Lecture 15 - Analytical Methods Flashcards
What does pharmaceutical analysis do?
determines the quality of drug products via analytical chemistry
What are the variety of analytical techniques?
spectroscopic, chromatographic and diffraction
What does each technique play a role in?
determining the quality of the pharmaceutical product and excipients
How to analyse pharmaceutical products?
UV/Vis spectroscopy
high pressure liquid chromatography
How to analyse the solid product?
Infrared spectroscopy
xray diffraction
UV/Vis spectrum?
radiate a UV light source through the sample, molecules absorb different wavelengths of radiation and the light which isn’t absorbed reaches a detector which allows us to plot absorbance vs wavelength
What is the lowest energy transitions?
rotational
What radiation induces transitions within rotational energy levels?
microwave raidation
What induces transitions within the vibrational energy levels?
Infrared radiation
What induces transitions within the electronic energy levels?
UV/Vis wavelengths
What are transitions?
electrons jumping from low energy states to high energy states
What else is related to electronic?
fluorescence and phosphorescence - related to changes in electronic structure
At room temp, what states are most molecules in?
ground vibrational and electronic states
How do molecules absorb radiation?
if polychromatic light of the correct wavelength interacts with the molecules
What happens to certain types of electrons?
they will be excited to higher energy states (electron transition)
What is the excitation accompanied by?
invariably accompanied by an increase in the energy levels of rotational and vibrational states
Where can transitions occur between?
between low electron energy levels to higher electron energy levels
can also get transitions from within the rotational energy levels, e.g. from first rotational level in the lower state to first in the higher state
What type of electronic transitions do not follow the specific pattern?
one that goes from the highest rotational energy level on the lower level, to the first rotational energy level on the higher level
How does a conventional spectrophotometer work?
polychromatic light source hits a diffraction grate, which splits the light up into its individual wavelengths (spectrum)
the exit slit then selects particular wavelengths of light that have come off the diffraction grate and allow it to pass through our sample
the sample absorbs some of the light and what isnt absorbed passes through to the PMT, which amplifies the signal
What is the PMT?
photomultiplier tube
How does a PDA spectrophotometer work?
polychromatic light passes through the whoe sample and the light is absorbed by the molecules contained
the light which is not absorbed passes through the diffraction grating, which seperates out the polychromatic light into a spectra
we then have a photodiode array which is a series of photosensitive diodes and each wavelength of light hits a specific diode
Benefits of PDA spectrophotometer?
increases the speed of analysis and allows us to asses the effect of interfering peaks more accurately
they are more expensive and have few moving components
Disadvantages of conventional spectrophotometer?
require more maintenance because they have many moving components
What is beer’s law?
the intensity of a beam of parallel monochromatic light decreases exponentially with the number of absorbing molecules
absorbance is proportional to the concentration
What does lambert’s law state?
absorbance is proportional to path length
What is the beer lambert law?
A = A(1%, 1 cm).b.c
what is the A11 value?
the specific absorbance or specific absorbance coefficient
What is c in BL?
concentration in g/100ml (%w/v)
what is A in BL?
absorbance
what is b in BL?
path length in cm
What is A(1%, 1 cm)?
the absorbance of a 1g/100ml solution in a 1cm cell at a specific wavelength
What does light scattering in a sample cause?
absorbance > true, we overestimate the absorbance
What is light scattering?
the light will hit small particles in the sample, and will be scattered, hitting internal surfaces of the UV spectrophotometer
the light is no longer making it to the detector and the detector things its been lost
How can we prevent light scattering?
by filtering our sample
What can the beer lambert law quantify?
the amount of sample present or the amount of analyte in a sample
What does ionisation cause?
structural changes that can cause changes in the electronic system, this changes the UV spectra
it causes a shift in the absorption spectra
What is an issue with UV/Vis?
its lack of specificity
it does not allow us to discriminate between closely related structures
What is absorbance?
additive, so total absorbance may be from more than one compound absorbing at that particular wavelength
What is chromatography?
a separation technique
How does chromatography work?
we spot our mixture of component on the TLC plate of an appropriate solvent, and immerse this within the tank
the bottom of the TLC plate sits within the mobile phase and through capillary action it would be drawn up the plate
as the mobile phase migrates up the plate, it drags along the components at different rates
What affects the rate the components are separated at?
the interaction with the stationary phase
if it interacts more strongly with the stationary phase, it will be more retained
What is column chromatograpy?
mixture is applied to the column in solvent
applying a flow of eluate to this compound allows the components to start to migrate through the column
what is the stationary phase typically in chromatography?
silica gel
What does column chromatography allow us to get?
a chromatogram, plotting response against time
What does the chromatogram allow?
to quantify the use of the components within the mixture
What is the frequency of use of tablets and capsules?
tabs = 46% capsules = 13%
What is the frequency of use of oral liquids and injections?
oral liquids = 16%
injections = 15%
What are the physiochemical properties of solid dosage forms correlated to?
the crystal structure of the raw materials
What does the final product affect?
the stability and the bioavailability
What is an IR spectrum?
graph of absorbance or % transmission against wavelength or wavenumber
%T = It/Io and A = log Io/It
What is the wavenumber?
number of waves in 1cm
units are cm-1
What is energy of IR proportional to?
E = 1/wavelength
IR has less energy than..?
UV/visible light
What does absorption of IR cause transitions in?
vibrational and rotational energy levels
For a compound to be active in IR what must absorption cause?
a change in dipole moment of the molecule
Stretching vibrations?
symmetric and asymmetric
result in a shift in the dipole of the molecule
Bending vibrations?
in plane rocking
in plane scissoring
out of plane wagging
out of plane twisting
What do bending vibrations result in?
A change of the dipole moment of the molecule
What is a strength of IR?
The specificity
What is the lower region of the IR spectrum?
the finger print region
specific to each individual molecule
What can we use the finger print region for?
to determine the exact structure of our analyte
What is polymorphism?
the ability of a compound to crystallise in more than one 3D packing arrangement
What do different polymorphs have?
distinct IR patterns if we look at the finger print regions
What can polymorphism cause?
different dissolution and bioavailability
What is the unit cell of a molecule?
the smallest repeating unit
What is Bragg’s law?
nλ = 2dsinθ
What do crystalline forms show?
diffraction
What do amorphous forms show?
they do not diffract
What types of X ray diffraction are available?
powder or single crystal X ray diffraction
What does powder X ray diffraction give us?
a similar finger print pattern as IR
what does single crystal X ray diffraction give?
absolute structure of particular analyte
