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: soluble in water, chloroform, ether, acetone, methanol, dimethyl formamide and in alkali hydroxides : high ceiling efficacy diuretic
Category
METHODS OF ANALYSIS:
1. Titrimetric method 2. Column chromatography 3. Thin layer chromatography 4. Gas chromatography 5. High performance liquid chromatography 6. Ultraviolet spectroscopy 7. Colorimetric method 8. Infrared spectroscopy
TITRIMETRIC METHOD:
Principle: Acid-base titration Reaction:
COOH NH O
H2NO 2S
NaOH
COONa NH O
H2NO 2S
Procedure:
0.5g of furosemide dissolve
Titrated with 0.1N NaOH End point is blue colour. Each ml of 0.1NaOH = 0.03308g of C12H11ClN2O5S
COLUMN CHROMATOGRAPHY:
Principle Column Adsorbent : Column adsorption chromatography : Stainless-steel column(15cm x 4.5cm) : Lichrosorb RP-18 or RP-8
Sample
: 0.5ml of plasma with 1ml methanol is centrifuged, a 50l of the aliquot of the supernatant liquid is used.
: 3hours
Drying time
HPLC:
Sl Stationary No phase
1. Bondapack C18 corasil (1.8x2mm) Bondapack C18 column (30x4mm) Lichrosorb RP8 (5)
Mobile phase
Retention time
3min
0.02M KCl-HCl 0.9ml/min U.V at buffer pH 2 280nm 10mM (NH4)2HPO4 in 25% methanol 2ml/min U.V at 254nm
2.
3min
3.
Methanol1ml/min 0.02M, phosphate buffer of pH 3.0(1:1) Methanol: water: acetic acid (40:57:3)
Flourimetric detection
3min
4.
3min
ULTRAVIOLET SPECTROSCOPY:
Sample : Lasix (furosemide preparation) Solvent : Water Absorbance maxima : 228nm E1%1cm : 945
COLORIMETRIC METHOD:
Furosemide produces blue colour when it reacts with butylamine,carbonyl chloride and acetic acid in a medium of anhydrous methanol.
INFRARED SPECTROSCOPY:
Frequency(cm-1) 3350-3400 1671 1576 1322 582 Type of vibration NH C=O NH -S=O Cl Assignment C-NH -COOH -NH2 -SO2 C-Cl
GAS CHROMATOGRAPHY:
Principle Sample : : Partition Trimethyl derivative of furosemide
Carrier gas :
Column
Nitrogen
Temperature : 245oC
Detector :
63Ni
electron-capture detector
SCOCH
Molecular formula : C24H32O4S Molecular weight : 416.59 g/mol Appearance : Crystalline powder
Colour
Odor
: Yellowish-white
: Faint mercaptan
METHODS OF ANALYSIS:
1. Thin layer chromatography 2. Ultraviolet spectroscopy 3. Colorimetric method 4. Infrared spectroscopy
5. Flourimetric analysis
Example 2: Solvent system : Benzene:Ethyl acetate:Methanol(73:25:2) Adsorbent : Silica gel G Detection : Spray with phosphomolybdic acid and heat at 80oC for 10 minutes Rf value : 0.67
INFRARED SPECTROSCOPY:
Wave number (cm-1) 1775 1670-1690 1620 Assignment 5 -membered,lactone carbonyl 3- ketone,7-thioester carbonyl 4,5-double bond
ULTRAVIOLET SPECTYROSCOPY:
COLORIMETRIC METHOD:
Reaction of spironolactone with methanolic hydroxylamine hydrochloride and ferric perchlorate yields a red ferric hydroxamate complex having an absorbance at about 515nm. Colour is stable upto 2hrs. Isonicotinic acid hydrazide may be reacted with spironolactone in methanolic solution,yielding a soluble yellow product having an absorbance at 575nm.
FLUORIMETRIC ANALYSIS:
Spironolactone
4,6-dienone,canrenone
AMILORIDE
Structure :
.HCl.2H2O
Mol formula : C6H8ClN7O.HCl.2H2O Mol weight : 302.12g/ml Description : Yellow to greenish yellow crystalline powder which is odorless. Solubility : Freely soluble in Dimethylsulfoxide, sparingly soluble in water, very slightly in ethanol. Brand name : MIDAMOR
METHODS OF ANALYSIS :
Non aqueous titration Thin layer chromatography UV spectroscopy HPLC IR spectroscopy
1. Non aqueous titration : Principle : Substances which are either too weakly basic or too weakly acidic to give sharp end point in aqueous solutions are determined by non-aqueous method. Procedure : Dissolve 450mg of sample in 100ml of glacial acetic acid Add 10ml of mercuric acetate. Add 15ml of dioxane. Titrate with 0.1N perchloric acid using crystal violet as indicator until a blue color. Blank determination is done to make necessary corrections. Each ml 0.1N perchloric acid = 26.61gm of Amiloride hydrochloride.
HClO
ClO 4
CH 3COOH HClO
CH 3COO
CH 3COOH
+ 2
ClO 4
2 CH 3COOH
O Cl N
NH2 Cl .HCl.2H 2O N
NH2
+
CN CNH 2
CN C NH
CH3COOH 2
H2N
NH2
H2N
NH2
Amiloride HCl
Amiloride hydrochloride is analyzed by Normal phase TLC on silica gel by employing two solvent system ;
Solvent system
Rf value
Sensitivity
0.01%
By UV at 254nm or 360nm.
UV spectroscopy :
Principle : Any molecule has either , and or a combination of these
electrons. Bonding ( and ) & non-bonding () electrons abosorb the characteristic radiation and undergoes transition from ground state to excited state. By this characterization absorption peaks are obtained. There by the nature of electrons present and hence the molecular structure can be elucidated.
of 0.01N aqueous hydrochloric acid. Absorption maxima is approximately at 212nm, 285nm and 362nm . Molar absorptivity of 1% drug solution in a 1cm cell is 642, 555 and 617 for the wavelengths of 212nm, 285nm and 362nm respectively.
5. IR spectroscopy : Principle :
For any molecule to absorb IR radiation it should posses, Dipole moment Applied frequency of IR must be equal to natural frequency of radiation. IR spectra is nothing but a fingerprint of a molecule.
Procedure :
1) IR spectrum in a KBr pellet was obtained using a Fourier-transform (FT IR ) spectrometer. 2) IR spectrum obtained in a mineral oil called mull ( nujol )
IR spectral assignments :
Frequency ( cm-1 )
3250 -3500 3150 1680 1640 1600 1240 770
Assignments
N-H stretch ( NH2) N-H stretch (NH) C=O stretch H2 deformation mode H2 deformation mode N- (C6H6) stretch C-H out of plane mode
REFERENCES :
Text book of Pharmacology by P.D Seth. Pg 292 303. Analytical Profiles of Drug Substances by Klaus Florey, vol 15, Pg 1 30. Clinical Pharmacology by P.N.Bennett and M.J.Brown. 9th edition, Pg 529 547. Modern Pharmacology by Charles.R.Craig and Robert.E.Stitzel,3rd edition, pg 270 270. The Pharmacological basis of Therapeutics by Goodmann and Gilman, 8th edition, vol 1, pg 708 730.