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2. Definition:
• Quantitative determination of the concentration of the substance in
stepwise reaction up to the End point.
• At the end point, one equivalent of each substance involved in titration
reacts with one equivalent of the other substance.
Titrant:
• The solution added or reagent generated in a titration.
• It is of known volume and concentration.
Calculation:
• Wt X V = Wt/ X V/
• Wt = Weight of sample used to form titrant.
• V = Volume of titrant.
• V/ = Volume of solution used to reach End point.
• Wt/ = Weight of sample to be weighted.

3. End point:
• The point in titration where sharp physical change (in the solution being
titrated) occurs at or near the end point.
• Detected by:
• Colour change: due to titrant, substance being titrated or Indicator.
• Disappearance or Appearance of precipitant high colored.
• Change in Electrical properties of solution.
Indicator:
• Supplementary chemical compound that show colour change as a result
of concentration change occurring near End point.

4. Theory of Indicator behavior:
• Acid Base Indicators are weak organic acids or bases which upon
dissociation or association give rise to alteration in color.
• H2O + H In (Acid color) H3O+ + In- (Base Color)
• OH- + In H+ (Acid Color) H2O + In (Base color)
• Red < 2.9 – 4.0 < Yellow Colorless < 8 – 9.8 < Red violet
• Topfer's indicator Just Pink
• Phenolphthalein indicator

6. Types of Titration:
1. Acid Base titration e.g.: gastric HCl + NaOH (Neutralization
reaction)
Strong Acid against Strong Base.
Strong Acid against Weak Base.
Weak Acid against Strong Base.
Weak Acid against Weak Base.
2. Oxidation reduction titration e.g.: uric Acid & K permanganate.
3. Precipitation titration e.g.: Silver Nitrate & BaCl.
Precautions of titration:
• When read in Burette, reading must be taken from lower point of
concave of fluid.
• Mix after each drop.
• No air bubbles at the end of burette.
• All Equipments should be clean.

7. Gastric Juice
• Pure gastric juice is clear watery colorless solution.
• Produced by 3 types of gastric mucosa:
1. Chief cells → Pepsin.
2. Parietal cells → HCl.
3. Columnar Epithelial cells → Mucin.
• 2000-3000 ml of gastric juice is secreted / day.
• Specific gravity 1.006-1.009
• PH 1 due to HCl which constitutes 0.2-0.5 % of gastric juice so it is the
most acidic secretion in the body.

8. Hydrochloric Acid (HCl) of Gastric Juice:-
• Parietal cells are the sole source of gastric HCl.
• Carbonic Acid is formed in parietal cells by hydration of CO2 by
Carbonic Anhydrase enzyme.
• Carbonic Acid is ionized to H+ and HCO3-.
• H+ is secreted into stomach lumen by active process (by membrane
located K+ - ATPase).
• HCO3- diffuses to plasma to replace movement of Cl- which is
transferred from blood side to stomach lumen.
Alkaline Tide:-
• Alkaline urine that follows ingestion of a meal.
• Due to arrival of excess Bicarbonates to blood during HCl secretion by
the stomach.

10. • In pure gastric juice, all HCl is nearly present as free HCl.
• Small amount of HCl is present in combined form.
• Combined HCl takes place through its reaction with proteins.
• Total Acidity = Free HCl + Combined HCl + Other Organic Acids.

11. Estimation of Free & Total
Acidity In Gastric Juice sample
•Gastric juice sample (colorless) + Topfer's indicator → Pink.
•Add 0.1 N-NaOH (X1) → Salmon Pink (Orange Yellow).
•This is the end point of Free acidity at PH 3 = all free HCl is
neutralized.
•Add phenolphthalein indicator → Colorless.
•Add 0.1 N-NaOH → Just Pink (Pink Yellowish gradually).
•This is the end point of total acidity at PH 8.
•The reading from the start till this second end point → (X2).

14. End Point of Total Acidity
Color: Pink
PH = 8
Phenolphethalein
NaOH: ??
End Point of Free Acidity
Color: Salmon Pink
PH = 3
Topfer’s
NaOH used: X1

16. Free Acidity:-
• Number of milliliters of 0.1 N-NaOH
required to neutralize free HCl in 100 ml of
gastric juice.
Total Acidity:-
• Number of milliliters of 0.1 N-NaOH
required to neutralize total acids present in
100 ml of gastric juice.
• Free Acidity = Mean X1 X 100/5 =
…………….. 0.1 N-NaOH %
• Total Acidity = Mean X2 X 100/5 =
…………….. 0.1 N-NaOH %

17. Fractional Test Meal:-
•The curve of normal people shows:
1. Fasting juice acidity is neutralized by 20-30 ml 0.1 N-NaOH per
100 ml of gastric juice.
2. 15-30 minutes after the meal, acidity falls to lower level due to:
Dilution of gastric juice with saliva and tea.
Neutralization of gastric juice by NaHCO3 of saliva,
proteins of the meal and mucin inside the stomach.
3. Then acidity rises again and reaches its maximal value after 90
minutes due to stimulation of gastric glands by the meal.
4. After 120 minutes acidity starts to decline gradually until the
fasting level is nearly reached at the end of 150 minutes due to:
Gastric secretion gets diminished.
Gastric contents are evacuated to the duodenum.
Gastric contents are neutralized by duodenal regurgitation.

19. Abnormal Curves:-
1. Hyperacidity (Hyperchlorhydria):-
•Acidity more than normal.
•As in cases of duodenal ulcers and gall bladder
diseases.
2. Hypoacidity (Hypochlorhydria):-
•Acidity is below normal as in gastric carcinoma.
3. Anacidity (Achlorhydria):-
•Complete Absence of HCl.
•The curve obtained is flat.
•Achlorhydria is compatible with Pernicious Anaemia.

21. Colorimetric Determination of total Plasma Proteins
•Principle plasma proteins include Albumin, Globulin,
Fibrinogen and Prothrombin.
•They are all synthesized in Liver except γ Globulins
synthesized in Reticulo Endothelial System (RES).
•Normal value: 6 – 8 g/dL.
This value decreases in:
1. Over hydration.
2. Liver disease (decreased synthesis).
3. Kidney disease (increased loss in urine as in nephropathy).
4. Diminished dietary protein intake.
5. Extensive burns.
6. GIT losses (Malabsorption).

22. Total protein increases in:
1. Dehydration.
2. Chronic inflammation.
3. Para proteinemia (abnormal Ig or part of Ig in blood or
urine).
Principle:
•Copper in alkaline solution reacts with peptide bonds of
proteins and polypeptides producing Violet color.
•The intensity of color is proportional to protein
concentration in the medium.
•Conc. of total serum proteins = X conc of
standard (10 g/dL) = ………… g/dL.
A Sample
A Standard

24. Determination of Serum Uric Acid
•Waste product of purines from diet or synthesized in body.
•Increased level in blood is found in Leukemia, Polycythemia,
intake of food rich in nucleoproteins and familial idiopathic
Hyperuricaemia.
Normal value:
•Male 3 – 7 mg /dL in plasma serum
•Female 2 – 6 mg /dL
•Average diet: 250 – 750 mg / 24 h.
•Low purine diet: up to 450 mg/ 24 h. in urine
•High purine diet: up to 1 gram/24 h.

25. Principle of Serum Uric Acid
Determination (Enzymatic method):

28. Principle of Na Colorimetry :
• Na ions + Excess uranyl acetate + Mg Acetate →
Na Mg uranyl acetate + Residual uranyl acetate
• Residual uranyl acetate + potassium ferricyanide → Colored
Complex
•The absorbance varies inversely with sodium concentration.
• Na in Sample = Aʀʙ - Asa x 150 = …… mmol/L
• Aʀʙ - Ast

33. Sample:
•Non -hemolysed serum or plasma.
Normal Value :
•135 – 150 mmol/L or mEq/L
 Reagents :
1) Acid Reagent
2) Color Reagent ( Potassium ferricyanide )
3) Precipitant Reagent ( uranyl acetate – Mg acetate )

34. Principle of K Colorimetry :
• K ions in protein free filtrate + Na tetraphenyl boron → colloidal
solution
• This colloidal solution can be measured colorimetrically
• K concentration = Asample x 5 = …… mmol/L
Astandard

38. Sample:
•Non -hemolysed serum or plasma.
Normal Value :
•3.6 – 5.5 mmol/L
 Reagents :
1) Acid Reagent : Trichloroacetic acid
2) Alkaline Reagent : NaOH
3) Boron Reagent : Na tetra phenyl boron