Experiment 2

Preparation

At least 8 1 test tube PPE

Wash and dry label Get reagent
test tubes rack per TS

Experiment
Test ph
Calibrate
Prepare a dropper for benzoic acid,
the pH
phenol, aniline, and naphthalene
meter

Add
TEST Place petroleum Add Add Add
benzoic
TUBE A ether (hm) phenol aniline naphthalene
acid

Remove aqueous layer and place in test tube B.1 Add sodium bicarbonate and mix

Tongs and alcohol lamp

TEST Get a TEST Add 10 Add 10

Add 3M Gently
TUBE small TUBE drops drops
HCl heat
B.1 portion B.2 ethanol 3M HCl

Gently
Smell the
Place aque- waft the
TEST Add 3M Gently fruity odor
ous sol’n in solution
TUBE A NaOH Mix
test tube C

Get a TEST Observe a

TEST Add 3M Add 5%
small TUBE blue to purple
TUBE C HCl FeCl3
portion C.1 discoloration

Place aque-
TEST Add 3M Gently
ous sol’n in
TUBE A HCl Mix
test tube D

Get a TEST Add Observe the for-

TEST Add 3M
small TUBE concentrated mation of needle
TUBE D NaOH -like crystals
portion D.1 sulfuric acid

Dry the solution Remove Get a TEST

TEST Evaporate Add 1mL
over anhydrous the sol- small TUBE
TUBE A to dryness KMnO4
CaCl2 pellets vent portion A.1

Observe and take note

 Post-Experiment Disposal

Test solution Dispose to

10% phenol Non-Halogenated waste

95% ethyl alcohol
0.1M acetic acid Dilute with water and
0.1 glycine dispose to sink
0.1M urea
Treat with 10% Na2CO3
0.1 M chloroacetic acid
and dilute with water.
0.1 M trichloroacetic acid
Then dispose to sink
Treat with dilute HCl and
0.1M ammonium hydroxide dilute with water.
Then dispose to sink
 Experiment 2
Structural Effects on Polarity

A. Preparation of TLC plates F. Visualization of the Spots

Cut a 1.5 cm x 6 cm TLC plate Using a sprayer, deliver ninhydrin solution
evenly to the TLC plates and observe for the
formation of violet spots
Heat the plate at 110°C for 10 mns prior to use

Outline the spots using pencil

B. Preparation of the Sample and Standards

Prepare 10mL of a 1% ethanolic solution of

Compute for the Rf values for each of the
the provided unknown sample
standards and the sample

D. Spotting the Plate Disposal

Measure 0.5 cm from the end of the plate
and lightly mark it with a pencil Excess standards
Throw in the sink
and samples

Using a micropipet, introduce 10µL for Discard in the Non-

each of the standards and sample Excess mobile phase halogenated waste
bottle
E. Developing the TLC plates

Using a 150-mL beaker of a screw

capped-jar, add enough of the mobile
phase, making sure that the solvent system
will not touch the spots

Place a filter paper inside the chamber.

Make sure that the chamber is saturated with
the mobile phase before placing the TLC plate

After the solvent has risen near the top of the

TLC plate, take the plate out.

Mark the solvent front with a pencil and allow

the solvent to dry.
 Experiment 6
Hydrocarbons
A. Preparation of Hydrocarbons
Methane
Equip a 250-mL distilling flask with a tightly
fitted one-holed cork or rubber stopper.
Fit a delivery tube (glass tubing) into the cork
stopper so that the evolved gas can be col-
lected by water displacement.
Add 36 grams of zinc dust
Add 60 mL of 95% ethyl alcohol
Add 36 mL of dichloromethane (fumehood?)
Add 12 mL cupric sulfate TS
Add 60 mL of 95% ethyl alcohol
Add 36 mL of dichloromethane (fumehood?)
Add 12 mL cupric sulfate TS and shake
Insert the stopper with the delivery tube
If reaction is fast, cool by immersing in cold water
Let the air be displaced from the apparatus
Immediately collect methane gas by water
displacement.
Disposal: Solids to a paper tare, and the
thrown into a trash bin. Dispose liquids to
the halogenated waste container.
Ethylene
Put 60 mL ethyl alcohol in the fusion tube.
Gradually add 150 mL concentrated sulfuric acid
Heat the mixture in the flask to 165-170°C
Regulate the heat to have
a steady flow of gas
After air is expelled, collect the
gas in the test tubes
Keep the test tubes lightly stoppered.
Disposal: Neutralize contents of the distil-
ling flask with sodium carbonate. Flush
the resulting mixture into the sink with
plenty of water.
Acetylene
Fill the separatory funnel with 150 mL water
Slowly add water to the flask
containing calcium carbide
Collect the gas evolved in 4 test tubes.
Add sufficient water to decompose the
remaining calcium carbide.
Disposal: Decant the aqueous layer into
the sink and dump the solid residue in the
waste bin
 Experiment 5
Hydrocarbons
A. Preparation of Hydrocarbons
Methane
Equip a 250-mL distilling flask with a tightly
fitted one-holed cork or rubber stopper.
Fit a delivery tube (glass tubing) into the cork
stopper so that the evolved gas can be col-
lected by water displacement.
Add 36 grams of zinc dust
Add 60 mL of 95% ethyl alcohol
Add 36 mL of dichloromethane (fumehood?)
Add 12 mL cupric sulfate TS
Add 60 mL of 95% ethyl alcohol
Add 36 mL of dichloromethane (fumehood?)
Add 12 mL cupric sulfate TS and shake
Insert the stopper with the delivery tube
If reaction is fast, cool by immersing in cold water
Let the air be displaced from the apparatus
Immediately collect methane gas by water
displacement.
Disposal: Solids to a paper tare, and the
thrown into a trash bin. Dispose liquids to
the halogenated waste container.
Ethylene
Put 60 mL ethyl alcohol in the fusion tube.
Gradually add 150 mL concentrated sulfuric acid
Heat the mixture in the flask to 165-170°C
Regulate the heat to have
a steady flow of gas
After air is expelled, collect the
gas in the test tubes
Keep the test tubes lightly stoppered.
Disposal: Neutralize contents of the distil-
ling flask with sodium carbonate. Flush
the resulting mixture into the sink with
plenty of water.
Acetylene
Fill the separatory funnel with 150 mL water
Slowly add water to the flask
containing calcium carbide
Collect the gas evolved in 4 test tubes.
Add sufficient water to decompose the
remaining calcium carbide.
Disposal: Decant the aqueous layer into
the sink and dump the solid residue in the
waste bin
 Experiment 5
Hydrocarbons

A. Properties of Hydrocarbons
3. Ignition test
Take note sa itetest na compound. Hold the test tube with the sample
downward, at arms length

1. Baeyer’s test
Shake 1 mL potassium permanganate con- Bring a flame to the mouth of the test tube
taining the test compounds
Observe the color of the flame etc.
Disposal: Discard brown precipitate to
trash can. NOT TO SINK
For liquid samples, put a drop or
2. Bromine in dichloromethane two on an evaporating dish
Measure 1 mL of the test compound

In a well ventilated area, heat gently at first

Add 2 mL of 5% bromine in dichloromethane. and then dull to redness.

Immediately add blue litmus paper. For naphthalene, ignite in solid form.

Record all your observations Observe the nature of the flame.

Disposal: Pour the reaction mixture into

the container for halogenated wastes.
 Experiment 7
Alkyl Halides

A. Beilstein’s Test Series 2: Ethanolic Silver Nitrate

To each test tube, add 2mL of 1% ethanolic

Heat a small loop of a copper wire until the AgNO3 solution.
flame is no longer green

Note the time of addition and note for the first

Cool the wire and dip in compound turbidity and for a definite precipitate.

Disposal
Heat on the edge of the flame

Add saturated NaCl solution

B. Reactivity of Halogen-Containing
Organic Compounds
Filter out precipitate and discard to the
Prepare two series of 4 test tubes. “metal residues” waste bottle.

Put 3 to 4 drops to each series of (1) benzyl Supernatant is disposed to sink and
chloride, (2) chlorobenzene, (3) 0.1M chloro- flushed with plenty of water.
acetic acid, and (4) 0.1 M trichloroacetic acid.

Series 1: Sodium Iodide in Acetone

To each test tube, add 2mL of sodium iodide

in acetone reagent.

Note the time of addition and observe

After 5 minutes, place tubes without precipitates

in a 50°C water bath.

Disposal: Dilute with 10-15mL water then

discard to sink
 Experiment 7
Alkyl Halides

C.1 Preparation of Alkyl Halides

Place 20.0g of NaBr in a Wash with 15 mL saturated

250-mL round bottom flask. sodium chloride solution. ??

Add 15 mL of water and 15 ml of 1-butanol Collect the organic layer and discard
the aqueous layer

Mix thoroughly and cool the flask

in an ice bath Wash the collected organic layer with
saturated sodium bicarbonate solution

SLOWLY add 15 mL of concentrated

sulfuric acid to the solution Add 15 mL saturated
sodium chloride solution

Remove the flask from the ice batch, add a

couple of boiling chips Collect the organic layer and discard
the aqueous layer

attach a reflux condenser

Transfer the cloudy I-bromobutane layer to a
small Erlenmeyer flask
Heat the flask in a heating mantle until most
of the salts have dissolved and the solution is dry it with anhydrous magnesium sulfate.
at a gentle reflux.

After 10 mins, filter the mixture and subject to

Note the appearance of two layers confirmatory test

Continue the reflux for 45 mins.

Cool the heterogenous mixture to

room temperature

transfer to a 250-mL separatory funnel

Add 25 mL of water and invert the funnel

gently with venting for several times

Collect the lower layer which contains

1-bromobutane
 Experiment 7
Alkyl Halides

C.2 Confrimatory test: Silver Nitrate test

Add 1 drop of the alkyl halide to 2 mL of a

solution of 0.1 M solution of silver nitrate in
95% ethanol in small test tube.

Stopper, shake and note any change (and

the time required

Observe for 5mns at room temp

If nothing happens, warm the mixture in a

steam bath and observe any change.
 Experiment 8
Alcohols, Phenols, and Ethers

A. Elemental Test for Oxygen A. Reaction for Alcohols

1. Ferrox test 1. Lucas test

Grab a stirring rod, and a dry test tube To a test tube, add 3 to 4 drops of the sample.

Grind together, with a glass stirring rod, a Add 2 mL of the Lucas reagent at 26°-27°C
crystal of ferric ammonium sulfate and a
crystal of potassium thiocyanate.
Stopper the tube and shake
The iron hexathiocyanatoferrate that is
formed sticks to the stirring rod. then allow the mixture to stand

In another test tube, dissolve 30 mg or 3

drops of the unknown in a minimal Note the time required for the formation of
amount of toluene. the alkyl chloride, which appears as an
insoluble layer or emulsion.

Use the stirring rod with the iron hexathiocya- Disposal: Add sodium carbonate to the
natoferrate solid to stir the unknown. test solution until foaming no longer oc-
curs and discard to sink.
If the solid dissolves and a reddish-purple
color develops, the compound contains
oxygen.

Place the mixture in the

NONHALOGENATED waste container.

Just in case kelangang gumawa ng reagent oki

 Experiment 8
Alcohols, Phenols, and Ethers

2. Esterification test A. Reaction for Phenols

1. Esterification
In a test tube, add 1mL Ethyl alcohol
In a test tube, place a pinch of phenol

Add 10 drops of acetyl chloride

Add 2mL of 10% KOH with crushed ice

Add 5 drops of concentrated sulfuric acid

Stopper the tube and shake

If the odor of acetic acid masks the odor of

the ester, neutralize with dilute NaOH sol’n Gradually add 1mL acetyl chloride
Acetic acid has a distinctive sour taste and pungent smell

Shake for a few minutes

Separate the two layers. ESTER LAYER to
Non-Halogenated waste bottle. AQUEOUS
LAYER diluted then discard to sink
3. Chromic Acid test
Disposal: Discard ester layer to the Non
Halogenated waste bottle. Neutralize
aqueous layer with 10% HCl, dilute with
water then dispose to sink.

Put 1mL of sample in a test tube

2. Ferric Chloride

Note: TRANSIENT REACTION

Add 5 drops of potassium dichromate
Place 1mL of the sample in a test tube
Add 5 drops sulfuric acid, then warm
Add 4 to 5 drops of 5% aqueous FeCl3
Note the color and odor of the solution

Discard all contents to the

*note the ease by which the alcohol is oxidized NonHalogenated waste bottle

Disposal: If Blue or Green, flush to the

sink with lots of water. If not, treat with Fe (III) forms complexes with phenols
ethanol until it turns blue or green. Warm
if necessary.

http://intranet.tdmu.edu.ua/data/kafedra/internal/zag_him/
metod_rozrobky/en/pharm/prov_pharm/ptn/organic%20chemistry/2%
20course/11.%20Phenols.%20Ethers.%20Small%20practicum.htm

Just in case for Chromic Acid test

 Experiment 8
Alcohols, Phenols, and Ethers

3. Bromine Water 4. Phenolphthalein formation

In a test tube, add 1mL of the sample In a test tube place a pinch of pthalic anhydride

Add bromine water Add a crystal of phenol

Add 5 drops of concentrated sulfuric acid Add 2 drops concentrated sulfuric acid

Immediately stopper. Heat gently over a small flame for 2 miinutes

Let sit for 15 minutes Shake the tube constantly

Discard to the Halogenated waste bottle Heat until the melt becomes dark red

Avoid heating strongly. Iitim yung material.

Cool. Then treat with 5mL water

Add 2 drops to a test tube with dilute HCl

Add 2 drops to a test tube with dilute NaOH

Paghaluin lahat, dilute with water, then

discard to sink
 1
Experiment 9
Aldehydes and Ketones

A. General tests for Aldehydes and 3. Tollen’s Test

Ketones
Dapat nahugasan at na-airdry yung test tube
1. Schiff’s Test

Place a drop of Place 0.5 mL of 1% silver nitrate

DO NOT LAGAY EXCESS AMMONIA Add 1 drop of 10% sodium hydroxide solution
Shriner, p. 284
observe within 3-4 mns. Upon formation of black precipitate, add 2%
ammonia solution, drop by drop
Neutralize with sodium carbonate. Dilute
with water. Dispose to sink
Shake constantly until precipitate dissolves
2. Phenylhydrazone Formation

Add 2 mL 95% ethyl alcohol to a test tube DO NOT LAGAY EXCESS AMMONIA

Add 2 to 3 drops of sample Add 1 drop of the test compound.

Add 3 mL of 2,4-dinitrophenylhydrazine Mix well and warm in water bath.

Shake well Disposal

Note the color of precipitate. (or wait 15 mns) Transfer test tube contents to beaker.

Throw to PHENYLHYDRAZONE WASTE

bottle Wash test tube with a few drops dilute HNO3

Pour washings to beaker

aldehyde Schiff’s reagent Add dilute HNO3

(colorless)

Add 5 mL of saturated NaCl

(violet-purple solution)

Discard AgCl precipitate to “metals resi-

due” waste bottle

aldehyde 2,4-dinitrophenylhydrazone
or ketone 2,4-dinitrophenylhydrazine
Discard the remaining liquid to sink
 2
Experiment 9
Aldehydes and Ketones

6. Sodium Bisulfite Addition Product

Add 0.5 mL of alcoholic sodium bisulfite

aldehyde Tollen’s reagent
Salt of the
Carboxylic Acid

Add 0.5 mL of the sample

aldehyde (blue) carboxylic

acid
(red, yellow, or
yellowish-green)
Stopper the test tube

4. Fehling’s test Shake vigoriously

In a test tube, add 1 mL Fehling’s A
Filter off the solid. Dissolve the solid in
water, then dispose to sink. Discard the
Add 1 mL Fehling’s B filtrate to the NONHALOGENATED waste
container

Add 1 drop of sample

Warm in a water bath aldehyde sodium bisulfite

or ketone sodium bisulfite
addition complex
Discard Supernatant liquid to
NONHALOGENATED waste bottle.
Discard precipitate to sink

5. Benedict’s Test
B. Test for Methyl Ketones
Add 0.5 mL of the test compound
1. Nitroprusside Test

Place 5 drops of acetone

Add 5 mL of Benedict’s reagent

Add 1 mL of water
Warm if necessary

Discard Supernatant liquid to Add a drop of sodium nitroprusside TS

NONHALOGENATED waste bottle.
Discard precipitate to sink
Add a drop of 3% NaOH and observe

Add a few drops 96% acetic acid and observe

aldehyde
Alkalinify with 10% NaOH and throw the
(blue) carboxylic acid (red, yellow, or
yellowish-green) contents to the CYANIDE waste bottle
 3
Experiment 9
Aldehydes and Ketones

2. Iodoform C. Autooxidation
On seprate 4 separate test tubes Add 1 drop benzaldehyde to watch glass

2 drops acetone, methanol, ethanol, Expose to atmosphere

isopropyl alcohol

Add 8 drops 10% NaOH Dissolve solid in 5% NaOH. Dilute with

water the dispose to sink

Add, drop-by-drop I2-KI solution until liquid

remains yellow

Warm test tube to 60°C

Add a few drops of acetone to destroy I2-

KI. Remove the iodoform by suction
filtration. Place in the HALOGENATED
waste container. Discard filtrate to sink.
 4
Experiment 9
Aldehydes and Ketones

P. 283

P. 312

P. 311
 5
Experiment 9
Aldehydes and Ketones

Fehling’s

iodoform
 Experiment 10
Experiment 10
Carbohydrates
Carbohydrates
Tests for Polysaccharides
Tests for Polysaccharides Iodine test [Starch]

Acid Hydrolysis 1% Starch

Sa dalawang mahabang test tube
Sa mahabang test tube
Run a blank test using distilled water
Add 10 mL 1% starch solution

Add tig-3 mL of starch

Add 2 mL 20% sulfuric acid

Sa isa lang, add several drops 40% NaOH

Boil for 10 minutes

Add very dilute iodine solution to each tube

Maintain the volume of liquid by adding water

Note the color.

Run a blank test using distilled water

Heat the tube na nagkakulay

Record your results

Dispose all contents to sink and flush Cool and note any change
with water

Compare with the blank tube

Experiment 10
[Glycogen, Cellulose, Gum Arabic, Agar]
Carbohydrates
Add 5 mL of 1% sample solution
General Color Reactions
Molisch test [Sucrose, starch, blank]
Add one drop of iodine solution
Add 0.5mL of sample solution

Observe and record results

Add 2 mL water
Dispose all contents to sink and flush
with water
Add 2 drops Molisch reagent

Mix well

Incline each tube and slowly add 3 mL con-

centrated sulfuric acid on the side of the tube
to form a layer below the sugar solution.

Treat with technical grade sodium

carbonate prior to disposal to sink
 Experiment 10 Experiment 10
Carbohydrates Carbohydrates
General Color Reactions Other Tests for Carbohydrates
Anthrone test [Glucose, Sucrose, Starch, Blank]
Phenyhydrazine test (Osazone formation)
Add one drop sample solution. [glucose, fructose, galactose, arabinose,
maltose, lactose, and sucrose]

Add 1 mL sample solution

Add 0.5 mL water

Label properly
Add 1 mL Anthrone reagent

Add 1 mL of phenylhydrazine eagent

Mix thoroughly by swirling

Mix the contents well

Heat mixture in boiling water bath or 3 minutes

Stopper with cotton loosely

Heat the tube na nagkakulay

Put all the tubes at the same time in a large

Cool and observe the color formed in each tube beaker of boiling water

If the solution is not clear, Shake the tube occasionally to avoid supersaturation
add more sample solution

Remove the tubes after 30 minutes and cool

Treat with technical grade sodium
carbonate prior to disposal to sink
Note the sugars that form osazones only after cooling
Experiment 10
Carbohydrates Refrigerate the tubes if the crystals will be
viewed at a later time
Tests for Polysaccharides
Tollen's Phloroglucinol Test
[galactose, xylose, blank]
Examine the crystals under low power microscope
Add 1 mL sample solution

add 1 ml of concentrated hydrochloric acid The crystals may be washed several times
with distilled water before examination
Add 2-3 drops of Tollen’s phloroglucinol reagent
Better crystals are obtained when allowed to
Maintain the volume of liquid by adding water cool in a water bath

Heat in a boiling water bath Draw the crystals as viewed under the microscope.

Dispose all the test tube contents in the

sink and flush with water. Dispose all contents to sink and flush
with water
 Experiment 10 Experiment 10
Carbohydrates Carbohydrates
Other Tests for Carbohydrates
Benedict’s test Other Tests for Carbohydrates
[glucose, sucrose, starch, blank]
Barfoed’s test [Glucose, Sucrose, Maltose, Blank]

Add 1 mL Benedict’s solution Add 1 mL of Barfoed's reagent

add exactly 2 drops of the sugar so!ution add 2 drops of the sugar solution
To compare the reactions of mono-
saccharides and disaccharides:

Mix well place the tubes at the same time in a boiling

water bath
place in boiling water bath for 3 minutes
Note the time when signs of
reduction appear in each tube
Allow the solution to cool spontaneously
If no precipitate was formed

continue boiling for 15 minutes and remove

Do not hasten cooling by immersion in cold water
If the sample tested positive, separate the
precipitate formed and dispose it in the
“solid waste" container. The filtrate may
be disposed into the sink. If the sample
tested negative, dispose the contents in
the sink.
Experiment 10
Carbohydrates
the tubes from the bath
Note the amounts of precipitate in each tube
after standing for 15 minutes
If the sample tested positive, separate the
precipitate formed and dispose it in the
“solid waste" container. The filtrate may
be disposed into the sink. If the sample
tested negative, dispose the contents in
the sink.

Tests for Polysaccharides

Bial’s Test for Pentoses [xylose, blank]

Add 3 mL of Bial's reagent

add 2 ml of 1% xylose solution

Heat until it just begins to boil

Maintain the volume of liquid by adding water

Record your results

Treat with technical grade sodium

carbonate prior to disposal to sink.
 Experiment 10 Experiment 10
Carbohydrates Carbohydrates
Other Tests for Carbohydrates
Seliwanoff’s test
Other Tests for Carbohydrates
[glucose, fructose, blank]
Barfoed’s test [Glucose, Sucrose, Maltose, Blank]

Add 5 ml of Seliwanoff's reagent Add 1 mL of Barfoed's reagent

add exactly 2 drops of the sugar solution add 2 drops of the sugar solution
To compare the reactions of mono-
place 1 ml of the sugar solution saccharides and disaccharides:

place the tubes at the same time in a boiling

Place them in a boiling water bath water bath
for exactly 1 minute
Note the time when signs of
Note the results reduction appear in each tube
If no precipitate was formed

Continue heating continue boiling for 15 minutes and remove

the tubes from the bath
Note the results
Note the amounts of precipitate in each tube
observe the color change at one-minute intervals after standing for 15 minutes
for 4 minutes
If the sample tested positive, separate the
If the sample tested positive, separate the precipitate formed and dispose it in the
precipitate formed and dispose it in the “solid waste" container. The filtrate may
“solid waste" container. The filtrate may be disposed into the sink. If the sample
be disposed into the sink. If the sample tested negative, dispose the contents in
tested negative, dispose the contents in the sink.
the sink.
Note and draw the appearance of any crystal
[galactose]

Evaporate to approximately 5 ml on the

carbonate prior to disposal to sink.

Treat with technical grade sodium
Add 5 ml concentrated nitric acid

steam bath UNDER THE HOOD

Do not evaporate to dryness.

formed under the microscope

Add 10 mL of 10% galactose
Tests for Polysaccharides
Experiment 10
Carbohydrates

Mucic Acid Test

sa beaker
 Experiment 10
Carbohydrates
Other Tests for Carbohydrates
Fermentation test: ethanol
[glucose]
Get a Einhorn Saccharometer
Introduce 10 ml of 20% Baker's yeast suspension
Add 4 ml of 10% glucose
Invert the tube several times until well mixed
Fill the closed end of the tube completely and
make sure that there are no air bubbles
The liquid must not fill the open arm (mouth) to
give room for water displacement
Stopper loosely with cotton
Observe the volume of the gas
Evolved at the closed end of the tub
If the sample tested positive, separate the
precipitate formed and dispose it in the
“solid waste" container. The filtrate may
be disposed into the sink. If the sample
tested negative, dispose the contents in
the sink.
Experiment 10
Carbohydrates
Other Tests for Carbohydrates
Barfoed’s test [Glucose, Sucrose, Maltose, Blank]
Add 1 mL of Barfoed's reagent
add 2 drops of the sugar solution
To compare the reactions of mono-
saccharides and disaccharides:
place the tubes at the same time in a boiling
water bath
Note the time when signs of
reduction appear in each tube
If no precipitate was formed
continue boiling for 15 minutes and remove
the tubes from the bath
Note the amounts of precipitate in each tube
after standing for 15 minutes
If the sample tested positive, separate the
precipitate formed and dispose it in the
“solid waste" container. The filtrate may
be disposed into the sink. If the sample
tested negative, dispose the contents in
the sink.
 Experiment 10
Carbohydrates
Other Tests for Carbohydrates
Fermentation test: ethanol
[glucose]
Get a Einhorn Saccharometer
Introduce 10 ml of 20% Baker's yeast suspension
Add 4 ml of 10% glucose
Invert the tube several times until well mixed
Fill the closed end of the tube completely and
make sure that there are no air bubbles
The liquid must not fill the open arm (mouth) to
give room for water displacement
Stopper loosely with cotton
Observe the volume of the gas
Evolved at the closed end of the tub
If the sample tested positive, separate the
precipitate formed and dispose it in the
“solid waste" container. The filtrate may
be disposed into the sink. If the sample
tested negative, dispose the contents in
the sink.
Experiment 10
Carbohydrates
Other Tests for Carbohydrates
Barfoed’s test [Glucose, Sucrose, Maltose, Blank]
Add 1 mL of Barfoed's reagent
add 2 drops of the sugar solution
To compare the reactions of mono-
saccharides and disaccharides:
place the tubes at the same time in a boiling
water bath
Note the time when signs of
reduction appear in each tube
If no precipitate was formed
continue boiling for 15 minutes and remove
the tubes from the bath
Note the amounts of precipitate in each tube
after standing for 15 minutes
If the sample tested positive, separate the
precipitate formed and dispose it in the
“solid waste" container. The filtrate may
be disposed into the sink. If the sample
tested negative, dispose the contents in
the sink.
 Experiment 11 Experiment 11
Carboxylic acids and derivatives Carboxylic acids and derivatives
Reactions of Monocarboxylic Acids Reactions of Monocarboxylic Acids
Ethanolic Silver Nitrate Test Ferric Chloride Test A

Add 1 mL of the dilute acetic acid Add 0.2 mL of 10% NaOH

add 1% ethanolic silver nitrate solution

add dilute acetic acid dropwise until the
solution is slightly acid to litmus
Observe for 5 mns in room temperature
Add a few drops of 5% ferric chloride
If no reaction occurs, heat to boiling
Observe the color of the solution
Watch for precipitation
Divide the solution into 3 equal parts
Add 2 drops dilute nitric acid

To the
Note the solubility of the precipitate. Heat one second por- Observe and
portion to tion, add a compare
boiling and few drops of with the third
Repeat the test using 1% benzoic acid note any pre- dilute HCI portion of the
cipitation. until the color solution
(1) changes. (the original
Add enough nitric acid to completely (2) portion)
dissolve the precipitate and discard the
contents of the test tube in the "metal
residue" waste bottle Combine contents into a beaker.
Neutralize with NaHCO3. Dispose to sink.
 Experiment 11
Carboxylic acids and derivatives
Reactions of Monocarboxylic Acids
NaHCO3 test
Add 1 mL of the dilute acetic acid
Experiment 11
Carboxylic acids and derivatives
Reactivity of Substituted Carboxylic
Acids (Limewater Test)
Add 1 mL of 0.1 M acetic acid to a test tube

Repeat the test using 1% benzoic

Heat slowly using the set-up
acid

Repeat using 0.1 M chloroacetic acid

Discard the contents of the test tube to
the sink.
Repeat using 0.1 M trichloroacetic acid

Treat the contents of the test tube

Experiment 11
Carboxylic acids and derivatives
Reactions of Monocarboxylic Acids
Ethanolic Silver Nitrate Test B
Add 1 mL of 1% benzoic acid
containing the test compound with 10%
Na2C03 until effervescence ceases; then
dilute with water prior to disposal to the
sink.
Dissolve the precipitate formed in the test
tube with limewater with dilute HCI; then
dispose the contents to sink.

add 10% ammonium hydroxide dropwise until

the solution is slightly basic to litmus

Boil off excess ammonia

add 2 drops of 5% ferric chloride

Note the color of the precipitate

add dilute HCI

note the solubility of the precipitate

Combine contents into a beaker.

Neutralize with NaHCO3. Dispose to sink.
 Experiment 11 Experiment 11
Carboxylic acids and derivatives Carboxylic acids and derivatives
Reactions of Acid Derivatives Reactions of Acid Derivatives
Hydroxamic Acid Test Esterification A

Get a mixture of 1 drop or a pinch of the test

Add 1 mL of ethyl alcohol
compound

add 10 drops of acetic anhydride

Heat to boiling

Add 1 mL of 0.5 M hydroxylamine Add 5 drops of concentrated sulfuric acid

hydrochloride in 95% ethanol
Warm
Add 0.2 mL of 6 M NaOH
if the odor of acetic acid masks the odor of
Slightly cool ester, neutralize the mixture with NaOH
solution

Note the solubility of the precipitate.

Separate the ester layer and discard In the
"NONHALOGENATED WASTE" bottle.
cautiously add 2 mL of 1 M HCI Dilute the aqueous layer With water and
discard to the Sink

Observe for cloudiness

If cloudy, add 2 mL of 95% ethanol Experiment 11

Carboxylic acids and derivatives
Add a drop of 5% ferric chloride solution Reactions of Acid Derivatives
Esterification B
If the color by the drop of ferric chloride
On a watch glass, add a pinch of benzoic acid
solution does not persist, continue to add fer-
ric chloride dropwise until the observed color
permeates the entire solution.
add 5 drops of methyl alcohol

observe Add 5 drops of concentrated sulfuric acid.

Perform this test on acetyl chloride, Take note of the odor

acetic anhydride. and ethyl acetate

Neutralize the contents with dilute NaOH

Neutralize the reaction mixture With 10% Discard the ester layer in the
NazC.03 until foaming ceases and dispose 'NONHACOGENATED WASTE" bottle and
into the sink the aqueous layer into the sink.
 Experiment 11
Carboxylic acids and derivatives
Reactions of Acid Derivatives
Schotten-Baumann Reaction

In a test tube, place 0.5 mL of ethanol

Add 1mL water

Add 10 drops of acetic anhydride

Add in proportions, with vigorous saking, 2

mL of 25% NaOH solution

Stopper the flask

shake the mixture for several minutes

test the solution with litmus paper to make

sure that it is still alkaline

Note the odor of the mixture and any layer

separation

Separate the water-immisciblel ayer and

dispose it into the "NONHALOGENATED
WASTE" bottle. Neutralize the aqueous
layer with dilute HCI prior to disposal to
the sink.
 Experiment 11 Experiment 11
Carboxylic acids and derivatives Carboxylic acids and derivatives

Reactions of Acid Derivatives Reactions of Acid Derivatives

Amide Hydrolysis Basic Hydrolysis of Esters

Add a pinch of benzamide in a test tube Transfer 4 mL of ethyl acetate to the flask.

add 5 mL of 10% NaOH add 5 mL of 25% NaOH

heat Observe the formation of two layers

Note the odor Reflux for 15 minutes. (reflux: allow to flow back)

Neutralize the contents with dilute HCI, Observe the results.

dilute with water prior to disposal to the
sink.
Neutralize the solution with dilute HCI

Warm the test tube

note the odor of the solution

Dilute the contents with water prior to

disposal to the sink. Dispose any water
immiscible layer into the
"NONHALOGENATED WASTE" container
 Dissolve about 17.5g of AgNO̬3 in
1000 mL and standardize

Dry enough sodium chloride at 110°

for two hours, such that at least
100 mg will remain

Transfer 100 mg dried NaCl to

150-mL beaker

Dissolve in 5 mL distilled water

Add 5 mL acetic acid

Add 50 mL methanol

Add 0.5 mL eosin

Stir

Titrate
 3
BALITON, Rebecca Aira Feb. 4, 2019

0.1 N Silver Nitrate

1000 mL

Dissolve about 17.5 g of AgNO3 in 1000 mL of water. (USPCI, 2012)

x g AgNO3
0.1 N AgNO3 = = 16.987 g AgNO3 (USPCI, 2012)
(169.87)(1)

Non-metallic container, protected from light. (British Pharmacopoeia, 2012)

The United States Pharmacopeia: USP 35: The National Formulary: NF30: By authority of the
United States Pharmacopeial Convention. (2012). Rockville, MD: United States Pharmaco-
peial Convention.
British pharmacopoeia 2012 (Vol. 2). (2012). London: Stationery Office.