Today scientists must deal with complex samples that either cannot be adequately separated using one-dimensional chromatography or that require an inordinate amount of time for separation. For these ...cases we need two-dimensional chromatography because it takes far less time to generate a peak capacity
n
c
twice in a row than to generate a peak capacity
n
c
2
once. Liquid chromatography has been carried out successfully on thin layers of adsorbents and along tubes filled with various adsorbents. The first type of separation sorts out the sample components in a physical separation space that is the layer of packing material. The analysis time is the same for all the components of the sample while their migration distance increases with decreasing retention. The resolution between two components having a certain separation factor (
α
) increases with increasing migration distance, i.e., from the strongly to the weakly retained compounds. In the second type of separation, the sample components are eluted from the column and separated in the time space, their migration distances are all the same while their retention times increase from the unretained to the strongly retained compounds. Separation efficiency varies little with retention, as long as the components are eluted from the column. We call these two types of separation the chromatographic separations in space (LC
x
) and the chromatographic separations in time (LC
t
), respectively. In principle, there are four ways to combine these two modes and do two-dimensional chromatographic separations,
LC
t
×
LC
t
,
LC
x
×
LC
t
,
LC
t
×
LC
x
, and
LC
x
×
LC
x
. We review, discuss and compare the potential performance of these combinations, their advantages, drawbacks, problems, perspectives and results. Currently, column-based combinations (
LC
t
×
LC
t
) are the most actively pursued. We suggest that the combination
LC
x
×
LC
t
shows exceptional promise because it permits the simultaneous second-dimension separations of all the fractions separated in the first-dimension, thus providing remarkable time saving.
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%.
Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at ...least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 μm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 μL.
A second part of this review illustrated briefly the effect of dead volume on column performance. The experiment evaluated the change in resolution and separation efficiency of some weak to moderately retained solutes on a 250 × 4.6 mm i.d. column packed with 5 μm particles. The data showed that reaction loops beyond 100 μL resulted in a very serious loss of performance.
Our study concluded that practitioners of PCD methods largely avoid the use of UHPLC-type column formats, so yes, very much, PCD is incompatible with the modern HPLC column.
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•Post-column derivatisation methods are a powerful class of detection techniques.•84 methods were reviewed, which were published since 2009.•Post column dead volume associated with PCD methods causes a serious loss in separation efficiency.•We discovered that most users do not use columns packed with particles less than 5 μm.
•Expired Conventional HPLC columns could be renewed using Active Flow Technology.•The expired columns had reduced plate heights of 3.5.•The active flow technology renewed column had a reduced plate ...height less than 2.•The renewed column was better than original.
The performance of a particle packed column will inevitably degrade through use or misadventure. ‘Active flow technology’ (AFT) is known to greatly improve the performance of pristine columns, but is as of yet untested when used on columns that have degraded significantly. In this study AFT was used to regenerate a degraded column, where the reduced plate height and asymmetry values were 3.5 and 1.25 respectively. Once the AFT fittings were fitted to the column outlet and the flow segmentation ratio adjusted to 28% from the radial central exit port, the reduced plate height decreased to 2.0, and the bands were almost perfectly symmetrical with asymmetry factors equal to 1.04. Subsequently, the performance of the degraded column with AFT fittings provided performance that was comparable to that of a new conventional column fitted with traditional end fittings.
The separation power of the degraded conventional column and that of the same column fitted with the AFT end fittings was then tested using the separation of oligostyrenes. In AFT mode, detection was undertaken at both the radial central exit port of the column and the peripheral exit port. The resulting separation that was achieved from the radial central exit port was superior to that observed on the conventional column, whereas, the separation observed from the peripheral port was very poor. It was subsequently determined that the reason for the degraded performance of the conventional column was a result of increased heterogeneity associated with the packing material in the wall region of the column.
•AFT columns yielded faster and more efficient separations than UHPLC columns.•The 5μm particle AFT columns operated at two-thirds the back pressure of the UHPLC columns.•Ultra-high speed HPLC–MS was ...achieved using AFT columns.
The performance of active flow technology chromatography columns in parallel segmented flow mode packed with 5μm Hypersil GOLD particles was compared to conventional UHPLC columns packed with 1.9μm 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 5–6mL/min have been recorded.
•Use of active flow management technology to monolithic columns.•Demonstrated improvement in efficiency by as much as 115%.•Demonstrated a reduction in peak asymmetry by as much as 26%.
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.
Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken ...assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses.
This is the first study to employ a reaction flow (RF) HPLC column of a short 5cm length to identify antioxidants in a variety of complex samples represented by tea, utilising simultaneous ...multiplexed detection techniques. The detection responses included: underivatised UV at 280nm, underivatised fluorescence detection (FLD) and a post column derivatisation (PCD) colorimetric response for antioxidants via the ferric reducing antioxidant power (FRAP) assay. Both similarities and differences in the chromatograms obtained using the three detection modes highlight the power of multiplexed detection. The main findings from the bioactive profiling include: the Fruit of the Forest tea contains the lowest level of antioxidants and is clearly distinct from the other three teas which are based on Camellia sinensis. The antioxidants detected in the green tea and the two black teas are similar. The green tea contains a higher concentration of the two main antioxidants that eluted at 4.0 and 5.7min.
The simplicity of sample analysis and chemical profiling using RF columns and selective detection was the focus of this study via the analysis of antioxidants in tea using the FRAP reagent. The main advantage of RF-PCD is the ability to split flows for simultaneous detection with minimal post column dead volume contributions, making peak matching between detectors much easier. To date, few studies exploit the power of RF-PCD multiplexed detection to differentiate and identify bioactive compounds in various complex samples. Applications of the approach developed in this study may include antioxidant screening and profiling of natural products, foods and beverages.
•A novel method to profile bioactive antioxidants in complex samples was developed.•Reaction flow (RF): post column derivatisation (PCD) without a reaction coil.•The use of an RF column of 5cm and multiple detection responses: UV, FLD & PCD.•Easy peak assignment between peaks detected in the UV, FLD and PCD profiles.•Applications: antioxidant screening of food, beverages or natural products.
