Effect of temperature on the selectivity of sulfonamides and trimethoprim on a carbon clad zirconium dioxide column
S. Giegold1,2, Thorsten Teutenberg1J. Tuerk1, T. K. Kiffmeyer1 , B. Wenclawiak2
1Institute of Energy and Environmental Technology (IUTA), Bliersheimer Straße 60, D-47229 Duisburg, Germany; 2University of Siegen, D-57068 Siegen, Germany
The use of high temperatures in liquid chromatography is paid more and more attention, since it has many advantages compared to conventional HPLC at low temperatures [1-3]. The content of the organic solvent in the mobile phase can be drastically reduced and separations can even be carried out with pure water on a reversed phase column due to a steady decrease of the dielectric constant of water. Altering the eluent temperature also allows a change in the selectivity of the phase system. This is very convenient because eluent temperature can be easily controlled provided that the heating system and the stationary phase are both suitable to generate and
Time (min) Time (min) Time (min)
to withstand high eluent temperatures. In this investigation, the elution behaviour of eight sulfonamides andtrimethoprim was studied on a zirconium dioxide based carbon clad column. Prior to these experiments the
Figure 5 (a to c): Chromatograms of the sulfonamide mixture including trimethoprim at 60 °C (a), 120 °C (b) and 180 °C (c). All other chromatographic parameters except temperature were kept constant. Flow rate of mobile phase: 1mL/min; Detection: UV at 270 nm. The
column was tested for long-term stability.
red arrow highlights the unexpected and unusual movement of trimethoprim which at some point moves backward.
In order to verify the unusual chromatographic behaviour of trimethoprim, each analyte was eluted on the carbon
clad column separately. A van’t Hoff plot for all analytes is given in Figure 6 showing that for all analytes except trimethoprim and AcSDD a linear relationship is obtained. 2.1 System set up For the column stability test (numbers 1 to 10 of Figures 1 to 4) a Shimadzu HPLC system consisting of two LC-
10ADVP pumps, a DGU-14 A degasser, an SIL 10ADVP autosampler and an SPD-M10AVP diode array detector (Shimadzu, Duisburg, Germany) was used. The system was controlled via an SCL-10ADVP controller. For data
acquisition and analysis, the Shimadzu Class VP software (version 6.12 SP3) was used. For all other measurements (numbers 11 to 43 of Figures 1 to 4 and all other data presented in this poster) an
Agilent 1100 HPLC system consisting of a G1312A binary pump, a G1379A degasser, a G1313A autosampler
and a G1315C diode array detector (Agilent, Waldbronn, Germany) was employed. For data acquisition and
analysis, the Agilent ChemStation for 3D LC software (version B01.03) was used.
The elution of the sulfonamides and trimethoprim was carried out on exactly the same column used for the column
stability test consisting of carbon clad zirconium dioxide (ZirChrom-Carb, 150 x 4.6 mm, 3 µm, 300 Å). HPLC
solvents (Merck, Darmstadt, Germany) were of HPLC gradient grade.
A homemade heating system was used for controlling eluent temperature. The heating system was developed for high temperature liquid chromatography and consists of three modules, whích can be independently controlled.
The heating range of this system extends from room temperature to 225 °C with maximum heating rates of
40°C/min. This system can be used for isothermal and temperature programmed operations and is described in
detail elsewhere . To keep the mobile phase in the liquid state, even at high temperatures, a 500 psi back
pressure regulator (GammaAnalysenTechnik, Bremerhaven, Germany) was connected behind the UVD. 2.2 Measurement of analytical parameters at high temperatures
For the column stability test the resolution, asymmetry and retention factors of test solutes were measured and
calculated according to the specified formulae below. Furthermore, the column back-pressure was monitored. The
data is depicted in Figures 1 to 4 (numbers 1 to 10). The column stability test was carried out by exposing the column to a mobile phase of pure deionized water at a temperature of 185 °C and a flow rate of 0.5 mL/min over a Figure 6: Van‘t Hoff plots of sulfonamides and trimethoprim. For all analytes except trimethoprim, linear van‘t Hoff-plots are obtained.
time of 50 hours. After every five hours the column was cooled down and a test mixture containing acetone as a void volume marker, p-cresol, ethylbenzene and nitrobenzene was measured at 60 °C and a flow rate of 1mL/min.
3.3 Measurement of peak areas
After this test period, the performance of the column was permanently monitored over the whole duration of this study when the column temperature exceeded 150 °C.
The next experiment was to determine the peak areas of each analyte in dependence on column temperature at otherwise constant chromatographic conditions. If the flow rate and the wavelength are not changed, the peak
The separation of the sulfonamide mixture as depicted in Figures 5 a to c was carried out at three different
area of a certain analyte should remain constant provided that there is no thermal degradation of the analyte on-
temperatures with an isocratic mobile phase containing 50 % acetonitrile and 50% deionized water.
column. The column temperature was successively raised from 60 °C to 180 °C in increments of 20 °C and the
Van‘t Hoff plots were generated by recording the dependence of the natural logarithm of retention factor of each
peak area was recorded for each analyte at a wavelength of 270 nm. The results are given in Figure 7. It can be
analyte against temperature. The results of these measurements are given in Figure 6.
seen that except for trimethoprim and AcSDZ the peak area for all substances is not constant at different
Finally, the peak area was measured for each analyte depending on the eluent temperature at a constant mobile
temperatures. The peak areas of sulfathiazole and sulfamethoxazole become bigger with increasing temperature.
phase composition, which is shown in Figure 7.
Another phenomenon is observed for the compounds sulfadiazine, sulfamethazine, sulfamerazine, AcSDD and AcSMR where peak areas pass through a minimum at around 100 °C to 120 °C and reach the original level at
higher temperatures. Taking the corresponding chromatograms (data not shown here) into consideration we found
out that for these substances excessive tailing occurrs over this temperature range leading to very broad peaks.
This could be due to a pH-shift of the mobile phase induced by temperature so that an on-column change of the
analytes‘ molecular structure takes place. If the temperature is raised further, the tailing is reduced and the peaks
ω1 = peak width at half height of first peak
ω2 = peak width at half height of second peak
Results and Discussion 3.1 Column stability test
The numbers 1 to 10 in Figures 1 to 4 depict the progression of the selected parameters for the monitoring of the column stability. It can be clearly seen that the column pressure is increasing after the fourth test measurement,
reaching a maximum of approx. 250 bar after the end of the column test study. This indicates that either a
continuous clogging of the column or a break down of the stationary phase takes place. Additionally, both the
retention factors and resolution of test analytes decrease which might suggest that there is a constant change of
the surface properties of the stationary phase.
After the stability test the column was thoroughly regenerated and stored for six months without further use.
Afterwards, the column was used again for all subsequent experiments and the same test mixture as for the
column stability procedure was run after each day the column temperature exceeded 150 °C (data points 11 to 43
in Figures 1 to 4). As can be seen the column pressure decreases and reaches the same level as was noticed before the column stability test. The other parameters do not change as much but remain rather constant over
time, suggesting that there is no further alteration of column properties during the complete duration of this study. Monitoring of total column pressure Monitoring of retention factors ethoprim ethazine ethoxazole Sulfadiazine Sulfathiazole number of measurements Figure 7: Peak areas of sulfonamides and trimethoprim at different column temperatures and constant chromatographic conditions. number of measurements p-Cresol Ethylbenzene Nitrobenzene Figure 1: Monitoring of column pressure. Figure 2: Monitoring of retention factors of test solutes. Conclusions Monitoring of resolution Monitoring of asymmetry
The column long-term stability test shows that a carbon clad zirconium dioxide column can be used over a very long period without a breakdown of the stationary phase. This is an important pre-requisite for establishing high-
temperature liquid chromatography as a routine method. Furthermore, analyte stability was estimated by using a
UV-detector at a fixed wavelength and measuring peak areas at different temperatures but constant
chromatographic conditions. The results indicate that a thermal degradation of analytes on-column does not
occur, but further investigations are clearly needed. We were able to show that one of the most important aspects
of using temperature as an optimization tool in liquid chromatography is that the selectivity of the phase system
can be controlled by simply changing eluent temperature. number of measurements number of measurements Acetone/p-Cresole p-Cresole/Ethylbenzene Acknowledgements Ethylbenzene/Nitrobenzene p-Cresol Ethylbenzene Nitrobenzene Figure 3: Monitoring of resolution of test solutes. Figure 4: Monitoring of asymmetry of test solutes.
The financial support by the German Federation of Industrial Cooperative Associations „Otto von Guericke“ e. V. (Pro Inno II, project number KF0087403FK5) is gratefully acknowledged.
3.2 Elution behaviour of a sulfonamide mixture
The Agilent 1100 HPLC system was provided by Scientific Instruments Manufacturer GmbH (SIM) for the duration of this study and is also gratefully acknowledged.
The next paragraph deals with the elution behaviour of the sulfonamide mixture on the carbon clad zirconium dioxide column. A DryLab optimization was attempted by varying column temperature and solvent composition.
Four experimental runs were carried out at two temperatures (60 °C and 180 °C) and two solvent gradient run times (30 minutes and 90 minutes). Unfortunately, peak tracking was not possible by using either peak area or
Greibrokk, T., Andersen, T.; “High-temperature liquid chromatography”; Journal of Chromatography A 2003, 1000, 743-755
UV-spectra of analytes, because the total sum of all peak areas was not constant at different temperatures and
Fields, S. M., Ye, C. Q., Zhang, D. D., Branch, B. R., Zhang, X. J., Okafo, N.; “Superheated water as eluent in high-temperature
the UV-spectra for the sulfonamides are quite similar.
high-performance liquid chromatographic separations of steroids on a polymer-coated zirconia column”; Journal of Chromatography A 2001, 913, 197-204
Thus, the temperature was raised in increments of 10 °C to track peak movements and monitor the change of the
Zhu, P. L., Dolan, J. W., Snyder, L. R.; “Combined use of temperature and solvent strength in reversed-phase gradient elution.
elution order. At some intermediate temperature, the interpretation of the chromatogram becomes very difficult
II. Comparing selectivity for different samples and systems”; Journal of Chromatography A 1996, 756, 41-50
which is underlined when looking at Figure 5 a to c. There, three chromatograms recorded at different
Teutenberg, T., Goetze, H.-J., Tuerk, J., Ploeger, J., Kiffmeyer, T.K., Schmidt, K.G., Kohorst, W. gr., Rohe, T., Jansen, H.-D.,
temperatures but otherwise constant chromatographic conditions are shown. The red arrow highlights the
Weber, H.; “Development and application of a specially designed heating system for temperature-programmed high-performance
unexpected and unusual movement of trimethoprim which at some point moves backward.
liquid chromatography using subcritical water as the mobile phase”, Journal of Chromatography A 2006, 1114, 89-96
Building reliable normal reference range, As an interpretation guide for clinical practices Clinical research team of Medicore Co., Ltd. Since Medicore Co., Ltd. completed the development of function of autonomic nervous system, which the SA-2000, the first HRV analyzer model in 2,000, we, accelerated various HRV studies to find out the by organizing the research team from neuropsychiatr
blockade ( 5 ). Other opportunistic 7. Smolen JS, Beaulieu A, Rubbert-Roth A, of deaths, especially among remote infections that have been reported in et al. Effect of interleukin-6 receptor in-hibition with tocilizumab in patients with jirovecii pneumonia, herpes zoster, rheumatoid arthritis (OPTION study): a have been reported ( 1 ), with even asymptomatic Mycobacterium avium– domised