Checking Gradient Performance

During the development of our OMA-based detector system, we performed a number of experiments checking the performance by measuring the absorbances of uniform solutions spun in the ultracentrifuge. It was observed that the flat absorbance profiles for the hundreds of points for each cell varied only by about 0.002 absorbance units and a plot of the average absorbance for each cell vs. the concentration was linear from 0 to 1 absorbance units and even higher ( 1,2). However, such a demonstration with uniform solutions does not prove that the correct absorbance profile is obtained from a cell with a concentration gradient. 

Operational Qualification (OQ) is a QA approved document designed to check that the individual components operate according to specification. Pumps will be tested with calibrated flow meters to confirm that they perform to the set specification. Calibration standards will be used to assess other instrumentation such as Refractive Index (RI) or Ultra Violet (UV) detectors. Gradient performance will be checked to confirm that the solvent delivery system and mixer is working to specification. Once complete and approved by QA and Technical Management, the equipment will now be made available for use. 

Multiple Column Experiments. The accuracy of the developed program was checked by performing three binary linear gradient, coupled column experiments. Prior to conducting the gradient experiments, four Isocratlc data points were acquired for the five test solutes. These Isocratlc data are presented In Table VII. 

For the investigation of a butadiene-styrene rubber, a set of three SEC columns in series was used 500 x 8 mm, d0 = 150 nm 500 x mm, d0 = 25 nm 800 x 8 mm, d0 = 4 nm, all three with dP = 10 pm. The flow rate was 1 ml/min. The injection amounted to 200 pi of a 2 % solution from which the carbon black had been removed. Although the additives of interest were separated from the polymer, they were still covered by an intense band from process oil. Hence, coupled-column chromatography with reversed-phase separation of SEC eluates became neccessary. 10 pi of the latter were injected into a C 8 column (250 x 2.2 mm dP = 10 pm) and analyzed at 0.5 ml/min flow rate through a water/acetonitrile gradient (rising from 20% B by 6%/ml). Here, UV detection was performed at 254 nm. The peaks of the additives could be clearly separated from the process oil band. The technique also proved useful for checking... 

A detailed model is required to evaluate distributions of temperature and current in the slack. Temperature gradients are particularly important in stack operation in order to avoid hot spots that can cause failures. In addition, temperature gradients must be checked in stack design because of their effects on the cell performance. To build such model some simplifications must be introduced. In the following section the main characteristics of a stack model are introduced. In this model the energy equation and the conservation of current are solved, while information about mass transfer is introduced. 

After extraction, these fractions should be dried to remove water. When dry, the extraction solvent is removed by evaporation and the sample is reconstituted with a solvent or mobile phase before injection. Care must be taken that these evaporated samples go completely back into solution. Sonicating the sample with your starting mobile phase is usually sufficient. However, at least the first time you perform an extraction, it is always good technique to sonicate the dry-down tube with a strong solvent and reinject this wash as a check that everything redissolved. For gradient work, the stronger of the two mobile phases is an excellent choice for this second sonication solvent. 

Figure 7-4 emphasizes that if it is important to know the hardware performance of the gradient system, the instrument should be checked out in a manner similar to that shown in the figure. For some systems where high sparging rates of solvents will be used, the compound methyl paraben should be used at a low concentration (approximately 0.1% or less) instead of ace-... 

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