Chemistry Reference
In-Depth Information
OH
CH
3
CH
3
OH
CH
3
CH
3
H
3
C
CH
3
H
3
C
Thymol
(IS)
Propofol
(a)
(b)
0
5
10
15
20
25
30
Retention time (min)
Figure 20.5
Chemical structures and chromatograms of propofol and thymol (IS) obtained from a
drug-administrated monkey serum (a), and a blank one (b), respectively. HPLC condition: column,
P(NIPAAm-
) modified column (4.6 mm i.d. × 150 mm); column temperature, 40°C; mobile phase,
H
2
O; flow rate, 1.0 ml min
−1
; fluorescent detection, excitation wavelength 267 nm, emission wavelength 310 nm.
co
-BMA5
%
20.5.2
Contraceptive drugs analysis using temperature gradient chromatography
In the isocratic elution of biological fluid containing solutes with a wide range of polarity, it is sometimes
difficult to achieve the desired resolution within a reasonable time. It may be necessary to use gradient
elution, where volumes of an organic solvent, composition of mobile phase, or other property of solvent, such
as pH or ionic strength are changed during the separation. Little attention has been paid to program the
temperature changes during chromatographic analysis. Because HPLC can use the solvent strength as a
functional time (solvent gradient), it has a much greater potential for changing the retention and selectivity
than dose temperature change. On an HPLC column packed with temperature-responsive polymer modified
silica, temperature programming can be used in lieu of a gradient solvent [33, 34]. By using a temperature-
responsive polymer modified HPLC stationary phase, the gradient elution-like effect can be achieved with a
single mobile phase by only controlling column temperature.
As an example, the simultaneous analysis of two kinds of oral contraceptive drugs was carried out using a
temperature step gradient. Figure 20.6 shows chromatograms of levonorgestrel and ethinylestradiol, which
were obtained at 10°C and 40°C, respectively. For levonorgestrel, with lower hydrophobicity, the retention
time hardly changed at both temperatures. On the other hand, for ethinylestradiol, with a higher hydrophobicity,
the retention time was increased too much, and influenced the hydrophobic interaction at 40°C. To move
strongly retained components of the contraceptive drugs and to optimize the analysis, a temperature-
programming technique was used. With a single mobile phase of water and by controlling the external
temperature from 40°C to 10°C, the analytical time was dramatically reduced. The excellent resolution of the
analyte was accomplished using a temperature step gradient mode and developed method could be applied to
