Chemical derivatization is an indirect quantitative analysis method used with UV-Vis spectrophotometry. It is needed because many compounds do not absorb in the UV-Vis region or absorb weakly. Derivatization reacts the compound with a chemical agent to form a derivative that has increased absorptivity and selectivity compared to the original compound. This allows analysis of compounds present in traces. Common derivatization methods include diazotization and coupling reactions, condensation reactions, reduction of tetrazolium salts, acid-dye methods, oxidation methods, and formation of metal-ligand complexes.

1. CHEMICAL DERIVATIZATION
Ravish Yadav

2. What is chemical derivatization
It is an INDIRECT METHOD OF QUANTITATIVE
ANALYSIS by UV-VIS spectrophotometry
NEED :
It is used because many compounds exhibit either very
weak or no absorbance in the UV or visible regions.
Hence such compounds can not be analysed by UV-Vis
techniques
.
.

3. What is chemical derivatization
NEED :
• INCREASES SENSITIVITY: Some compounds absorb
UV weakly but are present in traces eg impurities in drug
substance , drug in biological fluids like plasma , urine
etc. Hence the absorbance is low due to low
concentration. By converting in to a derivative that
absorbs strongly than parent , analyte can be determined
at low concentration
• INCREASES SELECTIVITY:
WHEN two or more uv absorbing species are present in
the sample then interference may occur.
.
.

4. •In order to analyse such compounds they need to be
converted to a DERIVATIVE by using CHEMICAL
REACTION
•Such methods usually involve adding an organic reagent/
derivatising agent which reacts with the parent molecule to
form a derivative. The resulting derivative has different
spectral properties than original analyte.
•SPECTRAL PROPERTIES means the ʎmax and
ABSORPTIVITY
•As the result of derivatization following changes occur:
•The ʎmax ----- of the derivative is higher than the original
molecule ----------Underoges bathochromic shift
•The absorbtivity ------- of the derivative is higher than the
original molecule- ----- HYPERCHROMIC SHIFT

5. What is chemical derivatization
ANALYTE
DERIVATISING
AGENT
DERIVATIVE
COLOURLESS
(ʎmax1,Ɛmax1)
By Chemical
reaction
COLORED
(ʎmax2,Ɛmax2)
ʎmaxof DERIVATIVE >>>> ʎmaX OF ANLAYTE
ƐmaxOF DERIVATIVE >>> Ɛmax OF ANALYTE
Hence the derivative absorbs more strongly and will give
a high absorbance even if present in traces

6. Examples of assay/ reactions based on chemical derivitisation
• Drugs used in Diazotization and coupling : Used for drugs containing
primary aromatic amine functional gp. E.G sulfadiazine
 Drug used in condensation reaction : isoniazide
 Drugs used in reduction of tetrazolium salts : corticosteroids
• Drugs used in acid – dye method : dye is methyl orange or
bromocresol
• Durgs used in oxidation method : ephedrine hcl
• Drugs used n metal – ligand complex :

7. DIAZOTIZATIONANDCOUPLING
Used for drugs containing primary aromatic amine functional gp. E.G sulfadiazine
Amin This is a two-step reaction:
STEP 1: DIAZOTISATION
Primary Amine of analyte is converted to diazonium salt by reaction with an aquoeus
soln of nitrous acid (Nitrous acid generated by the rxn of Hcl and sodium nitrite )
HCl + NaNO2 ------- HNO2
Diazonium salt is formed is colourless .
STEP 2 : COUPLING
Diazonium salt is treated with a suitable coupling reagent (Ar’-H)
Eg : phenol or aromatic amine to produce an azo derivative .

8.  azo deritatives are coloured .
 max absorption in the visible region .
 lambda max and emax is depends on the AR and Ar’
 Example of coupling reagents used : 1- napthol , 2- napthol and N-(1-napthyl)-
ethane -1,2-diammonium dichloride ) – the Brattons – marshall reagent which
gives high absorptivity .
 So , the sulfadiazine is coupled with brattons-marshall reagent which absorb
around 545nm.
 Due to its excessive conjugation .
.

9. OFFICAL APPLICATION OF THIS METHOD:
The sensitivity and selectivity method will permit the assay of low con
of impurities that a primary amine contain in the presence of amine
(parent sub)
Now, as per standards in British pharmacopeia some anime free
impurity tests are permitted like furosemide, iothalamic acid are
based upon diaztiosed and coupling . --- not clear explain

10. 2.CONDENSATION: involves loss of water
• Many colorimetric procedure are based on condensation Rxn
that occurs under suitable condition between amine and
carbonyl compound . The rxn invovle the nucleophilic attack
by the amine on the carbonyl carbon with the elimination of
water . So the substance containing a carbonyl group reacts
with a variety of reagents that containing an amine group :

11. • colorimetric assay of ketosterid is a selectivity rxn for steroid with keto group in
diffeerent position depending on the reagents .
• Reagent used : hydrazine or hydrazides
• Eg : ISONIAZIDE ( isonicotinic acid hydrazide ) reacts with 4-en-3-one in acidic soln to
form a derivative which gives yellow coloured with lamda max 400nm.
• As per standard the we can carried out the assay of nandralone deconoate injections
and betamethasone sodium phosphate injections as well as the tablets of
methyltstestoreone by using similar reagents reagents ie : 2’4-dinitrophenylhydrazine
.
• So , finally the hydrazone is formed on condensation of the 3, 17, 20 ketosteroids.
• The lipid soluble impurities is formed during rxn and is removed by the extraction
using chloroforms .

12. 3. REDUCTION OF TETRAZOLIUM SALTS
In the presence of steroid and alphaketone (21-hydroxy-20-
keto)
Tetrazoium salts are reduced to their coloured formazan
derivative.
corticosteroid assay using triphenyltetrazolium chloride . The
rxn is carried out in an alkaline medium
(tetramethylammmonium hydroxide) .
The absorbance occurred at 485nm of the red product .

13. the oxidation of the alphaketol group and the
reduction triphenyltetrazolium chloride to
triphenylformazan is take place .
As per standards the derivatives of corticosteroid
ie ( hydrocortisone acetate ) assay is carried out by
this method to yield a ester free 21 –
hydroxysteroid compound by removing esterified
group ,the precautions are taken in this procedure
is from light and oxygen

14. The acid dye method
• Here amine in its ionized form is added to an ionsed acid dye , eg:
methylorange or bromocresol purple , that yields a slt ( ion pair )
which is extracted into an organic solvent such as chloroform and
dichloroform .
• Indicator is added in excess, PH of aq soln is adjusted where both the
amines and dyes are in ionized form .
• Ion pair is seprated by organic solvent , and the absorbance is
measured with lamda max of the indicator of solvent .
• molar absorptivity is is high .
• More sensitive technique for amines . That absorps weakly into uv
region eg: hyoscine butylbromide.
• e max 202

15. The correct choice of PH is necessary it may permit the selective assay mix of an
amine and quaternary ammonium compound . .
as in the assay of a tertiary amine and a quaternary ammonium salts both the
substance are ionic at PH 3 , and the resultant absorbance of the ion pair is
extracted into an organic solvent which measusue total concentration , whereas at
PH 9 only the quaternary compound is ionised and form the extractable ion pairs ..
As , per the standards it is used for the assay of certain quaternary ammonium salts
or amine ie : biperadine lactate injection , clonidine hcl injection and
tablets

16. Oxidation method
•Oxidation of side chain of weakly absorbing compounds
containing a simple phenyl group produce a carbonyl
derivative that has a much greater absorptivity than the
parent compound.
•Oxidizing reagents are alkaline potassium permanganate
soln , acidified potassium dichromate soln or perchlorate
soln .
• eg : ephedrine or propanol amine to its corresponding
derivative of Benzaldehyde.,which exhibit absorption
around 240nm .

17. as per standards the assay of Ephedrine hydrochloric elixir , can be carried out it involves the
extraction of benzaldehyde into cyclohexane and measures the absorbance at lambda max
241nm .

18. Metal ligand complex
•Many organic reagents form complexes with metal atoms
by the formation of coordinate bonds
•Ligands with two or more donating group may share one
pair of electron and the chelate which formed by
multidentate ligands with metal atoms are often colored
and determined by visible spectroscopy .
• eg : STREPTOMYCIN INJECTION:
•Streptomycin is converted to maltol by alkaline hydrolysis
and maltol forms a PURPLE Colored complex with ferric
ions
• Absorbs at 520 nm