Normal phase HPLC

Unlike the more popular reversed-phase chromatographic mode, normal-phase chromatography employs polar stationary phases, and retention is modulated mainly with nonpolar eluents. The stationary phase is either (a) an inorganic adsorbent like silica or alumina or (b) a polar bonded phase containing cyano, diol, or amino functional groups on a silica support. Tlie mobile phase is usually a nonaqueous mixture of organic solvents. As the polarity of the mobile phase decreases, retention in normal-phase chromatography 

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright 2007 by John Wiley Sons, Inc. 

As is the solute cross-sectional area, Ae is the molecular area of the strong solvent, Ne is the mole fraction of the strong solvent in the mobile phase, ki is retention factor of the solute in the binary mobile-phase mixture, and k is the retention factor in the strong solvent alone. 

Yet another adsorption-based retention model similar to that of Snyder was proposed by Soczewinski [6] to describe the retention in NPC. It assumes that retention in NPC is the product of competitive adsorption between solute and solvent molecules for active sites on the stationary phase surface. The stationary-phase surface consists of a layer of solute and/or solvent molecules, but, unhke the former, the latter model assumes an energetically heterogeneous surface where adsorption occurs entirely at the high-energy active sites, leading to discrete, one-to-one complexes of the form 

A is an active surface site and q refers to the number of substituents on a solute molecule that are capable of simultaneously interacting with the active site. This equation takes into account the possibility of an analyte molecule s interaction with multiple sites. Based on this model, the solute retention factor can be expressed by the following equation, which is similar to Snyder s  

Natural nucleotides are too polar to allow chromatography in the normal phase mode but chemically modified nucleotides which are used as precursors for the chemical synthesis of DNA are much less polar. All the reactive sites in these synthetic molecules are chemically blocked and they are neutral, Upophihc compounds thus, the preferred analytical and preparative mode for their separation is normal phase sUica adsorption chromatography (Seliger et al., 1982). 

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We proposed using MLC for assay of azithromycin in tablets and capsules. As alternative conventional reversed-phase HPLC method MLC was used for analysis of Biseptol (sulfamethoxazole and trimethoprim) tablets and injection. The MLC was proposed to assay of triprolydine hydrochloride and pseudoephedrine hydrochloride in tablets as alternative normal-phase HPLC method described in USP phamiacopoeia. 

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

Figure 10.9 shows the ehromatograms of fortified eoeonut oil obtained by using (a) normal-phase HPLC and (b) GPC/normal-phase HPLC. As ean be seen from these figures, ehemieal interferenees due to lipid material in the oil were eliminated by using the MD system that was used for quantitative analysis of all of the eom-pounds, exeept DL-a-toeopheryl aeetate, where the latter was eo-eluted with a triglieeride eompound and needed further separation. 

Figure 10.9 Cliromatogi ams of foitified coconut oil obtained by using (a) normal-phase HPLC and (b) GPC/noimal-phase HPLC. Peak identification is as follows 1 (a,b), DL-a-toco-pheryl acetate, 2 (b), 2,6-di-tert-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 liigh-peiformance liquid cliromatography , pp. 1929-1933, copyright 1988, with permission from the American Chemical Society.

Different transfer techniques and type of interfaces have been developed. Most of the applications involve normal-phase HPLC conditions, although reversed-phase coupled with capillary GC has also been reported. 

The analysis of sterols, sterols esters, erythrodiol and uvaol, and other minor components of oils and fats, is usually carried out by normal-phase HPLC-HRGC by using a loop-type interface and the concurrent eluent evaporation technique, as reported in the papers cited by Mondello et al. (48) (up to 1995) and in more recent papers (49, 50). More recently, reversed-phase LC-GC methods have been... 

An application of an LC-SFC system has been demonstrated by the separation of non-ionic surfactants consisting of mono- and di-laurates of poly (ethyleneglycol) (23). Without fractionation in the precolumn by normal phase HPLC (Figure 12.18 (a)) and transfer of the whole sample into the SFC system, the different homologues coeluted with each other. (Figure 12.18(b)). In contrast with prior fractionation by HPLC into two fractions and consequent analysis by SFC, the homologues in the two fractions were well resolved (Figures 12.18(c) and 12.18(d)). 

Figure 12.18 LC-SFC analysis of mono- and di-laurates of poly (ethylene glycol) ( = 10) in a surfactant sample (a) normal phase HPLC trace (b) chromatogram obtained without prior fractionation (c) chromatogram of fraction 1 (FI) (d) chromatogram of fraction 2 (F2). LC conditions column (20 cm X 0.25 cm i.d.) packed with Shimpak diol mobile phase, w-hexane/methylene chloride/ethanol (75/25/1) flow rate, 4 p.L/min UV detection at 220 nm. SFC conditions fused-silica capillary column (15 m X 0.1 mm i.d.) with OV-17 (0.25 p.m film thickness) Pressure-programmed at a rate of 10 atm/min from 80 atm to 150 atm, and then at arate of 5 atm/min FID detection. Reprinted with permission from Ref. (23).

Another important issue that must be considered in the development of CSPs for preparative separations is the solubility of enantiomers in the mobile phase. For example, the mixtures of hexane and polar solvents such as tetrahydrofuran, ethyl acetate, and 2-propanol typically used for normal-phase HPLC may not dissolve enough compound to overload the column. Since the selectivity of chiral recognition is strongly mobile phase-dependent, the development and optimization of the selector must be carried out in such a solvent that is well suited for the analytes. In contrast to analytical separations, separations on process scale do not require selectivity for a broad variety of racemates, since the unit often separates only a unique mixture of enantiomers. Therefore, a very high key-and-lock type selectivity, well known in the recognition of biosystems, would be most advantageous for the separation of a specific pair of enantiomers in large-scale production. 

Normal-phase HPLC An HPLC system in which the mobile phase is less polar that the stationary phase. 

Figure 9. Reversed-phase HPLC analysis of PAH s extracted from SRM 1649, urban dust/organics, with UV detection, not preceded by normal-phase HPLC clean-up, (Reprinted from reference 72.

Knowledge of the identity of phenolic compounds in food facilitates the analysis and discussion of potential antioxidant effects. Thus studies of phenolic compounds as antioxidants in food should usually by accompanied by the identification and quantification of the phenols. Reversed-phase HPLC combined with UV-VIS or electrochemical detection is the most common method for quantification of individual flavonoids and phenolic acids in foods (Merken and Beecher, 2000 Mattila and Kumpulainen, 2002), whereas HPLC combined with mass spectrometry has been used for identification of phenolic compounds (Justesen et al, 1998). Normal-phase HPLC combined with mass spectrometry has been used to identify monomeric and dimeric proanthocyanidins (Lazarus et al, 1999). Flavonoids are usually quantified as aglycones by HPLC, and samples containing flavonoid glycosides are therefore hydrolysed before analysis (Nuutila et al, 2002). 

A normal-phase HPLC separation seems to be useful to separate major chlorophyll derivatives, but it is not compatible with samples in water-containing solvents an additional extraction step is required to eliminate water from the extract since its presence rednces chromatographic resolution and interferes with retention times. Besides that, the analysis cannot be considered quantitative due to the difhculty in transferring componnds from the acetone solution into the ether phase. On the other hand, an advantage of the normal-phase method is its efficacy to separate magne-sinm-chlorophyll chelates from other metal-chelated chlorophyll derivatives. ... 

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... 

Fused-silica column (15 cm x 4.6 mm) of Spherisorb S5W 0.1 mL chloroform-methanol-aqueous 25% ammonia (868 125 7) [1.5 mL/min] 254 nm Normal phase HPLC analysis in whole blood and urine. [96]... 

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

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. 

Normal phase HPLC consists of methods that utilize a nonpolar mobile phase in combination with a polar stationary phase. Adsorption HPLC actually fits this description, too, since the adsorbing solid stationary phase particles are very polar. (See discussion of adsorption columns in Section 13.5.3.) Normal... 

Distinguish normal phase HPLC from reverse phase HPLC. 

List some typical mobile and stationary phases for (a) reverse phase HPLC, and (b) normal phase HPLC. 

Answer the following with either polar or nonpolar. How would you describe the mobile phase for normal phase HPLC How would you describe the stationary phase for reverse phase HPLC ... 

Fig. 2.3.5. Normal-phase HPLC chromatograms of commercial standards of (b) f-octylphenol and (a) 4-nonylphenol (85%). Column 100 X 4.6 mm2 Hypersil 3 NH2 (3 pm), gradient elution with re-hexane-2-propanol-H20, detection fluorescence, excitation 230 nm...

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. 

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