NP chromatography

Even though NP chromatography is not common in peptide and protein separations, examples of applications for the analysis of peptides produced by the cleavage of membrane proteins are reported in the literature [122],... 

Both NP (428, 441, 442) and RP (427, 436, 437) chromatography has been applied to analysis of vitamin A and provitamin A. A Lichrosorb Si60 column (250 mm x 4.6 mm) has been used for NP chromatography using hexane and hexane/2-propanol as mobile phase [441], More widespread are C8, C18, and C30 column with acetonitrile, methanol, water, and mixtures of these as the most used mobile phase [424,427,428,443], Chavez-Servm et al. [442] reported the use of a short narrow-bore column (50 mm X 2 mm id), which enables less solvent consumption and higher mass sensitivity. 

NP chromatography enables the separation of the c -isomer from the active trans-isormi, nevertheless, it has been applied mainly as preparative chromatography using silica columns. RP chromatography instead is widely used as the separation step, even if the resolution between cis- and trans-isomer is not achieved. Menaquinone-4 (MK-4) [504,508-510] or 2, 3 -dihydrophylloquinone [517] are frequently used as an internal standard. 

NP chromatography is unable to separate vitamin D2 from vitamin Dj. So it is usually used as semipreparative chromatography [527-531]. Instead, RP chromatography is able to resolve vitamin D2 and Dj, thus it is widely applied as aualy tical chromatography. Mattila et al. [532] describe a two dimensioual LC procedure. The sample is saponified and an NP semipreparative column is used before the quantification in a tandem column set (Zorbax ODS x Vydac 201 TP54 CIS). 

In NP chromatography non-polar solvents (e.g. alkanes) are used as eluents in cases of insufficient eluting strength the polarity of the eluent can be raised by addition of more polar solvent. More polar sample components have longer residence times in NP chromatography, i.e. they leave the column later because they are retained longer. 

Sub-/supercritical fluid chromatography is essentially NP chromatography with the added advantage that the lower viscosity and higher diffusivity of the mobile phase results in higher column efficiencies allowing for rapid resolutions. The columns employed are the same as those utilized in conventional NP chromatography. Carbon dioxide is the most commonly used nonpolar eluent but requires a more polar modifier such as an alcohol for the elution of polar solutes. The modifier increases the polarity of the mobile phase and... 

RP chromatography generally offers a better selectivity than NPLC for the separation of molecules differing in hydrophobic parts of the molecules, but there are some practical reasons for selecting NP chromatography methods in specific cases, as follows ... 

Unlike RP chromatography, NP chromatography enables the direct injection of samples extracted into a nonpolar solvent. 

The previous chapter discussed the solvent and its interaction with the solute. To complete the chromatographic system the adsorbent has to be selected. As mentioned in Chapter 3.2.1 one has to distinguish between enantioselective and non-enantioselective adsorbents. Both groups of adsorbents are classified into polar, semi-polar and nonpolar adsorbents (see Tab. 4.4). This classification is based on the surface chemistry of the packing material. Interaction between mobile phase and adsorbent characterizes the phase system, which is distinguished between normal phase (NP) chromatography and reversed phase (RP) chromatography. This differentiation is historic and appointed by the ratio of the polarity of the adsorbent and the mobile phase. 

If the retention time is much higher than the time of the initial gradient (retention factor 10) the substances seem to be too lipophilic for RP separation on the initial adsorbent. The polarity of the adsorbent should be increased by the use of amino or diol phases. If the adsorbent is already polar the phase system must be changed to NP-chromatography (Section 4.3.2). 

Figure 4.6 (See colour insert following page 142.) Separation in NP-chromatography.

The first step in HPLC method development consists of selecting an appropriate separation mode. Many neutral compounds can be separated either by reversed-phase (RP) or by normal-phase (NP) chromatography. An RP system is usually the best first choice because it is likely to result in a satisfactory separation of a great variety of... 

In NP chromatography (also called adsorption or liquid-solid chromatography), the stationary phase is more polar than the mobile phase. The retention increases as the polarity of the mobile phase decreases, and polar analytes are more strongly retained than non-polar ones (i.e., the opposite of RP chromatography Fig. 1). The column packing is... 

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