Liquid chromatography (LC) is well-known in the fields of chemical separation, compound purification and chemical analysis. A liquid chromatograph generally includes a separation column that comprises a capillary tube that is packed with a permeable solid material that either is, itself, a chromatographic stationary phase or otherwise comprises or supports a chromatographic stationary phase. A mobile phase, which is a fluid mixture comprising a compound of interest for purification or separation as well as one or more solvents, is caused to flow through the column under pressure from an input end to an output end. Generally, the chemical properties of the stationary phase and the mobile phase solvents are such that the degree of partitioning of the compound of interest between the mobile phase and the stationary phase is different from the degree of partitioning of other compounds within the fluid. As a result, the degree of retention or time of retention of the compound of interest within the column is different from the degree or time of retention of the other compounds, thus causing a physical separation or at least partial purification of the compound of interest from the other compounds.
There are numerous solvents available for liquid chromatography. For instance, the HPLC solvents available under the Fluka® brand name from Sigma-Aldrich Corporation (3050 Spruce Street, St. Louis, Mo. 63103 USA) include: water, Acetonitrile, Benzene, 1-Butanol, 2-Butoxyethanol, tert-Butyl methyl ether, Carbon tetrachloride, 1-Chlorobutane, Chloroform, 2-Chloropropane, Cyclohexane, Cyclopentane, 1,2-Dichloroethane, Dichloromethane, Diethyl ether, 1,2-Dimethoxyethane, N,N-Dimethylacetamide, Dioxane, Ethanol, Ethanol, Ethyl acetate, Heptane, Hexane, Isooctane, Methanol, Methanol, Methyl acetate, Nitromethane, Pentane, 1-Propanol, 2-Propanol, 2-Propanol, Tetrachloroethylene, Tetrahydrofuran, and Toluene.
Within a chromatograph instrument or system, solvents or other reagents are generally made available to the various columns, pumps, valves and associated interconnecting tubing lines by means of a dedicated rack or compartment. The rack or compartment generally comprises a dedicated storage area for the set of solvents or other reagents that will routinely be needed or that may be needed by the chromatograph instrument or system during the course of several separations. The reagent rack is generally designed to be accessed by an operator or technician at such times that one or more solvents or reagents need to be replaced, having been depleted over the course of operation of the instrument or system.
Successful chromatographic separations depend on specific chemical interactions of the various analytes and other components with a stationary phase and with the various chemical constituents of a mobile phase. Because different analytes have different respective chemical properties, it is important that the correct set of solvents or reagents for an analysis at hand are mixed with a sample containing or potentially containing any particular analyte. Therefore, the various different solvents or reagents are provided in respective dedicated bottles or other containers within a reagent or solvent rack or compartment. The different containers or bottles either have respective assigned locations within the rack or compartment or are associated with different respective assigned draw tubes for aspiration of the solvent or reagent into the system.
Because of the specificity of solvents or other reagents required for any particular chromatographic analysis protocol, it is important that these materials are not confused with one another (or with completely different substances) or misplaced within a reagent or solvent rack or compartment. Although reagents, solvents and other required chemicals are generally supplied by manufacturers in well-labeled containers, these materials may be re-distributed into smaller containers within a laboratory environment. The smaller containers may be multi-purpose, initially-unlabeled vessels which require appropriate manual labeling upon initial receipt of material transferred from a manufacturer's original container. The manual label applied in a laboratory may be a non-permanent label. After manual labeling, the small transfer vessel may be handled within the laboratory many times and by many different users, since multiple replenishments from a large-volume manufacturer's “bulk” container may be required as the material within the vessel is routinely consumed. The same vessel may be re-inserted into a solvent or reagent rack many times.
Many opportunities for operator error will occur over the course of the multiple handlings of the transfer vessel or, occasionally, even a manufacturer's original container. For instance, a temporary label may be lost and replaced with an incorrect label. Even if the label is correct, the operator may transfer the wrong material into the transfer vessel. Even if the label and material are correct, the operator may mis-place the vessel within a reagent rack or compartment. Conventional chromatograph systems are designed to expect that particular solvents or reagents will be drawn into particular respective tubing lines. If an incorrect material is supplied, through any one or more of the errors listed above, the chromatograph will continue to perform the pre-programmed steps of an analysis protocol with the wrong material. This may lead to incorrect or poor-quality results, necessitating repetition of many faulty analyses. In a worst-case scenario, the error may never be discovered, and inappropriate actions may be taken based on the incorrect analytical results. Nonetheless, by comparing the properties of a solvent—such as viscosity and compressibility—with the expected values which can be obtained through user input or by means of a sensor mechanism, such as bar code, the solvent identity can be validated. Accordingly, there is a need in the art for an automated chromatograph system that can take automated procedural steps in an attempt to recognize unexpected solvents or reagents before analysis steps are performed unexpected material and that can raise an operator alert if any such errors are detected.
Liquid chromatography systems utilized in clinical laboratories or for purposes of drug discovery may remain in near continuous operation over long periods of time. As a result of wear, repeated handling, repeated pressurization, multiple replacements of samples, etc., occasional or periodic situations or conditions may occur which result in sub-optimal performance of or even instrumental malfunctions in chromatographic systems. For example, as a result of long term repeated pressurization of fluid lines and other fluidic components, leaks may develop which either lead to undesirable loss of fluids from a fluidic system or, perhaps, undesirable ingestion of air into the system. Repeated replacement of sample vials or fluid or solvent containers may lead to contamination of fluid lines by particulates or ingested air. Further, since many components such as pumps and valves undergo repeated mechanical operation, long term wear of such components may occur which, if not addressed, may lead to loss of precision, loss or pressure integrity or even total malfunction of one or more components. Finally, undesirable pressure imbalances may occur within fluidic systems comprising various fluidic sub-systems, each sub-system having its own respective pumps. Accordingly, there are needs in the art for methods for monitoring the performance of chromatographic systems for the purpose of detecting sub-optimal conditions, deterioration of performance, possible future failures, etc. and for warning users of the need to take corrective action or notifying users of estimated remaining useful lifetimes of components. Moreover, there is a need in the art for an automated chromatograph system that can perform such monitoring and provide such warnings or notifications automatically. Preferably, liquid chromatograph (LC) system self-diagnostics and monitoring should include self-diagnoses, validation and troubleshooting of i) the pump and ii) the LC system plumbing for leakage, air bubbles and fluid pathway blocking.
There is also a need in the art for methods for balancing pressures between different fluidic sub-systems. The compressibility of an LC solvent affects the flow rate which in turn affects the chromatographic performance. This effect is an issue for all high-performance (or high-pressure) liquid chromatography (HPLC) systems in general and for those that use syringe pumps in particular. This effect is one of the main drawbacks associated with the syringe type of pump, although such syringe pumps provide other advantages such as smooth gradients and a high degree of robustness. M. Martein, et. al (“The use of syringe-type of pumps in liquid chromatography in order to achieve a constant flow-rate”, Journal of Chromatography, 112, 1975) concluded that “[i]t is therefore not surprising that the syringe-type pumps have evolved into very sophisticated and expensive devices” in order to compensate the compressibility issue. Even so “the use of syringe-type pumps is often more difficult and less satisfactory than the use of other types of pumps.”