Argentation HPLC separation

Because the retention times of the different TGs within a PN differ, it is important to collect the whole peak at fractionation. One total fractionation, with an elution time of ca. 1.5 h, will provide enough material for the subsequent separation procedure based on argentation-HPLC (utilizing silver ions in the mobile phase), in order to separate geometrical isomers. 

Separation of Isophenlndamlne and phenlndamlne deserves special mention since an argentated HPLC mobile phase can bring about this difficult separation based on the position of double bond (8). 

In this connection reversed-phase preparative HPLC with UV spectroscopic detection (254nm) of fractions has proved of particular use for the separation on Develosil ODS-5 of the stereoisomers in urushiol with eluent MeCN-H20-MeC02H (ref.25) and these results are summarised in Table 9 (the % results are based on peak areas). The isolated comounds were identified by MS and H NMR. Extensive work by Japanese groups on several other different chromatographic procedures has been described including the argentation HPLC of urushiol diacetate. 

Argentation HPLC using silver nitrate-loaded silica stationary phases has been used for the analysis of the positional isomers of triglycerides including 2-unsaturated-l,3-disaturated (SUS), 1-un-saturated-2,3-disaturated (USS), fully saturated (SSS) and fully unsaturated (UUU) isomers (Smith et al., 1980 Hammond, 1981). In these separations the silica was loaded with 10% silver nitrate and the mobile phases used were either benzene (Smith et al., 1980) or toluene (Hammond, 1981). The resolution of the lipid species was found to... 

The most popular mobile phases used in argentation HPLC have been mixtures of chloroform (80-90%) and acetic acid <0.5%) with varying proportions of acetonitrile and/or methanol to optimally adjust chromatographic retention. Addition of methanol to the mobile phase is thought to reduce interactions with polar groups on the stationary phase (Powell, 1982) while acetonitrile reduces retention by direct interaction with silver ions bound to the stationary phase, thereby reducing interactions with the olefinic unsaturated bonds (Merritt and Bronson, 1977). Adjustment of the composition of the mobile phase allows the specific types of interactions between the mobile phase, sample molecules and stationary phase to be enhanced or reduced in this way an optimal chromatographic separation can be achieved. 

Having developed the argentation TLC/GLC procedure and proved its capability for measuring the erucic acid content of any foodstuff, in 1977 LGC considered adapting the method to utilise high-performance liquid chromatography (HPLC) in an attempt to combine the separation and quantitation procedures into one step and to make the method more rapid. There was very little reference in the literature to the use of silver in HPLC separations, but work proceeded with the basic premise that any separations on a TLC plate should also be possible on an efficient HPLC column. 

Prior to structural elucidation and possible eventual synthesis, the isolation of component phenolic lipids in a pure state is essential. The cold methods of thin layer chromatography (TLC), column chromatography (CC), flash chromatography, high performance liquid chromatography (HPLC) and hot methods (GC), often after derivatisation, are well established. Argentation versions of these separatory methods are less common but are desirable for the rapid separation of unsaturated constituents. 

Two further separation techniques have been described for the resolution of the oestranes reversed phase ion-pair partition (Hermansson, 1978) and argentation reversed phase HPLC (Tscheme and Capitano, 1977). In the former technique C2 or Cg columns were coated with 1-pentanol and used with tetrapropylammonium hydroxide as an ion-pairing reagent. In the latter technique an ODS stationary phase was used with a mobile phase of methanol-5% silver nitrate (60 40) to elute oestriol, equilin, oestrone and oestradiol. 

The CLA and 18 1 isomer composition in the milk and meat fat of ruminants is a mixture of numerous positional and geometric isomers, most of which are generated by specific rumen bacteria, or subsequently re-synthesized in tissues by specific enzymes. To understand their biosynthesis, with the aim of manipulating these biochemical processes, requires appropriate techniques to determine each of the individual isomers with confidence. Detailed chemical syntheses are presented in Chapter 3 to prepare appropriate standards Chapter 4 provides a summary of complementary gas-, adsorption-, and argentation-chromatographic techniques required for the analysis of all the CLA and trans- and af-18 l isomers Chapter 5 presents improved separations of the CLA isomers using modified silver ion and reverse phase HPLC techniques while Chapter 6 is devoted to a complete structural characterization of the methyl esters of CLA isomers using acetonitrile chemical ionization tandem mass spectrometry. 

However, there are serious limitations when relying only on Ag -TLC or Ag -HPLC results to analyze the TFA content of a fat-containing product. These are not independent methods but should be used to compliment GC results. The two argentation methods make it possible to analyze the mono TFA in greater detail, but they are not very suitable for the analysis of TFA with more than one double bond in the molecule. One would need to look at many other TLC bands (or HPLC peaks) to arrive at the total TFA content of a product. Analysis of all the TFA is crucial considering that we still do not know which TFA isomers are responsible for increased risk of CHD. Many of the more unsaturated FA containing trans bonds can be analyzed by GC and for that reason the results of both GC and argentation separations should be combined. Alternatively, TFA could be analyzed by FT-NIR (38), or as total TFA by FTIR (13). 

Argentation chromatography, in which silver is used as a IT complexing metal on a silica gel support, is usually employed for the separation of organic compounds with electron-donor properties because of the presence of unsaturated groups in the molecule of the analytes. TLC is particularly appropriate for applying silver complexation techniques because the instability of silver causes severe limitations to column lifetime and, therefore, to HPLC methods. 

Since its introduction by Morris and others in 1962, TLC on silica gel impregnated with silver nitrate has been of enormous value to the lipid analyst. It is sometimes termed "argentation" chromatography. The basis of the separation is the facility with which the double bonds in the alkyl chains of fatty acids form polar complexes reversibly with silver compounds. Fatty acids can be separated according to both the number and the configuration of their double bonds and sometimes, with care, according to the position of the double bonds in the alkyl chain. HPLC has been slow to make a mark in this area, because of problems in preparing stable columns, but many of the major difficulties now appear to have been resolved. However, most of the data on the elution characteristics of silver complexes of unsaturated fatty acids has been obtained by TLC. 

---