Professional Documents
Culture Documents
P Analysis
P Analysis
Pharmaceutical Analysis
Spectrophotometric Analysis High Performance Liquid Chromatography Gas Liquid Chromatography Radioimmunoassay Enzyme Multiplied Immunoassay Fluoroimmunoassay
Assay Steps
Separation (Dosage form - Biological Sample)
Centrifugation Extraction Chromatography Immunoassay
Quantitation
Spectrophotometric (absorbance, fluorescent) Radioactivity
Separation Step
Centrifugation
Excipients Macromolecules
Extraction
pH-pKa partition Lipid/Aqueous solubility
Chromatography
Adsorption, partitioning, molecular size, ion-exchange
Immunoassay
17 January 2001
Quantitation Step
Absorbance Fluorescence Radioactivity Electrochemical Other
Spectrophotometric Analysis
Theory
Beer-Lamberts Law
Equipment
Single Beam Double Beam
Application
Quality control HPLC
Absorption Spectroscopy
Absorption
Absorption by Molecules in Solution Electronic, Vibrational, and Rotational Energy
Bonding Electronic Transitions
* * * *
C O
Antibonding
17 January 2001
Energy Absorption
A b s o r b a n c e
Wavelength
0.6 0.4 Light Out 0.2 0 0 0.25 0.5 0.75 1 Pathlength Through Solution
Beer-Lamberts Law
Absorbance proportional to path length and concentration A=abc where A = absorbance and a = absorptivity (), b = pathlength, and c = concentration If b is 1 cm and c is in g/100 ml the absorptivity is 1% given as A1%cm at wavelength x. Absorptivity may also 1 be called the extinction coefficient or absorption coefficient
17 January 2001
Redrawn from: Bauer, H.H., Christian, G.D., and O Reilly, J.E. 1978 Instrumental Analysis, Figure 7.14, page 187
Application
Standard Curve
1.6 1.4 1.2 B J B
Absorbance
Concentration (% of stock)
17 January 2001
Fluorescence Spectroscopy
Molecule absorbs energy and immediately emits energy at a higher wavelength (lower energy) Excitation and emission wavelengths specific to the compound Emission measured at 90 to the excitation light path Emission proportional to drug concentration
Fluorescence Transitions
Excited State Absorbance Excitation Ground State
Fluorescence Emission
Fluorescence Instrument