Results and Discussion
The dependence of 100% BCS phase selectivity on elution temperature may
be exploited to precisely tailor the separation of all FAME that need to
be quantified for nutritional labeling purposes [10]. Raising the
elution temperature increases the relative retention of FAME with more
DB relative to those with fewer DB. Also, with a lower magnitude, it
increases the retention of FAME with DB in cis configuration
relative to their analogs with same DB but in transconfiguration. PHO and RVO are preferentially analyzed by isothermal
elution at 180°C because it provides suitable resolution oft 13/t 14-18:1 from c 9-18:1, and the elution ofc 11-20:1 in between t 9,c 12,c 15-18:3 and
18:3n-3 [4], but the separation of SCFA is sacrificed. Lowering the
elution temperature to separate SCFA would cause a progressively
stronger overlap between the trans -18:1 and cis -18:1 and
increase retention of c 11-20:1 with respect to 18:3. Golayet al. [7,8] developed a temperature gradient method capable
of simultaneously separating SCFA and 18:1 TFA contained in milk
products, infant formula, and adult/pediatric nutritional formula. In
this study, the applicability of this method to the analysis of PHO and
RVO was tested using a 2:1 blend of PHO and soybean oil. Starting the
temperature program at 60°C provided the separation of SCFA, then the
rapid temperature ramp to 165°C followed by a single ramp to 225°C
provided the elution of trans -18:1 ahead of c 9-18:1 (Fig.
1A). However, the elution of all C18 FAME with a single ramp to 225°C as
prescribed in the original method caused the overlap oft 9,c 12,c 15-18:3 with c 11-20:1. This
inconvenience may be resolved by tailoring the temperature program more
precisely. The addition of a temperature plateau at 184°C (method 1)
rephased the elution of c 11-20:1 in exchange for a minor loss of
separation of t 13/t 14-18:1 from c 9-18:1 (Fig. 1B).
The temperature of this plateau (184°C) may be finely tuned to account
for minor column to column variability: the elution of c 11-20:1
may be moved toward t 9,c 12,c 15-18:3 by increasing
this temperature, or toward 18:3n-3 by lowering it.
If a greater separation of t 13/t 14- and c9-18:1 is
necessary, the temperature of the plateau may be increased 190°C (Fig
1C). This change improved the separation of trans -18:1, but it
caused the partial overlap of t 9,c 12,c 15-18:3 withc 11-20:1. This co-elution may be resolved by adding a negative
temperature gradient to 175°C after the elution of trans -18:1
(Fig. 1D, method 2). If necessary, the time that the column is
maintained at 190°C may be slightly adjusted to optimize the separation
of c 11-20:1 in between t 9,c 12,c 15-18:3 and
18:3n-3 (i.e., c 11-20:1 may be eluted closer tot 9,c 12,c 15-18:3 by increasing the time at 190°C).
After the elution of 18:3n-3 the elution temperature is increased to
225°C to elute the remaining analytes. The retention time of 21:0 was
used as reference point to start the final temperature gradient to
225°C, which provided the elution of all FAME contained in the Supelco
37 mix in 71 minutes. To increase the reproducibility of separations the
samples were injected back-to-back with the column re-equilibration time
set to 5 min, and the column was conditioned 25 min at 225°C prior to
the first injection.
The negative temperature program modification (method 2) was applied to
the analysis of the Supelco 37 FAME mix, a canola oil, a PHO, a
shortening blend (containing PHO and palm oil), and butter (BCR 632A)
(Fig 2, 3, 4). The proposed modifications provided the separation of all
PUFA contained in butter (Fig 4); however, the final ramp may be more
precisely tuned if the resolution of other specific PUFA eluting after
18:3n-3 is necessary. While this adaptation of the Golay et al.method adds some complexity to the temperature program, the resolution
of the overlap between c 11-20:1 andt 9,c 12,c 15-18:3 provides a remarkable gain in
method versatility by providing accurate measurement of all TFA
occurring in PHO and RVO. If the GC is not capable of negative
temperature gradients, or more simplicity is desired, the introduction
of the 184°C plateau is a suitable compromise.