Optimization of conditions in on-line comprehensive two-dimensional reversed phase liquid chromatography. Experimental comparison with one-dimensional reversed phase liquid chromatography for the separation of peptides.

Comprehensive on-line two-dimensional liquid chromatography is an attractive technique to enhance peak capacity and deal with complex samples. Yet, its optimization is not straightforward and requires to take into account a huge number of parameters to finally obtain a good compromise between different criteria which are often conflicting. In this study, we propose a procedure for RPLC×RPLC separations able to define, for a given analysis time, the optimized LC×LC parameters for achieving the best compromise between high peak capacity and low dilution. This procedure makes use of both a prediction tool based on theoretical relationships and a Pareto-optimality approach to optimize these two criteria. The possibility to split the flow-rate of the first dimension before the interface is taken into account as well as the focusing effect in the second dimension. This optimization procedure is applied to the prediction of optimized conditions for the separation of peptides. The need for very short and efficient columns (typically 30 mm as column length and 1.3 µm as particle diameter) in the second dimension is clearly established whatever the geometry of the first dimension column. Furthermore it is shown that several sets of quite different conditions in the first dimension can lead to similar results and hence that additional quality descriptors are required to make a decision. RPLC×RPLC separations of a protein digest were carried out and the obtained results were found in very good accordance with the predicted ones. Our results were compared to the top results reported in the literature for 1D-RPLC separations of peptides. More than 4-fold increase in peak capacity (5100 vs 1150) at 200 min has been obtained. Furthermore a 40-fold gain in analysis time (1h vs 40 h) was obtained for achieving 1800 as effective peak capacity which is close to the highest peak capacity reported in 1D-RPLC for the separation of peptides.

Development of on-line comprehensive two-dimensional liquid chromatography method for the separation of biomass compounds.

Comprehensive on-line two-dimensional liquid chromatography (on-line LC × LC) was used for the characterization of bio-oils obtained by fast pyrolysis of lignocellulosic biomass. The resulting bio-oil contains a large number of oxygenated chemical families and must therefore be upgraded before being used as drop-in transportation biofuels. The good knowledge of its complex composition is essential for optimizing the mandatory bio-oil upgrading process to biofuels, thereby requiring powerful separation techniques designed to be hyphenated to mass spectrometry detection (LC × LC-MS). In this study, reversed phase conditions were optimized in both dimensions for the RPLC × RPLC separation of the aqueous fraction of bio-oils. The first step of method development consisted in searching for a suitable set of RP-conditions via the screening of a large number of RP-systems (made up of different stationary phases and/or mobile phases and/or temperature). The practical peak capacity and the degree of orthogonality were calculated for a sample of 38 representative compounds, both descriptors having been considered as selection criterion. Two different couplings were chosen and evaluated for the RPLC × RPLC separation of the 38 representative compounds. The best of both, in terms of real practical peak capacity, was further successfully applied to the separation of the aqueous phase of a partially dehydroxygenated bio-oil.

On-line comprehensive two dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part II: application to the separation of peptides.

In this second paper of a two-part series, on-line RPLC×HILIC is compared to on-line RPLC×RPLC through the separation of peptides. Our choices regarding the conditions are discussed. Injection effects and overloading effects are evaluated in both configurations. It is shown that whereas large volumes can be injected in the second dimension in RPLC×RPLC under HT-UHPLC conditions (>20% of the column dead volume), even small injection volumes (8% of the column the dead volume) have a detrimental effect on peak shapes in RPLC×HILIC. Advantages and limits of the two 2D-systems are compared through the 2D-separation of a tryptic digest of three proteins. A ten-fold gain in analysis time along with a significant gain in peak capacity are obtained with both systems compared to the most efficient one-dimensional separation of peptides recently published.

On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: orthogonality and practical peak capacity considerations.

Comprehensive on-line two-dimensional liquid chromatography is expected to generate impressive peak capacities, which makes it a method of choice for the analysis of complex samples such as pharmaceutical or biological ones. A comparative study of different sets of chromatographic conditions including stationary phase, mobile phase and column temperature was carried out with mixtures of representative solutes in order to find out the best two-dimensional analytical conditions for charged compounds. Our approach focused on ultra-fast gradient runs in second dimension using HT-UHPLC conditions. The choice of volatile buffers was intended for future coupling with mass spectrometry in order to get another relevant dimension. The potential of various pairs of chromatographic systems was examined by means of two-dimensional gradient data. An attempt is made to rationalize the concept of orthogonality and a method is proposed to assess, for a given pair of chromatographic systems, both the degree of orthogonality and the practical peak capacity. It is shown that the degree of orthogonality between both dimensions is a critical factor but it is not sufficient to definitely appreciate the potential of a given pair of systems. The combination of HILIC and RPLC (HILIC×RPLC or RPLC×HILIC), although providing a very high degree of orthogonality, is disappointing due to the poor peak capacities obtained in HILIC especially with peptide samples. RPLC systems offer a large variety of analytical conditions, some of them leading to appropriate degrees of orthogonality when they are combined. More importantly, due to high column efficiencies along with large separation power, some combinations of RPLC systems leads to very high practical peak capacities.

Effect of pH additive and column temperature on kinetic performance of two different sub-2 µm stationary phases for ultrafast separation of charged analytes.

The performance characteristics of separation were studied for small pharmaceuticals and larger charged molecules (peptides) in various mobile phase conditions on two 5-cm long narrow bore columns packed with 1.7 µm core-shell and totally porous particles respectively. The effect of temperature and pH additives (formic acid, trifluoroacetic acid, ammonium formate, ammonium acetate and ammonia) on column efficiency was investigated through a kinetic study based upon data obtained under gradient elution conditions. Sample peak capacities were calculated and compared in all studied conditions for a sample of ten representative peptides having masses ranging from 500 to 2000 Da. The elevation of temperature was found to be significantly beneficial. The effect of flow-rate on peak shape was also investigated. Ammonium acetate at neutral pH led to the best results in terms of both efficiency and peak capacity. It was found that column performance was strongly dependent on the type of stationary phase, especially in acidic medium.