16 Other Considerations
There are other practical considerations when dealing with elevated temperatures, in particular most detectors will be affected by the elevated temperatures of the mobile phase. It is therefore important to ensure that the solvent temperature when it reaches the detector is compatible with that detector. This invariably means that the temperature needs to be reduced to room temperature to ensure that the detector performs optimally [31-33]. However, as reported by Pereira [26], raising the temperature of the mobile phase can be used to improve the sensitivity of the detector system, in this case a mass spectrometer.
Another consideration when running at temperatures above 100°C with an aqueous mobile phase, is the phase transition that can occur within the column. Bubbling within the column can cause bed movement which will deteriorate the separation unless suitable precautions are taken. To avoid a phase transition occurring in the column, it is necessary to apply a pressure at the exit of the column; this can be achieved using a linear restrictor which has an associated pressure drop of 40 bars [34] which ensures that even at 200°C, the eluant will remain in a liquid state.
Equation 5. Variatiation of retention factor to temperature
One of the advantages of just using a single mobile phase component, and making the chromatography greener is that the method development can become a single variable, temperature. Since it is relatively easy to alter the temperature this becomes an ideal scenario for separation scientists. The example shown in Figure 6 demonstrates that organic solvents can be completely replaced to ensure green chromatography, simply by raising the temperature.
In
this example a comparable separation of 6 compounds is achieved, with a slight improvement in peak shape when using a green mobile phase.
Figure 5: Effect of varying the temperature on the retention time. Note that that the elution order changes with temperature.
Figure 4a. Chromatogram obtained from running an isothermal separation at an optimal temperature of 124°C: a = hydroxy-O-glucuronide, c = hydroxyl, d = hydroxyl, e = di-glucuronide, f = parent AZD5438, b = hydroxy-O-glucuronide and g = hydroxyl. Note no observation of e.
Figure 6: An example of an isothermal separation obtained on a Hypercarb 100 x 4.6mm 5µm, using two temperatures, separating cytosine (1), uracil (2), thymine (3), hypoxanthine (4), guanine (5), xanthine (6).
a. Temperature 50°C flow rate 0.8 mLs/min, mobile phase: water + 0.1% formic acid / ACN (85/15 v/v) b. Temperature 190°C , flow rate 2 mLs/min., mobile phase: water.
Gradient Studies
As with multi-solvent systems, an optimal time based separation is obtained when an elution strength gradient is used [33]. Using only water as the mobile phase, temperature programming can be used instead of solvent programming to alter the elution strength of the mobile phase. Figure 7 demonstrates how a temperature gradient can be applied to the successful separation of a series of purines and pyrimidines on a porous graphitic carbon column and only using water as the mobile phase.
Figure 4b. Chromatogram obtained from using the final thermal gradient. a = hydroxy-O-glucuronide, c = hydroxyl, d = hydroxyl, e = di-glucuronide, f = parent AZD5438, b = hydroxy-O-glucuronide and g = hydroxyl.
Isothermal Studies
One of the interesting aspects of using temperature as an active variable in method development is that the selectivity of the chromatographic separation can change as the temperature changes, which is shown in Figure 5. In the example given here the mobile phase is primarily water, with 0.1% formic acid added. This figure is based on the van’t Hoff equation (Equation 5) which relates the retention time of a component to the temperature. There is an assumption that the retention mechanism is consistent at different temperatures to obtain linearity, however non-linearity would indicate a change in the retention mechanism.
There are many other examples of where green chromatography has been successfully applied [34-38] either on a porous graphitic carbon column or on one of the columns mentioned previously, either isothermally or utilising a thermal gradient to ensure optimisation of the analysis time. These examples range from pharmaceutical analysis to petrochemical samples and demonstrate the wide applicability of the technique.
Conclusion
We have shown in this article that performing separations at elevated temperatures can be beneficial. In an environment where the use of organic solvents is increasing within separation science, the use of water coupled to temperature will improve the green credentials of this particular science. To progress this area of chromatography further, a wider range of thermally stable columns and a greater awareness of the available column oven technology needs to be developed. The capability to perform rapid temperature gradients will ensure that this technology can become mainstream and does not become a niche topic, with little industrial applicability but with a high academic following due to the peculiarity of using such relatively extreme conditions.
INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2013
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