CHROMATOGRAPHY e MASS SPECTROMETRY
5. RETENTION BEHAVIOR
5.1 FATTY ACIDS AND THEIR DERIVATIVES
explained by several temperature-related factors (Adlof, 2007): (1) solubility of the sample in the mobile phase, (2) solubility of acetonitrile in hexane, (3) changes in the flexibility/3D configuration of the analyte or stationary phase. Another possible explanation is that the number of unsaturated mol- ecules coordinated in the complex with the silver ion depends on the temperature, whereas only one unsaturated molecule forms the complex at 25C in comparison to two coordinated molecules at 0C (Winstein and Lucas, 1938). Temperature gradients (Adlof, 2007) could be used for the optimization of chromatographic resolution of complex FAME or TG, instead of the more common solvent compo- sition gradient, but in our best knowledge this idea has not yet been used in any published paper.
4. SILVER-ION HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
IN TWO-DIMENSIONAL HIGH-PERFORMANCE LIQUID
most cases they are separated as FAME or other derivatives (Nikolova-Damyanova, 2009). In general, retention times of FAs in Ag-HPLC are determined by the DB number, theircis/transconfiguration, and the positions of DB in fatty acyl chains. There is no direct proportionality between retention times of FA and DB number, but in general, retention times of FAs increase with the increasing number of DB. For FIGURE 4.2
Off-line two-dimensional chromatograms of blackcurrant oil using nonaqueous reversed-phase (NARP) in the first dimension and silver-ion mode in the second dimension after fraction collection each minute: (A) dot plot with the peak identification, double bond (DB), and equivalent carbon number (ECN) labeling, (B) contour plot showing peak intensities.Ag-HPLC, silver-ion high-performance liquid chromatography.
Reprinted with permission from Holcapek, M., Velı´nska´, H., Lı´sa, M.,Cesla, P., 2009. Orthogonality of silver-ion and non-aqueous reversed-phase HPLC/MS in the analysis of complex natural mixtures of triacylglycerols. J. Sep. Sci. 32, 3672e3680.
example, D9,12e18:2 is retained about 3 times longer than a corresponding D9e18:1 homolog, D9,12,15e18:3 is retained 2.5 times more thanD9,12e18:1, andD5,8,11,14,17e20:5 is retained 1.5 times more thanD5,8,11,14e20:4 (Nikolova-Damyanova et al., 1992).
The complex of silver ions withcis-DB is stronger than withtrans-isomers, and thereforecis-FA isomers are retained more strongly thantrans-isomers. The retention behavior of polyunsaturated FAs containing trans-DB is given by the number and positions of trans-DB in the fatty acyl chain.
Retention times of FAs decrease with the increasing number oftrans-DB (Toschi et al., 1993; Adlof, 1994; Adlof and Lamm, 1998; Christie and Breckenridge, 1989; Phillips et al., 1997). The retention order of 18:3 FA isomers as methyl (Adlof, 1994) and phenacyl esters (Juaneda et al., 1994) is the following (Fig. 4.3):
D9t,12t,15t<<D9t,12c,15t<D9c,12t,15t<D9t,12t,15c<<D9c,12t,15c
<D9t,12c,15c<D9c,12c,15t<<D9c,12c,15c.
The stability of FA complexes with silver ions during the chromatographic process is also influ- enced by their position(s) in the fatty acyl chain and the number of methylene units between two DB.
Conjugated dienes with various configurations and positions of DB in the fatty acyl chain are always eluted prior to cis-monoenes (Adlof and Lamm, 1998; Momchilova and Nikolova-Damyanova, 2000a,b,c). FAs with isolated DB are retained more strongly than FAs with methylene-interrupted DB
90 min 60
30 0
inj
1 3 2 SF
4
5 6 7
8
FIGURE 4.3
Separation of fatty acids phenacyl esters of C18:3cis/transgeometrical isomers by silver-ion high-performance liquid chromatography. Peak annotation: SFdsolvent front, 1dD9t,12t,15t, 2dD9t,12c,15t, 3dD9c,12t,15t, 4dD9t,12t,15c, 5dD9c,12t,15c, 6dD9t,12c,15c, 7dD9c,12c,15t, 8dD9c,12c,15c.
Reprinted with permission from Juaneda, P., Sebedio, J.L., Christie, W.W., 1994. Complete separation of the geometrical-isomers of linolenic acid by high-performance liquidechromatography with a silver ion column. HRCeJ. High Res. Chromatog. 17, 321e324.
(Nikolova-Damyanova et al., 1992). The series of positional isomers of C18:1 and C18:2 (Nikolova- Damyanova et al., 1992) or conjugated C18:2 (Delmonte et al., 2005) has been studied and their relative retention factors (k) were plotted against DB positions. The retention behavior of all series of isomers is similar, giving sinusoidal curves ofkvalues of individual isomers. In general, FAs with the first DB at C5eC7are the most strongly retained.
Although the fatty acyl chain length is supposed to have no effect in Ag-HPLC, FAs with longer chains are eluted slightly earlier than FAs with short chains (Juaneda et al., 1994). This phenomenon can be ascribed either to the normal-phase effect occurring in silica-based silver-loaded columns (Adlof, 1997) or to the lower complex stability for longer alkyl chains (Lucas et al., 1943; Winstein and Lucas, 1938). Similar behavior is reported for TG (Lı´sa et al., 2009a,b; Holcapek et al., 2010). The retention of various FA derivatives has been tested, i.e., short chain alkenyl esters (Nikolova- Damyanova et al., 1995a,b), benzyl and phenacyl esters (Nikolova-Damyanova et al., 1996), phenethyl, phenacyl and p-methoxyphenacyl esters (Momchilova et al., 1998), and 2-naphthacyl, 9-anthrylmethyl and 2-naphthylmethyl esters (Momchilova and Nikolova-Damyanova, 2000a,b,c).
The elution order is the same for all derivatives, but the type of derivative affects the selectivity, mainly for positional isomers of FAs.p-Methoxyphenacyl esters provide the best selectivity for positional isomers of FAs, enabling separation according to the position of DB and the chain length. Changing the composition of the mobile phase can reverse the retention order, as demonstrated by the example of p-methoxyphenacyl esters, when 2-propanol was replaced by acetonitrile in dichloromethane-based mobile phases (Momchilova and Nikolova-Damyanova, 2000a,b,c). Conjugated linoleic acid isomers have been separated by Ag-HPLC and further structurally characterized by the inline combination of ozonolysis reaction and MS study of their fragmentation behavior (Sun et al., 2013).