Liquid chromatography (LC) is a technique which employs a stationary phase and a liquid mobile phase to separate the solubilized components of a mixture into its various parts. A separation column generally consists of uniformly-packed small particles of stationary phase in a tube and the separation power of a separation column depends on the uniformity of the stationary phase. Particulate from the mobile phase and or samples can block flow passages formed from the interspaces of the packed stationary particles, causing high backpressures and disturbing the uniformity of stationary phase. Stationary phases have different surface chemistries and can irreversibly absorb some soluble components. The irreversible absorption will degrade the column capacity, disturb the uniformity of stationary phase, and increase column backpressure. Both irreversibly-absorbed soluble components and insoluble particulate will degrade column performance and reduce column life. A means for protecting liquid chromatography columns from contamination of particulate and soluble contaminants is critical for good performance and long service life of a separation column.
The most effective means to prevent column contamination is to place a guard device in front of a separation column. The guard device should allow analytes to pass through while retaining both particulate and soluble contaminants which would be adsorbed onto the column stationary phase irreversibly. For this reason, the guard device should have the same properties as the separation column. Though many types of guard devices exist in the market, they can be generally viewed as one special type of LC column packed with various stationary phases similar to that of the separation column. The guard device is a semi-disposable component of the liquid chromatography system and should be much less expensive.
Guard devices in liquid chromatography may be classified as guard columns, guard cartridges and guard discs according to the shape and the embodiment of the devices. A guard column refers to a device containing a short packed tube, a pair of frits on each end of the tube to hold the packing material in the tube white allowing liquid mobile phase to pass through, and two endfittings for fixing the frits and the tube together and for installation of the guard column between an injector and a separation column in a LC system. A guard cartridge contains a packed tube, a pair of frits on the ends of the tube to prevent the packing material from leaking out, and a means to keep the frits and the tube together. A guard cartridge usually contains no end fittings and a cartridge holder is needed in order to install the guard cartridge into chromatography flow line. A guard disc refers to a short guard cartridge and its commercially available form is a protection pad of stationary phases impregnated in PTFE (Teflon) matrix.
A guard column contains the same stationary phase as a separation column and is manufactured in the same way as the separation column. The only difference between a guard column and a separation column is the length-guard columns are short and separation columns are long. The production cost of a guard column is comparable to that of a separation column and renders guard columns the most expensive and the lest favorable protection means for separation columns.
Besides cost, guard columns may degrade the separation efficiency of separation columns. Column efficiency may be affected by the flow path taken when a guard column is used. Typically, the liquid mobile phase travels from a sample injection valve through a tube having a small inside diameter to the guard column which has a relatively large inside diameter, and then back to a connecting tube having a small inside diameter and finally to the separation column which again has a relatively large inside diameter. This change in diameter of the flow path produces a broadening effect on the sample zone and decreases overall column efficiency. The only way to overcome this effect is to place a guard device of the same diameter as the separation column in direct contact to the stationary phase of the separation column. The general configuration of guard columns makes this option impossible.
Guard columns contain chromatographic stationary phase and can retain samples slightly. This effect will change the retention time of the sample components. The change in retention time is a serious problem, especially for chromatography systems employing computer data acquisition systems. These systems rely on peak time windows to locate particular peaks. It also increases the difficulty of identifying certain components by matching their retention times to the published retention times in literature, which is an important technique for the characterization of unknown compounds.
In order to maintain their performance it is not recommended that guard columns be opened by users, The only way to determine the effectiveness of a guard column is by observing the chromatographic separation, such as peak broadening or retention time shifting. But chromatographic separations do not always indicate whether a guard column is adequately protecting a separation column. Contamination of a separation column can take place well before there are noticeable changes in plate number, pressure and resolution. Well-defined criteria for guard column replacement are missing.
Guard cartridges were developed as cost effective protection devices for separation columns. Guard cartridges need cartridge holders for in line installation. The functions of a guard device are divided between a guard cartridge and its cartridge holder. The guard cartridge removes contaminants and is a disposable part. The cartridge holder accommodates the cartridge and connects the cartridge in-line with the chromatography flow path. The cartridge holder is considered as a permanent part. In this way guard cartridges are simpler than guard columns and usually have no endfittings on their ends. However, they must have frits on both ends to retain the packing material. The frit design and its incorporation onto the packed cartridge is one of the most important considerations for guard cartridges. The frits should prevent packing material in the cartridge from leaking out; The frits should be permeable to the liquid mobile phase; The frits should be assembled onto the cartridge in such a way that once the guard cartridge is placed in a cartridge holder and installed in-line under high pressure, the liquid mobile phase should not leak out.
U.S. Pat. No. 5,482,628 describes a typical guard cartridge system which consists a cartridge and a holder. To prepare a guard cartridge, a tube of a given length is packed with the stationary phase. Two specialized frits, which comprise a porous center section and a plastic outer ring, are placed on the ends of the packed tube to hold the stationary phase in the tube and to permit the liquid mobile phase to pass through. The frits and the tube should have the same outer diameter. To fix the frits on the ends of the packed tube, a housing tube, or sleeve, is required. The housing tube has an internal diameter slightly smaller than the outer diameter of the packed tube. The two frits and the packed tube are forced and crimped into the housing tube. Though many types of frits have been used for guard cartridges, they all contain a porous center section permeable to liquid and a nonporous outer section for adequate sealing of the interface between the packed tube and the frits.
Though guard cartridges are more advantageous than guard columns from cost point of view, they still have many of the same limitations. Most guard cartridges are installed in the LC flow line in the same way as guard columns and suffer the diameter changes in flow path of liquid mobile phases as previously described; A used guard cartridge can not be opened to examine its effectiveness and no clear criteria or guidelines about when a guard cartridge should be replaced; Due to their configuration most guard cartridges are prepared 10 millimeters or longer in length and as such interfere with performance and retention time as previously described; The crimping process increases the possibilities for frit damage and leakage. Guard cartridges require relatively expensive frits of specific configuration which renders them more costly than a disposable item should be.
To overcome the limitations of guard columns and guard cartridges, a guard disc was invented and is described in U.S. Pat. No. 5,338,448. The guard discs are circular pads made of packing materials immobilized by membrane and are relatively inexpensive. The design allows the disc to be placed on the head of a separation column to eliminate flow disturbance. The stationary phase is the pad itself. The guard holder can be opened and the stationary phase can be visualized directly. If contaminants have color, the color change of the disc is an indication of its effectiveness. The disc is so thin that retention time for an analyte on a separation column will not be influenced. However, these discs are too thin, and a large portion of the disc mass is the impregnating membrane material itself. For this reason, the capacity of a guard disc is limited, which is a major disadvantage as a protection device. More importantly, the stationary phase of the disc has completely different physical structure from that of a packed guard column or a guard cartridge. In the case of the guard column or guard cartridge, the stationary phase is packed in a tube and the packed bed has microporous pores inside stationary particles and macro channels at the interspaces of the particles. The interspaces will range up to several micrometers. Particulate and soluble contaminants will stay on the surface of stationary particles and in the interspaces. Since the interspaces are large and will not be blocked completely by contaminants, the backpressure will not change very much after prolonged use. The pad disc is prepared by impregnating stationary particles into a PTFE membrane and the interspaces between the particles are filled with membrane polymer and are not available for liquid mobile phase to pass through. The only pores available for liquid flow are the micropores on the membrane which are very easily blocked. The consequence of the pad disc structure is rapid pressure build-up when contaminants block the surface pores of the pad membrane and cut off the flow passage for the liquid mobile phase. Though the pad disc still retains a large percentage of its initial adsorption capacity, it is not usable due to the resulting high backpressures. A special experiment showed that when the pad disc was placed in front a column and eluted with 50% acetonitrile, the pad backpressure increased from 1 bar to 100 bars within a few hours. However, a packed guard cartridge showed no significant change in backpressure under the same conditions.
As discussed above, there is a need to provide a guard device which does not significantly affect the efficiency or retention time of a chromatography column. The guard device should be able to be placed on the head of a chromatography column to minimize flow disturbance. It should be easy to examine the effectiveness of the guard device to ensure adequate protection of the separation column. Yet as a protection means the guard device must also have a reasonable capacity. Lastly, as a disposable element a guard device should be relatively inexpensive.