Method and instrumentation for comprehensive multidimensional chromatography separations using a micro flow modulator

#This application is a 371 of PCT/EP2011/058404, filed May 24, 2011, which claims the benefit of priority from Italian Patent Application No. ME2010A000011, filed May 27, 2010, the contents of each of which are incorporated herein by reference.

#The present invention refers to a modulator, to be used for comprehensive multidimensional chromatography separations, to entrap and release sample solute fractions (entrapped in a capillary loop of fixed or variable volume), deriving from a capillary column with an internal diameter ranging from 0.01 mm to 0.53 mm, onto another capillary column with an internal diameter ranging from 0.01 mm to 0.53 mm. The micro-device has been integrated in a gas chromatographic system, composed of two ovens for the independent temperature control of the two columns; the micro-device is characterized, internally, by a system of channels that enable the controlled splitting of gas flow, entering the second capillary, to generate an optimum gas linear velocity and to release the pressure in excess, with the objective of attaining the maximum separation efficiency in the second column. Furthermore, the system is equipped with a second device, to divide the flow exiting the second column in two different detectors that operate at the same or at different pressures. Gas chromatography (GC) is generally achieved through the use of a capillary column for the separation of the constituents of a mixture. Comprehensive multidimensional gas chromatography (GC×GC) is certainly the most revolutionary and innovative technique in the gas chromatography field. In an ideal comprehensive multidimensional gas chromatography system the peak capacity is equal to the product of the two single peak capacities (n1×n2) in the two dimensions. Although such a value is optimistic, the peak capacity generated by GC×GC systems is certainly unprecedented and is particularly suitable for the separation of complex mixtures of volatiles, prior to the detection system. The advantages of these methods, compared to conventional ones (one-dimensional) are mainly three: increased separation power enhanced sensitivity formation of two-dimensional chromatograms with regions occupied by chemically-similar compounds. This aspect is particularly important for the identification of unknown compounds, in the absence of mass spectral libraries or when mass spectra are very similar (for example, homologous series of compounds such as fatty acids, terpenes, etc.).

#Amongst all comprehensive multidimensional chromatography methods, GC×GC has been probably the most applied and developed. A GC×GC separation is normally obtained on two capillary columns, linked in series, and with a transfer device, defined as modulator, positioned between the two columns. The function of the modulator is to isolate, re-concentrate and introduce fractions of effluent from the primary column onto the secondary capillary column, continuously throughout the analysis. The time required to complete this process is defined as the modulation period (generally between 1 and 10 seconds). The primary column is generally apolar and, hence, the separation is based on the boiling-point differences (not only on these) between the different analytes. Each modulation generates fractions which undergo a further rapid separation on the second column, generally of polar chemistry: isovolatile compounds are resolved on the basis of polarity-based interactions (dipole-dipole, hydrogen bond, polarization effects).

#FIGS. 1 and 2 show a modulator for comprehensive multidimensional chromatography for samples collected in a capillary loop of variable volume.

#FIG. 3 shows a modulator for comprehensive multidimensional chromatography for samples collected in a capillary loop of fixed volume.

#FIGS. 2A and 3A show a detail of FIG. 1 while valve 8 is in the “normally-closed” position.

#The present invention relates to a device that functions as modulator used in chromatography, and in particular, in comprehensive multidimensional chromatography, comprising: a port that receives effluent from a chromatography column; one or two external capillary sample loops, or one or two sample loops integrated in the modulation device; two independent ports that receive a flow from one or more electronic pressure and/or flow control devices; three channels, either integrated, or located externally with respect to the modulation device, to split, in a controlled manner, the flow exiting the sample loop and to release the pressure in excess within the device and into the second column with a different objective with respect to the system introduced by Tranchida et al (Analytical Chemistry, vol. 81(20), 2009, pages 8529-8537) where the aim of the split-flow device was only to reduce the linear velocity in the second dimension. a needle valve, either integrated or external to the device, that regulates the flow exiting the accumulation capillary; a system of integrated channels that connects the different ports in an appropriate manner.

#In a preferred embodiment, said one or two external capillary sample loops, easy to interchange and to vary the accumulation volume, are characterized by a ratio between capillary length and internal diameter within 5 and 40, to avoid the re-mixing of components separated in the first dimension.

#The device, object of the present invention, can comprise one or two internally integrated accumulation channels, with a fixed volume.