Examples reversed phase liquid chromatography

In pharmaceutical development, the determination of APIs and counterions are two important assays. Due to the charge and/or hydrophobicity differences, APIs and counterions are usually analyzed by different chromatographic techniques that require different separator columns and/or detection methods. For example, reversed-phase liquid chromatography is most commonly used for analyzing APIs with intermediate to higher hydrophobicity, but it fails to provide adequate retention for hydrophilic counterions. In contrast, ion chromatography provides a selective and highly sensitive solution for the analysis of counterions. 

One of the first examples of the application of reverse-phase liquid chromatography-gas chromatography for this type of analysis was applied to atrazine (98). This method used a loop-type interface. The mobile phase was the most important parameter because retention in the LC column must be sufficient (there must be a high percentage of water), although a low percentage of water is only possible when the loop-type interface is used to transfer the LC fraction. The authors solved this problem by using methanol/water (60 40) with 5% 1-propanol and a precolumn. The experimental conditions employed are shown in Table 13.2. 

Figure 4.29 An example of the use of ternary solvents to control mobile phase strength and selectivity in reversed-phase liquid chromatography. A, methanol-water (50 50) B, tetrahydrofuran-water (32 68) C, methanol-tetrahydrofuran-water (35 10 55). Peak identification 1 - benzyl alcohol 2 phenol 3 3-phenylpropanol 4 2,4-dimethylphenol 5 benzene and 6 -diethylphthalate. (Reproduced with permission from ref. 522. Copyright Elsevier Scientific Publishing Co.)...

The column methods are much faster and are automated so that a much larger number of samples can be processed per unit time. An example of this technology, described in more detail in Chapter 10 by Lubman and coworkers, is shown in Figure 1.2, where the first dimension is from a chromatofocusing column, which gives separations in pI much like isoelectric focusing, only here the p/ axis is in bands instead of continuous pI increments. The second dimension is by reversed-phase liquid chromatography (RPLC). 

One column can be used for different types of liquid chromatography by changing the eluent components. As an example, a column packed with octadecyl-bonded silica gel has been used for size-exclusion liquid chromatography with tetrahydrofuran (THF), normal-phase liquid chromatography with n-hexane, and reversed-phase liquid chromatography with aqueous acetonitrile. Examples of the chromatograms are shown in Figure 1.4. 

A quantitative analysis of the structure-retention relationship can be derived by using the relative solubility of solutes in water. One parameter is the partition coefficient, log P, of the analyte measured as the octanol-water partition distribution. In early work, reversed-phase liquid chromatography was used to measure log P values for drug design. Log P values were later used to predict the retention times in reversed-phase liquid chromatography.The calculation of the molecular properties can be performed with the aid of computational chemical calculations. In this chapter, examples of these quantitative structure-retention relationships are described. 

It seemed possible to correlate the elution order in reversed-phase liquid chromatography with the octanol-water partition coefficient, logP. For example, the partition coefficients calculated by Rekker s method showed a... 

Reversed-phase liquid chromatography shape-recognition processes are distinctly limited to describe the enhanced separation of geometric isomers or structurally related compounds that result primarily from the differences between molecular shapes rather than from additional interactions within the stationary-phase and/or silica support. For example, residual silanol activity of the base silica on nonend-capped polymeric Cis phases was found to enhance the separation of the polar carotenoids lutein and zeaxanthin [29]. In contrast, the separations of both the nonpolar carotenoid probes (a- and P-carotene and lycopene) and the SRM 869 column test mixture on endcapped and nonendcapped polymeric Cig phases exhibited no appreciable difference in retention. The nonpolar probes are subject to shape-selective interactions with the alkyl component of the stationary-phase (irrespective of endcapping), whereas the polar carotenoids containing hydroxyl moieties are subject to an additional level of retentive interactions via H-bonding with the surface silanols. Therefore, a direct comparison between the retention behavior of nonpolar and polar carotenoid solutes of similar shape and size that vary by the addition of polar substituents (e.g., dl-trans P-carotene vs. dll-trans P-cryptoxanthin) may not always be appropriate in the context of shape selectivity. 

Whilst gas chromatography has been used for the analysis of many of the lycoctonine-based alkaloids [52], the larger, less volatile, and more thermally labile MSAL compounds require analytical procedures such as TLC and HPLC for separation and detection. For example, both normal phase liquid chromatography [53] and reversed phase liquid chromatography [54] with UV detection have been used for separation, detection, and quantitation of alkaloids from Delphinium species associated with livestock poisonings in the western US and Canada. The introduction of API techniques has allowed the analysis of all types of diterpene alkaloids by direct MS methods and with MS methods coupled to liquid chromatography. 

From the literature there is evidence that in GC on polar phases and in normal-phase (adsorption) liquid chromatography (HPLC and TLC) the chemically specific, molecular size-independent intermolecular interactions play the main retention-determining role. For example, the HPLC retention parameters determined for substituted benzenes on porous graphite are described by QSRR equations comprising polarity descriptors but containing no bulk descriptors [93-95]. Because, in general, it is difficult to quantify the polarity properties precisely, the QSRR for GC on polar phases and for normal-phase HPLC are usually of lower quality than in the case of GC on non-polar phases and in the case of reversed-phase liquid chromatography. 

A convenient point of departure is that of the increasingly popular quantitative structure activity relationships (QSAR) mentioned above [696,699,11], which derive adsorbate-adsorbent interaction indices from, for example, water solubility data, molecular connectivities [697], n-octanol-water partition coefficients, reversed-phase liquid chromatography capacity factors [723], or linear solvation energy relationships (LSER). 

The first report on the analytical use of an aqueous solution of a surfactant, above its critical micellar concentration (CMC), as mobile phase in reversed-phase liquid chromatography (RPLC) was published in 1980. The technique, named micellar liquid chromatography (MLC), is an interesting example of the modification of the chromatographic behavior taking advantage of secondary equilibria to vary both retention and selectivity. 

In addition to the three classical separation methods mentioned above, reversed-phase liquid chromatography (RPLC) is becoming increasingly popular for the separation of highly polar and ionic species, respectively. Long-chain fatty acids, for example, are separated on a chemically bonded octadecyl phase after protonation in the mobile phase with a suitable aqueous buffer solution. This separation mode is known as ion suppression [18]. 

Reversed-phase liquid chromatography and gas-liquid chromatography have been applied to studies on the complexation of cyclodextrin with isomeric dime-thylnaphthalenes (DMN) 19-22. Both / - and y-CyD have been found to form inclusion complexes with them. With the exception of the complex of 1,8-DMN 22 with yS-CyD, all other complexes under investigation are of weak stability. The stability constant of the former complex is always about 1.5-2.0 orders of magnitude greater than the corresponding value for the other isomers [47]. For example, at 40 °C in 40% of ethanol in water solvent, the stability constants of the complexes of yS-CyD with 1,8-DMN 22 and 1,3-DMN 19 are equal to ca. 500 and ca. 8 respectively. 

Figure 10.9 shows an example of peptide separations obtained with reversed-phase liquid chromatography. Tte sample is the tryptic digest of bovine... 

The rapid separations offered by capillary electrophoresis have made it amenable as a detector in hyphenated techniques. For LC-CE, the total analysis time is usually governed by the LC separation, which generally takes minutes. However, capillary electrophoresis detection adds more peak capacity because of a second and orthogonal dimension for separation, and shorter separation conditions for LC can often be tolerated. For example, a 2.5 min reversed-phase liquid chromatography gradient was used in conjunction with 2.5 s CE separations for the detection of a tryptic digest of cytochrome c. ... 

There are several examples of significant improvements on the limits of detection (LODs) of compounds in MLC with fluorimetric detection, with respect to conventional Reversed-Phase Liquid Chromatography (RPLC). The LODs of a series of polycyclic aromatic hydrocarbons (PAHs) obtained in SDS micellar and methanol-water... 

When one has an interest in the separation of different types of compound, silica or alumina, with the appropriate mobile phase, can readily accomplish this. Also, isomer separation frequently can easily be accomplished with adsorption chromatography for example, 5,6-benzoquinoline can be separated from 7,8-benzoquinoline with silica as the stationary phase and 2-propanol hexane (1 99). This separation is difficult with reversed-phase liquid chromatography. "... 

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