Detection efficiency by means of electrospray mass spectrometry is concentration dependent so the best sensitivity is achieved when samples are eluted in volumes that are as small as possible. This is done by using chromatographic columns of narrow inner diameters and low flow rates for the mobile phase. However, small column volumes provide fewer binding sites on the stationary phase such that the loading capacity is low. Overloading columns with sample results in poor resolving power, broad and asymmetric peaks and eventually deteriorating detection efficiency.
In liquid chromatography, the equilibrium distribution between analyte that is immobilized on the stationary phase and analyte dissolved in the mobile phase is a function primarily of the compositions of the mobile and stationary phases but also many other physical/chemical parameters may have a pronounced effect. One such parameter is temperature and it is common practice to use column ovens (and more rarely column-coolers) in order to maintain a stable temperature of the columns during analysis since temperature stability greatly enhances the retention time reproducibility of repeat experiments. In addition, it is often observed that eluting peaks are slightly narrower and more symmetrical when column temperatures are raised slightly above ambient temperature. Typical temperature ranges used for separation columns are 30-50° C. In nano-LC (which is liquid chromatography that uses a flow range of typically 50 nL/min to 500 nL/min) and in proteomics applications in particular, the use of column temperature control is less common than in most other application areas and when using larger flow ranges.
Frequently, trapping columns (or “pre-columns”) are used to capture samples during sample loading where such trap-columns are shorter and often of slightly wider ID than the analytical (or “separating”) columns. By virtue of being shorter and wider, the flow rate through pre-columns when loading and desalting samples can be much higher than when samples are applied directly onto analytical columns. This saves analysis time. Compounds eluted from the pre-column move onto the analytical column and are separated there. A primary negative effect caused by the combined use of trapping columns in-line with separating columns, is that peak widths are much (typically 1.3 to 5 times) wider than when samples are loaded directly onto separating columns (entirely omitting trap-columns). This reduces resolving power and absolute sensitivity. Typically the ID for trap columns is in the range of 100% to 130% of the ID of the separating columns and lengths of trap columns are typically from 0.5 cm to few centimeters whereas the lengths of separating columns typically range from 5 cm to 100 cm. The larger the trapping column ID (and hence volume) is, the more pronounced will the peak broadening effect typically be.
Currently, experimental conditions for LC-MS in proteomics are chosen to match the requirements for sensitivity, analysis speed, dynamic range, and resolution. Each of these parameters is highly important and much effort goes into finding ways to improve and optimize these parameters, albeit, one cannot optimize all parameters simultaneously because of counteracting effects.
The chromatographic retention and resolution, i.e., the separation of the different peaks in the chromatogram associated with different substances, are to some extent influenced by the system temperature. It is known that the system temperature can be manipulated to optimise conditions for a specific separation task. And it is widely practised to ensure reproducible chromatographic results, by holding the temperature constant during the separation process and from analysis to analysis. To this end, a thermally stabilized housing is often employed in liquid chromatography in order to keep the chromatographic bed temperature in the separation column constant. By adjusting temperatures, it is also possible to optimize the separation performance of a separation column for a given analyte and given set of mobile phases. Various forms of thermally stabilized housings of the cited type are known, in which the temperature within the housing is essentially constant, such as a water bath, circulating-air heating/cooling, a heating jacket, etc.
The prior art includes various apparatus for thermal stabilization in liquid chromatography.
U.S. Pat. No. 4,404,845 discloses a thermal regulator for liquid chromatographs. This regulator comprises a thermostat arrangement for the mobile phase and the separation column in a liquid chromatograph, whereby the thermostat has a heat transformer through which the mobile phase passes. The heat transformer comprises a heating and/or a cooling element and is positioned between a sample injection device and the inlet of the separation column. The heat exchanger serves to selectively heat or cool the ambient air surrounding the separation column. Thermal regulation of the mobile phase and the separation column prevents disadvantageous temperature gradients.
DE8536810 discloses an oven for setting the temperature of separation columns for high pressure liquid chromatography. In order to avoid a fluctuation of the retention times due to temperature deviations, temperature regulation of the separation column is proposed. The temperature of the separation column in this case is set by an oven consisting of a thermally heatable and coolable block of a metal with high thermal conductivity and with a well for accepting one or more separation columns. A Peltier element is arranged on the heatable and coolable block to allow working temperatures to be set below room temperature.
EP438618 discloses an apparatus for thermally stabilizing a mobile phase in a liquid chromatograph. This apparatus comprises a chromatographic column to which an ingoing capillary tube and an outgoing capillary tube are attached, this chromatographic column being positioned in a receptacle element that can be adjusted to a desired temperature using a temperature control unit. In order to avoid excessive temperature of the liquid directed to a detector through the outgoing capillary tube, which would lead to inaccurate measurement, it is proposed in this case to route the ingoing and outgoing capillary tubes parallel with one another for a predefined distance in order to achieve a heat exchange between the higher-temperature capillary tube leading to the detector and the lower-temperature ingoing capillary tube leading to the separation column.
In addition to the aforementioned apparatus with one separation column, there are known liquid chromatography systems that work with two columns, a pre-column and a separation column. In transferring the substances to be analyzed from the pre-column to the separation column, however, a more strongly elutropic eluent is required for the pre-column than for the separation column, even if the same packing material is used in both columns. This causes the dispersion in the pre-column to act as an “elutropic injection volume”, whereby even well-packed pre-columns considerably reduce the overall efficiency of the liquid chromatography system. Of added disadvantage in this case are pre-columns with low efficiency and high capacity.