WO2003087756A2

WO2003087756A2 - Chemical analysis of samples

Info

Publication number: WO2003087756A2
Application number: PCT/IB2003/001407
Authority: WO
Inventors: Egmont Richard Rohwer; André VENTER
Original assignee: University Of Pretoria
Priority date: 2002-04-16
Filing date: 2003-04-15
Publication date: 2003-10-23
Also published as: WO2003087756A3; AU2003219362A1; AU2003219362A8

Abstract

The invention relates to a method of analyzing a sample, and to an apparatus for such analysis. The method includes subjecting a liquid sample to high pressure fluid chromatography in a first chromatographic column (12) operating at a first temperature in order to effect partial analysis of the sample. The eluent fraction produced by the first separation is then passed through a pressure-reducing zone in fluid flow connection with the first chromatographic column, after which the eluent fraction is fed through a second chromatographic column (48) which is in fluid flow connection with the pressure-reducing zone. The temperature of the second chromatographic column (48) is then ramped independently of the first chromatographic column (12) in order to effect fast gas chromatographic separation of the eluent fraction into further fractions of differing volatilities.

Description

CHEMICAL ANALYSIS OF SAMPLES

THIS INVENTION relates to the chemical analysis of samples. More particularly, it relates to a method of, and apparatus for, analysing a sample. Still more particularly, the invention relates to a method of, and apparatus for, analysing a sample by means of a comprehensive two- dimensional chromatographic technique.

Multidimensional chromatography is a chromatographic technique wherein a plurality of chromatographic separation systems having different selectivities is coupled together. Each dimension comprises a stationary phase and a mobile phase which passes along the stationary phase, with the components of a sample to be analysed partitioning between the two phases. Each component thus moves along with the mobile phase at a characteristic speed, defined by its partitioning behaviour. An example of multidimensional chromatography is two-dimensional column chromatography, wherein a fraction of the eluent from a first chromatographic column is fed into a second chromatographic column, with high performance liquid chromatography (HPLC) typically being used for both separation/analysis steps, ie in both columns. Such two- dimensional column chromatography is also referred to by the designation HPLC-HPLC. In a similar manner, separation/analysis of samples may be achieved using two-dimensional gas chromatography (GC-GC). If each fraction of the first column is transferred to the second column for analysis by the second column, the technique is termed comprehensive two- dimensional chromatography and is designated GCxGC.

GCxGC generally uses, as its first dimension, a non-polar column to separate and analyse samples according to volatility. Consecutive small portions of the resultant chromatogram are refocused and introduced into a short polar column, where fractions are then separated according to differences in polarity, and thereby analysed further. The polar column is thus used for the second chromatographic dimension.

The range of samples that can be analysed by GCxGC is often restricted by the upper temperature limit of the polar column which is used as the second dimension. The maximum attainable final boiling point (FBP) of analysable samples is thus usually limited to 400°C. This limits the types of stationary phases that can be used for the columns, which therefore restrains the scope of GCxGC analysis. Typically, both GC columns are located in a single oven, with the temperature of the oven (and thus also both the columns simultaneously) being ramped to enable the full boiling point range of compounds in a sample to be analysed.

Supercritical fluid chromatography (SFC) generally operates at a lower temperature. It is particularly suited for the analysis of polarity classes of chemical compounds, for chiral separations or separation according to molecular shape. Apart from chromatographic advantages over HPLC, SFC generally uses C0₂ as its mobile phase, with the low boiling point of C0₂ making it comparatively easy to be removed from the analytes following separation. After decompression the C0₂ may be used as the gaseous mobile phase in a subsequent gas chromatographic dimension, when a two-dimensional SFCxGC technique is employed. While strong on simplicity, this technique limits the range of compounds that may be analysed and leaves much to be desired with regard to separation efficiency and speed, particularly in the second dimension or column, as, due to slow diffusion coefficients, C0₂ has limitations when used as the mobile phase in GC applications where rapid separation and analysis are important. Known SFCxGC systems typically comprise a single oven containing both the SFC column and the GC column, with the columns being kept at the same temperature, such as has been described hereinbefore for the GCxGC technique.

To aid the transfer of partially analysed samples from the first (SFC) column to the second (GC) column, a thermal modulator can be used. By varying the temperature in different zones of the thermal modulator, transfer of a sample is effected in a step-wise fashion from the first column to the second column. The transfer of the sample is effected by temperature variation and only very narrow, focused bandwidths can successfully be analysed or resolved by the second column. The thermal modulator thus transfers a pulse of a focused sample of the analytes eluted from the SFC column to the GC column, in order to permit adequate separation and analysis of the sample in the second column.

It is to be appreciated that during supercritical fluid chromatography (SFC) analysis, the carrier fluid may, at certain stages during the analysis, be at a subcritical or near-critical pressure. Thus, reference is hereinafter made to 'high exit pressure fluid chromatography' or 'HPFC, rather than to SFC; however, it is to be understood that high exit pressure fluid chromatography or HPFC is thus effectively the same as SFC, but the carrier fluid, while mostly being at supercritical pressure, may for some analyses be at subcritical. or near-critical pressure. It thus also includes fluidity-enhanced liquids. The distinction between HPFC and liquid chromatography is the employment of a restrictor that maintains the pressure throughout the column to prevent boiling of the volatile mobile phase.

Thus, according to a first aspect of the invention, there is provided a method of analysing a sample, which method includes subjecting a sample to be analysed to high pressure fluid chromatography (HPFC) in a first chromatographic dimension operating at a first temperature, thereby partially analysing the sample according to a first sample property and producing an eluent; passing a first discrete fraction of the eluent through a pressure- reducing zone which is in fluid flow connection with the first chromatographic dimension; thereafter passing that eluent fraction through a second chromatographic dimension which is in fluid flow connection with the pressure-reducing zone and which operates at a second temperature; ramping the second temperature independently of the first temperature, so as to effect fast gas chromatographic (GC) separation of the eluent fraction into further fractions of differing volatilities, in the second chromatographic dimension; subsequently passing at least one further discrete fraction of the eluent from the first chromatographic dimension through the pressure- reducing zone and through the second chromatographic dimension; and subjecting each such further discrete fraction of the eluent to GC separation in the second chromatographic dimension, thereby to obtain a comprehensive two-dimensional analysis of the sample.

The first chromatographic dimension may effect polar analysis or separation of the sample, with polar analysis thus being the first sample property. The first chromatographic dimension may be provided by a first chromatographic column, with which group type analysis of the sample can be achieved. Accordingly, the first chromatographic column may be a silica gel column.

It will, however, be appreciated that any other suitable column can be used, dependent upon the type of analysis required, provided that it operates by means of HPFC.

The first temperature may be room temperature, ie the first chromatographic column may function at room temperature.

Alternatively, the first temperature may be above room temperature. The first chromatographic column may then be subjected to temperature programming independently of the second chromatographic dimension.

C0₂ may be used as a mobile phase for the sample in the first chromatographic column.

The second chromatographic dimension may be provided by a second chromatographic column. The volatility separation in the second chromatographic column may be effected by fast temperature programmed GC.

A carrier gas with a high diffusion coefficient may be used as a mobile phase for the eluent fractions in the second chromatographic column. H₂ may be used as the carrier gas in the second chromatographic column, for enhanced speed and separation efficiency, after allowing for escape of the C0₂ used as the mobile phase in the first column.

The mobile phase composition may be exchanged by way of an interface between the first and second chromatographic columns. The discrete eluent fractions obtained from the first column thus pass sequentially through the second column upon heating of the second column. The heating may occur by passing an electric current through the column body, thereby resistively heating the body of the second column and the eluent fraction contained therein. The programming rate of the column must be fast, typically 5-10°C per second, to allow a speedy analysis of the complete fraction that may contain compounds spanning a wide range of volatilities.

The discrete eluent fractions may be formed by means of flow modulation of the eluent from the first chromatographic column.

Thus, the flow modulation may be effected by allowing the sample to pass through and exit from the first chromatographic column for a time interval; collecting the eluent from the first chromatographic column; interrupting the passage of the sample through the first chromatographic column so that the eluent which has formed during the passage of the sample through the first chromatographic column prior to the interruption constitutes the first discrete eluent fraction; focussing the first discrete eluent fraction on the head of the second chromatographic column by means of its passage or transfer through the pressure-reducing zone; subjecting this focused fraction to said GC by heating the second chromatographic column independently of the first chromatographic column; thereafter again allowing the sample to exit from the first chromatographic column for a time interval; again interrupting the passage of the sample through the first chromatographic column so that the eluent which has formed during the passage of the sample through the first chromatographic column prior to this interruption constitutes a further or second discrete eluent fraction; focussing the second discrete eluent fraction on the head of the second chromatographic column where it is subjected to said GC by heating the second chromatographic column independently of the first column; and so on, until all the sample has passed through the first and second chromatographic columns.

Alternatively, the sample transfer and the carrier gas exchange may be arranged without interruption of the flow in the first column. Sample transfer and carrier gas exchange will thus then be effected in a continuous fashion with an additional focusing step selectively to re- concentrate sample components in the interface.

The flow modulation thus has the effect of cutting small consecutive sections from a first chromatogram, refocusing the individual sections or fractions on the second chromatographic column at its starting temperature, followed by a rapid heating of this column in order to produce a second chromatogram for every cut made from the first chromatogram.

The time intervals may all be the same, and may be selected to provide at least one transfer for every peak created by the first chromatographic column. Typically, however, from 5 to 10 transfers may be made from the first to the second chromatographic column for each peak of the first chromatographic column. Thus, the eluent fraction collection time may be between 1 and 15 seconds, preferably from 3 to 10 seconds, eg about 5 seconds, when the first chromatographic column is operated in stop-flow mode.

The focussing of the eluent fractions from the first chromatographic column on the head of the second chromatographic column may be pressure-drop focussing effected by passing the fractions through a restrictor, with the restrictor being in fluid flow connection with the first and second chromatographic columns. By 'pressure-drop focussing' is meant that the drop in pressure when the carrier gas and sample exit the restrictor serves to focus the sample fraction on the head of the second chromatographic column. Subsequently, the temperature of the second chromatographic column is ramped or increased independently of the first chromatographic column, thereby serving to drive the focused sample fraction through the second chromatographic column, and thus effecting further separation and analysis of the sample fraction. Once^' the focused sample fraction has been passed through the second chromatographic column, the column temperature is dropped in anticipation of the next focused sample fraction being passed from the first chromatographic column through the restrictor and onto the head of the second chromatographic column. The cyclic temperature ramping and cooling of the second chromatographic column allows volatility analysis of the discrete eluent fractions obtained from the first chromatographic column through the second chromatographic column.

Interruption of the passage of the sample through the first chromatographic column in the stop-flow mode of operation may additionally be effected by closing a valve in an eluent transfer line from the first chromatographic column to the second chromatographic column. The valve may typically be an electrically actuated low dead volume valve.

According to a second aspect of the invention, there is provided an apparatus for analysing a sample, the apparatus including a first high pressure fluid chromatographic column; a . second gas chromatographic column which is rapidly heatable independently of the first chromatographic column; pressure-reducing means between the first and the second chromatographic columns and in fluid flow connection with both chromatographic columns; and eluent transfer means for transferring eluent from the first chromatographic column to the second chromatographic column.

The eluent transfer means may be adapted to provide either stop-flow or continuous operation.

The apparatus may include exchanging means for exchanging a mobile phase in the first chromatographic column with another mobile phase having a different chemical composition, in the second chromatographic column.

The exchanging means may allow for the exchange of C0₂, after expansion, with H₂, thereby selectively retaining analytes on the head of the second column.

The apparatus is thus a multidimensional chromatographic apparatus, which utilizes comprehensive multidimensional chromatography in which two chromatographic separations with different selectivities are coupled together.

Furthermore, the apparatus may include a flow modulator associated with the eluent transfer means, with the modulator adapted to permit sequential passage of discrete fractions of eluent from the first chromatographic column to the second chromatographic column at selected time intervals.

The flow modulator may be a valve, typically an electrically actuated valve. The valve may, in particular, be a low dead volume valve, such as a port valve. The valve may be programmable to allow the collection of eluent fractions from the first chromatographic column at time intervals of between about 1 and 15 seconds, preferably from 3 to 10 seconds, eg about 5 seconds.

The apparatus may include a flow restrictor associated with the eluent transfer means. In use, the sample fractions produced by the flow modulator will pass through the restrictor and be focused on the head of the second chromatographic column, prior to the second chromatographic column being resistively heated to effect the transfer of the fraction through the second chromatographic column.

The first chromatographic column may be connected to a supply of C0₂ so that, in use, it contains C0₂ as a carrier fluid or mobile phase for the sample. The second chromatographic column may be connected to a supply of H₂ so that, in use, it contains H₂ as a carrier gas.

The invention will now be described in more detail with reference to the accompanying diagrammatic drawings.

In the drawings,

Figure 1 is a schematic representation of a multidimensional chromatographic apparatus according to the invention, for analysing a sample; Figure 2 is a more detailed schematic representation of the interface system between the first and second columns of the apparatus of Figure 1 ; Figure 3 is a chromatogram of a complex mixture of alkanes, mono- aromatics and di-aromatics, obtained by means of the apparatus of Figures 1 and 2;

Figure 4 is a plot showing, in respect of the resistively heated temperature programmable GC column chromatograph 40 of the apparatus of Figures 1 and 2, the effect of decreasing analysis time by increasing flow rate, while keeping the normalised heating rate constant, in order to obtain the maximum peak capacity in the shortest time;

Figure 5a shows an unmodulated chromatogram obtained with the packed silica gel column 12 of the apparatus of Figures 1 and 2; and

Figure 5b shows a modulated chromatogram obtained with the packed silica gel column 12 of the apparatus of Figures 1 and 2, wherein the eluent fraction collection time was 5 seconds and the GC cycle time was 60 seconds.

In the drawings, reference numeral 10 generally indicates a multidimensional chromatographic apparatus according to the invention, for analysing a liquid sample.

The apparatus 10 includes a packed silica gel column 12 in which polarity separation of samples by means of HPFC can be performed. A piston pump 14 is operatively connected, by means of a flow line 15, to the head 11 of the column 12. The piston pump 14 is also operatively connected to a supply of C0₂ (not shown) so that C0₂ can thus be used as a mobile phase in the column 12. An electrically actuated internal loop injector 16 is provided in the flow line 15. A sample to be analysed can thus be introduced into the column 12 through the injector 16, with the sample being mixed with C0₂ and travelling through the column 12 at a rate determined by the partitioning behaviour of the analytes.

The apparatus 10 includes eluent transfer means, generally indicated by reference numeral 20, for transferring eluent from the outlet 13 of the column 12. The eluent transfer means includes a flow line 22 leading from the outlet 13 of the column 12.

The flow line 22 leads into a multiport stop-flow valve 24, with a flow line 26 leading from the valve 24 to a low dead volume T-connector splitter 28. The splitter 28 is coupled to integral restrictors 30, 32. The restrictor 30 is connected to a first flame ionisation detector ('FID') 34.

The apparatus 10 includes a resistively heated temperature programmable GC column assembly or chromatograph generally indicated by reference numeral 40. The assembly 40 includes a split/splitless injector 42 to which the restrictor 32 is connected.

The apparatus 10 also includes a multipurpose input/output PC board 36 to which the valve 24 is operatively connected and by means of which switching of the valve 24 is effected.

The split/splitless injector 42 includes a frit 44, and is mounted to the head 46 of a GC column 48. A pressure control valve or regulator 50 is operatively connected to the injector 42, as are a needle valve 52 and a solenoid split valve 54.

The assembly 40 includes a second FID 56. A power supply 57 is connected to column heater connectors 58, 60 on an injector leg 62 and on a detector (FID) leg 64 respectively.

As can best be seen in Figure 2, graphite ferrules 66 establish electπcal contact between the injector leg 62 and the metal exterior of the GC column 48. Although not shown, electrical contact between the FID leg 64 and the metal exterior of the column 34 is also effected by graphite ferrules in a similar fashion. The FID leg 30 and injector leg 32 of the splitter 28 are electrically isolated from the body of the GC column 48 by way of an electrical insulator 68, as shown in Figure 2. The electrical current through the GC column 48 is controlled from the input/output board 36 by adjusting the base voltage on a power transistor 69 as shown in

Figure 1.

The functioning of the apparatus 10 will be described hereinafter with reference to the specific example of the apparatus 10 which is described in detail. EXAMPLE

In a specific example of the apparatus 10, polar separation of samples by HPFC is performed in the silica gel column 12. A piston pump 14 (Model 501 , Lee Scientific, Salt Lake City, Utah) is used to deliver SFC grade C0₂ (obtainable from Airproducts (Pty) Limited, Sandown, Johannesburg,

South Africa) at 200 atm to the 2.1 mm x 250 mm column 12 packed with silica gel (SFC group separation column, Agilent Technologies). The electrically actuated internal loop injector 16 (C14-W, Vici, Switzerland) with an internal injection volume of 0.2 μL is used for sample injection. The column 12 is coupled through the low dead volume T-connector splitter 28 (ZT1C, Vici, Switzerland) to the two integral restrictors 30 and 32. The restrictor 30 is connected to the flame ionisation detector (FID) 34 while the other restrictor 32 is coupled to the split/splitless injector 24 on a Varian 3300 gas chromatograph 40 (Varian Instrument Corp.). The first FID 34 can be used to determine the sample quality of the fractions eluted from the HPFC column 12. The HPFC polarity analysis is allowed to progress through the column 12 for 5 seconds, after which the flow of eluent is stopped in the case of the so-called stop-flow operation.

Flow modulation is achieved with the electrically actuated six port stop- flow valve 24 (CW6-K, Vici, Switzerland) which has all but two adjacent ports closed off. Valve switching is controlled with a TTL pulse from the multipurpose input/output board 36 (PCI 6024E, National Instruments, Texas, USA). The valve 24 is located between the splitter 28 and the HPFC column outlet 13. The 5 second cut from the HPFC eluent is splitlessly transferred to the split/splitless injector 42 on the Varian 3300 chromatograph 40 through the HPFC restrictor 32, passed through the frit 44, and focused at a sub-ambient temperature on the head 46 of the gas chromatograph (GC) column 48. The split valve 54 is opened and closed by means of a solenoid valve (not shown) controlled from the input/output board 36 by TTL pulse. The needle valve 52 forms part of the split/splitless injector 42. The needle valve 52 is used to control the flow throughout the split valve 54 and is opened when the six-port stop-flow valve 24 is closed. This relieves C0₂ pressure inside the injector 42 and ensures that the gas chromatography is performed using H₂ gas supplied by the H₂ pressure regulator 50. The entire GC column 48 is cooled down to the ramp starting temperature (which is normally below 0°C) with C0₂ using the temperature control (not shown) of the Varian 3300 chromatograph 40. After pressure-drop focusing of a discrete eluent fraction (not shown) obtained from the first column 12 on the head 46 of the GC column 48, a 5 second equilibration time is allowed for the pressure in the injector 42 to normalize. As can best be seen in Figure 2, the pressure control valve 50 together with the needle valve 52 is used to effect equilibration. Typically, during the pressure-drop focusing of the eluent fraction, the eluent fraction pressure is dropped from about 200 atm to about 5 psi. The pressure-drop focusing is effected through the integral restrictors, which can be in the form of small diameter glass nozzles.

The resistively heated GC column 48 comprises a one metre length of a 0.25 mm internal diameter SE-30 stainless steel column (Quadrex Corp.

SS Ultra alloy), tightly coiled in a spiral but without its loops or turns touching each other. The coil has a diameter of 1 .5 cm. The GC column 48 is connected to the split/splitless injector 42 and to the second FID 56 on the Varian 3300 chromatograph 40. A 30V power supply 57 is connected to the heated column connectors 58, 60 on the injector leg 62 and detector (FID) leg 64 respectively.

A very small thermocouple (not shown) is constructed from type K thermocouple wire having a diameter of 25 μm (Goodfellow, Cambridge GB). The thermocouple is glued to the exterior of the GC column 48 with a drop of polyamide resin (Alldrich) (not shown). The temperature is controlled through PID feedback on the thermocouple signal. A program written in LabVIEW (Version 5.1.1 ) with the LabVIEW PID control kit is used to control the temperature, using the input/output board 36.

The 1 metre long metal GC column 48 is resistively heated at 450°C/min from -50°C to 300°C, depending on the type of sample to be analysed. The temperature ramp serves to pass the focused sample from the head 46 of the GC column 48, through the column 48, after which the eluent from the column 48 is analysed by the second FID 56. At the end of the

GC run the host GC fan (not shown) and temperature control (not shown) are switched on and the temperature of the column is returned to -50°C in less than 30 seconds. The host GC fan and temperature control (not shown) is switched off before the next heating cycle starts. These steps are repeated for the duration of the HPFC run. Flexible heater tape (not shown) is coiled around both the FID leg 30 and the injector leg 32 of the splitter 28 to ensure that they reach the upper temperature of the ramp.

Chromperfect (Version 3.7.4.0) data acquisition software (Justice Innovations, California, USA) is used for data acquisition. Each GC run is recorded as a separate chromatogram. After the HPFCxGC run is completed, the data from the different chromatograms are compiled into a single text matrix by a program written using LabVIEW (National

Instruments, Texas, USA). The text matrix is then imported into Transform (Version 3.4, Fortner Software LLC) for visualization.

Two modulation stages are thus utilised to ensure that no sample components or fractions reach the GC column 48 unfocused and to ensure that a definite starting time is created for each second dimension, ie GC, chromatogram. With the apparatus 10, the first modulation stage is achieved with the stop-flow valve 24, thereby serving to create a definite starting time for the second separation/analysis relative to the progression of the first separation time axis. The second modulation stage is provided by focusing of cut analytes obtained from the HPFC column 12 on the head 46 of the GC column 48, which is stabilized at the sub-ambient starting temperature of the temperature ramp. Focusing of the eluent fractions obtained from the first column 12 is achieved by means of a pressure-drop focusing technique, in which samples eluted under pressure from the HPFC column 12 experience a sharp drop in pressure when exiting an end 70 of the restrictor 32, with the end 70 being located inside the split/splitless injector 42, thereby serving to focus the eluted HPFC fraction on the head 46 of the GC column 48.

Figure 3 shows an HPFCxGC chromatogram of a petrochemical standard obtained by means of the apparatus of the Example described above. The total analysis time is approximately one to two hours for a petrol or diesel sample. However, the time scales of the two axes may differ. In a GCxGC analysis, the initial volatility separation is equal to the run time of the temperature ramp. This is typically one to two hours. The subsequent polar separation is typically completed in 10 seconds or less. In HPFCxGC sample analysis using the stop-flow approach according to the invention, a 10 minute unmodulated SFC run requires 120 cuts which, with a one-minute fast programmed GC cycle time, takes two hours to complete a full HPFCxGC sample analysis. The cycle time is dependent on the temperature range of the GC column ramping and the final temperature. These temperatures may differ for different sample types.

In optimizing the conditions of the second dimension, ie of the GC column 48, the basic recommendation is that 10°C/tm (tm=void time) is a good general normalized ramp rate for all separations. However, 18°C/tm is specifically recommended for low pressure-drop conditions. Since the

C0₂ expanding from the HPFC restrictor 32 may potentially be used to drive the GC separation, flow and ramp rates for both H₂ and C0₂ must be examined. Figure 4 shows the effect of decreasing analysis time by increasing flow rate while keeping the normalized heating rate constant. For hydrogen, a maximum in peak capacity (n) of 60 is obtained when analysis time is equal to 0.6 minutes. Analysis time is taken as the retention time of tetracosane, the last eluting peak in the sample, n=56 is obtained at a flow rate of 100 cm/s (tm=1.33 sec) and heating rates of 450°C/min for the normalized heating rates of 10°C/tm.

These conditions are used for all subsequent GC analyses. The difference in peak capacity for the two normalized heating rates for hydrogen is not significant. A slightly more pronounced effect is observed for C0₂. Figure 4 clearly demonstrates the advantage of exchanging carbon dioxide for H₂ as the mobile phase, namely that, by using C0₂, the same peak capacity can only be approached at more than twice the analysis time required when using H₂ as the mobile phase.

The peak capacity of comprehensive multidimensional techniques approaches the product of the individual peak capacities of each separation. With the stop-flow conditions used for flow modulation, the peak capacity of the HPFC separation is equal to the peak capacity of the "new" chromatogram that is obtained after the average of flow and no-flow conditions have been obtained. Figure 5a shows an unmodulated chromatogram of the HPFC separation. Figure 5b shows the modulated chromatogram with the calculated average chromatogram superimposed. A peak capacity of 8 and 12 was calculated for the modulated and unmodulated chromatograms. The total peak capacity that was obtained for this HPFCxGC arrangement approached 500.

With the apparatus 10 little or no synentropy or cross information in respect of the selectivities of the HPFC and GC separations takes place.

Any such synentropy is wasteful. Thus, the separation mechanisms of the apparatus 10 are orthogonal to each other, and produce a rectangular separation space with compounds evenly distributed across the plane.

In the apparatus 10, the entire sample eluting from the first separation is analysed by the second separation, and the resolution achieved in the first analysis is conserved throughout subsequent analysis steps.

The Inventors are of the belief that independent fast temperature programming in the second dimension with a low temperature first dimension allows less correlation between the separation mechanisms of the first and second dimensions than with conventional systems. The two dimensions of the SFCxGC apparatus of the invention thus achieve greater orthogonality than existing GCxGC or SFCxGC instrumentation.

Furthermore, low temperature operation of the SFC dimension in this application increases selectivity to chemical differences in sample components such as polarity, chirality or molecular shape given a suitable stationary phase.

The apparatus of the invention also allows for less critical injection times into the second dimension than for existing GCxGC or existing SFCxGC instrumentation. In addition, temperature modulation in the SFCxGC interface is not required due to the focusing effect of the low temperature of the second dimension column.

The independent fast temperature programming in the second dimension also allows efficient separation of volatile organic modifiers, such as a low percentage methanol in C0_2, from analytes allowing use of the FID and Mass Spectrometer (MS) with such modifiers in the first dimension mobile phase. In addition, only the polydimethylsiloxane stationary phases that are able to withstand high temperatures need to be heated, seeing as the polar analysis is achieved at room temperature and does not rely on using polar phases such as polyethyleneglycol at elevated temperatures where they become unstable. This implies that samples with higher final boiling points can be analysed using the method and apparatus of the invention than with conventional GCxGC instrumentation.

Furthermore, thermally labile compounds are exposed to high temperatures for a much shorter time and are therefore less likely to decompose than with existing GCxGC or SFCxGC instrumentation where both columns are operated at high oven temperatures for a prolonged time. This invention thus expands the scope of compounds that may be analysed to include less stable compounds.

As regards stop-flow modulation, the Inventors are of the belief that the method and apparatus of the invention provides greater flexibility in the operational parameters of each dimension, allowing each dimension to be optimised independently of the other.

Secondly, the slower second dimension allows easier interfacing with conventional Mass Spectrometry (MS) than current GCxGC or SFCxGC instrumentation.

In addition, the need for complex thermal modulation of the interface is eliminated.

As regards the change in mobile phase composition, the Inventors are of the belief that the method and apparatus of the invention allows the use of a first dimension mobile phase that is incompatible with, or interferes strongly with, detection in the second dimension. Organic mobile phases or C0₂ with organic modifiers can thus be used together with FID or MS detection.

In addition, the change in mobile phase composition allows faster second dimension separations, thereby improving total analysis time by reducing the second dimension cycle time. This cycle is repeated many times during an analysis and thus contributes additively to the reduction of the total run time when stopped flow operation is used.

Furthermore, faster second dimension analysis allows the first dimension to be run faster while still allowing the transfer of ample cuts to the second dimension for further analysis. This assists in preserving the resolution already obtained in the first dimension and allows the analysis to be completed in a shorter time. Peak capacity production per unit time is thus improved.

Claims

1. A method of analysing a sample, which method includes subjecting a sample to be analysed to high pressure fluid chromatography (HPFC) in a first chromatographic dimension operating at a first temperature, thereby partially analysing the sample according to a first sample property and producing an eluent; passing a first discrete fraction of the eluent through a pressure- reducing zone which is in fluid flow connection with the first chromatographic dimension; thereafter passing that eluent fraction through a second chromatographic dimension which is in fluid flow connection with the pressure-reducing zone and which operates at a second temperature; ramping the second temperature independently of the first temperature, so as to effect fast gas chromatographic (GC) separation of the eluent fraction into further fractions of differing volatilities, in the second chromatographic dimension; . subsequently passing at least one further discrete fraction of the eluent from the first chromatographic dimension through the pressure- reducing zone and through the second chromatographic dimension; and subjecting each such further discrete fraction of the eluent to GC separation in the second chromatographic dimension, thereby to obtain a comprehensive two-dimensional analysis of the sample.

2. A method as claimed in Claim 1 , wherein the first chromatographic dimension effects polar analysis or separation of the sample, which is thus the first sample property.

3. A method as claimed in Claim 1 or Claim 2, wherein the first chromatographic dimension is provided by a first chromatographic column, with which group type of analysis of the sample can be achieved.

4. A method as claimed in Claim 3, wherein the first chromatographic column is a silica gel column.

5. A method as claimed in Claim 3 or Claim 4, wherein the first temperature is room temperature.

6. A method as claimed in Claim 3 or Claim 4, wherein the first temperature is above room temperature, with the first chromatographic column being subjected to temperature programming independently of the second chromatographic dimension.

7. A method as claimed in any one of Claims 3 to 6 inclusive, wherein the second chromatographic dimension is provided by a second chromatographic column.

8. A method as claimed in Claim 7, wherein the volatility separation in the second chromatographic column is effected by fast temperature programmed GC.

9. A method as claimed in Claim 7 or Claim 8, wherein C0₂ is used as a mobile phase for the sample in the first chromatographic dimension.

10. A method as claimed in Claim 9, wherein a carrier gas with a high diffusion coefficient is used as a mobile phase for the eluent fractions in the second chromatographic dimension.

11. A method as claimed in Claim 10, wherein the carrier gas is H₂.

12. A method as claimed in Claim 10 or Claim 11 , wherein the mobile phase composition is exchanged by way of an interface between the first and second chromatographic dimensions.

13. A method as claimed in any one of Claims 7 to 12 inclusive, which includes forming the discrete eluent fractions by means of flow modulation of the eluent from the first chromatographic column.

14. A method as claimed in Claim 13, wherein the flow modulation is effected by allowing the sample to pass through and exit from the first chromatographic column for a time interval; collecting the eluent from the first chromatographic column; interrupting the passage of the sample through the first chromatographic column so that the eluent which has formed during the passage of the sample through the first chromatographic column prior to the interruption constitutes the first discrete eluent fraction; focussing the first discrete eluent fraction on the head of the second chromatographic column by means of its passage or transfer through the pressure-reducing zone; subjecting this focused fraction to said GC by heating the second chromatographic column independently of the first chromatographic column; thereafter again allowing the sample to exit from the first chromatographic column for a time interval; again interrupting the passage of the sample through the first chromatographic column so that the eluent which has formed during the passage of the sample through the first chromatographic column prior to this interruption constitutes a further or second discrete eluent fraction; focussing the second discrete eluent fraction on the head of the second chromatographic column where it is subjected to said GC by heating the second chromatographic column independently of the first column; and so on, until all the sample has passed through the first and second chromatographic columns.

15. A method according to Claim 14, wherein the time intervals are all the same and are selected to provide at least one transfer for every peak created by the first chromatographic column.

16. A method as claimed in Claim 15, wherein from 5 to 10 transfers are made from the first to the second chromatographic column for each peak of the first chromatographic column.

17. A method as claimed in Claim 16, wherein the eluent fraction collection time is about 5 seconds.

18. A method as claimed in any one of Claims 14 to 17 inclusive, wherein the focussing of the eluent fractions from the first chromatographic column on the head of the second chromatographic column is pressure- drop focussing effected by passing the fractions through a restrictor, with the restrictor being in fluid flow connection with the first and second chromatographic columns.

19. An apparatus for analysing a sample, the apparatus including a first high pressure fluid chromatographic column; a second gas chromatographic column which is rapidly heatable independently of the first chromatographic column; pressure-reducing means between the first and the second chromatographic columns and in fluid flow connection with both chromatographic columns; and eluent transfer means for transferring eluent from the first chromatographic column to the second chromatographic column.

20. An apparatus as claimed in Claim 19, wherein the eluent transfer means is adapted to provide either stop-flow or continuous operation.

21. An apparatus as claimed in Claim 19 or Claim 20, which includes exchanging means for exchanging a mobile phase in the first chromatographic column with another mobile phase, having a different chemical composition, in the second chromatographic column.

22. An apparatus as claimed in any one of Claims 19 to 21 inclusive, which the apparatus includes a flow modulator associated with the eluent transfer means, with the modulator adapted to permit sequential passage of discrete fractions of eluent from the first chromatographic column to the second chromatographic column at selected time intervals.

23. An apparatus as claimed in Claim 22, wherein the flow modulator is an electrically actuated valve.

24. An apparatus as claimed in Claim 23, wherein the valve is a low dead volume valve.

25. An apparatus as claimed in Claim 23 or Claim 24, wherein the valve is programmable to allow the collection of eluent fractions from the first chromatographic column at time intervals of from 1 to 15 seconds.

26. An apparatus as claimed in any one of Claims 19 to 25 inclusive, which includes a flow restrictor associated with the eluent transfer means.

27. An apparatus as claimed in any one of Claims 19 to 26 inclusive, wherein the first chromatographic column is connected to a supply of C0₂ so that, in use, it contains C0₂ as a carrier fluid or mobile phase for the sample.

28. An apparatus as claimed in any one of Claims 19 to 27 inclusive, wherein the second chromatographic column is connected to a supply of H₂ so that, in use, it contains H₂ as a carrier gas.

29. A method as claimed in Claim 1, substantially as herein described and illustrated.

30. An apparatus as claimed in Claim 19, substantially as herein described and illustrated.

31. A new method or a new apparatus, substantially as herein described.