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.

Comparing the selectivity and chiral separation of d- and l- fluorenylmethyloxycarbonyl chloride protected amino acids in analytical high performance liquid chromatography and supercritical fluid chromatography; evaluating throughput, economic and environmental impact.

The chiral separation of d- and l- FMOC amino acids was undertaken using the Lux Cellulose-1 polysaccharide based chiral column in HPLC (normal phase and reverse phase) and SFC conditions. This was done to compare the relative selectivity and separation between the three separation modes and to evaluate the potential benefits of SFC separations with regards to resolution, throughput, economic and environmental impact. It was established that the separation of d- and l- FMOC amino acids in SFC displayed behaviours that were similar to both normal phase and reversed phase, rather than distinctly one or the other. Additionally, although reversed phase conditions yielded significantly higher resolution values between enantiomers across the range of amino acids studied, improvements in selectivity in SFC via the introduction of higher concentrations of formic acid in the mobile phase allowed for better resolution per unit of time. Moreover since the SFC mobile phase is composed mostly of recyclable CO2, there is a reduction in organic solvent consumption, which minimises the economic and environmental costs.

Improving quantification using curtain flow chromatography columns in the analysis of labile compounds: a study on amino acids.

The performance of curtain flow chromatography column technology with MS detection was evaluated for the analysis of labile compounds. The curtain flow column design allows for separations that are faster and/or more sensitive than conventional columns, depending on how exactly the curtain flow column is configured. For example, when mass spectral detection is employed, the curtain flow column can yield separations that are 5-times faster than conventional columns when the curtain flow and the conventional columns have the same internal diameter. Or when the internal diameter of the conventional column is reduced in order to yield the same analytical through-put as the curtain flow column, the sensitivity on the curtain flow column can be as much as 66-fold higher than the conventional column. As a consequence of the higher analytical through-put less standardization is required in the analysis of labile compounds because less sample degradation is apparent. Consequently the sample integrity is preserved yielding data of a higher quality.

High through-put and highly sensitive liquid chromatography-tandem mass spectrometry separations of essential amino acids using active flow technology chromatography columns.

A preliminary investigation was undertaken to assess the performance of a new chromatography column technology in applications involving liquid chromatography coupled to mass spectrometry. The new column design allows mobile phase and solute to be extracted from the radial central region of the column, which reduces the solvent load to the mass spectrometer and improves separation efficiency. Effectively the column functions as a 'wall-less' column. The advantages of this design is that the analysis through-put can be increased by a factor of five, while at the same time there is a reduction in baseline noise, which results in an increase in the signal to noise response by up to 10-fold in comparison to standard columns with the same internal diameter and approaching 66-fold in comparison to standard columns with the same virtual internal diameter.

Reaction flow chromatography for rapid post column derivatisations: the analysis of antioxidants in natural products.

The analysis of antioxidants from complex samples is conveniently achieved using liquid chromatography, which provides sample fraction, coupled with an on-line antioxidant assay, which provides detection. One particularly useful on-line antioxidant assay that has routinely been coupled with HPLC involves the diphenylpicrylhydrazyl radical (DPPH), which provides a positive test for phenolic antioxidants through a decolorisation of the DPPH reagent. A limitation of this assay, however, is the need to employ a reaction coil, which is often large with respect to the peak volume, consequently adding substantial band broadening to the separation. In this study we introduce a new concept that can be employed for systems requiring post column derivatisations, such as the DPPH assay. We have termed this 'reaction flow' chromatography, whereby, the derivatisation reagent can be added directly into one of the outlet ports of a parallel segmented flow column. Subsequently, the mixing between the derivatising reagent and the solute is very efficient removing the need to employ reaction coils. The concept is tested here using the DPPH assay for the analysis of antioxidants in samples derived from natural origin.

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.

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.

Anomalies in evaporative light scattering detection.

A two-dimensional (2-D) "heart-cutting" HPLC system was used to fractionate oligostyrenes into the respective diastereoisomers. For samples of known composition, the response of an ultraviolet (UV) absorbance detector followed the anticipated pattern. The response of an evaporative light-scattering (ELSD) detector on the other hand indicated quite different concentrations for the two diastereoisomers, relative to what was anticipated and what was indicated by the UV detector. Whereas approximately the same concentration was indicated by UV, ELSD in some cases indicated no detection of the later eluting isomer. The magnitude of the errors depended on both the molecular weight and the tacticity of the diastereomers. These anomalies appear to be an artifact of power transform functions imbedded within the firmware processor of the ELSD, invisible to the user.

Application of power functions to chromatographic data for the enhancement of signal to noise ratios and separation resolution.

Chromatographic detection responses are recorded digitally. A peak is represented ideally by a Guassian distribution. Raising a Guassian distribution to the power 'n' increases the height of the peak to that power, but decreases the standard deviation by radicaln. Hence there is an increasing disparity in detection responses as the signal moves from low level noise, with a corresponding decrease in peak width. This increases the S/N ratio and increases peak to peak resolution. The ramifications of these factors are that poor resolution in complex chromatographic data can be improved, and low signal responses embedded at near noise levels can be enhanced. The application of this data treatment process is potentially very useful in 2D-HPLC where sample dilution occurs between dimension, reducing signal response, and in the application of post-reaction detection methods, where band broadening is increased by virtue of reaction coils. In this work power functions applied to chromatographic data are discussed in the context of (a) complex separation problems, (b) 2D-HPLC separations, and (c) post-column reaction detectors.

Reproducibility of the finger pattern in viscous fingering.

The complex pattern of viscous fingering (VF) appears to be chaotic. Its early evolution seems predictable but, on a long time-scale, it is not so and the transition is complex. Detailed experimental observations of fingering systems have been hindered by various limitations. We present a new method for visualising VF in particulate beds. Our results show that the onset of VF and its initial evolution are reasonably reproducible at very low Reynolds numbers (Re < 0.005). The transition to irreproducibility of the fingering pattern develops progressively over long migration distances. When the flow velocity increases, changes in the finger distribution take place over shorter migration distances, become more important, and the fingers evolve faster. Understanding this new aspect might allow improvements in the efficiency of processes governed by VF, e.g. injection of large, concentrated samples or transfer of large fractions between two streams having different viscosities.

A column capacity study of single, serial, and parallel linked rod monolithic high performance liquid chromatography columns.

The loading capacity of rod monolithic C18 columns was found to be sensitive to the injection volume, but essentially insensitive to the mass loading for a separation of oligostyrenes. When rod monoliths were coupled in series the injection volume loading increased, as too did the resolution of the oligomers, but at the expense of separation time. The volume load capacity of these serially connected monoliths was, however, not directly proportional to the number of columns connected. The volume load capacity was, however, directly proportional to the number of columns when the monoliths were connected in parallel and the flow stream split between each of the monolithic channels. When the number of monoliths in each channel equaled the number of monoliths that were connected in a single channel serial system the peak capacity and retention time was equivalent for both systems, but the volume load capacity of the parallel system was twice that of the serial connection each time the number of channels doubled. The results of this study indicate that parallel connection of rod monolithic columns may be useful for preparative scale and multidimensional separations where it is important that the volume load capacity is high.

Isolation of the active constituents in natural materials by 'heart-cutting' isocratic reversed-phase two-dimensional liquid chromatography.

A multidimensional heart-cutting reversed-phase HPLC separation approach, where two columns were operated independently via two six-port two-position switching valves, was employed in the isolation of a major bioactive found in the ethanol-water (80:20) crude extract of Clerodendrum floribundum. In this mode of operation, the specific productivity of the multidimensional approach under overload conditions was twice that of conventional gradient methods with the same loading factor. Isolated sample purities were greater than 99% with recoveries of 95%. The independent operation of each of the two columns employed in the multidimensional approach allowed the cycle time to be less than 7 min, compared to 23 min in the gradient elution single-dimension mode of operation.

Visualization of solute migration in chromatographic columns influence of the frit porosity.

The dual-perspective, on-column detection method previously described was used to observe the effects of the inlet frit on the profiles of chromatographic bands. Visualization of bands of iodine was achieved by injecting its dilute solutions in carbon tetrachloride into a glass column packed with a C18-bonded silica and eluted with carbon tetrachloride, which has the same refractive index as the packing material. The bands were photographed on-column with two standard 35-mm SLR cameras oriented at right angle. The photographs were scanned and the digitized images of the sample bands analyzed with proper software. A number of columns, as similar as possible, were fitted with different 2- and 10-microm porosity stainless steel frits. Subsequent analysis of the digitized band images revealed irregularities in the band shape resulting from frit contributions to band dispersion. The 2-microm frits produced more dramatic effects overall than the coarser frits. Local axial dispersion coefficient values, expressed as local reduced plate height, were calculated. The results demonstrate the possibly damaging effects of the frit on the band shape.

Xanthine oxidase inhibitory activity of selected Australian native plants.

Twenty-eight extracts from 17 species of Australian native plants traditionally used as general anti-inflammatory medicines by Australian Aboriginal people were examined for inhibition of the enzyme xanthine oxidase (XO). The extracts from nine species were found to have more than 25% inhibition at a concentration of 100 microg/ml in the assay mixture. Extracts from three species Clerodendrum floribundum R. Br. (Verbenaceae), Eremophila maculata (Ker Gawler) (Myoporaceae) and Stemodia grossa Benth. (Scrophulariaceae) showed the greatest activity with inhibitions of 84, 61 and 57%, respectively, at 50 microg/ml, with four other species having more than 40% inhibitory activity at this concentration.

Physical evidence of two wall effects in liquid chromatography.

Using optical on-column visualization for the study of the migration of sample bands, the radial variations of the local migration rate were studied in the region near the column wall. Photographs of small sample bands migrating along the column at various radial locations were obtained. On-column chromatograms extracted from these photographs showed evidence of two wall effects. The first of these effects was present only within the immediate vicinity of the wall. It is a direct result of the inability of the packing material to form a close packed configuration against the rigid column wall surface. The second wall effect causes a systematic variation of the migration rate of the sample band in the region of the wall, this rate increasing from the wall to the central region of the column. The corresponding images portrayed the classical "wall effect" that chromatographers have long discussed. They also show that this effect extends further into the column than anticipated. As to what are the relative contributions to the results of our observations of the wall effect and of a frit effect discussed in previous publications, this could not be ascertained.

Evaluation of the secondary consolidation of columns for liquid chromatography by ultrasonic irradiation.

The consolidation of packed analytical chromatography columns was carried out under ultrasonic irradiation. Columns were first packed using a conventional high pressure downward slurry method. Then, they were subjected to further bed consolidation in the presence of ultrasonic vibration. This process of further bed consolidation is referred to as secondary consolidation. Secondary consolidation was observed to occur more readily in solvents of low viscosity and at low flow-rates (low pressures). Column efficiency was not observed to be a factor affecting the process of secondary consolidation of the packed bed.

Visualization of solute migration in chromatographic columns quantitation of the concentration in a migrating zone.

The concentration distribution across a zone of iodine migrating along a column made of glass, packed with C18-bonded silica, and eluted with carbon tetrachloride was derived from a quantitative analysis of the photographs of the zone. The photographs were scanned and turned into digital images. The intensity distributions obtained from the measurement of the grayscale intensity were converted into concentration profiles using a calibration method. This procedure is illustrated and suitable corrections are introduced to account for the transverse variation of the optical path length, as a result of using a cylindrical detector cell (the column itself), and for the refraction of light due to the differences between the refraction indices of the glass wall and the liquids involved. An error analysis is also reported. It shows that the method can reliably produce results with a precision of a few percent, allowing on-column evaluation of column performance and the derivation of the radial distributions of the column efficiency, the migration velocity of the zone, and the sample distribution at the head of the column.

On-column visualization of sample migration in liquid chromatography.

A quantitative on-column visualization technique for evaluating solute migration in liquid chromatography columns was described. This technique employed a matched refraction index phase system in high-pressure glass columns. In this case, the mobile phase was carbon tetrachloride and the stationary phase was a C18 silica. Because the refractive indexes of the two phases were the same, the column bed, otherwise opaque, was transparent to the eye. Zones of colored solutes could be injected (iodine for instance) and their migration studied along the column. Photographing the sample band during its migration allowed chromatographic information to be extracted using digital technology. As an example of applications for this technique, we show the sample entry through various inlet fittings. The results demonstrate that chromatographers should pay more attention to the selection of inlet frits. Importantly, the frit porosity should be matched to the particle size of the packing.

Visualization of sample introduction in liquid chromatography columns. The effect of the frit diameter.

A previously developed on-column detection technique using 35 mm SLR cameras [J. Chromatogr. A 826 (1998) 1] was employed to visualize colored sample bands as they elute through frits of differing diameter. Head fittings containing a 4.0 mm frit and a 15.9 mm frit were mounted in a 17 mm I.D. glass column packed with C18 silica with an average particle size of 21 microns. A carbon tetrachloride mobile phase of matching refractive index to that of the silica provides clarity along the column diameter during band migration. The photographs of the migrating sample zones were scanned and analyzed with appropriate imaging software. The smallest diameter frit induced severely parabolic sample distributions at the column inlet compared to the larger frit. Local axial dispersion coefficient values, expressed as local reduced plate height, were calculated as well as local zone velocities at the column inlet. The results demonstrate clearly the need to match the diameter of the inlet frit to the I.D. of the column so as to avoid the initial onset of zone broadening due to the frit.

Visualization of viscous fingering in high-performance liquid chromatographic columns. Influence of the header design.

Using an on-column visualization technique, band profiles of solutes migrating along an HPLC column were studied. The study showed that, under conditions where viscous fingering is prevalent, the design of the inlet header has little influence on the outcome of the viscous fingers. Two types of headers were studied. The first contained a small diameter inlet frit, which localized the majority of the sample in or near the central region of the column. The second header contained a wide frit and produced a more uniform radial distribution of the sample. In both cases, the extent of viscous fingering was essentially the same.