CHROMATOGRAPHY e MASS SPECTROMETRY
5. RETENTION BEHAVIOR
5.2 TRIACYLGLYCEROLS
(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).
where symbols are defined as Sa for saturated, Mo for monounsaturated, D for diunsaturated, and T for triunsaturated FAs. Positional isomers are not distinguished. To avoid the confusion of using the same symbol in two meanings (i.e., S for both stearic acid and saturated, M for both myristic and mono- unsaturated), we use the symbol Sa for saturated and Mo for monounsaturated unlike the annotation used in most previous works. The retention behavior observed in our experiments in hexanee2- propanoleacetonitrile mobile phases (Lı´sa et al., 2009a,b; Holcapek et al., 2009, 2010) is mostly in a good agreement with published data with few exceptions. In mobile phases consisting of hexaneeacetonitrile (Adlof, 1995; Dugo et al., 2004), heptaneeacetonitrile (Macher and Holmqvist, 2001), or hexanee2-propanoleacetonitrile (Lı´sa et al., 2009a,b; Holcapek et al., 2009, 2010), the retention order of all TG follows the rule that an increasing number of DB means longer retention, while one exception is reported above for DMoMo (4)<DDSa (4)SaSaT (3). The explanation of changed retention patterns probably lies in the different mobile-phase composition, which affects the retention mechanism. The alkyl chain length has a small effect on the retention, but it does not change the retention order.Fig. 4.4highlights this effect for TG with saturated FAs from C7:0 to C22:0, where TG with longer chains elute earlier (Holcapek et al., 2010). For TG of the LLSa type found in sun- flower oil, the retention decrease in the LLSa series isw0.4 min per two methylene units (Lı´sa et al., 2009a,b). The analysis of a randomized mixture containing six different TGs (PPP, SSS, OOO, LLL, LnLnLn, and AAA) and of numerous plant oils (Lı´sa et al., 2009a,b; Holcapek et al., 2009, 2010)
FIGURE 4.4
Ag-HPLC/APCI-MS chromatogram of monoacid triacylglycerols standards. Numbers correspond to the double bonds number. Inset zoom shows the resolution of saturated TG from C7:0 to C22:0. Concentrations of all standards are identical except for C16:0, C18:0, and C20:0 with doubled concentrations.Ag-HPLC, silver-ion high-performance liquid chromatography;APCI, atmospheric pressure chemical ionization;MS, mass spectrometry.
Reprinted with permission from Holcapek, M., Dvor a´kova´, H., Lı´sa, M., Giro´n, A.J., Sandra, P., Cvacka, J., 2010. Regioisomeric analysis of triacylglycerols using silver-ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry:
comparison of five different mass analyzers. J. Chromatogr. A 1217, 8186e8194.
enabled generalization of the retention order of TG, including positional isomers, valid in hexanee2- propanoleacetonitrile mobile phases (Fig. 4.5):
SaSaSa (DB¼0)<<SaMoSa (1)<SaSaMo (1)<<SaDSa (2)<SaSaD (2)SaMoMo (2)<
MoSaMo (2)<<SaTSa (3)SaDMo (3)SaMoD (3)<SaSaT (3)MoSaD (3) MoMoMo (3)<<SaDD (4)<SaTMo (4)MoDMo (4)DSaD (4)MoMoD (4)SaMoT (4)<MoSaT (4)<<SaTD (5)MoDD (5)DMoD (5)SaDT (5)MoTMo (5)MoMoT (5)DSaT (5)<<DDD (6)<MoTD (6)MoDT (6)DMoT (6)SaTT (6)<TSaT (6)<<
DTD (7)DDT (7)<MoTT (7)<TMoT (7)<<DTT (8)TDT (8)<<TTT (9).
We believe that this order is fully applicable for similar mobile phases containing acetonitrile (or propionitrile) in hexane (or heptane) with or without the addition of 2-propanol.
The randomized mixture containing monoacid TG differing only in the geometry of their DB (oleic acid,D9c-C18:1, O vs. elaidic acid,D9t-C18:1, E) shows (Holcapek et al., 2009) that thesecis/trans regioisomers cannot be separated in NARP mode, but the partial separation of regioisomers and the full separation of other isobaric TG is achieved with Ag-HPLC/MS (Fig. 4.6) with the following retention order:
EEE<<EOE<OEE<<OOE<OEO<<OOO.
This retention order is identical to that already published (Dobson et al., 1995), but additionally with the partial resolution of regioisomers. Physicochemical properties of trans-FAs are closer to FIGURE 4.5
Ag-HPLC/APCI-MS chromatogram of the randomized mixture of PPP, SSS, OOO, LLL, LnLnLn, and AAA.
Peak annotation:D, diunsaturated;DB, double bond number;Mo, monounsaturated;Sa, saturated;T, triunsatu- rated.Ag-HPLC, silver-ion high-performance liquid chromatography;APCI, atmospheric pressure chemical ionization;MS, mass spectrometry.
Redrawn 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.
saturated FAs because of the linear arrangement of alkyl chains containingtrans-DB. Therefore, the retention of TG containingtrans-FAs is slightly higher in NARP mode, but significantly lower in Ag-HPLC, compared tocis-FAs. This effect is more pronounced for outersn-1/3 positions, because of better steric availability.
The effect of DB position in TG can be illustrated by the example of linolenic (Ln,D9,12,15-C18:
3) versus gamma-linolenic (gLn, D6,9,12-C18:3) acids. LnLnLn has higher retention than gLngLngLn in most mobile phases [acetoneeacetonitrile (Leskinen et al., 2008), chlorinated solvents (Laakso and Voutilainen, 1996)], whereas the reverse order is reported for the hexanee2- propanoleacetonitrile mobile phase (Holcapek et al., 2010). This example shows that the retention mechanism in Ag-HPLC is rather complex and not fully understood so far. If only one FA in a TG molecule differs in the DB position, then only peak broadening is observed because of small differ- ences in retention times of isomers differing in the DB position only. Unlike FAs and their derivatives, the systematic study of the retention behavior of TG containing unusual DB positions is still missing.
Neff et al. (1994)have shown that TG containing a triple bond (crepenynic,D9,12triple-C18:2) elute later in comparison to analogous TG containing DB at the same position (linoleic,D9,12-C18:2).