Separation methods reversed phase liquid chromatography

Einarsson et al. [106] introduced (-t-)-l-(9-fluorenyl)-ethyl chloroformate (41) as a versatile reagent for the simple chiral derivatization of primary and secondary amino acids to give stable, highly fluorescent products, which are readily separated by reversed phase liquid chromatography, or by GC after methylation with diazomethane. The authors also illustrated a selective method using this reagent for the determination of imino acids in the presence of primary amino acids. 

The OPA reagent was first reported in 1971 by Roth as a postcolimm fluorogenic reagent for amines [5] and has been widely used for the sensitive determination of primary amino compounds. However, the fluorescent derivatives are not sufficiently stable, and it is sometimes difficult to obtain reproducible results using the postcolumn derivatization system. A precolumn derivatization technique has also been developed using OPA in the presence of alkylthiol compounds such as 2-mercaptoethanol. OPA rapidly reacts with primary amino compounds within 2 min at room temperature, and the derivatives can be separated by reversed-phase liquid chromatography [20]. Fluorescence detection of the derivatives is performed at 440 nm (emission wavelength) with excitation at 330 nm. Because OPA does not react with secondary amino compounds, proline and hydroxyproline can not be determined by this method. Replacement of 2-mercaptoethanol with other thiols, such as 2-ethanethiol [21] and 3-mercaptopropionic acid [22], produced more stable fluorescent derivatives. 

In 2000, Nohta et al. described a method for the determination of biologically active polyamines by intramolecular excimer-forming derivatization with 4-(l-Pjo ene)butyric acid N-hydroxysuccinimide ester (PSE) [34], By this method, dipyrene-labeled putrescine, cadaverine, spermidine, and spermine could be separated by reversed-phase liquid chromatography and specifically detected by the excimer fluorescence at 475 nm with excitation at 345 nm. The excimer fluorescence-emission wavelength is far different from that of the monomer fluorescence-emission wavelength (375 nm) derived from the excess PSE reagent, the hydrolysate product (4-(l-pyrene)butyric acid), and other monopyrene-labeled derivatives. In real biological samples, various monopyrene-labeled derivatives are formed by reaction with PSE and severely interfere with the determination of polyamines. 

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). 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

Normal-phase liquid chromatography is thus a steric-selective separation method. The molecular properties of steric isomers are not easily obtained and the molecular properties of optical isomers estimated by computational chemical calculation are the same. Therefore, the development of prediction methods for retention times in normal-phase liquid chromatography is difficult compared with reversed-phase liquid chromatography, where the hydrophobicity of the molecule is the predominant determinant of retention differences. When the molecular structure is known, the separation conditions in normal-phase LC can be estimated from Table 1.1, and from the solvent selectivity. A small-scale thin-layer liquid chromatographic separation is often a good tool to find a suitable eluent. When a silica gel column is used, the formation of a monolayer of water on the surface of the silica gel is an important technique. A water-saturated very non-polar solvent should be used as the base solvent, such as water-saturated w-hexane or isooctane. 

Matthijs, N., Perrin, C., Maftouh, M., Massart, D.L., Vander Heyden, Y. Definition and system implementation of strategies for method development of chiral separations in normal- or reversed-phase liquid chromatography using polysaccharide-based stationary phases. 7. Chromatogr. A 2004, 1041, 119-133. 

Reverse-phase liquid chromatography is now virtually the only method used in the analysis of the TG mixtures. The first paper on TG-HPLC analysis was published in 1975 by Pei et al. (81). Triglycerides were separated on a VYDAC reverse-phase (35 - 44 /xm) column and eluted with methanol-water (9 1). Since Pei et al. first applied RP-HPLC to the separation of triacyl-glycerols, a number of reverse-phase systems have been developed as rapid and efficient resolution of complex triacylglycerol mixtures can be achieved. 

Hultman et al. [130] developed a LC/MS/MS method for the quantitative determination of esomeprazole and its two main metabolites 5-hydro-xyesomeprazole and omeprazole sulfone in 25 /il human, rat, or dog plasma. The analytes and their internal standards were extracted from plasma into methyl ferf-butyl ether-dichloromethane (3 2). After evaporation and reconstitution of the organic extract, the analytes were separated on a reversed-phase liquid chromatography column and measured by atmospheric-pressure positive ionization mass spectrometry. 

The liquid chromatographic procedure [622] uses reversed phase liquid chromatography with fluorescence detection to separate all 16 PAHs completely. The method is sensitive and so selective as often to allow the method to be applied without clean-up procedure. For gas chromatographic methods, detection limits are about lpg L 1 whereas for the liquid chromatographic methods, limits are between 1 and lOOpg L 1 for two- and three-ring aromatics and below lpg L 1 for the four-, five- and six-ring compounds. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

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. 

Reverse phase liquid chromatography has typically been used for the separation of PFCs, employing either Cg or Cig columns [96], although the use of perfluoroalkyl columns has also been reported [115]. Mobile phases are typically mixtures of methanol-water or acetonitrile-water and are often modified with ammonium acetate to improve chromatographic separation and MS sensitivity. Both isocratic and gradient elution methodologies have been employed [96]. LC-MS/MS methods [116, 117] have also been developed for the separation of PFSA and PFCA isomers and generally employ linear perfluorooctyl stationary phases and acidified mobile phases. 

RP-HPLC The most commonly used methods for the analysis of SFs are reverse-phase liquid chromatography with UV-detection, which allow the separation of SFs with different sterol moieties. The number of components separated is naturally dependent on the type of column used, but most reverse-phase columns provide a full separation of at least five to six components of y-oryzanol. Cl 8-columns are most abundantly applied for the separation of SFs, but other reverse-phase... 

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]. 

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