Chemistry Reference
In-Depth Information
influenced by a difference in the construction of PNIPAAm modified surfaces due to the temperature [29]. It
is considered that it maintains high mobility by fixing the PNIPAAm molecule at the end on the modified
terminal, and quickly responds to any temperature change.
20.4
Temperature-responsive chromatography for green analytical methods
Using a column packed with PNIPAAm-modified silica, the HPLC analysis of steroids was carried out with
only water as a mobile phase by changing the column temperature. Typical chromatograms of steroids
analysis are shown in Figure 20.3 at a variety of temperatures (10
40°C), and extensions of retention times
were observed with increasing temperature. With increasing temperature, an increased interaction between
the solute components and PNIPAAm-grafted surfaces of the stationary phase was also observed [11, 17]. It
was considered that a hydrophobic interaction exists between the steroids and the PNIPAAm. In contrast, in
the conventional RPLC system, the opposite chromatographic behavior of the shortened retention times was
observed with increasing temperature due to the solvent viscosity. A temperature-dependent resolution was
achieved using only water as a mobile phase on the PNIPAAm-modified column. A drastic and reversible
change in the physicochemical property of a PNIPAAm-terminally grafted silica surface from hydrophilic
to hydrophobic is caused from a rapid alternation in the polymer hydration state around its transition
temperature. The retention strength of steroids exhibits a linear relationship with the log
P
values (the
partition coefficients in a 1-octanol/water system). The log
P
values for the steroids were 1.61 of
hydrocortisone, 1.62 of prednisolone, 1.83 of dexamethasone, 2.30 of hydrocortisone acetate and 3.32 of
testosterone. The more hydrophobic steroids show longer retention times. These results indicate that a strong
hydrophobic interaction is the primary driving force for partitioning steroids into a PNIPAAm-modified
surface. Since the phase transition of PNIPAAm results from the stability of the hydrophobic groups along
the polymer chain in aqueous media, the LCST of the polymer should decrease with increasing polymer
hydrophobicity.
A plot of the retention factor (ln
k
) versus the reciprocal temperature (1/
T
) normally shows a linear
regression, with the slope representing the enthalpy change involved for the retention reaction. Figure 20.4
shows a plot for the steroids on the PNIPAAm-modified column. The slope of the plots on the PNIPAAm-
modified column is negative, and it is opposite to that observed in common chromatography. This provides
evidence that the interaction between steroids and the temperature-responsive surface becomes stronger at
elevated temperature. Additionally, linearity in the plots is commonly observed for commercially available
RPLC columns under the standard analytical condition with narrow temperature ranges. On the PNIPAAm-
modified column, however, a deviation from linearity was found between ln
k
values and 1/
T
. The slope of the
plots of each analyte on the PNIPAAm-modified column changed markedly at the LCST boundary. This
phenomenon corresponds to a phase transition of the polymer modified on the surface, and it is indicated that
the retention mechanism can be controlled by changing temperature.
∼
20.5
Biological analysis by temperature-responsive chromatography
Therapeutic drug monitoring (TDM) has become a well-established clinical specialty [30]. Though the
immunoassay auto-analyzer is well utilized for TDM, it is generally expensive, and subject to potential cross-
reactivity and interference by drug metabolites and endogenous components. Furthermore, measurable drugs
depended on the commercially available immunoassay kit. On the other hand, the HPLC method has the
advantage of specificity and the application. In this background, HPLC, especially RPLC analysis is widely
used for the simultaneous determination of a variety of drugs and accurate measurements. As previously
