Liquid chromatographic separation modes

The principle of adsorption chromatography is known from classical column and thin-layer chromatography. A relatively polar material with a high specific surface area is used as the stationary phase, silica being the most popular, but alumina and magnesium oxide are also often used. The mobile phase is relatively non-polar (heptane to tetrahydrofuran). The different extents to which the various types of molecules in the mixture are adsorbed on the stationary phase provide the separation effect. A non-polar solvent such as hexane elutes more slowly than a medium-polar solvent such as ether. 

Rule of thumb polar compounds are eluted later than non-polar compounds. 

Note Polar means water-soluble, hydrophilic non-polar is synonymous with fat-soluble, lipophilic. 

The stationary phase is covalently bonded to its support by chemical reaction. A large number of stationary phases can be produced by careful choice of suitable reaction partners. The reversed-phase method described above is the most important special case of chemically bonded-phase chromatography. 

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In any chromatographic process, separation results from molecular interactions between the solute molecules and the stationary and mobile phases. Various types of forces maybe involved in such mechanism in the case of lipids, hydrophobic interactions play a major role, while ionic, dispersive, and polar interactions are involved to a greater or lesser extent. It depends on the liquid chromatographic separation mode, finally. 

The need to measure concentrations in very small volumes is not restricted to biological systems. For example, open tubular columns for liquid chromatographic separations offer the advantage of increased resolution, but because their internal diameters may be as small as 15 pm, the amount of material in the eluted peaks is very small. Thus, the use of these columns requires detectors that can be used with low concentrations in small volumes. Jorgenson and co-workers showed that this could be accomplished by the insertion of a 10-pm-diameter, cylindrical electrode made from a carbon fiber into the end of the column [4]. The close fit between the column wall and the fiber ensured that a large fraction of the eluting molecules were electrolyzed. When the electrochemical data were collected in a voltammetric mode, the resolved compounds could be classi-... 

The modes of liquid chromatographic separation can be classified as follows ... 

An example for the separation for flavonoids with HP-RPC is the screening method employed for the systematic identification of glycosylated flavonoids and other phenolic compounds in plant food materials by Lin et al20 These authors used an analytical 4.6 mm x 250 mm 5 pm C18 silica column at 25 °C with linear gradient elution (eluent A (0.1% FA in water and eluent B 0.1% FA in ACN) at 1.0 ml min-1. DAD was performed at 270, 310, 350, and 520 nm to monitor the UV/VIS absorption. The LC system was directly coupled to an ESI mass spectrometer without flow splitting and the mass spectra acquired in the positive and negative ionization mode. The same analytical scheme (aqueous MeOH extraction, reversed-phase liquid chromatographic separation, and diode array and mass spectrometric detection) can be applied to a wide variety of samples and standards and therefore allows the cross-comparison of newly detected compounds in samples with standards and plant materials previously identified in the published literature. 

The efficiency of a real GPC system depends above all on the rate of mass transfer between mobile phase and gel phase, as well as on the extent of secondary processes. Quantitatively, the efficiency can be expressed by the terms like width (w) or deviation (o) of the chromatographic peak, as well as by other terms of the theoretical plate concept. Since the diffusion rate of solute molecules decreases with an increase of their dimensions, one has to expect generally lower efficiency in gel chromatography of macromolecules in comparison with any other mode of liquid chromatographic separation of low molecular substances. 

Figure 4 Examples of retention mode, stationary phase, and typical mobile phase used in MIP-based liquid chromatographic separations (MeCN acetonitrile AcOH acetic acid).

Two-dimensional liquid-chromatographic separations can be performed in the hnear ( heart-cut ) format or in the comprehensive mode. In the former case, one (or a few) fractions are isolated from the sample and these are subsequently subjected to a second separation. An advantage of this approach is that the specific fraction(s) can be subjected to two (lengthy) high-resolution separations. A great disadvantage is that only one or a few small fractions of the sample are extensively characterized. In comprehensive two-dimensional LC the entire sample is subjected to two different separations. The word comprehensive is justified if the final (two-dimensional) chromatogram is representative of the entire sample (Schoenmakers et al, 2003). The recommended notation for linear ( heart-cut ) two-dimensional LC is LC-LC, whereas comprehensive two-dimensional LC is commonly denoted by LCxLC (Schoenmakers etaL, 2003). 

Fast atom bombardment, liquid-SIMS (secondary ion mass spectrometry), electrospray (ESI), and matrix assisted laser desorption (MALDI) ionization modes have been applied successfully for the investigations of biomolecules.However, ESI and MALDI are the two most frequently adopted techniques for investigations of biopolymersDetails involving the principles and application of all of these techniques can be found elsewhere. The samples may be introduced either directly or after liquid chromatographic separation. All of the above techniques, with the exception of MALDI, have been adopted for the LC/MS experiments. " Although most of the reported LC/MS investigations involved the electrospray ionization of the molecules, continuous flow-FAB ionization techniques have also been found useful. 

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

On the basis of the preceding discussion, it should be obvious that ultratrace elemental analysis can be performed without any major problems by atomic spectroscopy. A major disadvantage with elemental analysis is that it does not provide information on element speciation. Speciation has major significance since it can define whether the element can become bioavailable. For example, complexed iron will be metabolized more readily than unbound iron and the measure of total iron in the sample will not discriminate between the available and nonavailable forms. There are many other similar examples and analytical procedures that must be developed which will enable elemental speciation to be performed. Liquid chromatographic procedures (either ion-exchange, ion-pair, liquid-solid, or liquid-liquid chromatography) are the best methods to speciate samples since they can separate solutes on the basis of a number of parameters. Chromatographic separation can be used as part of the sample preparation step and the column effluent can be monitored with atomic spectroscopy. This mode of operation combines the excellent separation characteristics with the element selectivity of atomic spectroscopy. AAS with a flame as the atom reservoir or AES with an inductively coupled plasma have been used successfully to speciate various ultratrace elements. 

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