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Possibilities of High Temperature and Temperature Programming in Conventional LC

by Gerd Vanhoenacker and Pat Sandra, Research Institute for Chromatography, and Jody Clark, Selerity Technologies, Inc.

Although often neglected, temperature plays an important role in HPLC since the majority of the chromatographic properties are a function of temperature. Nevertheless, to this date the possibilities of temperature to improve LC separations have not been fully investigated and the majority of liquid phase separations are performed in the temperature region between 20 C and 40 C or without thermostatting. We have recently reviewed the use of elevated temperature and temperature programming in HPLC.¹

The main advantages of using temperature in LC are:

1. Speed

The current trend is to use shorter columns with smaller particle sizes, typically less than 2 µm, for increasing the speed. This has the disadvantage that the pressure drop over such columns is drastically increased. Furthermore, to fully exploit the potential of such ‘sub-two-micron’ columns, high linear velocities have to be used. The combination of the intrinsic high backpressure and the required high flow rate causes analysts to reach the upper pressure limits of conventional LC systems. The recent introduction of ultra-performance or ultra high pressure LC (UPLC) equipment has partially overcome this problem.

Figure 1. Plate height (H) versus linear velocity (u) for propylparaben (k~3.6 at 30 C) on a Blaze 200 column (150-mm L 4.6-mm ID, 5-m dp). Mobile phase: water/ACN 60/40, detection: DAD 210 nm. Click to enlarge.

In nearly all reversed phase separations, an increase in temperature will also cause a decrease in retention. Additionally, decreased solvent viscosity at elevated temperature leads to lower back pressure. This allows the use of higher flow rates using standard equipment. Since high temperature leads to a flatter van Deemter curve, it enables the use of higher flow rates without hampering efficiency (Figure 1). An increased flow rate is even favorable to fully benefit from the increased temperature.

The application of high temperature enables the use of columns packed with ‘sub-two-micron’ particles at high flow rates. In this way it is possible to exploit the advantages of these columns using conventional LC equipment.

2. Efficiency and Resolution

The efficiency that can be realized on one particular analytical setup is relatively independent of analysis temperature provided that adequate preheating of the mobile phase prevents radial temperature gradients. At elevated temperature however, the solute transfer from the mobile phase to the stationary phase is more efficient. This results in high efficiency, even at elevated flow rates.

The lower back pressure due to the decreased solvent viscosity at elevated temperature allows the use of smaller particle sizes and/or longer columns to increase efficiency and resolution. An example of this approach is shown in Figure 2 where a mixture of drug substances is analyzed. First, the analysis was carried out on a 25-cm-long column packed with 5-m particles. The same analysis was performed on a set of five columns coupled in series giving a total column length of 125 cm. This set should provide five times the initial efficiency and therefore should increase the resolution with a factor slightly above two.

From the chromatograms, it is clear the resolution has increased significantly, enabling complete separation of compounds 4 and 5. Additionally, more impurities eluting between compounds 1 and 2 are resolved in comparison to the one column experiment. This approach has also been used for a high resolution analysis of tryptic digest. A peak capacity of ca. 900 was obtained using a set of eight 25-cm columns at 60 C.²

Figure 2. Analysis of a pharmaceutical mixture with impurities. Analysis was carried out using a 1200 Series HPLC (Agilent Technologies, Waldbron, Germany) and a Polaratherm Series 9000 column oven (SandraSelerity Technologies, Kortrijk, Belgium). Temperature: 60 C, mobile phase: formic acid in water/acetonitrile, 50/50 isocratic, 1 mL/min, detection: UV 254 nm, column: Zorbax 300StableBond C18, 250 4.6 mm, 5 m. Click to enlarge.

The example in Figure 2 also demonstrates the possibilities of increased efficiency and resolution in preparative LC. Instead of multiple collection sessions to completely resolve all of the compounds, a single method with increased resolution can provide a more elegant and productive alternative.

3. Selectivity

Selectivity in LC is determined by the stationary phase, mobile phase pH, organic modifier and several other parameters. Temperature is a parameter that also plays an important role in this aspect and has been reviewed by Dolan in 2002.³ This is especially true for polar and ionizable compounds since ionization equilibria are temperature dependent. Even more, because it is an instrumental setting, temperature is one of the easiest and most straightforward parameters to change and control in order to tune chromatographic selectivity. This has been reported frequently in the literature, yet its potential role in method development is still often underestimated.

4. Lower Consumption of Organic SolventsGreen Chromatography

By increasing the temperature, the amount of organic solvent in the mobile phase can be reduced to maintain retention. Roughly, a temperature increase of ca. 4 C or 5 C has a similar effect on retention as a 1% increase in methanol and acetonitrile, respectively. Superheated water at 200 C has a similar eluting power as methanol at ambient temperature. Additionally, since the back pressure is reduced at elevated temperature, ethanol becomes a practical alternative for toxic solvents such as methanol and acetonitrile. Mobile phases composed of water, ethanol, and additives like ammonia and acetic acid can be considered non-toxic and can be used for green chromatography.

5. Improved Detectability

The reduced amount of organic solvent in the mobile phase also results in additional advantages. The mobile phase UV transparency in the low UV range is improved. Alternative detection techniques such as flame ionization detection (FID) also become an option when using solvent-free mobile phases.

Additionally, an improved peak shape for basic solutes is frequently encountered. The ionic strength and buffer pH required for good peak shape of these solutes can therefore often be changed to levels less harmful for the column and chromatographic system.

6. Temperature Programming

Since the temperature of the column and mobile phase influences the retention, programming the temperature in time can be used to elute compounds from the column. If the system is capable of covering large temperature ranges, a temperature gradient can be used as in gas chromatography and in many instances, can replace the solvent gradient. This enables the use of temperature programmed elution on detectors that are restricted to isocratic operation such as a refractive index detector.

On the other hand, there are practical considerations to be made when using elevated temperature in LC.

1. Analyte Stability

This is a major concern when dealing with high temperature in LC. However, many compounds considered thermally labile do not degrade when being analyzed at higher temperatures. If degradation occurs, as we experienced for some thermolabile pharmaceuticals, the amount of breakdown depends not only on the time spent at the higher temperatures, but above all on the nature and quality of the packing material (e.g. presence of trace metals). ⁴

2. Column Stability

When using water in the mobile phase as in most reversed phase type separations, loss of the bonded phase from the silica support due to hydrolysis is enhanced at high temperatures. Therefore, traditional silica-based stationary phases are only stable at temperatures up to 60 C. With enhanced bonding chemistry the functional group can be sterically protected and in some instances be stable up to at least 90 C. In the past, the absence of suitable stationary phases for high temperature LC held back the expansion of this technique.

At this time, new temperature stable silica-based columns and alternative stationary phases are available which enable the application of high temperature over a long period of time while maintaining column performance. A new generation of a silica-based phase that is stable at temperatures up to 200 C under certain reversed phase conditions has recently been introduced.⁵

Table 1. Commercially Available LC Columns for High Temperature Operation. Click to enlarge.

For columns intended to be used at temperatures of 120 C or higher, care has to be taken that the PEEK present in the column hardware is replaced by stainless steel. An overview of commercially available stationary phases is shown in Table 1.

3. Equipment

In the past, there was a lack of dedicated equipment for high temperature LC. The main shortcomings were the oven/column temperature range along with the absence of an efficient mobile phase preheating system, and the lack of an integrated post-column cooling/thermostatting unit.

The temperature of the incoming mobile phase should be within 6 C of the oven/column temperature to minimize band broadening by radial temperature gradients. This is not a concern when using (packed) capillary columns at high temperatures. However, efficiently heating aqueous mobile phases to significantly higher temperatures with typical analytical flow rates of several hundred L/min up to several mL/min is more of a concern. Many of the literature references made use long pieces of stainless steel tubing to heat the LC columns and to preheat the incoming mobile phase, respectively. The column effluent was subsequently cooled by a water or ice bath.

Recently, instrumentation for elevated temperature and temperature programmed LC with conventional and semi-prep column dimensions became available with the introduction of the Polaratherm Series 9000, 9010 and 9020 from SandraSelerity Technologies, Inc (Kortrijk, Belgium). These systems actively heat the entering mobile phase to the same temperature as the column/oven temperature. They can heat up to 200 C and the Series 9000 can also be used for sub-ambient separations down to -20 C. The active preheating implies that the amount of energy delivered by the system is capable of adapting to the changing mobile phase flow rates and compositions. This enables the use of fast temperature programming (20 C/min) with conventional LC columns and flow rates. After the separation, the effluent is thermostatted to a fixed temperature to protect the detector and to stabilize the signal. If necessary, a backpressure regulator can be installed after the detector to prevent boiling of the mobile phase.

For more information, contact Professor Dr. Pat Sandra, Research Institute for Chromatography, at pat.sandra@richrom.com or by phone at +32-56-204031.

References