ULTRAVIOLET-VISIBLE

SPECTROPHOTOMETRY
(APPLICATION TO PHARMACEUTICAL ANALYSIS)

Submitted By Submitted To
Ch.Devadasu
Paritala Jagadeesh Asst. Prof
B.Pharm Vignan Pharmacy
Guntur
College,
S. No. Name of the drug Solvent λmax nm
1 Isoxuprine HCl Water 250
2 Paracetmol 0.1N NaOH (PH 13) 257
3 Ibuprofen 0.1N NaOH (PH 13) 260
4 Testosterone propionate Ethanol 241
5 Phenylephrine HCl injection Ammonium sulphate 273
6 Phenobarbitone 0.1N NaOH (PH 13) 255
7 Indomethacin Water 318
8 Tubocurarine HCl Water 280
9 Nicotinamide 0.1N NaOH (PH 13) 263
10 Isoniazid 0.1 M HCl 263
11 Sulphonamides Alkali 251
12
13
 CHEMICAL DERIVATIZATION
• Conversion of the analyte to a derivative that has a longer λmax
and / or a higher absorptivity.
• When an excess of the reagent is used, the absorbance of the
derivative is usually proportional to the concentration of the
analyte.
• For example, sugars which do not absorb significantly above
220nm can be determined spectrophotometrically by heating
with anthrone in concentrated sulphuric acid and measuring the
absorbance of the coloured derivative at 625nm.
 Diazotization and coupling of
primary aromatic amines:
• The amine is first diazotized with an aqueous solution of
nitrous acid (generated in situ by the reaction of hydrochloric
acid and sodium nitrite) at 0-5°)

H+
NH 2 + HNO2 N N Cl + 2H2O

Nitrous acid
Primary
aromatic amine Aryl diazonium chloride

Sulphamic acid or ammonium sulphamate is added to the

solution of the diazotized amine before the coupling stage to
destroy the excess nitrous acid, which inhibits the coupling
reaction.
HNO2 + NH2SO3H → N2 + H2SO4 + H2O
The colourless diazonium salt is very reactive and when treated with a suitable
coupling reagent (Ar’ – H), e.g. a phenol (1- naphthol or 2-naphthol) or aromatic
amine(B.M. reagent), undergoes an electrophilic substitution reaction to produce
an azo derivative.

N N Cl + NH

Aryl diazonium chloride

NH2

N-(1-naphthyl) ethylenediamine dihydrochloride

Bratton-Marshall reagent

N N NH

NH 2

Diazo derivative with extensive conjugation

λmax is about 525-550nm purple colour

• British Pharmacopoeia tests for free amine impurities in
Frusemide, Iothalamic Acid and Iodipamide Meglumine Injection
are based upon diazotization and coupling.

• Substances that can be hydrolyzed (e.g. Bendrofluazide,

Chlorothiazide) or reduced (e.g. chloramphenicol) to a derivative
containing a primary aromatic amine group can also be assayed
spectrophotometrically by diazotization and coupling of the
amine derivative.
 Condensation reactions:
• Many colorimetric procedures are based on the rapid
reaction that occurs under suitable condition
between amines and carbonyl compounds.

• The reaction involves the nucleophilic attack by the

amine on the carbonyl carbon with the elimination of
water.

• Substances containing a carbonyl group react with a

variety of reagents containing an amino group:
R' R'
C O + NH2R''' C NR''' + H 2O
R'' R''

• Where R’’’ = Alkyl or aryl, the product is a Schiff’s base

• = NH2 (hydrazine), the product is a hydrazone

• = NHCONH2 (semicarbazide), the product is a

semicarbazone
• = OH (hydroxylamine), the product is an oxime.
Isoniazid (isonicotinic acid hydrazide) reacts with 4-en-
3-one and 1, 4-dien-3-one steroids in acidic solution to
form yellow derivatives with λmax around 400nm.

N CONHNH 2 + A N CONHN

O
YELLOW CONDENSATION
ISONIAZID KETOSTEROID PRODUCT

The reagent is used in the assays of Nandralone Decanoate

Injection and Betamethasone Sodium Phoaphate Injection.
• 2, 4-dinitrophenylhydrazine is used in the assay of
Methyltestosterone Tablets.
• The hydrazone formed on condensation of the 3-,
17-, or 20-ketosteroid with (carboxymethyl)
trimethylammonium chloride hydrazine (Girard’s
reagent T) is water-soluble owing to the quaternary
ammonium group.
• Alternatively, the hyrdazone itself may be extracted
into dichloroethane as the ion-pair with
bromothymol blue.
• Amine compounds can be assayed
spectrophotometrically using a suitable carbonyl
reagent.
• 4-dimethylaminobenzaldehyde (Ehrlich’s reagent).

• Assay of procaine hydrochloride , the condensation

product which absorbs at 454nm.
• Concentrated sulphuric acid is used to maintain acidic
conditions in this reaction.
 Condensation of Procainamide with
p-dimethylamino benzaldehyde

H
N(H 3C) 2 C N COOHCH 2CH 2N(C 2H5)2

Yellow coloured schiff’s base

 Reduction of tetrazolium salt:

• In the presence of a steroid with an α-ketol (21-hydroxy-20-

keto) side-chain group, tetrazolium salt are reduced to their
coloured formazan derivatives.

• Several formulation containing corticosteroids are assayed

using triphenyltetrazolium chloride.

• The reaction is carried out in an alkaline medium

(tetramethylammonium hydroxide) at 30-35° for 1-2h and the
absorbance of the red product is measured around 485nm.
The oxidation of the α-ketol group and the reduction of
triphenyltetrazolium chloride to triphenylformazan

CH2 OH C6 H 5 +Cl- C 6H 5 CH
N N
C O C O
+
N N
OH OH
C C6 H5
C6 H5 + HN N C N N + HCl
C 6H 5 C 6H 5
Triphenyltet
steroid with razolium
an α-ketol (21- chloride Triphenyl formazan
hydroxy-20- (red coloured)
keto)

Steroids esterified in the 21-position, e.g. Hydrocortisone Acetate, hydrolyze in the

alkaline solution to yield the free 21-hydroxysteroids and are also determined by this
procedure.