Spectrophotometry Lecture 4 PDF

New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Instrumental Analysis (PMC 205) Semester 3 - Level II Students Spectrophotometry...

New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Instrumental Analysis (PMC 205) Semester 3 - Level II Students Spectrophotometry [Lecture 4] Prof. Dr. Rania El-Shaheny Professor of Pharmaceutical Analytical Chemistry  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ ◆ Objectives of the lecture After this lecture, you should know: ◼ Applications of spectrophotometry in qualitative analysis. ◼ Applications of spectrophotometry in quantitative analysis of organic and inorganic compounds. ◼ Selected examples for determination of pharmaceutical compounds by spectrophotometry  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Applications of UV-VIS spectrophotometry  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Several types of compounds absorb ultraviolet and visible radiation. This can be used for several qualitative and quantitative analyses. Absorption by Organic species: Organic compounds containing chromophores (e.g. carbonyl (C=O), azo (N=N), nitro (N=O), conjugated diene (C=C-C=C) and aromatic ring) exhibit characteristic absorption bands in UV or Visible range. In addition, conjugation between two or more chromophores tends to shift absorption maxima to longer wavelengths and increase in the intensity of absorption.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Absorption by Inorganic species: Absorption spectra for most of inorganic absorbing ions or molecules are similar to those of organic compounds. Generally, absorption by inorganic species depends on: (1) oxidation state of the element, (2) its position in the periodic table (3) type of the ligand bonded to its ions. Several inorganic compounds are colored and absorbs light in the visible region.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Absorption by Charge-Transfer Complexes: Charge-transfer absorption occurs for many inorganic and organic complexes. A charge-transfer complex is a strongly absorbing species that consists of an electron-donor group bonded to an electron-acceptor. Upon light absorption, an electron is transferred from the donor to an orbital in the acceptor. Ferric-thiocyanate complex Ex.: Ferric-thiocyanate complex is a charge-transfer complex having blood-red color.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ UV-VIS molecular absorption spectrophotometry is probably more widely used in chemical and clinical laboratories throughout the world than any other single method due to the following important advantages of spectrophotometric methods: 1- Wide applicability: they can be applied on a large group of organic and inorganic compounds as well as several biochemical species. 2- Easy, simple and rapid: in comparison with other sophisticated techniques. 3- High accuracy: they have very low relative errors encountered in concentration lie in the range from 1% to 3%. 4- Moderate to high selectivity: depending on the absorption wavelength. 5- High sensitivity ranges from 10-6 – 10-4 M  Applications of UV-VIS spectrophotometry Quantitative Qualitative Quantitative Quantitative determination of determination of organic species inorganic species  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ What is that Molar absorptivity () or specific absorbance A (1% - 1cm),  substance? max, and UV/VIS absorption spectrum are useful for identification of compounds. However, UV spectra are not enough to confirm the identity of the compound. Therefore, qualitative spectrophotometric data must be supplemented with other chemical or physical evidences such as infra-red (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS) as well as solubility, melting point and boiling point information.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Absorption characteristics of some common organic chromophores: Chromophore Example max (nm)  Alkene R.CH=CH2 177 13,000 Conjugated alkene CH2=CH.CH=CH2 214 21,000 Carbonyl CH3.CO.CH3 186 1000 Carboxyl CH3.COOH 204 41 Azo R.CH2.N=N.CH3 339 5 Aromatic Benzene 204 7,900 254 215  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ ◆ The spectrum is a physical constant, which along with melting & boiling points, refractive index beside other investigations (IR, NMR,& MS) may be used for characterization of compounds (analytes). Example: An absorption band at 254 nm with characteristic fine forked like structures may be an evidence for existence of aromatic structure (i.e: benzenoid structure). Benzene ring 220 240 260 280 Wavelength, nm  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Applications of UV-VIS spectrophotometry Quantitative Qualitative Quantitative Quantitative determination of determination of organic species inorganic species  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ ◼ UV-visible molecular absorption spectrophotometry is one of the most useful tools available for quantitative analysis. ◼ It has a great importance in several pharmaceutical and biological fields. Spectrophotometry Determination of drug concentrations  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ UV-Visible spectrophotometry can be applied to both: a) Absorbing compounds: by direct measurement. b) Non-absorbing compounds: by derivatization through their reaction with chromophoric reagents to produce products that absorb strongly in the ultraviolet or visible regions.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Quantitative determination of inorganic species 1-Determination of copper via ammine complex. Cu2+ + 4 NH3 [Cu(NH3)4 ]2+ (Azur Blue color) 2-Determination of ferric via thiocyanate charge transfer complex. Fe3+ + SCN- [ Fe(SCN)]2+ (Blood red color)  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 3-Determination of ferrous ion via 1,10-phenanthroline complex. 1,10-phenanthroline Red color  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Applications of UV-VIS spectrophotometry Quantitative Qualitative Quantitative Quantitative determination of determination of organic species inorganic species  Quantitative determination of organic compounds Spectrophotometry is used for quantitative determination of organic compounds such as drugs. Spectrophotometry is commonly used for the determination of drugs in pharmaceutical preparations like tablets-capsules-ampoules-vials. Also, Spectrophotometry is commonly used for the determination of drugs in biological fluids like plasma and urine.  Steps for a spectrophotometric determination 2- Drawing the Standard Calibration 1- Determination of max by drawing the Curve: Absorption Spectrum: Relation between A & c Relation between A &  using constant  (max) using constant c Absorbance Absorbance concentration λmax Wavelength 3- Determination of unknown sample concentration from the standard calibration curve: by measuring its absorbance and find the corresponding concentration from the calibration curve. 21  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ A) Direct determination of conjugated compounds: EX: UV determination of caffeine λmax = 273nm  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ B) Spectrophotometric determination of pharmaceuticals after derivatization. Chemical derivatization means reacting the analyte with certain reagent to give a colored product. This is usually performed for weakly absorbing drugs to increase the sensitivity and selectivity of the analysis. Derivatization depends on the chemical structure of the drug; nature of functional groups.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Weakly absorbing Derivatizing drug reagent Colored reaction product Higher sensitivity Measured by Higher selectivity spectrophotometer  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ ◆ Requirements for colored product to be used for spectrophotometric determinations: 1- Should be of strong color, to increase the sensitivity. 2- Should be unaffected by pH or the pH must be adjusted. 3- Should be stable with time. 4- The reaction of its formation, must be rapid and quantitative. 5- The colored product, should obey Beer-lambert’s law, i.e on plotting A versus C at fixed b, we obtain straight line passing through the origin.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Examples of derivatizing reagents for spectrophotometric determination of pharmaceutical compounds  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 1) 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) This is a chromogenic reagent for derivatization of primary and secondary amines. NBD-Cl Primary amines. -NH2 -NH- Secondary amines. NBD-Cl  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Example: Application of NBD-Cl for determination of the non-chromophoric drug pregabalin (anti-convulsant).  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 2) 2,4 dinitrofluorobenzene (DNFB) This is a chromogenic reagent for derivatization of primary and secondary amines. DNFB -NH2 -NH-  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Example: Application of DNFB for determination of the non-chromophoric drug alendronate (antiosteoporosis).  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 3) 1,2-Naphthoquinone-4-Sulphonic Acid Sodium Salt (NQS) This is a chromogenic reagent for derivatization of primary and secondary amines. NQS -NH2 -NH-  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Example: Application of NQS for determination of the non-chromophoric drug gabapentin (anticonvulsant). Gabapentin  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 4) Analysis of aromatic amines by diazotization and coupling This reaction involves 2 steps: 1- diazotization of the aromatic amine (drug) with NaNO2/HCl. 2- coupling reaction with Bratton-Marshall reagent Step 1: diazotization Diazonium salt Step 2: coupling reaction Bratton-Marshall reagent Azo dye  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Examples for drugs containing aromatic amine that can be determined by spectrophotometry after diazotization and coupling reaction: Procaine (local anesthetic) Sulfanilamide (antimicrobial)  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 5) Formation of metal complex Metal complexation of some drugs results in hyperchromic effect, and sometimes bathochromic shift takes place. So, metal complexation can be used for spectrophotometric determinations. Example: The use of complex formation for spectrophotometric analysis of gatifloxacin drug (antibiotic) based on Co(II), Ni(II) and La(III) ions Co2+ La3+ Ni2+  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ 6) Oxidation-reduction reaction This method depends on the ability of drug to act as oxidizing or reducing agent in a chemical reaction to give a colored substance that can be used for colorimetric determination.  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Example: Paracetamol is oxidized by ferricyanide. Ferricyanide is reduced to ferrocyanide. Ferrocyanide react with Fe3+ to give the highly colored complex Prussian Blue (ferric ferrocyanide).  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ ◆ Today we learnt together: ◼ Applications of spectrophotometry in qualitative analysis. ◼ Applications of spectrophotometry in quantitative analysis of organic and inorganic compounds. ◼ Selected examples for determination of pharmaceutical compounds by spectrophotometry  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________  New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ I- Complete the following sentences: - Ferrous ions can be determined colorimetry after reaction with ………… to produce ……. - ferric ions can be determined colorimetry after reaction with ………… to produce ……. II- Mention the requirements of a colored product to be used in spectrophotometric determinations. III- Mark () or (): 2- Derivatization increase the sensitivity, and not increase selectivity of the analysis. ( )