1. Isolation of Caffeine from tea powder.

Aim: To isolate and characterize caffeine from tea powder.

Theory:
Caffeine (C8H10N4O2) is a bitter white alkaloid compound which is found especially in tea and
coffee plants and is a stimulant of the central nervous system.
Tea leaves contain approximately three percent caffeine content by weight; but factors like soil
chemistry, type of tea plant and whether the tea leaves are dry or wet can affect caffeine content.

Chemicals required:
Tea (25 gms), Calcium carbonate (25 gms), Chloroform (25 ml)

Procedure:
Place 25 gm of dry tea leapowder, 100ml of water and 25 gm of powdered CaCO 3 in a 500 ml
beaker. Heat the mixture with constant stirring for 30 minutes.
After heating filter the hot mixture using Buchner funnel.(Use a large cork stopper to press out as
much of water from the solid as possible).Cool the filtrate to room temperature and extract it
twice with 25ml of chloroform.(ethyl acetate)
Place the combined chloroform extract in a dry beaker and evaporate to dryness.
The purity of the obtained caffeine is checked using TLC.

Result:

Yield: ----------------
2. Identification of carbohydrates:
Molisch’s test, Iodine test, Fehling’s test and Benedict’s test

Aim: To detect the presence of carbohydrate in the given sample by Molisch’s test, Iodine test,
Fehling’s test and Benedict’s test.

Preparation of reagent:
a) Molisch’s test:
Molisch Reagent: α-naphthol (C10H8OH) dissolved in ethanol (C2H5OH) is Molisch’s
reagent.

b) Preparation of Fehling’s solution:

Fehling's solution is prepared by combining two separate solutions, known as Fehling's A
and Fehling's B. Fehling's A is aqueous solution of copper (II) sulphate, which is deep
blue. Fehling's B is a colorless solution of aqueous potassium sodium tartarate (also
known as Rochelle salt) made strong alkali, commonly with sodium hydroxide.

Test Observation Inference

1.Molisch’s test
5ml of solution taken in a test
tube. Two to three drops of
molisch reagent is added.
Solution is mixed gently for
some time. Test tube is inclined
and 1ml of concentrated
sulphuric acid is added.

Test Observation Inference

2.Iodine test:
Small amount of sample is taken
in a test tube. One drop of
(brown) iodine solution is added
in the test.

2.Fehling’s Test:
15 ml of Fehling’s A and 15 ml
of Felhing’s B solution is taken
in a test tube. To this mixture 3
drops of compound is added.
This mixture is kept in a water
bath at 600C.
3. Estimation of saponification value of oil:

Aim: To calculate the number of milligrams of potassium hydroxide required to completely

saponify 1 gm of oil.

Theory: Saponification value indicates the average molecular weight of a fat or oil. The
saponification value may be defined as the number of milligrams of caustic potash required to
neutralize the fatty acids obtained by complete hydrolysis of 1 g of oil or fat. Thus saponification
value gives us information weather an oil or fat contains high proportions of lower or higher fatty
acids. For eg., butter has a large proportion of lower fatty acids than lard and tallow, and has high
saponification value. Coconut oil also has a comparatively higher saponification value.
saponification value gives us an idea about the molecular weight of fat or oil.

Oil X Saponification value = Amount of lye needed to make soap

Procedure:
1. Weigh 0.5 grams of oil and transfer into the RB flask.
2. Add 10 ml of 0.5 N alcoholic KOH to the RB flask. Add 5 ml of ethanol for washing.
3. Follow the above procedure without taking oil for blank titration.
4. Reflux both RB flasks for 1 hr. using water condenser.
5. After reflux allow both the RB flask to cool.
6. Titrate both the samples using 0.5 N HCl. with phenolphthalein indicator.
7. The disappearance of pink color indicates the end point.

Calculation:

i)Solution in the burette : 0.5N HCl solution

ii) Solution in the conical flask : 10 ml of Alcoholic KOH (with and without oil)
iii) Indicator used : Phenolphthalein
iv) Color change : Pink to colourless.

Tabulation: For Blank titration

Readings Pilot I II III IV
Final in ml

Initial in ml
Difference in ml
Tabulation: For main titration
Readings Pilot I II III IV
Final in ml

Initial in ml
Difference in ml

Saponification value=
(Titre value of blank in ml – Titre value of sample in ml) X 0.5N KOH X Equivalent weight of
KOH (56.11 g)

Weight of the sample=

Saponification value=

Result:
The saponification value of the given oil sample is =

4. Estimation of sugars:

Aim: To estimate the amount of glucose present in the given unknown solution using
Benedict’s quantitative reagent.

Principle: This test is also used to differentiate between reducing and non reducing sugars.
Benedict’s reagent contains blue coloured cupric sulphate which is reduced to red copper oxide
thus oxidising aldehydes to carboxylic acid. The final colour of the test is green to brick red
depending on the number of copper(II) ions.

Procedure: 10 ml of Benedict’s reagent was pippeted out into a clean conical flask. About
600mg. of anhydrous sodium carbonate was added to provide the required alkalinity with a few
porcelain bits and heated to boiling over a moderate flame. Standard glucose solution is taken in
a burette. When the Benedict’s solution boils continuously, glucose solution is added drop by
drop (1 drop/sec) till last trace of blue colour disappears. The volume of glucose rundown is
noted and the titrations are repeated to concordant values. The given unknown sugar solution
was made up to 100ml in a standard flask with distilled water. Then the burette was filled with
unknown sugar solution and the Benedict’s reagent was titrated as before. The volume of sugar
solution rundown was noted and titrations are repeated for concordant values.
Tabulation:
Standardisation of benedict’s reagent:
i)Solution in the burette
ii) Solution in the conical flask
iii) Indicator used
iv) Color change
: 0.05 N glucose solution
: Benedict’s reagent
: Self Indicator
: Blue to Reddish orange

Readings Pilot I II III IV

Final in ml

Initial in ml
Difference in ml

Normality of Benedict’s reagent= N(glucose)X CBR

10

Determination of glucose in unknown sample:

i)Solution in the burette : Unknown glucose solution

ii) Solution in the conical flask : Benedict’s reagent
iii) Indicator used : Self Indicator
iv) Color change : Blue to Reddish orange

Readings Pilot I II III IV

Final in ml

Initial in ml
Difference in ml

Normality of unknown glucose solution=

N(Benedict’s solution)X 10

CBR

Amount of glucose in 1 litre of the solution= N X 180

Amount of glucose in 100 ml of the given solution= N X 180

10
Result: The amount of glucose present in 100ml of given unknown solution is -----------mg.
5. Estimation of proteins by Biuret method:

Aim: To estimate the protein using Biuret method.

Principle: The –CO-NH- bond (peptide) in polypeptide chain reacts with copper sulphate in
an alkaline medium to give a purple colour which can be measured at 540 nm.

Reagents Required:
1. Biuret Reagent: Dissolve 3 g of copper sulphate (CuSO4.5H2O) and 9 g of sodium
Potassium tartarate in 500 ml of 0.2 mol/liter sodium hydroxide; add 5 g of potassium
Iodide and make up to 1 liter with 0.2 mol/liter sodium hydroxide.
2. Protein Standard: 5 mg protein sample/ml.

Apparatus and Glass wares required: Test tubes, Pipettes, Colorimeter, etc.,

Procedure:
1. Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labeled
test tubes.
2. Pipette out 1 ml of the given sample in another test tube.
3. Make up the volume to 1 ml in all the test tubes. A tube with 1 ml of distilled water
serves as the blank.
4. Now add 3 ml of Biuret reagent to all the test tubes including the test tubes labeled 'blank'
and 'unknown'.
5. Mix the contents of the tubes by vortexing / shaking the tubes and warm at 37 ºC for 10
min.
6. Now cool the contents to room temperature and record the absorbance at 540 nm against
blank.
7. Then plot the standard curve by taking concentration of protein along X-axis and
absorbance at 540 nm along Y-axis.
8. Then from this standard curve calculate the concentration of protein in the given sample.
Observations and Calculations:

Nature of the graph:

Result: The given unknown sample contains ----mg protein/ml.

6. Estimation of potassium in dry fruits:

Aim: To estimate the amount of potassium in the given dry fruit.

Principle: Potassium (K) is the major cation found inside of cells. The proper level of
Potassium is essential for normal cell function. An abnormal increase of potassium
(hyperkalemia) or decrease of potassium (hypokalemia) can profoundly affect the nervous
system and heart, and when extreme, can be fatal. The normal blood potassium level is 3.5 - 5.0
millimoles/liter (mmol/l). Potassium also plays an important role to mental function as well as to
physical processes. It helps to promote efficient cognitive functioning by playing a significant
role in getting oxygen to the brain. In view of the nutritional and health benefits of dry fruits,
their daily need in our diet and the effects of anti nutritional factors, this study was designed to
determine the Potassium ion in highly nutritive dry fruits.

Preparation of Standard solution of Potassium:-

100mg of potassium chloride was dissolved in deionised water and was diluted up to 100 ml with
the same (1mg /ml).
10ml of above 1mg/ml solution was pipetted out into a clean volumetric flask using a micro-
pipette and diluted to 100 ml with deionised water (100μg/ml).
1ml of above 100mg/ml solution was diluted to 10ml with deionised water (10μg/ml).Similarly
series of standards were prepared by pipetting out 2ml, 3ml, 4ml,......9ml of above 100μg/ml
solution and diluting each of them to 10ml with deionised water (20μg/ml,
30μg/ml,40μg/ml.....90μg/ml).

Procedure: Dry fruits are washed with distilled water and external moisture is removed with a
dry cloth. The individual fruit was separated, dried in hot air oven at 1000C for 1 h. The dried
samples were then powdered in blander and sieved and 10 g of each of fruit sample powder was
weighed and subjected to dry ashing in a well-cleaned silica crucible for about 1 to 2 h. The
resultant ash is dissolved in 5 ml HNO3 / HCl / H2O (1:2:3), heated gently on a hot plate until
brown fumes disappeared. To the remaining residue in each crucible, 5 ml of distilled water is
added and heated till colorless solution is obtained. The mineral solution in the crucible is
transferred into 100 ml volumetric flask by filtration through a Whatman filter paper No. 42 and
the volume was made up to the mark with DIW. The solution is used for the determination of
potassium by using a flame photometer.

Graph: 100
80
60
ppm

40 Y-Values
20 Fertilizer
0
0 20 40 60 80 100

concentration of K in µg/ml
7. Estimation of potassium in junk food:

Aim: To estimate the amount of sodium in junk foods by flame photometry.

Principle: Junk foods are generally very high in sodium. Hence by analysis of such type of
foods a general awareness about impact of it on health can be given. Accordingly the occurrence
of serious health problems also can be prevented.

Apparatus: Conical flasks, Beakers, Pipettes, Measuring cylinders, Standard flasks,

Deionized water.

Preparation of Standard solution of Potassium:

Refer experiment no. 6.

Procedure: Prepare an HCl solution by diluting 500ml of concentrated HCl with 220ml of
water. The sample is prepared by weighing 5g of each junk food (diced or ground) into 500ml
Erlenmeyer flasks. Add 50ml of the above HCl solution. Bring each to a boil on a hot plate, and
then simmer for 5 min. Cool and filter each through Whatman filter paper into flasks. Now
quantitatively transfer the supernatant for each to separate 100ml volumetric flasks. Dilute to the
mark with distilled water and shake. This extract is used to prepare the appropriate dilutions
required for the experiment.

Calculation: Prepare a
1000ppm of standard solution contains 254 mg of NaCl which contains 100mg of Na alone
X ppm of sample solution contain?
X × 100mg
Amount of sodium = ------------------------
1000

Therefore, amount of Sodium = y mg/100mL

8. Estimation of sodium in crude salt by flame photometry:

Aim : To estimate the amount of sodium in crude salt by flame photometry.

Procedure:

Preparation of standard solution of sodium:

 100mg of sodium chloride was dissolved in deionised water and was diluted up to 100 ml
with the same (1mg /ml).
 10ml of above 1mg/ml solution was pipetted out into a clean volumetric flask using a
micro-pipette and diluted to 100 ml with deionaised water (100μg/ml).
 1ml of above 100mg/ml solution was diluted to 10ml with deionised water (10μg/ml).
 Similarly series of standards were prepared by pipetting out 2ml, 3ml, 4ml,......9ml of
above100 μg/ml solution and diluting each of them to 10ml with deionised water
(20μg/ml, 30μg/ml,40μg/ml.....90μg/ml).

Series of Standards for Sodium (Na)

Concentration Intensity
0µgm/ml 0
10µgm/ml 8
20µgm/ml 18
30µgm/ml 28
40µgm/ml 38
50µgm/ml 46
60µgm/ml 55
64
70µgm/ml
74
80µgm/ml
84
90µgm/ml 94
100µgm/ml
 Standard graph for Sodium (Na)
100
90
intensity of emitted light 80
70
60
50
40
30
20 Series1
10
0

concentration of sodium