PRACTICAL

3rd Sem

1. Cereals: Rice (habit sketch, study of paddy and grain, starch grains, micro-chemical tests).

2. Legumes: Soybean, Groundnut, (habit, fruit, seed structure, micro-chemical tests).

3. Sources of sugars and starches: Sugarcane (habit sketch; cane juice- micro-chemical
tests),Potato(habit sketch, tuber morphology, T.S. tuber to show localization of starch grains,
w.m. starch grains, micro-chemical tests).

4. Spices: Black pepper, Fennel and Clove (Macromorphology).

5. Beverages: Tea (plant specimen, tea leaves), Coffee (plant specimen, beans).

6. Sources of oils and fats: Coconut- T.S. nut (photograph), Mustard-plant specimen, seeds; tests
for fats incrushed seeds.

7. Essential oil-yielding plants: Habit sketch ofRosaandEucalyptus- specimens/photographs.

8. Rubber: specimen, photograph/model of tapping, samples of rubber products.

9. Drug-yielding plants: Organoleptic study of specimens ofAndrographisand Catharanthus.

10. Woods: Tectona, Pinns'. Specimen, Section of young stem.

11. Fiber-yielding plants: Jute (specimen, transverse section of stem, test for lignin on transverse
section of stem and fiber)

Practical
1. Study of instruments used to measure microclimatic variables: Soil thermometer, maximum
and minimum thermometer, anemometer, psychrometer/hygrometer, rain gauge and lux
meter.

2. Determination of pH of various soil and water samples (pH meter, universal indicator and
pH paper)

3. Analysis for carbonates, chlorides, nitrates, sulphates, organic matter and base deficiency
from two soil samples by rapid field tests.

4. Determination of organic matter of different soil samples by Walkley & Black rapid titration
method.

5. Determination of dissolved oxygen of water samples from polluted and unpolluted sources.

6. Ecological adaptations of some species: Ipomoea aquatica stem, Phyllode of Acaccia

 auriculiformis, Nerium leaf and Vanda root

7. Determination of minimal quadrat size for the study of herbaceous vegetation in the college
campus, by species area curve method (species to be listed).

8. Field visit to familiarize students with ecology of different sites.

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The University of Burdwan
BSc Botany (Hons.) Sem-III Practical
Chemistry Portion :: Dr. Kalyan Kumar Rana

How do you test for Carbonate (CO3-2) or CaCO3 present in Soil Sample?
Experiment: At first take a sample of soil (~15g-20g) in a 100 mL beaker. Ground it well
and add ~10-15mL (little more than half test tube) distilled water, stir the mixture using a
glass rod. Filter the mixture in a test tube using a filter paper, add few drops of conc. HCl.
Observation: Fizzing of effervescence
Inference: The soil sample contains carbonate (CO3-2)

How do you test for Chloride (Cl—) present in Soil Sample?

Experiment: At first take a sample of soil (~15g-20g) in a 100 mL beaker. Ground it well
and add ~10-15mL (little more than half test tube) distilled water, stir the mixture using a
glass rod. Filter the mixture in a test tube using a filter paper and acidify the filtrate with
2-3 drops of HNO3, add few drops of AgNO3 solution available in laboratory.
Observation: Dense white precipitate
Inference: The soil sample contains chloride (Cl—).

How do you test for Nitrate (NO3—) present in Soil Sample?

Two tests are suggested:
Ring Test—
i. Experiment: At first take a sample of soil (~15g-20g) in a 100 mL beaker. Ground it well
and add ~10-15mL (little more than half test tube) distilled water, stir the mixture using a
glass rod. Filter the mixture in a test tube using a filter paper, add dil. FeSO4 solution. Now
slowly add concentrated sulphuric acid such that the acid forms a layer below the aqueous
solution.
Observation: A brown ring is formed at the junction of two layers
Inference: The soil sample contains nitrate (NO3—).
[This test is sensitive up to 2.5 micrograms and a concentration of 1 in 25,000 parts.]

ii. Experiment: At first take a sample of soil (~15g-20g) in a 100 mL beaker. Ground it well
and add ~10-15mL (little more than half test tube) distilled water, stir the mixture using a
glass rod. Filter the mixture in a test tube using a filter paper, add powdered aluminium,
shake well the test tube. Immediately, hold a piece of pale red litmus paper ~ 1 inch down
the inner portion of the test tube.
Observation: The litmus paper turns blue (due to evolution of ammonia)
Inference: The soil sample contains nitrate (NO3—).
[Nitrate (NO3—) is reduced to Ammonia NH3 in presence of Aluminium]

How do you test for sulphate (SO4-2) present in Soil Sample?

Experiment: At first take a sample of soil (~15g-20g) in a 100 mL beaker. Ground it well
and add ~10-15mL (little more than half test tube) distilled water, stir the mixture using a
glass rod. Filter the mixture in a test tube using a filter paper, add dil. HCl and a few drops
of Barium Chloride (BaCl2) solution
Observation: A white precipitate (of barium sulphate)
Inference: The soil sample contains sulphate (SO4-2).

How do you test for organic matter present in Soil Sample?

The organic matter of soils is generally determined by converting it to carbon dioxide by
means of moist and dry combustion. The carbon dioxide thus evolved is then calculated to
organic matter by the use of a conventional factor, 0.471.

Determination of organic matter of different soil samples by Walkley & Black

rapid titration method

1) Take 2 g air dried soil into a conical flask.

2) Add 10 ml 1N K2Cr2O7 solution and carefully add 10 ml concentrated H2SO4 and mix
thoroughly with slight rotating.
3) Allow the mixture to cool for half an hour with occasional slight shaking. (If the colour
of the mixture appears green then add an additional 10 ml of 1N K2Cr2O7 solution. Green
colour is an indication that all oxidizing agent added is used up to oxidize organic
carbon.)
4) Add 150 ml dw, 10 ml H3PO4 (85%) and 0.2 g NaF in sequence.
5) Add 3 ml diphenylamine (Dissolve 0.25 g indicator into 10 ml dw and slowly add 50 ml
concentrated H2SO4 into it) indicator prior to titration. The mixture appears deep violet in
colour.
6) Titrate excess K2Cr2O7 solution left in the flask with FeSO4.7H2O solution (1N).
7) Run a blank titration (This step is essential to standardize FeSO4.7H2O solution
against standard K2Cr2O7 solution).
C in soil (%) = [(B-T) × S × 0.003 × 1.3 × 100] ÷ W
Where,
B = Amount of FeSO4 required in blank titration.
T = Amount of FeSO4 required in soil titration.
S = Strength of FeSO4 (from blank titration).
W = Weight of the soil.
Organic matter in soil (%) = % organic C × 1.724

Determination of dissolved oxygen of water samples from polluted and

unpolluted sources.

OBJECTIVE: To determine dissolved oxygen in a sample of water.

Apparatus required: Burette, pipette, conical flask, beaker, measuring cylinder
Chemicals required: MnSO4, KOH, KI, Na2S2O3, Starch, and NaN3
Theory: It is based on oxidation of potassium iodide. The liberated iodine is titrated against
standard hypo solution using starch as a final indicator. Since oxygen in water is in
molecular state and not capable to react with KI, an oxygen carrier manganese hydroxide
is used to bring about the reaction between KI and O 2.Manganous hydroxide is produced
by the action of potassium hydroxide and manganous sulphate.
Chemical reaction:
2KOH+MnSO4 → Mn(OH)2+K2SO4
2Mn(OH)2+O2 → 2MnO(OH)2
M O(OH)2+H2SO4→ MnSO4+2H2O+[O]
2KI+ H2SO4+ [O] →K2SO4+H2O+I2
I2+2Na2S2O3→2NaI+Na2S4O6
Sodium tetrathionate
Starch+I2→ Starch iodide complex
(Blue in colour)

Procedure:
1. Take 500ml of water in a D.O bottle.
2. Add 10ml of alkaline KI and 10 ml of MnSO4 into it.
3. Stopper the bottle and shake it well.
4. Keep the bottle in dark for 5 min and add conc H 2SO4 till the brown precipitates are
dissolved.
5. Take 100 ml of the above solution in a conical flask. Titrate against hypo till the color
changes to light Yellow.
6. Add 3-4 drops of starch in to it and the color changes to blue.
7. The blue color solution is titrated against hypo solution till blue color disappeared.
8. This is end point of the titration. Repeat this process till to get three concordant reading.

No of Volume of Initial Final Burette Difference in Remark

observation water in mL Burette Reading in mL
Reading in mL
mL

Calculation:
1000 ml of 1N Na2S2O3 =8 gm of O2
“V” ml of N/40 Na2S2O3 = 8V / 40 1000gm of O2
= 8V 1000 / 401000 mg of O2
= V / 5 mg of O2
100 ml of water sample contain=V/5 mg of O2
 1 lit of water sample contains =2V mg of O2 =…………………..ppm
Conclusion:
The amount of dissolved oxygen in a sample of water is found to be ppm.

[[ Micro-chemical test of Cereals:

The iodine test is used to test for the presence of starch. Starch turns into an intense
"blue-black" colour upon addition of aqueous solutions of the triiodide anion, due to the
formation of an intermolecular charge-transfer complex. In the absence of starch, the
brown colour of the aqueous solution remains.

Test for fats in crushed seeds

Ethanol Emulsion Test for Fats and Oils
The Ethanol Emulsion Test is a food test which determines the presence of a broad
group of naturally occurring compounds known as lipids. Lipids consist of fats and oils.
Other lipid tests include the Grease Spot Test and the Sudan Stain Test. The Grease
spot test is performed on fats - lipids which are solid at room temperature. Sudan stain
colours lipids red, but is a less common bench reagent than ethanol. The Ethanol Emulsion
Test is the most common test amongst the three.
Procedure
Solid sample
1. Crush the food sample and place in a dry test tube.
2. Add ethanol to about 2 cm3 above the level of the sample and shake thoroughly.
3. Allow the solid to settle (about 3 min) to allow the lipid to be extracted.
4. Decant the ethanol into another test tube.
5. Add 2 cm3 of deionized water to the second test tube
6. Make observations.
Liquid sample
1. Add a few drops of the liquid food sample to a dry test tube.
2. Add 2 cm3 ethanol and shake it thoroughly
3. Add 2 cm3 of deionized water.
4. Make observations.
Observations & Interpretation
Observation Interpretation
Solution remains colourless. No emulsion is formed. Lipids are not
present
A layer of cloudy white suspension forms at the top of the
solution. (Upon close inspection you can see the tiny globules of
fat suspended in the solution. This an emulsion. Foods with high Lipids are present
lipid content have a ‘higher’ layer than foods with less).
Principle of the Ethanol Emulsion Test

The solubilities of lipids and ethanol are exploited in this test.

Lipids are non-polar organic compounds. Hence they are

 soluble in organic solvents such as ethanol (alcohol), but
insoluble in water.

Ethanol is an organic substance and so dissolves other organic

substances; it is frequently used as an organic solvent.

However ethanol is also miscible in water due to the presence

of the hydroxyl (-OH) functional groups and the shortness of its
chain (2C). The hydroxyl group participates in hydrogen
bonding with water.

The hydrophobic interaction of the carbon in the short chain

with water is not great and is overcome by the hydrogen bonding.

Ethanol extracts the lipid from the crushed solid sample. As

ethanol is miscible with lipids no change is seen upon its addition
to the solid and liquid samples.

The lipid spontaneously comes out of solution when water is

added and is dispersed as micelles (small droplets) throughout
the solution of ethanol and water.( This happens as hydrophobic
portion of the lipid molecules project inwards and excludes the
aqueous environment; the hydrophilic portion (-COOH) group
faces the aqueous environment.)

A layer is formed at the top as lipids are less dense than water.
The droplets diffract light, appearing cloudy white.

CONCLUSIVE TEST
A positive test shows conclusively that lipids are present - and not the other major
biological molecules.
1. Carbohydrates
 Reducing sugars and non- reducing sugars - slightly soluble in ethanol and soluble in
water
 Starch – insoluble in both ethanol and water
2. Proteins – insoluble in ethanol (the addition of ethanol is used to precipitate proteins)
3. Nucleic Acids – are insoluble in ethanol and soluble in water.
No change is seen in a negative test as there are no lipids to come out of solution.
Test for Lignin
Acid-insoluble lignin and acid-soluble lignin are determined according to TAPPI method
T222 om-98, and CPPA G.8 and G.9, respectively. Carbohydrate content is calculated
using the following formula:
Carbohydrate, % =100 – acid-insoluble lignin – acid-soluble lignin – ash
-Lignin moisture and ash contents are determined gravimetrically according to the ASTM
D 2974-87 Standards Test Methods, or ash content can be tested according to TAPPI
method T211 om-93. ]]
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