Automated mobile phase preparation

Mixing errors automated mobile phase preparation. Problems encountered with an automated system can still be the result of analyst error. For example, the wrong component may have been dispensed into the HPLC solvent reservoir. Where there is an apparent issue in relation to the retention time of a compound, the mobile phase should always be checked for accuracy of preparation. Proportioning valve errors can also cause a problem with automated systems. When the valve ceases to dispense the correct amount of either solvent, the retention time of the compounds of interest will vary. 

Mobile phase. Many of the problems associated with the mobile phase will depend on the HPLC instrument configuration and, as a consequence, whether the mobile phase preparation is manual or automated. 

If selectivity tuning of certain separations is possible by changing pH or changing temperature, the question arises what is the preferred way. It is obviously more convenient to change the temperature. This can be easily set at the instrument and does not require further mobile phase preparation work. Moreover, there is limited flexibility for pH changes with a given buffer type, especially if the buffer capacity has to be maintained. These considerations clearly promote the temperature as the first choice, and this optimization experiment can easily be automated. 

A completely automated system with reinjection/sample collection and solvent recovery allows the separation of up to 10 g of extract a day. The Cjq is obtained in very high purity and the recovery is nearly 100%. A simple benchtop method for the enrichment of preparative amounts of Cjq, C7Q and higher fullerenes (up to Cjqq) from a crude fullerene mixture is based on a single elution through a column of poly(dibromostyrene)-divinylbenzene using chlorobenzene as mobile phase [208]. 

Untreated silica column can be advantageously used for HPLC preseparation of PAHs from triglycerides. The capacity of a silica column to retain fat (for columns of the same particle size) depends on the column size, the mobile phase composition, as well as the type and by-products (free acids and polymerized material) of the fat injected [706,713]. Off-line HPLC-HPLC, employing silica column (250 X 4.6 mm i.d., 5 pm of particle size) for sample preparation before RP-HPLC and spec-trofluorometric detection, was successfully applied for PAH determination in edible oils [659,691] and fish [714]. After PAH elution, the silica column needs to be backflushed with dichloromethane to remove the fat. The entire sample preparation step can be automated by using a backflush valve and a programmable switching valve box [691]. 

Another feature of modern HPLC systems that makes them desirable for both analytical and preparative applications is the complex mobile-phase gradients that they are capable of producing. Many systems come equipped with a pump integrator or controller (computer) that allow a number of different mobile-phase solvents to be simultaneously mixed and delivered to the stationary phase. Since this process is automated, complex gradients used for a particular application are quite reproducible. 

Inverse gas chromatography (IGC) refers to the characterization of the chromatographic stationary phase (polymer) using a known amount of mobile phase (solvent). The stationary phase is prepared by coating an inert support with polymer and packing the coated particles into a conventional gas chromatography column. The activity coefficient of a given solvent can be related to its retention time on the column. The equipment itself is commercially available, easily automated, and extremely versatile. 

Mechanistic considerations (e.g., the extensive work published on brush-type phases) or the practitioner s experience might help to select a chiral stationary phase (CSP) for initial work. Scouting for the best CSP/mobile phase combination can be automated by using automated solvent and column switching. More than 100 different CSPs have been reported in the literature to date. Stationary phases for chiral pSFC have been prepared from the chiral pool by modifying small molecules, like amino acids or alkaloids, by the deriva-tization of polymers such as carbohydrates, or by bonding of macrocycles. Also, synthetic selectors such as the brush-type ( Pirkle ) phases, helical poly(meth) acrylates, polysiloxanes and polysiloxane copolymers, and chiral selectors physically coated onto graphite surfaces have been used as stationary phases. 

An important feature and advantage of on-line SPE, compared with off-line SPE, is direct elution of the analyte from the SPE cartridge into the mobile phase of the LC system. The time-consuming off-line steps of evaporation, reconstitution, and preparation for injection are eliminated and make the online SPE more efficient and fully automated. Since the entire volume of eluate is analyzed, maximum sensitivity for detection is obtained. Some other advantages of this on-line approach include ... 

Another development in HPLC analysis is the interfacing of on-line sample clean-up technologies at the front end of the HPLC analytical column. Using a switching valve, complex samples can be injected directly onto a sample preparation column, the stationary phase of which is designed to bind the analyte(s) of interest to the exclusion of the rest of the matrix, which runs to waste. Then the valve is switched and the mobile phase flows through the sample preparation column where it picks up the analyte(s) and carries them onto the analytical column and through to the detector in the normal way. This automation of sample preparation saves time and effort and reduces errors. There are many applications of HPLC assays in conjunction with on-line sample clean-up methods in the literature ... 

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