Reagents are used for the qualitative and quantitative determination of the pharmaceutical preparations, as they react with particular groups and increase their detectability by impart colour to the compounds.

1. Reagents and Reactions
E. Udaya Rajitha
I M. Pharmacy
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2. CONTENTS
 Introduction
 Reactions
 Reagents
 Preparation
 Mechanism
 Applications
2

3. Introduction
Reagents are used for the qualitative and quantitative
determination of the pharmaceutical preparations, as they
react with particular groups and increase their detectability
by impart colour to the compounds.
3
Reaction Reagent
Diazo coupling reaction BM
Oxidative coupling reaction MBTH
Condensation reaction PDAB, PDAC
Oxidation / Reduction reaction FC

4. 4
Preparation: 100mg of BM reagent powder
100ml of mixture [ acetone : water (7:3) ]
Keep in dark place & protect from light
Mechanism:
Diazotization
Coupling
BM reagent:
The compounds which contains primary
aromatic amine groups are react with the BM regent and
form red colour azo dye which shows λmax at 550nm.
Diazo coupling reaction:

5. Diazotization:
5
NaNO2
HCL HNO2
HNO2 NH2
+
0-50C
+ Aromatic Ammonium molybdate+ Diazonium salt
NHCH2
CH2
NH2
Diazonium salt + Red azo dye
Coupling:
BM reagent

6. Applications:
6
Analysis of sulphonamides, paracetamol, dapsone,
isoniazid, procainamide, nifidipine, metronidazole…etc.,
OH
N NCl NHCH2
CH2
NH2 NHCH2
CH2
NH2
N OHN
+
+
Analysis of Paracetamol:
OH
NHCOCH3
OH
NH2
NaNO2
+HCL
OH
N NCl
Hydrolysis
+
Paracetamol
Step:1
Para amino phenol Diazonium salt
Step:2
BM reagent
Red azo dye

7. 7
Oxidative coupling reaction:
MBTH reagent:
Compounds like phenols, amines,
aldehydes, indoles, catechol amines reacts with the
MBTH reagent and forms orange-red colour product.
Preparation:
200mg of MBTH in 100ml of water
It gives 0.2% strength
Mechanism:
Oxidation
Coupling

8. 8
CH3
S
N-NH2
N
FeCl3
/CAS
CH3
S
N-NH
N
+
CH3
S
N-NH
N
OH
CH3
S
N
N-N O
+
+
Oxidation:
Coupling:
MBTH reagent
Phenol
Red azo dye

9. 9
CH3
S
N-NH2
N
FeCl3
/CAS
CH3
S
N-NH
N
+
CH3
S
N
N-N
CH3
S
N-NH
N HCHO
C
H
H
+
+
Applications:
Analysis of aldehydes, indoles, carbazoles,
nimesulide, diclophenac, oxiphenbutazole, primaquine,
metronidazole…etc.,
Analysis of Formaldehyde:
Step:1
Step:2
MBTH reagent
Red azo dye
Formaldehyde

10. Condensation reaction:
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PDAB reagent:
Compounds like primary aromatic amines,
primary alkyl amines and compounds contains phenolic
–OH groups react with the PDAB reagent and give
yellow colour product.
Preparation:
Dissolve 125mg of PDAB reagent
cool
65ml of conc.H2SO4,
35ml of water

11. Mechanism:
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NC
O
H
CH3
CH3
NC
CH3
CH3
H
Ar-NAr-NH2
H2
SO4
+
Schiff's base formation:
PDAB form coloured condensation products with
primary aromatic amines in the presence of conc.H2SO4.
Here C=O is converted to C=N.
PDAB reagent Schiff's base

12. 12
NC
O
H
CH3
CH3
NHNH2
NC
CH3
CH3
NHN
H
+
Analysis of phenyl hydrazine:
Applications:
Determination of phenyl hydrazine, nimesulide,
nifidipine, indole and pyrrole derivatives, amoxicillin,
metronidazole and estimation of urobilinogen…etc.,
PDAB reagent Phenyl hydrazine Schiff's base

13. Oxidation / Reduction reaction:
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FC reagent:
10gm of sodium tungstate + 2.5gm of sodium molybdate
add 70ml of water and 10ml of conc.HCl
add 5ml of 80% phosphoric acid and 15g of lithium sulphate
reflux for 10 hrs
Add 5ml of water and 1 drop of bromine
reflux for 15 min
Cool to room temperature
Preparation:

14. 14
FC reagent is a oxidising agent, so it itself reduces and
oxidise the analyte, probably drug effects reduction of 1 or
more oxygen atoms from tungstate or molybdate in the FC
reagent, there by producing one or more possible reduced
species which have characteristic intense blue colour.
Mechanism:
Drug + FC reagent
Molybdenum blue /
Tungstic green
Alkali

15. Applications:
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Estimation of Omeprazole, Aceclofenac, assay of Folic
acid, Acyclovir, Diclofenac, Antibiotics…etc.,
+
Omeprazole FC reagent
↓ Na2CO3
Blue colour complex

16. 16
Other reagents:
• Folins reagent
• Gibbs reagent
• NBS
• FeCl3, Potassium ferri cyanide, 1,10- Phenanthroline
• Froehde Reagent
• 4-(Dimethylamino) Cinnamaldehyde
• Phosphomolybdic acid
• Zwikker Reagent

17. 17
Common applications:
Reagents Drugs
BM, MBTH Procainamide
BM, PDAB Nifidipine, Sulphonamides,
Metronidazole
PDAB, MBTH Indole, Nimesulide
MBTH, Gibb’s Vitamin B6
Folins, PDAB, NBS Amoxicillin
BM, NBS, FeCl3, K.Fe(CN)6,
1,10-phenanthroline Paracetamol
PDAB, MBTH, FC, FeCl3,
K.Fe(CN)6, 1,10-phenanthroline Diclofenac

18. Conclusion
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19. References:
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• Text book of pharmaceutical analysis by P. D. Chaithanya sudha,
chapter VII (Reagents), Pg. no. 348-369.
• A review article on Chemical derivatization methodologies for
Uv‐Visible Spectrophotometric determination of pharmaceuticals by
Olajire A. Adegoke.
• A review article on Role of Chemical and Analytical Reagents in
Colorimetric Estimation of Pharmaceuticals by K. Sushma,
Somsubhra Ghosh et al.,

20. THANK YOU
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