Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

University of Amsterdam

A new column technology - termed parallel segmented outlet flow was employed here to illustrate gains in separation performance that are achievable by the active management of flow as it exits from the outlet of the chromatography column. Parallel segmented outlet flow requires a column be fitted with an outlet fitting that separates flow from the central region of the column from that of wall region. Each region of flow is able to be processed independently, such that post column detection emulates end column localised detection. As a result of this flow segmentation and the subsequent more efficient means of detection, column efficiency was observed to increase by more than 20%, with gains in sensitivity by as much as 22%, and a decrease in peak volume by up to 85%.

Enhanced separation performance using a new column technology: parallel segmented outlet flow.

Active Flow Management is a new separation technique whereby the flow of mobile phase and the injection of sample are introduced to the column in a manner that allows migration according to the principles of the infinite diameter column. A segmented flow outlet fitting allows for the separation of solvent or solute that elutes along the central radial section of the column from that of the sample or solvent that elutes along the wall region of the column. Separation efficiency on the analytical scale is increased by 25% with an increase in sensitivity by as much as 52% compared to conventional separations.

The design of a new concept chromatography column.

Active flow management in the form of curtain flow sample introduction and segmented outlet flow control has been shown to enable sample to elute through a chromatography column under the principles of the "infinite diameter column". Such an elution process avoids the detrimental effects of the heterogeneity of particle-packed chromatographic columns by injecting the sample directly into the radial core region of the column, thus avoiding wall effects. The process described herein illustrates how the principles of the infinite diameter column can be applied using conventional injection devices suitable for long-term analysis that requires robust protocols. Using this approach, sensitivity in separation was 2.5 times greater than conventional chromatography, yielding a product at twice the concentration. Benefits of curtain flow injection are thus relevant to both preparative-scale and analytical-scale separations.

Active flow management in preparative chromatographic separations: a preliminary investigation into enhanced separation using a curtain flow inlet fitting and segmented flow outlet fitting.

Speed of analysis is a significant limitation to current high-performance liquid chromatography/mass spectrometry (HPLC/MS) and ultra-high-pressure liquid chromatography (UHPLC)/MS systems. The flow rate limitations of MS detection require a compromise in the chromatographic flow rate, which in turn reduces throughput, and when using modern columns, a reduction in separation efficiency. Commonly, this restriction is combated through the post-column splitting of flow prior to entry into the mass spectrometer. However, this results in a loss of sensitivity and a loss in efficiency due to the post-extra column dead volume.

The use of parallel segmented outlet flow columns for enhanced mass spectral sensitivity at high chromatographic flow rates.

We describe a new approach to multiplex detection for HPLC, exploiting parallel segmented outlet flow - a new column technology that provides pressure-regulated control of eluate flow through multiple outlet channels, which minimises the additional dead volume associated with conventional post-column flow splitting. Using three detectors: one UV-absorbance and two chemiluminescence systems (tris(2,2'-bipyridine)ruthenium(III) and permanganate), we examine the relative responses for six opium poppy (Papaver somniferum) alkaloids under conventional and multiplexed conditions, where approximately 30% of the eluate was distributed to each detector and the remaining solution directed to a collection vessel. The parallel segmented outlet flow mode of operation offers advantages in terms of solvent consumption, waste generation, total analysis time and solute band volume when applying multiple detectors to HPLC, but the manner in which each detection system is influenced by changes in solute concentration and solution flow rates must be carefully considered.

Parallel segmented outlet flow high performance liquid chromatography with multiplexed detection.

Multi-dimensional chromatographic techniques, such as (comprehensive) two-dimensional liquid chromatography and (comprehensive) two-dimensional gas chromatography, are increasingly popular for the analysis of complex samples, such as protein digests or mineral oils. The reason behind the popularity of these techniques is the superior performance, in terms of peak-production rate (peak capacity per unit time), that multi-dimensional separations offer compared to their one-dimensional counterparts. However, to fully utilize the potential of multi-dimensional chromatography it is essential that the separation mechanisms used in each dimension be independent of each other. In other words, the two separation mechanisms need to be orthogonal. A number of algorithms have been proposed in the literature for measuring chromatographic orthogonality. However, these methods have their limitations, such as reliance on the division of the separation space into bins, need for specialist software or requirement of advanced programming skills. In addition, some of the existing methods for measuring orthogonality include regions of the separation space that do not feature peaks. In this paper we introduce a number of equations which provides information on the spread of the peaks within the separation space in addition to measuring orthogonality, without the need for complex computations or division of the separation space into bins.

Michelle Camenzuli

Van′t Hoff Institute for Molecular Sciences

University of Amsterdam

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

Michelle Camenzuli

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Van′t Hoff Institute for Molecular Sciences

University of Amsterdam

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

Van′t Hoff Institute for Molecular Sciences