Patent Publication Number: US-2002008058-A1

Title: Tapered bore column for high performance liquid chromatography

Description:
FIELD OF THE INVENTION  
       [0001] The following invention relates to analytical high performance liquid chromatography where components of a sample are chromatographically separated before analytically determining the nature of the components of the sample. More particularly, this invention relates to columns for use in analytical high performance liquid chromatography systems which are configured to rapidly and precisely separate components of a sample to be analyzed.  
       BACKGROUND OF THE INVENTION  
       [0002] Liquid chromatography (LC) has been in use for nearly a century, for separation of a wide range of inorganic, organic and biological chemicals. Classical LC used large bore (10-50 mm ID) glass columns packed with large particle supports (silica gel, polymeric beads, etc.) using gravity flow to elute samples slowly (hours to days) from the column. LC can separate chemicals based on a wide variety of physical and chemical properties including size, charge and polarity.  
       [0003] High performance liquid chromatography (HPLC) was developed over thirty years ago, by packing smaller, more uniform particles in metal columns of constant bore (2-5 mm ID) and using high pressure pumps to flow liquids through the column. HPLC resulted in faster (minutes to hours), higher resolution separations. In the past decade, improvements in HPLC columns have included the use of more inert column tubes (316 stainless steel, glass-lined stainless steel, fused silica and solvent resistant high-pressure plastics).  
       [0004] Known prior art LC and HPLC columns have been packed in cylindrical tubes of consistent ID (same ID at the inlet and outlet of the column). HPLC columns are available in ID&#39;s from 0.05-50 1  mm. Conventional HPLC separations for analytical applications use tubular columns with ID&#39;s from 2-5 mm and lengths from 30-300 mm. Separations are usually run at an optimum linear velocity (˜5 cm/min) using a constant mobile phase (isocratic elution) or a continuously changing mobile phase (gradient elution) with total run times from 5-120 minutes, depending on the complexity of the samples being analyzed. Analytical HPLC accounts for over 90% of the use of HPLC, while more specialized techniques such as preparative HPLC for purification of large amounts of various chemicals of interest (columns with ID&#39;s from 10-50+ mm) and micro/capillary HPLC for trace level isolation and identification of various chemicals of interest (columns with ID&#39;s from 0.05-1.0 mm) make up the majority of the remaining uses of HPLC.  
       [0005] Although there are several different modes of HPLC (size exclusion, ion exchange, normal phase, reversed phase, etc.), reversed phase HPLC is used in over 90% of the analytical HPLC methods currently in use. In reversed phase HPLC, the stationary phase (bonded to a silica or polymeric packing) is non-polar and the mobile phase (pumped under high pressure through the column) is polar. Reversed phase HPLC was named because the initial mode of liquid chromatography (normal phase) used a polar stationary phase and a non-polar mobile phase.  
       [0006] The major area of application of HPLC is in the pharmaceutical industry. Over the past ten years, pharmaceutical companies have been developing techniques to synthesize greater numbers of chemicals in the continuing search for new pharmaceuticals to improve human health care and promote longer, healthier human life spans. This has resulted in the rapid growth of a new field called combinatorial chemistry, where a single chemist can design and automate the synthesis of hundreds to thousands of compounds per day (compared with an average of hundreds per chemist per year a decade ago). Paralleling the development of combinatorial chemistry has been the development of two related biochemical fields: 1) genomics—the characterization of the entire genetic make-up (DNA) of humans and other living organisms and 2) proteomics—the characterization of human proteins that are the key targets for pharmaceuticals that can help to cure various human diseases (Cancer, AIDS, Alzheimer&#39;s, Multiple Sclerosis, etc.). All three of these fields have put a huge demand on analytical chemists who are required to separate, purify, analyze, quantitate and characterize these vast arrays of chemicals and biochemicals with a range of techniques including HPLC.  
       [0007] Accordingly, a need exists for systems and devices which can more rapidly perform high pressure liquid chromatography, while maintaining the necessary accuracy. With this invention, a column is provided which can be used with existing high pressure liquid chromatography equipment and which can increase by five times or more the speed with which chromatographic separation of components of a sample can occur.  
       [0008] The column of this invention has a bore which tapers from a larger diameter to a smaller diameter to achieve highly accurate, more rapid chromatographic separation of sample components. Prior art columns and related fluid handling devices are known which include tapering profiles and/or outlets larger than inlets. Such prior art devices are described in detail in the following patents:  
                                                   Inventor   Patent Number                          Dalton   3,492,396           Fraser   3,771,659           Eisenbeiss   3,791,522           Randau   3,855,130           Hara   4,289,620           Golias   4,341,635           Ruijten   4,554,071           Shalon   4,719,011           Donald   4,787,971                      
 
       [0009] However, these prior art devices are not applicable in analytical HPLC systems. Either they are low pressure separation devices unrelated to HPLC or they are large volume preparative HPLC devices.  
       SUMMARY OF THE INVENTION  
       [0010] This invention provides an improved column for use in analytical high performance liquid chromatography systems. Rather than having a constant diameter cylindrical bore passing through the column, the column of this invention has a bore with a side wall which tapers. Specifically, the bore includes an inlet which is larger than the outlet and which has a tapering side wall defining the bore passing through the column. Tapering of the bore has been shown to accelerate chromatographic separation of components of the sample which passes through the bore. Tapering of the bore has also been shown to axially concentrate the individual sample components, resulting in enhanced resolution and sensitivity. The bore is packed with a chromatography separation medium which is appropriate for any one of the high pressure liquid chromatography separation modes, such as size exclusion, ion exchange, normal phase, reversed phase, etc.  
       [0011] The column preferably includes frits at ends thereof that retain the separation medium within the bore. The resulting packed bore column and frit cartridge can be easily placed into a holder for location within a high performance liquid chromatography system between a sample injector and a component detector. According to this invention, the column can take on many different forms, so long as the bore within the column tapers from a larger size at the inlet to a smaller size at the outlet, either with a constant rate of taper or with various other non-constant tapering configurations.  
       OBJECTS OF THE INVENTION  
       [0012] Accordingly, a primary object of the present invention is to provide a column for high performance liquid chromatography (HPLC) which provides faster analysis times than a standard cylindrical bore HPLC column with a similar inlet inner diameter.  
       [0013] Another object of the present invention is to provide a HPLC column which provides better sensitivity than an analogous standard bore HPLC column.  
       [0014] Another object of the present invention is to provide a HPLC column which provides a lower back pressure than a standard bore HPLC column at a common linear velocity.  
       [0015] Another object of the present invention is to provide a tapered bore HPLC column which has a similar sample capacity as a standard bore HPLC column.  
       [0016] Another object of the present invention is to provide a tapered bore HPLC column which provides lower back pressure than a standard bore HPLC column at a common flow rate.  
       [0017] Another object of the present invention is to provide a tapered bore HPLC column which provides a higher sample capacity than a standard bore HPLC column having an inner diameter equal to an outlet inner diameter of the tapered bore column of this invention.  
       [0018] Another object of the present invention is to provide a HPLC column which can achieve higher throughput than a standard bore HPLC column.  
       [0019] Another object of the present invention is to provide a higher sensitivity HPLC column than a standard cylindrical bore HPLC column.  
       [0020] Another object of the present invention is to provide a tapered bore HPLC column which can fit within existing HPLC equipment without significant modification of such equipment.  
       [0021] Another object of the present invention is to provide a HPLC column which can be formed from injection molded plastic materials and withstand pressures in excess of 5,000 psi. 
     
    
    
     [0022] Other further objects of the present invention will become apparent from a careful reading of the included drawing figures, the claims and detailed description of the invention.  
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0023]FIG. 1 is a full section view of a holder and column assembly similar to that which would be inserted into an analytical HPLC system between a sample injector and a component detector.  
     [0024]FIG. 2 is a perspective view of the column of this invention.  
     [0025]FIG. 3 is a full section view of that which is shown in FIG. 2 revealing the tapering bore passing therethrough and the frits located adjacent the inlet and the outlet of the bore.  
     [0026]FIG. 4 is a left end view of that which is shown in FIG. 2.  
     [0027]FIG. 5 is a right end view of that which is shown in FIG. 2.  
     [0028]FIG. 6 is a block diagram revealing how the column of this invention is interposed between a sample injector and a component detector as part of an overall analytical high pressure liquid chromatography system.  
     [0029]FIG. 7 is a graph depicting detection of peaks for different components within a sample over time at a given flow rate and pressure using the tapered bore HPLC column of this invention.  
     [0030]FIG. 8 is a graph analogous to that which is shown in FIG. 7 but utilizing a standard bore HPLC column having an inner diameter matching the inlet diameter of the tapered bore HPLC column of this invention at a similar flow rate, revealing how a significantly greater amount of time is required to achieve a similar amount of component detection resolution when compared to the tapered bore HPLC column of this invention.  
     [0031]FIG. 9 is a graph similar to that which is shown in FIGS. 7 and 8 but representing a higher pressure higher flow rate standard bore HPLC column which exhibits decreased resolution and sensitivity when compared to the detection resolution shown in FIG. 7 for the tapered bore HPLC column when given a similar amount of time.  
     [0032]FIG. 10 is a graph depicting five different gradient elution assays utilizing the tapered bore HPLC column of this invention and revealing how satisfactory separation resolution is achieved after approximately one minute as opposed to the three to six minutes required for similar gradient elution procedures utilizing a standard bore HPLC column.  
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
     [0033] Referring to the drawings, wherein like reference numerals represent like parts throughout the various drawing figures, reference numeral  20  (FIGS. 2 and 3) is directed to a column for use within a holder  10  (FIG. 1) as part of an overall analytical high performance liquid chromatography (HPLC) system (FIG. 6). The column  20  has a tapering bore  50  passing therethrough in contrast to cylindrical bores passing through prior art columns. The tapering bore  50  allows the column  20  to more rapidly and more precisely separate components of a sample for later detection.  
     [0034] In essence, and with particular reference to FIG. 3, the primary structural features of the column  20  of this invention are described. The column  20  is an elongate cylindrical structure which has a tapering bore  50  passing therethrough. An inlet ring  30  surrounds a first end  22  of the column  20 . The inlet ring  30  supports an inlet frit  38  overlying an inlet  52  of the tapering bore  50 . An outlet ring  40  is located adjacent a second end  24  of the column  20  and supports an outlet frit  48  adjacent an outlet  54  of the tapering bore  50 . While the tapering bore  50  can have various different configurations, the tapering bore  50  is preferably frusto-conical in form with a side wall  56  which tapers at a constant rate from the inlet  52  to the outlet  54 . The inlet  52  is thus larger in cross-sectional area than the outlet  54 .  
     [0035] More particularly, and with particular reference to FIG. 1, details of the holder  10  are described. Preferably, the column  20  is not placed directly into a HPLC device/system. Rather, the column  20  is first inserted into a holder  10 . The holder  10  with column  20  therein is then inserted into the HPLC system. The system typically additionally includes a sample injector  1  (FIG. 6) upstream from the column  20  and a detector  60  downstream from the column  20 . If necessary, the holder  10  can take on various different configurations to properly adapt to the dimensional requirements of a variety of different HPLC systems. The preferred holder  10  includes a supply manifold  12  which supports an inlet conduit  13  passing therethrough and is surrounded by a collar  14  (FIG. 1). An outlet manifold  16 , supporting a discharge conduit  17  therein, threadably engages the collar  14  with the column  20  between the outlet manifold  16  and the collar  14 .  
     [0036] In this way, the column  20  is securely supported within the holder  10  with the first end  22  of the column  20  directly adjacent the inlet conduit  13  of the supply manifold  12  and the second end  24  of the column  20  directly adjacent the discharge conduit  17  of the outlet manifold  16 . If necessary, the holder  10  can additionally assist the column  20  in withstanding forces associated with high pressure within the tapering bore  50  within the column  20 . While not shown in FIG. 1, the column  20  would typically have an inlet frit  38  (FIG. 3) and an outlet frit  48  appropriately inserted to maintain a chromatographic separation medium packed within the tapering bore  50  of the column  20 .  
     [0037] With particular reference to FIGS.  2 - 5 , details of the column  20  are described. The column  20  is preferably formed from a rigid mass of material in a unitary fashion with a generally cylindrical shape having the tapering bore  50  passing along a central axis of the column  20 . Preferably, the column  20  is formed by injection molding from a polymeric hydrocarbon material having the ability to withstand pressures up to 5,000 psi and which is highly non-reactive with a variety of different materials which might be present as components within samples passed through the column  20 . Most preferably, the column  20  is formed from a poly ether ether ketone material.  
     [0038] The column  20  preferably has a cylindrical outer wall  26  extending between a first end  22  and a second end  24 . The first end  22  supports an inlet ring  30  integrally formed with the first end  22 . The second end  24  includes an outlet ring  40  integrally formed with the second end  24  of the column  20 .  
     [0039] The inlet ring  30  is dimensioned to bear against portions of the holder  10  both radially and axially to properly position the column  20  where desired within the holder  10 . Specifically, the inlet ring  30  includes an inlet rim  32  which is flat and defines the axial extremity of the first end  22  of the column  20 . An outside wall  33  defines a radial extremity of the outlet ring  40 . The outlet wall  43  is preferably cylindrical and extends further from the central axis of the column  20  than the outer wall  26 . A frit recess  34  extends in from the inlet rim  32 . An inside wall  35  defines a perimeter of the inlet frit recess  34 . A face  36  of circular form is surrounded by the inside wall  35 .  
     [0040] The inside wall  35  is preferably substantially cylindrical but actually slightly tapering. This slight taper allows an inlet frit  38  to be press fitted into the inlet frit recess  34  with an interference fit between the inlet frit  38  and the inside wall  35 . The inlet frit  38  is preferably formed from titanium and has fenestrations therein which are smaller than a particle size of the chromatographic separation medium packed within the tapering bore  50  of the column  20 . The inlet frit  38  thus helps maintain the separation medium within the bore  50 .  
     [0041] The outlet ring  40  is substantially similar in form to the inlet ring  30 . Hence, the outlet ring  40  includes an outlet rim  42 , outside wall  43 , outlet frit recess  44 , inside wall  45  and face  46 . An outlet frit  48  of similar construction to the inlet frit  38  is press fitted into the outlet frit recess  44 . The outlet frit  48  thus assists in holding the separation medium within the tapering bore  50  of the column  20 . Typically, the inlet frit  38 , outlet frit  48  and separation medium are not considered part of the column  20 . Rather, when the column  20  is packed with the separation medium and the frits  38 ,  48  are press fitted in place, the assembly of column  20 , inlet frit  38 , outlet frit  48  and separation medium are referred to as a cartridge. This packed cartridge is then placed into the holder  10  for eventual use within a HPLC system.  
     [0042] The tapering bore  50  extends entirely from the first end  28  of the column  20  to the second end  24  of the column  20 . The tapering bore  50  includes an inlet  52  adjacent the first end  22  and an outlet  54  adjacent the second end  24 . A side wall  56  extends between the inlet  52  and the outlet  54 . The side wall  56  is preferably frusto-conical in form having a constant rate of taper between the inlet  52  and the outlet  54 . The inlet  52  thus maintains a larger diameter than the outlet  54 .  
     [0043] The particular dimensions for the inlet  52 , outlet  54  and rate of taper of the side wall  56  can be varied depending on the particular needs of the user. In at least one application it has been shown to be effective to provide an inlet  52  of circular cross-section with a 2.0 millimeter diameter and an outlet  54  with a circular cross-section of 0.5 millimeter diameter, and with a side wall  56  which tapers at a constant rate between the inlet  52  and the outlet  54  on a column approximately 1.0 inches long. Alternatives for the configuration of the tapering bore  50  include allowing the side wall  56  to taper in steps between the inlet  52  and the outlet  54 , or to have an accelerating or decelerating rate of taper, such that a greater or lesser slope away from a central axis of the bore  50  is exhibited closer to the inlet  52  or the outlet  54 .  
     [0044] With particular reference to FIG. 6, details are described of the system in which the tapered bore column  20  of this invention can be utilized. A typical HPLC system utilized for sample analysis includes a sample injector  1  located upstream from the column  20 . The sample is directed, along arrow A, to the inlet  52  of the column  20 . The sample then passes through the tapered bore  50  of the column  20 , along arrow B where the sample is separated into its components by an appropriate separation medium packed into the tapering bore  50 . The components of the sample are then sequentially discharged out of the outlet  54  of the tapering bore  50  and are passed onto the detector  60 , along arrow C. The detector  60  can be any of a variety of different devices including an ultraviolet spectrophotometer, a mass spectrometer or other device capable of determining either mere presence of the components of the sample, the identity or characteristics of the components of the sample, and/or the specific quantity of one or more sample components. This information can then be used in accordance with the needs of the user, such as to verify the purity of the sample being analyzed.  
     [0045] The particular efficacy of the tapered bore column  20  of this invention is particularly illustrated by consideration of the following examples. In conventional HPLC the separation of the sample components (resolution) is a function of the column length, linear velocity of the mobile phase (optimum at ˜5 cm/min, independent of the ID for conventional tubular columns), temperature, column packing particle diameter and selectivity of the column packing stationary phase. Simple samples (1-10 different sample components of similar polarity) are usually separated in 5-30 minutes on columns from 50-300 mm long, packed with particles from 3-10 microns in diameter using a mobile phase of constant composition (isocratic elution). More complex samples (10-100+ different sample components of widely varying polarity) often require separation time of 30-300+ minutes using a continuously changing mobile phase (gradient elution) from polar to non-polar solvents in reversed phase HPLC.  
     [0046] To illustrate the particular efficacy of this invention, a sample containing four distinct components, (methyl, ethyl, propyl and butyl paraben), was analyzed in a HPLC system (Magic 2002 HPLC, Michrom BioResources, Inc., Auburn, Calif.) including the tapered bore column  20  of this invention. A flow rate of 200 micro liters per minute and a pressure of 200 psi were utilized. The sample was completely analyzed after 1.3 minutes with an appropriately high level of resolution and sensitivity.  
     [0047] In a second experiment (FIG. 8) the same sample was utilized in the same HPLC system but with a standard bore HPLC column utilized rather than the tapered bore column  20  of this invention. The column had a 2.0 millimeter inner diameter along its length which matched the inlet inner diameter of the tapered bore column  20  utilized in the first example (FIG. 7). The same flow rate of 200 micro liters per minute was utilized with a pressure of 100 psi only being necessary to push the sample through the standard bore HPLC column. A similar level of separation resolution and sensitivity was obtained as that provided by the tapered bore column  20 , but required over six minutes to complete. Hence, an approximately five-fold increase in time was required to analyze the sample with a similar degree of resolution and sensitivity when utilizing a standard cylindrical bore HPLC column.  
     [0048] With reference to FIG. 9, a different approach to high speed chromatographic separation of sample components utilizing a standard bore HPLC column was compared to the results obtained with the tapered bore column  20  of this invention. Specifically, the same sample was utilized in the same HPLC system, but the flow rate was raised to 1,000 micro liters per minute and the pressure raised to 500 psi. The sample was fully analyzed after 1.3 minutes just as when utilizing the tapered bore column of this invention. However, the sensitivity obtained in this higher pressure higher flow rate standard bore HPLC column example was significantly degraded. Accordingly, the test data depicted in FIGS.  7 - 9  show that the tapered bore column  20  of this invention provides either higher sensitivity in a similar amount of time at lower flow rates and lower pressure or higher speed with similar sensitivity when compared to standard bore HPLC columns. In either case, an advantage of approximately five times was evidenced.  
     [0049] Another technique for maximizing the speed of analytical HPLC, especially when the sample contains many components (i.e. ten or more) involves utilizing a gradient elution HPLC process. In gradient elution HPLC, resolution is usually improved (at the cost of time) by increasing the time it takes to go from the polar starting solvent to the non-polar final solvent. This improvement in resolution is due to the fact the column bed volume is changed more times during a long gradient than during a short gradient. This concept is shown in FIG. 10, where the number of column volumes (CV) per minute is increased from 5 to 60, and the resulting resolution and peak capacity (the total number of component peaks that can be separated over the gradient time) improve in direct proportion to the number of column volumes displaced during the gradient.  
     [0050] When the data is generated using a conventional cylindrical bore HPLC column with a 2.0 mm ID and a 25 mm length, running at a flow rate of 1,000 ml per minute (a linear velocity of 25 cm per minute or five times the optimum linear velocity). Optimum resolution is achieved in the 30-60 CV/min range, but using a short, conventional cylindrical bore column (constant ID at five times the standard flow velocity), this level of resolution requires gradient times of 3-6 minutes, and total analysis times of 5-10 minutes.  
     [0051] Because of the tapered bore of the column  20  of this invention, the total column bed volume is only 20% of the volume of a conventional HPLC column of the same length with a constant ID equal to the inlet ID of the column  20  of this invention (i.e. 2.0 mm). At the same flow rate as the conventional HPLC column (1,000 ml/min), the column  20  of this invention would displace 30-60 CV in 36-72 seconds (FIG. 10), resulting in total analysis times between 1-2 minutes, a five-fold improvement.  
     [0052] In addition to the improvements in resolution per unit time gained using the column  20  of this invention due to its lower column volume, the tapering bore  50  also helps to improve resolution by axially concentrating the individual sample component bands as they traverse the length of the column  20 . This axial compression results in narrower sample peaks at the outlet  54  of the bore  50 , further improving resolution and peak capacity for complex separations. In effect, the sample component concentration at the outlet  54  of the bore  50  is sixteen times greater than at the inlet  52  (due to a four time reduction in ID from the inlet  52  the outlet  54 ). Although the same concentration could theoretically be achieved using a conventional HPLC column with an ID equal to the ID at the outlet  54  of the bore  50  (i.e. 0.5 mm), the pressure required to run such a column at the same flow rate (i.e. 1,000 ml/min) would be prohibitively high (much greater than the 5,000 psi upper pressure limit of most HPLC columns and systems).  
     [0053] A third advantage of the tapered bore design of the column  20  of this invention for complex samples is that the total sample loading capacity is equal to that of a conventional column with a constant ID equal to the inlet ID of the column  20  of this invention. This is especially important for quantitative assays of pharmaceutical compounds and their metabolites in physiological fluids. In order to achieve the high throughput and high sensitivity required by these quantitative assays (in vitro metabolism studies, pharmacokinetic studies, etc.), large volumes of sample are injected onto the HPLC column. Although decreasing the column ID will result in better sensitivity for single component standards, this is not true for physiological fluid assays because the volume of sample injected must be proportional to the ID of the column to prevent overloading and rapid destruction of the column. Using the column  20  of this invention, the sample capacity at the inlet  52  is the same as for a conventional HPLC column of the same ID, but as the components in the sample are separated during the gradient elution process, the individual amount of each component is much less than the total and therefore the capacity at the outlet  54  ({fraction (1/16)} the area of the inlet  52 ) is sufficient to prevent overloading by the separated individual sample components.  
     [0054] This disclosure is provided to reveal a preferred embodiment of the invention and a best mode for practicing the invention. Having thus described the invention in this way, it should be apparent that various different modifications can be made to the preferred embodiment without departing from the scope and spirit of this disclosure. When structures are identified as a means to perform a function, the identification is intended to include all structures which can perform the function specified.