Normal phase LC

Normal-phase LC tends to separate according to solute polarity since the stationary phase is polar and retention is often dominated by hydrogen bonding. Thus, normal-phase LC is useful in sorting out classes of materials according to the polarity of the solutes. Fatty acids are easily separated from monoglycerides, but the separation of individual saturated fatty acids from each other on the basis of their carbon... 

Figure 10.13 GC clrromatogram obtained after on-line LC-GC(ECD) of a human milk sample analysed for PCBs (attenuation X 64). Peak identification is as follows (1) PCB 28 (2) PCB 118 (3) PCB 153 (4) PCB 138 (5) PCB 180 (6) PCB 170 (7) PCB 207. Reprinted from Journal of High Resolution Chromatography, 20, G. R. van der Hoff et al, Determination of organochlorine compounds in fatty matiices application of normal-phase LC clean-up coupled on-line to GC/ECD , pp. 222-226, 1997, with permission from Wiley-VCH.

The first bioanalytical application of LC-GC was presented by Grob et al. (119). These authors proposed this coupled system for the determination of diethylstilbe-strol in urine as a replacement for GC-MS. After hydrolysis, clean-up by solid-phase extraction and derivatization by pentafluorobenzyl bromide, the extract was separated with normal-phase LC by using cyclohexane/1 % tetrahydrofuran (THE) at a flow-rate of 260 p.l/min as the mobile phase. The result of LC-UV analysis of a urine sample and GC with electron-capture detection (ECD) of the LC fraction are shown in Ligures 11.8(a) and (b), respectively. The practical detection limits varied between about 0.1 and 0.3 ppb, depending on the urine being analysed. By use of... 

The low polarity of CO,-based eluents makes SFC a normal phase technique. Therefore, it is not surprising that most of the successful applications of chiral SFC have utilized CSPs designed for normal phase LC. Flowever, some exceptions have been noted. Specific applications of various CSPs are outlined in the next sections. 

The nature of the modifier and the modifier concentration impact both retention and selectivity in packed column SFC. SFC offers considerable flexibility in modifier selection because nearly all commonly used organic modifiers, including methanol and acetonitrile, are miscible with CO,. In contrast, methanol and acetonitrile are rarely used as modifiers in normal phase LC because they are immiscible with hexane [68]. 

In the analysis of complex PAH mixtures obtained from environmental samples, reversed-phase LC-FL typically provides reliable results for only 8-12 major PAHs (Wise et al. 1993a). To increase the number of PAHs determined by LC-FL, a multidimensional LC procedure is used to isolate and enrich specific isomeric PAHs that could not be measured easily in the total PAH fraction because of interferences, low concentrations, and/or low fluorescence sensitivity or selectivity. This multi-dimensional procedure, which has been described previously (Wise et al. 1977 May and Wise 1984 Wise et al. 1993a, 1993b), consists of a normal-phase LC separation of the PAHs based on the number of aromatic carbon atoms in the PAH, thereby providing fractions containing only isomeric PAHs and their alkyl-substituted isomers. These isomeric fractions are then analyzed by reversed-phase LC-FL to separate and quantify the various isomers. 

Alternatively, the LC dimension of LC-GC may be replaced by packed-column SFC, in order to improve the compatibility between two mobile phases and to allow the FID to be used for both separations. Because of the relatively nonpolar nature of scCCL. SFC-GC is particularly recommended as a substitute for many normal-phase LC-GC analyses. The techniques developed for solvent evaporation at the LC-GC interface are often not required in SFC-GC, because the solutes are deposited at the front of the GC column when CCL decompresses into a gas at the end of the SFC column. 

SFE-GC and SFC-GC may replace normal-phase LC-GC. SFC-SFC may be adopted for a wide range of applications, particularly for solutes that are not amenable to GC and as a replacement for some coupled LC-LC separations. However, no applications to additives in polymers can be mentioned. 

Quaternary ammonium cationic surfactants, such as DTDMAC, were determined in digested sludge by using supercritical fluid extraction (SFE) and FIA-ESI-MS(+) after separation by normal phase LC. Standard compounds—commercially available DTDMAC— were used to check the results. The DTDMAC mixture examined showed ions at m/z 467, 495, 523, 551, and 579 all equally spaced by A m/z 28 (-CH2-CH2-) resulting from the ionisation of compounds like RR N (CH3)2 X (R = / RO as shown in Fig. 2.12.11(a) and (b) [22],... 

Normal phase LC-ES-MS was used for the analysis. In a previous work by the same authors, an interesting comparison was made between LC-FL, LC-UV and LC-ES-MS analysis of the same (spiked and unspiked) marine sediments [29]. The concentrations measured by LC-FL and LC-UV were consistently higher by about a factor of two than concentrations analysed by LC-ES-MS (however, with LC-UV analysis, for the unspiked sediments concentrations were all below the detection limit). This was attributed to the limited specificity of the LC-FL and UV techniques. 

A representative gas chromatogram with ECD of the analysis of various polar chlorinated pesticides isolated from cod liver oil [179] is shown in Fig. 13. Determination of the polar chlorinated pesticides in cod liver oil required clean up of the lipid matrix with a dimethylformamide/water/hexane liquid-liquid partitioning procedure followed by isolation using a normal-phase LC procedures, and final analysis by GC-ECD [179]. 

Various PCB congeners and lower polarity pesticide fractions analyzed from cod liver oil is shown in Fig. 15 [179]. Measurement of the PCB congeners and pesticides in the cod liver oil required clean-up of the lipid matrix with a di-methylformamide/water/hexane liquid-liquid partitioning procedure followed by isolation of the PCBs and pesticides using a normal-phase LC procedures. The normal-phase LC procedures separate the analytes into two fractions, one containing the PCBs and the lower polarity chlorinated pesticides (HCB, 2,4 -DDE, and 4,4 -DDE) (Fig. 15) and the second containing the more polar chlorinated pesticides. The separation of PCBs and pesticides reduces the possible coelution of many of the pesticides with PCB congeners of interest. These two fractions were then analyzed by GC-ECD. 

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. 

Ding, J., Desai, M., and Armstrong, D.W., Evaluation of ethoxynonafluorobu-tane as a safe and environmentally friendly solvent for chiral normal-phase LC-atmospheric pressure chemical ionization/electrospray ionization-mass spectrometry, J. Chromatogr. A, 1076, 34, 2005. 

When APCI in used in combination with the normal phase LC, the nitrogen molecular ion will enter into a charge-transfer reaction with the organic solvent. Ion-molecule reactions lead to protonated solvent clusters that will react by proton transfer with the analyte molecules, forming [M + H]+ ions. 

The number of detectors that are sensitive and selective enough to be applied online with LC is limited because the solvents used are not compatible, as in the case of immunochemical detection after reversed- or normal-phase LC. The technology of coupling is still under development and not yet available in a large number of laboratories not specialized in techniques such as LC-MS. Therefore, LC separations are frequently followed by offline detection. Confirmatory analysis of suspected liquid chromatographic peaks can be made possible by coupling liquid chromatography with mass spectrometry. Atmospheric-pressure chemical ionization LC-MS has been employed for the identification of six steroid hormones in bovine tissues (448). 

Over the past 10 years, liquid chromatography coupled with ultraviolet detection appears to have become the method of choice for the determination of corticosteroids, offering tlie analyst both satisfactory selectivity and sensitivity. Both reversed-phase (544-547) and normal-phase (548) chromatography have been applied to the determination of dexamethasone in plasma, coupled with ultraviolet (UV) detection generally at 254 nm, and in bovine tissues (528, 535). A series of both reversed- and normal phase LC systems have also been used for the simultaneous determination of dexamethasone and other steroids. Two different liquid chromatographic separations have been described for the isolation and simultaneous separation of steroids in serum (549). 

High-performance LC was used to determine residue levels of p,p-DDE in marine mammal blubber. This compound can absorb UV well enough at shorter wavelengths. With normal-phase LC after derivatization to convert the p,p-DDE to / ,/ -DCPB (dichlorobenzophenone), a semiquantitative determination of this compound was done in the presence of PCBs (62). 

In normal"-phase LC systems, die solid phase is a polar solid such as silica gel (most common) or alumina and die liquid is generally an organic solvent of low polarity. In such a case, polar compounds bind more strongly to die polar silica gel surface and thus travel more slowly along the surface, whereas... 

M. Lechner, C. Bauer-Plank and E. Lorbeer, Determination of acylglycerols in vegetable oil methyl esters by on-line normal phase LC-GC , 7. High Resolut. Chromatogr. 20 581-585(1997). 

G. R. van der Hoff, R. A. Baumann, P. van Zoonen and U. A. Th. Brinkman, Determination of organochlorine compounds in fatty matrices application of normal-phase LC clean-up coupled on-line to GC/ECD , 7. High Resolut. Chromatogr. 20 222-226(1997). 

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