Determination of ammonia concentration by titration

Presented by Dr Nada Soliman

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Quantitative method: titration Definitions types Equipments Precautions Biological substance: ammonia Normal level Sources and fates Importance Principle of ammonia titration and calculation of concentration

Biological substance of unknown concentration and of known volume (sample)

To determine its conc A standard solution: titrant

1. 2. 3. 4.

Another substance Known concentration React with the analyte Volume: measured during the experiment

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

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

According to type of the reaction, we have 3 types of titration: 1. Acid-base: HCL with NaOH 2. Precipitation: silver nitrate with barium chloride 3. Oxidation-reduction: uric acid with K permenganate

How we know that the reaction is complete? By observing physical change: the end point Could 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 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 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. catabolism Glutamine (glutaminase) NH3 Monoamine epinephrine Polyamine (purine & pyrimidine) Intestinal Bacteria (urease)

a.a (Non essential) Glutamine (glutamine synthase)

90%

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

urea

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.
BLOOD URINE

glutaminase
glutamine H2CO2 NH3

Glutamic + acid

NaCL

HCO3- + H+

ClNH4CL

NaHCO3

Na+

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 acid estimated by titrating against a standard alkali. This reaction is as follows:4 NH4Cl + 6 HCHO 4 HCl + 6 H2O + (CH2)6N4

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 (R2R1) is the volume of standard alkali (R) equivalent to the NH3 content of 10ml of urine.

R1
10 ml urine + phenolphth alein

NaOH
colorless

2ml formalin Till just pink

colorless

NaOH R= R2-R1 Volume of 0.1 N NaOH equivalent to ammonia in 10 ml urine

R2 Till just pink

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

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