Stationary phases modes

Direct enantiomer separation methodologies circumvent the rather laborious formation of covalent diastereomers, but instead exploit subtle energetic differences of reversibly formed, noncovalent diastereomeric complexes for chiral recognition. Direct chromatographic enantiomer separation may be achieved in two different modes, the chiral mobile phase additive and the chiral stationary phase mode. 

This resulted in a three-way array X (6 x 6 x 8) of retention factors with mode one holding the stationary phase, mode two the mobile phase and mode three the solutes. The purpose of the analysis was two-fold (i) to investigate the differences between the types of stationary-phase material and (ii) to develop a calibration model for transfer of retention factors from one stationary phase to another one. Only the first part of the investigations will be discussed. 

After the above-mentioned transformations, the data were mean centered across the stationary phase mode, because investigating the differences between stationary phases was the primary goal of the analysis. No scaling was applied, because all the values were measured in the same units and were of the same order of magnitude. 

The first analyses performed were principal component analyses on the three matricized arrays X(/x/x), X,-/ 1K] and XiK u, to get an impression of the complexity in the different modes. The explained variation of these three PCA models is shown in Figure 10.35. For models with two principal components, all explained variations are already high. The highest one is for the stationary-phase (/) mode, and the lowest one for the mobile-phase (J) mode. This is in agreement with the a priori knowledge (see earlier), where two sources of variation were expected between stationary phases and three between the mobile phases. All in all, two or three components seem sufficient to describe the systematic variation in all the models. 

Figure 10.39. Loading plots of the two-component PARAFAC model of X. (a) the stationary-phase mode, (b) the mobile-phase mode and (c) the solute mode.

Models having two components in the stationary-phase mode can be compared to neighboring models having one extra stationary-phase component (e.g. comparing (2,2,2) with (3,2,2)). There are four such comparisons and they all indicate that going from two to three components in the stationary-phase mode does not improve the model. Similar comparisons... 

Compounds Stationary phase Mode Mobile phase Reference... 

Nonpolar organic mobile phases, such as hexane with ethanol or 2-propanol as typical polar modifiers, are most commonly used with these types of phases. Under these conditions, retention seems to foUow normal phase-type behavior (eg, increased mobile phase polarity produces decreased retention). The normal mobile-phase components only weakly interact with the stationary phase and are easily displaced by the chiral analytes thereby promoting enantiospecific interactions. Some of the Pirkle-types of phases have also been used, to a lesser extent, in the reversed phase mode. 

Cyclodextrin stationary phases utilize cyclodextrins bound to a soHd support in such a way that the cyclodextrin is free to interact with solutes in solution. These bonded phases consist of cyclodextrin molecules linked to siUca gel by specific nonhydrolytic silane linkages (5,6). This stable cyclodextrin bonded phase is sold commercially under the trade name Cyclobond (Advanced Separation Technologies, Whippany, New Jersey). The vast majority of all reported hplc separations on CD-bonded phases utilize this media which was also the first chiral stationary phase (csp) developed for use in the reversed-phase mode. 

Except for the high molecular weight range, nearly all substances can be separated by reversed-phase (RP) HPLC. The many different separation mechanisms in RP HPLC, based on hydi ophobic, hydi ophilic and ion-pairing interactions, and size exclusion effects together with the availability of a lai ge number of high quality stationary phases, explain its great populai ity. At present approximately 90% of all HPLC separations are carried out by reversed-phase mode of HPLC, and an estimated 800 different stationai y phases for RP HPLC are manufactured worldwide. 

Let us consider the separation of polymethylmethacrylate (PMMA) on a nonmodified silica column as an example. In THE (medium polar eluent) the PMMA eludes in size exclusion mode because the dipoles of the methylmethacrylate (MMA) are masked by the dipoles of the THE. Using the nonpolar toluene as the eluent on the same column, the separation is governed by adsorption because the dipoles of the carbonyl group in the PMMA will interact with the dipoles on the surface of the stationary phase. The separation of PMMA in the critical mode of adsorption can be achieved by selecting an appropriate THF/toluene mixture as the eluent. In this case all PMMA samples... 

GPC has many uses and is a powerful analysis technique for acrylate polymers. With care in selecting solvents and stationary phases, one finds that many polymers can be analyzed successfully. Opportunities always exist to use analytical GPC columns in nonstandard ways (semiprep, HDC, pseudo-ElPLC combined with GPC ) to the benefit of the analyst, but the analyst must always be keenly aware of which mode of operation is dominating when practicing such nonroutine analyses. 

In many respects, the coupling of GC columns is well suited since experimentally there are few limitations and all analytes may be considered miscible. There are, however, a very wide variety of modes in which columns may be utilized in what may be described as a two-dimensional manner. What is common to all processes is that segments or bands of eluent from a first separation are directed into a secondary column of differing stationary phase selectivity. The key differences of the method lie in the mechanisms by which the outflow from the primary column is interfaced to the secondary column or columns. 

When multiple development is performed on the same monolayer stationary phase, the development distance and the total solvent strength and selectivity values (16) of the mobile phase (17) can easily be changed at any stage of the development sequence to optimize the separation. These techniques are typically fully off-line modes, because the plates must be dried between consecutive development steps only after this can the next development, with the same or different development distances and/or mobile phases, be started. This method involves the following stages ... 

Figure 8.19 illustrates another example of the versatility of multidimensional OPLC, namely the use of different stationary phases and multiple development ("D) modes in combination with circular and anticircular development and both off-line and on-line detection (37). Two different stationary phases are used in this configuration. The lower plate is square (e.g. 20 cm X 20 cm), while the upper plate (grey in Figure 8.19) is circular with a diameter of, e.g. 10 cm. The sample must be applied on-line to the middle of the upper plate. In the OPLC chamber the plates are covered with a Teflon sheet and pressed together under an overpressure of 5 MPa. As the mobile phase transporting a particular compound reaches the edge of the first plate it must-because of the forced-flow technique-flow over to the second (lower) stationary phase, which is of lower polarity. 

One potential problem associated with column coupling in reversed phase is relatively high back-pressure ( 2600 psi at 1 mL miir ). This will place a limit on the flow rate, which in turn limits the further reduction of analysis time. Also, compared to the new polar organic mode, the retention in reversed phase on coupled columns is deviated more from the average retention on the individual stationary phases. 

A general phenomenon observed with chiral stationary phases having hydrophobic pockets is that a decrease of flow rate results in an increase in resolution. This change has significant impact mostly in reversed-phase mode (see Fig. 2-10). 

Typical normal-phase operations involved combinations of alcohols and hexane or heptane. In many cases, the addition of small amounts (< 0.1 %) of acid and/or base is necessary to improve peak efficiency and selectivity. Usually, the concentration of polar solvents such as alcohol determines the retention and selectivity (Fig. 2-18). Since flow rate has no impact on selectivity (see Fig. 2-11), the most productive flow rate was determined to be 2 mL miiT. Ethanol normally gives the best efficiency and resolution with reasonable back-pressures. It has been reported that halogenated solvents have also been used successfully on these stationary phases as well as acetonitrile, dioxane and methyl tert-butyl ether, or combinations of the these. The optimization parameters under three different mobile phase modes on glycopeptide CSPs are summarized in Table 2-7. 

At the current time, there is considerable interest in the preparative applications of liquid chromatography. In order to enhance the chromatographic process, attention is now focused on the choice of the operating mode [22]. SMB offers an alternative to classical processes (batch elution chromatography) in order to minimize solvent consumption and to maximize productivity where expensive stationary phases are used. 

It is important for the analyst to be able to select the best stationary phase to use for a particular chromatographic analysis. Silica gel can be used in two modes of chromatographic separations as a stationary phase in normal elution development or as a stationary phase in exclusion chromatography. 

General Description. Liquid chromatography encompasses any chromatographic method in which the mobile phase is a liquid (c.f. gas chromatography). A variety of stationary phases and retention mechanisms are available such that a broad range of modes of separation are possible. It is worthwhile to briefly describe the important modes that find use in clinical chemistry. 

Adsorption and ion exchange chromatography are well-known methods of LC. In adsorption, one frequently selects either silica or alumina as stationary phase for separation of nonionic, moderately polar substances (e.g. alcohols, aromatic heterocycles, etc.). This mode works best in the fractionation of classes of compounds and the resolution of isomeric substances (J). Ion exchange, on the other hand, is applicable to the separation of ionic substances. As to be discussed later, this mode has been well developed as a tool for analysis of urine constituents (8). 

Other modes of LC operation include liquid-liquid partition chromatography (LLC) and bonded phase chromatography. In the former, a stationary liquid phase which is immiscible with the mobile phase is coated on a porous support, with separation based on partition equilibrium differences of components between the two liquid phases. This mode offers an alternative to ion exchange in the fractionation of polar, water soluble substances. While quite useful, the danger exists in LLC that the stationary phase can be stripped from the column, if proper precautions are not taken. Hence, it is typical to pre-equil-ibrate carefully the mobile and stationary phases and to use a forecolimn, heavily loaded with stationary phase 9). 

By far the most popular phase system at the present time is the one in which R n-octadecyl. Since the stationary phase is hydrophobic and nonpolar, while the mobile phase is relatively polar, this mode of operation is frequently called reverse phase... 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

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