Normal phase elution

In contemporary HPLC. gradient elution is by far most frequently practised in reversed-phase systems for a plethora of sample types. Special precautions required in gradient-elution normal-phase chromatography discussed in Section 1.5.5 are usually not necessary. In RPC systems where the retention Eq. (1.18) applies. Eqs. (1.34) and... 

Single elution normal phase TLC Materials and equipment... 

In summary, for the chromatographic resolution of sterols it is advisable that reversed phase techniques should be tried initially and in the event of co-elution normal phase chromatography on silica should be tried as an alternative option. For very specific separations where the aforementioned techniques have failed, either NARP or argentation chromatography could be attempted although it is probable that if reversed phase or normal phase failed to elicit a separation, the remaining techniques would also be unsuccessful. 

Jandera P, Petranek L, and Kucerova M (1997) Characterization and prediction of retention in isocratic and gradient-elution normal-phase high-performance liquid chromatography on polar bonded stationary phases with binary and ternary solvent systems. Journal of Chromatography A 791 1-19. 

Figure 3.8 Experimental van Deemter plot of H (cm) vs u (cm.s ) for isocratic elution (normal phase) of hexamethylben-zene with a mobile phase of 4.8 % (w/v) ethyl acetate in n-decane. The column was 25 cm long, 9 mm in diameter and packed with 8.5 pm silica gel. The curve fitting procedure gave values for the van Deemter constants, and thus the separate contributions to the curve from the multipath dispersion, longitudinal dispersion and the resistance to mass transfer were calculated as shown. Reproduced from Scott, http //www.chromatography-online.org/, with permission.

The evaporative light scattering detector is well suited to gradient elution normal phase chromatography and eliminates the need to form derivatives. However, the 1- and 2-mole adducts are generally too volatile for quantification by this method (117,124,125). 

APE, E= 1-18 ethoxy distribution River and waste water and sediment Ion exchange cleanup, SPE on Cijj (MeOH elution) Normal phase HPLC-fluorescence detection (229 nm excitation, 310 nm emission) 106,196... 

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. 

Similar to the new polar organic mode, the retention of analytes in normal phase is not difficult to predict. For all the compounds, the average of the retention on individual columns is fairly close to the retention on the coupled columns. The selectivity of most compounds on coupled columns is an average of the selectivities of individual columns (Fig. 2-9). However, it was found that the elution order for some compounds was reversed on ristocetin A and teieoplanin or vancomycin. As a result. 

Fig. 2-18. Normal phase retention of the first eluted and seeond eluted enantiomer of mephenytoin on vaneomyein CSP (250 x 4.6 mm). The flow rate was 1.0 mL min at ambient temperature (23 °C).

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

For samples that meet the solubility requirements of the SEC approach, analyses were also reported for additives in polymers such as PVC and PS [28,29]. Direct SEC analysis of PVC additives such as plasticisers and thermal stabilisers in dissolution mode has been described [28,30,31 ]. In the analysis of a dissolved PS sample using a SEC column of narrow pore size, the group of additives was separated on a normal-phase column after elution of the polymer peak [21]. Column-loading capacity of HPSEC for the analysis of additives, their degradation products and any other low-MW compounds present in plastics has been evaluated for PS/HMBT, PVC/TNPP and PVC/TETO (glyceryl tri[l-14C] epoxyoleate) [31]. It was shown that HPSEC can be used to separate low-MW compounds from relatively large amounts of polymers without serious loss of resolution of the additives the technique has also been used for the group analysis of chlorohydrin transformation products of the TETO model compound [32]. 

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