Normal-phase HPLC chromatography

Analytical-Scale HPLC Separations. Reverse-phase HPLC chromatography favors the distribution of the semi- and nonpolar constituents of a sample of residue organics, whereas normal-phase HPLC chromatography favors the distribution of semipolar constituents (32). This approach is illustrated in Figure 2 by the chromatograms of residue organics from a waste water sample separated by both reverse-... 

The most common approaches to sulfonylurea determinations involve high-performance liquid chromatography (HPLC). The earliest reported methods utilized normal-phase liquid chromatography (LC) with photoconductivity detection this type of detector demonstrated undesirably long equilibration times and is no longer... 

When the predominant functional group of the stationary phase is more polar than the commonly used mobile phases, the separation technique is termed normal-phase HPLC (NPLC), formerly also called adsorption liquid chromatography. In NPLC, many types... 

NP-HPLC, NPLC Normal-phase liquid chromatography... 

The stationary phases available for HPLC are as numerous as those available for GC. As mentioned previously, however, adsorption, partition, ion exchange, and size exclusion are all liquid chromatography methods. We can therefore classify the stationary phases according to which of these four types of chromatography they represent. Additionally, partition HPLC, which is the most common, is further classified as normal phase HPLC or reverse phase HPLC. Both of these are bonded phase chromatography, which was described in Chapter 11. Let us begin with these. 

ASE using dichloromethane has been applied to extract alkylphenols and short-chain NPEO from sediment [8,47]. Samples of 2-5 g were extracted in two cycles of 30 mL at 100°C and 69 atm. Clean-up was performed using size exclusion chromatography to remove high molecular weight lipids, and then using normal phase HPLC. 

Chiral separations result from the formation of transient diastereomeric complexes between stationary phases, analytes, and mobile phases. Therefore, a column is the heart of chiral chromatography as in other forms of chromatography. Most chiral stationary phases designed for normal phase HPLC are also suitable for packed column SFC with the exception of protein-based chiral stationary phases. It was estimated that over 200 chiral stationary phases are commercially available [72]. Typical chiral stationary phases used in SFC include Pirkle-type, polysaccharide-based, inclusion-type, and cross-linked polymer-based phases. 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

Fig. 3.8 Normal-phase-HPLC chromatograms of PA fractions generated by a combination of solvent extraction and column chromatography on Toyopearl resin (protocol 2). a monomer-rich, b ohgomer-rich, c dimer-rich, and d polymer-rich fractions. Compounds were detected with post-column derivatization using DMACA...

Caude, M. J. and lardy. A., Normal-phase liquid chromatography, in Handbook of HPLC, Katz, E., Eksteen, R., Schoenmakers, R, and Miller, N. (Eds.), Marcel Dekker, New York, 1998, pp. 325-363. 

Many applications have been found for reversed-phase chromatography in HPLC. The composition of the stationary phase is more easily controlled than with the TLC methods, and thus provides more reproducible separations. The use of bonded non-polar phases enables gradient elution to be carried out in a reversed-phase system. This approach has been useful for the analysis of polar compounds and gives improved separations compared with normal-phase HPLC. These methods usually involve separation with systems consisting of Carbowax, C -polymer or similar phases bonded or physically coated on the support. 

Gel permeation chromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard reference material (SRM) samples of a fortified coconut oil (SRM 1563) and a cod liver oil (SRM 1588). The on-line GPC/normal-phase procedure eliminated the long and laborious extraction procedure of isolating vitamins from the oil matrix. In fact, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC columns used for the vitamin determinations were a 10 im polystyrene/divinylbenzene gel column and a semipreparative aminocyano column, with hexane, methylene chloride and methyl te/t-butyl ether being employed as solvent. 

Figure 10.9 Chromatograms of fortified coconut oil obtained by using (a) normal-phase HPLC and (b) GPC/normal-phase HPLC. Peak identification is as follows 1 (a,b), DL-a-toco-pheryl acetate, 2 (b), 2,6-di-terf-butyl-4-methylphenol 2 (a) and 3 (b), retinyl acetate 3 (a) and 4 (b), tocol 4 (a) and 5 (b), ergocalciferol. Reprinted from Analytical Chemistry, 60, J. M. Brown-Thomas et al., Determination of fat-soluble vitamins in oil matrices by multidimensional high-performance liquid chromatography , pp. 1929-1933, copyright 1988, with permission from the American Chemical Society.

There are numerous analytical techniques besides reversed-phase HPLC that can be used to analyze stress test samples. Some of the more common ones include normal-phase HPLC, thin layer chromatography (TLC), capillary electrophoresis (CE), and gas chromatography (GC). The following paragraphs contain brief discussions on the use of these techniques for analysis of stress test samples. 

Normal phase (NP) separations are comparatively rarely used in environmental analysis. Again, the reasons lie in the range of analytes amenable to this mode of separation, and in the limited compatibility of typical normal phase HPLC (NP-HPLC) mobile phases with mass spectrometric detection (this also applies to IC). Not only for this reason has interest recently grown in hydrophilic-lipophilic interaction chromatography (HILIC), which represents a viable alternative to the separation of very polar compounds with mobile phases that have a much better compatibility with MS detection, for example, acetonitrile/water with a low water content, typically below 10%, 32 Nonetheless, NP chromato-graphy retains its important role in sample preparation, particularly for the cleanup of complex environmental samples. In the off-line approach, fractions are collected and the relevant one is injected into the reversed phase HPLC (RP-HPLC) system, often after solvent exchange. 

Stewart et al. have also reported the efforts at Molecular Nature Ltd. to generate a pure natural product library [41], Compounds for this library were isolated utilizing parallel, normal phase column chromatography followed by C-18 and/or ion exchange chromatography. To be accepted into the library, the compounds must be > 90% pure with structural verification via a combination of HPLC, NMR, MS and GC/MS. 

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. 

Normal-phase HPLC explores the differences in the strength of the polar interactions of the analytes in the mixture with the stationary phase. The stronger the analyte-stationary phase interaction, the longer the analyte retention. As with any liquid chromatography technique, NP HPLC separation is a competitive process. Analyte molecules compete with the mobile-phase molecules for the adsorption sites on the surface of the stationary phase. The stronger the mobile-phase interactions with the stationary phase, the lower the difference between the stationary-phase interactions and the analyte interactions, and thus the lower the analyte retention. 

As opposed to normal-phase HPLC, reversed-phase chromatography employs mainly dispersive forces (hydrophobic or van der Waals interactions). The polarities of mobile and stationary phases are reversed, such that the surface of the stationary phase in RP HPLC is hydrophobic and mobile phase is polar, where mainly water-based solutions are employed. 

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