Pharmacognosy lab Manual 1

INTRODUCTION TO EXTRACTION

 

 

 

 

 

 

 

Percolator

 

 

 

 

 

INTRODUCTION TO EXTRACTION

DEFINATION:

Extraction means withdrawal of desired constituents from crude drug through the use of selective solvents in which desired constituents are soluble.

                                                                        OR

The process in which animal or plant tissues are treated with specific solvents whereby medically active constituents are dissolved out, most of inactive or inert components and cell tissues remain undissolved.

MENSTRUM:

            The solvent used for extraction purpose is called menstrum.

MARC:

                The residue left after extracting the desired constituents is called marc.

REASONS FOR EXTRACTION

·         Potency is more readily controlled.

·         Deterioration by enzyme action is diminished.

·         Preparations of drug are more easily formulated, more stable, more palatable and more elegant.

·         Tableting of crude material may not be possible.

·         Injection of crude material may be undesirable or dangerous.

·         Smaller bulk facilitates storage and transport.

Solvents used for extraction are:

·         Water

·         Alcohol i.e. Ethanol

·         Hydro alcoholic mixtures

·         Glycerin

·         Dilute HCl

·         Acetic acid

·         Organic solvents i.e. Ether

The products so obtained from plants by extraction are relatively impure liquids, semi solids or powders intended only for oral or external use.

These include different preparations like infusions, decoction, fluid extracts or tinctures. Such preparations have been called ‘galenicals’.

The product thus obtained may be used as a medicinal agent in the form of tinctures and fluid extracts, it may be further processed to be incorporated in any dosage form such as tablets or capsules.

 

 

METHODS OF EXTRACTION OF MEDICINAL PLANTS

INFUSION:

An infusion is a dilute solution of readily soluble constituents of crude drugs.

Fresh infusions are prepared by macerating the crude drug for short period of time with either cold or boiling water.

DECOCTION:

            It is also called heating or boiling process.

“A process in which water soluble or stable constituents of hard and woody crude drugs are extracted out by heating and boiling the water.”

For example: Boiling of leaves in water to make tea is a decoction process.

DIGESTION:

The modified form of maceration in which the extraction of drug is carried out by applying gentle heat.

This method is applicable only to those drugs where moderately elevated temperature is not objectionable.

MACERATION:

                The term maceration means ‘to soak’ or ‘to wet’.

“It is a process in which properly comminuted drug is permitted to soak in the menstrum until the cellular is softened and penetrated by menstrum and soluble constituents are dissolved.”

The whole or coarsely powdered crude drug is placed in a stoppered container with the solvent and allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved. The mixture then is strained, the marc is pressed and the combined liquids are clarified by filtration or decantation after standing.

PERCOLATION:

            It means to drain through a porous material or filter.

“The process by which comminuted drug is extracted from its soluble constituents by slow passage of a suitable solvent through a column of drug.”

Percolator:

                The process of percolation is carried out in a vessel called percolator.

Percolate:

                The collective extractives are called percolate.

Example: 

·         Tincture of Belladonna

·         Strong tincture of Cardamom

·         Preparation of Coffee

This is the procedure used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts.

The solid ingredients are moistened with an appropriate amount of specified menstrum and allowed to stand for approximately 4 hours in a well closed container after which the mass is packed and the top of percolator is closed. Additional menstrum is added to form a shallow layer above the mass and the mixture is allowed to macerate in closed percolator for 24 hours.

The outlet of percolator, then is opened and the liquid contained therein is allowed to drip slowly.

Additional menstrum is added as required, until the percolate measures about 3 quarters of required volume of finished product. The marc is then pressed and the expressed liquid is added to the percolate. Sufficient menstrum is added to produce the required volume and the mixed liquid is clarified by filtration or by standing followed by decanting.

Advantage:

The advantage of this method, compared to previously described methods, is that large amount of drug can be extracted with a much smaller quantity of solvent, it is most economical in terms of time, energy and consequently financial inputs.

            If the therapeutic values lies in the non-polar constituents, a non-polar solvent may be used.

 

 

 

 

 

 

 

 

 

 

 

 

 

ALKALOIDS

DEFINITION

“Chemically alkaloids are defined as organic nitrogenous substances of plant origin that are ionic in nature.”

These alkaloids are insoluble in water but their salts are freely soluble in water.

The free alkaloids are soluble in ether, chloroform or any other non-polar solvents.

EXAMPLES

·         Atropine

·         Morphine

·         Cocaine

·         Caffeine

·         Nicotine

·         Emetine

·         Vinblastine

·         Theophylline

·        Scopolamine

TYPES OF ALKALOIDS

·         Pyridine-piperidine Alkaloids       

e.g.Lobelia,Tobacco Nicotine

 

·         Purine Alkaloids               

e.gTea,coffee(Caffiene,Theophylline,Theobromine)

 

·         Tropane Alkaloids

e.g.Hyoscyamus,Stramonium,Belladona   ( Hyoscyamine,scopolamine,Belladonine)

 

·         Quinoline Alkaloids                      

e.g.Cinchona bark(Quinine, Cinchonine,Cinchonidine)

 

·         Iso-quinoline Alkaloids                 

e.g. Opium(morphine,codiene),Ipecac(Emetine)

 

·         Indole Alkaloids                            

e.g.Nuxvomica(Strychnine,Brucine) ,Rauwolfia(Reserpine),Catharanthus(Vinblastine,Vincristine)

l        Steroidal Alkaloids e.gVeratrum(Veratridine)

l        Imidazole alkaloids e.gPilocarpus(pilocarpine)

l Alkaloidal amines e.g Ephedra (ephedrine,psuedoephedrine)

   EXTRACTION OF CAFFEINE (PURINE ALKALOID) FROM TEA                        

 

Caffeine act as CNS stimulant. It is naturally occurring alkaloid found in certain bark of plants and in fruits, e.g. Coffeaarabica, camellia sinensisetc

BOTANICAL NAME:                     Camellia sinensis

FAMILY:                                              Theaceae

CHEMICAL CONSTITUENTS:    Caffeine ,Theophylline,Tannins

PART USED:                            Leaves

USES:                                       CNS stimulant

                REQUIREMENTS

APPARATUS

Beakers, burner, tripod stand, conical flask, funnel, filter paper, separating funnel, weighing balance and water bath.

CHEMICALS

·         4 tea bags

·         Distilled water

·         Dichloromethane

·         Sodium carbonate (Na₂CO₃)

EXTRACTION THEORY:

Solvent extraction is a process of purification involving the use of two solvents or solutions that are immiscible with one another. It is based on the differential solubility of compounds in water and an immiscible organic solvent (typically diethyl ether, chloroform, dichloromethane, ethyl acetate). This allows the desired organic product to be separated from inorganic or very other polar by-products. This is the basis of an older industrial method for "decaffeinating" coffee.

PROCEDURE

                PART 1: DISSOLUTION OF CAFFIENE IN WATER

·         Weigh 15 g of tea bags and place them in a beaker. Record actual weight of tea bags (W₁).

·         Add 300 ml of Distilled water to the beaker and 5g of Na₂CO₃.

·         Boil water containing tea bags for 10-15 minutes while stirring continuously.

·         After the boiling period is over, remove the beaker from heat and allow it to cool for 15 minutes.

·         After solution has cooled, squeeze the tea bags to remove all the liquid.

·         Dispose of the bags.

·         Filter the solution with regular filter paper to remove any solid particle left.

PART 2: TRANSFER OF CAFFIENE FROM WATER TO DICHLOROMETHANE

·         Transfer the above filtrate to a separating funnel and add 20 ml DCM in it and shake gently.

·         Allow DCM to settle to bottom. Carefully drain the DCM layer in flask. Repeat it 3 times with 20 ml DCM.

·         Dispose of the aqueous layer.

·         Filter the solution.

 

PART 3: CRYSTALLIZATION OF CAFFIENE

·         Place the DCM solution containing caffeine over boiling water.

·         Evaporate the solution down to about 20 ml and remove from heat.

·         Weigh a clean watch glass and weigh it (W₂).

·         Place the watch glass on the flame and let it cool.          

·         Now weigh the watch glass (W₃).

·         What is the weight of Caffeine? (W₃–W₂=W₄)  

·         What is your percentage yield of Caffeine?  (W₄/W₁ x 100)

 

 

CALCULATION:

 % yield = Weight of caffeine/Weight of sample × 100

 

 

MICROSCOPIC EXAMINATION:

- Caffeine + drops of mercuric chloride HgCl2 -----------àexamine under microscope------àneedle-shape Crystals.

 

 

 

 

 

 

 

 

 

 

Result Sheet

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

EXTRACTION OF STRYCHNINE & BRUCINE (INDOLE ALKALOIDS) FROM NUXVOMICA

   VERNACULAR NAME:            Kutchla

BOTANICAL NAME:                     Strychnosnuxvomica

FAMILY:                                              Loganiaceae

CHEMICAL CONSTITUENTS:    Strychnine(1.25%), Brucine(1.5%)

PART USED:                            Dried seeds

USES:   Strychnine: In physiologic research,CNSstimulant,Vermicide,Respiratorystimulant,Circulatory stimulant in case of shock

Brucine: Alcohol denaturant

 APPARATUS:

 *Petri dishes      * Beakers     *Funnel     * Separating Funnel        *Test tubes     *Tripod stand   

  *Stirrer      *Filter paper                                                                                 

CHEMICALS:

Aluminium hydroxide, Chloroform, Sulphuric Acid, Potassium dichromate

PROCEDURE:

l   10g of powdered drug is taken in a conical flask.Add water in it.Filter the solution.Filtrate is taken in a test tube .

l   Add 0.5ml of Al(OH)₃ and 2-3 ml of chloroform  & shake well.

l   Transfer the solution to separating funnel and leave it for sometime till the 2 layers is formed.

l   Upper layer is aqueous layer and lower layer is organic layer.

l   Perform the tests on organic layer:

 

EXPERIMENT	OBSERVATION	INFERENCE
1.Test for Brucine To a solution in a test tube, add few ml of HNO3	Crimson yellow color appear	Brucine Alkaloid present
2.Test for Strychnine To a solution in a test tube add few crystals of K2Cr2O7 & few ml of H2SO4 are added.	Brown color appear	Strychnine Alkaloid present

 

EXTRACTION OF NICOTINE (PYRIDINE-PIPERIDINE ALKALOIDS) FROM

TOBACCO LEAVES

Chemistry:                                                                       

              Nicotine is hygroscopic oily liquid that is miscible with water in its base form. As a nitrogen base, nitrogen nicotine from salts with a acid that are usually solid and water soluble. Nicotine easily penetrates the skin.

Toxicology:

      The LD50 of nicotine is 50mg/kg for rats and 3mg/kg for mice.40-60mg/kg can be lethal dosage for adult human being. This makes it an extremely deadly poison. It is more toxic than other alkaloids, such as cocaine, which have lethal dose of 1000mg.

Principle:

 The extraction depends on isolation of base by dissolving the cigarettes in NaOH. Then extract nicotine from the filtrate by ether. After evaporation of ether you will get nicotine oil.

 The factories of cigarettes remove large quantities of nicotine from cigarette leaves because of high toxicity. This is why the produced oil is very little. To get nicotine crystals, saturated solution of picric acid is added to form nicotine di picrate yellow crystals.

Aims of Experiment:

                In this experiment you will extract nicotine from cigarette with ether and precipitate as nicotine di-picrate salt.

Materials:

              1. Tobacco leaves.

              2. Ether.

              3. NaOH solution (5%)

              4. Beaker 250ml, separating funnel.

              5. Conical flask

              6. Saturated picric acid solution in methanol.

Procedure:

1. Weigh 10g of cigarette/tobacco leaves in a beaker.

2. Add 100ml of NaOH solution and stir very well for 15mins.

3. Filter it and press cigarette very well by using other beaker.

4. Transfer cigarette again to beakers.

5 .Add 30ml of distilled water, stir and filter it again

6. Collect the filtrate together.

7. Transfer the filtrate to separating funnel and extract by 25ml of ether.

8. Repeat extraction three times.

9 .Gather the filtrate into conical flask.

10. Dry by using one teaspoon anhydrous potassium carbonate. Filter it.

11.Evaporate ether on water bath. (Avoid extra heat because nicotine is hydrolyzed by extreme heating).

12- After evaporation of ether add 4ml methanol to dissolve the

resulted oil.

13- Add 10ml saturated picric acid solution.

14- Cool in an ice bath to precipitate the nicotine di picrate crystals.

15.Filter; allow drying and weighing the product.

 

RESULT:

% yield =  weight of oil extracted from crude drug /weight of crude drug used × 100

 

MICROSCOPIC EXAMINATION:

 

Nicotine + Mercuric Chloride HgCl₂ = Examine under microscope = Flowery-shape Crystals

 

 

 

 

 

 

 

 

 

 

 

 

ISOLATION OF AN ALKALOID :ISOLATION OF PIPERINE

FROM BLACK PEPPER

Introduction:

Piperine is an alkaloid found naturally in plants belonging to the Piperaceaefamily, such as Piper nigrumL, commonly known as black pepper, and Piper longumL, commonly known as long pepper.

Piperine is a solid substance essentially insoluble in water. It is a weak base that is tasteless at first, but leaves a burning aftertaste.

In this experiment we will extract the alkaloid components that give black pepper its physiological properties including carminative (relief of intestinal gas), diuretic (increases urine output) as well as the well-known “taste bud” effects.

As we know black pepper contains about ;

·-3% of volatile oils (terpenes),

·-10% of the alkaloids piperine.

·-13% starch.

·the remaining is cellulose & water.

·To get the pure piperine, all other components must be removed, so the starch can be dissolved and volatile oils must soapnification so it can be water soluble.

Among these, piperine is the major component while piperanine and pipercide are only present in minute amounts.

 

Materials:

• KOH solved in ethanol (2M).   

• Ethanol (95%) 300 ml.

• HCL (6 M).

• RAD condenser

• Buchner Funnel.

Method:

-Weight 50gm of black piper. (Note; the net result for the piperine extracted should be 5gm. (since it contain10% of piperine).

- Add 300ml ethanol 95% to the 50gm piper and hold the condenser above the bottle.

- Heat the mixture gradually until it boiled and keep it on heater for 3 hours then cool it.

- Filtrate the mixture in funnel.

-Distillate the filtrate in distillation device to remove the extra ethanol until the volume inside reached 25 ml.

-Then do soapnificationby adding 25 ml of KOH solved in ethanol (2 M) to this solvent and then mix it and hold the condenser above the bottle then boil the mixture for exactly 5min.

-After this add 35-40 ml water until the solvent become muddy then put it into a

beaker then in Ice bath and scratch the wall with a glass rode.

- Leave it to the next day.

-Filtrate the solution in Funnel.

- Piperine will expected to be 3gm but if you find it 2.7 gm which is

considered to be very close to perfect accuracy.

Warning;

- If boiling period after soapnificationstill more than 5min, the weak bond between N & C will be broken by the entrance of KoH and so it is hydrolyzed to piperidine + piperic acid which is undesirable.

 

 

 

 

 

 

 

 

GLYCOSIDES

DEFINITION

“Glycosides are compounds which upon hydrolysis give rise to one or more sugars (glycones) and a compound which is not a sugar (aglycone or genin).”

They are colorless, crystalline compounds.

Anthracene/Anthraquinone glycosides are red or orange colored compounds.

They are soluble in water and alcohol, but insoluble in other organic solvents like chloroform, petroleum ether.

EXAMPLES

·         Saponin glycosides e.g. Senegal

·         Cardiac glycosides    e.g. Digitalis, Strophanthus

·         Anthraquinone glycosides e.g.Senna, Aloe Vera

·         Cyanophore glycosides         e.g. Bitter Almond

·         Isothiocyanate glycosides  e.g. Mustard plant

·         Flavanol glycosides e.g.Rutin

·         Alcohol glycosides   e.g. Salix purpurea

·         Aldehyde glycosides               e.g. Vanilla planifolia

 

 

 

 

 

 

 

 

 

 

 

 

 

EXTRACTION AND IDENTIFICATION OF ANTHRAQUINONE GLYCOSIDES FROM SENNA LEAVES

BOTANICAL NAME:              Cassia acutifolia, Cassia angustifolia

FAMILY:                                 Leguminoseae

PART USED:                           Dried leaves

CHEMICAL CONSTITUENTS

Senna contains

·         Anthraquinone glycosides i.e. Sennoside ‘A’ and Sennoside ‘B’

·         Aloe-emodinglycoside,mucilage,resins.

APPARATUS

Beakers, Test tubes, Funnel, Tripod Stand, Burner, Pipette, Stirrer, Spatula, Filter Paper, Test tube holder, Weighing Balance

CHEMICALS

*Conc. H₂SO_{4                        *} NaCl                 *Ethanol              *Copper Sulphate

*Ammonia Solution    * Bromine Water     * Conc. HNO₃

PROCEDURE

·         Take about 0.6g Of Senna leaves.

·         Prepare 10% conc. H₂SO₄ and boil the leaves in H₂SO₄ for 15 minutes.

·         Filter the solution and allow it to cool.

·         Perform the confirmatory tests on this filtrate.

EXPERIMENT	OBSERVATION	INFERENCE
1.Borntragers Test: Took 2ml of extract in a test tube, added 2ml of Ammonia solution to it.	Reddish brown color appears.	AnthraquinoneGlyscosideare present.
2. Bromine water Test: Took 2ml of extract in a test tube, added 1-2 drops of BROMINE WATER.	Pale yellow color appears	AnthraquinoneGlyscosideare present.
3.Nitric acid Test: Take 2ml of extract, added 1-2ml of nitric acid	Pale brown color appears	AnthraquinoneGlyscosideare present.
4.KlungsIsobarbaloin Test: Take 2ml extract, added 1-2 drops CuSo4 +1gm NaCl+10ml 90% alcohol	Green color appears.	AnthraquinoneGlyscosideare present.

USES :1. Senna is used as purgative and cathartic. Also used as stimulant laxative.

2.The drug is used in acute constipation & in all cases in which defecation with soft   stool is required.

 

EXTRACTION AND IDENTIFICATION OF ANTHRAQUINONE GLYCOSIDES FROM ALOE VERA

BOTANICAL NAME:              Aloe barbadenis

FAMILY:                                 Liliaceae

PART USED:                           Leaves

CHEMICAL CONSTITUENTS

Aloe contains:

·         Aloin

·         Resin

·         Aloe-emodin

·         Volatile oils

·         Barbaloin

APPARATUS

Beakers, Test tubes, Funnel, Tripod Stand, Burner,Pipette, Stirrer, Spatula, Filter Paper, Test tube holder, Weighing Balance

CHEMICALS

    * Conc. HNO₃   *NaNo2(sodium nitrite)  *Acetic acid *CuSo4 solution *NaCl * Bromine water 90% Alcohol

PROCEDURE

·         Boil 1gm of drug with 100ml of water

·         Add little silica gel and filter it.

·         Perform the confirmatory tests on this filtrate.

EXPERIMENT	OBSERVATION	INFERENCE
1.Borntragers Test: Aloe (0.1g) drug is boiled with dil.HCl (5ml) for 5min. The solution is cooled,filtered and the filtrate is sahken with benzene.The benzene layer is separated, ammonia solution is added to this .	Pink  color appears.	AnthraquinoneGlyscosideare present.
2. Bromine water Test: Took 2ml of extract in a test tube, added 1-2 drops of BROMINE WATER.	Pale yellow color appears.	AnthraquinoneGlyscosideare present.
3.Nitric acid Test: Take 5ml of extract, added 1-2ml of nitric acid	Pale brownish yellow color appears.	AnthraquinoneGlyscosideare present.
5.Klungssobarbaloin Test: Take 20ml extract, added 1-2 drops CuSo4 +1gm NaCl+10ml 90% alcohol	Purple color appears.	AnthraquinoneGlyscoside are present.(Isobarbaloin)
6.Nitrous Acid Test: To aq. Solution of Aloe small amount of sodium nitrite and few ml of acetic acid are added	Pink color appears.	AnthraquinoneGlyscoside are present
7.Schontetens Test: To a solution(5ml) borax(0.2g) is added and is heated to dissolve completely. Few drops of liquid are poured in a test tube filled with water.	A Green fluorescence appears.	AnthraquinoneGlyscoside are present

 

USES :

1. Aloeis used as purgative and given in constipation.

2. Ointment of aloe-gel is used to cure burns caused by heat,sun or radiation and skin irritations.

3. It is also used to cure many skin disease and tonic for stomach.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TANNINS

DEFINITION

“Tannins are complex organic non.nitrogenous substances of polyhydroxy benzoic acid”.

They  are the complex compounds that are capable of combining either the animal proteins resulting in their precipitation.

·         Soluble in water ,alcohol and acetone.

·         Insoluble in organic solvents such as ether and chloroform.

·         They have molecular weight ranging from 500 to over 3,000(gallic acid esters) and upto 20,000(proanthocyanidins)

TYPES:

Hydrolysable Tannins:- These consist of gallic acid or related polyhydric compounds esterified with glucose because such esters are readily hydrolysed to yield the phenolic acid and sugar so they are referred as hydrolysable tannins. E.g clove

Condensed Tannins :-These tannins results from the condensation of two or more flavon-3-ols, such as catechin.When treated with hydrolyted agents, these tannins tends to b polymerited yielding insoluble red colour products known as phalobaphenes. E.g cinnamon, tea.

Pseudo Tannins:-They have properties of both hydrolysable and condensed tannins.Extraction of gallicacid,catecholchlorogenic acid is obtained .E.grhubarb,coffee.

 

 

 

 

 

 

 

 

 

 

 

 

 

Extraction and Identification Of Tannins From Cinnamon

 

BOTANICAL ORIGIN:-Cinnamomumloureirii

FAMILY:-Lauraceae

PART USED:-Dried bark

CONSTITUENTS:-Cinnomic aldehyde, eugenol, mannitol.

 

REQUIREMENTS:

Drug extract, test tubes, separating funnel, beakers, pipette, 5% Ferric chloride solution, bromine water.

PROCEDURE:

Take about 5g of drug and than add 100ml of water and boil it for about 10-15min.Filter the solution and cool it and perform identification test.

USES:

 

·         Tannins are an important ingredient in the process of tanning leather.

·         Wood adhesive.

·         Antiparasitic effects.

·         Antibacterial, antiviral.

·         Precipitate proteins.

·         Astringent

 

IDENTIFICATION TESTS FOR CINNAMON:

 

EXPERIMENT	OBSERVATION	INFERENCE
1. Took 2ml of extract in a test tube, added 2ml of distilled water and than added 1-2 drops of FERRIC CHLORIDE SOLUTION.	Light Greenish color was appeared.	Identification of condensed tannins.
2. Took 2ml of extract in a test tube, added 1-2 drops of BROMINE WATER.	Dark orange color was appeared.	Identification of condensed tannins.

 

 

 

 

 

 

CARBOHYDRATES

 

Carbohydrates are heterogeneous organic compounds. Except for polymers, most carbohydrates have a sweet taste, hence they are called sugars. They are also called saccharides due to their white crystalline appearance.

Carbohydrates maybe defined as “poly-hydroxy aldehydes” or “poly-hydroxy ketones”.

 

Classification

 

Monosaccharide:

They are the simplest Carbohydrates; if they are further broken, doesn’t possess the characteristics carbohydrates.Monosaccharides are colorless, crystalline substances which have a sweet taste and are soluble in water.Examples: Glucose (aldo-hexose), Fructose (keto-hexose).

Disaccharides:

They are formed by the condensation of two monosaccharide molecules. The monosaccharides are connected by aglycosidic linkage. Some common disaccharides are:Maltos, Lactose,Sucrose

Polysaccharides:

They are formed by the condensation of a large number of monosaccharide molecules and have a high molecular weight, hence are slightly soluble in cold water. They form a colloidal solution when heated with water. E.g starch

 

 

 

 

 

 

 

 

 

 

 

 

 

EXTRACTION& IDENTIFICATION OF STARCH FROM POTATO

 

BOTANICAL ORIGIN: Solanumtuberosum

FAMILY: Solanaceae

THEORY: Starch is a plant carbohydrate. It is a mixture of two structurally different polysaccharides which are:

Amylase: Linear molecules composed of 250-300 units of glucose uniformly linked by  1,4α glycosides linkage.

Amylopectin: Consist of 1000 or more molecules of glucose connects with 1,4 linkage and a number of  1,6 linkage are also present about 4%.

MATERIAL:

 Pestle and mortar, Beaker, Potato

PROCEDURE:

 Take 10g of peeled potato, wash them and cut into small pieces. Now crush them with water in the form of paste. Now add 200 ml of water in it and allow it to stand for 20 minutes. Now filter the water to remain the cell debris. The filtrate contain the starch .Allow it to stand for 2 hours. The starch will settle down and is separated, dried and identified through tests.

EXPERIMENT	OBSERVATION	INFERENCE
Molishs tests: Take two test tubes and label them S for sample and B for blank. Take 3ml of distilled water in test tube B and 3ml of given solution in test tube S. Add 2 to 3 drops of Molisch’s reagent in both test tubes. Mix thoroughly and incline the test tubes and run 3ml of concentrated sulphuric acid along the wall of test tubes. .	Appearance of reddish violet colored ring at the junctions of two liquid	Indicates the presence of carbohydrate (mono,di-,or polysaccharides)
Iodine test: Take 3ml of carbohydrate solution in a test tube. Acidify it with one drop of concentrated HCl. Now add one small drop of iodine solution in this test tube and mix it.	Blue color appears	Presence of Starch is confirmed
Benedicts test: Take 5 ml of benedict reagent in a test tube and add 8 drops of given solution. Mix and boil over a flame or in a boiling water bath for 2 minute and cool the solution.	Appearance of green, yellow, orange or red precipitate No change in color	Reducing sugars present     Non-Reducing sugars present

 

USES:

As dusting powder.

As base for enemas.

As antidote for iodine poisoning.

As binder and disintegrating agent in tablets.

As nutrient.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chromatography

 

Chromatography is usually introduced as a technique for separating and/or identifying the components in a mixture. The basic principle is that components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent. It is a powerful method in industry, where it is used on a large scale to separate and purify the intermediates and products in various syntheses.

Basic principle

All chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase). The techniques rely on one of the following phenomena: adsorption; partition; ion exchange; or molecular exclusion.

Partition

 In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin layer (or film) on the surface of an inert solid. The mixture to be separated is carried by a gas or a liquid as the mobile phase. The solutes distribute themselves between the moving and the stationary phases, with the more soluble component in the mobile phase reaching the end of the chromatography column first . Paper chromatography is an example of partition chromatography.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PAPER CHROMATOGRAPHY

Paper chromatography is most effective for the identification of unknown substances when known samples are run on the same paper chromatograph with unknowns

 

This is probably the first, and the simplest, type of chromatography that people meet. A drop of a solution of a mixture of dyes or inks is placed on a piece of chromatography paper and allowed to dry. The mixture separates as the solvent front advances past the mixture. Filter paper and blotting paper are frequently substituted for chromatography paper if precision is not required. Separation is most efficient if the atmosphere is saturated in the solvent vapour

Some simple materials that can be separated by using this method are inks from fountain and fibre-tipped pens, food colourings and dyes. The components can be regenerated by dissolving them out of the cut up paper.

 The efficiency of the separation can be optimised by trying different solvents, and this remains the way that the best solvents for industrial separations are discovered (some experience and knowledge of different solvent systems is advantageous).

Paper chromatography works by the partition of solutes between water in the paper fibres (stationary phase) and the solvent (mobile phase).

Common solvents that are used include pentane, propanone and ethanol. Mixtures of solvents are also used, including aqueous solutions, and solvent systems with a range of polarities can be made. A mixture useful for separating the dyes on Smarties is a 3:1:1 mixture (by volume) of butan-1-ol:ethanol:0.880 ammonia solution.

As each solute distributes itself (equilibrates) between the stationary and the mobile phase, the distance a solute moves is always the same fraction of the distance moved by the solvent.

This fraction is variously called the retardation factor or the retention ratio, and is given the symbol R or Rf:

(Rf )Retention ratio = distance moved by solute

                                       distance moved by solvent

 

So as long as the correct solvent and type of chromatography paper are used, a component can be identified from its retention ratio

 

It is possible that two solutes have the same Rf values using one solvent, but different values using another solvent (e.g this occurs with some amino acids). This means that if a multi component system is not efficiently separated by one solvent the chromatogram can be dried, turned through 90C, and run again using a second solvent

SEPARATION OF COMPONENTS FROM A MIXTURE OF RED AND BLUE INKS

Apparatus:

 

 

 

 

 

 

 

 

 

 

 

Lab Procedure

• Take a Whatman filter paper strip and using a pencil draw a horizontal line 4cm from one end of the paper. Then draw another line lengthwise (verticallly) from the centre of the paper. Name the point at which the two lines intersect as P.
• Using a fine capillary tube, put a drop of the mixture of red and blue inks at the point P. Let it dry in air.
• Put another drop on the same spot and dry again, so that the spot is rich in the mixture.
• Pour equal amounts of isopropyl alcohol and distilled water into a chromatographic chamber and mix it well using a glass rod. This is used as the solvent.
• Suspend the filter paper vertically in the chromatographic chamber containing the solvent in such a way that the pencil line remains about 2cm above the solvent level.
• Close the jar with its lid and keep it undisturbed.
• Notice the rising solvent along with the red and blue inks. After the solvent has risen about 15 cm you will notice two different spots of blue and red colors on the filter paper.
• Take the filter paper out of the jar and using a pencil mark the distance that the solvent has risen on the paper. This is called the solvent front.
• Dry the filter paper and put pencil marks at the centre of the red and blue ink spots.
• Measure the distance of the two spots from the original line and the distance of the solvent from the original line.
• Calculate the Rf values of the red and blue inks using the formula,

·         Rf=distance travelled by the component /distance travelled by solvent

 

WORK SHEET

 

Observation

Observations can be recorded as shown.

SI No.	Components	Distance travelled by the component from the original line (cm)	Distance travelled by the solvent from the original line (cm)	Rf value
1.	Red
2.	Blue

 

Inference

• R_f value of red ink = ……………
• R_f value of blue ink = …………..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SEPARATION OF PIGMENTS FROM THE EXTRACT OF SPINACH LEAVES

Materials Required

 

 Lab Procedure

• Take a Whatman filter paper strip and using a pencil draw a horizontal line 4cm from one end of the paper. Then draw another line lengthwise (verticallly) from the centre of the paper. Name the point at which the two lines intersect as P.
• Using a fine capillary tube, put a drop of the extract of spinach leaves at the point P. Let it dry in air. 
• Put another drop on the same spot and dry again, so that the spot is rich in the leaf extract.
• Pour equal amounts of isopropyl alcohol and distilled water into a chromatographic chamber and mix it well using a glass rod. This is used as the solvent.
• Suspend the filter paper vertically in the chromatographic chamber containing the solvent in such a way that the pencil line remains about 2cm above the solvent level.
• Close the jar with its lid and keep it undisturbed.
• Notice the rising solvent along with the coloured components of the leaf extract.
• After the solvent has risen to about 15 cm you will notice two different spots of coloured components on the filter paper.
• Take the filter paper out of the jar and using a pencil mark the distance that the solvent has risen on the paper. This is called the solvent front.
• Dry the filter paper and put pencil marks at the centre of each spot.
• Measure the distance of each spot from the original line and the distance of the solvent front from the original line.
• Calculate the Rf values of different components of leaf extract by using the formula,

Precautions

• Use good quality pencil for drawing the reference line so that the mark does not dissolve in the solvent in which the chromatography is carried out.
• Always use a fine capillary tube.
• Keep the jar undisturbed and covered during the experiment.
• The spot should be small and rich in mixture.
• Allow the spot to dry before putting the strip in the jar.
• Keep the strip erect. Do not let it curl.
• Do not allow the spot to dip in the solvent. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

WORK SHEET

Observation

Observations can be recorded as shown

SI.No	Component	Distance travelled by the component from the original line (cm)	Distance travelled by the solvent from the original line (cm)	Rf value
1.	Orange (Carotene)
2.	Yellow (Xanthophyll)
3.	Light green (Chlorophyll a)
4.	Dark green (Xanthophyll)

 

Inference

• R_f value of orange (Carotene) = ……………
• R_f value of Yellow (Xanthophyll) = …………..
• R_f value of Light green (Chlorophyll a) = …………..
• R_f value of Dark green (Xhlorophyll b) = …………..

 

 

 

 

 

 

 

 

 

 

 

PREPARATION OF TLC PLATES

 

Thin layer chromatography is a chromatography technique used to separate non-volatile mixtures.

Thin layer chromatography is performed on a sheet of glass,plastic,oraluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide or cellulose. This layer of adsorbent is known as the stationary phase.

 

Principle :

TLC is based on the principle of separation. The separation depends on the relative affinity of compounds towards stationary and mobile phase. The compounds under the influence of mobile phase( driven by capillary action) travel over the surface of stationary phase. During this movement the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus separation of components in the mixture is achieved.

Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography. However ,instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbent. For even better resolution and to allow for quantification, high-performance TLC can be used.

 

Preparation of TLC plates:

l  Weigh approx. 2gm of silica gel.

l  Mix the adsorbent (Silica gel) with a small amount of inert binder calcium sulphate(gypsum) and water.

l  This mixture is spread as thick slurry on glass slide or thick aluminium foil.

l  The resultant plate is dried and activated by heating in an oven for thirty minutes at 110ºC.

l  The thickness of absorbent layer is typically around 0.5-2mm for preparative TLC.

 

 

 

 

 

THIN LAYER CHROMATOGRAPHY

 DETECTION OF CAFFEINE IN VARIOUS SAMPLES

 

Materials Needed:

TLC equipment:

1 5-cm x 8-cm TLC plate, 2 capillary micropipettes, TLC tank and lid, ruler, UV lamp

Extraction Equipment:

Spatula, 2 small test tubes, Pasteur pipet and bulb

 

Chemicals:

Caffeine, ethanol, dichloromethane, TLC solvent (5% acetic acid in ethyl acetate)

 

Three of the following samples for analysis: Analgesic pill, instant coffee, tea

Purpose

In this experiment, thin-layer chromatography (TLC), a standard technique for the qualitative analysis of mixtures, will be introduced. First, a sample of caffeine will be authenticated by measuring its melting point.

 

The authentic caffeine will then be used as a standard for the TLC analysis one of the following items: a diet soda, tealeaves, instant coffee, cocoa powder, or an analgesic pill.

 

The caffeine present in these substances must first be separated from dissimilar materials in the substance using a process called extraction. Extraction simply involves treating the substance with an organic solvent that will dissolve out the caffeine and other soluble organic materials but leave insoluble matter (such as cellulose in leaves and inorganic binders in a pill) behind.

 

 

 

 

Laboratory Procedures

 

Melting Point Procedure.Use the capillary melting point technique to obtain the mp range of pure caffeine(238 C).

Carefully note your observations on what the sample looks like as it heats up and then melts.

Each group should extract and analyze one instant beverage sample, one analgesic tablet, and one liquid pop sample.

 

Extraction Procedures:

Solid samples – instant beverages and analgesic pills. Use 500 mg of the sample. Make sure to record the actual mass used. If a solid sample is not a fine powder to begin with, then you will need to grind it to a powder, preferably by using a mortar and pestle. Put each powder into a small, labeled test tube. Add 2.0 mL ethanol to each test tube and shake to mix thoroughly. Heat the tubes gently on a hot water bath for 2-3 minutes and then allow the contents to cool and settle. Not all of the powder will dissolve; the inorganic buffering agent (if present) and binder are insoluble in ethanol. However, any organic compounds present will dissolve. Remove the ethanol solution from undissolved solids using a pipet. Use this solution to spot the TLC plate.

Liquid samples.Measure 4 mL of the liquid into a large test tube. Wear gloves during the rest of this step. Add 5 mL of CH2Cl2 to the tube. Cap the tube with a tight fitting rubber sleeve stopper or cork and shake gently at first, frequently removing the stopper to allow venting of any pressure build up. Shake well then allow the layers to separate. Caffeine is much more soluble in CH2Cl2 than in H2O so any caffeine present goes into the CH2Cl2 layer. Use a Pasteur pipet to transfer most of the lower CH2Cl2 layer to a small test tube. Add a few CaCl2 pellets to this tube and let stand for 5-10 minutes – the solution should clear up as the CaCl2 absorbs any remaining water (“dries the solution”). Use this solution to spot the TLC plate.

 

 

TLC Procedures

 

l  Put approximately 25 mg of caffeine in a small, labeled test tube. Add 4.0 mL of ethanol mix, and heat gently to dissolve the caffeine.

l  Obtain a TLC plate. The plate should be handled using forceps so as to avoid contamination. A light pencil line should be drawn using a straight edge about 1 cm from one end of the plate.

l  The instructor will demonstrate the spotting technique. Use a capillary micropipette to make a small spot of the pure caffeine solution (made in step 3) on the plate. Place the spot 1 cm from the left edge along the pencil line you drew.

l  Also spot the TLC plate with each of the solutions prepared in steps 1 and 2. Make these three spots at 1-cm intervals to the right of the caffeine spot. In order to avoid confusion make sure to label the spots lightly in pencil bellow the line.

l  Develop the TLC plate by placing it in a beaker that has been filled with developing solvent (5% acetic acid in ethyl acetate) to a level of less than 1 cmhigh (the spot on the TLC plate should be above the level of the solvent). Cover the beaker with aluminum foil immediately after the TLC plate is immersed. Allow the solvent to migrate up the TLC plate until it is about one centimeter from the top. Do not allow the solvent line to reach the top of the plate.

l  Remove the TLC plate and mark the level to which the solvent rose with a pencil. Allow the solvent to evaporate off of the plate in the hood and then visualize the plate under UV light. Outline all spots with a pencil.

l  Measure the distance the solvent moved as well as the distances of all spots. Carefully sketch the TLC plate in the space provided on the report sheet.

 

Post-Lab Calculations

Calculate the Rf of all of the spots.

Rf of compound = Distance moved by spot / Distance moved by solvent

 

 

 

Z y   x

Ddd

 

 

 

Pic.

 

 

 

 

Calculating the Rf of Spots in TLC (a) before development (b) after development

 

 

PRE-LABORATORY QUESTIONS

 

 TLC AND MELTING POINT DETERMINATION: DETECTION OF CAFFEINE IN VARIOUS SAMPLES IN-LAB OBSERVATIONS/DATA

 

Name ____________        Section ______________      Date___________

 

Team Name/Partners ____________________

 

 1. Give the structure of caffeine in the space below. Explain why it is considered an amine.

 

Pure Caffeine

General Observations_________________________________________________

Melting point range _______________Literature mp ____________ Reference________________________________

 

Samples analyzed data and observations

Sample #1(instant beverage) ________

Sample #2 (liquid soda)  ___________

Sample #3 (analgesic pill) ___________

 

Amounts taken : Caffeine ______ Sample 1 ______ Sample 2 _______Sample 3________

 

Observations on solutions used to spot TLC plate:

Caffeine_________________________________

Sample #1 _______________________________

Sample#2________________________________

Sample#3________________________________

Observations on TLC ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

 

TLC Results:

 

SKETCH OF DEVELOPED TLC PLATE

Distances moved:

 

Solvent __________________

 

Caffeine _________________

 

Spots in sample#1 ____________________

 

Spots in sample#2 ____________________

 

Spots in sample#3 ____________________

 

 

 

 

 

 

 

 

 

 

 

 

 

DETECTION OF CAFFEINE IN VARIOUS SAMPLES

REPORT SHEET

 

 

I. Melting Point of Caffeine

 

Mp _______________   Literature Mp _________________

 

II. TLC Results Table

 

Solution	Rf value (s)	Approximate Relative Darkness and Size of Spot (compared to caffeine standard)
Caffeine
Sample 1:   ______________________
Sample 2 :   ______________________
Sample 3:   ______________________