The disclosure relates to a gradient twin column recycling chromatography method that is used to separate a mixture containing closely eluting compounds. In one embodiment, a sample includes a primary organic compound and one or more impurities that closely elute with the primary organic compound. A gradient mobile phase is initially used to remove unwanted early eluting and late eluting impurities from the sample. After the gradient removal of some of the impurities is complete, the remaining mixture of the primary organic compound and the closely eluting impurities are separated using recycle chromatography methodology with an isocratic mobile phase.

A method for coupling a tubing to a liquid chromatography column comprises: passing an end of the tubing through a coupling apparatus comprising: (i) at least one chamber; (ii) a first spring within the at least one chamber configured to transmit a spring force to the tubing; (iii) a second spring within the at least one chamber; and (iv) a deformable sealing member configured to receive a second force from the second spring; inserting the tubing end into a receptacle of an end fitting of the column; and moving the coupling apparatus towards the chromatography column such that the first spring urges the tubing into the receptacle whereby a pressure of the tubing end against the end fitting exceeds a maximum operating fluid pressure of the column and, further, whereby the second spring causes the deformable sealing member to form a fluid seal between the column and the receptacle.

A sealing element for sealing a fluidic connection between a coupling element and a tubular element and thereby providing a sealed flow path through the tubular element and between the coupling element and the tubular element in a longitudinal direction, the sealing element comprising: a recess extending in the longitudinal direction, the recess configured to receive the tubular element; a transverse wall defining an extent of the recess in the longitudinal direction, the transverse wall having a through hole.

The disclosed system and method concentrates and enriches a chemical sample while removing water and/or CO2 prior to analysis, improving detection limits and repeatability of quantitative chemical analysis without the need for cryogenic or sub-ambient cooling. The system can include a valve system, a dewpoint control zone, and a multi-capillary column trapping system (MCCTS). During a first time period, the valve system can couple the dewpoint control zone to the MCCTS. During a second time period, the valve system can couple the MCCTS to the chemical separation column such the dewpoint control zone is bypassed. Excess water included in the sample can condense in the dewpoint control zone as the sample transfers to the dewpoint control zone and MCCTS. When the sample is transferred from the MCCTS to the chemical separation column, the condensed water in the dewpoint control zone is not transferred to a chemical separation column.

Exemplary embodiments integrate a tee or valve into an outlet end fitting of a liquid chromatography column. The tee or valve is suitable for providing additional fluidic flow paths to ports of the end fitting and eliminates the need for post-column fluidic conduits connecting to tees or valves to insert fluidic inputs or divert flow to outputs. This integration decreases the distance that eluent from the liquid chromatography column has to travel to reach a detector relative to systems that use external tees or valves while providing tee/valve functionality and reducing the fluidic volume post-column. As a result, the exemplary embodiments help decrease sample dispersion.

The exemplary embodiments provide chromatography column positioning assemblies that can ensure that the distance between face seals or other sealing surfaces/mechanisms at the respective ends of a liquid chromatography column is a desired distance (i.e., the length of the liquid chromatography column). The exemplary embodiments can adjust the separation between the face seals to accommodate different length liquid chromatography columns. For example, a chromatography column positioning assembly of an exemplary embodiment can set the distance between face seals to accommodate a 25 mm column, a 50 mm column or a 100 mm column.

Disclosed herein are methods of analyzing a biological sample comprising: separating components of the biological sample via reversed-phase (RP) chromatography to obtain an elute; subjecting the elute to separation via ion-exchange (IEX) chromatography or mixed-mode IEX chromatography; and detecting the separated compounds to determine the components of the biological sample. Also disclosed are devices comprising a reversed-phase (RP) chromatography column in communication with an ion-exchange (IEX) chromatography column or mixed-mode IEX chromatography column, wherein there is no switching valve between the columns.

Disclosed herein are methods of analyzing a biological sample comprising: separating components of the biological sample via reversed-phase (RP) chromatography to obtain an elute; subjecting the elute to separation via ion-exchange (IEX) chromatography or mixed-mode IEX chromatography; and detecting the separated compounds to determine the components of the biological sample. Also disclosed are devices comprising a reversed-phase (RP) chromatography column in communication with an ion-exchange (IEX) chromatography column or mixed-mode IEX chromatography column, wherein there is no switching valve between the columns.

A streamlined method for purifying alpha-1-antitrypsin (AAT) from an AAT-containing protein mixture, such as Coh fraction IV precipitate, is provided. In the method of the invention, contaminating proteins are destabilized by cleavage of disulfide bonds with a reducing reagent, such as dithiol, which does not affect AAT. The destabilized proteins are then preferentially adsorbed on a solid protein-adsorbing material, without the addition of a salt as a precipitant. Separation of the solid absorbent from the solution leaves a purified AAT solution that is directly suitable for chromatographic purification, without the need for extensive desalting as in prior art processes. A process incorporating this method, which provides pharmaceutical grade AAT in high yield on a commercial scale, is also described.

An on-line system that performs concentration and solvent exchange in order to improve a detection level of analytes by a liquid chromatography (LC) is provided. The on-line system comprises: a first pump and a second pump for supplying a solvent; a liquid chromatography (LC) including a separation column (SC) connected to the first pump; a trapping column (TC) for collecting the analytes separated from the separation column; a concentration column (CC) for concentrating the analytes collected in the trapping column (TC); a detector; and first to third switching valves that communicate fluid with at least one of the first or second pumps. An analysis method using the same is also provided.