TOPICS TO BE DISCUSSED
Measurement of compounds
Solution and its strength
Preparation of imp. solutions
Acids and bases
Henderson Hasselbalch reaction
MEASUREMENT OF COMPOUNDS
MOLE: One mole of a compound is grams of
that compound equal to its molecular weight.
EQUIVALENT WEIGHT: One gram
equivalent is weight of an element or
compound represent its capacity to bind or
replace 1 mole of Hydrogen.
weight/Total no. of +ve valency
CAN YOU TELL THE NUMBER OF
MOLECULES IN ONE MOLE OF ANY
WHY ARE WE LEARNING EQUIVALENT
Because every chemical reaction what ever the
type may be always occurs in equivalence.At any
point of time same equivalent of reactant/s react
to form same equivalent of product.
STRENGTH OF SOLUTIONS
% CONC.- Parts per 100
Most frequently used is W/V.
e.g. Normal saline contains 0.9% NaCl.It means
100ml of NS contains 0.9 gram NaCl.
Parts Per Million(ppm): Part of substance in
one million part of the solution.
e.g. 10 ppm chloride solution means 10
microgram of chlorine in 1 gram of water.
MOLARITY: No. of MOLES of solute per litre
e.g.NaCl has mol.wt of 58.5. So to get 1(M) solution
of NaCl 58.5 gram of it should be dissolved in water
to make final volume of 1 litre.
Q.Di Abietes had an elevated blood
glucose level of 684 mg/dL. What is
the molar concentration of glucose
in Di’s blood? (Hint: the molecular weight of
glucose (C6H12O6) is 180 grams per mole.)
MOLALITY: No . Of moles of solute per 1000
So it is easy to prepare molal solutions.
NORMALITY: It depends on equivalent
weight of the solute.
It is the gram equivalent no. of solute dissolved
in one litre of solution.
e.g. 1 (N) NaOH contains one gram equivalent
i.e. 40 gram NaOH in one litre solution.
APPLICATION OF THIS
FUNDAMENTALS IN LABORATORY
PREPARATION OF 1N HCL
1. Conc. HCl. AR
2. Indicator- Methyl Orange
3. 1N Sodium Carbonate
4. Glasswares:250ml beaker, 25ml conical
flask, 5.0ml graduated pipette,1.0ml of
volumetric pipette,graduated cylinder,
volumetric flask, analytical balance,reagent
bottle of 125ml.
1) By using a measuring cylinder transfer 100ml of
distilled water in 250ml beaker.
Add 10ml of conc.HCl ,mix well.
Titration: Pipette2.0ml of diluted HCl in conical
flask ,by using a volumetric pipette.
Add one drop of methyl orange.
Titrate against 1N sodium carbonate till colour of
the reaction mixture changes from red to yellow.
Note the titration reading.
Take two more reading.
Calculate the Normality by using the formula
PREPARATION OF NORMAL SALINE
1. 2L conical flask
2. 2L measuring cylinder
3. 1L volumetric flask
4. Sodium Chloride AR
5. Distilled Water
6. Analytical Balance
7. Butter paper
8. 1L reagent bottle
9. Magnetic Stirrer
1. Weigh 9.0 g NaCl on an analytical balance by
using a butter paper.
Transfer it to 2L conical flask.
Add 900ml of distilled water.
Mix it by using Magnetic Stirrer.
Transfer it to 1L volumetric flask & add distilled
water upto the mark.
Mix well & store in a clean and dry reagent bottle.
Label it appropriately and store at room
By using watch glass ,weigh 4.0 g of NaOH on an analytical
Dissolve it in about 90 ml of distilled water. By placing the
pelletes in a glass beaker.
Titrate it against standard 1 N HCl solution.
Take atleast 3 titration reading.
Find out normality of NaOH.
If normality appears less than 1.0N then add few more
Adjust normality by titrating against 1.0N HCl.
Store at room temperature in a polyethylene bottle
1. Take 97ml of distilled water in a beaker.
2. Add slowly with constant stirring 2.7ml of conc. Sulfuric acid
and mix thoroughly .
Titrate against 1N NaOH.
Find out the normality of the prepared solution.
If normality is>1.0, then dilute the solution by adding
If normality is <1.0,then add little more conc. Sulfuric acid.
Measure the quantity of 1N sulfuric acid : X ml.
Add X/2 ml of distilled water to X ml of 1N sulfuric acid.
Store in a reagent bottle at room temperature.
The Color Change of the Indicator Bromthymol Blue
What are Acid and Bases?
• Definition of Svante Arrhenius (Sweden) in 1884
“An Acid is a substance that can release a proton or
hydrogen ion (H+) when dissolved in water”
H+ + Cl-
A Base is a substance that can release a Hydroxyl ion
when dissolved in water”
• According to Thomas Lowry (England) or J.N. Bronsted
(Denmark) working independently in 1923:
“An Acid is a material that donates a proton:
H+ + ClCH3COO- + H+
NH3 + H+
“A Base is a material that can accept a proton
OH- + H+
CH3COO- + H+
NH3 + H+
Every ion dissociation that involves a hydrogen or
hydroxide ion could be considered an acid- base reaction
• The G.N. Lewis (1923) idea of acids and bases is broader
than the Lowry- Bronsted model.
The Lewis definitions are:
“Acids are electron pair acceptors.
H+ + eH
“Bases are electron pair donors.
OHOH + e-
Some Properties of Acids
Produce H+ (as H3O+) ions in water (the hydronium ion is a hydrogen
ion attached to a water molecule)
React with bases to form a salt and water
pH is less than 7
Turns blue litmus paper to red “Blue to Red A-CID”
Produce OH- ions in water
Taste bitter, chalky
Feel soapy, slippery
React with acids to form salts and water
pH greater than 7
Turns red litmus paper to blue
KEY FACTS ABOUT pH
What does it mean?
What does it measure?
What ‘p’ denotes?
Why we require pH?
Who did this pioneering job?
Derivation of pH=-log[H+]
Significance of change of pH.
How to measure it of a given fluid?
The pH scale is a way of
expressing the strength
of acids and bases.
Instead of using very
small numbers, we just
use the NEGATIVE power of
10 on the Molarity of the
H+ (or OH-) ion.
7 = neutral
Over 7 = base
‘p’ denotes ‘negative logarithm of’ to the base 10
SORENSEN brought this pioneering concept in the
year of 1909.
A small change in pH!!!!
See what happens in reality?
Q.The laboratory reported that Di
Abietes’ blood pH was 7.08 (reference
range7.37-7.43) What was
the [H] in her blood compared with the concentration
at a normal pH of 7.4?
From inspection, you can tell that
her [H] is greater than normal, but
less than 10 times higher. A 10-fold
change in [H] changes the pH by 1 unit. For
Di, the pH of 7.08-log [H], and therefore
her [H] is 1x 107.08. To calculate her [H],
Express- 7.08 as- 8+ 0.92. The antilog to
the base 10 of 0.92 is 8.3. Thus, her [H] is
8.3x 10-8 compared to 4.0 x10-8 at pH 7.4,
or slightly more than double the normal
MEASUREMENT OF pH
Based upon change of colour
of acid/base indicator.
Most commonly used for
1.Methyl Red (pH range:4.46.2,color change:Red to
range 8.0-9.6,color change:
yellow to blue)
Test area is compared with
corresponding color chart.
Basically it is a Galvanic cell.
Very thin 0.1 mm.It contains
0.1mol/lit HCl connected to a
pt. wire via Ag-AgCl
Glass tube containing
saturated KCl connected to pt
wire through Hg-HgCl2 paste.
Principle of pH meter
When a pair of electrodes or a combined
electrode( glass &calomel electrode) is dipped in
an aqueous solution,a potential is developed
across the thin glass of the bulb.The e.m.f. of
complete cell(E) formed by linking of this two
electrodes at a given solution temperature is
E=Eref – Eglass
Eref is the potential of stable calomel electrode
which at normal room temp is +0.250V
Eglass is the potential of glass electrode which
depends on the pH of the solution under test.
The resultant small e.m.f. can be recorded
potentiometrically by using vaccum tube
amplifier.Variations of pH with E may be recorded
directly on the potentiometer scale graduated to
read pH directly.
The Henderson-Hasselbalch Equation
Take the equilibrium ionization of a weak acid:
HA(aq) + H2O(aq) = H3O+(aq) + A-(aq)
Solving for the hydronium ion concentration gives:
[H3O+] = Ka x [HA]
Taking the negative logarithm of both sides:
-log[H3O +] = -log Ka - log [HA]
pH = -log Ka - log [HA]
Generalizing for any conjugate acid-base pair :
pH = -log Ka + log
p H = -log [H ]
pH = pKa + log
a useful concept:
[H-A] = [ A ]
pKa = pH + log (1)
Biological fluids are often buffered (constant pH)
and it is useful to know the predominant species
present at a given pH.
Definition: Buffer is a solution that resists change
of pH value on addition of small amount of
strong acid or alkali.
Chemically a buffer system consists of a weak
acid and its salt mixture, alternatively weak
bases and their salt.
e.g. carbonic acid and sodium bicarbonate
One important point to note that buffers do not
remove H+ ions from body.It only acts
temporarily as shock absorbent to reduce free
H+ ion. The H+ ion ultimately eliminated by renal
Why buffer system required?
An average metabolic activity daily produces almost 22000meq of
acid. If all the acid is dissolved in body fluids at a point of time, pH of
body would be <1.0 However pH of body is strictly maintained
between 7.36-7.44 under normal condition, it needs to be buffered
Principal buffers of ECF
Principal buffers of ICF
Mechanism of action of
BICARBONATE BUFFER SYSTEM
Most important buffering system of our body.
Proper lung functioning is important as
bicarbonate buffer system is directly linked up
CO2 (d) + H2O
CO2 (d) means dissolved carbon dioxide.
Both the reaction is catalyzed by carbonic anhydrase.
How bicarbonate buffer acts
A strong and nonvolatile acid is converted into weak[less dissociable] and
volatile acid at the expense of NaHCO3.
Alkali reserve is represented by amount of
sodium bicarbonate in blood that has not yet
combined with strong and nonvolatile acid. It
denotes the buffering strength of bicarbonate
at that point of time.
Normal ratio of NaHCO3: H2CO3 = 20: 1
HENDERSON HESSELBALCH EQUATION
CALCULATION OF pH from ABG REPORT
pH=pKa + log [base]/[acid]
pKa = 3.5
So for bicarbonate buffer pH = pKa + log[HCO3]/[H2CO3]
[H2CO3] is best estimated as [CO2]d /400
pH= 3.5+ log400[HCO3-]/[CO2]d
=3.5+ log 400 + log [HCO3]/[CO2]d
=6.1 + log [HCO3]/[CO2]d
Because only 3% of the gaseous CO2 is dissolved,[CO2]d = 0.03PaCO2;so
pH= 6.1+ log[HCO3]/0.03PaCO2
[HCO3-] as mEq/ml PaCO2 as mm of Hg.
HEMOGLOBIN in RBC
Buffering capacity of Hb ,as of many
protein,depends on number of dissociable
buffering group viz. acidic –COOH gr ,basic –
NH2 gr, guanidino gr,and most importantly
With the pH range of 7.0 to 7.8 most of the
physiological buffering system of Hb is due to
the “imidazole” group of amino acid “HISTIDINE”
1.Fe++ containing group- carrying of O2
2.Imidazole N2 group which can give up or
accept H+ depending upon pH of medium.
Thus buffering capacity of Hb is due to
the presence of “Imidazole” nitrogen
group which remains dissociated in
acidic medium and conjugate base
Association of Hb and HCO3buffer in RBC
cooperate each other in buffering blood and
transporting co2 to the lungs.
although no CA is present in plasma and
interstitial tissue fluid ,RBC contain high
amount of this enzyme and CO2 rapidly
converted to H2CO3.
BICARBONATE is transported out of RBC
into the blood in exchange of Cl- ions
As the RBCs approach lungs alveoli
equilibrium reverses.CO2 is released from
RBCs causing more H2CO3 to dissociate into
CO2 and H2O.
THUS HCO3- BUFFER SYSTEM IS
INTIMATELY LINKED TO THE DELIVARY OF O2
TO THE TISSUES.
The PHOSPHATE buffer
Acts to regulate intracellular pH
Normal plasma ratio is 4:1
It plays a major role as intracellular buffer in the
RBCs and other type of cells where conc. Is
much higher than the blood or ICF.
When a strong acid enters;
When an alkali enters;
Thus a healthy KIDNEY is necessary .
The PROTEIN buffer
Blood contains high amount of extracellular
protein e.g. Albumin which contribute to its
Na+Pr- / H+Pr- = [salt]/[acid]
In acidic medium: NH2 group takes up H+ ions
and protein becomes positively charged.
In alkaline medium: here protein acts as an
acid.Acidic COOH gr dissociates & gives H+ .It
combines with added OH- to form Water.
Proteins becomes negatively charged.
Text of BIOCHEMISTRY
Text of BIOCHEMISTRY- VASUDEVAN
Text of BIOCHEMISTRY- CHATTERJEA &
CLINICAL BIOCHEMISTRY: MARK’S
GODKAR’S LAB. TECHNOLOGY