Reducing pressure and increasing peak capacity by incorporating an adaptable flow stream splitting platform to high-performance liquid chromatography columns: A study on C18 silica-based monoliths

In this study, an adaptable end-column platform was fitted to a commercially available monolith, which enabled the column to be fitted with a flow-splitting device. A variety of flow-splitting adapters could be incorporated into the platform, and in this study, a radial flow stream splitter was utilized. The advantage of the radial flow stream spitter was that it overcame issues relating bed density variations that could cause bands to distort in the radial cross-section of the column. Using propylbenzene as a test standard in isocratic elution mode, height equivalent to a theoretical plate curves were constructed across ten flow rates, and it was found that the column efficiency improved by as much as 73%. Furthermore, the dual outlet flow splitter enabled a very substantial reduction in column back pressure, with the decrease being consistently between 20 to 30% depending on the column length. Additionally, sensitivity increased by 45%, consistent with the observed increase in efficiency. The adaptable end-column platform could be retrofitted to almost any commercial column with the expectation of gaining efficiency, sensitivity, and reducing back pressure

High through-put liquid chromatography : mass spectrometry requires new strategies for the management of fluid at the interface

The ever increasing demand on regulation in bioanalysis, whether that be the routine testing of athletes to ascertain compliance with drug regulation, or the analysis of metabolites in biological samples to determine rapid response to drug treatment protocols, or any of the many other tests that regulation authorities demand, have, as such, placed a very high reliance on analytical testing. Many of these analyses are undertaken using chromatographic separations coupled with mass spectrometry, and there is substantial evidence that suggests HPLC–MS is the quintessential analytical technique for quantification and sample identification employed in bioanalysis. Furthermore, there will be continued growth in this area of analytical sciences because of the substantial information obtained, and as such, the mass spectrometer may well end up being the ‘go-to’ detector

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 μL 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

Segmented flow and curtain flow chromatography : overcoming the wall effect and heterogeneous bed structures

The variation in mobile phase velocity as a function of the column radius has been shown to be a major limitation in the efficiency of HPLC columns. One contributing factor to the variability in the flow velocity stems from the heterogeneity in the radial packing density, leading to what has been described as the 'wall-effect'. The wall-effect generates parabolic-type elution profiles, which dilutes the sample and creates tailing bands. In this communication a new column technology is discussed that has been designed to overcome the wall effect, minimising the limitations associated with packing heterogeneity. This technology has been referred to as active flow technology and consists of two types of column designs, parallel segmented flow and curtain flow. In both these column designs sample that elutes through the column in the radial central region of the bed is separated from the flow that elutes along the wall region. Hence, the sample that elutes through the most efficiently packed region of the bed is collected to the detector. As a consequence more theoretical plates are obtained, and sensitivity is increased since the sample is not diluted by the diffuse tail. Sensitivity is enhanced further in the curtain flow design. The benefits of these new columns are discussed

Recent advances in column technology

There have to date no advances in column technology that are able to provide the benefits of increased sensitivity, efficiency, and speed in a single design format. Usually, gains in sensitivity come at the cost of speed, since separations need to be run at a flow rate near the minimum in a HETP curve. Curtain flow chromatography, and even PSF chromatography, overcomes this limitation. Likewise, the cost of column efficiency is usually paid for in the currency of time. But AFT columns can provide higher efficiency than conventional columns, especially as the flow rate increases. Hence, the AFT columns function at their best when separations are run fast. Another factor that usually makes the cost of speed too much to bear is the limitation in detection processing, especially if the detector is a mass spectrometer. With AFT columns, less solvent enters the MS, in fact, in direct proportion to the segmentation ratio. Thus, ultrafast, highly efficient, and very sensitive assays can be undertaken using AFT columns with MS detectors

Solvent viscosity mismatch between the solute plug and the mobile phase : considerations in the applications of two-dimensional HPLC

Understanding the nature of viscosity contrast induced flow instabilities is an important aspect in the design of two-dimensional HPLC separations. When the viscosity contrast between the sample plug and the mobile phase is sufficiently large, the phenomenon known as viscous fingering can be induced. Viscous fingering is a flow instability phenomenon that occurs at the interface between two fluids with different viscosities. In liquid chromatography, viscous fingering results in the solute band undergoing a change in form as it enters into the chromatography column. Moreover, even in the absence of viscous fingering, band shapes change shape at low viscosity contrasts. These changes can result in a noticeable change in separation performance, with the result depending on whether the solvent pushing the solute plug has a higher or lower viscosity than the solute plug. These viscosity induced changes become more important as the solute injection volume increases and hence understanding the process becomes critical in the implementation of multidimensional HPLC techniques, since in these techniques the sample injection plug into the second dimension is an order of magnitude greater than in one-dimensional HPLC. This review article assesses the current understanding of the viscosity contrast induced processes as they relate to liquid chromatographic separation behaviour

Pentafluorophenyl bonded phase separation : the assay of Australian antioxidant formulation of ascorbic acid, rutin and hesperidin

This work describes two novel validated HPLC methods for the separation and determination of potent antioxidant formulation. The compounds of interest are ascorbic acid (ASC), rutin (Rut) and hesperidin (HES). Two pentafluorophenyl bonded phases (PFP) of different geometries and particle-type (fully porous and core-shell) were used and both methods yielded linear response over the ranges 80-800, 20-220 and 20-200 μg mL-1 for ascorbic acid, rutin and hesperidin, respectively. UV detection was accomplished at 260 nm using DAD. The developed methods were validated using ICH guidelines and were successfully applied to the determination of the ternary mixture in their pharmaceutical formulation in the Australian market with acceptable percent recoveries. Results obtained by the proposed methods were statistically compared to those of the reported methods regarding the mean percent recoveries and variances showing no significant difference. However, the analyses undertaken on core shell particles provided separations that were complete within 36 seconds

Using active flow technology columns for high through-put and efficient analyses : the drive towards ultra-high through-put high-performance liquid chromatography with mass spectral detection

The performance of active flow technology chromatography columns in parallel segmented flow mode packed with 5. μm Hypersil GOLD particles was compared to conventional UHPLC columns packed with 1.9. μm Hypersil GOLD particles. While the conventional UHPLC columns produced more theoretical plates at the optimum flow rate, when separations were performed at maximum through-put the larger particle size AFT column out-performed the UHPLC column. When both the AFT column and the UHPLC column were operated such that they yielded the same number of theoretical plates per separation, the separation on the AFT column was twice as fast as that on the UHPLC column, with the same level of sensitivity and at just 70% of the back pressure. Furthermore, as the flow velocity further increased the performance gain on the AFT column compared to the UHPLC column improved. An additional advantage of the AFT column was that the flow stream at the exit of the column was split in the radial cross section of the peak profile. This enables the AFT column to be coupled to a flow limiting detector, such as a mass spectrometer. When operated under high through-put conditions separations as fast as six seconds, using mobile phase flow rates in the order of 5-6. mL/min have been recorded

A new approach to live reaction monitoring using active flow technology in ultra-high-speed HPLC with mass spectral detection

A new type of chromatography column referred to as a parallel segmented flow (PSF) column enables ultra-high-speed high-performance liquid chromatography-MS to be undertaken. This occurs because the separation efficiency obtained on PSF columns has been shown in prior studies to be superior to conventional columns, and the flow stream is split radially inside the outlet end fitting of the column, rather than in an axial post-column flow stream split. As a result, the flow through the column can be five times higher than the flow through the MS. In this work, the degradation of amino acids in dilute nitric acid was used to illustrate the process. Separations were obtained in less than 12 s, although the reinjection process was initiated 6 s after the previous injection. The degradation rate constant of tryptophan, in the presence of tyrosine and phenylalanine, was determined

Sample introduction for high performance separations

In this review we discuss how viscosity contrasts between the injection plug and the mobile phase may lead to loss in separation performance, especially in UHPLC columns or SFC environments. Firstly, the wall effect is discussed, and how it can amplify viscosity contrast effects. We then illustrate how viscosity contrasts lead to the phenomenon known as viscous fingering, and we detail the destructive effects of this phenomenon. We expand on the viscous fingering component, however, demonstrating that viscosity contrast effects begin to deteriorate performance long before the conditions are such that viscous fingering occurs. Subtle changes in band-shape are apparent even with very low viscosity contrasts. Lastly we illustrate how viscosity contrast effects lead to severe peak distortions in SFC. Analysts who seek high efficiency separations must make every effort to eliminate, or at least minimise the viscosity contrast between the injection plug and the mobile phase