Buffer selection

Mistakes can occur when trying to control the pH of the mobile phase and it is not unusual to find methods where the buffer choice and pH range are completely incompatible with the intended separation and there is little or no buffering capacity. For example, it is quite common to see that the aqueous portion of the mobile phase has been adjusted to pH 7 with acetic acid with the expectation that the solution is therefore buffered at pH 7. This is not the case and can lead to confusion. What this means is that the pH of the solution is at 7 before the introduction of the sample. Upon addition of the sample, the pH of the solution may change this introduces many imnecessary complications that may lead to an increased level of unpredictability in terms of retention time and separation from run to rim. 

When the buffer is chosen, it is necessary to ensure that the pH range is within 1 pH unit of the pKa value quoted for that buffer. Buffer selection can involve a substantial amount of method development work and it is 

The pH of the buffer should be selected depending on the pKa values of the components in the mixture and the separation requirements. For any given separation system, there is an attraction between the solute (sample compound) and the stationary and mobile phases. This attraction can be influenced through ionisation or ion suppression of the acidic and basic compounds undergoing separation. Using reversed phase HPTC as an example where there is a nonpolar stationary phase and a polar mobile phase, it would be expected that a nonpolar basic compound would have a high affinity for the stationary phase. 

Doing this effectively reduces the attraction for the stationary phase and, in doing so, shortens the retention time. The compound will become charged and acts like a more polar molecule. Amphetamine is a weak base with a pKa of 10.1 and can be used to illustrate this point. As the pH in the mobile phase is reduced, the amphetamine begins to ionise and we would expect to see a reduction in retention time. As the drop in pH approaches the pKa, the change in retention time is quite marked until such time as we reach a state where all of the amphetamine is fully ionised. At this point (pH 2-3), small changes in pH will have less of an effect on the retention time. In summary, for basic compounds, the retention time will decrease as the pH of the mobile phase is decreased. 

The reverse also applies when the basic compounds may not be resolved sufficiently. It may be desirable to increase the retention time of the compounds to improve the separation. In this case, it is necessary to maximise the affinity for the stationary phase this can be achieved by adjusting the 

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Sample adsorption to the silica wall is a problem in HPCE, one that is highly undesirable. As we mentioned earlier, adsorption can be minimized by proper buffer selection, additives, or chemical modification of the surface. The selection of pH is one of the simplest separation parameters to manipulate and is critical to the success of all electrophoretic separations. The pH of the media will determine the charge of the sample and the charge of the silica surface. At low values of pH, the capillary wall is protonated, the EOF... 

A variety of buffers is used in electrophoresis. The selected buffer must contain ions to carry the current. Other than current-carrying capacity, the most critical criterion for buffer selection is the stability of the sample to be analyzed. Many proteins are unstable in acidic pHs, so alkaline buffers are frequently employed. Tris-(hydroxymethyl)amino methane (TRIS or THAM), sodium acetate, and ethylenedi-aminetetraacetate (EDTA) are common solutes in buffers, with pHs between 7.9 and 8.9 typical. (Refer to Chapter 5 for a discussion of buffers.) These buffers also work well with nucleic acid fragments. In addition, phosphate buffers, e.g., 10 mMK3P04, are often used with nucleic acid fragments (1.0 mM = 0.0010 M). 

Usually UV absorption of eluent is high because of counter-ion and buffer. Select suitable wavelength or change detector... 

The choice of native electrophoresis system depends on the particular proteins of interest. There is no universal buffer system ideal for the electrophoresis of all native proteins. Both protein stability and resolution are important considerations in buffer selection. Recommended choices are the Omstein-Davis discontinuous system21-24 and McLellan s continuous buffers.25... 

Add 0.5 -1 ml of protein sample, dialyzed against the respective buffer, to the wet media. Incubate for 15 - 30 min at that temperature which should be used for separation. Separate by centrifugation or filtration after incubation and determine the amount of target protein in the liquid. For binding buffer select that buffer at which the target protein disappears. 

From the following available buffers, select the best buffer, and then state what color change will be observed at the end point. Explain your answer. 

A pH electrode should be calibrated with two (or more) standard buffers selected so that the pH of the unknown lies within the range of the standards. Standards in Table 15-3 are accurate to 0.01 pH unit.16... 

The use of chemiluminescence reactions for the detection of metal ions by liquid chromatography was recently reported [59,60]. The detectors made use of the chemiluminescence produced in the reaction between luminol and hydrogen peroxide which is catalyzed by transition metals. The column effluent was mixed with the reagents in order to yield the chemiluminescence. The reaction was fast and was carried out at room temperature. By varying the pH of the buffer, selectivity towards certain metals was also achieved. For example, at pH 10-11 nickel could be analyzed but lead and aluminium were inactive at pH 13-14, the converse was true [59]. Aminco-Bowman has marketed a liquid chromatographic system in which amino acids and amines are analyzed by means of the fluorescence produced on reaction with the reagent fluorescamine. Fluorescamine does not fluoresce, but it does react with primary amino groups to produce fluorescent derivatives. The reaction is instantaneous and may be carried out at room temperature, usually at pH 9. This detection system promises to be far more sensitive than the ninhydrin detection system and is much more easily adapted to HPLC. 

In addition to buffer selection and effective pH control, a number of buffer additives can be used to optimize a separation (for more detailed discussion, see Section IV.A). 

Thormann et al. (1993) have published an overview of the strategies for using MEKC to monitor drugs in body fluids (serum, urine, saliva) they discuss buffer selection and sample preparation (direct injection, ultrafiltration, solid phase extraction. 

Oxidation of chroman-4-one and its thio analogue with Mn(OAc)3 gives the 3-acetates and subsequent basic hydrolysis yields the 3-hydroxychroman-4-one. Enzymatic hydrolysis of the 0-heterocycle using Amano PS lipase in a phosphate buffer selectively cleaved the (+)-isomer <03TA1489>. Enol ethers derived from chroman-4-one are converted into the 3-hydroxy-chromanone with high enantioselectivity, optimal with the pentyl ether, using a modified Sharpless asymmetric dihydroxylation reaction <03JOC8088>. 

Figure 3. Anion exchange chromatogram with Particle Beam MS detection eluting with ammonium acetate buffer (selected ion monitoring, isobutane PCI) of daminozide (125 rjg) (top trace), compared to a sample of commercial apple juice extracted with a SAX SPE cartridge equivalent to 100 ppb daminozide contamination (bottom trace).

For ionizable compounds, pH adjustment is often one of the most important ways to improve solubility. Sometimes, solubility data for salts may be needed. For example, when developing a solution formulation, the buffer selected should not form a less soluble salt with the drug substance. However, the actual salt may not be readily available. In these cases, the in-situ salt screening method may be useful in estimating the solubility of various salts (Tong and Whitesell, 1998). 

Guide to HPLC and LC-MS Buffer Selection, Advanced Chromatography Technologies, Aberdeen, free at... 

The objective of the present study was to determine whether cotton fabrics treated with alkcdine agents can be protected from degradation during accelerated oven aging. The buffers selected were cxies vhich have been used in paper ocxiservation. Mori line was of particular interest because it can be a( lied from the vapor phase. 

The most likely source of phosphate contamination would be ATP, ADP, or the enzyme. ATP and ADP may be unstable and undergo hydrolysis in aqueous solution, over time. The enzyme stock buffer must not contain phosphate, excluding common buffer selections such as PBS. If necessary, test individual component of the assay at appropriately diluted assay-level concentrations in ColorLock reagents for phosphate contamination. Sec Note 1 for additional details. 

This must be the species N=3, and possibly N=2. The characteristic point of maximum curvature occurs at about -195mv (x s in Figure 21), and it is not possible to fix a reliable mid-point because of the nearness of the other oxidation process. Since N=3 images are predicted to be less stable than N=2, we offer the hypothesis for future work that this transition goes at least from N=3 to N=2, and possibly on to complete destruction. We have neither a reliable third buffer selected at this moment, nor any experimental coatings to allow us to proceed farther. 

Binding buffer Selection buffer supplemented with 2pg/ mL of yeast tRNA (Invitrogen) and 125 pg/mL of BSA (Bovine serum albumin from Sigma-Aldrich). 

Elution buffer. Selection buffer supplemented with 500 mM of imidazole (BioShop). 

Good s Buffers - organic buffers selected by Dr. Norman Good because of then-favorable characteristics in biology and biochemistry... 

In the majority of practical applications, glass electrodes cells are calibrated by a two-point calibration, or bracketing, procedure using two standard buffer solutions, with pH values, pH(Sj ) and pH(S2), bracketing the unknown pH(X). Bracketing is often taken to mean that the pH(Si) and pHfSj) buffers selected from Table 2 should be those that are immediately above and below pH(X). This may not be appropriate in all situations and choice of a wider range may be better. 

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