Constant pressure mode of operation in the second dimension of two-dimensional liquid chromatography: A proof of concept.

The use of two-dimensional liquid chromatography (2D-LC) continues to grow as the advantages over 1D-LC become increasingly clear in specific application areas, and the number of experienced 2D-LC users increases. As with any technique, however, there is always room for innovation that could improve the performance of 2D-LC. In recent years the technical aspects and potential benefits of a volume-based mode of operation were studied in detail for 1D-LC. The salient features of this approach that are immediately interesting for use in 2D-LC are two-fold. First, the ability to maintain a nominally constant pressure in the second dimension by dynamically adjusting the flow rate to compensate for changes in the viscosity of the fluid in the 2D flow path provides a means to more fully utilize the pressure capability of the pumping system, and accelerates separations in the second dimension (2D). Second, constant pressure operation minimizes physical stress on the system components and the 2D column. In this paper we discuss the aspects of volume-based operation of LC that are particularly relevant to 2D-LC systems. The proof-of-concept experiments illustrate the viability of the constant pressure mode of operation for the second dimension of 2D-LC. In the described separations the throughput improvement is on the order of 10%; this gain will be strongly application-dependent, and may be as large as several tens percent in some cases. Future work will involve a detailed investigation of the impact of the constant pressure mode on robustness of 2D separations.

Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography.

Two-dimensional liquid chromatography (2D-LC) is increasingly being viewed as a viable tool for solving difficult separation problems, ranging from targeted separations of structurally similar molecules to untargeted separations of highly complex mixtures. In spite of this performance potential, though, many users find method development challenging and most frequently cite the "incompatibility" between the solvent systems used in the first and second dimensions as a major obstacle. This solvent strength related incompatibility can lead to severe peak distortion and loss of resolution and sensitivity in the second dimension. In this paper, we describe a novel approach to address the incompatibility problem, which we refer to as Active Solvent Modulation (ASM). This valve-based approach enables dilution of 1D effluent with weak solvent prior to transfer to the 2D column but without the need for additional instrument hardware. ASM is related to the concept we refer to as Fixed Solvent Modulation (FSM), with the important difference being that ASM allows toggling of the diluent stream during each 2D separation cycle. In this work, we show that ASM eliminates the major drawbacks of FSM including complex elution solvent profiles, baseline disturbances, and slow 2D re-equilibration and demonstrate improvements in 2D separation quality using both simple small molecule probes and degradants of heat-treated bovine insulin as case studies. We believe that ASM will significantly ease method development for 2D-LC, providing a path to practical methods that involve both highly complementary 1D and 2D separations and sensitive detection.

Restriction capillaries as an innovative mixing unit for intermediate mobile phase exchange in multidimensional analysis.

A novel mixing unit is proposed for the serial coupling of orthogonal columns to analyze polar and non-polar compounds in a single run. The principle relies on the isolation of unretained peaks eluting from a first dimension column in a sample loop, before directing them to a second column for separation. Since the mobile phases employed in highly orthogonal separations are not directly compatible, a mixing unit is required to alter the mobile phase composition before executing the second dimension separation. The mixing unit proposed in this work is based on the use of two restriction capillaries with different flow resistances to dilute the mobile phase eluting from the first dimension with a solvent appropriate for the second dimension separation. The restriction capillaries are implemented in an ultra-high performance liquid chromatography set-up using three high-pressure switching valves and two T-pieces. It is demonstrated that the dilution ratio can be adequately predicted using the law of Hagen-Poiseuille and can be adjusted easily by changing the dimensions of the restriction capillaries. The dilution volume required to obtain acceptable recoveries is investigated and the use of different column diameters in the first and second dimension is proposed to increase the sensitivity of the analysis. Under optimum dilution conditions, recoveries ranging between 82% and 99% are always obtained, while repeatability values are excellent. The proof-of-concept of the different set-ups is demonstrated for the separation of 20 pharmaceuticals with log D-values ranging between -5.75 and 4.22.