Determination of ammonia concentration by titration

Presented by

Dr Nada Soliman

contentQuantitative method: titration

DefinitionstypesEquipmentsPrecautions

Biological substance: ammoniaNormal levelSources and fatesImportance

Principle of ammonia titration and calculation of concentration

Biological substance of unknown concentration andof known volume (sample)

To determine its conc

A standard solution: titrant

Knowing the volume of the titrant needed to react completely with the analyte, helps to determine its concentration

The titrant is added drop by drop until the reaction is complete

According to type of the reaction, we have 3 types of titration:1. Acid-base: HCL with

NaOH2. Precipitation: silver nitrate

with barium chloride3. Oxidation-reduction: uric

acid with K permenganate

1. Another substance2. Known concentration3. React with the analyte4. Volume: measured during

the experiment

Definition of titrant: the solution of known concentration added during titration

How we know that the reaction is complete?By observing physical change: the end pointCould be:

changes in color Appearance or disappearance of ppt Change in electrical properties

Definition of end point:Point in titration where physical change manifests itself

For color changes, we use an Indicator:Supplementary chemical compound that changes its color with changes in the medium at the end point

Definition of titration

A quantitative determination of a substance occurs in a stepwise manner till reaching the end point.

Equipments Burettes holders. Pipettes volumetric flasks beakers

Precautions before starting titration

1. All equipments are clean.

2. The zero level is determined from the bottom of the concavity of the fluid inside the burette.

3. No air bubbles in the terminal end of the burette.

4. Be careful that during titration no drops are coming out of the beaker

5. Each drop is mixed well with the contents of the beaker

Ammonia sources and fates

a.a. catabolismPolyamine

(purine & pyrimidine)

Monoamineepinephrine

Glutamine(glutaminase)

IntestinalBacteria (urease)

NH3

ureaGlutamine(glutamine synthase)

a.a(Non essential)

Traces in the blood(10-20 ug/dl)

90%

importance The ammonia content of the blood in renal veins exceeds

that in renal arteries, indicating that the kidneys produce ammonia and add it to the blood.

glutamine

glutaminaseNH3 +

Glutamicacid

H2CO2 + H+

Na+

Cl-HCO3-

NaHCO3

NH4CL

BLOOD URINE

NaCL

In acidosis: increase glutaminase enzyme activity and increase excretion of NH4CL in urine

In alkalosis: decrease glutaminase enzyme activity and decrease excretion of NH4CL in urine

Principle of ammonia titration

If formalin (formaldehyde) is added to a known volume of urine, it combines with the ammonium salt present, liberating an equivalent amount of acid, which can be estimated by titrating against a standard alkali.

This reaction is as follows:-

4 NH4Cl + 6 HCHO 4 HCl + 6 H2O + (CH2)6N4

Type of titration: acid-base titrationTitrant: 0.1 N NaOHEnd point: color change (just pink)Indicator used: phenolphthalein,

coloreless at pH< 8Pink at pH = or > 8

Procedure

1. Fill a burette with 0.1 N-NaOH solution

2. Pipette 10 ml of the urine into a beaker (using a 10 ml pipette).

3. Add 2 drops of phenolphthalein indicator to the content of the beaker.

Before converting ammonium salt into acid, we must first neutralize the organic acid present normally in urine (as lactic, oxalic and glucuronic acid)

4. Titrate the urine in the beaker against the standard alkali in the burette until it is just pink. Take the reading of the burette (=R1).

5. Add to the contents of the beaker 2 ml of 40% (neutralized) formaldehyde. The pink color disappears.

6. Titrate again with the standard alkali until the pink color just reappears. Take the burette reading (=R2).

7. The difference between the second and first reading (R2-R1) is the volume of standard alkali (R) equivalent to the NH3 content of 10ml of urine.

10 ml urine +

phenolphthalein

colorless

NaOH

Till just pink

2mlformalin

colorless

NaOH

Till just pink

R1

R2

R= R2-R1Volume of 0.1 N NaOH

equivalent to ammonia in 10 ml urine

Calculation of ammonia concentration

R= ……ml 0.1 N NaOH/10 ml urine

mg ammonia 100 ml urine

1ml of 0.1 N-NaOH is equivalent to 1.7 mg of ammonia.

1ml 0.1 N NaOH 1.7 mg ammonia

R ? Ammonia conc

So to determine ammonia conc, multiply R by 1.7 And then to calculate the amount in 100 ml urine multiply by

10

AMMONIA CONCENTRATION= R X1.7X10 MG/DL