1.
CALCULATIONS FOR PREPARATION OF
SOLUTIONS
CHEMICALS USED IN THE LABORATORY
• All the chemicals used in the laboratory should be of
standard grade
• Chemicals are of two grades
1. Laboratory reagents (LR)
– Purity is about 95-98 percent.
– Mostly used for ordinary solutions
2. Analytical reagents (Anal R, A.R or guaranteed
reagents)
– Purity is about 100 percent.
– Mostly used for standard solutions

2.
Examples of some standard firms
manufacturing the chemicals
• British Drug House
• E.Merk
• Sigma
• Sarabhai Merck ( S.M.) etc.

3.
• The solutions of the reagents
must be accurately prepared in
distilled water and
• Preserved as prescribed in the
analytical procedure.
Preparation of solutions

4.
1. Percent solution
• If the chemical is solid it is prepared by
weight by volume and
• If liquid volume by volume
• According to the required percent, ‘X’
grams of the solid reagent is dissolved
in 100 ml of water or other solvent
• similarly for liquids ‘X’ ml of the liquid is
diluted to 100 ml water or other solvent
Types of solutions required in
the laboratory

5.
2. Molar solution
• It is prepared by dissolving one molecular
weight of the chemical in 1 litre of the solvent
• Some times fractions of molar solutions
such as milli mole (10-3M) or micromole (10-6M)
solutions are required
• If one ml of molar solution is diluted to 1 litre
it will be one milli mole
• If 1 ml of milli mole solution is diluted to 1
litre it will be one micro molar solution

6.
3. Normal solutions
• When one equivalent weight of a substance is dissolved in 1
litre of water, it is called one normal solution.
• For the majority of substances the equivalent weight is
calculated as follows:
Eq. wt. = Molecular wt. of the substance
Replaceable number of H or OH ions
For example, the Eq. wt. of
• NaOH = 40 / 1 : = 40g
• KOH = 56 / 1 : = 56 g
• Na2CO3 = 106 / 2 = 53 : = 53g
• HCl36.45 / 1 : = 36.45 g
• H2SO4 = 98 /2 : = 49 g

7.
Standardization of acid solutions
• The exact normal solutions of solid unhygroscopic
substances can be prepared by dissolving one
equivalent weight of the substance in one litre of
solvent
• In biochemical tests generally normal solutions of
alkali ( NaOH, KOH ) and acids ( HCl, H2 SO4 ) are
required
• Since alkalis are hygroscopic and acids in liquid form,
their exact normal solutions can not be prepared
directly
• For ordinary purposes their approximate normal
solutions are prepared and used but for exact
normality they are standardized by titration

8.
• The volume of concentrated acid to be diluted
to 1 litre with water for preparing approximate
one normal solution can be calculated in two
ways:
(i) If the specific gravity and percentage of
purity of the acids are known, the volume is
calculated by the formula
VoI of acid required for 1N = Equivalent wt. of the acid x100
Percent purity x Sp. gr.
For Example, 1N H2 SO4 = 49 x 100 = 27.89 ml = 28.0ml
95.6 x 1.84
Acid solutions of approximate normality

9.
(ii) Divide 1000 by the normality of the
concentrated acid and the volume
obtained is diluted to 1 litre with water
• e.g., the normality of conc. HCl is 11
• So 1000 / 11 = 91 ml which is diluted to 1 litre
for 1 N solution.

10.
Table: approximate normal acid solutions
• .
Chemicals Sp. gr. Normality of
concentrated
chemical
Vol. of conc.
Chemical (ml)
to be diluted
to 1 litre with
water
Sulphuric acid 1.84 36 28
Hydrochloric acid 1.18 11 91
Nitric acid 1.42 16 63
Perchloric acid 1.54 9 111
Acetic acid 1.05 17 59
Ammonia 0.88 17 59

11.
Precautions
• Concentrated acids and liquor ammonia
should be handled with great care
• The bottles should be opened near window
where ventilation is available
• While diluting concentrated acids,
particularly sulphuric acid, they must be
added to water gradually with care
• Water should never be added to
concentrated sulphuric acid

12.
Preparation of exact 1 N -HCl
• For common use 1N –HCL is prepared by diluting
91 ml of concentrated HCL to 1 litre with water.
For exact 1N-HCL, more than calculated volume
of HCL is diluted to 1 litre and titrated against a
base solution as follows:
(1) Dilute 100 ml of conc. HCL with water to 1 litre,
mix well.
(2) prepare exact 1 N solution of sodium carbonate
by dissolving 5.30g anhydrous sodium carbonate
in 100ml water (Eq wt. 53g)

13.
(3)Phenolphthalein indicator
• Dissolve 250mg indicator in 50ml of 50 percent
alcohol.
(4)Titration
• Take 10ml of acid solution and 10ml water in a small
beaker
• Add 2-3 drops of the indicator and
• Titrate with sodium carbonate solution from a 25ml
burette till a faint red colour is obtained
• Note the volume (x ml) of base consumed at the end
point.
Preparation of exact 1 N –HCl Contd…

14.
(5)Calculate the exact normality of the acid
by formula
• Normality of base x vol. of base = Normality
of acid x volume of acid
• So, the normality of HCL=1 x X / 10
• Normality of base is 1 volume of base is x ml.
Preparation of exact 1 N –HCl Contd…
•After calculating the exact normality of the
acid, it is proportionally diluted with water to
obtain exact 1 normal solution.

15.
Exact 1N –NaOH solution
• The equivalent wt. of sodium hydroxide is
40g
• So 40g is dissolved in 1 litre of water for
approximate 1N solution and used.
• But for exact normality it is titrated against
oxalic acid solution.

16.
Titration against oxalic acid solution
1. Dissolve 50g sodium hydroxide in 1 litre of water
and mix well.
2. Prepare exact 1N solution of oxalic acid by
dissolving 6.3g of it in 100ml water.
3. Take 10ml of oxalic acid and 10ml water in a beaker,
add 2-3 drops of phenolphthalein indicator. Titrate
against the sodium hydroxide from a burette till a
faint red colour is obtained.
4. Calculate the exact normality of sodium hydroxide
solution as in the case of acid and dilute
proportionately with water to obtain exact 1N
solution

17.
• If the equivalent weight of a substance is x gram
and if x mg of the substance is dissolved in 1 litre
of water it is called one mEq/L solution.
• In biochemical tests the results of anions and
cations of blood are expressed in mEq/L.
(i) To convert the results obtained in mg/100 ml to
mEq/L the formula given below is used:-
mEq/L = mg /100ml x 10 x valency of ion
atomic weight of ion
Milli equivalent solution -(mEq/L)

18.
Divide the values in mg/100 ml by the following factors:
Sodium(mg/l00ml) by 2.3
Potassium(m g/l 00 ml) by 3.9
Calcium (mg/l00 ml) by 2.0
Magnesium(m g/l 00 ml) by 1.2
Chloride( mg chloride! 10 0 ml) by 3.55
Chloride(NaCVI00ml) by 5.85
Bicarbonate(volume/l00 ml) by 2.24
Phosphate (mg phosphorus/l 00 ml) by 1.72
Protein( g/100 ml) by 0.41
ii) To convert mEq/L values into mg per 100 ml, multiply
by the above factors.

19.
Isotonic solution
• When two solutions are separated by a semi
permeable membrane, the solvent will flow
from the solution of low osmotic pressure to
the solution of higher osmotic pressure until
both solutions have equal osmotic pressure.
• Such pair of solutions which have same
osmotic pressure ( osmolarity) are called
isotonic solutions.
• When isotonic solutions are mixed together
there is no exchange of their contents
between the two solutions.

20.
Standardization of method
 Prepare the reagents accurately in distilled
water and preserve them as prescribed.
 The standard solutions must he prepared
with Anal. pure grade chemicals.
 Set up 10 tubes with same volume of the
standard solution and develop colour as
given in the procedure.
 Measure the absorbance.
 Calculate mean and standard deviation of the
absorbance and keep in the record for the
future check up.

21.
3. Beer's law –
 Prepare a set of standard solutions of
different concentrations e.g. glucose 50, 100,
150,200 and 250 mg/l00 mI.
 Develop the colour as given in the procedure
and measure their absorbance.
 Prepare a curve by plotting the absorbance
against the concentration.
 A straight line is found upto that
concentration to which Beer's law is obeyed.
(4)
 Analyse a number of food samples
 Check that the values obtained are in
agreement with the normal range reported in
literature

22.
• Once the method is standardized, the main
requirement is to maintain a careful and satisfactory
performance of the routine analysis.
• The standard should be set up with every batch of
tests and it should be noted that the absorbance of
the standard obtained is practically the same which
was obtained during standardization and does not
vary significantly from batch to batch or day to day.
• The constant value of the absorbance of the
standard effects that the reagents, instrumental
conditions and performance of the tests are
satisfactory and the results reproducible.
• If the absorbance of the standard varies
significantly, this signals that some thing is wrong
and the cause of it should be checked.

23.
Quality control
• The laboratory tests often vary
significantly from day to day and differ
from laboratory to laboratory even by
the same method
• In order to maintain an acceptable
degree of precision and accuracy of
the results, certain measures are
adopted in the analysis which is called
quality control

24.
Common practice of quality control in laboratory
Use of standard solutions
• This is a very common and convenient practice
• With each batch of test samples, a known standard
solution is assayed and the results of unknown
samples are calculated against the absorbance of
standard solution
• The standard solution may be prepared of a single
substance or a mixture of several standard substances
used for different tests
• The idea behind the use of mixture control is to take in
to account the effect of various substances normally
present in the samples on the analysis of a particular
substance although it is not always feasible
• If the standard solution is treated in the test exactly
like test samples, it is a better safeguard against error
than the standard solution which is used for colour
development only in the final stage of the analysis