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Friday, 27 June 2014

Preparation of Simulated Intestinal Fluid (SIF)


Preparation of Simulated Intestinal Fluid (SIF)

Simulated Intestinal Fluid is a synthetic fluid use in analysis of pharmaceuticals in the laboratory. The extent to which the analysed pharmaceutical dissolve and release it actives in the simulated intestinal is a good indicator of how the pharmaceutical will be available for absorption into the system from the villi in the intestine. The Simulated Intestinal Fluid is use to determine bioavailabilty of a particular drug when taken.

Procedure

1.      Weigh 6.8g of monobasic potassium phosphate into a 500ml beaker

2.      Add 250ml of distilled water and mixed to dissolve.

3.      Add 77ml of 0.2N NaOH.

4.      Transfer the solution into 1000ml volumetric flask

5.      Add 300ml of water

6.      Add 1g of Pancreatine to the solution.

7.      Measure the pH (The pH will be in region of 4.5-4.6 at 25oC)

8.      Correct the pH of the solution to pH 6 by adding 0.2N NaOH to increase the pH value to 6.0 (0.2N HCl can also be used if the solution is basic).

9.      Make up the solution to the 1000ml mark.

Monday, 23 June 2014

Preparation of 0.5M Acetate buffer


Preparation of 0.5M Acetate buffer

1.      Weigh 2.99g of sodium acetate trihydrate into a beaker

2.      Transferred into 1000ml volumetric flask with half filled with distilled water

3.      Add 1.66ml of glacial acetic acid

4.      Make up to the mark with distilled water.

5.      The pH of the solution should be measured with a pH meter and should be 4.51  at 37C.

Friday, 20 June 2014

Preparation of Simulated Gastric Fluid (SGF)


Preparation of Simulated Gastric Fluid (SGF)

 

Simulated Gastric Fluid (SGF) is a synthetic form of the gastric fluid in the stomach. This fluid will show the effect of the gastric juice in the stomach on a particular drug in the stomach.

Drugs made for dissolution and disintegration in the small intestine must not disintegrate or dissolve in the in the Simulated Gastric Fluid (SGF). On the other hand, drugs designed to act in the stomach will dissolve in this synthetic solution of Simulated Gastric Fluid (SGF).

 

Procedure

To prepare Simulated Gastric Fluid (SGF)

1.      Weigh 2g of NaCl and 3.2g of Pepsin derived from stomach mucosa with an activity of about

2.      800 to 1200 units per milligram

3.      Add 7ml concentrated HCl to dissolve

4.      Transfer into 1000ml volumetric flask

5.      Add deionized/distilled water into 1000ml volumetric flask in drops

6.      Make up to the mark with deionized/distilled water.

7.      Test the pH with a pH meter and the pH should be a pH of 1.199.

Wednesday, 18 June 2014

Preparation of 0.2N Phosphate buffer of pH 5.8


Preparation of 0.2N Phosphate buffer of pH 5.8

1.      Weigh 54.44g of potassium dihdro-orthophosphate (KH2PO4) into a beaker

2.      Transfer and washed into a 2000ml volumetric flask with deionized/distilled water

3.      Make up to the mark with deionized/distilled water.

4.      Measure 1250ml of the solution into 5000ml beaker to read the pH.

5.      The pH value should be measured with a pH meter and the pH should be in the region of 4.21

6.      Increase to 5.8 with 0.2M NaOH in drops with continuous stirring.

7.      Transferred the solution into a 5 litre volumetric flask

8.      Make up to the mark with deionized/distilled water (pH 7).

PRAPARATION OF FEHLING’S SOLUTIONS


PRAPARATION OF FEHLING’S SOLUTIONS

Fehling solutions are vital reagent for Biochemist and Chemists for qualitative and quantitative analysis of carbohydrates in the laboratory. It was first prepared by Hermann Von Fehling.

The following are the standard procedure for the preparation of Fehling I and II solutions.

Preparation of Fehling I solution

APPARATUS: 500ml Beaker, stirrer, 1000ml volumetric flask

REAGENT: Distilled water, CuSO4 powder

PROCEDURE:

1.      Weigh 69.3g of CuSO4 powder into a 500ml beaker, add 200ml distilled water and stir to dissolve

2.      Transfer to 1000ml volumetric flask, make up to the mark with distilled water and shake to homogenise

 

Preparation of Fehling II solution

REAGENT: Distilled water, NaOH pellets, Potassium sodium tartarate powder

PROCEDURE:

1.      Weigh 100g of NaOH pellets into a 500ml beaker, add 200ml distilled water and stir to dissolve

2.      Weigh 346g of potassium sodium tartarate into another 500ml beaker, add 300ml distilled water and stir to dissolve

3.      Transfer both solutions to 1000ml volumetric flask, make up to the mark with distilled water and shake

NB: The above preparations are for the preparation of 1 litre of the resulting solution.

Thursday, 5 June 2014

PREPARATION OF 0.1M AgNO3


PREPARATION OF 0.1M AgNO3

AgNO3 also called silvertrioxonitate(iv) is a vital salt in analysis in inorganic chemistry.

Silvertrioxonitrate is a good reagent in qualitative analysis of chloride ions in solution of their salts.

Procedure

Weigh 17g of the AgNO3 powder into a 5oml beaker, dissolve with little amount of distilled water, transfer into 1000ml volumentric flask and make up to the mark with distilled water.

NB: Do not allow the AgNO3 to touch any part of your body and the preparation must be carried out in a dark room.

Monday, 2 June 2014

PREPARATION OF 4% BORIC ACID WITH SCREENED METHYL RED


PREPARATION OF 4% BORIC ACID WITH SCREENED METHYL RED

This is a reagent used in the analysis of protein in the Kjedahl method for collecting the condensed ammonia during distillation.

It is a useful weak acid for analysis of organic substances

Procedure

The procedure to prepare 1000ml 4% Boric acid with screened methyl red is as follows:

APPARATUS: 500ml Beaker, stirrer, 1000ml volumetric flask

REAGENT: Deionized water, Boric acid powder, Bromo cresol green, methyl red

PROCEDURE:

1.      Weigh 40g of Boric acid (B(OH)3) powder into a 500ml beaker, add 200ml deionized water and stir to dissolve

2.      Transfer to 1000ml volumetric flask,

3.      Add 10ml Bromocresol green, 7ml Methyl red, and make up to the mark with deionized water and shake.

Friday, 30 May 2014

KJELDAHL METHOD OF PROTEIN ANALYSIS


KJELDAHL METHOD OF PROTEIN ANALYSIS

The Kjedahl method of protein analysis is a general method of calculating the concentration of protein in substances. The protein analysed from a sample through Kjedahl method are said to be crude and commonly called Total Crude Protein. This is because this method does not account for the presence of nitrogen from non-protein substances e.g. secondary metabolites, vitamins and nucleic acids in the sample.

Protein is one of the four major macro molecules (others are Fat, Carbohydrate, and Nucleic Acid). Proteins are composed of α-L- amino acids that are joined by peptide bonds to form linear chains or branched chain through formation of disulphide bonds.

In addition to carbon, hydrogen, and oxygen, proteins contain approximately 16% nitrogen by weight.

The digestive process breaks down proteins to their constituent amino acids, which enter the blood. The complete oxidation of proteins to CO2, H2O, and NH4+ in the body yields approximately 4kcal/g.

The official method for analysis of crude protein in solid and powdered samples is the Kjeldahl method.

AIM: To determine the percentage protein through percentage Nitrogen

APPARATUS: Kjeldahl flask, ash less filter paper, Kjeldahl instrument, 250ml conical flask.

REAGENT: Concentrated H2SO4, 50% NaOH, 4% Boric Acid, Screened Methyl Red, Kjeldahl Tablet (Se2SO4) and distilled water

PRINCIPLE: The heating of the sample with sulfuric acid, decomposes the organic nitrogen present to ammonium sulphate. In this step there is also addition of  mercury oxide or copper sulphate which serves as catalyst to speed up the rate of the decomposition. Chemical decomposition of the sample is complete when the medium has become clear and colorless solution (initially black).

The solution is then distilled with sodium hydroxide which converts the ammonium salt to ammonia which is trapped with boric acid solution. The concentration of ammonia present (hence the amount of nitrogen present in the sample) is determined by titration, a type of titration known as back titration.

Equations for various Steps

1.      DIGESTION OF THE SAMPLE

Protein + H2SO4           (NH4)2SO4(l) + SO2(g) + CO2 + H2O(g)

2.      DISTILLATION

(NH4)2SO4(l) + NaOH           Na2SO4(s) + NH3(g) + H2O(l)

Capturing of ammonia by boric acid

B(OH)3(l) + NH3(l) + H2O(l)         NH4+(l) + B(OH)4-(l) (greenish colouration)

3.      TITRATION

 This is the final stage of the reaction, this involve the titration of the solution obtained from distillation against 0.1M HCl or 0.05M H2SO4.

NH4+ + B(OH)4- + HCl         NH4Cl + B(OH)3(l) + H2O(l)

NH4+ + B(OH)4- + H2SO4         (NH4)2SO4(l) + B(OH)3(l) + H2O(l)

PROCEDURE:

i) Weigh 0.2g of the sample in an ash less filter paper and transfer into the Kjeldahl flask

ii) Add 25ml concentrated H2SO4

iii) Add half Kjeldah tablet (catalyst)

iv) Placed in the Kjeldahl Digestion compartment and turned on the Heater. Heat till solution turn colourless, then turn off the heater and allow the solution to cool to room temperature.

v) Add 200ml of distilled water to the cool dilute the acidic solution

vi) Measure 50ml of 4% Boric acid (B(OH)3) into 250ml conical flask and add 3 drops of screened methyl red and place the solution in the ammonia outlet of the Kjedahl Apparatus.

vii) Add 75ml of 50% NaOH gently, glass beads, granulated zinc (anti-bumping agent) and 50ml of distilled water to the solution in step (v).

vii) Transfer immediately to the Kjeldahl distillation compartment and set up the apparatus, turn on the heater and distil to the 250ml mark in step (vi).

The distillate (whole 250ml) was titrated against 0.1M HCl (or 0.005M H2SO4) in the burette to a light reddish colour end point.

CALCULATIONS:

Percentage Nitrogen = Titre value *0.0014 * 100%

Weight taken

Percentage Protein = % Nitrogen * Factor

NB: Factors are derived from the compositional amino acids in the sample. Therefore different samples have different factors. The factors are 5.7 for flour, 6.38 for milk and 6.25 for other foods. Most time 6.25 is generally used.

 

Wednesday, 28 May 2014

PRAPARATION OF FEHLING’S SOLUTIONS


PRAPARATION OF FEHLING’S SOLUTIONS

Fehling solutions are vital reagent for Biochemist and Chemists for qualitative and quantitative analysis of carbohydrates in the laboratory. It was first prepared by Hermann Von Fehling.

The following are the standard procedure for the preparation of Fehling I and II solutions.

Preparation of Fehling I solution

APPARATUS: 500ml Beaker, stirrer, 1000ml volumetric flask

REAGENT: Distilled water, CuSO4 powder

PROCEDURE:

1.      Weigh 69.3g of CuSO4 powder into a 500ml beaker, add 200ml distilled water and stir to dissolve

2.      Transfer to 1000ml volumetric flask, make up to the mark with distilled water and shake to homogenise

 

Preparation of Fehling II solution

REAGENT: Distilled water, NaOH pellets, Potassium sodium tartarate powder

PROCEDURE:

1.      Weigh 100g of NaOH pellets into a 500ml beaker, add 200ml distilled water and stir to dissolve

2.      Weigh 346g of potassium sodium tartarate into another 500ml beaker, add 300ml distilled water and stir to dissolve

3.      Transfer both solutions to 1000ml volumetric flask, make up to the mark with distilled water and shake

NB: The above preparations are for the preparation of 1 litre of the resulting solution.

Monday, 26 May 2014

PROTEIN ANALYSIS


PROTEIN ANALYSIS

Protein is an essential component of all living things with various functions ranging from growth, repair of worn out tissues, immunoglobulins biosynthesis, hormones, transports. Protein is also one of the four major macro molecules (others are Fat, Carbohydrate, and Nucleic Acid). Proteins are composed of α-L- amino acids that are joined together by peptide bonds through condensation to form linear chains or branched chain through formation of disulphide bonds.

In addition to carbon, hydrogen, and oxygen, proteins contain approximately 16% nitrogen by weight.

The digestive process breaks down proteins to their constituent amino acids, which enter the blood. The complete oxidation of proteins to CO2, H2O, and NH4+ in the body yields approximately 4kcal/g.

The official method for analysis of crude protein in solid and powdered samples is the Kjeldahl method.

KJELDAHL METHOD OF PROTEIN ANALYSIS

 

AIM: To determine the percentage protein through percentage Nitrogen

APPARATUS: Kjeldahl flask, ash less filter paper, Kjeldahl instrument, 250ml conical flask.

REAGENT: Concentrated H2SO4, 50% NaOH, 4% Boric Acid, Screened Methyl Red, Kjeldahl Tablet (Se2SO4) and distilled water

PRINCIPLE: The heating of the sample with sulfuric acid, decomposes the organic nitrogen present to ammonium sulphate. In this step there is also addition of  mercury oxide or copper sulphate which serves as catalyst to speed up the rate of the decomposition. Chemical decomposition of the sample is complete when the medium has become clear and colorless solution (initially black).

The solution is then distilled with sodium hydroxide which converts the ammonium salt to ammonia which is trapped with boric acid solution. The concentration of ammonia present (hence the amount of nitrogen present in the sample) is determined by titration, a type of titration known as back titration.

Equations for various Steps

1.      DIGESTION OF THE SAMPLE

Protein + H2SO4           (NH4)2SO4(l) + SO2(g) + CO2 + H2O(g)

2.      DISTILLATION

(NH4)2SO4(l) + NaOH           Na2SO4(s) + NH3(g) + H2O(l)

Capturing of ammonia by boric acid

B(OH)3(l) + NH3(l) + H2O(l)         NH4+(l) + B(OH)4-(l) (greenish colouration)

3.      TITRATION

 This is the final stage of the reaction, this involve the titration of the solution obtained from distillation against 0.1M HCl or 0.05M H2SO4.

NH4+ + B(OH)4- + HCl         NH4Cl + B(OH)3(l) + H2O(l)

NH4+ + B(OH)4- + H2SO4         (NH4)2SO4(l) + B(OH)3(l) + H2O(l)

PROCEDURE:

i) Weigh 0.2g of the sample in an ash less filter paper and transfer into the Kjeldahl flask

ii) Add 25ml concentrated H2SO4

iii) Add half Kjeldah tablet (catalyst)

iv) Placed in the Kjeldahl Digestion compartment and turned on the Heater. Heat till solution turn colourless, then turn off the heater and allow the solution to cool to room temperature.

v) Add 200ml of distilled water to the cool dilute the acidic solution

vi) Measure 50ml of 4% Boric acid (B(OH)3) into 250ml conical flask and add 3 drops of screened methyl red and place the solution in the ammonia outlet of the Kjedahl Apparatus.

vii) Add 75ml of 50% NaOH gently, glass beads, granulated zinc (anti-bumping agent) and 50ml of distilled water to the solution in step (v).

vii) Transfer immediately to the Kjeldahl distillation compartment and set up the apparatus, turn on the heater and distil to the 250ml mark in step (vi).

The distillate (whole 250ml) was titrated against 0.1M HCl (or 0.005M H2SO4) in the burette to a light reddish colour end point.

CALCULATIONS:

Percentage Nitrogen = Titre value *0.0014 * 100%

Weight taken

Percentage Protein = % Nitrogen * Factor

NB: Factors are derived from the compositional amino acids in the sample. Therefore different samples have different factors. The factors are 5.7 for flour, 6.38 for milk and 6.25 for other foods. Most time 6.25 is generally used.

 

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