Preparation of BUFFER

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Preparation of BUFFER

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Buffers

• Buffers are the solutions which resist changes in
  pH when small
• amounts of acid or alkali is added to them.
• - A buffer is a pair of weak acid and its salt.
• - Buffers are of main importance in regulating
  the pH of the body fluids and tissues.
• - Many biochemical reactions including those
  catalyzed by enzymes require pH control which is
  provided by buffers.

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Buffers

• Mammalian tissues in the resting state have a pH
  of about 7.4
• In order to maintain the required pH in an
  invitro biochemical experiment a buffer is always
  used
• The pH of a buffer is given by Handerson-Hasselbal
  ch equation

pH pKa log A- HA
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Buffers

• pKa -log Ka
• Ka is the dissociation constant of the acid
• A- is the concentration of the base
• HA is the concentration of the acid

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Buffers

• - A buffer consisting of a mixture of the weak
  acid(CH3COOH) and its salt (CH3COONa), and
• Will undergo the following changes on the
  addition of acid or base
• If the base is added (OH) to the solution it
  will be buffered by the following reaction with
  acetic acid
• CH3COOH OH?CH3COO- H2O
• So the pH will not change significantly

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Buffers

• If acid (H) is added, it will be buffered by
  another reaction, this time using the salt
  (CH3COO-)
• CH3COO- H ? CH3COOH
• The pH will not alter significantly because
  the CH3COOH
• formed is a weak acid
• Addition of more base increases A- and decreases
  (HA) and this doesnt alter the pH much until
  A-gtgtgtgtHA

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Buffers

• The buffering power is greatest when pHpKa ,
  i.e. when the acid and the salt are at the same
  concentration

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A compound can buffer the pH of a solution when

• Its concentration is sufficient
• The pH of the solution is close (within about one
  pH unit) to its pKa. 

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To make a buffer

• Choose a compound whose pKa is close to the
  pH for the solution
• Determine what the buffer concentration should
  be. 

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Three practical methods to prepare a buffer

• 1- First Method By the Titration, in the
  presence of one of the two buffer forms with
  strong base or acid
• Prepare a buffer composed of an acid and its salt
  by adding a strong base(e.g. NaOH) to a weak acid
  (e.g. Acetic acid) until the required pH is
  obtained
• If the other form of buffer is available (in
  this case sodium acetate), a strong acid is added
  (e.g. HCl) until the required pH is obtained.
• CH3COONaHCl?CH3COOHNaCl 
• So acetate buffer is formed(CH3COOH/CH3COONa)

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• Advantages
• Easy to understand.
• Useful when only one form of the buffer is
  available (in this case acetic acid)
• Disadvantages
• Slow. 
• May require lots of base (or acid). 

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• 2- Second Method Using the buffer pKa ,
  calculate the amounts (in moles) of acid/salt or
  base/salt present in the buffer at the desired
  pH.
•  
• If both forms (i.e., the acid and the salt) are
  available, convert the amount required from moles
  to grams ,using the molecular weight of that
  component, and then weigh out the correct amounts
  of  both forms. Or convert moles to volume if the
  stock is available in the liquid form.
•      

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• Advantages
• Fast. 
• Easy to prepare. 
• Additional pH adjustment is rarely necessary, and
  when necessary, the adjustment is small.
• Disadvantages
• Requires the buffer pKa
• and solving two equations.

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• .3- The Thired Method Using table
• Find a table of the correct amounts of acid/salt
  or base/salt required for different pH's
• Dissolve the components in slightly less water
  than is required for the final solution volume. 
• Check that the pH and correct if necessary.
• Add water to the final volume.    

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• Advantages
• Easy to do (with appropriate table). 
• Convenient for frequently prepared buffers.
• Disadvantages
• May be impossible to find table. 
• Table may be incorrect. 
• Requires both forms of buffer. 
• Component amounts from table will need to be
  adjusted to give the buffer concentration and
  volume in your solution.

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- Decide on the Buffer Properties

• Before making a buffer you must know
• what molarity you want it to be
• what volume to make
• what the desired pH is.
• Most buffers work best at concentrations between
  0.1 M and 10 M.
• The pH should be within 1 pH unit of the acid/
  conjugate base pKa.

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In this lab

• We will prepare acetate buffer
• By two ways
• 1- Titration of week acid. Titration of acetic
  acid with strong
• base.
• 2- Using the known Pka value through
  Handerson-Hasselbalch
• equation.

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1- Titration curve of week acid acetic acid.

• Titration curves are produced by monitoring the
  pH of given volume of a sample solution after
  successive addition of acid or alkali
• The curves are usually plots of pH against the
  volume of titrant added or more correctly against
  the number of equivalents added per mole of the
  sample

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Titration

• At the starting point the acid form predominates
  (CH3COOH).
• As strong base is added (e.g. NaOH), the acid is
  converted to its conjugate base.
• At the mid point of the titration, where pHpK,
  the concentrations of the acid and the conjugate
  base are equal.
• At the end point(equivalence point), the
  conjugate base predominates, and the total
  amount of OH added is equivalent to the amount of
  acid that was present in the starting point.

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Titration.
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Titration.

• Determination of pKa values
• pKa values can be obtained from the titration
  data by the following methods
• The pH at the point of inflection is the pKa
  value and this may be read directly
• By definition the pKa value is equal to the pH at
  which the acid is half titrated. The pKa can
  therefore be obtained from the knowledge of the
  end point of the titration.

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2- Handerson-Hasselbalch equation

• Method
• You are provided with 0.1M sodium acetate and
  0.1M acetic acid solution.
• Prepare 50ml of the buffer solution (0.1 M) by
  mixing the two standard solutions
• Use the following info to calculate the
  composition of the buffer
• pH of the buffer 4.86
• pKa of acetic acid at 25C 4.76

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• Determine the Ratio of Acid to Base.
• Use the Henderson-Hasselbalch equation (below)
  to determine what ratio of acid to base is
  required to make a buffer of the desired pH.

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• Use the pKa value nearest your desired pH and the
  ratio will refer to the acid-base conjugate pair
  that correspond
• to that pKa.
• HH Equation

pH pKa log (Base/Acid)
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b. Substitute for Baseand Solve for Acid

• The desired molarity of the buffer is the sum of
  Acid Base.
• For a 1 M buffer, Base Acid 1 and
  Base 1 - Acid
• By substituting this into the ratio equation,
  from step 2, you get
• gt Acid value

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• c. Solve for Base
• Using the equation Base 1 - Acid, you can
  calculate that
• Base value

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• d. Mix the Acid and Conjugate Base
• e. Check the pH
• Use a pH meter to confirm that the correct pH for
  the buffer is reached. Adjust slightly as
  necessary.
• f. Correct the Volume
• Once the desired pH is reached, bring the volume
  of buffer to ___L.
• g. Dilute as Desired
• This same buffer can be diluted to create buffers
  of 0.5 M, 0.1 M, 0.05 M or anything in-between.