Sohag university

Faculty of Science

Chemistry Department

EXPERIMENTAL ORGANIC
CHEMISTRY

For
B.Sc.Students

1
Part 1
Introduction 5
Volumetric analysis 5
Classification of volumetric method 6
Estimation of an aliphatic acid 14
Estimation of an aromatic acid 19
Estimation of purity of aniline hydrochloride 25
Estimation of an aliphatic amino acid 30
Estimation of hydroxyl groups in alcohols and phenols 36
Disussion of selected methods for the determination of hydroxyl
groups in alcohols
Disussion of selected methods for the determination of hydroxyl
groups in phenol
Estimation of amino groups in amines 47
Estimation of formaldhyde in sample of formalin solution 54
Estimation of acetone 61
Part II
Chromatography 68

Column Chromatography 68
Paper Chromatography 74
Thin-layer Chromatography 81
Exercises 86

2
3
 Introduction
Volumetric analysis

 Volumetric analysis is one of the most useful techniques in

analytical chemistry for quantative analysis .
 This technique is rapid and very good accuracy can be obtained .
 Since the concentration is known and since the reaction between
analyte and titrant is known , SO the amount of analyte can be
calculated.
 Volumetric analysis depends on measuring the volume of titrant
required to just completely react with the analyte .

Titration process

 In this titration, the test substance (analyte) reacts with a reagent added
(standard solution).
 The titration process need to burette and conical flask .

Standard solution (titrant)

That solution has a known concentration and added from the burette.

4
Analyte

That solution has an unknown concentration and placed in conical flask.

The requirement of titration

l- Reaction should be stoichiometric .

2- Reaction should be quantitative .
3- Reaction is rapid and specific .
4- No side reaction occurs during the titration .
 If the interference substance occur, these must be removed before
start titration .
5- It must be a change in the colour of the solution .
 A colour change usually brought by addition of the indicator ,
 The colour is dependent on the properties of the solution e.g. pH .

Equivalence point

Is the point at which an equivalent or stoichiometric amount of titrant is

added , It can be determined using an indicator .

CLASSIFICATION OF VOLUMETRIC METHOD

The reactions employed in volumetric analysis classified into two main
classes :-

A-Those in which no change in valence occurs , these are dependent upon

the combination of ions .

 This type of reaction divided into three main classes.

I-Neutralization reactions :-

 These include the titration of free bases or those formed from salts of
weak acids by hydrolysis with a standard acid (acidimetry) .
 And the titration of free acid or those formed by the hydrolysis of
salts of weak bases, with a standard base (alkalimetry) .

5
 These reactions involve the combination of hydrogen and hydroxide
ions to form water.

H+ + OH- → H2O

HCl + NaOH → NaCl + H2O

II-Complex formation reactions :-

 These depend upon the combination of ions, other than hydrogen or

hydroxide ions, to form a soluble slightly dissociated ion or compound
as in the titration solution of a cyanide with silver nitrate or of chloride
ion with mercuric nitrate solution
 Also ethylene diamine tetra acetic acid (EDTA) is very important
reagent for complex formation titrations.
Ag+ + 2CN- → [ Ag(CN)2]-

III-Precipitations reactions :-

 These depend upon the combination of ions, other than hydrogen or

hydroxide ions, to form a simple precipitate as in the titration of silver
ion with a solution of chloride.

Ag+ + Cl- → AgCl ↓

White p.p.t

B-Oxidation reduction reaction

 These involve a change of valence or otherwise expressed, transfer of

electrons for example :-

I2 + 2 Na2S2O3 → 2 NaI + Na2S4O6

Types of standard substance: :

 It's classified into :-

1- Primary standard material .
2- Secondary standard material .

6
  Properties of primary standard substances :

1-It must have high purity .

2-It must have a high molecular weight .
3-It must be stable in concentration during the time .
4-It must change the properties during the titration .
Examples:
Na2CO3 K2Cr2O7 C2O4H2.2H2O

Sodium carbonate pot.dicromate oxalic acid dehydrate

 Properties of secondary standard substances :

There are one or more conditions which must be found in primary

standard substance, in a mistake .

For example if the material is not enough pure, a solution is prepared to give
approximately the desired concentration and it standardized by using primary
standard solution .

Examples:-
HCI NaOH KMnO4
Hydrochloric acid sodium hydroxide pot.permanganate

* Standardization of sodium hydroxide by using standard oxalic acid.

** Standardization of hydrochloric acid by using standard sodium carbonate.
Preparation of standard solution:-

We can prepare several standard solution in different types of

concentrations by using volumetric flasks.

Volumetric flasks funnel Beaker

7
Concentration of solutions

1- Molarity :-
Number of moles of solute which dissolve in one litre of solvent.

g=

2- Normality :-

Number of equivalent weight of solute which dissolve in one litre of solvent.

g=

3- g/ L (gram per litre) :-

Number of grams of solute which dissolve in one litre of solvent.

4- % (percentage) :-

Number of grams of solute which dissolve in 100 ml of solvent.

Molar solution (1M) :

It's the solution in which one mole (gram molecular weight) of the solute is
contained per litre (1000 ml) of the solvent.

Normal solution (IN) :-

It's the solution in which one gram equivalent of the solute is contained per
litre (1000ml) of the solvent.

Molecular weight (M.wt) :-

It's summation of atomic weight for substance.

Equivalent weight (Eq.wt) :-

Eq.wt of acid =

Eq.wt of base =

8
 Eq.wt of salt =

Eq.wt of oxidizing agent =

Eq.wt of reducing agent =

(N×V)analyte = (N\ V\ )titrant

[ ] [ ]

9
***Write the M.wt and Eq.wt for the following compounds :
1 -Oxalic acid dehydrate = ………………………………………………………………

2-0xalic acid anhydrous = ………………………………………………………………

3-Sodium hydroxide = ………………………………………………………………

4-Sodium carbonate = ………………………………………………………………

5-Hydrochloric acid = ………………………………………………………………

6-Tartaric acid = ………………………………………………………………

7-Succinic acid = ………………………………………………………………

8-citric acid anhydrous = ………………………………………………………………

9-citric acid monohydrate = ………………………………………………………………

10-Acetic acid = ………………………………………………………………

11-Formic acid = ………………………………………………………………

12-Malonic acid = ………………………………………………………………

13-Benzoic acid = ………………………………………………………………

14-Phthalic acid = ………………………………………………………………

15-Cinnamic acid = ………………………………………………………………

16-Salicylic acid = ………………………………………………………………

17-Aniline hydrochloride = ………………………………………………………………

18-Glycine = ………………………………………………………………

19-Formaldehyde = ………………………………………………………………

20-Phenol = ………………………………………………………………

21-Catechol = ………………………………………………………………

22-Pyrogallol = ………………………………………………………………

23-Aniline = ………………………………………………………………

24- O-phenylenediamine = ………………………………………………………………

10
Write on titration curves for acid-base titration and its types
of indicator used.

11
 1- Estimation of an aliphatic acid

Theoretical bases
 The formula of an aliphatic acid is R(COOH)n , where n is the number
of basicity.
 We can calculate the eq.wt of an aliphatic acid by direct titration in
aqueous solution.
 The most direct method for the determination of water-soluble
carboxylic acids (aliphatic acids) is titration with standard alkali
( sodium hydroxide) using phenolphthalein as indicator.
 Standard sodium hydroxide solution may be employed and is prepared
from the solid of analytical reagent purity and standardize using
standard reagent.

R(COOH)n + n NaOH → R(COONa)n + n H2O

strong base

 The pH at the stoichiometric end point or the equivalence point of

the neutralization reaction will naturally depend upon the
ionization constant of the carboxylic acid.

 Neutralization produces the salt of the weak acid and a strong base,
consequently the resulting solution will be slightly alkaline, having
a pH greater than 7.

 Most carboxylic acids are neutralized at a pH of about 8.4 ;

rendering phenolphthalein a satisfactory indicator (pH interval
8.3-10.1 ; colorless to red ).

 In general, the choice of an indicator for a titration is dictated by

the acid to be determined, i.e., the colour change interval of the
indicator must include the equivalence point.
strong base = Aliphatic acid

SO,
12
 (N.V)aliphatic acid = (N\.V\)NaOH
OR …

[ ] [ ]

So,

The neutralization equivalent is the number of grams of acid required to

neutralize one litre of normal alkali .
Procedure

1-Prepare a solution of 0.1N sodium hydroxide in 250 ml Volumetric flask.

2- Prepare a solution of 0.1N oxalic acid in 100 ml volumetric flask.

3-Standardize sodium hydroxide by using oxalic acid in presence of

phenolphthalein as indicator.

4-Weight out accurately 1.5 : 2 gm of sample (theoretical wt) and dissolve it

in 100 ml or 250 ml volumetric flask.

5-Transfer 10 ml or 25 ml of sample to a 250ml conical flask, and then titrate

with standard sodium hydroxide in presence of phenolphthalein as indicator.

6-Repeat step 5 about 3-5 times and calculate mean volume (M .V.).

7-Calculate eq.wt of an aliphatic acid from this expression :-

[ ] [ ]

Where , Factor =

NOTE

Also, we can calculate all the following :-

1-Calculate M.wt of an aliphatic acid from this expression :
M.wt = Eq.wt n , n= number of basicity

13
2-Calculate number of basicity of an aliphatic acid from this
expression :-
n=

3-Calculatethe the purity of sample, when sample is known from

these expressions :

[ ] [ ]

Calculation
1- Preparation of …… N sodium hydroxide in ….ml volumetric flask

2- Preparation of …… N oxalic acid in ….ml volumetric flask

3- Standardization of sodium hydroxide using ….N oxalic acid

No. S.P. E.P. M.V. indicator

Normality of sodium hydroxide = ……………….. N

14
 UnKnown no. (………)
calculate the eq.wt of an aliphatic acid
 Wt. of sample = ….. gm , dissolved in ….. ml volumetric flask
 ….. ml of sample + ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

Eq.wt of sample = ……………………

UnKnown no. (………)

calculate the M.wt of an aliphatic acid
 Wt. of sample = ….. gm , dissolved in ….. ml volumetric flask
 ….. ml of sample + ph.ph ….. N sodium hydroxide .

No. S.P. E.P. M.V. indicator

M.wt of sample = ……………………

15
 UnKnown no. (………)
calculate the number of basicity of an aliphatic acid
 Wt. of sample = ….. gm , dissolved in ….. ml volumetric flask
 ….. ml of sample + ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

Number of basicity of sample = ……………………

UnKnown no. (………)

calculate the purity of …………… acid
 Wt. (theoretical) = ….. gm , dissolved in ….. ml volumetric flask
 M.wt of ………acid = ………. , n= ………
 Eq.wt of ……...acid = ………..
 ….. ml of sample + ph.ph ….. N sodium hydroxide .

No. S.P. E.P. M.V. indicator

Wt. practical of sample = ……. g

% purity = ………………………..

16
 2- Estimation of an aromatic acid
Theoretical base
 The formula of an aromatic acid is Ar(COOH)n , where n: is the
number of basicity.
 We can calculate the eq.wt of an aromatic acid by back titration
(indirect method) in aqueous solution, why?.
The indirect method for the determination of water insoluble carboxylic
acids (aromatic acid) is considered by dissolving it in excess of aqueous
alkali (sodium hydroxide); the excess of alkali is back titrated with
standard hydrochloric acid .

Ar(COOH)n + NaOH Ar(COONa)n + n H2O + NaOH HCl

Strong base excess

Strong acid (HCI) ≡ Sodium hydroxide (excess)

So, volume of sodium hydroxide (excess) V\ can be calculated

from this expression :
(N.V)HCl = (N\.V\)NaOH excess
so, volume of sodium hydroxide V, that equivalent to aromatic
acid calculated as :
VNaOH = [ Vtotal – V\excess] ≡ Aromatic acid
so, the eq.wt of an aromatic acid gives by this expression :

[ ] [ ]

Another method
Also acids which insoluble in water may be dissolved in
aqueous or absolute methanol or ethanol and titrated with
standard alkali (sodium hydroxide) using phenolphthalein as
indicator .

17
These types of titrations are recommended in which a blank
titration be carried out using the same amount of solvent and
indicator;
 the difference between the two titrations gives the volume
of alkali consumed by the organic acid

Ar(COOH)n + n NaOH Ar(COONa)n + n H2O

Strong base

Procedure
1-Prepare a solution of 0.5N sodium hydroxide in 250 ml
volumetric flask.
2- Prepare a solution of 0.5N oxalic acid in 100 ml volumetric
flask.
3-Prepare 0.25N hydrochloric acid in 250 ml volumetric flask.
4-Standardize sodium hydroxide by using oxalic acid (0.5N) in
presence of phenolphthalein as indicator.
5-Standardize hydrochloric acid by using sodium hydroxide in
presence of phenolphthalein as indicator.
6-Weight out accurately 0.2-0.5 gm of sample (theoretical wt),
place it in a 250 ml conical flask
 and add 20 ml from the prepared sodium hydroxide
solution with shaking until all the sample dissolve,
 Then titrate the mixture by using hydrochloric acid or
oxalic acid (0.25N) in presence of phenolphthalein as
indicator.
7-Calculate V\ and V of sodium hydroxide from these
expressions :
(N.V)HCl = (N\.V\)NaOH excess
VNaOH = [ Vtotal – V\excess] ≡ Aromatic acid

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 8- Calculate the eq.wt of an aromatic acid from this expression :
[ ] [ ]

Note
Also, we can calculate all the following:
1 -Calculate the M.wt of an aromatic acid from this expression :

M.wt = Eq.wt × n ; n= number of basicity

2-CalcuIate the number of basicity of an aromatic acid from this expression

n=

3-Calculate purity of sample, when sample is known from these expressions :

[ ] [ ]

Calculation
1-Prepare ……N sodium hydroxide in ….. ml volumetric flask
2-Prepare …...N oxalic acid in ……….. ml volumetric flask
3-Prepare …...N hydrochloric acid in ……….. ml volumetric flask
4-standerdization of sodium hydroxide using ……N oxalic acid

 10 ml of …N oxalic acid + ph.ph ….N sodium hydroxide

No. S.P. E.P. M.V. indicator

SO, Normality of sodium hydroxide = …………….N

19
5-standerdization of hydrochloric acid using ……N sodium
hydroxide

 10 ml of ……N hydrochloric acid + ph.ph ….N sodium

hydroxide
No. S.P. E.P. M.V. indicator

Normality of hydrochloric acid = …………..N

UnKnown no. (………)

calculate the eq.wt of an aromatic acid
 Wt. of sample = ….. gm , dissolved in V(total)NaOH= ….. ml
+ ph.ph ….. N hydrochloric acid .
S.P. E.P. V Indicator

Eq.wt of sample = ……………………

UnKnown no. (………)

calculate the M.wt of an aromatic acid
 Wt. of sample = ….. gm , dissolved in V(total)NaOH= ….. ml
+ ph.ph ….. N hydrochloric acid .
S.P. E.P. V Indicator

M.wt of sample = …………………× n

20
 UnKnown no. (………)
calculate the number of basicity of an aromatic acid
 Wt. of sample = ….. gm , dissolved in V(total)NaOH= ….. ml
+ ph.ph ….. N hydrochloric acid .
S.P. E.P. V Indicator

Number of basicity of sample =

UnKnown no. (………)

calculate the purity of ……………acid
 Wt. (theoretical) of sample = ….. gm , dissolved in
V(total)NaOH= ….. ml + ph.ph ….. N hydrochloric acid .

S.P. E.P. V Indicator

M.wt of ……. acid = ………… , n = …….

Eq.wt of …… acid = …………
Wt(practical) of sample = ……..gm ; %purity = ………..

21
 3-Estimation of purity of aniline hydrochloride
Theoritical base
The amine salt (e.g., aniline hydrochloride) Is a salt
formed from a very weak base (aniline) and a strong acid
(hydrochloric acid).

C6H5NH2 + HCl → C6H5NH2 . HCl

Aniline hydrochloride is strongly acidic, so the titration may

be carried out directly with standard sodium hydroxide solution
in an aqueous medium.
This is because considerable hydrolysis occurs in aqueous
solution and progressive neutralization of the free acid results
ultimately in complete hydrolysis to the amine and strong acid.

C6H5NH2 . HCl C6H5NH2 + HCl NaOH

Strong base
SO ,
Strong base ≡ Hydrochloric acid ≡ Aniline hydrochloride

[ ] [ ]

 When eq.wt of aniline hydrochloride =………….. ;

SO , (practical wt ) of sample can be calculated.
 The percentage purity of the amine salt is calculated from the
formula :

22
 Procedure
1-prepare a solution of 0.IN sodium hydroxide in 250 ml
volumetric flask.
2-prepare a solution of 0.1 N oxalic acid in 100 ml volumetric
flask .
3-standardize sodium hydroxide by using oxalic acid in
presence of phenolphthalein as indicator.
4-Weight out accurately (1.5-2.0) gm of sample (theoretical
wt.) and dissolve it in 100 ml or 250 ml volumetric flask.
5-Transfer 10 ml or 25 ml of sample to a 250 ml conical flask,
and then titrate with standard sodium hydroxide solution in
presence of phenolphthalein as indicator.
6-Repeat step 5 about (3-5) times and calculate mean volume
(M.V.).
7-Calculate (practical wt.) of sample from this expression :

[ ] [ ] × Factor

Where ; Factor =

8-Calculate the percentage purity of sample from the following

expression :

NOTE THAT :
[ Eq.wt = M.wt ]aniline hydrochloride

23
 Calculation
1- Preparation of ……. N sodium hydroxide in …. ml
volumetric flask .
2- Preparation of ……. N oxalic acid in …. ml volumetric
flask .
3- Standerization of sodium hydroxide using …. N oxalic
acid
 10 ml of ….. N oxalic acid + ph.ph ….. N sodium hydroxide
No. S.P. E.P. M.V. indicator

Normality of sodium hydroxide = ………….. N

UnKnown no. (………)

 Wt. (theoretical) = ….. gm , dissolved in ….. ml volumetric
flask
 ….. ml of sample + ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

% Purity = ……………………%

24
 UnKnown no. (………)
 Wt. (theoretical) = ….. gm , dissolved in ….. ml volumetric
flask
 ….. ml of sample + ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

% Purity = ……………………%

UnKnown no. (………)

 Wt. (theoretical) = ….. gm , dissolved in ….. ml volumetric
flask
 ….. ml of sample + ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

% Purity = ……………………%

25
 UnKnown no. (………)
 Wt. (theoretical) = ….. gm , dissolved in ….. ml volumetric
flask
 ….. ml of sample + ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

% Purity = ……………………%

26
 4- Estimation of an aliphatic amino acid

Theoretical bases
The formula of an aliphatic amino acid is :
H2N-CHR-COOH
Aliphatic amino acids are present in aqueous solution largely
as zwitter ions and cannot be titrated directly with standard
alkali owing to the buffering effect of the zwitter ions.

H2N-CHR-COOH H3N+-CHR-COO –
zwitter ion

Estimation by formal titration :

The aqueous neutral solution of the amino acid is treated with
an excess of a carefully neutralized solution of formaldehyde,
it can then be titrated with a solution of a strong alkali (sodium
hydroxide).
H3N+-CHR-COO – + CH2O → H2C=N-CHR-COOH → NaOH
Amino acid neutral formalin formyl amino acid strong
Solution base
neutral

Strong base ≡ Formyl amino acid ≡ Amino acid

so,
(N.V)amino acid = (N\.V\)NaOH
OR ;

[ ] [ ]

So ; we can calculate the eq.wt of an amino acid.

27
 Proceudre
1-Prepare a solution of 0.1N sodium hydroxide in 250 volumetric flask
2-prepare a solution of 0.1 N oxalic acid in 100 ml volumetric flask
3-standardize sodium hydroxide by using oxalic acid in presence of
phenolphthalein as indicator
4-For preparation of neutral formalin solution :
 place 20 ml of commercial formalin solution (35-40 %
formaldehyde) in a conical flask .
 Add 2 drops of phenolphthalein as indicatcor .
 Titrate with sodium hydroxide until the solution has a pale pink
colour.
5-For preparation of neutral amino acid solution sample :
 Weight out accurately (1.5-2.0 ) gm of sample (theoretical wt. )
 Dissolve it in 10 ml distilled water, add one drop of
phenolphthalein,
 Add sodium hydroxide drop wisely using burette until the
solution has a pale pink colour,
 Then complete the sample by distilled water into 100 ml or 250
ml volumetric flask.
6-Transfer 10 ml or 25 ml of the solution of the neutral amino acid to
a 250 ml conical flask,
 Add 5 ml of the neutral formaldehyde solution,
 Then titrate with standard sodium hydroxide in presence of
phenolphthalein as indicator.
7-Repeat step 6 about ( 3-5 ) times and calculate mean volume (M.V.).
8-Calculate the eq.wt of an amino acid from the expression :-

[ ] [ ] × Factor

Where ; Factor =

28
NOTES :

Also , we can calculate the percentage purity of known amino acid as

the following :
By Known eq.wt of an aliphatic amino acid , we can determine
practical wt. of sample by this expression :

[ ] [ ] × Factor

SO ; we can determine the percentage purity of all amino acids of each

all these types :

Glycine (Gly) Alanine (Ala) Aminobutyric Acid (Abu)

Valine (Val) Isoleucine (Ile) Leucine (Leu)

Methionine (Met) Norleucine (Nle) Norvaline (Nva)

Phenylalanine (Phe) Threonine (Thr) Serine (Ser)

29
 Calculation

1- Preparation of ……. N sodium hydroxide in …… ml volumetric

flask .
2- Preparation of ……. N oxalic acid in …… ml volumetric flask

3-standardization of sodium hydroxide by using …… N oxalic acid

 10 ml of ….. N oxalic acid + ph.ph ….. N sodium hydroxide

No. S.P. E.P. M.V. indicator

Normality of sodium hydroxide = …………… N

UnKnown no. (………)

calculate the eq.wt of an amino acid
 Wt. of sample = ….. gm , dissolved in ….. ml volumetric flask
 ….. ml of neutral sample + 10 ml of neutral formaldehyde +
ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

Eq.wt of sample = ……………………

30
 UnKnown no. (………)
calculate the Eq.wt of an amino acid
 Wt. of sample = ….. gm , dissolved in ….. ml volumetric flask
 ….. ml of neutral sample + 10 ml of neutral formaldehyde + ph.ph
….. N sodium hydroxide .

No. S.P. E.P. M.V. indicator

Eq.wt of sample = ………………..

UnKnown no. (………)

calculate the percentage purity of …………
 Wt. of sample = ….. gm , dissolved in ….. ml volumetric flask
 ….. ml of neutral sample + 10 ml of neutral formaldehyde +
ph.ph ….. N sodium hydroxide .
No. S.P. E.P. M.V. indicator

% purity = ……………………

31
 5-Estimation of hydroxyl groups in alcohols and phenols

A –Discussion of selected methods for the determination of hydroxyl

groups in alchol

Theoretical base:

The formula of alcohols is : R-(OH)n

 Alcohols are present as simple or polyhydric alcohols

(adjacent hydroxyl groups — Sugar compounds).
 The procedures commonly used for the determination of
hydroxyl groups in alcohols include acetylation , phthalation
and oxidation with periodates.
 The last-named is applied to polyhydric alcohols containing
adjacent hydroxyl groups .

I-Estimation of hydroxyl groups by acetylation with a solution of

acetic anhydride in pyridine :

 The acetylation method involves the replacement of the

hydrogen on an alcoholic hydroxyl group by acetyl.
 The reagent consists of a solution of acetic anhydride in
pure pyridine and is used in excess .
R(OH)n + n(CH3CO)2O R-(OCOCH3)n + n CH3COOH

 The addition of water converts the excess of acetic

anhydride into acetic acid.
 The total free acetic acid is then titrated with standard
sodium hydroxide solution :
(CH3CO)2O + H2O 2 CH3COOH NaOH
Strong base

A control or blank experiment is performed simultaneously

Identical with the above except that the addition of sample of the
hydroxyl compound is omitted .

32
  The difference between the volumes ∆V (VBlank – Vactual) of
sodium hydroxide solution required in two experiments is
equivalent to the difference in the amount of acetic acid formed,
i.e., to the acetic acid derived from the acetic anhydride consumed
in the actual acetylation of the sample.
so,
∆V (VBlank – VActual) ≡ Acetic acid ≡ Alchol
Stong base

[ ] [ ]

 We can calculate the eq.wt of alcohol .

 If the molecular weight of the compound is known, the
number of hydroxyl groups in the compound can be
calculated.
n (number of hydroxyl groups) =

Also, the percentage of hydroxyl in the sample is calculated from

the formula :

% OH =

The advantages of control or blank determinations are two fold :

l- The absolute concentration of the reagent (here the exact

concentration of acetic anhydride in the pyridine solution) need
not be determined.
 If the same volume of reagent is used in the actual and in
the blank experiment, the difference gives the actual amount
used.
2- Indeterminate losses of the reagent (due to slight chemical
action of the glass vessels, slight absorption by the corks, etc.) are
almost identical in the actual and blank experiments and therefore

33
 do not affect the difference in result between the two experiments.

Importance of pyridine:
 It may be mentioned that pyridine is used as a
solvent in acetylation because it inactive towards the
reagents .
 It removes the acid products by salt formation ,
 And it also serves as a catalyst .

The reactions can be represented as follows:

Acetyl pyridinium acetate intermediate

34
 II-Estimation of hydroxyl groups by phthalation with phithalic
anhydride in pyridine :
 The phthalation method is essentially similar to that of
acetylation.
 The reagent is a solution of phthalic anhydride in pyridine

 The acetylation procedure is more rapid than phthalation

 A large excess of phthalic anhydride is therefore needed
for completing the reaction.
 Neither procedure can be used with tertiary alcohols
because the alcohol is dehydrated.

B-Discussion of selected methods for the determination of

hydroxyl groups in phenols :
Theoretical bases
The formula of phenols are :
Ar-(OH)n
The following methods may be used :-
I-Estimation by acetylation with a solution of acetic
anhydride in pyridine :
Ar(OH)n + n (CH3CO)2O Ar(OCOCH3) + n CH3COOH
 The procedure is similar to that described for the
acetylation of alcohols.
Notes :
Phenols are not phthalated , so that alcohols can be
determined in the presence of phenols.

35
 II- bromination with excess of a standard bromate-
bromide solution in presence of hydrochloric acid :

KBrO3 + 5 KBr + 6 HCl→ 3 Br2 + 6 KCl + 3H2O

 The excess of bromine is determined by the addition

of potassium iodide solution
 And titration of the liberated iodine with standard
sodium thiosulphate solution , using starch as
indicator .

Br2 + 2 KI → I2 + 2 KBr → Na2S2O3

 The variables in the bromination procedure include

temperature ,excess of bromine and time of reaction .
Procedure
Estimation of hydroxyl groups in alcohols or phenols by
acetylation with a solution of acetic anhydride in pyridine

1-Prepare a solution of 0.2N sodium hydroxide in 250 ml

volumetric flask.
2-Prepare a solution of 0.2N oxalic acid in 100 ml volumetric
flask.
3-Standardize sodium hydroxide by using oxalic acid in
presence of phenolphthalein as indicator.
4-For preparation of sample in actual flask :
 Weight out accurately 0.5 gm (theoretical wt) from sample
(liquid alcohols, sugars, or phenols), and place it in pure, dry
25 ml or 50 ml round-bottom flask or round flat-bottom flask,
 Then add 2 ml of Ac2O and 8 ml of dry pyridine.
 Heat the flask on a steam bath for 45 minutes .
 Add 6 ml of water to the top of condenser, so that the
water rinses down the condenser and the walls of the
flask.
36
 Heat for more 10 minutes.
 Cool the flask under cold water or in ice.
 Add 5 ml of n-butanol, and then complete the sample by
distilled water into 100 ml or 250 ml volumetric flask.
5- preparation of blank flask :
 In dry 25 ml or 50 ml round-bottom flask or round flat-bottom
flask,
 add the reagents simultaneously and similar in all respects
except for the addition of the sample as in step 4 .
6- Transfer 10 ml or 25 ml of the solution of the sample to a
250ml conical flask , and then titrate with standard sodium
hydroxide and phenolphithalin as indicator .
7- Repeat step 6 about 3-5 times and calculate the mean
volume .
M.V. ≡ V1 ≡ Vactual
8- Transfer 10 ml or 25 ml of the solution of the blank to a
250 ml conical flask , and then titrate with standard sodium
hydroxide and phenolphithalein as indicator .
9- Repeat step 8 about 3-5 times and calculate mean volume .
M.V. ≡ V2 ≡ Vblank
10- Calculate the Eq.wt of the sample ( alchols or phenols )
from the expressions :
(V2 – V1 )NaOH ≡ CH3COOH reacted ≡ Sample

[ ] [ ] × Factor

Where ; Factor =

11-Calculate the number of hydroxyl groups in the sample from the

formula :

37
 n (number of hydroxyl groups) =

12-Calculate the percentage of hydroxyl groups in the sample from

the formula :
% OH = × Factor

It must be that :

1- factor of sample = factor of blank

2- Normality of sodium hydroxide is the same for titration of
sample and blank .
 If there is a change in normality , it must be change the volume of
sample or blank according to the new normality as follows :

( N × V )before = ( N×V)after for sample or blank

Also, we can calculate the percentage purity of known alcohols

or phenols as the following :
 By known the eq.wt of sample (alcohol or phenol), we can
determine practical wt. of sample by this expression :

[ ] [ ] × Factor

 Notes :
Compound which yield highly coloured solution can be
titrated potentiometrically by the use of a pH meter with
glass and calomel electrodes.

38
 Conical flask

Condenser Heater

Round- bottom flask measuring or volumetric flask

39
 Unknown no. (………)
 Calculate the eq.wt , n and percentage of hydroxyl groups
of …………..

1. Preparation of ……. N Sodium hydroxide in …..ml

volumetric flask .
2. Preparation of ……. N oxalic acid in …… ml volumetric
flask .
3. Standardization of sodium hydroxide by using …..N oxalic
acid .
 10 ml of …… N oxalic acid + ph.ph ……. N sodium hydroxide
No. S.P. E.P. M.V. indicator

Normality of sodium hydroxide = ……………. N

4-Titration of sample by using …… N sodium hydroxide in

presence of phenolphthalein as indicator :
…….. ml of sample + ph.ph ….. N sodium hydroxide
No. S.P. E.P. M.V. indicator

V1 = ……….. ml
Factor = ………….

40
 5-Titration of blank by using …… N sodium hydroxide in
presence of phenolphthalein as indicator :

 ….. ml of blank + ph.ph …… N sodium hydroxide

No. S.P. E.P. M.V. indicator

V2 = ……… ml
Factor= …………….
6-Calculation of the eq.wt of sample:
Eq.wt = ……….
7- Calculation of the number of hydroxyl groups in a
sample:
n = ………..

8- Calculation of the number of hydroxyl groups in a

sample:
%OH = …………….%

41
 6- Estimation of amino groups in amines
Discussion of selected methods for the determination of amino
groups in amines :

 Theoretical base
The formula of amines is :
R-NH2 RR\-NH RR\R\\-N
Primary amines secondary amines tertiary amine
\ \\
Where , R,R ,R may be aliphatic or aromatic .

Estimation of amino groups by acetylation with a solution of

acetic anhydride in pyridine :
The acetylation method with an excess of a solution of
acetic anhydride in pyridine :

R-NH2 + ( CH3CO)2O R-NHCOCH3 + CH3COOH

R\R\\-NH + ( CH3CO)2O R\R\\-NCOCH3 + CH3COOH

The residual excess of acetic anhydride is decomposed

by the addition of water and the total free acetic acid is
determined with standard sodium hydroxide solution :
(CH3CO)2O + H2O 2 CH3COOH NaOH
Strong base
A Control or blank experiment is conducted
simultaneously, identical with that used for the sample
except that the addition of the amine is omitted.

The procedure is similar to that described for the

acetylation of alcohols and of phenols.

42
 NOTES:
1- This procedure is applied only to primary and secondary
amines , and it is difficult to tertiary amines.
2- with some primary amines, the presence of a group such
as : NO2 , -CH3 or –Br ortho to the amino group may lead
to the formation of a diacetyl compound.
so, a check determination is therefore advisable if no
detailed information is available.
II- Bromination with the excess of a standard bromate-
bromide solution in the presence of hydrochloric acid :
The procedure is similar to that described for the
bromination of phenols.
Proceudre
Estimation of amino groups in amines by acetylation
with a solution of acetic anhydride in pyridine :
1-Prepare a solution of 0.2N sodium hydroxide in 250 ml
volumetric
flask.
2-prepare a solution of 0.2N oxalic acid in 100 ml
volumetric flask.
3-Standardize sodium hydroxide by using oxalic acid in
presence of phenolphthalein as indicator.
4-For preparation of sample in actual flask :
 weight out accurately 0.5 gm (theoretical wt.) from amine
(liquid or solid), and place it in pure, dry 25 ml or 50 ml round-
bottom flask or round flat-bottom flask.
 Then add 2 ml of AC2O and 8 ml of dry pyridine.
 Heat the flask on a steam bath for 45 minutes.
 Add 6 ml of water to the top of condenser, so that the water
rinses down the condenser and the walls of the flask.
 Heat for more 10 minutes.
 Cool the flask under cold water or in ice.
43
 Add 5 ml of n-butanol .
 And then complete the sample by distilled water into 100 ml or
250 ml volumetric flask.
5-For preparation of blank flask :
 In dry 25 ml or 50 ml round-bottom flask or round
flat-bottom flask.
 Add the reagents simultaneously and similar in all
respects except for the addition of the amine as in
step 4.
6- Transfer 10 ml or 25 ml of the solution of the sample to
250 ml conical flask.

 And then titrate with standard sodium hydroxide in

presence of phenolphithalein as indicator.
7- Repeat step 6 about 3-5 times and calculate the mean
volume.
M.V. ≡ V1 ≡ Vactual
8- Transfer 10 ml or 25 ml of the solution of the blank to a
250 ml conical flask,
 And then titrate with standard sodium hydroxide and
phenolphithalein as indicator .
9- Repeat step 8 about 3-5 times and calculate mean
volume.
M.V. ≡ V2 ≡ Vblank
10- Calculate the Eq.wt of amine from the expressions:
(V2 – V1 )NaOH ≡ CH3COOH reacted ≡ Amine

[ ] [ ] × Factor

44
Where ; Factor =

11-Calculate the number of amino groups in the sample from the

formula :

n (number of amino groups) =

12-Calculate the percentage of amino groups in the sample from

the formula :

% NH2 = × Factor

It must be that :

1-factor of sample = factor of blank .

2-Normality of sodium hydroxide is the same for titration of

sample and blank .

 If there is a change in normality , it must be change the volume

of sample or blank according to the new normality as follows :

( N × V )before = ( N×V)after for sample or blank

Also, we can calculate the percentage purity of known

amines as the following :
By knowing the eq.wt of amine , we can determine
the practical wt. of amine by this expression :

[ ] [ ] × Factor

45
 Unknown no. (………)
 Calculate the eq.wt and percentage of amino groups in
amine

1- Preparation of ……. N Sodium hydroxide in …..ml

volumetric flask.
2- Preparation of ……. N oxalic acid in …… ml
volumetric flask.
3- Standardization of sodium hydroxide by using …..N
oxalic acid.
 10 ml of …… N oxalic acid + ph.ph ……. N sodium hydroxide
No. S.P. E.P. M.V. indicator

Normality of sodium hydroxide = ……………. N

4-Titration of sample by using …… N sodium hydroxide in

presence of phenolphthalein as indicator :
…….. ml of sample + ph.ph ….. N sodium hydroxid
No. S.P. E.P. M.V. indicator

V1 = ……….. ml
Factor = ………….

46
 5-Titration of blank by using …… N sodium hydroxide in
presence of phenolphthalein as indicator :

 ….. ml of blank + ph.ph …… N sodium hydroxide

No. S.P. E.P. M.V. indicator

V2 = ……… ml
Factor= …………….
6-Calculation of the eq.wt of amine:
Eq.wt = ……….
7- Calculation of the number of amino groups in a sample
of amine:
n = ………..

8- Calculation of the percentage of amino groups in amine:

%NH2 = …………….%

47
 7- Estimation of formaldehyde in sample of formalin solution

theoretical bases
 The formula of formaldehyde is :
H-CHO

 This compound is usually encountered as an aqueous solution

"formalin" containing about )33 to 37( % by weight of
formaldehyde.
 Strong solutions polymerize spontaneously to
paraformaldehyde.
Two methods will be described for the determination of
formaldehyde in the commercial aqueous solution :
A-Formaldehyde quantitatively oxidized to formic acid by excess of
iodine in alkaline solution:

The effective oxidizing agent is probably sodium hypoiodite,

and the formic acid formed in neutralized by the alkali present :
I2 + 2 NaOH → NaOI + NaI + H2O
HCHO + NaOI → HCOOH + NaI
HCOOH + NaOH → HCOONa + H2O

HCHO + I2 + 3 NaOH → HCOONa + 2 NaI + 2H2O

When the oxidation is complete, the solution is acidified with

hydrochloric acid, and the liberated iodine (the excess of iodine
not utilized in the oxidation) is titrated with standard sodium
thiosulphate solution in presence of starch as indicator :

48
NaOI + NaI + 2HCl → 2 NaCl + I2 + H2O
↓
Na2S2O3
AL ,
NaI + HCl → HI + NaCl
HCOONa + HCl → HCOOH + NaCl

 A blank experiment is used with the same volume of

iodine titrated with standard sodium thiosulphate solution
in absence of sample of the fomaldehyde solution.
 Carry out a blank determination to check the normality of
the iodine solution and also to deduce the net volume of
standard thiosulphate solution equivalent to the sample .
 The difference between the volumes ∆V ( Vblank – Vactual )
of sodium thiosulphate solution required in two
experiments is equivalent to the amount consumed of
iodine necessary to oxidize the sample of formaldehyde to
formic acid.
so,
∆V ( Vblank – Vactual ) ≡ Iodine ≡ Formaldehyde
sodium thiosulphate

 The percentage of formaldehyde in the formalin

solution is calculated using the relationship:
1 Litre 0.1 N iodine ≡ 1 Litre 0.1 N Na2S2O3

≡ HCHO / (2 x 10)

≡ 1.5013 gm HCHO

% HCHO =

49
Where ,

Vblank = volume (ml.) of thiosulphate solution used for blank

Vactual = volume (ml.) of thiosulphate solution used after reaction

of sample with iodine solution
N = normality of sodium thiosulphate solution
M.wt = molecular weight of formaldehyde (30.026) ; and
Wt = weight (gm.) of sample.
Notes:
l- The above procedure is applicable only to dilute solution of
formaldehyde (concentration < 1 %).
2- Other aldehydes and most ketones must be absent.
B-Determination of an aqueous neutral solution of formaldehyde by
hydroxylamine hydrochloride :

The liberated acid liberated in the reaction is titrated with

standard sodium hydroxide solution.
CH2O + NH2OH.HCl → CH2=NOH + HCl + H2O
↓
NaOH (Strog base)

SO,
NaOH ≡ HClFree ≡ NH2OH.HCl ≡ HCHO

 The percentage of formaldehyde in the formalin

solution can be calculated as shown :

% HCHO = [ ]NaOH × [ ]HCHO

50
 Procedure :
Esimation of formaldehyde in sample of formaline solution by
oxidizing quantitatively to formic acid by excess of iodine in
alkaline solution:
1-Prepare a solution of 0.5N sodium hydroxide in 100 ml
volumetric flask.
2-Prepare a solution of 0.5N hydrochloric acid in 100 ml
volumetric flask.
3-Prepare 0.1N sodium thiosulphate in 250 ml volumetric flask
4-Prepare 0.1N iodine solution in 250 ml volumetric flask
5-For preparation of sample:
Weight out accurately 1 gm from formalin solution, and
complete it by distilled water into 250 ml volumetric flask
6-Transfer 10 ml from sample solution to a 250 ml conical
flask,

 Add 20 ml of ca, 0.1N iodine solution.

 Immediately, add ca. 0.5 N sodium hydroxide solution until
the liquid becomes pale yellow in colour (V ml), and wait for
10-15 minutes with shacking.
 After that time, acidify with ca. 0.5N hydrochloric acid
(3V ml)
 Titrate the excess of iodine with standard 0. I N sodium
thiosulphate solution, using starch as indicator.
7-Repeat step 6 about 3-5 times and calculate mean volume
M.V. ≡ V1 ≡ Vactual

51
 8-Titration of blank :

 Transfer 20 ml of iodine solution to a 250 ml conical flask

 And then titrate with standard 0.1N sodium thiosulphate
solution in presence of starch as indicator.
9-Repeat step 8 about 3-5 times and calculate mean volume.
M.V. ≡ V2 ≡ Vblank
10-Calculate the percentage of fomaldehyde in sample of
formalin solution from the following formula :

% HCHO = Factor

Where,

Factor =

Calculation:
1- Preparation of 0.5 N sodium hydroxide in 100 ml volumetic flask
2- Preparation of 0.5 N hydrochloric acid in 100 ml volumetric flask
3- Preparation of 0.5 N sodium thiosulphate in 250 ml volumetric
flask
4- preparation of 0.1 N iodine in 250 ml volumetric flask
5- Titration of sample :
No. S.P. E.P. M.V. indicator

V1 = ………………………

52
 6- Titration of blank :

20 ml I2 Na2S2O3 (0.1 N ) + starch Na2S2O3 (0.1 N)

No. S.P. E.P. M.V. indicator

V2 = …………….

7- Calculation the percentage of formaldhyde in sample of formalin

%HCHO = …………………%

53
 8- Estimation of acetone

 Theoretical base

The formula of acetone is :

CH3COCH3

Acetone reacts with iodine in the presence of sodium

hydroxide solution to yield iodoform and sodium acetate :

CH3COCH3 + 3 I2 + 3 NaOH → CH3COCI3 + 3 NaI + 3H2O

CH3COCI3 + NaOH → CHI3 + CH3COONa

CH3COCH3 + 3 I2 + 4 NaOH → CHI3 + CH3COONa + 3 NaI + 3H2O

 A dilute aqueous solution of the sample is added to a known volume

of 0.1 N sodium hydroxide , followed by an excess of standard 0.1 N
iodine solution.
 After acidification , unreacted iodine is determined by titration with
standard 0.1 N sodium thiosulphate solution , and starch as indicator

A blank experiment is used with the same volume of iodine titrated

with standard sodium thiosulohale solution in absence of sample of the
acetone solution.

The difference between the volumes ∆V = (Vblank – Vactual ) of sodium

thiosulphate solution required in two experiments is equivalent to the
amount consumed of iodine which reacts with sample of acetone.

SO,

∆V = (Vblank – Vactual ) ≡ Iodine ≡ Acetone

sodium thiosulphate

54
 the percentage of acetone can be calculated using the relationship

1 Litre 0. 1N iodine ≡ 1 Litre 0.1 N Na2S2O3

≡ CH3COCH3 / (6 x 10)

≡ 0.9680 gm CH3COCH3

% CH3COCH3 =

where,

Vblank = volume (ml.) of thiosulphate solution used for blank

Vactual= volume (ml.) of thiosulphate solution used after reaction

of sample with iodine solution ;

N = normality of sodium thiosulphate solution ;

M.wt = molecular weight of acetone (58.079) ;

Wt = weight (gm.) of sample.

Notes :

1- The above procedure is sometimes termed as Messinger's

method.

2- Aldehydes, compounds which contain an acetyl group, or a

group oxidisable by hypoiodite to an acetyl group , interfere.

3- Compounds containing a -CH=CHC=O group (e.g., acrolein

or furfuraldhyde ) will consume iodine and therefore interfere .

3- Methyl and ethyl alcohols should also be absent.

55
Procedure

1-Prepare a solution of 0.5N sodium hydroxide in 100 ml

volumetric flask.

2-Prepare a solution of 0.5N hydrochloric acid in 100 ml

volumetric flask.

3-Prepare 0.1N sodium thiosulphate in 250 ml volumetric flask.

4-Prepare 0.1N iodine solution in 250 ml volumetric flask.

5-For preparation of sample :

Weight out accurately 0.5 gm from acetone solution, and
complete it by distilled water into 250 ml volumetric flask
6-Transfer 10 ml from sample solution to a 250 ml conical
flask,

56
  Dilute to 70 ml with distilled water (i.e. add 60 ml distilled
water )
 Then add 20ml of 0.5 N sodium hydroxide solution , mix
well and allow to stand at room temperature for 5 minutes
 Add 20 ml of 0.1 N iodine solution whilst shaking the
conical constantly with swirling motion .
 Allow the mixture to stand for 15 minutes at room
temperature
 After that time , acidify with ca. 0.5 N hydrochloric acid
(26 ml or more ) and titrate the excess of iodine with
standard 0.1 N sodium thiosulphate solution , using starch
as indicator .
7-Repeat step 6 about 3-5 times and calculate mean volume
M.V. ≡ V1 ≡ Vactual

8-Titration of blank :

 Transfer 20 ml of iodine solution to a 250 ml conical flask

 And then titrate with standard 0.1N sodium thiosulphate
solution in presence of starch as indicator.
9-Repeat step 8 about 3-5 times and calculate mean volume.
M.V. ≡ V2 ≡ Vblank
10-Calculate the percentage of acetone in sample of formalin
solution from the following formula :

% CH3COCH3 = Factor

Where , Factor =

57
Calculation:
1- Preparation of 0.5 N sodium hydroxide in 100 ml volumetric flask

2- Preparation of 0.5 N hydrochloric acid in 100 ml volumetric flask

3- Preparation of 0.1 N sodium thiosulphate in 250 ml volumetric

flask

4- Preparation of 0.1 N iodine in 250 ml volumetric flask

58
 5- Titration of sample :
No. S.P. E.P. M.V. indicator

V1 = ………………………

6- Titration of blank :

20 ml I2 Na2S2O3 (0.1 N ) + starch Na2S2O3 (0.1 N)

No. S.P. E.P. M.V. indicator

V2 = …………….

7- Calculation the percentage of acetone in sample

% CH3COCH3 = …………………%

59
60
 Chromatography

 Definition

Chromatography is the modern technique used for qualitative

and quantitative analyses of organic and inorganic compounds.

The most important uses of chromatography are :

purification and separation of mixtures

In this technique the components of a mixture can be separated

by allowing the sample to be distributed between two phases, one
of them is stationary (stationary phase), while the other moves
(mobile phase).

The stationary phase may be solid or liquid supported on a solid

 Types of chromatographic techniques in this study :

l- Column chromatography

2- Paper chromatography.

3- Thin layer chromatography (TLC technique}

I- Column Chromatography

A column is a long narrow tube with dimensions depend on the

weight of the sample, e.g. the column with 20-30 cm. long and 1-3
cm diameter packed with 50-100 gm of stationary phase supported
on a plug of cotton or glass wool, may retain several grams of the
sample.

Column used in different chromatographic techniques, one of

them named adsorption chromatography.

Adsorption chromatography :

 This technique of chromatography is based upon the selective

adsorption from solution to the active surface.
 When related substances exhibit different degree of adsorption, the
separations take place.

61
Column chromatography technique depend on spread the
components of a mixture through a column of two phases:
solid Stationary phase and mobile phase. So this technique is named
― Liquid-Solid chromatography

Liquid – Solid Chromatography Technique

62
For Example ,

 A solution of leaf pigments in an organic solvent is passed slowly

through a tube packed with a white adsorbent, such as alumina, the
individual pigments tend to be retained in different coloured
zones, the most strongly adsorbed components being retained at
the top, and those with less affinity for the adsorbent at lower
levels.
 The initial separation of the different coloured layers is usually not
very distinct, but by passing a fresh portion of the original solvent
or of another solvent through the tube, the zones become more
sharply defined and may spread over the length of the whole
column.
 The resolution into chlorophyll-b (yellowish-green), chlorophyll-a
(bluish-green), xanthophyll (yellow), and carotene (yellow) is
clearly visible.

63
Choice of stationary phase ( Adsorbents ) :

The separation process for organic mixture is successfully occur ,

when solid stationary phase has these conditions :

1- Chemically inert.
2- Must be adsorbent material.

The most widely used adsorbent is activated aluminium oxide

―alumina ― and silica gel .

Other adsorbents include magnesium oxide , magnesium carbonate ,

magnesium trisilicate , calcium carbonate , barium carbonate , calcium
hydroxide , calcium sulphate , glucose , lactose , starch and cellulose .

They are all employed in the form of uniform white powders , the
inorganic compounds may usually be improved by heating at 200-230 c

Choice of mobile phase :

Mobile phase is the moving phase " Eluent ", which separate the
mixture to its components.

The choice of solvent will naturally depend in the first place upon the
solubility relations of the substance.

The solvents generally employed possess boiling points between 0

and 100 C

The most widely used medium is light petroleum (b.p. not above 0 );
others are cyclohexane, carbon disulphide , benzene, chloroform, carbon

64
tetrachloride, methylene chloride, ethyl acetate, ethyl alcohol, acetone,
ether, pyridine and acetic acid.

Solvents have a triple role:

1- They serve to introduce the mixture to the column.

2- They effect the process of development by which the zones of the

chromatogram are separated to their fullest extent.

When used for this purpose, the solvents are termed developers.

The developer is generally a solvent in which the components of the

mixture are not too soluble and is usually a solvent of low molecular
weight.

3- They remove the required content of each zone from the mechanically
separated parts of the column.

Solvents utilized for removing the various components of a mixture as

separated on a column are called eluents.

Reference has already been made to the choice of solvent for

introducing the mixture to the column. Adsorption takes place most
readily from non-polar solvents , such as petroleum ether or benzene, to
highly polar solvents such as alcohols, esters and pyridine.

The choice of an eluent is governed by a few simple and obvious rules.

 It should be a liquid which is a good solvent for the components to

be eluted.
 The eluent may be well adsorbed itself, so that its solvent action
is assisted by its displacing action at the interface; sometimes some
strongly adsorbed substance may be added to the eluent to promote
this displacement.
 The eluent should be easily removable from the desorbed
component ; low- boiling eluents may be used to elute high-boiling
substances; basic or acidic solvents may be employed to elute
stable neutral copmpounds ; and neutral solvents to elute acidic or
basic subtances .

65
66
 Paper Chromatography

Definition:
paper chromatography is the cellulose filter paper, that has capillary
action, let it be known direction.

Uses :

This is a very simple form of chromatography that is used widely for

qualitative and quantitative analyses.

Theory :
A sample mixture is spotted onto a strip of filter paper with a
micropipette, and the chromatogram is "developed" by placing the
bottom of the paper (but not the sample spot) in a suitable solvent.

67
 The solvent" mobile phase- eluent" is drawn up the paper " stationary
phase" by capillary action and the sample components move up the paper
at different rates depending on :

1-Their solubility .

2- Their degree of retention by the paper.

Following development, the individual solute spots are noted or are

made visible by treatment with a reagent that forms a colored derivative.
The spots will generally move at a certain fraction of the rate at which
the solvent moves and they are characterized by the Rf value
(Retention factor = Retardation factor) :

Rf = 1

So,

(Rf ) A = , (Rf ) B = , (Rf ) C =

The distance which the solvent moves is measured from the starting
line to the maximum solvent front, whereas the distance the solute moves
is measured from the starting line to the center of the solute spot .

The Rf value , then, is characteristic for a given paper and solvent

combination .

68
 Notes :

1-The mechanism of separation :

 The cellulose filter paper used is very hydrophilic and will normally
have a thin coat of water adsorbed from the air.
 So the mechanism of separation is a liquid-liquid chromatography
(partition chromatography) in which the sample distributes between the
stationary water phase and the developing solvent.
 The developing solvent is usually a mixture of an organic solvent with
water and sometimes, a water-immiscible solvent is used to develop the
chromatogram.
2-Two-dimensional paper chromatography :
 A principal advantage of this technique is that greater separating power
can be achieved by using two-dimensional paper chromatography;
 A large square piece of paper is used, and the sample is spotted at a
bottom corner of the paper.
 After development with a given solvent system, the paper is turned (90
deg ) and further development is obtained with a second solvent system.
Thus if two or more solutes are not completely resolved with the first
solvent, it may be possible to resolve them with a second solvent .

69
 Two-dimensional paper chromatography is required for complex
mixtures, such as protein hydrolyses. Almost any mixture of organic
components can be separated.

Separation of chlorophyll mixture by using paper chromatography

Extraction of chlorophyll components from green plants :

There are two methods :

1-By grinding green plants with sand in mortar, using chloroform as

solvent

2-By refluxing green plants in methanol or chloroform in round-

bottom Flask .

mortar

Choice of solvent :

The most choice of eluent for large separation by using paper

chromatography is :

chloroform : petroleum ether (40-60 C) as 2 : 8 ratio

procedure

1-A pencil line is drawn across the paper a few centimeters from the
bottom and the sample is spotted on it.

The spot must be made as small as possible for maximum separation

and minimum tailing .

70
 3- It is best done dropwise with a blower to evaporate the solvent
after each drop .
4- The paper is placed in chamber (Jar) with its end dipping in the
developing solvent, a Closed chamber must be used to saturate the
atmosphere with the solvent and prevent it from evaporating .
5- The developing may take time, but it requires no operator time.

The amount of development will depend on the complexity of the mixture

being separated. Several samples and standards can be spotted along the
bottom and developed simultaneous .

6- Dry the paper

7- 1f the solutes fluorescence , they can be detected by an ultraviolet
light
8- Apencil line is drawn around the spots for permanent
identification, color developing reagent are often used; for
example, amino acids and amines are detected by spraying the
paper with a solution of ninhydrin, which is converted to a blue or
purple color.
9- Afier the spots identified, they may be cut out and the solutes
eluted to determined quantitatively by a micro method, and then
determined qualitatively by Rf values.

Calculation:

 Number of components = …………

 Distance solvent front move "E" =……… Paper

chromatography

1- Distance solute moves ―a‖ = …………. cm

( Rf ) a = ……………..

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2- Distance solute moves ―b‖ = …………. cm

( Rf ) b = ……………..

3- Distance solute moves ―c‖ = …………. cm

( Rf ) c = ……………..

4- Distance solute moves ―d‖ = …………. cm

( Rf ) d = ……………..

5- Distance solute moves ―e‖ = …………. cm

( Rf ) e = ……………..

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 Thin-layer Chromatography (TLC)

Definition

Thin-layer chromatography (TLC) is very similar to paper

chromatography except that the stationary phase is a thin layer of
finely divided adsorbent supported on glass or aluminum plate,
plastic strip, etc.

Uses :

This technique is used for separation of mixtures or purification

of different chemical compounds.
Separations include vitamins, cholesterol, fatty acids, lipids in
serum, amino acids, dyes, glycerides, pesticides, and sugars

Preparation of TLC plates :

 The most commonly used adsorbents, include alumina, silica gel, and
cellulose.
 A slurry of the powdered substance is formed and spread on a
plate with an adapter to assure uniform thickness.
Often a binder such as plaster of paris (chloroform or distilled
water) is added to improve the adhesive properties of the material
 After being dried, it is activated by heating in an oven at 110 C for
several hours.
 Commercially prepared plates and strips on plastic are available.

Theory :

 The technique of development is the same as for paper

chromatography.
 The detection of the spots is sometimes easier than with paper
chromatography because more universal techniques can be used.
 A sample mixture is spotted onto a plate of TLC with a micropipette,
and the chromatogram is "developed" by placing the bottom of the plate
(but not the sample spot) in a suitable solvent.
 The solvent "mobile phase – eluent " is drawn up the plate of TLC
"silica gel – stationary phase " by capillary action and the sample
73
 components move up the plate at different rates so the mechanism of
separation is liquid—solid chromatography.

Qualitative analysis :

The spots will generally move at different rate and they are
characterized by Rf value (Retention factor = Retardation factor) :

Rf = 1

So,

(Rf ) A = , (Rf ) B = , (Rf ) C =

The common technique for organic compounds is spraying the

plate with sulfuric acid and then heating it to char the Compounds
and develop black spots .

Quantitative analysis

The spots can be scraped off the plate and the solutes eluted for
quantitative determination.
The TLC plates must be have less thickness and must be
powder silica gel used to give good accuracy.

Separation of chlorophyl components using TLC -

chromatography

74
Extraction of chlorophyl components from green plants:

There are two method :

I- By grinding green plants with sand in mortar, chloroform

as solvent.
II- By refluxing green plants in methanol or chloroform in round
bottom flask.

mortar

Choice of solvent :

The most choice of eluent for large separation by using TLC

chromatography is :

chloroform : methanol
as 10 : 0.5 ratio

Procedure

1- Preparation of silica-gel TLC plate :

A slurry of the powdered silica gel with distilled water (1:2 ratio), is
formed and spread on the plate; and then dried, activated by heating in an
oven at 110 C for several hours.

2- By using a pencil, TLC plate marked a few centimeters from the

bottom in any side (right or left) of plate without make a mistake
isurface of silica gel.
 And the sample is spotted on this for future reference in Rf
measurements. The spot must be made as small as possible for
maximum separation and minimum tailing.
3- It is best done dropwise with a blower to evaporate the solvent
each drop.

75
 4- The TLC plate is placed in a chamber (Jar) with its end dipping in
the developing solvent, a closed chamber must be used to saturate
the atmosphere with the solvent and prevent it from evaporating
from the surface of the silica gel as it moves up.
5- The developing may take time, but it requires no operator time
6- Several samples and standards can be spotted along the bottom
and developed simultaneously .
7- Dry the TLC plate horizontal .
8- The spots for identification components were detected.
9- After the spots identified, they may be cut out and the solutes
eluted and determined quantitatively by a micro method, and then
determined qualitatively by Rf values.

Calculation:

 Number of components = …………

 Distance solvent front move "E" =………

1- Distance solute moves ―a‖ = …………. cm

( Rf ) a = ……………..

2- Distance solute moves ―b‖ = …………. cm

( Rf ) b = ……………..

3- Distance solute moves ―c‖ = …………. cm

( Rf ) c = ……………..

4- Distance solute moves ―d‖ = …………. cm

( Rf ) d = ……………..

76
 5- Distance solute moves ―e‖ = …………. cm

( Rf ) e = ……………..

Exercises

1- When 2 gm. of citric acid monohydrate was dissolved and

completed into 100 ml by distilled water . Titration of 10 ml of
this solution, required 20 ml sodium hydroxid (0.1 N). Calculate
the purity of sample.

2- Calculate the M.wt of aliphatic acid , when 3 gm of the sample

(purity = 82 %) dissolved and completed into 100 ml by distilled
water. The titration of 13 ml of this solution, required 21.3 ml of
sodium hydroxide (0.2 N) .
( number of basicity = 2 )

3- Calculate the purity of' aniline hydrochloride , when 2 gm of the

sample dissolved in 100 ml distilled water , and titration of 10 ml
of sample solution ,required 3.3 ml of sodium hydroxide (0.45 N) .
( M.wt of aniline hydrochloride = 129.59 )

4- Titration of 30 ml of an aliphatic acid solution ( 0.5 gm of the acid

in 250 ml distilled water) , required 7.6 ml of sodium hydroxide
(0.1 N).
Calculate number of basicity of the acid .
( M.wt of acid = 158 )

5- Calculate the eq .wt for aliphatic and aromatic acids in a mixture

( the sample wt. = 4 gm )

The mixture was dissolved in 100 ml distilled water, and filtered off the
solution mixture .

77
 10 ml of the filtrate was titrated with 15 ml of 0.5 N sodium hydroxide
solution.

The precipitate (Wt. = 1.5 gm) Was dissolved in 30 ml 0.5 N sodium

hydroxide solution . the excess sodium hydroxide was titrated with 12 ml
0.32 N hydrochloric acid .

6- Calculate the percentage of formaldehyde in sample of formalin

solution, when 2 ml of sample was diluted into 500 ml by distilled
water ; and 25 ml of this solution was treated with ((50 ml I2 , 0.1
N) , 5 ml NaOH , 2N ) , and then acidified with HCl 2 N , the
liberated iodine titrated with sodium thiosulphate solution in
presence of starch as indicator, the volume consumed was 32 ml.
(10 ml iodine react 9.4ml sodium thiosulphate)

7- For that reaction :

R-(NH2)2 + (CH3CO)2O …………….

Answer the following

i- complete the equation.

ii- give reason for important use of pyridine.
iii- give reason for important use of blank for determination of
eq.wt of amine .
iv- when 2 gm of sample are used for that reaction, and the
excess of acetic anhydride converted into acetic acid, and
the reaction
mixture is completed into 250 ml by dist. water.
The titration of 25 ml of this solution required 30 ml of 0.1 N
sodium hydroxide.
Calculate M.wt, Eq.wt and percentage of amino groups in
sample .
( 25 ml of the blank required 67 ml of 0.1 N NaOH). (Exa. 2002)

8- Titration of 12 ml of an aliphatic acid solution (0.5 gm of the acid

in 100 ml dist. water) required 7.6 ml of NaOH (0.1 N). Calculate
the M.wt of the acid
78
 (basicity of acid 2) (Exa. 2002)

9- Calculate the eq.wt of amino acid, when 3 gm of the sample

dissolved and completed into 100 ml by dist. water.
Titration of 13ml of this solution after neutralization and addition
of 10 ml of neutralized formaldehyde solution, required 16.6 ml of
NaOH (0.2N).
(purity of sample = 76 %). (Exa. 2003)

10- For pure wt. gm of hydroquinone, the percentage of hydroxyl

groups = ………. (Exa. 2004)
11- Calculate the percentage of vinegar, when 15 ml of sample
diluted to 100 ml. Titration of 10 ml of this solution, required 13.5 ml
of sodium hydroxide (0.1 N).
(density of sample = 0.9 gm/cm3),(Exa. 2004)
12- When 2 gm of crude citric acid have purity 76 %, was dissolved
and completed into 100 ml by dist. water, and titration of 10 ml of
this solution, required 20 ml of NaOH (0.1 N). Calculate the
number of hydrated water in sample.
(Exa. 2005)
13- Calculate the following :
1- purity of ß-naphthylamine in sample .
2- percentage of amino groups in crude ß-naphthylamine sample.
3- percentage of amino groups in pure ß-naphthylamine sample.
When 3 g of crude ß-naphthylamine dissolved in 20 ml pyridine
and reacted with 4 ml acetic anhydride, and the excess of acetic
anhydride hydrolyzed by water into acetic acid . Then the reaction
mixture was completed into 250 ml by distilled water. Titration of
30 ml of this solution required 51 ml of 0.12 N NaOH.
(Titration of 30 ml of the blank prepared at the same condition,
required 39 ml of 0.2 N NaOH) , (Exa. 2006)

14- A mixture (3.5 g) of aliphatic acid and aniline hydrochloride was

dissolved in 100 ml distilled water. Titration of 13 ml of this solution
requires 15.2 ml NaOH (0.428N) using phenolphthalein as indicator,
while titration of 12 ml of this solution requires 13.9 ml NaOH (0. 1N)
using methyl orange as indicator.
79
 Calculate the Number of basicity of this aliphatic acid .
M.wt of aniline hydrochloride =129.59
M.wt of an aliphatic acid = 104

80
 Unknown ( ……………)
………………………………………………….

81
 Unknown ( ……………)
………………………………………………….

82
 Unknown ( ……………)
………………………………………………….

83
 Unknown ( ……………)
………………………………………………….

84