary phase under various solvent conditions was investigated. An analytical method for the detection of the presence of solubilized Chiralpak AD coating was developed using CD spectroscopy (CD signal at 245 nm). In addition, NMR analysis of the solubilized polymer revealed a characteristic signal for the 3,5-dimethylphenyl carbamate methyl protons at around 2.5 ppm. Both of these methods may be helpful in detecting contamination by the Chiralpak AD polymer or in the study of CSP solvent compatibility.