Buffers and samples

In preparative continuous free flow electrophoresis, continuous buffer and sample feed are introduced at one end of a thin, rectangular electrophoresis chamber. A schematic is presented in Fig. 11-5. The sample stream is usually introduced through a single port while buffer is introduced through several ports, essentially producing a buffer curtain . Because the buffer streams are introduced independently, it is fairly easy to establish a variety of gradients (e.g., pH, density, ionic strength) across the buffer curtain . 

The washing of capillaries with dilute alkaline solution is advisable before analysis. The alkaline solution can be followed by deionized water and buffer. Capillaries can be washed between runs too. Samples can be introduced into the capillary by hydrodynamic and electro-kinetic methods. The hydrodynamic method applies a pressure difference (5-10 sec) between the two ends of the capillary. The pressure difference can be achieved by overpressure, vacuum or by creating a height difference between the levels of the buffer and sample reservoirs. In the case of electrokinetic injection, the injection end of the capillary is dipped into the sample for a few seconds and a voltage of some thousand volts is applied. 

CE instruments are thermostated to dissipate excessive Joule heat. Generally that covers only the main part of the capillary, and not, e.g., the autosampler with the buffer and sample vials. In some instruments, it is difficult to control the autosampler temperature due to the near presence of extraneous heating sources such as the detector lamp. Also, some labs... 

Stability and shelf life of buffers and sample solutions Reproducibility between capillaries... 

The pH of the standard buffer and sample can vary with temperature due to shifts in their chemical equilibrium. Since the actual pH of the most common... 

The difference in the conductivity of the calibration buffers and sample can cause a very large error on the sample measurement, due to junction potentials in different environments. Solid samples should be dissolved in purified water. It is necessary that the water be carbon dioxide-free. The presence of dissolved carbon dioxide will cause significant bias in the measurement of samples with low buffering capacity. For pH measurements with an accuracy of 0.01 to 0.1 pH unit, the limiting factor is often the electrochemical system (i.e., the characteristics of the electrodes and the solution in which they are immersed). 

Reagents. Cyclic nucleotides (3, 5 -cyclic adenosine monophosphate (c-AMP), 3, 5 -cyclic guanosine monophosphate (c-GMP), and 3, 5 -cyclic inosine monophosphate (c-IMP)) sodium tetraborate hydrochloric acid and potassium hydroxide were purchased from Sigma Chemical Company, St. Louis, Missouri). Millex disposable filter units (0.22 pm) were obtained from Millipore Corporation (Bedford, Massachusetts). Triply distilled and deionized water was used for the preparation of buffer solutions. Both buffers and samples were routinely degassed with helium after filtration (using microfilter units). 

Contrary to classical packed beds, fluidization of beads provides a practical option to process very crude material containing particles in suspension such as protein aggregates, cells, or cell debris. In this separation mode, microbeads are lifted inside a column by an upward liquid stream generated by buffers and sample solutions. The particles leave larger empty zones between beads where the feedstock and particles in suspension pass through. 

Fig. 9.7.18). The external sample source was left to float electrically (no grounding necessary), which was to make a more compatible interface with real external process sampling equipment such as a microdialysis probe head for bioreactor sampling. In addition, a gated injection method was introduced to accommodate this conhguration. In this design, the buffer and sample streams flow towards each other in orthogonal directions in the microchannels, then meet at the cross in a fashion similar to Fig. 9.7.7.

A variation of the above method is crossed immunoelectrophoresis with an intermediate gel. The first electrophoretic steps including plate preparations, running buffer and sample application are the same as with the conventional procedure of Laurell s. The modification with the intermediate gel has been worked out to find out which of the peaks appearing on the complex electrophoretic profile of serum... 

It is critical that pH electrodes are calibrated often as the slope of their response curve will change with temperature, as defined by the Nernst equation [2], Calibration should be performed with at least two certified pH calibration buffers that bracket the expected pH of the sample. Calibration is performed as described in the manufacturer s instructions provided with the pH meter. Calibration should be checked by measuring the pH of the first calibration buffer. The result should be no more than 0.02 pH units different from the certified pH of the buffer after adjusted for temperature. pH measurements are best performed when calibration buffers and samples are held at a constant temperature. 

Because of the small injection volumes used in CE, peak area reproducibility is poorer than HPLC. The use of high sample concentrations and internal standards makes CE precision values more comparable to HPLC. The precision of solute migration times can also suffer because of variations within the capillary between injections, affecting the EOF. This can be alleviated by careful preparation of buffers and samples and the use of dynamic capillary coating systems. 

Initial conditions that must be specified for a simulation include (1) the distribution of all components, (2) the pK and mobility values of the buffer and sample constituents, (3) the diffusion coefficients and charge tables of the proteins (GENTRANS only), (4) the electroosmotic input data [constant velocity... 

The PASS model requires description of electromigration, diffusion, and advection of sample ions as well as BGE ions. The general system of equations is highly coupled and nonlinear and, therefore, difficult to solve. However, the concentration of sample ions is much smaller than the buffer ions (typically p,M sample ions concentration or less versus order 1 mM buffer ion concentrations). Therefore, we can decouple the buffer and sample ion concentration fields. Using this approach, Bharadwaj and Santiago [4] have developed a dynamic model for PASS in a flat-plate... 

Mobile Phase Preparation, the Use of Buffers, and Sample Preparation... 

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