Silver-ion HPLC

Recently, Mondello et al. [13] used silver ion HPLC to develop a comprehensive LC (LCxLC) system. The results obtained on rice oil sample led to the separation of a number of isomers that cannot be separated by the usual monodimensional HPLC. The use of APCI-MS as a detection system provided a more affordable method for the identification of different isomers. 

Additional analytical methods are appropriate when a more complete characterization of the CLA isomers in biological samples is required. Most often, a combination of GC and silver ion-HPLC is used and permits excellent separation and identification of positional and geometrical isomers of CLA (see Adlof, 2003, and Kramer et al., 2004, for detailed reviews of this approach). In addition, the use of gas chromatography-mass spectrometry (GC-MS) has become increasingly popular and represents a very powerful technique for identification of the position of double bonds in fatty acids (see Dobson, 2003), and the orientation of those bonds in CLA isomers (Michaud et al., 2003). 

The product quality was analysed using silver-ion-HPLC and gradient elution [14]. This method was developed from an isocratic method [15]. The kind (cis/ trans) and the amount of the FAME was determined. From these data the iodine value (IV) was calculated. 

Different methods of analysis will give different and often incomplete information about such a mixture. GC analysis will separate molecular species by carbon number (sum of fatty acid chain lengths). Silver-ion HPLC will separate by number of double bonds. Stereospecific analysis measures the proportions of fatty acids at the sn-1, sn-2, and sn-3 positions, but it does not detect individual molecular species. 

In addition to normal- and reversed-phase chromatography, silver ion HPLC, RP ion-pair HPLC, chiral separation, and supercritical fluid chromatography have been used for analysis of lipids [3]. In silver-ion HPLC, the counterion of an ion-exchange column such as sulfonate is exchanged with silver ions. Only the degree of unsaturation in the lipid molecule determines retention. In RP ion-pair HPLC, different ion-pairing agents, such... 

The saturated CFAM, isolated by silver-ion HPLC from high-oleate and normal sunflower oils used in a small-seale frying operation of potato strips (7,23), were shown by GC to be composed of two different groups of CFAM (Fig. 6). The first group (q-x) was presumably derived from oleate and constituted a greater proportion of the total CFAM in high-oleate ( 67%) compared with normal ( 25%) sunflower... 

As methyl esters, GC separation from other fatty acids can be achieved on polar capillary columns (Fig. 5.3 Christie, Brechany and Shukla, 1989) and would probably be adequate on non-polar columns, as this is possible for dimethyloxazoline (DMOX) derivatives (Zhang et al, 1989). If necessary, prior isolation of cyclic monoenoic and dienoic fractions, separated from straight-chain saturates, monoenes and dienes, may be obtained by means of silver-ion HPLC (Christie, Brechany and Shukla, 1989). In this way, minor components were concentrated for subsequent GC-MS analysis as the pico-linyl (3-hydroxymethylpyridinyl) esters, and the possibility of inadequate resolution from straight-chain esters on non-polar columns, necessary for eluting these relatively involatile derivatives, was avoided. Presumably the use of modern high-temperature polar phases for GC-MS would eliminate possible resolution problems with picolinyl esters. 

Until recently, a complete analysis of CFAMs from heated oils was a daunting task first, because of the complexities of the mixtures and, second, because of the difficulties in obtaining full structural information, especially with respect to double bond positions. However, recently this has been made possible by combining fractionation of a CFAM mixture (obtained by urea adduction) by silver-ion HPLC with GC-MS analysis of the picolinyl esters or DMOX derivatives of the fractions (Fig. 5.10 Christie et al, 1993 ... 

Total CFAM phenacyl esters Silver-Ion HPLC CFAM phenacyl ester fractions... 

Delmonte, P, M.P. Yurawecz, M.M. Mossoba, C. Cruz-Hernandez, andJ.K.G. Kramer. Improved Identification of Conjugated Linoleic Acid Isomers Using Silver-Ion HPLC Separations,/..(4CMC/wremrfr. 87 563—568 (2004). 

Yurawecz, M.E, and K.M. Morehouse. Silver-Ion HPLC of Conjugated Linoleic Acid... 

Improved Identification of Conjugated Linoleic Acid Isomers Using Silver-ion HPLC Separations,/AQ(4C7 r. 87, 563-568 (2004). 

Initial separation of commercial CLA can be achieved by GC but, depending on the sample s complexity, other chromatographic methodologies such as selected-ion recording (SIR) GC-MS with DMOX derivatives or silver-ion HPLC might be necessary to achieve full identification and quantification of the isomers. 

Much better resolution is possible by silver-ion HPLC using columns packed with ion-exchange media loaded with silver ions. Indeed, positional and geometri-... 

Silver ion HPLC appears to be the best method for separation and quantification of positional and geometrical isomers of CLA when a definitive analysis is required of a particular sample. Conmiercial CLA preparations can be analyzed without difficulty. However, natural CLA isomers may require some concentration by appropriate methods to ensure that sufficient material is available for detection of minor components (see below). 

GC-MS of DMOX derivatives has been utilized in identifying the isomers in commercial CLA samples. Several commercial CLA samples were found to vary widely in CLA composition (6). In some samples, the only positional isomers were 9,11- and 10,12-18 2 (9c,llf and 10f,12c isomers were major components with less of the corresponding c,c and t,t isomers), whereas in others 8,10- and 11,13-18 2 (8f,10c and llc,13f isomers with less of the corresponding c,c and 1,1 isomers) were additionally present. Under harsher conditions, minor amounts of other isomers (e.g., ll,9l and l2l,lAl) have been observed by HPLC and GC (52), but their presence was not confirmed by GC-MS. The identities of CLA peaks separated by silver-ion HPLC in a commercial CLA sample were also confirmed by GC-MS (4). 

The identities of CLA isomers derived from heat-treated vegetable oils, and isolated by silver-ion HPLC, were confirmed using MTAD adducts (75). The trans,trans isomers, 9t,llt- and 10f,12i-18 2, were major componoits. Fatty acid mixtures containing natural CLA appear not to have been analyzed as the MTAD adducts by GC-MS. It has been observed that MTAD may react with methylene-interrupted uusaturat-ed fatty acids if conditions are too harsh (73), and therefore further investigation might be warranted to assess the applicability to natural CLA samples, bearing in mind that CLA, compared with other fatty acids, is at low levels in such samples. 

In a number of other studies, GC-MS of DMOX derivatives has been utilized to determine the CLA isomer distribution from a variety of sources. The structures of pure isomers of 9c,llt-18 2 and 10f,12c-18 2, isolated by crystallization of a CLA mixture prepared by alkali-isomerization of linoleate, were confirmed (67). The presence of 9c,llt-18 2 was established in chocolate (49). In conjunction with GC-FTIR, all possible geometrical isomers of 9,11-18 2 (c,i > t,t > c,c and t,c) were detected in human adipose tissue (10). In dehydrated castor oil, although the 9,11 isomers (c/i, c,c and t,t) appeared to be the most abundant, 7,9- and 8,10-18 2 (c/t and but not c,c) were also detected with the aid of SPA (46). The presence of It, 9c-18 2 (as well as lower levels of 7c,9c-, lt,9t- and possibly 7c,9f-18 2) was confirmed in cow s milk, cheese, beef, and human milk and adipose tissue (9). Together with silver-ion HPLC, the isomer distribution in different tissues of pigs fed commercial CLA was determined (2). The CLA content of lactic acid bacteria (44), and the nature of the CLA isomers formed as a result of add-catalyzed methylation of allylic hydroxy oleates (secondary hpid autoxidation products) (47) were also established. 

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