Abstract:
This invention is a method and device for use with multi-dimensional chromatography that utilizes partial modulation. An analyte-bearing sample is subjected to a first dimension of chromatography. Thereafter the separated analyte-bearing sample is diluted with a modulated second carrier such at the analyte-bearing sample is not stopped or its temperature altered. The partially modulated analyte-bearing sample then feeds into a secondary column where the analyte-bearing sample is further separated.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims the benefit of U.S. Provisional Patent Application No. 60/498,821 entitled, “Partial Modulation via Pulsed Flow Modulator for Comprehensive Two-Dimensional Liquid or Gas Chromatography,” filed on Aug. 29, 2003 in the U.S. Patent and Trademark Office. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002]     Not Applicable.  
       BACKGROUND OF THE INVENTION  
       [0003]     1. Field of the Invention  
         [0004]     The present invention relates to two-dimensional chromatography. Specifically this invention relates to devices and method for performing two-dimensional liquid or gas chromatography with partial modulation.  
         [0005]     2. Description of the Related Art  
         [0006]     Two-dimensional chromatography is known in the art. The fields of both gas and liquid chromatography utilize two-dimensional separation techniques to analyze sample matrices contained in a sample analyte.  
         [0007]     Comprehensive two-dimensional liquid or gas chromatography utilizes two orthogonal columns connected in series to separate compounds within a sample. The term “orthogonal” as used herein means that the columns separate compounds within a sample based on two different properties of the compounds. Ideally, the different properties are independent of each other, resulting in a lack of correlation between the retention time of the compound in the first column and its retention time in the second column. The stationary phase in the second column should yield a faster separation than that of the first column. In the prior art, a sample is injected into the injection port and integrated with the mobile phase. The mobile phase is necessary to introduce and transport the sample through the column. In gas chromatography, the mobile phase is generally an inert gas and is often referred to as the carrier gas. In liquid chromatography, the mobile phase is a liquid of low viscosity and is often referred to as the carrier fluid. Injection of the sample may be by a syringe or operation of a valve or valves connected to a source or loop, among other methods. As the mobile phase transports the sample through the first column, the compounds in the sample are separated based on a first property. In the prior art, the first-column separated sample-bearing carrier exits the first column and is thereafter trapped and held by operation of a modulator, which releases the first column separated sample-bearing carrier in “plugs” or “packets” to the second column. The period during which a packet of first column separated sample-bearing carrier enters the second column is typically measured in seconds and is referred to as the secondary retention time. A detector at the exit of the second column measures the intensity of compounds in each packet at the conclusion of the second separation.  
         [0008]     Primary retention time and secondary retention time identify each compound in the sample-bearing carrier in three dimensions. Modeling of the data with intensity in three-dimensions displays quantitative and qualitative properties of the compounds within the sample.  
         [0009]     In prior art two-dimensional gas chromatography, the sample-bearing carrier was fully modulated. U.S. Pat. No. 5,196,039 issued to Phillips et al. on Mar. 23, 1993 discloses a thermal modulator device and method of performing comprehensive multi-dimensional chemical separation using a first dimension of a two-dimensional chromatograph to generate a chromatogram in a time comparable to or even faster than prior practice while the second dimension generates multiple chromatograms each in a time comparable to the fastest prior art chromatography. The transfer of sample portions from the first dimension to the second dimension is by any one of several sample-bearing carrier modulation techniques wherein portions of sample-bearing carrier are accumulated between the first and second dimensions and transferred as very sharp concentration pulses.  
         [0010]     An article entitled, “Time-resolved Cryogenic Modulation for Targeted Multidimensional Capillary Gas Chromatography Analysis” by Philip J. Marriott et al. was published in the  Journal of Chromatography  in 2000. The article discloses a method incorporating two directly coupled columns and employing a longitudinally modulated cryogenic trap located between the columns. A method termed “selected zone compression pulsing” is used. All of the first column effluent is passed to the second column. The times at which the modulation of the trap is performed determines which target solutes will be selected for enhanced separation, allowing almost instantaneous separation of selected zones in the second column.  
         [0011]     U.S. Pat. App. Pub. No. US 2002/0148353 by Seeley published on Oct. 17, 2002 discloses a two-dimensional gas chromatograph with a primary column and dual secondary columns. Flow rates in the primary column are less than those in the secondary column due to an accumulation valve. The accumulation valve operates to accumulate the sample as it exits the primary column, and introduce the accumulated sample to the dual secondary columns. Typically the ratio of the combined dual secondary columns flow capacity to the primary column flow capacity is approximately between 10 to 1 and 30 to 1.  
         [0012]     U.S. Pat. No. 6,007,602 issued to Ledford, Jr. et al. on Dec. 28, 1999 discloses an apparatus and a method for forming a chemical modulation of a substance present in a fluid stream. The apparatus utilizes a movable device, such as a movable heater, to induce changes in the retention of a chemical substance flowing through the modulator tube.  
         [0013]     U.S. Pat. No. 6,702,989, issued to Sacks et al. on Mar. 9, 2004 and U.S. Pat. Nos. 6,706,534, and 6,706,535 both issued to Sacks et al. on Mar. 16, 2004 disclose a gas chromatography system having a computer-controlled pressure controller that delivers pressurized pulses to a column junction point of two series-coupled columns having different stationary-phase chemistries. Each pressurized pulse causes a differential change in the carrier gas velocities in the two columns, which lasts for the duration of the pressurized pulse.  
         [0014]     Comprehensive 2-D chromatography with full modulation of the sample-bearing carrier is not without drawbacks as to temperature, size and power requirements of equipment, and time required for secondary dimension analysis. Each column is operated throughout a temperature range, which may be beyond the range of the modulator. In such cases, the sample-bearing carrier is removed from the higher-temperature environment of the first column to a second environment where full modulation occurs, then reintroduced to the higher temperature environment to pass through the second column. Such cooling and heating may alter the compounds within the sample-bearing carrier, skewing the results. Such a full modulator may require cryogenic cooling, restricting the size and portability. Finally, the secondary retention time for full modulation should be long enough for a full separation in secondary dimension.  
         [0015]     It would be an improvement to the art to be able to sufficiently modulate an analyte-bearing sample to permit comprehensive 2-D liquid or gas chromatography which would not alter the chemical properties of primary separation and which would permit a shorter secondary retention time.  
       BRIEF SUMMARY OF THE INVENTION  
       [0016]     Accordingly, it is an object of the present invention to provide a partial modulation device and method for use in multi-dimensional chromatography that: 
        possesses both a primary and secondary signal;     maintains resolution of the primary signal; and     permits a shorter secondary retention time.        
 
         [0020]     This invention is a method and device for use with multi-dimensional gas or liquid chromatography that utilizes partial modulation. A sample-bearing first carrier is subjected to a first dimension of chromatography. Thereafter the primary-column separated sample-bearing first carrier is partially modulated, which occurs by modulation between two concentrations by dilution with a second carrier, which will be a carrier gas in gas chromatography and a liquid in liquid chromatography, such that the primary-column separated sample-bearing carrier is not stopped or its temperature altered. The second carrier may be, but is not required to be, of the same composition as the first carrier. The partially-modulated primary-column separated sample-bearing first carrier then feeds into a secondary column where the primary-column separated sample-bearing first carrier is further separated. A detector at the end of the secondary column is used to determine the intensity of compounds exiting the column. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0021]      FIG. 1  is a schematic of the inventive chromatograph with partial modulation.  
         [0022]      FIG. 2  is a top view of three-way connector.  
         [0023]      FIG. 3  is a schematic of the modulator.  
         [0024]      FIG. 4A  depicts three-way connector with primary-column separated sample-bearing first carrier and higher flow rate second carrier entering connector.  
         [0025]      FIG. 4B  depicts three-way connector with lower flow rate second carrier entering connector.  
         [0026]      FIG. 4C  depicts formation of partially-modulated primary-column separated sample-bearing first carrier when pulsed higher flow rate carrier enters three-way connector.  
         [0027]      FIG. 5A  is a series of graphs of a fully modulated sample-bearing first carrier undergoing two-dimensional chromatography.  
         [0028]      FIG. 5B  is a series of graphs of a partially modulated analyte-bearing sample undergoing two-dimensional chromatography.  
         [0029]      FIG. 6A  depicts the results of chromatography on a partially-modulated analyte-bearing sample in the negative pulse mode.  
         [0030]      FIG. 6B  depicts the results of chromatography on a partially-modulated analyte-bearing sample in the positive pulse mode. 
     
    
     DESCRIPTION OF THE INVENTION  
       [0031]     Structure and Method  
         [0032]     Referring to  FIG. 1 , a chromatograph with partial modulation is depicted as  10 . The chromatograph  10 , for liquid or gas, comprises an injector  20 , a primary column  30 , a modulator  50 , a three-way connector  60 , a secondary column  70 , and a detector  80 .  
         [0033]     The sample-bearing first carrier  100  to be analyzed is introduced into primary column  30  by injector  20 . In the preferred embodiment, for gas chromatography, primary column  30  is located within a thermal chamber  40 , which maintains sample-bearing first carrier  100  at a predetermined temperature.  
         [0034]     As sample-bearing first carrier  100  transverses primary column  30 , various compounds within sample-bearing first carrier  100  linger in primary column  30  for differing lengths of time. The length of time in which a compound remains in primary column  30  depends upon its affinity for the stationary phase in that column. Compounds with less affinity for the stationary phase in primary column  30  will progress faster than compounds having an affinity for the stationary phase. Thus, various compounds exit primary column  30  at different points in time after their injection into primary column  30 .  
         [0035]     From primary column  30 , a now primary-column-separated sample-bearing first carrier  100  enters three-way connector  60 . Three-way connector  60  interfaces between primary column exit  34  and secondary column entrance  72 . Preferably, three-way connector  60  is under the same conditions as primary column  40 . In the preferred embodiment, for gas chromatography, three-way connector  60  is located in thermal chamber  40 .  
         [0036]     As depicted in  FIG. 2 , in the preferred embodiment, three-way connector  60  is a press-on Y-connector having two entrance ports  64  and  66  and an exit port  62 . There is an angle  68   a  between entrance port  64  and exit port  62  and an angle  68   b  between entrance port  66  and exit port  62 . Angles  68   a  and  68   b  are greater than 90 degrees, that is each entrance port  64  and  66  is located at an obtuse angle from exit port  62 .  
         [0037]     Referring to  FIGS. 1 and 2 , primary column exit  34  is connected to one entrance port  64 . Exit port  62  is connected to secondary column entrance  72 . Therefore, the flow of primary-column-separated sample-bearing first carrier  110  is unobstructed between primary column  30  and secondary column  70 .  
         [0038]     Referring to  FIG. 3 , modulator  50  is depicted. In the preferred embodiment, modulator  50  includes a T-connector  59 , through which a second carrier  150  is divided into two second carriers  152  and  154 . The flow rate of second carrier  152  is adjusted downstream to be slower than the flow rate of the second carrier  154 . Regulators  57  and  58 , located along the path of second carrier  152  and  154 , respectively, are adjusted to provide a slower flow rate to second carrier  152  than the flow rate of second carrier  154 . In the preferred embodiment, regulators  57  and  58  regulate pressure for gas chromatography.  
         [0039]     Each second carrier  152  and  154  passes through a cross connector  55  and  56  respectively. In the preferred embodiment, pressure gauges  53  and  54  are also connected to cross connectors  55  and  56  respectively to monitor the pressure of second carrier  152  and  154  respectively for gas chromatography. Each cross connector  55  and  56  also connects to a valve  51  or  52  respectively, the purpose of which is discussed below and which are needle valves in the preferred embodiment. The remaining terminal of each cross connector  55  and  56  connects to a valve  90 .  
         [0040]     Valve  90 , which may be a fast solenoid switch, alternates the flow into a modulator exit  92  between slower second carrier  152  and faster second carrier  154 . Modulator exit  92  connects directly to entrance port  66  of three-way connector  60 .  
         [0041]     Needle valves  51  and  52  are used to vent second carrier  152  and  154 , respectively. When slower second carrier  152  is directed to modulator exit  92  by valve  90 , faster second carrier  154  is vented through needle valve  52 . Likewise, when faster second carrier  154  is directed to modulator exit  92 , slower second carrier  152  is vented through needle valve  51 .  
         [0042]     As previously discussed, the difference in flow rates between the slower second carrier  152  and the faster second carrier  154  is adjusted so that the flow rate of slower second carrier  152  is less than that of faster second carrier  154 . The flow rate of slower second carrier  152  is set at a rate sufficient to prevent upstream contamination by primary-column-separated sample-bearing first carrier  110  in entrance port  66 . Specifically, the flow rate of slower second carrier  152  should stop any part of primary-column-separated sample-bearing first carrier  110  from entering entrance port  66 .  
         [0043]     Modulator  50  may have alternative structures so long as the second carrier flow rate can be cycled between a faster and a slower flow rate. Alternative embodiments of modulator  50  include a pump (not shown), an impeller (not shown), or a valve that cycles between differently sized orifices (not shown). Other structures that allow cycling of the flow rate of the second carrier may also be used as a modulator ( 50 ).  
         [0044]     Referring to  FIGS. 4   a  through  4   c , the result of introducing modulated faster second carrier  154  and slower second carrier  152  to three-way connector  60  is depicted in a negative pulse mode.  FIG. 4   a  shows faster second carrier  154  and primary-column-separated sample-bearing first carrier  110  entering three-way connector  60  through entrance ports  66  and  64  respectively. Within exit port  62 , primary-column-separated sample-bearing first carrier  110  and faster second carrier  154  mingle to form more-diluted primary-column-separated sample-bearing first carrier  160 .  
         [0045]      FIG. 4   b  depicts slower second carrier  152 , which flows at a rate sufficient to prevent upstream contamination into entrance port  66 , and primary-column-separated sample-bearing first carrier  110  entering entrance ports  66  and  64  respectively. The flow rate of slower second carrier  152  should be low enough to permit detection of compounds in the sample by detector  80 .  
         [0046]      FIG. 4   c  shows the effect of re-introduction of the faster second carrier  154 . Primary-column-separated sample-bearing first carrier  110  is again diluted to form more-diluted primary-column-separated sample-bearing first carrier  160 . Less diluted primary-column-separated sample-bearing first carrier  110  becomes packet  165 . Packet  165  is bounded by volumes of higher dilution caused when faster second carrier  154  is cyclically added to primary-column-separated sample-bearing first carrier  110 .  
         [0047]     Because the second carrier  150  is fed into three-way connector  60  with alternate flow rates and because primary-column-separated sample-bearing first carrier  110  continuously flows from primary column  30  through three-way connector  60 , the resulting stream is continuous. As a result, primary-column-separated sample-bearing first carrier  110  is now partially modulated between volumes of more-diluted primary-column-separated sample-bearing first carrier  160  and packet  165 , collectively forming partially-modulated carrier  170 .  
         [0048]     Negative pulse mode is characterized by cycling the second carrier  150  flow rate from a high flow rate for a long duration of time to a low flow rate for a short duration of time. The short duration of time slower second carrier  152  enters entrance port  66  results in a small packet  165  of less diluted primary-column-separated sample-bearing first carrier  110  exiting three-way connector  60 .  
         [0049]     Positive pulse mode is characterized by cycling the second carrier  150  flow rate from a low flow rate for a long duration of time to a high flow rate for a short duration of time. The short duration of time over which faster second carrier  154  enters entrance port  66  results in a longer packet of less dilute primary-column-separated sample-bearing first carrier  110 .  
         [0050]     Partially-modulated carrier  170  enters secondary column  70 . In the preferred embodiment, secondary column  70  contains a stationary phase different from the stationary phase of primary column  30 , such that primary column  30  and secondary column  70  are orthogonal and the separation in secondary column  70  is faster than the separation in primary column  30 . Alternatively, different temperatures in the first column  30  and second column  70  could achieve the desired difference in separation speed. Through secondary column  70 , the compounds in partially-modulated carrier  170  further separate. The partially-modulated, second-column-separated partially-modulated carrier  180  exits column  70 .  
         [0051]     At the exit of secondary column  70 , a detector  80  reads intensity levels of the compounds in second-column-separated partially-modulated carrier  180  at a predetermined frequency. The frequency of the detector  80  readings should be adjusted so that there are sufficient readings per modulation. More readings result in improved accuracy in interpreting the results of the two dimensional chromatography.  
         [0052]     Results of Partial Modulation Compared with Full Modulation  
         [0053]      FIG. 5A  graphically shows the results of two-dimensional gas chromatography with full modulation. Analog graph  300  shows peaks of multiple compounds at the conclusion of a first dimension of separation. The x-axis  305  indicates the primary retention time, or the time in which a particular compound is retained in the first column. The y-axis  308  indicates the intensity of the compounds detected. Interpretation of the data at this point is difficult because the primary retention times of the analyzed compounds are similar. The distributions of the intensity of the compounds present after single dimension gas chromatography overlap, making it difficult to discern the curve of one compound from that of the other compound. Two peaks  302  and  304  can be discerned, however the individual curves are indistinguishable.  
         [0054]     In the prior art, the analyte-bearing sample, which has been separated in the first column, is fully modulated. Modulated graph  310  shows the result of fully modulating the analyte-bearing sample between the exit of the primary column  30  and the entrance of secondary column  70 . The x-axis  315  represents the primary retention time and the y-axis  318  represents the intensity of the detected compounds. Two peaks  302  and  304  are still discernable, but the curves associated with the individual compounds are still obscured.  
         [0055]     The fully modulated analyte-bearing sample enters secondary column  70 , where the compounds in the analyte-bearing sample are further separated by an orthogonal means of chromatography. Detector  80  senses the intensity of compounds exiting secondary column  70 . The results of the chromatogram after two dimensions of separation and modulation of the analyte-bearing sample are shown on detector graph  320 . The x-axis  325  and y-axis  328  still represent retention time and intensity as in the previous graphs. Because the sample analyte-bearing sample underwent an orthogonal means of separation, peaks  302  and  304  are easier to distinguish, as is most of the curve associated with each peak.  
         [0056]     Further improvement in the interpretation of data may be found by looking at the data generated as depicted in contour graph  340 . The x-axis  345  of contour graph  340  represents the primary retention time, or the time the sample was retained in the chromatograph. The y-axis  348  represents the secondary retention time, or the time between modulations. Each of the contour lines  346  represents different intensity levels. The area inside the center-most contour line represents the peak  302  or  304  of the detected compound. By graphically depicting the detected intensity levels in two time dimensions, the chromatographer may more easily discern the compounds present and detected in the sample.  
         [0057]      FIG. 5B  graphically shows the results of two-dimensional chromatography with partial modulation. Analog graph  400  has the same characteristics as analog graph  300 , where the x-axis  405  represents retention time and the y-axis  408  represents intensity of the detected compounds. Two peaks  402  and  404  are somewhat discernable, but the curves are overlapping to such an extent that they are not clearly identifiable.  
         [0058]     The analyte-bearing sample is partially modulated by alternately introducing a faster and slower second carrier. The result of partially modulating the analyte-bearing sample is depicted in partial modulation graph  410 .  
         [0059]     The partially modulated analyte-bearing sample is passed through a second dimension of chromatography, where the compounds in the analyte-bearing sample are further separated. Detection graph  415  shows that the analyte-bearing sample exiting the second column contains diluted compound alternated with packets of concentrated compound. Spikes occur where the detector processes the sharper pulses, or packet, of concentrated compound.  
         [0060]     The partially modulated analyte-bearing sample contains the same information as the fully modulated analyte-bearing sample. The contour graph  440  resulting from partial modulation depicts the same information as contour graph  340  resulting from full modulation.  
         [0061]     The use of partial modulation with comprehensive two-dimensional chromatography allows faster second dimension separation than the use of full modulation. The speed in which more-diluted primary-column-separated sample-bearing first carrier  160  and packet  165  are transferred through the second column  70  may be faster than that possible with full modulation of the analyte-bearing sample.  
         [0062]      FIGS. 6A and 6B  depict the difference in the detection of compounds between partial modulation in negative pulse mode and positive pulse mode. After separation in second column  70 , detector  80  outputs signals associated with compounds present in the analyte. The signal associated with each compound is separated by the signal associated with the second carrier.  
         [0063]      FIG. 6A  shows the chromatographic result of using negative pulse mode partial modulation. In negative pulse mode, the intensity of the signal associated with each compound in concentrated primary-column-separated sample-bearing first carrier  110  will be greater than and in addition to the signal associated with second carrier  150  and highly diluted primary-column-separated sample-bearing first carrier  110 .  
         [0064]      FIG. 6B  shows the chromatographic result of using positive pulse mode partial modulation. In positive pulse mode, the intensity of the signal associated with each compound in concentrated primary-column-separated sample-bearing first carrier  110  will be less than and in opposition to the signal associated with second carrier  150  and highly diluted primary-column-separated sample-bearing first carrier  110 .  
         [0065]     The foregoing description of the invention illustrates a preferred embodiment thereof. Various changes may be made in the details of the illustrated construction within the scope of the appended claims without departing from the true spirit of the invention. The present invention should only be limited by the claims and their equivalents.