Drug analysis (2) Flashcards
Some causes for deviation from the Beer-Lambert law
Sample
- Contamination
- Precipitation
- Degradation (photolysis)
- Fluorescence
- Tautomerisation
- pH effects
- Temperature
Some causes for deviation from the Beer-Lambert law
Other
- Stray light
- Non-monochromatic light source
- Mismatched cells
- Sensitivity A-0.002
- Solvent absorption
- NB- Must establish the validity of the Beer-Lambert law for each drug under the measurement conditions to be used over an appropriate concentration range => calibration curves
Calibration curves
- Use at least 5 standard solutions spanning the working concentration range
- Measure in duplicate in a matched pair cells against the solvent as reference
- Ideal absorbance range optimum for a modern spectrophotometer: 0.1-1.0
- Exceptionally this can be pushed up to an absorbance 1.5

Instrumentation- single-beam spectrophotometers
- Intense source of UV light (Deuterium or hydrogen lamp)
- Prism of diffraction grating monochromator
- Capable of high precision (0.1-1.0 abs.unit)
- Used for absorbance determination at a fixed wavelength, not to obtain a spectrum
Instrumentation: double-beam spectrophotometers
- Similar to single beam instrument but
- Radiation split into 2 beams by rotating merror
- One beam passes through the sample
- The other beam passes through the reference cell
- The 2 beams are compared to give the absorbance suitable for fixed wavelength readings and whole spectra
Spectral bandwidth
- In some assays the minimum desirable resolution must be specified since changes in the spectral bandwidth (or monochromator slit width) can effect the absorbance of sharp peaks
- The BP (1980)- the spectral bandwidth used should be such that further reduction does not give an increase in absorbance
- Important for drugs having aromatic or strongly conjugated systems e.g. diphenhydramine and phenoxymethylpenicillin
Stray light
- Increase with instrument age
- Needs to be checked
- BP (1980)- absorbance at 200nm of a 1.2% w/v KCl aq must be >2 absorbance units relative to water as a reference
Quantitative applications
Pharmacological applications
- Single drugs
- Mixtures of drugs
- Colourimetric methods
- Tablet dissolution
- Limit tests for impurities
- Assays for bulk drugs or extracts
Other applications- physicochemical measurements
- pKa
- Velocity constants in enzymatic reactions
Single component systems
- Single component systems
- Establish linear range for compliance with the Beer-Lambert law using the centre of a broad maximum (calibration curve)
- Adjust the drug concentration within the optimum instrument range
- Measure the sample under identical conditions to the reference
- Problem- non-specific absorbance =>
- Difference spectrophotometry
- Second derivative spectrophotometry
- Chemical or physical transformation of the drug to shift the lambda max to a longer wavelength
Multicomponent systems
- The absorption spectra often overlap
- If the components obey the Beer-Lambert law and the law of additivity of absorbance applies then (see equation)

Multicomponent systems
- Simultaneous equations (one per component)
- Need
- Accurate absorptivity values
- Non-overlapping lambda max regions for the components
- The errors are very great for similar components
- Make derivatives for coulourimtric analysis
Derivative for colourimetric analysis
*

Derivative spectroscopy
- The absorbance (A) of a sample is differentiated with respect to wavelength (lambda) by computer
- Zero order- A=f(lambda)
- 1st derivative- dA/d(lambda)= f(lambda)
- 2nd derivative- dA2/d(lambda)2= f(lambda)
- Subtle changes of gradient in the normal spectrum (zero order) are observed as distinctive bipolar features
- Broad bands are suppressed relatice to sharp bands
- Increases with increasing order of differentiation
- This give selective rejection of broad additive spectral interferences such as rayleighs scattering
Derivative spectroscopy
- First derivative
- Represents the gradient at all points
- Can locate ‘hidden’ peaks (dA/d(lambda)= 0 at peak maxima
- Even-order derivatives
- The bipolar function of the alternating sign at the centroid (2nd- 4th+)
- Centroid coincides with the original peak maximum
- Centroid peak with decreases with increasing order of differentiation (useful for resolution of overlapping peaks)
- Satellite peaks become increasingly complex

Example 1


Example 2


Fluorescence spectrophotometry
- Very sensitive- better than absorption spectrophotometry (Io/I difficult at low concentrations)
- Measured against a dark background
- Selective- fluorescent drugs and their metabolites may be analysed more readily than by conventional spectrophotometry
- Format
- Scan Lambdaemmisions at fixed Lambdaexcitation
- Scan Lambdaexcitation at fixed Lambdaemmision
- Scan Lambdaexcitation and Lambdaemmision synchoronously

Fluorescence spectrophotometry
Signal intensity
- Generally more sensitive to the environment than absorbance measurements
- The signal intensity may be affected by
- pH (ionisable groups)
- Temperature (T, increased collisional quenching- use a thermostat)
- Quenching (formation of complexes between the sample and another species
- Interfering substances- often limiting in the analysis of biological samples; can be reduced by pre-treatment of the sample, use pure solvents, clean glassware
- Solvents
- Interference from Rayleigh and Raman scattering
Quantitative applications of fluorescence spectrophotometry
In dilute solution
- The total absorbance of the system (Epliso b c) must not exceed 0.005 absorbance units
- At high drug concentrations, ground-state molecules may absorb the fluorescence emitted by excited molecules
- => negative deviations from linearity
