Argentation HPLC

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. 

In summary, argentation HPLC provides a useful alternative to conventional reversed phase chromatography for the isolation and quantitation of prostaglandins. The major advantages of this technique are that it provides a better resolution of compounds which... 

Fig. 4. Argentation HPLC of two /ra 5-C18 l fatty acids as phenacyl esters. Nucleosil lOSA-AgNOj, column length 250 mm, i.d. 4mm, Temp. -15 C. Flow rate 0-4ml/min. CHCI3 hexane, 1 13. UV detection 243nm.

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. 

Synthesis has been useful in the chemistry of phenolic lipids for structural confirmation and in enabling structure/property correlations to be made by the general applicability of syntheses to a range of non-natural isomers. With the polyunsaturated constituents synthesis is valuable since either argentation chromatography or preparative HPLC have usually proved laborious on the natural products and on transformed isomeric compounds required for evaluatory purposes in structure/activity studies. 

The sterols include that group of compounds which contain an alcoholic hydroxyl group at C3 and a branched aliphatic chain of at least eight carbon atoms at C17. These compounds, which include cholesterol and its derivatives, can occur as free alcohols or as long-chain fatty acid esters. A number of HPLC techniques have been used in the analysis of the sterols including reversed phase, non-aque-ous reversed phase, normal phase, argentation and combinations of the above. 

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. 

There are a few techniques related to normal phase chromatography that are worth mentioning. Silica can be coated with the salts of heavy metals. This results in unique selectivities for analytes that form complexes with the metal ions. An example of this is argentation chromatography (20). The silica is coated with silver nitrate, which gives it a spedal sel ivity for compounds with al atic double bonds. The technique has acquired a broad range of applications. However, while Ag -coated TLC plates are commercially available, HPLC columns have to be prepared by the user. Other metal salts can be used in a similar manner for different applications. 

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. 

TLC is often used as a preparative technique to isolate individual lipid fractions for subsequent studies by a variety of analytical procedures. Preparative isolation of the sterol (Chitwood et al., 1985 Shetty et al., 1990) and triacylglyc-erol (Horutz et al., 1993) fractions have been reported. Preparative isolation of sterols may allow these lipids to be analyzed further by argentation TLC (Ditullio et al., 1965 Morris, 1966) or by GLC (Shetty et al., 1990). Moreover, preparative isolation of triacylglycerols will allow for TLC analysis of glyceryl ethers (Snyder, 1971) and HPLC analysis of specific triacylglycerols (Horutz et al., 1993). Experiment 7 is concerned with a preparative procedure for the analysis of sterols by argentation TLC. 

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. 

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. 

In the present state of scientific knowledge, control of the amount of long-chain fatty acids in the human diet is desirable. Suitable— though not rapid, easy or inexpensive—methods of analysis have been developed for the determination of erucic acid in foods. For laboratories which have the capability of capillary GLC, this method would appear to be the best and most rapid, since no sorting test would be necessary. However, as an official reference method, the argentation TLC/GLC procedure is preferred because it requires less specialised apparatus, less experimental art, and is more easily defined in documentary form. When it is further developed, HPLC will also play a part in certain laboratories. 

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