_____________________________________________

ORGANIC CHEMISTRY LABORATORY

Name: Date: ____________________

Year & section: Instructor: Melvin Reyes,RPh.

ACTIVITY NO. 3: FUNCTIONAL GROUP ANALYSIS

Objectives:
• At the end of the activity the student must be able to:

1. Define the different test for functional group

NOTE:

Organic Chemistry has a series of reactions which are characteristic of functional

groups, and therefore it is possible to characterize almost any functional groups
presents in the molecules. Sometimes, various functional groups can give the same
reaction, so that it will require some other characteristic reaction to be sure of their
nature, or some polyfunctional compounds may even give different reactions to each
function separately.

I. Write the procedures of the following test and identify what functional group
correspond to each, give the color of the positive result of each test.

1. BROMINE IN CCL4 TEST (ALKENE)

positive result: BROWNISH / BROWN

Dissolve 2 drops (approximately 50 mg) of the

product in 1 ml of CCl4, and add dropwise a solution
of bromine in CCl4 (2%) until the bromine color
persists. If more than 2 drops must be added so that
the color (brownish) persists, the test is considered
positive.

2. BAEYER TEST (BAEYER PERMANGANATE) (ALKENE)

positive result: VIOLET

Dissolve a drop (25 mg) of the compound in 2 ml of

water or acetone in a test tube. Dropwise, with
vigorous stirring, add an aqueous solution of 1%
potassium permanganate. The result is positive if it is
necessary to add additional drops of permanganate
solution to maintain the violet color. If positive, the
initially violet solution turns brown
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

3. ALKALINE MERCURIC IODIDE TEST ( ALKYNE )

positive result: WHITE OR OFF-WHITE PRECIPITATE

Prepare the reagent dissolving 6.6 g of mercuric chloride

in a solution of 16.3 g of potassium iodide in 16.3 ml of
water, and then add 125 ml of 10% NaOH. Cool the
mixtures and dissolve the alkyne to 20-fold its volume
of Ethanol. Add this solution dropwise to 2 ml of the
above reagent. The appearance of a white or off-white
precipitate is indicative of terminal alkyne. Stir for 2 to
3 min, filter out and wash with 50% ethanol to get a
mercuric derivative, and handle very carefully when dry
because many of these derivatives are explosive when
dry.

4. NITRATION TEST

Add 500 mg of product to about 3 ml of concentrated H2

SO4 and then an equal volume of concentrated HNO3
dropwise, stirring the mixture after each addition. The
reaction is heated 5–10 min in a water bath at 60 ◦C.
Remove the tube from the bath and shake frequently. Cool
it and pour over ice water. Filter the precipitate under
vacuum, and wash it with water. The product can be
purified by recrystallization from aqueous ethanol.

5. AROYLBENZOIC ACID TEST

In a flask with reflux condenser, place 400 mg of phthalic

anhydride, 10 ml of carbon disulfide, 800 mg of
anhydrous aluminum chloride, and 400 mg of the
aromatic hydrocarbon. Heat the mixture for 30 min in a
water bath, and then cool it with a water bath. Slowly
decant the carbon disulfide layer. Add cooling 5 ml of
concentrated HCl and then 5 ml of water. Shake the
mixture vigorously until solid. Collect the solid by
filtration and wash it with cold water. If oil forms, if not
crystallized, extract it with diluted ammonium hydroxide
and then neutralize with HCl (conc.).
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

6. SILVER NITRATE TEST

positive result: SILVER MIRROR

Add a drop of halogenated compound to 2 ml of 2%

ethanolic silver nitrate. If after 5 min no reaction is
observed, heat the solution for several minutes until
boiling; note the color of the precipitate and add 2
drops of 5% HNO3 solution.

7. SODIUM IODIDE IN ACETONE TEST

Two drops (or 100 mg) of the product are dissolved
in a small amount of acetone for 1 to 2 ml of reagent
(15 g of sodium iodide in 100 ml of acetone) by
letting the solution stand for 3 or 4 min at r.t. If any
change occurs, heat the test tube to 50 ◦C for 6 min.
Cool to r.t. and record the observed changes.

8. ALKYL HALIDE CHARACTERIZATION:

PICRATES OF “S”-ALKYLTHIOURONIUM

Alkyl halide (1 mmol) and thiourea (150 mg) in 5 ml

of ethylene glycol are dissolved in a flask equipped
with a condenser. The mixture is heated for 30 min.
Primary alkyl iodides require heating to a temperature
of approximately 65 ◦C, while the other halides
require temperatures about 120 ◦C. Then add 1 ml of
saturated picric acid in Ethanol and heat for 15 min.
Cool the reaction mixture and add 5 ml of cold water.
Let stand in an ice bath and filter the precipitate under
vacuum.

9. LUCAS TEST (ZNCL2 /HCL (CONC.)

positive result:
Primary – clear
Secondary – cloudy
Tertiary – brownish
The reagent is prepared by dissolving 16 g of
anhydrous zinc chloride in 10 ml of concentrated
HCl, cooling the mixture to avoid loss of HCl. Seal
the flask reagent, because the ZnCl2 is strongly
hygroscopic and loses effectiveness. Add 3 or 4
drops of alcohol to 2 ml of Lucas’ reagent in a
small test tube. Close the tube and shake
vigorously for 15 s, and let stand at r.t
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

10. OXIDATION TEST (K2CR2O7 /H2 SO4)

positive result: SILVER MIRROR

The reagent is prepared by dissolving 16 g of

anhydrous zinc chloride in 10 ml of concentrated
HCl, cooling the mixture to avoid loss of HCl. Seal
the flask reagent, because the ZnCl2 is strongly
hygroscopic and loses effectiveness. Add 3 or 4
drops of alcohol to 2 ml of Lucas’ reagent in a
small test tube. Close the tube and shake
vigorously for 15 s, and let stand at r.t

11. (SCHOTTEN-BAUMANN REACTION)

positive result: FRUITY ODOR

After placing 0.5 ml of alcohol in a dry test tube,

carefully add dropwise 0.3 ml of acetyl chloride,
observing the reaction that occurs by heat release.
Pour the solution onto 10 ml of ice/water and smell
the sample (esters from simple alcohols typically
have fruity odors).

12. BORAX TEST

positive result: PINK COLOR
Prepare 100 ml of borax (sodium tetraborate) 10%
with enough phenolphthalein to produce a pink
color. Add one or two drops of glycol to 0.5 ml of
this reagent, observing the disappearance of the
color. The color reappears on heating and dims as
the solution cools.
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

13. PERIODIC ACID OXIDATION (H5 IO6)

positive result: WHITE PRECIPITATE

In a test tube containing 2 ml of reagent, a add 1

drop of HNO3 (conc.) and shake vigorously. Add
a drop of compound to test and stir for about 15 s.
Add 1 or 2 drops of AgNO3 solution 3%. The
instantaneous formation of a white precipitate of
silver iodate means a positive test result.

14. BROMINE WATER TEST

positive result: WHITE PRECIPITATE

Add to an aqueous 1% solution of phenol a

saturated solution of bromine water (5 ml of
bromine per 100 ml water) dropwise until the
bromine color per sists. A positive result is given
by the appearance of a precipitate caused by the
formation of bromophenol and strongly acidic
reaction mixture.
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

Name: Date:____________________

Year & section: __________________________Instructor: Melvin Reyes,RPh.

ACTIVITY NO. 5: CHROMATOGRAPHIC TECHNIQUES

Objectives:
• At the end of the activity the student must be able to:

1. Define chromatography
2. Enumerate and describe the different types of chromatography

NOTE:

Chromatography is a separation technique with extraordinarily

versatile variants. In all chromatographic separations, there are two
phases: a mobile phase and a stationary phase, which move relative to
one another while maintaining contact. The sample is introduced into
the mobile phase, and the sample components are distributed between
the stationary and mobile phases. The components of the mixture to
be separated spend different times in each phase, and therefore
separation occurs. If a component spends most of the time in the
mobile phase, the product moves quickly, whereas if it spends the
main time in the stationary phase, the product is retained and its
output is much slower

The principal objectives of obtainable through the use of

chromatography are:

1. Resolution of mixtures into constituent parts

2. Determination of homogeneity
3. Comparison of substances suspected of being identical
4. Purification
5. Concentration of substances from dilute solution
6. Identification and control of technical products
7. Qualitative separation from complex mixtures
8. Indication of molecular structure
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

COLUMN CHROMATOGRAPHY (CC)

A method frequently used for purifying substances and separating

mixtures at a preparative scale. Use a chromatography column as the main
device. The column is filled with an adsorbent that acts as a stationary phase,
usually silica gel or alumina. The sample is introduced into the column and
a solvent or solvent mixture (eluent) is passed to separate the impurities of
desired product or the components of a mixture, due to the different retention
time on the stationary phase to separate. The eluent can be run throughout
the column either by gravity or by exerting pressure with an inert gas or by
an air pump. This type of CC is called flash chromatography.

Simplest type of chromatographic column consists of a suction flask

and a cylindrical glass or quartz tube considered at one end.
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

GEIGER MULLER COUNTER-

determine the position of the
component that are radioactive.

ELUTION CHROMATOGRAPHY
- The column is washed with suitable solvent called Eluants until each
compound is successively obtained in the effluent fluid known as the
Eluate.

ADSORPTION CHROMATOGRAPHY
- Separation of mixtures through a competitive process in which the
molecules of the mobile phase compete with analyte molecules for
polar adsorption sites on the adsorbents.

- LIQUID SOLID CHROMATOGRAPHY

Mobile phase: LIQUID

PARTITION CHROMATOGRAPHY

- MOBILE AND STATIONARY is liquid

- LIQUID – LIQUID CHROMATOGRAPHY

PAPER PARTITION CHROMATOGRAPHY

- Differences in partition coefficients of substances between two

immiscible liquids.
- If the solid adsorbent is filter paper (CELLULOSE) the process is
referred PPC

Three main methods or procedures for the preparation of paper

partition paper chromatogram

1. DESCENDING CHROMATOGRAPHY:
- accomplished by allowing the mobile phase to flow downward on
the paper strip.
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

2. ASCENDING CHROMATOGRAPHY:

- Mobile phase is allowed to rise upward on the paper by capillary

attraction.

3. RADIAL CHROMATOGRAPHY:

- Mobile phase moves out in concentric circle from the center of a circle
piece of paper.

REVERSED PHASE CHROMATOGRAPHY

- Is also based on the partition phenomenon but as the name implies,

the on polar solvent is fixed to the paper or solid column and act as
the stationary phase and polar is used as the mobile phase.

MOLECULAR EXCLUSION CHROMATOGRAPHY

- AKA: GEL FILTRATION OR GEL PERMEATION

CHROMATOGRAPHY
- Separation procedure in which differential migration of solute
molecules is based on molecular size.

GAS CHROMATOGRAPHY

- Mobile phase an inert gas commonly called carrier gas.

HIGH PRESSURE LIQUID CHROMATOGRAPHY

The advantages of using HPLC as compared to classical

chromatographic methods are greater speed, precision, accuracy and
ease of operation.

THIN-LAYER CHROMATOGRAPHY (TLC)

This is one of the most popular solid-liquid chromatography variants,19

and its most common uses are:
• To identify the components of a mixture.
• To identify two substances.
• To follow the course of a reaction.
• To determine the appropriate conditions for the separation of mixtures
by Column chromatography
• To follow the development of a Column chromatography
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

a) Adsorption process

The sample is applied to the layer and is adsorbed on the material surface
by the action of electrostatic forces (van der Waals forces, hydrogen bonds,
inductive effects, etc.). Later, when the plate is exposed to a flow of solvent
by capillarity, the substance undergoes a competition between the active site
of the adsorbent and the solvent.

b) Adsorbents

The most common adsorbents in TLC are:

• Silica gel (SiO2 ) (used in 80% of separations).

• Alumina (Al2O3 ) (acid, neutral, or basic).

• Siliceous earth or Kieselguhr.

• Cellulose (native or microcrystalline).

• Polyamid

a) Chromatographic plates: These are used as the adsorbent-carrier sheets

of glass, plastic, or aluminum. The sizes (TLC foils) of conventional TLC plate
are 20×20, 10×20 and 5×2 (in cm). There are plates containing a fluorescent
indicator (F254 or F366).

b) Capillaries and Pasteur pipettes: The sample is applied dissolved onto

the plate whether the target is preparative or analytical. For preparative
purposes, the sample is dissolved and applied with a Pasteur pipette; this is
called band application. If the aim is to perform the technique for analytical
purposes, the sample dissolved is applied using a glass capillary. In this case,
it is called point or spot application. The sample should be applied at a
distance from the edge of the plate. The height and the precise location where
the sample is placed can be marked with a pencil to avoid damaging the
adsorbent
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

c). Choice of eluent: Choosing a solvent in which the components of the

mixture present an Rf average around 0.3–0.5 is recommended. The search
for a suitable eluent requires testing several solvents of different polarities or
mixtures

d) Elution tank (chromatography tanks): This is a closed container used

for the development of TLC plates. Its atmosphere is saturated with eluent
vapors. There are various types of tanks on the market (see Figure 4.34). Flat-
bottom tanks with different designs, dual compartment tanks (with
advantages the former of less solvent consumption and no waste, allowing
the environment to be saturated with different vapor-eluting solvent), etc.
Closed bottles and even a beaker with a watch glass is also used. The tank is
filled with the eluent to a height that does not reach the area where the
products are located. Usually, it is a good practice to place in the elution tank
a piece of filter paper that reaches the mouth of the tank so that the eluent
rises by capillarity, and the tank becomes saturated with eluent vapors. This
facilitates proper development of the plate. When the solvent front has
reached the upper edge of the plate, this tank is removed and then revealed

ION-EXCHANGE CHROMATOGRAPHY

Ion-exchange chromatography (or ion chromatography) allows the separation

of ions and molecules with a net charge. Ionic resins are used as stationary
phase. Depending on the load of the sample to separate or purify, two types
of anion and cation exchange are distinguished. Buffered aqueous solutions
are used as a mobile phase