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Thin Layer Chromatography
The samples were applied to the plates as bands by means of an automated sample
applicator (Linomat IV, Camag, Muttenz, Switzerland): volume 5 μL, bandwidth
7 mm, distance between the middle of the bands 15 mm, delivery rate 1 μL/10 s.
Ascending development was performed at room temperature in a CAMAG doubletrough
chamber without previous saturation. Plates were developed to a distance
of 70 mm with 0.05 M oxalic acid/methanol = 0.80:0.20 as a mobile phase. After
development, the plates were air dried and the chromatograms were visualised
under UV light at = 254 nm and = 366 nm.
Video densitometry was performed with a highly sensitive 3CCD colour video camera
HV-C20 (Hitachi Denshi, Japan). The parameters of video densitometry were as
follows: close-up lens +2 Dpt, zoom lens 9.5 mm, integration period-exposure time 1
frame (= 20 ms) at 254 nm and 15 frames at 366 nm, frame accumulation-off mode,
aperture (F-stop number) 2.0. Imaging, processing and archiving were controlled
via VideoStore2 2.30 documentation software. The extended version of Camag
VideoScan 1.01 software was used for quantitative evaluation of the stored TLC
chromatograms. For quantification purposes, detection was carried out at 366 nm,
and TMP was measured at 254 nm. Method was validated for selectivity, linearity,
limit of detection (LOD), limit of quantification (LOQ), precision and recovery.
RESULTS AND DISCUSSION
Optimization of the mobile phase was performed by systematic variation of the
composition of the basic mobile phase (methanol and 0.05 M oxalic acid). The quality
of chromatographic separation was evaluated by chromatographic response function
on the basis of the predicted R F
values. In this work, the MRF criteria (De Spiegeleer
and De Moerloose, 1987) was used as the separation criterion, and maximum value
of MRF function was found using the genetic algorithm approach (Babic et al., 2005).
Optimum mobile phase composition obtained with these parameter settings was
0.05 M oxalic acid/methanol = 0.80:0.20. The obtained results indicate excellent
agreement between experimental and calculated retention factors (R F
-values), as
well as very good overall resolution (Table 1 and Figure 1).
Table 1:
Experimental and calculated R F
values of pharmaceuticals at
optimal mobile phase composition
Pharmaceutical Calculated Experimental
NOR 0.16 0.15
OTC 0.31 0.31
TMP 0.38 0.37
SDIAZ 0.45 0.44
SMETH 0.56 0.54
PGP 0.62 0.65
SGUA 0.71 0.71
For qualitative purposes, the method was evaluated by taking into account the
precision in the R F
-value and selectivity. Comparing the chromatogram of extracts
with the chromatogram of standard solution (Figure 1a and b) a coincidence of
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