Ross A Shalliker

Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a ‘virtual’ narrow bore column☆

Narrow bore columns find advantage in HPLC applications when volumetric flow is important, For example, for detection processes that are volume limited. Yet there are significant drawbacks to narrow bore columns. Due to their small column volume relative to analytical scale columns, narrow bore columns are more affected by system dead volume. In addition the wall effect and the variation in packing density from the centre to the wall are more significant in these columns relative to larger scale analytical columns. In this study we operate a 4.6mm i.d. parallel segmented flow column in such a manner that it emulates 2.1mm i.d. and 3.0mm i.d. columns. By using a parallel segmented flow column in this way, it was possible to combine the benefits of narrow bore and analytical scale columns.

The separation of diastereoisomers of polystyrene oligomers in reversed phase HPLC

SummaryThe separation of diastereoisomers from oligomers of low molecular weight polystyrene was achieved using a carbon clad zirconia stationary phase and an acetonitrile mobile phase. The selectivity of the C18-methanol system separated the polystyrene oligomers based on molecular weight while the carbon clad zirconia surface in combination with an acetonitrile mobile phase allowed the expression of the isomeric sample dimensionality. Consequently, full utilisation of the different retention mechanisms on each surface greatly improved the isomeric separation from oligomers of low molecular weight polystyrene.

Understanding the importance of the viscosity contrast between the sample solvent plug and the mobile phase and its potential consequence in two-dimensional high-performance liquid chromatography.

The effect of solvent viscosity mismatch on elution performance in reversed-phase HPLC was studied using moment analysis. Two conditions were tested: (1) the mobile phase viscosity was less than the injection plug viscosity, and (2) the mobile phase viscosity was greater than the injection plug viscosity. Under the first condition, retention time and elution performance decreased as the viscosity contrast between the mobile phase and injection plug increased. The effect on performance was more marked as the injection volume increased. A decrease in performance of 12% for compounds with retention factors up to 2.8 was apparent even when the viscosity contrast was only 0.165 cP. In the second set of conditions, elution performance was actually observed to increase, by as much as 25% for a 40 microL injection, as the viscosity contrast between the mobile phase and the solute plug increased. No change in the retention factor was observed. This behaviour was attributed to the shape of an injection plug as it enters into the column, whereby a low viscosity plug permeates away from the wall when the column contains a higher viscosity mobile phase, and vice a versa for a high viscosity plug entering a low viscosity mobile phase. At no stage was either a band splitting or shoulders observed with viscosity contrasts up to 1.283 cP, as could have been expected.

Gradient elution chromatography with segmented parallel flow column technology: A study on 4.6 mm analytical scale columns☆

A new column format known as parallel segmented flow has recently been introduced, whereby improvements in column performance are observed. These improvements are achieved via the separation of eluent from the column core from that of the column wall region. The segmentation of flow is accomplished immediately as the eluent exits the column through the use of a multi-channel end fitting. The ratio of flow exiting through the column central port relative to the peripheral ports, known as the segmentation ratio, can be tuned to optimise chromatographic performance. Investigations into the use of parallel segmented flow chromatography columns have demonstrated increased sensitivity and theoretical plates in analytical scale isocratic separations, but so far no studies have detailed the performance of these columns in gradient elution. The current study addresses the performance of parallel segmented flow columns in gradient elution, detailing the reproducibility of the gradient at various segmentation ratios and compares the performance to conventional columns. The study found that there was no observable difference in the gradient shape, or reproducibility of the gradient profiles generated at any segmentation ratio, tested on three different types of stationary phases. A separation of an 11-component test mixture verified that the primary advantage of parallel segmented flow columns was that the peak volume was reduced in proportion to the segmentation ratio.

Packing procedures for high efficiency, short ion-exchange columns for rapid separation of inorganic anions

An optimised packing procedure for the production of high efficiency, short, particle-packed ion-exchange columns is reported. Slurry-packing techniques were applied to a series of interconnected short columns, with the columns situated intermediate between the inlet and outlet ends of the series being used for separations. The fast separation and determination of inorganic anions was achieved using short (4mm ID, 30mm long) columns packed with Dionex AS20 high-capacity anion-exchange stationary phase. Seven inorganic anions (bromate, chloride, chlorate, nitrate, sulfate, chromate and perchlorate) are separated in 2.6min using a hydroxide gradient and a flow-rate of 1.8mL/min (total analysis time including re-equilibration was 3.5min). Under isocratic conditions, the home-packed columns exhibited efficiency values of 43,000N/m for chloride at a flow-rate of 0.3mL/min, compared to 54,000N/m for a commercial 250mm AS20 column at the same flow-rate. However, the short columns gave approximately a threefold higher sample throughput. The short, home-packed columns could be produced reproducibly and gave consistent performance over extended periods of usage.

Enhancing the separation performance of the first-generation silica monolith using active flow technology: parallel segmented flow mode of operation.

Active flow technology (AFT) columns are designed to minimise inefficient flow processes associated with the column wall and radial heterogeneity of the stationary phase bed. This study is the first to investigate AFT on an analytical scale 4.6mm internal diameter first-generation silica monolith. The performance was compared to a conventional first-generation silica monolith and it was observed that the AFT monolith had an increase in efficiency values that ranged from 15 to 111%; the trend demonstrating efficiency gains increasing as the volumetric flow to the detector was decreased, but with no loss in sensitivity.

Ferric reducing antioxidant potential (FRAP) of antioxidants using reaction flow chromatography

High performance liquid chromatography coupled with post column derivatisation (HPLC-PCD) may be used to profile the antioxidant content of a sample. There are, however, drawbacks in the use of HPLC-PCD setups; namely the high volume reaction coils that are typically used lowering the observed separation efficiency. Reaction flow chromatography has the ability to overcome these inefficiencies by using a more efficient mixing technique inside the outlet fitting itself, post column reaction loops can be removed with resulting improvement in signal to noise response, plus the separation efficiency is maintained. We assessed two methods of HPLC-PCD antioxidant analysis based on the ferric reducing antioxidant power (FRAP) assay in both conventional and reaction flow HPLC-PCD modes. It was found that the reaction flow technique demonstrated significant advantages over the conventional technique in terms of signal to noise, linear range, precision and observed separation efficiency.

Investigating retention characteristics of a mixed‐mode stationary phase and the enhancement of monolith selectivity for high‐performance liquid chromatography

The synthesis and chromatographic behavior of an analytical size mixed-mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3-cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π-type bonding within a single separation environment. Selectivity studies were undertaken using n-alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π-type selectivities afforded by the two different ligands on the mixed-mode column.

ACTIVE FLOW MANAGEMENT IN CHROMATOGRAPHIC SEPARATIONS: A PRELIMINARY INVESTIGATION INTO ENHANCED PREPARATIVE SCALE SEPARATION USING A PARALLEL SEGMENTED OUTLET FLOW DISTRIBUTOR

The detrimental effect of heterogeneity on the efficiency of particle packed columns has been a long standing issue in chromatographic separations. This heterogeneity arises from the difference in packing density between the wall and central regions of the chromatographic bed and imparts radial variance in mobile phase velocity through the column. In conjunction with the uneven distribution of the sample band through frits and distributors at the head of the column, the resultant effect is a decrease in efficiency due to the formation of a parabolic-“like” sample band. We introduce the concept of “Active Flow Management” to counteract the effects of column heterogeneity via the separation of the flow eluting from the center of the column from that of the flow eluting near the peripheral wall region. The flow streams from the two aforementioned regions are separated by the use of a specialized column outlet fitting. By varying the ratio of flow eluting from the center to the peripheral region it was possible to increase the efficiency of a 100 × 21 mm column by up to 57% compared to a column operated in the conventional manner.

TARGETED ISOLATIONS FROM COMPLEX SAMPLES USING TWO-DIMENSIONAL HPLC. PART 1: ANALYTICAL SCALE ANALYSIS

The practical aspects in the optimization of targeted isolations using two-dimensional HPLC in a heart cutting mode with emphasis on analytical scale analysis were investigated. Assessment of the experimental parameters that affect the purity of an isolated component from a complex mixture were evaluated by employing two different strategies; (1) recovering the target component from the central area of the band in both dimensions; and (2) recovering the target component from off-center of the band in the first dimension and from the central area of the band in the second dimension. For this particular sample, the results reveal that the highest purity product was a result of off-centering the heart-cut away from the central peak maxima in order to minimize contamination from the limiting impurity. However, the highest purity product was obtained at the lowest recovery.