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Chemical derivatization cont...

 Condensation reaction
 Reactions between amines and carbonyl compounds.
 Hydrazine and hydrazide reagents are used in many colorimetric
assays of ketosteroids giving yt " rivatives (Amax = 400 nm).

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Reduction of tetrazolium salts

 Alpha-ketol containing drugs are oxidized by tetrazolium salts to diketo-
compounds and the salt is converted to a red compound that absorbs at
485 nm.

°
N--e SHREYASI GHOSH RAY

•
Cl OH
1 N HN. + HCI
ON

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Acid-dye method

 Amine containing drugs can be converted to salts upon reaction with

acid-dyes.
 Acid-dyes are added in excess and the resulting salts are washed with
organic solvents to remove unreacted dyes.
 The ion-pairs thus formed have high absorptivities and thus suitable
for analysis of amines with weak UV absorptions.
 E.g. Clonidine HCI,injection, Biperidine Lactate lnj., hyoscine
butylbromide.

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Oxidation methods

 Absorptivity of compounds with carbonyl groups attached to benzene

rings are higher than corresponding alcohols.
 Thus compounds like ephedrine are oxidized to benzaldehyde
derivatives which have strong absorbance at 240 nm.

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Metal-ligand complexes

 Many drugs contain nucleophilic metal-chelating centers. Often

chelation is associated with changes in the oxidation state of the
metal ions and the chelate absorbs in the UV-visible region with
high
,
absorptivities.
 E.g. chelation of Iron(11) salts (ferrous) by catechols (ortho-hydroxy
phenols). Adrenaline added to buffered ferrous sulfate solution gives
purpule complex (Amax = 540 nm).
 Procaine and Adrenaline inj., lignocaine and adrenaline inj.

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Optimization of spectrophotometric
measurements/assays
 While developing a new analytical method following aspects are
•
considered.
 Sample preparation should be robust, reproducible and demonstrate
extraction of analyte from the drug rc, -mulation.
 Instrumental conditions need to be standardized. It

 Accuracy and precision of the method depends on stray light, solvent,

concentrations and path length.

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 Sample conditions
 Solvent — hydrophilicity, hydrophobicity, reactivity, functional groups
 Solubility of the analyte 4 log P, log D, pH.
 Water is ideal solvent, transparent above 180 nm, easy to purify and
cheap, pH must be controlled.
 Organic solvent absorb below 220 nm due to n 4 n* transitions (e.g.
ethanol, acetone), 11 4 rc* e.g. benzepe, toluene.
 UV cut off of some solvent ( ethanol: 205 nm, methanol: 210, ACN,
hexane, cychex: 210, Et2O: 220, CHCI3: 245, CCI4: 265, Toluene: 280,
Acetone: 330.

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 Instrument parameters
 Slit width
 Larger slit width leads to lower absorbance. Ideal is 1/10th of natural width of the absorption
•
band (which is width at half max-A).
 Scanning speed
 Faster scanning speed may lead to saturation of the response of the detector. Optimum is 1-
10 nm/s.

 Stray light
 Stray light due to scattering and reflection due to imperfect monochromators surfaces leads
to falsely low absorbances.
 Especially when A is high effect of stray light is more.
 Chemical effects k-

 Benzoic acid has different Xmax values for ionized (268 nm) and unionized (273)
states. Thus unbuffered solutions will show different A at different concerations.

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Structural analysis

 Absorbance of light in UV-visible region is due to the presence of

chromophores.
 Classically used for unsaturated compounds, but nowadays also used
for compounds with multiple c'„ onds —ene, -yne and carbonyl
groups.
Compound Chromophore Amax nm 125
Methane 185 8000
- 4
Oct-3-ene  C=C- 188, 279 900, 15

Acetone  C= 190 5000

GUDDATI SRI SOWMYA has left the meeting 0-
Ci me
C=N-

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