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INTRODUCTIN Buffer are compounds or mixture of compounds that, by their presence in solution, resist changes in pH upon addition of small quantities of acid or alkali. The resistance to a change in pH is known as buffer action. A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid.

Criteria of a good buffer : 

The buffering capacity in the desired pH range with the ability to maintain constant pH during fixation. Suitable ionic concentration so that materials are neither extracted nor precipitated during fixation. Maximum solubility in water and minimum solubility in all other solvents. Reduced ion effects. Dissociation of buffer least influenced by buffer concentration, temperature and ionic composition. Criteria of a good buffer

Suitable osmolarity so that cells neither swell nor shrink during fixation. Resistance to oxidation (stable). No reaction with fixation. Inexpensive and easy to prepare.


TYPE OF BUFFER SOLUTIONS Acidic buffer solutions Alkaline buffer solutions Acidic buffer solutions:- An acidic buffer solution is simply one which has a pH less than 7. Acidic buffer solutions are commonly made from a weak acid and one of its (conjugate base) salt. A common example would be a mixture of acetic acid and sodium acetate in solution.

Alkaline buffer solutions : 

Alkaline buffer solutions An alkaline buffer solution has a pH greater than 7 Alkaline buffer solutions are commonly made from a weak base and one of its salts. A frequently used example is a mixture of ammonia solution and ammonium chloride solution. If these were mixed in equal molar proportions, the solution would have a pH of 9.25.

Common Buffers : 

Common Buffers Phosphate Buffer (Sorenson's buffer) pH 5.8-8. Advantages: 1.   Most physiological of common buffers.  2.     Non-toxic to cells. 3.     pH changes little with temperature. 4.     Stable for several weeks at 4 C Disadvantages: 1.     Precipitates more likely to occur during fixation.  2.     Becomes slowly contaminated with microorganisms.

Preparation of Buffer :- 1 Phosphate buffer : 

Preparation of Buffer :- 1 Phosphate buffer Mix X ml of 0.2M dibasic sodium phosphate with Y ml monobasic sodium phosphate.  Dilute to 100 ml with H20   pH (25 C)                                                      X ml                                          Y ml   5.8                                                                  4.0                                          46.0 6.0                                                                  6.15                                        43.75 6.2                                                                  9.25                                        40.75 6.4                                                                13.25                                        36.75 6.6                                                                18.75                                        31.25 6.8                                                                24.5                                          25.5 7.0                                                                30.5                                          19.5 7.2                                                                36.0                                          14.0 7.4                                                                40.5                                            9.5 7.6                                                                43.5                                            6.5 7.8                                                                45.75                                          4.25 8.0                                                                47.35                                          2.65

Acetate buffer (sodium acetate- acetic acid) pH 4 – 5.6 : 

Acetate buffer (sodium acetate- acetic acid) pH 4 – 5.6 Sodium acetate             0.2M = 27.2 gm/1 CH3CO2Na*3H20          (MW - 136.09)   Acetic acid                    0.2M   CH3COOH                   (MW = 60)   Citrate Buffer (sodium citrate-citric acid buffer) pH 3-6.2 Sodium citrate                           0.2M = 58.8 gm/1 Na3C6H507*H20                          (MW = 294.12)   Citric acid                                  0.2M = 42.02 gm/1  C6H807*H20                                (MW = 210.14)

Other example of buffers : 

Other example of buffers Borate Buffer                 pH 7.4-9.2 Dimethylglutarate Buffer                          pH 3.2-7.6  Succinate Buffer                        pH 3.8-6 Maleate Buffer   pH 5.2-6.8  Imidazole Buffer  pH 6.2-7.8 Veronal-acetate Buffer  (Michaelis buffer)  Cacodylate Buffer (arsenate buffer) pH 5-7.4

Mechanism of action of buffer solution : 

Mechanism of action of buffer solution Acidic buffer solutions We'll take a mixture of acetic acid and sodium acetatete as typical. Acetic acid is a weak acid, and the position of this equilibrium will be well to the left: Adding sodium acetate to this adds lots of extra acetate ions.

The solution will therefore contain these important things: : 

The solution will therefore contain these important things: Lots of un-ionised acetic acid; Lots of acetate ions from the sodium acetate; Enough hydrogen ions to make the solution acidic. Adding an acid to this buffer solution: The buffer solution must remove most of the new hydrogen ions. Hydrogen ions combine with the acetate ions to make acetic acid.

Although the reaction is reversible, since the acetic acid is a weak acid, most of the new hydrogen ions are removed in this way. Since most of the new hydrogen ions are removed, the pH won't change very much - but because of the equilibria involved, it will fall a little bit.

Adding an alkali to this buffer solution : 

Adding an alkali to this buffer solution Because most of the new hydroxide ions are removed, the pH doesn't increase very much.


BUFFER EQUATION Buffer equation for weak acid: The pH of a buffer solution and the change in pH upon the addition of an acid or base may be calculated by use of the buffer equation. If the acid is weak and ionizes only slightly, the expression [HAc] may be considered to represent the total concentration of acid, and it is written simply as [acid]. In the slightly ionized acidic solution, the acetate concentration[Ac-] may be considered as having come entirely from the salt.

Since 1 mole of sodium acetate yields 1 mole of acetate ion, [Ac-] is equal to the total salt concentration and is replaced by the term [salt]. Hence, the equation is written as, H3O+ = Ka [Acid]/ [Salt] Equation may be expressed in logarithmic form, with the sing reversed,as -log [H3O+ ] = -log Ka + log [Salt] -log [Acid] or pH = p Ka+ log [Salt]/ [Acid] This equation are known as Hendersion Hasselbach equatin, for weak acid and its salt:

Buffer equation for a waek base and its salt : 

Buffer equation for a waek base and its salt The buffer equation for solution of weak bases and the corresponding salt may be derived in a similar manner to that for the weak acid buffer. Accordingly, [OH-] = Kb [base]/ [Salt] and using the relationship, [OH-] = Kw/ [H3O+ ] , the buffer equation becomes pH = pKw - pKb - log [base]/[salt]

Some factors influencing the pH of buffer solution : 

Some factors influencing the pH of buffer solution The addition of neutral salts to buffers changes the pH of the solution by altering the ionic strenghth. Changes in inonic stregth and hence in the pH of a buffer solution may also be brought about by dilution. The addition of water in moderate amounts, while not changing the pH, may cause a small positive or negative deviation because it alters activity coefficients. Bates has expressed this quantitatively in terms of a dilution value, which is the change in the pH on diluting the buffer solution to one half its original strength.

A positive dilution value signifies that the pH rises with dilution, and a negative value signifies that the pH decreases with dilution of the buffer. Temperature also influences buffers. The pH of a buffer solution is change with change in temperature. The pH of acetate buffer was found to increase with temperature, whereas the pH of boric acid-sodium borate buffers decreased with temperature.

Buffer capacity : 

Buffer capacity The magnitude of the resistance of buffer to pH changes is referred to as the buffer capacity ß. It is also known as buffer efficiency, buffer index and buffer value. Van slyke introduced the concept of buffer capacity and defined it as the ratio of the increment of strong base or acid to the small change in pH brought about by this addition. β = Δ B/ ΔpH

Where ∆B is the small increment in gram equivalent per liter of strong base or acid added to the buffer solution to produce a pH change. The buffer has its greatest capacity before any base is added where [salt]/[acid] = 1 { pH=pKa}. Van slyke’s equation for buffer capacity:- β = 2.3C*Ka*[H3 O+]/(Ka+[H3 O+])2 Where C is the total buffer concentration , i.e. , the sum of the molar concentrations of the acid and the salt.

Maximum buffer capacity : 

Maximum buffer capacity The maximum buffer capacity occurs where pH = pKa , or , in equivalent terms, where [H3 O+]= pKa . Substituting [H3 O+] for Ka in both the numerator and denominator of equation, β max= 2.303C*[H3 O+]2/(2[H3 O+])2 β max= 2.303*C/4 β max= 0.576*C In which C is the total buffer concentration.

Isotonic solution : 

Isotonic solution Tonicity is a measure of the osmotic pressure of two solutions separated by a semipermeable membrane. Like osmotic pressure, tonicity is influenced only by solutes that cannot cross the membrane, as only these exert an osmotic pressure. Solutes able to freely cross the membrane do not affect tonicity because they will always be in equal concentrations on both sides of the membrane.

Osmotic pressure is the pressure that must be applied to a solution to prevent the inward flow of water across a semipermeable membrane. Isotonic solution:- A isotonic solution are thoes solutin in which its solute concentration is the same as the solute concentration of another solution with which it is compared. A solution which has the same tonicity as the other solution with which it is compared.

Isotonic solutions cause no swelling or contraction of the tissues with which they come in contact . And produce no discomfort when instilled in eye, nasal tract , blood or other body tissues. Isotonic 0.9g sodium chloride solution is a familiar pharmaceutical example of such preparation. Measurement of tonicity The tonicity of solution may be deyermined by one of two method.

Hemolytic method:- In the hemolytic method, the effect of various solutions of the drug is observed on the appearance of the red blood cells suspended in the solutions. If a small quantity of blood is mixed with solution containing 0.9g Nacl per 100 ml, the cell retain there normal size. The solution has essentially the same salt concentration and hence the same osmotic pressure as the red blood cell contents, and is said to be isotonic with blood.

If the red blood cells are suspended in a 2% Nacl solution, the water within the cells passes through the cell membrane in an atempt to dilute the surrounding salt solution until the salt concentrations on the both side of the erythrocyte membrane are identical. This outward passage of water causes the cells to shrink and become wrinkled or crenated. The salt solution in this instance is sad to be hypertonic with respect to RBC contents.

If the blood is mixed with 0.2% Nacl solution or with distilled water, water enters the blood cells causing them to swell and finally brust, with liberation of hemoglobin This phenomenon is known as hemolysis, and the weak salt solution is said to be hypotonic with respect to the blood. The second approach used to measure tonicity is based on any of the mathod that determine the colligative properties.

Freezing point depression method : 

Freezing point depression method This method is based on the measurement of the slight temperature difference The freezing point of blood and lacrimal fluid is -0.52° The reference solution for the freezing point- depression method is 0.9% Nacl, which has a freezing point depression of ∆Tf = 0.52° This temperature corresponds to the freezing point of a 0.9%Nacl solution, which is therefore considered to be isotinc with both blood and lacrimal fluid.

Calculating tonicity using L(iso) values L(iso) = ∆Tf/c C is concentratin that is isotonic with body fluids. ∆Tf is the freezing point depression. The Liso value for a 0.9% (0.154M) solution of Nacl , which has a freezing point depression of 0.52 and is thus isotinc with body fluids, is 3.4 Liso = ∆Tf/C Liso = 0.52/0.154 = 3.4

Methods of adjusting tonicity One of several methods may be used to calculate the quantity of Nacl, dextrose , and other substance that may be added to solution of drug to render them isotonic. The method are divided in to two classes. In the class 1 methods, Nacl or some other substance is added to the solution of the drug to lower the freezing point of the solution to -52° and thus make it isotonic with body fluids. Under this class are included the cryoscopic method and the sodium chloride method.

Cryoscopic method : 

Cryoscopic method The freezing point depression of drug solution that have not been determined expermentlly can be estimated from the theoretical considerations, knowing only the molecular weight of the drug and the L(iso) value of the ionic class. Sodium chloride equivalent method E ≡ 17 L(iso)/ M Where M is the molecular weight

Class ll Method : 

Class ll Method In the class ll methods, water is added to the drug in a sufficient amount to form an isotonic solution The two method White-Vincent method and the sprowls method are included in this class. White –Vincent method:- V = w*E*111.1 V is the volume in ml of isotonic solution that may be prepared by mixing the drug with water W is weight of drug in grams And E the sodium chloride equivalent obtained from table.

The Sprowls Method : 

The Sprowls Method A further simplification of the method of White and Vincent was introduced by Sprowls. He recogonised that equation could be used to construct a table of values of V when the weight of the drug w was arbitrarily fixed. Sprowls chose as the weight of drug 0.3g . The volume V of isotonic solution that can be prepared by mixing 0.3 g of a drug with sufficient water may be computed for drugs commonly used in opthalmic and parentral solutions.

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