Separation techniques liquid chromatography

Helfrich et al " developed a reliable method for the size characterization of Au nanoparticles based on the combination of two different separation techniques (liquid chromatography and gel electrophoresis), coupled on-line to ICP-MS. Separation by liquid chromatography shows good reproducibility with size-dependent behavior for retention (RSD<1%). The results are in a good agreement with complementary methods like dynamic light scattering (DLS) and transmission electron microscopy (TEM). 

The versatility of chiral stationary phases and its effecitve application in both analytical and large-scale enantioseparation has been discussed in the earlier book A Practical Approach to Chiral Separation by Liquid Chromatography" (Ed. G. Sub-ramanian, VCH 1994). This book aims to bring to the forefront the current development and sucessful application chiral separation techniques, thereby providing an insight to researchers, analytical and industrial chemists, allowing a choice of methodology from the entire spectrum of available techniques. 

Natural products and natural-like compounds, generally coming from microbes, plants, sponges and animals [2, 3] may be fully identified and quantified by means of modem and advanced analytical techniques, such as high-performance liquid chromatography (HPLC) coupled to various detectors - from the most common UV/Vis to mass spectrometry and tandem mass spectrometry (HPLC-MS and HPLC-MS/MS). The role of MS is to provide quantitative and qualitative information about mixtures separated by liquid chromatography [4],... 

Pesticides (14, 85), polychlorinated biphenyls (86, 87) and herbicides (88) are usually separated by this technique also. In analytic work, however, the detection sensitivity of the selective detectors used in gas chromatography could not be achieved (59). Nevertheless, sUch substances can be separated by liquid chromatography with no attendant decomposition problems and no derivatization, making the procedure significantly simpler. i... 

The number of articles dealing with the on-line coupling of the two most widely used separative techniques, liquid and gas chromatography, are few. Many analyses of complex mixtures or trace analyses in complex matrices utilize a liquid chromatograph for sample cleanup or for analyte concentration prior to gas chromatographic analysis. 

In order to analyse a complex mixture, for example natural products, a separation technique - gas chromatography (GC), liquid chromatography (LC) or capillary electrophoresis (CE) - is coupled with the mass spectrometer. The separated products must be introduced one after the other into the spectrometer, either in the gaseous state for GC/MS or in solution for LC/MS and CE/MS. This can occur in two ways the eluting compound is collected and analysed off-line or the chromatograph is connected directly to the mass spectrometer and the mass spectra are acquired while the compounds of the mixture are eluted. The latter method operates on-line. Reviews on the coupling of separation techniques with mass spectrometry have been published in the last few years [1-4]. 

As a result of the development of special bonded phases, carbohydrates or their derivatives are usually separated by liquid chromatography. However, certain carbohydrate samples are still analyzed by GC due to the inherent high efficiencies obtainable from the technique and to the associated short elution times. In addition, gas chromatography-mass spectrometry (GC-MS) is a particularly powerful analytical technique for carbohydrates, especially for their identification. As a consequence, appropriate derivatives must be formed to render them sufficiently volatile but stiU easily recognizable from their mass spectra. 

Adjusting the selectivity of the chromatographic system, as measured by a, is often a useful technique in improving separations in liquid chromatography. Such adjustments need to be made with consideration of the second term in Eq. (5), which is (a - l)/o . When a = 1, the term is equal to zero, resulting in no resolution. This indicates that the chromatographic system must exhibit some selectivity toward the components of the mixture before any separation is possible. The term... 

The increased need for stereoselective analyses has induced a tremendous development of analytical techniques resolving enantiomers. Among these techniques, liquid chromatography, and more recently capillary electrophoresis (CE), are recognized as methods of choice for the chiral separation of pharmaceutical compounds. Chiral discrimination by CE is generally achieved with the direct separation method where the chiral selector is simply added to the background electrolyte (BGE). 

In liquid chromatography, two dimensional separations in the vast majority of cases are not comprehensive. While comprehensive 2D-LC separations (LC X LC) can be accomplished and have been demonstrated (e.g., Refs. 30 and 31), the technique is not very popular. Probably one of the main reasons for this is the inability to perform very fast separations in liquid chromatography. In GC X GC, a typical second dimension separation can be completed in a few seconds. In LC, the separation time required is much longer. The problem can be overcome by stopping the flow in the first dimension column while the second dimension separation proceeds, but this causes the overall analysis times to be very long. 

Full automation of separations by liquid chromatography requires automation of the sample introduction process. This includes automation of scheduled injections and automation of sample processing when required to isolate the analytes of interest from the sample in a form suitable for separation and detection. An example of the latter approach is on-line solid-phase extraction-liquid chromatography (SPE-LC) discussed in section 5.3.2. Automation of time scheduled injections increases accuracy and precision by removing human error [1,18,39]. Typical precision for manual valve injections is about 0.5% for complete-fill and 1-2% for partial-fill loop injections. Typical precision for automated injection is about 0.25% for complete-fill and 0.5% for partial-fill loop techniques. 

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