A buffer is a solution that resists changes in pH by the action of its acid-base conjugate components. Buffers act by releasing hydrogen ions when a base is added and absorbing hydrogen ions when acid is added, with the overall effect of keeping [H+] relatively constant. The key buffer in human blood is an acid-base pair consisting of carbonic acid (acid) and bicarbonate (base). These two substances interact in a pair of reversible reactions. First, CO2 and H2O join to form carbonic acid (H2CO3), which in a second reaction dissociates to yield bicarbonate ion (HCO3-) and H+. If some acid or other substance adds H+ to blood, the HCO3- acts as a base and removes the excess H+ by forming H2CO3. Similarly, if a basic substance removes H+ form the blood, H2CO3 dissociates, releasing more H+ into the blood. The forward and reverse reactions that interconvert H2CO3 and HCO3- thus stabilize the pH of blood.

Buffers consist of a weak acid (HA) and its conjugate base (A-) or a weak base and its conjugate acid. Weak acids and bases do not completely dissociate in water, and instead exist in solution as a equilibrium of dissociated and undissociated species. Consider acetic acid. In solution, acetate ions, hydrogen ions and undissociated acetic acid exist in equilibrium. This system is capable of absorbing either H+ or OH- due to the reversible nature of the dissociation of acetic acid (HAc). HAc can release H+ to neutralize OH and form water. The conjugate base, A-, can react with H+ ions added to the system to produce acetic acid. In this way, pH is maintained as the three species constantly adjust to restore equilibrium. (Chapter 5, Buffers for biochemical Reactions” Protocls & Applications Guide, 2004-212, Promega Corporation).

All buffers have an optimal pH range over which they are able to moderate changes in hydrogen ion concentration. This range is a factor of the dissociation constant of the acid of the buffer (Ka) and is generally defined as the pKa (-logKa) value plus or minus one pH unit. pKa can be determined uising the Henderson-Hasselbalch equation. (Chapter 5, Buffers for biochemical Reactions” Protocls & Applications Guide, 2004-212, Promega Corporation).

Preparation of Buffers

In general, solvents are mixed in terms of their relative volumes (V/V) or relative weights (w/w). Buffer solutions are prepared by combining a weak acid and its sale (e.g., sodium salt) or a weak base and its salt. Commonly used preparation methods include 1) dripping the acid (or base) into an aqueous solution of the salt while measuring the pH value with a pH meter and 2) rendering the acid as an aqueous solution with the same concentration as the salt’s aqueous solution and mixing the two while measuring the pH value with a pH meters ((“Tips for practical HPLC analysis –Separation Know-how – Shimadzu LC World Talk Special Issue Volume 2, which also lists how to prepare many different buffer solutions).

Commonly Used Buffers

Bis -tris methane: (Bis-Tris or BTM): is an organic tertiary aminewhich is an effective buffer between pH 5.8-7.2. It has a chemical formuls C8H19NO5.

Tris (tris(hydroxymethyl)aminomethane: is an organic compound with the formula C4H11NO3 and commonlyly used in buffer solutions such as in TAE and TBE buffers. It contains a primary amineand thus undergoes the reactions assocaited with typical amines such as condensations with aldehydes.

Conductivity of Commonly Used Buffers

Sodium citrate:

(Falkenstein (US 14/934866 published as US 2107/006814) lists the conductivity for various concentrations of buffer containing sodium citrate in addition to sodium citrate and NaCL.

Sodium Free Buffers:

No Salts: (acid/base pair and sodium salt conjugate base)

Goklen (WO 2012/135415) discloses a method for purifying a protein using simplified sodium chloride-free buffer system that includes two components (acid and base pairs) for appropirate solution pH control and a thrid component for ionic strenght content where the thrid component is the sodium salt conjugate base. For edample, goklen discloses equilibrating a Protein A with a buffer that includes 55 mM tris base, 45 mM acetic acid at about pH 7.5, (b) adsorbing the protein from the contaminated solution to the Protein A and (c) removing contaminants by washing with a wash buffer that includes 55 mM Tris Base, 45 mM acetic acid, 300 mM sodium acetate at about pH 7.5 and recovering the protein with an elution buffer that includes 1.8 mM sodium acetate, 28.2 mM aetic acid, at about pH 3.6 wherein all the buffers are made without the addition of NaCl. Other chromatographies include AEX and CEX.