Contains procedure examples and also applications and principles

1. BY
Ch.lakshmi kalyani
Y14MPH432
CHALAPATHI INSTITUTE OF PHARMACEUTICL
SCIENCES
PRINCIPLES AND APPLICATIONS OF
MBTH, FC AND BM REAGENTS

2.  INTRODUCTION TO REAGENTS
 REAGENTS
a. MBTH REAGENT
b. FC REAGENT
c. BM REAGENT
 REAGENT PROFILE
 PRINCIPLE AND MECHANISIM
 PROCEDURE
 EXAMPLES
 APPLICATION
 REFERENCES
 CONCLUSION

3. • Reagents are the substances used for the detection or
determination of another substances by chemical are
microscopical means.
• Reagent is a substance or compound that is added to system
in order to bring a chemical reaction.
• These are mainly used for both qualitative and quantitative
analysis.
• There are a number of reagents are used for determination of
different pharmaceuticals some of them are MBTH ,FC ,PDAB
,NQS and BM etc…

4.  It is chemically called as phosphomolybdotungistic
acid.
 Also known as hetero poly acids.
 Used for colorimetric estimation of drugs containing
phenols and amines.
 It is a mixture of phosphomolybdate and
phosphotungstate.
 It consist of :
Sodium tungstate
Sodium molybdate
85% phosphoric acid

5.  Synonym : Folin ciocalteu / Folin phenol / Folin
denis/
phosphomolybdotungistic acid
 Color : Clear bright yellow
 Storage : Tightly capped at room temperature
Hexavalent phosphomolybdotungistic acid complexes with
the following structures formed in the solution.
3H₂O₅.P₂O₅13WO₃.5MoO3.10H₂0
3H₂O.P₂O₅.14WO₃.4MoO3.10H₂O

6.  When FC reagent reacts with drug in presence of reducing
agents like sncl₂, ascorbic acid hydrazine, probably drug
effects reduction of 1 or more oxygen atoms from tungstate
or molybdate in the f-c reagent, there by producing one or
more possible reduced species which have characteristic
intense blue color.
 Reaction:-
Phosphomolibdo tungstate reducing agents Molybdic /tungstic
blue.

8.  Used as a spray reagent in many chromatographic
procedures.
Used in Lowry method for determining protein
concentration.
It is also reactive towards thiols and many vitamins.
Used for estimation of several drugs like
Aspirin
Piroxicam
Acetazolamide
Pyridoxine HCL
Omeprazole
And also spectrophotometric determination of dastinib at
765nm and Cefadroxil at 970nm.

10. Kept aside for 10 minutes and scan the spectrum
measure the absorbance
From the above solution pipette out 0.2,0.4,0.6,0.8,1.0 to
that add 0.8ml of f.c reagent+4ml of Na₂co₃ solution
Weigh a powder of 10 mg and dissolved in 10 ml of sod.
Hydroxide solution(1000μg/ml) from that prepare
100(μg/ml).

12.  IUPAC name : 3-methyl-2- benzothiazoline hydra
zone
 Molecular formula : C₈H₉N₃S.HCL
 molecular weight : 233.72
 Color : white
 Solubility : freely soluble in distilled water
 Synonym : MBTH
Mbth monohydrate
Sawicki’s reagent
Besthorns hydrazone

13.  MBTH first reacts with aldehyde to from azine. Only if there
is remaining MBTH, it is oxidized to another species which
combines with the azine to form formazon.
 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 MBTH, the amount of aldehyde
can be tested.
Less aldehyde - more blue color
More aldehyde - less blue color

14.  With phenols:
With phenols under suitable conditions MBTH loses two
electrons and one proton to form the electrophillic intermediate.
This undergoes electrophillic substitution with phenols and other
groups to form the colored product.
Mechanism involved is
1. oxidative coupling
2. dehydration

20.  MBTH is used in the analysis of drinking surfaces saline
waters, domestic and industrial waste.
 Used in the estimation of drugs like acyclovir, ganiciclovir,
ceftazimide and Cefadroxil, nicornadil.
 Used in identification of groups such as
 Aldehydes
 Amines
 Phenols
 Aryl amines
 Used for estimation of disinfectants.

23. Preparation of stock
solution - Dissolve 10
mg of crude drug in 10
ml of methanol
Construction of
calibration curve – From
stnd solutions pipette
out 0.2,0.4,0.6,0.8 to
that add 2ml of
0.2%fecl₃ and MBTH
reagent kept aside for 10
mins n make up with
water n measure the
absorbance.

24.  It is chemically n-1- naphthyl ethylene diamine
dihydrochloride.
 Widely used for the determination of drugs and
pharmaceuticals containing free primary aromatic amino
group.
 At present it is employeed for the determination of sulpha
drugs and local anesthetics.

25.  Iupac name : N-1-naphthyl ethylene diamine
dihydrochloride
 Molecular formula : C₁₂H₁₆CI₂N₂
 Molecular formula : 259.18
 Color : White to light tan or gray crystalline solid.
 Synonym : Monomethanolate / Bratton reagent
It contains - N-1-naphthyl ethyelenediaminedihydrochloride.0.05%w/v
Ammonium sulphate0.5%w/v
Sodium nitrite 0.5%w/v
Hydrochloric Acid 1.5M

26.  The primary aromatic amine group is first diazotized with
sodium and HCL. The excess nitrous acid (HNO₂) IS neutralized
by treating with ammonium sulphamate reagent. Finally the
diazonium ion is allowed to couple with BM reagent to produce
a highly colored azodye complex measured at 550nm.
HCL Coupling with reagent
R-NH₂+HNO₂ R-N⁺ = N Cl⁻ . diazonum
compound

27. Dissolve 100mg of N-1-
naphthyl ethylene
diamine dihydrochloride
In 100ml of mixture
I,e 7 parts of acetone
and 3 parts of water

28.  Used for spectrophotometric determinations of amino
phenols, phenylenediamines,
dinitroanilines,choloroanilines,thiols, and sulfonamides.
 Used for estimation of
chloromphenicol,sulphamethoxazole,dapsone,folic acid.

29.  ESTIMATION OF DAPSONE BY BM REAGENT:-
PREPRATION OF
STOCK
SOLUTION:-
dissolve 10mg of
dapsone in 10ml
methanol(1000μg/
ml) from that
100μg/ml
preparation of
calibration curve:-
from 100μg/ml
pipette out
0.1,0.2,0.3,0.4,0.5 to
that 1ml 5n hcl+1ml
0.1% Nano₂ maintain
cool conditions for 10
mins after to that 1ml
of 0.5% ammonium
sulphate and 1ml Bm
reagent at 543nm

30.  It is chemically called as 1,2 napthaquinone-4-
sulphonate sodium.
 It is a chromogenic reagent used for estimation of
primary aromatic amines and it is a derivative of o-
quinone.
 It will produce a bright red color in alkaline solutions and
is also fluorescent.

31.  IUPAC name : sodium-3,4 dioxo-3,4
dihydro
napthalene -1-
sulphonate
 Molecular formula : C₁₀H₅NaO₅S
 Molecular weight : 260.2g
 melting point : 289˚c
 Synonyms : Beta napthaquinone-
4- sulphonate
sodium salt/ NQS
Reagent.

32.  The mechanism of action of the NQS reagent involves
when the NQS treated with the any amine-containing
compound that will release the hydroxyl group and the
sodium sulphonate group is replaced with the aromatic
amine group.

33.  NQS used to determine drugs which contains amines and
amino acids.
 Determination of aromatic amines in industrial waste
water;
 It is used in determination of Amphetamine, meta
amphetamine in urine by RP-HPLC.
 By using visible spectrophotometric method, flurometric
method, and HPLC method pharmaceuticals can be
estimated in reactions with NQS.

34. After make up with distilled water scan and measure the
absorbance.
From the stock prepare 0.1,0.3,0.5,0.7,0.9ml to that add
1ml of NAOH, 1Ml of NQS regent and kept aside for 10
mins
10 mg of Cefadroxil is dissolved in 10 ml distilled water
(1000μg/ml)

35.  Here by it can be concluded that when reagents are
used in optimized concentration they be successfully
used for the estimation of drugs in pharmaceutical
preparation both quantitatively and qualitatively .

36.  www.ijmca.com.
 www.pharmaresearchlibrary.com/ijmpr.
 http:// www.safaribooks
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