Simple automated liquid chromatographic system for splitless nano column gradient separations (original) (raw)
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Analytical Chemistry, 1999
An automated liquid nano-separation system has been developed for nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) using both isocratic and gradient elution. One fused-silica nanocolumn, typically 75 µm i.d. × 39 cm (25 cm effective packed length), packed with Spherisorb ODS 1, 3 µm particle size, can be used with either technique without having to remove the column upon switching from one mode to the other. The mobile phase is delivered by two reciprocating micro-LC pumps at a flow rate of 30 µL/min to a postinjection splitter that houses the nanocolumn inlet. The splitter is directly connected to a micro-injection valve with a 0.5 µL injection volume. In the CEC mode, pressure is not applied (no restriction on splitter) to the column inlet or outlet and the voltage is continuously applied during sample injection and mobile phase delivery. In the nano-LC mode, the restrictor is coupled to the splitter. Using the same nanocolumn under isocratic conditions, the repeatabilities of retention time and peak area for nano-LC were better than 0.2% and 4%, respectively, and those for CEC were better than 0.6% and 6%, respectively. On average, column efficiency was 57% higher in CEC compared to nano-LC. Gradient elution separations of parabens and polynuclear aromatic hydrocarbons (PAHs) were accomplished by CEC. Capillary electrochromatography (CEC) is an electrokinetic technique that is receiving significant attention in the separation community because of the combination of attractive features of liquid chromatography (LC) and capillary electrophoresis (CE). Combining the selectivity of LC with the high efficiency of CE leads to an exciting technique that will allow separation scientists to drive complex sample characterization, hyphenation, miniatur-ization, and mass spectrometry detection to new frontiers. Recent studies on the optimization of CEC focused on the fabrication of frits 1-3 that are designed to hold the packing material in the separation column, the minimization of air bubble formation in the column, 4,5 and the generation of gradient separations. 6-9 Commonly used frits seem to be those formed by sintering the stationary phase within the column. 3,10 In our laboratory, we have found that frits formed of silanized glass wool work as well as the sintered frits, are much easier to construct, and can be transferred from column to column. 11 Since air bubbles result in loss of current, and thus electroosmotic flow, most CEC separations are accomplished by applying a constant pressure to both the inlet and outlet buffer reservoirs. The application of pressure reduces the risk of air bubble formation in both the packed and unpacked regions of the separation capillary. It is also common to apply pressure to the inlet end of the column, which results in the combination of pressure and electrically driven flows. 1,12-17 Tsuda et al. first reported this combination of flows using microbore
Despite the fact that strong routine separation methodologies can give reliable specificity and validity at usual working pharmaceuticals concentrations, they may fail at very low concentration levels. This poses considerable challenges for researchers inves-tigating product purity and therapeutic drug monitoring. Sensitivity enhancement pro-cedures are thus required to maximize the performance of separation techniques. Large volume injection, solid phase extraction/solid phase enrichment (SPE/SPEn), pre-, post-, and in-column derivatization, as well as the use of sensitive detection devices are the simplest strategies for improving sensitivity of the separation-based analytical techniques. Large volume injection of samples with online SPE/SPEn coupled with separation techniques increased sensitivity and improved detection as well as quantification limits without affecting peak shape and system performance. Although the primary purpose of derivatization is to improve sensitivity and...
Strategy for On-Line Preconcentration in Chromatographic Separations
Analytical Chemistry, 2001
In chromatographic separations, the heights of peaks are proportional to the concentrations of sample components present in an injected mixture. In general, an increase in the peak height cannot be achieved by simply increasing the injection time or the sample plug length. An exception occurs if some form of on-line preconcentration is possible. We present a new strategy for achieving on-line preconcentration by the use of a porous chromatographic material that acts as a solid-phase extractor as well as a stationary-phase separator. We are able to realize significant on-line preconcentration using capillary columns filled with a photopolymerized sol-gel (PSG). More than 2-cm plugs of sample solution can be loaded into the capillary and concentrated using a running buffer that is the same as the injection buffer (to avoid solvent gradient effects). As a demonstration, mixtures of three different polycyclic aromatic hydrocarbons, eight different alkyl phenyl ketones, and five different peptides in solutions of aqueous acetonitrile have been injected onto the PSG column and separated by capillary electrochromatography. The preconcentration is marked in terms of peak heights, with up to 100-fold increase for the PAH mixture, 30-fold for the alkyl phenyl ketone mixture, and 20-fold for the peptide mixture. Preconcentration takes place because of the high mass-transfer rates possible in the highly porous structure, and the extent of preconcentration follows the retention factor k for a given analyte.
Journal of Chromatography A, 2010
This paper describes a multivariate approach to study the effect on chromatographic conditions and to optimize such conditions in capillary liquid chromatography when high injection volumes are required. Several separations have been evaluated by using isocratic and gradient solvent elution, as well as isocratic elution combined with temperature programming. In this study, easily ionisable organic compounds with low log P have been used as representative analytes. Injection volume and nature of the injection solution have been evaluated in order to increase the sensitivity (peak area) and column performance (N values). The equations obtained by multiple linear regressions and response surfaces allow achieving the optimum on-column focusing conditions for chlorophenoxy acids, carbamates and heterocyclic amines.
Talanta, 2006
A simplified preconcentration method for a range of ultra-trace level pharmaceuticals in natural waters has been developed. Solid phase extraction was performed on-line using a micro-reversed-phase monolithic silica column, allowing for very rapid trace enrichment from large volume (500 ml) samples with minimal sample handling. Acceptable recoveries of >70% were obtained for the majority of compounds investigated and the monolithic columns could be washed and conditioned on-line with no sample carryover and used reproducibly for up to eight extractions each. The on-line SPE-LC-UV method was coupled to electrospray ionisation ion trap mass spectrometry (ESI-MS) to increase both selectivity and specificity. Detection limits were determined in spiked river and tap water samples and found to lie in the low ng/l region using sample volumes of 500 ml, loaded at a flow rate of 10 ml/min, and therefore, were suitable for ultra trace analysis. (B. Paull). pharmaceuticals from various matrices include C 18 silica , crosslinked poly(styrene divinylbezene) , C 2 silica [10], immunosorbents and hydrophilic lipophilic balanced polymers .
Analysis of applying different solvents for the mobile phase and for sample injection
Journal of Chromatography A, 2005
Overloading a chromatographic column with a compound possessing low solubility in the mobile phase has been investigated. In order to increase the concentration of injection a strong solvent for dissolving the feed was used. The injection of such concentrated samples brings the risk of triggering undesired crystallisation processes. A model system has been investigated with ethanol-water as the mobile phase and dl-threonine as the sample dissolved in pure water. Under extreme overloaded conditions band splitting was observed. Measurements of the adsorption isotherms and systematic solubility studies were carried out. For the process analysis a simplified mathematical model was applied. The simulations of the band profiles were compared with the experimental data.
Critical Reviews in Analytical Chemistry, 2014
Sequential injection chromatography was proposed in 2003 to perform a simple, rapid, reagentsaving, environmentally benign, on-site, and instrumentally inexpensive separation procedure. Sequential injection chromatography is a version of sequential injection analysis, which is the second generation in the family of flow injection techniques. Despite its advantages over high-performance liquid chromatography, sequential injection chromatography has confronted some challenges. Furthermore, the applications of sequential injection chromatography in its first five years are almost all limited to pharmaceutical analysis. Interestingly, in its second five years, various developments in sequential injection chromatography technology were achieved. The developments have enhanced the efficiency of sequential injection chromatography and hence its applications have extended to biological, food, and environmental analyses. The main objectives of this review are to examine recent developments (2008-2013) in sequential injection chromatography and to describe how these developments improve the efficiency of the technology. The sequential injection chromatography methodologies reported during that period are also discussed along with controlling conditions and analytical results. The review also describes the principles, instrumentation, and procedure behind sequential injection chromatography.
Gradient elution chromatography with microbore columns
Analytical Chemistry, 1983
of many possible approaches to vaporization from a solid surface. As mentioned in the introduction, methods such as SIMS, FAB, etc. show real promise and may eventually allow the moving belt LC/MS interface to become practical for virtually any compound class.
European Journal of Pharmaceutics and Biopharmaceutics, 2007
Liquid chromatography (LC) is currently considered as the gold standard in pharmaceutical analysis. Today, there is an increasing need for fast and ultra-fast methods with good efficiency and resolution for achieving separations in a few minutes or even seconds. A previous article (i.e. method transfer for fast LC in pharmaceutical analysis. Part I: isocratic separation) described a simple methodology for performing a successful method transfer from conventional LC to fast and ultra-fast LC in isocratic mode. However, for performing complex separations, the gradient mode is often preferred. Thus, this article reports transfer rules for chromatographic separations in gradient mode. The methodology was applied for the impurity profiling of pharmaceutical compounds, following two strategies.