The document discusses various reagents used in pharmaceutical analysis. It begins by classifying reagents based on their reaction mechanisms such as oxidation, condensation, diazotization, and others. Several examples of reagents are provided for each classification including MBTH, Folin-Ciocalteu reagent, PDAB, and 2,4-DNP. Their chemical properties and applications in estimating specific drugs are described. The document concludes by providing an example estimation procedure using MBTH reagent.

1. PRINCIPLES & PROCEDURES INVOLVED IN
USAGE OF REAGENTS IN
PHARMACEUTICAL ANALYSIS
• KAPIL KUMAR PATIDAR
• M.PHARM (ANALYSIS)
• 2015-2016
• GT Road (NH-95), Ghal Kalan, Moga(142001), Punjab, India

2. • Introduction
• Classification based on their reaction
mechanism
• Various types of reagents
• Applications
• Conclusion
Contents

3. Introduction
• Most of the pharmaceuticals are colorless in nature and can be estimated by UV
spectrophotometry easily.
• But there are some drugs which do not show good results in UV region due to
following reasons
weak absorption in UV region
effect of interferences
• Hence to estimate these type of drugs a technique called chemical derivatization
is employed.
• In this technique, various spectrophotometric reagents are used.
• These reagents on reacting with drugs produces a colored complex depending
upon their concentration which can be easily estimated by visible
spectrophotometry. eg: Folins reagent, gibbs reagent etc.

4. Classification of reagents based on their mechanism of reaction
• Oxidation
eg : Folin-ciocalteau reagent
tetrazolium salts
• Oxidative coupling
eg : MBTH
• Condensation:
eg : P- dimethyl amino benzaldehyde (PDAB)
p- dimethyl amino cinnamaldehyde (PDAC)
2,4-dinitro phenyl hydrazine (2,4 DNP)
Folins reagent (1,2-naphthoquinon-4-sulfonate)
Gibbs reagent (2,6-dichloro-4-chlorimide)

5. • Diazotisation :
eg : sodium nitrite & HCl / α - naphthol
sodium nitrite & HCl / Bratton Marshall Reagent
sodium nitrite & HCl / Resorcinol
sodium nitrite & HCl / p-aminophenol
sodium nitrite & HCl / aniline
• Oxidation followed by complexation :
eg : Ferric chloride and 1,10 phenanthroline
Ferric chloride and 2,2 1 bipyridine
Ferric chloride and potassium ferricyanide
• Complexation due to charge transfer:
eg : 2,3-dichloro-5,6-dicyano- 1,4-benzoquinone (DDQ),
2,4-dinitrophenol (DNP)
2,4,6-trinitrophenol (picric acid)
p – chloranilic acid Chloranil

6. MBTH is a chromogenic reagent used for colorimetric
estimation of drug containing phenolic, aromatic amines and
active methylene groups

7. Reagent Profile:
IUPAC name : 3-methyl-2- benothiazolinone hydrazone
Molecular Formula : C8H10N3S. H2O
Molecular Weight : 233.72
Color :White to half white powder
Solubility : freely soluble in distilled water

8. MBTH first reacts with aldehydes to form azine. Only if there is remaining
MBTH, it is oxidized to another species which combines with the azine to form
formazan.
However if there is enough aldehyde, all the MBTH is converted to azine and
there is no formation of blue color.
Thus by using the limiting agent MBTH, the amount of aldehyde can be tested.
Less aldehyde produce more blue color
More aldehyde produce less blue color

9. With Phenols
With phenols under suitable conditions MBTH loses two
electrons and one proton to form the electrophilic intermediate.
This undergoes electrophilic substitution with phenols and other
groups to form the colored product
Mechanism Involved
1. Oxidative coupling
2. Dehydration

10. Oxidative Coupling Reactions:
Step-I
Formation of oxidation form of MBTH as an
Intermediate

11. STEP-2
Formation Of ColoredComplexes:
a) Reaction With Aldehydes:

12. b) Reaction With Phenols
CH3

13. c) Reaction with Amines:

14. a) Reaction With aldehydes:

15. b) Reaction With Citrals:

16. Applications
Applications It is used to estimate various drugs like phenols, aromatic amines,
heterocyclic bases, polyhydroxy compounds, aldehydes, amino hetero aromatic
compounds like indoles, carbazones, phenothiazines, glycerides, glyco
aminoglycones
Drug name Colour λ max Beers range
Ethacridine Violet 535nm 2-8µg/ml
Gancyclovir Bluish green 611.8nm 50-250µg/ml
Montelukast Green 610nm 2-10µg/ml
Moprolol Green 629nm 10-35µg/ml
Pregabalin Green 668nm 50-350µg/ml

17. Example
Estimation of Lamotrigine in given pharmaceutical
formulation
Principle:
The principle involved in this method is Ion catalysed coupling reaction
of MBTH. In presence of FeCl3, MBTH loses 2 electrons and one
proton forming an electrophilic intermediate and which is proton active
coupling species.
The intermediate formed undergoes electrophilic substitution with the
drug to form green colored product, that is estimated colorimetrically
at 663nm.

19. PPn of stock soln
• Dissolve 100mg of crude
drug in 100ml of
methanol(100mcg/ml)
Construction of
calibration curve
• From std soln pipette
out 0.2, 0.4, 0.6, 0.8 ml
• +2ml of 0.2%
MBTHreagent +2ml of
0.2% FeCl3
• Keep aside for 20mins
• Make upto 10ml with
distilled water
• Measure abs at 663nm
Procedure

20. Assay Of Lamotrigine Tablets
Dissolve equivalent to 10mg powder in 10ml methanol
From above solution 60mcg/ml of solution was taken, to
this +2ml of 0.2% MBTH +2ml of 0.2% FeCl3. Allow
to stand for 20mins
Make up the volume to 10ml with distilled water.
Measure absorbance at 663nm

21. Reagent compile with following test:
• 2ml aldehyde free methanol
• Add 60 ml of 0.01%w/v soln of propionaldehyde
• 5ml of 0.4 w/v soln of reagent MBTH
• Allow to stand for 30 min.
• 25ml of 0.2% w/v soln of Fecl3.6H2O
• Dilute to 100 ml with acetone
• Absorbance at 660 nm (0.60)
•

22. Folin ciocalteau Reagent
• Also known as Folin -phenol / Folin denis reagent
• It is a mixture of phosphomolybdic acid and phosphotungstic acid
3H 2 O.P 2 O 5 .13WO 3 .5MoO 3 .10H 2 O
3H 2 O.P 2 O 5 .14WO 3 .4MoO 3 .10H 2 O
• Physical properties
Appearance: Clear bright yellow solution Boiling point:101.43°C
Solubility: Easily soluble in cold water, hot water. Soluble in methanol

23. Method of Preparation
• Dissolve 10g of sodium tungstate and 2.5g sodium molybdate in 70 ml water.
• Add 5 ml 85% phosphoric acid and 10 ml concentrated hydrochloric acid, reflux
for 10 hr.
• Add 15g lithium sulfate, 5 ml water and 1 drop bromine. Again reflux for 5mins.
• Cool to room temperature and bring to 100 ml with water.
Reaction mechanism :
FC reagent is an oxidizing agent.
It oxidizes the drug and itself gets reduced.
This reduced form of FC reagent is blue colored in nature (initially
yellow colored) and can be estimated by colorimetrically.
The reaction is carried out in alkaline medium.

24. Applications
• It is used to estimate phenolic drugs and non phenolic drugs which can be
reduced.
• It can also be used as a spraying reagent for chromatographic techniques.
• The color development is due to the transfer of electrons at basic pH and
hence reducing the FC reagent.
Drug name Color λ max Beers range
Hydralazine
hydrochloride
Blue 640nm o.1-1µg/ml
Ganciclovir Blue 764.7nm 50-250µg/ml
Ritodrine Blue 760nm 0.8-12µg/ml
Metolazone Blue 725nm 50-250µg/ml
Memantine Blue 760nm 4-12µg/ml

25. Example:
Estimation of Omeprazole in given pharmaceutical formulation
Principle:
Omeprazole affects reduction of 1, 2 & 3 oxygen atoms from tungstate and
molybdate in Fc reagent, thereby producing one or more of possible reduced
species which have a characteristic blue color, that can be estimated
colorimetrically at 742nm
+ 3H2O.P2O5 13WO3. 5MoO3. 10H2O
3H2O xP2O5. 14WO3. 4MoO3. 10H2O
Blue colored complex

26. Preparation Of Standard Stock Solution
To 100mg of crude drug + 10ml of 0.1N NaOH, made upto 100ml
with distilled water (1000mcg/ml) SOLUTION -1
From above solution, pippete out 10ml and make to 100ml with distilled
water(100mcg/ml) SOLUTION-2
From solution-2 pipette out 0.2, 0.8, 1.4, 2.0, 2.6 ml of solution +
2ml of Fc reagent + 9ml of sod. Carbonate, make to 25ml with
distilled water
Keep aside for 10mins. Measure Abs at 742 nm

27. Assay Of Omeprazole Capsules:
Weight of powder equivalent to 10mg was dissolved in 10ml
0f sodium hydroxide solution
From above solution pipette out 2ml and add 2ml of FC
reagent, 9ml of Sod. Carbonate, make to 25ml with distilled
water
Keep aside for 10mins. Measure absorbance at 742nm

28. •It is an aromatic compound and chromogenic in nature.
•Used for colorimetric estimation of amine group containing drugs.
Reagent Profile:
Synonym : Ehrlich’s reagent
IUPAC Name : 4-(dimethyl amino)-benzaldehyde
Molecular Formula : C9H11NO
Molecular Weight : 149.9
Color : White or offwhite
Solubility : soluble in alcohol, 0.1N HCl, water

29. The primary amine group which is present in the structure of drug
reacts with carbonyl group in the PDAB reagent and forms schiff
base which can be measured colorimetrically.
The principle in the formation of schiff’s base is nucleophilic
addition reaction. Here the nucleophile is amine group.

30. Physical properties:
Melting point: 72-75 °C
Boiling point: 176-177 °C
Density: 1.10 g/ mL
Solubility: soluble in water (0.3 g/L)
Reaction mechanism:
The reaction is carried out in acidic medium.
On reaction with amines, it results in the formation of a condensed product
called Schiffs base along with a water molecule.
This schiffs base is colored in nature.
Depending upon the reactants, it produces the following products
Amines - imines
Hydroximes - oximes
Hydrazines - hydrazones

31. General Reaction
•More suitable for amines
oximes.
•NH2, NR2, NR3 are strong auxochromes.
•But in aromatic compounds it is 100% colored.

33. Example:

34. Preparation Of PDAB Reagent:
To 400mg of PDAB
+2ml of conc H2SO4
+10ml alcohol
Finally, make upto 100ml
with distilled water

35. Applications Of PDAB Reagent
PDAB reagent is used in:
•Micro determination of urea in urine samples.
•Detection of sulphonamides
Amines
Ergot alkaloids
•Used as TLC stain.
•Used for spectrophotometric determination of Ranitidine,
Chloramphenicol, Metronidazole, Sulphamethoxazole.

36. Applications
• It is mainly used to determine primary amines
• Used to estimate various drugs like reserpine , ergot alkaloids, atropine, tetracycline,
antipyrine , paracetmol , isoniazid , oxyphenbutazone , terbutaline etc.,
Drug name Color λ max Beers limit
Abacavir sulfate Yellow 455nm 50-250 µg/ml
Ganciclovir Greenish yellow 401nm 80-200 µg/ml
Mesalamine Yellow 450nm 10-50 µg/ml
Acenocoumarol Yellow 441.5nm 5-25 µg/ml
Ornidazole Orange 390nm 25-162.5 µg/ml

37. Example:
Estimation of Chloramphenicol in given pharmaceutical
formulation:
Principle:
The aromatic nitro group present in the chloramphenicol is
reduced to aromatic amine group by refluxing with zinc dust in
presence of HCl for 10mins.
The resultant amine group of chloramphenicol reacts with the
carbonyl group of PDAB reagent and forms schiff base that is
estimated colorimetrically at 441nm.

38. Reaction:

39. PPn of stock
soln
• To100 mg ofdrug
• +500mg of zinc
dust +2ml of 5N
HCl, boil for 30mins
• Filterate was made
upto 10ml with
methanol
Determination
of λmax
•Pipette out 0.1ml
from stock soln +
2ml of PDAB
reagent
•Make upto 10ml
with methanol
•Scan from 400-
800nm, and λmax
was found to be at
441nm
PPn of
calibration std
•From stock pipette
out 0.5, 1.5, 2.0,
2.5 ml
•+2ml of PDAB
reagent
•Make upto 10ml
with methanol

40. PDAC
It is chemically para dimethyl amino cinnamaldehyde .
Also known as DMACA reagent.
Molecular structure:
Molecular formula:C 11 H 13 NO
Molecular weight: 175.23
Physical properties:
Melting point: 138-140 °C
Appearance: Dark yellow powder
Solubility: Soluble in ethanol, dioxane , insolube in water

41. Reaction mechanism:
The reaction is carried out in acidic medium.
On reaction with amines, it results in the formation of a condensed product
called Schiffs base along with a water molecule.
This schiffs base is colored in nature.
Depending upon the reactants, it produces the following products
Amines - imines
Hydroximes - oximes
Hydrazines - hydrazones

42. Applications
It is used for estimation of aromatic amines, isocarboxazid , benzodiazepines,
flavanoids , cysteine , cystine , methyl dopa , ranitidine, mefenamic acid,
indomethacin, nimesulide , nimodipine etc .,
Drug name Color λ max Beers range
Dapsone Red 548nm 4-12 µg/ml
Metoclopramide
.HCl
Red 553nm 4-24 µg/ml
Modafinil Reddish brown 443nm 6-14 µg/ml
Sumatriptan
succinate
Purple red 560nm 20-60 µg/ml
Nateglinide - 420nm 20-140 µg/ml

43. 2,4-DNP
It is chemically 2,4 dinitro phenyl hydrazine.
Also known as Brady's reagent
Molecular structure:
Molecular formula: C 6 H 6 O 4 N 4
Molecular weight: 198.14g

44. Physical properties
Melting point: 197-200 °C
Density: 0.843 g/ mL
Solubility: Soluble in 50% sulfuric acid & slightly soluble in water
Reaction mechanism:
2,4-DNP first attaches at the carbon-oxygen double bond (the addition
stage) to give an intermediate compound which then loses a molecule
of water (the elimination stage) and results in formation of a condensed
chromogen

45. It is mainly used to determine aldehydes and ketones .
Used to determine drugs like corticosteroids, flavanones , atorvastatin , ezetimibe ,
isoxsuprine , valproic acid etc.,
Applications
Drug name Color λ max Beers range
Tolperisone Pink 520nm 2.5-15 µg/ml
Didanosine Orange red 424nm 20-80 µg/ml
Salbutamol Red 540nm 2.5-17 µg/ml
Ritodrine Red 510nm 2-29 µg/ml
Amoxicillin Red 520nm 4-33 µg/ml

46. • Preparation of reagent:
• 4gm ninhydrin
• in 100 ml of 2methoxy
ethanol
• Shake with 1gm cation
exchanger region filter it
• Add 100ml 0.16%w/v of
(PH-5.5) stanous chloride
in acetate buffer
Ninhydrin
• Ninhydrin is a chemical used to detect
ammonia or primary and secondary
amines.
• When reacting with these free amines, a
deep blue or purple color known as
Ruhemann's purple is produced.
Formula: C9H6O4
Molar mass: 178.14 g/mol
Density: 862.00 kg/m³
Melting point: 250 °C
IUPAC ID: 2,2-Dihydroxyindane-1,3-dione

47. Mechanism
Application
1. Ninhydrin is primarly used to detect ammonia 1&2 amines.
Ninhydrine react with free amines to give a deep blue colour.
2. Growth of peptide chain is maintained by ninhydrin
A deproted peptide chain yield blue colour where as
a coupled peptide chain yield yellow colour
3. Amine Carbamates &amides can be detect by ninhydrine in TLC

48. Carr-Price reagent (sbcl3)
Sbcl3 in Hcl (22.5 gm /100ml cHcl3)
Mol. wt. 228.13
Reaction of antimony trichloride with Vit.A to yield a brillant Blue colour.
Carotenoid can also be estimated
Carotenoids accumulate in the golgi app. Of neuron can be estimated
Carotenoids –( Tetraterpenoids i.e. contain 40 C atom ) carotenoids are isoprenoids.
They are polyenes.
Generally yellow colour pigments.
Vit.A –Retinol i.e. vit. A when react with sbcl3 a lewis acid in CHCL3 a blue colour is
developed which may detected spectrometrically with ɻ max 620 nm.
Carotenoids & vit, A can be estimated by calorimetric assay using carr- price reagent.
Mechanism of action
The polyene system of a Retinoid & carotenoid is protonated by strong a line sbcl3 in
organic solvent to give a characteristic blue colour of high intensity.
Short coming of carr-price reagent:
1. Age of the reagent affects development & fading of blue colour.
2. Increase temprature affect stability of colour
3. Different batches of sbcl3 give discontent results.

49. Conclusion….
Here by it can be concludedthat when reagents are usedin optimized
volume and optimizedconcentration theybe successfully usedfor the
estimationof drugsin pharmaceutical preparations bothquantitativelyand
qualitatively

50. THANK YOU…