The Heat Is On

The Heat Is On

High-temperature liquid chromatography (LC) is a hot topic in separation science at the moment. David Collins from the Irish Separation Science Cluster based at Dublin City University, Ireland, describes a new column heater design that aims to enhance the efficiency of high temperature separations.

LCGC Europe Editor Alasdair Matheson spoke to him to find out more.
Why is moderating temperature control important in LC and what are the benefits of performing separations at elevated temperatures?
Temperature control is vital in LC, even at room temperature, because it can have quite an effect on the efficiency and repeatability of the separation. Everyone is familiar with the van Deemter equation, and both the B and C terms are influenced by temperature. To achieve maximum efficiency the rates of longitudinal diffusion and mass transfer between the stationary and mobile phases should be kept constant along the entire length of the column.

Temperature variations along the column can cause the B and C terms to vary and this will negatively affect the quality of the separation. A real-world example of temperature mismatch along the column would be when using ultrahigh-pressure liquid chromatography (UHPLC), where high back pressures can cause considerable frictional heating within the column. This is just one aspect of the importance of temperature control with respect to LC.

Perhaps, more interestingly, temperature can be used as a very effective and powerful chromatographic tool, and it has only been in the last few years that the full potential of temperature programming for LC is being realized. The application of temperature to the chromatographic process can have profound effects on the separation, including reduced run times as a result of reduced retention, increased peak efficiency, increased resolution, increased analyte signal-to-noise ratio, the ability to change peak elution order, and reduced mobile phase consumption.  

Higher temperatures will also generally reduce the viscosity of the mobile phase, reducing back pressure, and allowing the application of higher flow rates, combined with a reduction in retention. For reversed-phase chromatography this means that run times can be significantly shortened.

With so many benefits it’s easy to see why high-temperature chromatography has developed so much research interest. This is particularly evident in the quest for faster analysis, specifically when it comes to capillary and microscale separations because of their low thermal mass, high thermal conductivity, and, therefore, fast thermal equilibration times. Fast separations and rapid chromatography are one of the most powerful driving forces behind the continuous development of the field. High-temperature chromatography is also a favorable technique for green chromatography because, in addition to reducing solvent consumption, it can also provide the capability of using only supercritical water as the mobile phase.

What problems can occur with current approaches to modifying temperature for LC in terms of column thermostating and also as a chromatography tool?
There are a number of problems with the current methods where temperature is applied (1,2). During column thermostating, the idea is that the column is kept at an isothermal point. In other words the entire column is kept at the same temperature. This would be used, for example, in cases where a column was exhibiting longitudinal temperature variation as a result of frictional heating.

In some examples of UHPLC, the eluent temperature at the exit of the column might be 10 °C or 15 °C hotter than that at the inlet side, and so the column compartment is heated to perhaps 40 °C or 50 °C, which brings the entire column up to the same temperature. This is an adequate solution in most cases. However, it can become a real problem if the user is using thermally unstable analytes or if the stationary phase is thermolabile. In such a situation it can be very difficult to adequately and accurately control temperature along the length of the column. Current commercially available column ovens, by their design, are only capable of providing one temperature at any point in time or any point on the column.

As such they cannot effectively thermostat a column without heating the entire column to a temperature higher than the eluent at the column exit. Most column ovens are also limited by the fact that they cannot cool below ambient, or below 5 –10 °C less than ambient temperature.

As a tool for chromatography, most column ovens also fall short of the mark in terms of response time. In other words, they are simply too slow, especially when it comes to rapid separations (3–6). They are also limited in terms of the temperature range that they offer. As mentioned above, the lower limit is usually 5–10 °C below ambient temperature, while the upper limit of most ovens is around 85 °C, with some manufacturers supplying ovens capable of over 100 °C. There are one or two types of heater available that offer temperatures higher than 200 °C with exceptionally fast ramp rates, but they are for capillary scale only. There are some disadvantages associated with these systems too because they must be wound onto the column and are, therefore, quite laborious to use and they don’t provide anything close to a uniform heat profile. When applying temperature profiles with fast ramp rates, these systems generally have a considerable overshoot.

Why is your approach novel and what was the main obstacle you had to overcome?
Our approach is novel both in terms of design and performance.

When we began to develop this system our initial goal was to build a direct-contact column heater that had a segmented contact block and so could heat various parts of the column to different temperatures if desired. This was a powerful tool in research because it could provide complex temperature profiles and rapid dynamic gradients, providing some interesting insights into the band-broadening processes within a column. However, as we developed the system further, we began to realize that it had great potential.

The segmented zones can be both heated and cooled through the use of Peltier modules. Essentially these are semiconductors that allow you to heat one side of the chip while cooling the other. By simply controlling the current and the polarity of the chip, it is possible to heat or cool any individual segment of the heater. The segmented design also ensures that the mass of each thermal element is minimized and so the response time of the system is extremely fast at over 400 °C/min-. Compare this with commercial air bath column ovens at 10 °C/min and direct contact units at around 30 °C/min. Each segment has individual closed-loop control so even with such a fast response there is minimal overshoot and the steady state fluctuation is only ±0.2 °C. The system response can be seen in Figure 1, where the system is cycled between 16 and 40 °C. The temperature range of the system is also very broad, from -20 °C to over 200 °C.

Figure 1: System response during cycling between 16 and 40 °C.

What we have now is a very fast column heating/cooling platform capable of operating across a broad temperature range while also capable of providing the user with rapid, complex gradients and longitudinal profiles. In terms of column size, the heater is designed to take capillary and microscale columns up to 16 cm in length. Good thermal conductivity is provided by a soft thermally conductive silicon layer on the heating/cooling segments that molds to the shape of the column and fittings. The column heater has been designed with an integrated eluent pre-heater and fitting heater to ensure full column thermostating, and for high temperature work it is possible to post cool the eluent before it reaches the detector. The result is an extremely versatile, compact, column heater with a footprint smaller than an A4 page and only 15 cm high. There is onboard data acquisition, and it has remote I/O ports for talking to other instrumentation and for exporting data. The system has received quite a lot of industrial interest, and earlier in 2011 we submitted a patent application for the device.

There were numerous problems that we faced with the design and development of the system, although the main obstacle was probably designing the system to handle such temperature extremes and manage the heat flow and dissipation within the system itself. We went through a few iterations of the design and heat exchanger before we got it right.

 What benefits does this offer chromatographers in practice? Can you illustrate this with a practical example?
This system could offer many benefits to chromatographers apart from the usual advantages of applied temperature, such as increased separation efficiency, variation of peak selectivity, and decreased run times. Because of its segmented design, it can provide a longitudinal temperature gradient and so can be used for thermostating in UHPLC.

The segmented design also allows the spatial application of heated or cooled zones for potential on-column thermally controlled trap-and-release applications, analyte focusing on the head of the column, or to apply instant or dynamic temperature gradients to the column, the latter of which could be utilized to provide insights into frictional heating-related band-broadening processes within capillary columns. Precolumn heating ensures that there is no thermal mismatch between the eluent and column, and the provision of postcolumn cooling ensures that vapor problems associated with high-temperature chromatography can be avoided without the need for restrictors between the detector and column, which cause additional band broadening.

The fast response time of the oven makes it ideal for use in rapid separations because it can provide very rapid temperature gradients. Finally, such a heating/cooling platform can also find use as a tool in various hyphenated techniques that demand minimal extracolumn band broadening and require either high or low temperatures that are outside the normal operating envelope of most conventional column heaters.

We have also used the platform for the fabrication of polymer monolithic columns, and because of the segmented design, the system is particularly useful for discrete polymerization. In other words, you can form monoliths in areas of capillaries or microfluidic chips with excellent precision. We have even formed monoliths with gradients of pore size.

Is this approach being implemented in commercial instruments?
The short answer is no. There are currently no segmented commercial heaters available, at least none where the segments can be set to individual temperatures. In addition, to date there are only a few commercial instruments using Peltier modules, and the majority of these systems use them simply for cooling air streams in air bath systems. It isn’t straightforward to apply Peltier technology to both heating and cooling, and the semiconductor material must be specified very carefully otherwise the module will degrade over time.

Are you working on developing this technology further?
The development of this heating/cooling platform is still ongoing, and we have applied the technology to different chromatographic applications. We have built several versions of the platform including a circular design for very long capillary columns, such as gas chromatography (GC) columns, and we hope to continue to push the boundaries of the technology while making the platform as versatile and flexible as possible.

I would also like to thank my collaborators in this project, Professor Brett Paul, and Dr. Ekaterina Nesterenko, who have been instrumental to the design of the system, and who remain involved in the development of the technology.

David Collins graduated in 2000 with a B.Eng in Mechatronic Engineering from Dublin City University, and most of his experience has been gained in the Aerospace and Pharmaceutical/Medical Device industries. In 2005 David was awarded his M.Eng for his thesis on laser-based metrology. David started his own engineering business in 2007, offering mechanical, electrical, and control system services to the Pharmaceutical sector. In 2009 he joined the Irish Separation Science Cluster and is heavily involved in multidisciplinary research within the group, focusing on instrumentation.

Further reading

[1] Collins, D., Nesterenko, E., Connolly, D., Vasquez, M., Macka, M., Brabazon, D., Paull, B., Anal. Chem., 2011, 83 (11), pp 4307–431.

[2] Collins, D., Connolly, D., Macka, M., Paull, B., Chromatography Today, 2010, Aug/Sep 2010, pp 30-31.

[3] http://www.jascoinc.com/Products/Chromatography/HPLC-Systems/HPLC-Components/HPLC-Column-Ovens.aspx

[4] http://www.zirchrom.com/pdf/news5.pdf

[5] http://www.sim-gmbh.de/index.php?option=com_content&task=view&id=64&Itemid=502&lang=en

[6] http://www.selerity.com/main/main_products_hplc_9000.html