Abstract:
Devices for heating and cooling chromatographic columns, transfer tubing, fittings and accessories and can also be placed next to the mass spectrometer&#39;s inlet region are disclosed. These devices have the advantage of allowing the user to dramatically reduce the post-column dead-volume while using the heating or cooling devices, a necessity for low-flowrate liquid chromatography. These devices also do not require the user to heat or cool the entire column with fittings to achieve optimized benefits for chromatography.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    The present application claims the benefit of U.S. patent application Ser. No. 61/520,032, filed Jun. 3, 2011 and U.S. patent application Ser. No. 61/521,568 filed Aug. 9, 2011, which are hereby incorporated by reference in their entirety. 
     
    
     TECHNICAL FIELD 
       [0002]    The present application relates to devices that improve the performance of low-flow-rates high-performance liquid chromatography. 
       BACKGROUND OF THE INVENTION 
       [0003]    High performance liquid chromatography (HPLC) is a technique used widely to separate a mixture of chemical species in a liquid mobile phase based on their interactions with the stationary phase of the particles packed into a tube or capillary called a column. For many separation applications, it is desirable to have the temperature of the chromatographic column elevated above room temperature to gain the benefits of faster peak elution, better chromatographic resolution, better peak shape especially for hydrophobic eluting species, better retention times reproducibility, lower back pressure, reduced carry-over, etc. In the art, the most common column heater is in the form of an oven which is an enclosed, well insulated space typically much larger than the column itself. In another instance of the art when the column is a fused silica capillary, the column heater may be an extremely flexible slender cylindrical sleeve not much larger in diameter but longer than the fused silica capillary column itself that may damage the sharp nanospray emitter during column insertion into the sleeve if the emitter is an integrated front end of the capillary column. Still another kind of column heater in the art is of the “blanket” type which wraps around the column and also its end fittings. These blanket-style column heaters are also much larger in size than the capillary column itself. 
         [0004]    The large sizes of the oven-style or blanket-style column heaters are undesirable since the column contained within these heaters have to be placed further away from the mass spectrometer inlet than is desirable. In the case of the ovens which are typically integrated into their respective liquid chromatography (LC) pumps, the distance between the heated column and the mass spectrometer inlet where the eluates are detected is large because the LC pump cannot be placed close enough to the mass spectrometer. The large sizes of both the oven-style and the blanket-style heaters are primarily due to the large amount of insulation used to maintain a constant temperature within the heater, and the perceived need to encase the entire column and its connection fittings inside the oven proper. For nano-liquid chromatography (nanoLC) where the flow rates for the eluate are typically under 1 microliter/minute, it is desirable to have the LC column placed as close to the detector, a mass spectrometer, as possible to eliminate post-column “dead volume” that broadens the chromatographic peaks. 
         [0005]    It is an object of the present application to disclose devices that heat or cool capillary columns to achieve excellent chromatographic results by heating or cooling the entire column with its connection fittings, or just a part of the column. Columns of different lengths and outside diameters may be heated or cooled in the same device if desired; and the devices, because of their small sizes and built-in column positioning features, allow these columns to be placed at their optimized positions in front of the mass spectrometers for detection. Moreover, these disclosed devices are not prone to damaging the columns during column insertion into the devices. It is also an object of the present application to disclose devices that allow the column temperature to be changed, for example, from an elevated temperature to ambient temperature, quickly for certainly applications such as hydrogen/deuterium exchange. It is also the object of the present application to disclose devices that allow the column and associated plumbing and attachments to be heated to above room temperature or cooled to below room temperature in the same device. 
       SUMMARY OF THE INVENTION 
       [0006]    The invention discloses devices for heating or cooling a capillary column and also fittings and tubing commonly used in a liquid chromatography system to a preset temperature constant to about 0.2° C. The devices comprise a structurally stiff or bendable tube or two-dimensional surfaces which are heated or cooled by the appropriate active heating or cooling elements. A capillary LC column or a part of a capillary column, and in some instances, its fittings and tubing when placed in close thermal contact of the heating or cooling surfaces, or surrounded by the heated or cooled surfaces, attain the temperature of the heated and cooled surfaces. The disclosed devices are small enough so that the capillary column within can be placed in a position in front of the mass spectrometer detector to optimize sensitivity and peak shape by reducing post-column dead volume. The small sizes of the devices result from adequate but not excessive amount of thermal insulation and the realization that not the entire capillary column needs to be heated to obtain the full benefits of column heating. The heating or cooling surface, which is preferably made of copper or aluminum, is resistively heated by means known in the art, or thermoelectrically heated or cooled, and covered with a relatively thin layer of thermal insulation material such as fiberglass or a plastic material. The near constant temperature is achieved both by the finite amount of thermal insulation and the temperature controller with a proportional-integral-differentiation (PID) type of algorithm known in the art. In one embodiment of the invention, a narrow slit about 0.06 inch in width or larger along the full length of the temperature-varying tube device allows a capillary column with fittings or connectors at both ends to be inserted into a heater or cooler device sideways through the slit but with the fittings exposed to the ambient and not heated. One or more outer concentric sleeves having a similar slit in each sleeve and are made of insulating materials surround the heating tube so that all the slits of the concentric tubes can be aligned for the insertion of the column into the heater tube through the slits. After the insertion of the column, the outer sleeves can be rotated with respect to the heater tube so that the slit of the heater tube is covered by the insulating sleeves. 
         [0007]    In another embodiment of the invention, the disclosed device regulates the temperature surrounding a capillary column to a preset temperature constant to about 0.2° C. of the preset temperature, which may be above or below ambient temperatures. The device comprises at least one temperature varying plates that are at least partially planar and are a few cm in extent, preferably from about 2 to 8 cm in width and may be up to tens of cm in length. The thickness of the temperature-varying plates can be from 0.001 inch to 0.03 inch, with the preferred range of thickness to be from 0.005 to 0.02 inch. In good thermal contact with the temperature-regulating plates are one or more temperature-varying elements such as one or more resistive heaters, a adiative heaters, thermoelectric element, or a combination of these and other similar temperature-varying elements. The plate may be flat, or may be bent or rolled in some portion to accommodate columns of a different diameter or columns with fittings that need to be heated or cooled also. In the preferred embodiment of the device, the device comprises at least one heated or cooled plate and a second plate with or without an active temperature-varying element. A gap space is formed between these two plates into which a capillary column as well as other LC fittings and tubing can be inserted to be heated or cooled. In another embodiment, the second plate is absent so that the gap space is formed by the first heated or cooled plate and thermal insulation. The width of the widest part of the gap space may be from about 0.02 inch for a bare fused silica capillary column, to up to 0.5 inch if the fittings of the column are to be heated also. If both a bare fused silica column and a column with fittings are to be heated or cooled in the same device as is often the case when a trap column is used in conjunction with a capillary column in a separation, then the gap space may have a width or diameter of up to 0.4 inch in one portion of the device and a gap space width of 0.02 to 0.06 inch in another portion of the device. An adequate but not excessive amount of thermal insulation materials for a heater and a cooler is applied to cover the temperature-varying element side of the temperature-varying plates and also surrounding the gap space. For a thermoelectric element is used in the device, an adequate heat sink an fan has to be used in addition to the thermal insulation. Fittings or apertures for securing the spray tip end of the column are also built into the cover housing of the device so that the spray tip end of the capillary LC column placed inside this device can be securely and reproducibly positioned in its optimized position in front of the mass spectrometer detector. The first and second temperature-varying plates may reside in two separate thermally insulated structures so that the two structures may be hinged or mechanically clamped together appropriately to form the gap space, or they may reside in a single folded plate with the gap space forming an opening for the insertion of the capillary column. Because the capillary column can be coiled or looped and placed into the device, the device can accommodate capillary columns many times longer than the smallest dimension of the area of the plate. In still another embodiment of the invention, a cooling aid such as a fan is attached to the housing cover of the device to help cool the temperature-varying plate and the air above it quickly to quench the temperature of the capillary column. In still another embodiment of the invention, one type of temperature-varying element resides on the first plate, while a different type of temperature varying element resides on the second plate. For example, a resistive heater is in good thermal contact with the first plate, and the cooling side of a thermoelectric heater is in good thermal contact with the second plate. Such a device can be used to heat the column to a temperature not typically achievable by using a thermoelectric element as a heater, and also cool the column to below ambient temperature. In yet another embodiment of the invention, the temperature-varying plate of the disclosed device is in thermal contact with a thermoelectric cooling element that heats or cools the disclosed device using appropriate electronic control known in the art. The near constant temperature is achieved both by the finite amount of thermal insulation and the temperature controller with a proportional-integral-differentiation (PID) type of algorithm known in the art. All the embodiments of the column heater and cooler disclosed in this invention maintain a near constant temperature in a space in which one or more capillary analytical column and the trap column of a different length and diameter for chromatographic separation can be placed with minimal risk of having any fragile parts of the column damaged, and the heater or cooler can be placed in a position in front of the mass spectrometer which is optimized for LC-MS detection. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING FIGURES 
         [0008]    The present invention will be understood and appreciated more fully from the following detailed description of preferred embodiments of the present invention, taken in conjunction with the following drawings in which: 
           [0009]      FIG. 1  is a schematic drawing of a column heater device in the form of a tube for capillary columns with end fittings. 
           [0010]      FIG. 2 : is a schematic cross-section of the column heater device for capillary column with end fittings and the outermost insulating sleeve is rotated to cover the slit for insertion of the capillary column. 
           [0011]      FIG. 3 : is a schematic drawing of a cross-section of the device comprising planar or largely planar temperature-varying plates with the exemplary components and their configuration in the device. 
           [0012]      FIG. 4 : is a schematic model of the device comprising planar or largely planar temperature-varying plates showing the invented device in a one-piece construction and an opening for inserting the column into the gap space of the device. 
           [0013]      FIG. 5 : is a cross-sectional schematic drawing of the view of the invented device comprising planar or largely planar temperature-varying plates device used for heating the column and then for fast quenching the temperature of the column after the heat has been turned off. 
           [0014]      FIG. 6 : is a schematic drawing of the temperature-varying plates of an example of the invented device that can accommodate columns of dramatically different lengths and diameters, including columns with end fittings. 
       
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0015]    In one embodiment of the invention as shown in  FIG. 1 , the heater device  20  is more or less tubular in shape and is used for heating capillary columns with end fittings. Only the portion of the capillary between the end fittings is inserted into the heater device  20 . At both ends of the device  20 , rotatable caps  300  made preferably of PEEK are used to limit the exposure of the ends of the heater tube  200  to the ambient. The rotatable cap  300  has a slot  301  through the body of the cap  300  so that the capillary column can be inserted into the slot  301  when the slot  301  is aligned with the slits  201 ,  221 , and  241  described below. Referring to the cross sectional drawing in  FIG. 2 , the heater tube  200  inside the heater  20  runs the length of the heater  20  and has a slit  201  about 0.06 to 0.1 inch down the length of the heater tube  200  which has a diameter of about 0.15 inch to 0.25 in. The heater tube  200  is resistively heated and is covered with thermal insulation  210  made of materials such as fiberglass, heat-resistant polymers such as Teflon® or Kapton® or high temperature silicone rubber, or a combination of these materials. A temperature sensor not shown in the figure is in good thermal contact with the heated air inside the heater tube by attaching the thermal sensor to a cutout in the heater tube  200 . The insulation  210  does not cover the slit  201 . A second tube  220  preferably made of Tefon® or PEEK also with a slit  221  from about 0.05 inch to about 0.1 inch down the length of the tube  220  is sleeved snugly over the insulation  210  so that the slit  201  and the slit  221 , once they are aligned to give access  240  to the heater cavity  230 , will not rotate against each other to lose the access  240 . The wall thickness of the tube  220  may be from 0.01 inch to 0.09 inch, with the preferred wall thickness in the range of 0.01 inch to 0.04 inch. A third tube  240  also with a slit  241  down the whole length of the tube  240  is sleeved over the structure comprising tube  200 , insulation,  210 , and the second tube  220  so that the tube  250  can be rotated freely over the second tube  220 . The slit  241  is aligned with the slits  201  and  221  for inserting the capillary column. Once the column is in the heater cavity  205 , the tube  250  is rotated against the tube  220  so that the slit  241  is no longer aligned with the slits  201  and  221 . Both the thermal energy in the heater cavity  205  and the capillary column are now kept in place inside the heater cavity  205 . With this heater  20 , capillary column with fixed fittings at both end can be slipped into the heater  20  with the fittings exposed to the ambient. The device  20  can be made with an overall outside diameter of about 0.5 inch. 
         [0016]    In another embodiment of the invention as shown in  FIG. 3  in cross-section, the device  500  for varying the temperature of one or more capillary chromatography columns is at least in part planar in shape. The device  500  comprises a first plate  1000  and a second plate  2000 . The plates  1000  and  2000  may be two separate plates, or may be two leaves of a plate that is folded so that a gap space  3000  is formed between the surfaces of the folded plate  1000 . 
         [0017]    The temperature of the surface  1100  of the plate  1000  can be varied through a variety of means in good thermal contact with the plates  1000  such as conduction heating from a heated filament  1300 , radiative heating via a hot filament or an infrared lamp placed close to the plate  1000  but not in contact, or cooling through the cold side of a thermoelectric Peltier plate, a tubing carrying a coolant in good thermal contact with the plate  1000 , or any other appropriate heating or cooling means known in the art. The materials that are most suitable for making the plate  1000  are thermally conducting materials such as copper, aluminum, anodized aluminum, stainless steel, and thermally conducting ceramics and the like, and the preferred materials are a copper or an aluminum plate from 0.001 inch in thickness to 0.03 inch in thickness. The preferred range of thickness of the plate  1000  is from about 0.002 inch to about 0.02 inch. A temperature sensor  140  such as a thermocouple or a similar device is attached appropriately such as with a thermal-bonding substance, a piece of temperature-appropriate tape and the like to the surface  1100 , or to the other surface  1200  of the plate  1000 . The second plate  2000  also contains a thermally conducting surface  2100  whose temperature can be varied by a second temperature varying element  2300  which may be similar to the temperature varying element  1300  or a different temperature element. For example, the temperature-varying element  1300  may be a resistive heater that can raise the temperature to 100° C. or higher, and the element  2300  may be the cooling side of a Peltier plate that can cool the device to 0° C. or lower. A second temperature sensor may be used but is not necessary for the plate  2000 . The plates  1000  and  2000  are brought close together so that the surfaces  1100  and  2100  are brought into close proximity of each other preferably from about 0.02 inch to 0.07 inch to form a gap space  3000 . Surrounding the plates  1000  and  2000  on the side of the surfaces  1200  and  2200  are thermally insulating materials  4800  which may be made of a combination of materials such as air, fiberglass, silicone, ceramic or structural plastic materials chemically and mechanically stable to over 200 degrees C. such as polytetrafluoroethane (PTFE), PTFE-derived materials, PEEK and the like if the temperature-varying elements  1300  and  2300  are heaters, and heat sink materials such as copper or aluminum blocks with fin-type structures for efficient radiative heat loss. A fan for improving air-cooling of the heat sink is usually installed to further improve the performance of the cooler. When the temperature-varying elements  1300  and  2300  are turned on by a temperature controller such as one using a PID method for setting and maintaining a specific temperature below or above ambient temperatures, the gap space  3000  between the surfaces  1100  and  2100  attains the predetermined temperature indicated by the temperature sensor  1400  so that when one or more capillary columns or coiled capillary columns  6000  are inserted into the gap space  3000 , the inserted column or columns  6000  will attain the same predetermined temperature to within 0.5 degree C. In  FIG. 3 , the housing  4100  for the plate  1000  of the device  500  is made of structural plastic materials and contains mechanical fastening fixtures  4500  for securing the end  6001  of the capillary column  6000  inserted into the gap space  3000 . The housing  4100  and  4200  may also be made of thermally conducting materials such as metals as long as the housing is well insulated from the temperature-varying plates  1000  and  2000 . The housing  4100  and  4200  may also be made of fabric-like material such as fiberglass, polyimide sheet, Teflon sheet or a combination of these materials to form a flexible and compact device  500 . In this case, exits and entrances to the device  500  can be created by simply cutting the fabric cover and the insulation in the appropriate locations of the housing  4100  and  4200  to allow access to the gap space  3000  of the device  500 . If the end  6001  of the capillary column is shaped into a tip for nanospray-mass spectrometry, the device  500  can be placed in front of the mass spectrometer inlet and the column end spray tip  6001  can be positioned optimally in front of the mass spectrometer for spraying the eluates into the mass spectrometer directly with minimal post-column dead volume. The fixtures  4500  may be a simple aperture or contains mechanical threads that mate with the nut and ferrule used for connecting capillary columns in the art. More than one fixture  4500  can be incorporated into the housing  4100  to accommodate both ends of the column in the gap space  3000 , or more than one column in the same gap space  3000 . The fixtures  4500  can also be designed for securing to the device other chromatographic parts such as the union or Tee used for applying high voltage for electrospraying the eluates from the column or columns. The ability of the device  500  to incorporate these fixtures into its body greatly improves the utility of the device  500  for low flow rates liquid chromatography. 
         [0018]    In the embodiment shown in  FIG. 3 , the insulating materials surrounding the plates  1000  and  2000  except for the surfaces  1100  and  2100  may form two units: one unit  4100  housing the plate  1000  and the second unit  4200  housing the plate  2000 . The two units  4100  and  4200  may be stacked together so that the surface  1100  and the surface  2100  face each other and form the gap space  3000 , and the units may be clamped together by usual means known in the art. The two units  4100  and  4200  may also be hinged at one end to facilitate easier column installation into the gap space  3000 . It should also be obvious to one skilled in the art that the housing of the device may be made of one piece  4000  with an opening  4900  for insertion of the column into the gap space  3000 , as depicted in  FIG. 4 . 
         [0019]    In another embodiment of the invention shown in the cross-sectional schematic drawing in  FIG. 5 , the distance between the surfaces  1100  and  2100  forming the gap space  3000  is from about 0.03 to about 0.25 inch so that the temperature of the capillary column placed within this space can be quenched quickly to ambient temperatures when the heating elements are turned off or when the unit  4200  is removed from its original position of being in contact with the unit  4100 . In this embodiment, the plate  2000  may not be present in the unit  4200 , i.e, the unit  4200  contains only thermally insulating materials, which may be air in some cases if the maximum temperature required to be provided by the device is relatively low, for example around 50 degrees C. To help accelerate the quenching of the gap space temperature, the unit  4200  has a large hole  4400  which may constitute up to 30% of the area of the unit  4200 . The hole  4400  allows a substantial amount of air from the gap space  3000  to go freely into and out of the gap space  3000 . A fan  4600  may be mounted on the outer surface  4200  to draw ambient air through the space  3000  to quench the temperature of the column placed in the gap space  3000 . This embodiment is especially useful for applications such as hydrogen/deuterium exchange in proteins in which the device is placed within a cold box and the materials inside the capillary undergo temperature cycling. 
         [0020]    In still another embodiment of the invention as shown in  FIG. 6  without the housing cover  4100  and  4200 , the plates  1000  and  2000  are not flat but are rolled into a semi-circular form with a diameter of up to 0.5 inch. In this embodiment, the gap space  3000  is not more or less uniform in width but contains two regions where one region  3100  has a width of about 0.02 inch to 0.25 inch, and the second region  3200  has a gap width of about 0.25 inch to 0.5 inch. The gap space region  3200  can accommodate a trap column  6100  with its fittings. For thin plate material, for example, plate thickness between 0.003″ to 0.02″, used in fabricating plate  1000  and  2000  and with the housing  4100  and  4200  made of flexible fabric materials, it may not be necessary to roll the plates  1000  and  2000  into specific shapes since the plates  1000  and  2000  and the housing cover  4100  and  4200  can conform to the shape of the fittings. In this embodiment the device  500  can be used for heating columns of different diameters and different lengths, and also the fittings attached to the end of the column. The gap space regions of  3100  and  3200  may also be created by folding a single plate  1000  appropriately. 
         [0021]    In still another embodiment of the invention, the plate  2000  is a Peltier element with the surface  2100  being the cold side of the element. Heat sink materials such as metal blocks and finned metal blocks are used to remove the heat generated on the surface  2200 . External fan or fans are usually needed to facilitate the heat removal from the heat sink materials. 
         [0022]    While the invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. 
       Utility 
       [0023]    The invention described in this application can be used to heat one or more than one capillary liquid chromatography columns of various lengths to a temperature of about 10° C., which may be quenched very quickly to ambient temperatures. The device in this invention can also be configured to cool the capillary column or columns placed in the device to temperatures substantially below ambient temperatures. The disclosed devices can be used to vary the temperatures of one or more chromatographic columns, fittings, accessories for low flow-rate liquid chromatography without having to remove the column from its usual position in front of the mass spectrometer.