Buffers and pH

Buffers are solutions that resist a change in pH when we add an acid or base. A buffer contains both a weak acid (HA) and its conjugate base (A-). The acid part will neutralize any base added and the base part of the buffer will neutralize any acid added to the solution. We may calculate the hydronium ion concentration of a buffer by rearranging the Ka expression to yield the Henderson-Hasselbalch equation, which we can use to calculate the pH of a buffer  

These equations allow us to calculate the pH of the buffer solution knowing the Ka of the weak acid or Kb of the weak base and the concentrations of the weak acid and its conjugate base. If we know the desired pH along with the Ka of the 

There are two ways to solve this problem. Assume x = [OH ] = [NH4] that comes from the reaction of the ammonia. Ammonia is the conjugate base (A ) of the ammonium ion (HA). 

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Dissolve the protein to be reduced at a concentration of l-10mg/ml in 20 mM sodium phosphate, 0.15M NaCl, pH 7.4. Other buffers and pH values also may be used. A strong denaturant may be added (6M guanidine or 8M urea) to this solution to promote protein unfolding and make buried disulfides more accessible. 

Dissolve the antibody to be biotinylated in 50 mM sodium bicarbonate, pH 8.5, at a concentration of 10 mg/ml. Other buffers and pH conditions between pH 7 and 9 can be used as long as no amine-containing buffers like Tris are present. Avoid also the presence of disulfide reducing agents that can cleave the disulfide group of the biotinylation reagent. 

D. D. Perrin 8e B. Dempsey (1974) Buffers and pH and Metal Ion Control, Chapman and Hall, London. 

Incubate 0.5 ml of the protein dissolved in a buffer with elevated ionic strength, e.g., 1M ammonium sulfate or sodium chloride or potassium chloride, 50 mM phosphate buffer, and pH 7.0, with 0.5 ml of HIC media of different hydrophobicity, e.g., ethyl (C2), butyl (C4), hexyl (C6), and octyl (C8) substituted agarose, at 0 °C for 30 min. Centrifuge and analyse the supernatant for the protein of interest. 

The buffer and pH itself may induce phlebitis during injection. A detailed study indicated that pH range from 3 to 11 may not induce phlebitis upon bolus injection... 

Hold buffer at 30°C and aerate with a stream of bubbles via a fine capi llary tube. An aquarium aerator is a convenient way to achieve this. Select the buffer and pH to suit the enzyme being investigated. Citrate/phosphate (Mcllvaine s) buffer (appendix 2A)in the pH range of 4.0 to 6.5 is usually satisfactory for the assay of most DPOs. Recipes for a wide range of buffer solutions may be found in appendix 2a or in any standard textbook of practical biochemistry (for example, Dawson et al 1986). 

Weak Acid and Weak Base Equilibria. Buffers and pH Control. The... 

DNPH is often susceptible to formaldehyde or acetone contamination. It should, therefore, be crystallized with acetonitrile to remove any impurities. Repeated crystallization may further be performed to achieve the desired level of purity for DNPH. A 100-mL aliquot of aqueous sample is buffered with a citrate buffer and pH adjusted to 3 0.1 with HC1 or NaOH. The acidified sample is then treated with DNPH reagent and heated at 40°C for an hour under gentle swirling. The DNPH derivatives of aldehydes and ketones formed according to the above reaction are extracted with methylene chloride using liquid-liquid extraction. The extract is then solvent exchanged to acetonitrile for HPLC determination. 

The enzymatic assay is then described, including buffers and pH, the method for initiating the reaction, and the process used for termination. Next, the methods used in the preparation of the sample for HPLC analysis are described, including centrifugation, filtration, or any type of purification preceding injection into the HPLC system. For many of the assays, time span and range of protein concentration for which the reaction is linear are also indicated. 

Weak acid and weak base equilibria. Buffers and pH control. The pH of salt solutions. 

CSPs based on SOs with charged functional groups can be classified and operated as chiral ion exchangers if oppositely charged functional groups are present in the SA to be resolved. Inherently connected to this mode of separation is the use of buffered and pH-controlled mobile phases, to adjust and to control the adsorption-desorption processes. Accordingly, the primary mode of operation is in the reversed-phase mode or, alternatively, with polar-organic mobile phases. 

Media include acidic media (e.g., 0.1 N HCl) or simulated gastric fluid, USP without enzymes, pH 4.5 buffer and pH 6.8 buffer of simulated intestinal fluid, USP without enzymes (From FDA Draff Guidance, Jan, 1999.)... 

The use of buffers and pH adjustment is an important consideration in lens care products. It is a general practice that all products which are likely to come in direct contact with ocular tissues should be buffered for ocular comfort around physiologic pH and preferably in the range 6-8.0. The most commonly used buffers in contact lens care products are phosphates and borates. Buffers used occasionally are acetate, citrate, and others. Besides buffers, sodium hydroxide and hydrochloric acid are generally used to achieve a desirable pH in the final product. They are also used to adjust the final pH in products, which do not have any buffering system. The selection of an appropriate buffering system should consider the pH necessary for optimal performance of the product, as well as products... 

Table II. Effect of Various Concentrations of Citrate Buffer and pH on HPLC Retention Time and Chromatographic Pattern for Ascorbic Acid and Metaphosphoric Acid...

To determine the stability of ascorbic acid under the various concentrations of citrate buffer and pH, the following experiment was performed with the use of a double wavelength, double beam, spectrophotometer (Perkin-Elmer 557). 

Auxiliary salt Ionic strength and conductivity Buffering and pH control... 

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