Phosphate Buffer:

A phosphate buffer solution is a handy buffer to have around, especially for biological applications. Because phosphoric acid has multiple dissociation constants, you can prepare phosphate buffers near any of the three pHs, which are at 2.1 , !."! and 12.#2. $he buffer is most commonly prepared at pH % using monosodium phosphate and its con&ugate base, disodium phosphate. phosphate buffered saline 'PB() at pH %.*.
Phosphate Buffer '(orenson+s buffer) pH ."," Advantages: 1. 2. #. *. -ost physiological of common buffers. -imics certain components of e.tra cellular fluids. /on,to.ic to cells. pH changes little with temperature. (table for several wee0s at * 1.

2isadvantages: 1. 2. Precipitates more li0ely to occur during fi.ation. $ends to form precipitates in presence of calcium ions. Precipitates uranyl acetate and tends to react with lead salts. Becomes slowly contaminated with micro,organisms

Phosphate Buffer -aterials

monosodium phosphate disodium phosphate water phosphoric acid to ma0e the pH more acidic or sodium hydro.ide to ma0e the pH more al0aline pH meter glassware hot plate with stirring bar Prepare the Phosphate Buffer 1. 2ecide on the concentration of the buffer. -ost buffers are used at a concentration between 3.1 - and 13 -. 4f you ma0e up a concentrated buffer solution, you can dilute it as needed. 2. 2ecide on the pH for your buffer. $his pH should be within one pH unit from the p5a of the acid6con&ugate base. (o, you can prepare a buffer at pH 2 or pH %, for e.ample, but pH 7 would be pushing it. 3. 8se the Henderson,Hasselbach e9uation to calculate how much acid and base you need. :ou can simplify the calculation if you ma0e 1 liter of buffer. (elect the p5a value that is closest

to the pH of your buffer. ;or e.ample, if you want the pH of your buffer to be %, then use the p5a of !.7: pH < p5a = log '>Base?6>Acid?) ratio of >Base?6>Acid? < 1.37! $he molarity of the buffer is the sum of the molarities of the acid and con&ugate base or the sum of >Acid? = >Base?. ;or a 1 - buffer 'selected to ma0e the calculation easy), >Acid? = >Base? <1 >Base? < 1 , >Acid? substitute this into the ratio and solve: >Base? < 3. 2# moles6@ /ow solve for >Acid?. >Base? < 1 , >Acid? so >Acid? < 3.*%% moles6@ *. Prepare the solution by mi.ing 3.*%% moles of monosodium phosphate and 3. 2# moles of disodium phosphate in a little less than a liter of water. . 1hec0 the pH using a pH meter and ad&ust the pH as necessary using phosphoric acid or sodium hydro.ide. !. Ance you have reached the desired pH, add water to bring the total volume of phosphoric acid buffer to 1 @. %. 4f you prepared this buffer as a stoc0 solution, you can dilute it to ma0e up buffers at other concentrations, such as 3. - or 3.1 -. $B buffer: $B buffer is a commonly used buffer solution in molecular biology, especially in procedures involving 2/A or C/A. D$BD is derived from its components: $ris, a common pH buffer, and B2$A, a molecule that chelates cations li0e -g2=. $he purpose of $B buffer is to solubiliEe 2/A or C/A, while protecting it from degradation. Recipe A typical recipe for ma0ing 13:1 $B buffer is:

13 m- $ris, bring to pH ".3 with H1l 1 m- B2$A

$B buffer is also called as $13B1 Buffer, and read as D$ ten B one bufferD. $o ma0e a 133 ml solution of $13B1 Buffer, 1 ml of 1 - tris H1@ 'pH ".3) and 3.2 ml B2$A '3. -) and ma0e up with double distilled water up to 133ml. Based on nuclease studies from the 17"3+s, the pH is usually ad&usted to %. for C/A and ".3 for 2/A. $he respective 2/A and C/A nucleases are supposed to be less active at these pH values, but pH ".3 can safely be used for storage of both 2/A and C/A. B2$A further inactivates nucleases, by binding to metal ions re9uired by these enEymes. $AB buffer: $AB buffer is a buffer solution containing a mi.ture of $ris base, acetic acid and B2$A. 4n molecular biology it is used in agarose electrophoresis typically for the separation of nucleic acids such as 2/A and C/A.>1?4t is made up of $ris,acetate buffer, usually at pH ".3, and B2$A, which se9uesters divalent cations. $AB has a lower buffer capacity than $BB and can easily become e.hausted, but linear, double stranded 2/A runs faster in $AB. Cecently, Brody F 5ern simplified electrophoretic buffers by substituting $BB and $AB buffers for a more efficient and ine.pensive conductive media in gel systems.

Uses $AB '$ris,acetate,B2$A) buffer is used as both a running buffer and in agarose gel. 4ts use in denaturing gradient gel electrophoresis methods for broad,range mutation analysis has also been described.$AB has been used at various concentrations to study the mobility of 2/A in solution with and without sodium chloride.However, high concentration of sodium chloride 'and many other salts) in a 2/A sample retards its mobility. $his may lead to incorrect interpretations of the resulting 2/A banding pattern. 1ompared with $BB buffer, $AB buffer offers advantages in subse9uent enEymatic applications for the 2/A sample. ;or e.ample, if a 2/A sample is going to be used in a cloning e.periment, the step that follows its running on an agarose gel is to ligate 'covalently lin0) to a cloning vector 'most li0ely a plasmid). 2/A sample from $AB buffer is suitable for this purpose, while 2/A from $BB buffer is not. Borate in the $BB buffer is a strong inhibitor for many enEymes. $his enEyme inhibiting property made $BB buffer very popular in its realm for two reasons. ;irst, a 2/A sample run in a $BB buffer can better 0eep its integrity. $he other main reason is that the purpose for many agarose gel electrophoreses is to analyEe the siEe of 2/A molecules. 4n particular, the 0inds of 2/A analyses shown on $G and other public media are more li0ely run in $BB buffer. Applications of buffers:

Buffer solutions are necessary to 0eep the correct pH for enEymes in many organisms to wor0. -any enEymes wor0 only under very precise conditionsH if the pH moves outside of a narrow range, the enEymes slow or stop wor0ing and can denature, thus permanently disabling their catalytic activity.>2? A buffer of carbonic acid 'H21A#) and bicarbonate 'H1A#I) is present in blood plasma, to maintain a pH between %.# and %.* . 4ndustrially, buffer solutions are used in fermentation processes and in setting the correct conditions for dyes used in colouring fabrics. $hey are also used in chemical analysis >1? and calibration of pH meters. $he ma&ority of biological samples that are used in research are made in buffers, especially
$ris buffer Advantages: 1. Jood buffering capacity at higher pH re9uired for some tissues and some cytochemical procedures.

2. D-ore or lessD physiologically inert. 2isadvantages: 1. 2. pH changes with temperature. -ust be measured at desired temperature. pH must be measured with certain type of electrode.

Acetate Buffer: 'sodium acetate,acetic acid buffer) pH *, .! (odium acetate 1H#1A2/aK#H23 Acetic acid 1H#1AAH 3.2- < 2%.2 gm61 '-L , 1#!.37) 3.2'-L < !3)

Add sodium acetate to acetic acid to give desired pH. 2ilute with ddH 23 to desired molarities.
Acetate Buffer pH 4 for Chlorine Acetate Buffer pH 4 for Chlorine Acetate Buffer pH 4 for Chlorine Acetate Buffer pH 4 for Chlorine Acetate Buffer pH 4 for Chlorine