The Column Oven

The oven controls the temperature of the fluid entering the separation column. 

This includes not only the eluent but the sample that is injected into the system. 

The fluid entering the oven compartment is normally cooler than the oven and there is a time lag before this new fluid comes to the oven temperature. In most HPLC ovens, the fluid never does reach the set point of the oven. In DNA and RNA separation instrumentation, a pre-heat tube is used to bring the fluid to column temperature before it reaches the column. The oven temperature should be accurate, should not drift and should be precise, i.e. it should come to the same temperature each time it is directed by the run method to go to a specific temperature. 

The oven remains one of the most critical and difficult parameters to control in the DNA chromatograph. The reader should consult the HPLC manufacturer for information on oven use, calibration and upkeep. Selerity Technologies (Salt Lake City, UT) has developed an active eluent preheater that uses a feedback technology to maintain a set temperature for the eluent prior to entering the column. This unit appears to be compatible with different HPLC instrumentation. 

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For GC, the injector is most frequently a small heated space attached to the start of the column. A sample of the mixture to be analyzed is injected into this space by use of a syringe, which pierces a rubber septum. The injector needs to be hot enough to immediately vaporize the sample, which is then swept onto the head of the column by the mobile gas phase. Generally, the injector is kept at a temperature 50 C higher than is the column oven. Variants on this principle are in use, in particular the split/splitless injector. This injector can be used in a splitless mode, in which the entire injected sample goes onto the column, or in a split mode, in which only part of the sample goes onto the column, the remainder vented to atmosphere. For other less usual forms of injector, a specialist book on GC should be consulted. 

Other thermal zones which should be thermostated separately from the column oven include the Injector and detector ovens. These are generally insulted metal blocks heated by cartridge heaters controlled by sensors located in a feedback loop with the power supply. Detector blocks are usually maintained at a temperature selected to minimize detector contamination from condensation of column bleed or sample components and to optimize the response of the detector to the sample. The requirements for i injectors may be different depending on the injector design and may include provision for temperature program operation. 

Figure 3.8 Alternative designs for cold on-column injectors, h, Injector with a duck bill valve (Hewlett-Packard), B, an injector with provision for secondary cooling of the column inlet (Carlo-Erba), and C, a temperature- programmable on-column injector with its own oven isolated from the column oven (Varian Associates).

The only disadvantage to the use of hydrogen as a carrier gas Is the real or perceived explosion hazard from leaks within the column oven. Experience has shoim that the conditions required for a catastrophic explosion may never be achieved in practice.. However, commercially available gas sensors will automatically switch off the column oven and carrier gas flow at air-hydrogen mixtures well below the explosion threshold limit [143]. 

Copper, aluminum, stainless steel, nickel, or glass tubes bent into various shapes to fit the dimensions of the column oven provide the container for column packings [126]. Neither copper mor aluminum tubing is recommended as both metals are readily SKlditsd active, oxide-coated films formed on the inner walls promote decomposition or tailing of labile and polar solutes. Ptalnless steel is adequate for nonpolar samples but its catalytic activity precludes the analysis of labile solutes. Nickel, after acid passivation, and glass are the most inert column materials. 

The column oven is generally a forced circulation air thermostat of sufficient size to allow comfortable installation of the longest columns normally used. In the design of a column oven it is important to ensure a uniform temperature throughout the column coil region. The temperature uniformity depends on the geometry of the oven, the Ideation of the heater and sensor, and the pattern of mixing and circulation of air. A temperature... 

A schematic diagram of a chromatograph for SFC is shown in Figure 6.10. In general, the instrument components are a hybrid of components developed for gas and liquid chromatography that have been subsequently modified for use with supercritical fluids. Thus, the. fluid delivery system is a pump modified for pressure control and the injection system a rotary valve similar to components used in liquid chromatography. The column oven and... 

Whether you realize it or not, the GC column has its own heater—the column oven. If you turn the temperature up, the compounds hotfoot it through the column very quickly. Because they spend less time in the stationary phase, they don t separate as well, and the GC peaks come out very sharp but not well separated. If you turn the temperature down some, the compounds spend so much time in the stationary phase that the peaks broaden and overlap gets very bad. The optimum is, as always, the best separation you need in the shortest amount of time. There are two absolute limits, though. 

Examine the instrument to which you are assigned. Locate the source of the carrier gas and trace the line to the instrument. If an FID is to be used, also locate the source of the hydrogen and air, and trace the lines of each to the instrument. Locate the injection port. Note any gauges and controls on the front of the instrument, and try to identify their functions. Open the column oven and locate the column. Note the proximity of the inlet end of the column to the injection port. Note the outlet end of the column and locate the detector. 

A precise control of the column temperature is not only a must but also a requisite, whether it is intended to maintain an invariant-temperature or to provide a programmed-temperature. Importantly, the temperature of the column oven must be controlled by a system that is sensitive enough to changes of 0.01°C and that maintains an accurate control to 0.1 °C. In normal practice, an air-bath chamber surrounds the column and air is circulated by a blower through the thermal compartment. However, separate temperature controls are very much desirable for the vaporizer block as well as the detector-oven. 

Erba model 4160 (Erba Science (UK) Ltd, Swindon, Wilts) with a sub-ambient attachment was fitted with a 25m BP1 flexible silica capillary column (SGE Ltd, Milton Keynes, Bucks.). Following 10 minutes at 10°C the column oven was temperature programmed at 3°C min-1 up to 150°C. 

Turn on the column oven if required. Rinse the column with strong solvent (e.g., 100% MeOH) first then equilibrate column for 5-10 min with the new mobile phase until the baseline and pressure are stable. 

Turn off the column oven. Flush the buffer out of the system and stop the flow. Keep the idle column in methanol or methanol/water. 

This chapter reviews the principles and strategies used for HPLC system calibration that includes the pump, the detector, the autosampler, and the column oven. A case study is used to illustrate the development of the calibration procedures for all system modules and the rationale of setting up acceptance criteria that balance productivity and compliance. 

A temperature accuracy test of the column oven measured with a calibrated thermal probe is used. An acceptance criterion of 35 2°C is adopted. 

Inside the column oven, the solvent flows through 0.75 m of 0.009 in. I.D. conditioning coil, through a low dead-volume tee containing a thermocouple to monitor solvent temperature, and then to the column. The column oven, with a 425 0 maximum temperature, is heated by two 2-kilowatt wire wound heaters which are controlled with a Gulton Model 2GB Controller which provides either Isothermal or programmed temperature control. 

The injection temperature can be a signiflcant issne for thermally unstable samples or where samples are stored for hours in an antosampler prior to injection. For this reason, most manufacturers sell autosamplers with optional thermostated sample compartments. This can be done either by placing the sample tray in an air bath oven or by a condnctive temperature control of the sample rack. The need to keep samples cool prior to injection when conpled with elevated temperature separation increases the complexity of the flow system reqnired. For such application, a separate mobile phase pre-heater with a low volnme placed between the injector and the column is a good choice. Alternatively, the injector valve wonld need to be monnted ontside the antosampler or in the column oven to insure preheating of the mobile phase before the colnmn. 

GC and GC-MS Analyses. Glass capillary columns were prepared in our laboratories as described briefly elsewhere (3). Aliquots (l-2 ul) of the PAH fraction dissolved in a small volume of chloroform were injected without stream splitting into the Hewlett Packard 5dk0A gas chromatograph. Injection port temperature was held at 250°C, and the column oven temperature was started at 100°C. Two minutes after injection a multistep temperature program was initiated final temperature was 290°C. Nitrogen was the carrier and make up gas. 

Column compartment The column oven should be capable of maintaining a temperature range of 5 °C above ambient to 60 °C. The oven should be within 3°C of the set temperature and the temperature precision should be <2.0%. 

The column oven should be free from the influence of changing ambient temperatures and line voltages. The difference between the maximum and minimum temperature observed over a long period of time at any one fixed point in the oven (sometimes referred to as thermal noise) must be minimal—less than... 

Operation of the column oven at 50°C or lower has been a problem in earlier chromatographs because of the difficulty of completely isolating the column oven from other heated components, such as the detector, injection port, and splitter, and still having a usable oven. The processor controller described overcomes this problem by mixing controlled amounts of room air into the column oven and can control very adequately at temperatures of about 30°C without cryogenic cooling. A further advantage of the processor controller is that the processor normally also can handle the temperature control of the other heated zones—inlet, detector, valves, and so on. 

The mechanical programmers were replaced by electronic programmers. These depended mainly on different solid state time-logic RC circuits to increase the power to the column oven to supply the desired heating rates. This allowed multilinear pro-... 

The column oven temperature will now rise as rapidly as the controller can supply the heat (Figure 6.17A). This type of programming is usually used for "cooking-out" or conditioning columns after they have been in use for some time. 

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