17 January 2001

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

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Energy Absorption
A b s o r b a n c e

Wavelength

Peaks are broadened by vibrational and rotational energy transitions

Absorption Through a Solution

Light in 1 0.8
Absorbance

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

Double Beam Instrument

Redrawn from: Bauer, H.H., Christian, G.D., and O Reilly, J.E. 1978 Instrumental Analysis, Figure 7.14, page 187

Single Beam Instrument

Application
Standard Curve
1.6 1.4 1.2 B J B

Absorbance (540 nm) Absorbance (570 nm)

J J J J

Absorbance

1 0.8 0.6 0.4 0.2 J 0 0 20 40 60 80 100 J

Concentration (% of stock)

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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