Pharmaceutical Analysis Flashcards
What are the 3 classic separation methods?
- Electrophoresis
- Chromatography
- Membrane Separation
What is chromatography?
When components of a mixture are separated based on differences in the rate at which they are carried through a stationary phase by a gaseous or liquid mobile phase
What is the stationary phase?
Fixed in a place either in a column or on a planar surface
What is the mobile phase?
Moves over or through the stationary phase, carrying with it the analyte mixture
What are the 4 different examples of chromatography?
- Thin Layer Chromatography
- Column Chromatography
- High Pressure Liquid Chromatography
- Gas Chromatography
How is chromatography used to separate?
- Mobile or stationary phase
- Both have two different compounds, two different sets of functional groups
- One will interact with it in a different way from the other with the stationary phase
What is elution? And what does it mean when it runs quicker down through the column?
- The process of washing samples components through the stationary phase by continuous flow of the mobile phase
- Less interaction with solvent
- When component A or B gets to the detector, it makes a reading so we can see a graph
What is a chromatogram?
- A peak that appears when the saturated component reaches the detector at the end of a column
- Components are then identified by unique retention time under a certain set of separation conditions
- Factors such as tR (retention time) include:
- velocity (flow rate) of mobile phase
- chromatographic retention
What does retention time normally depend on?
- Amount of time the component spends in the stationary or mobile phase
- More time in mobile means it passes through faster so the partition co-efficient is smaller
- More time in stationary phase then partition co-efficient is larger
What is the aim of resolution and where is it found?
- Found in the chromatograph
2. It’s aim is how well it can separate two peaks
What is baseline resolution?
When the detector goes to zero between the two peaks which is good resolution as you can clearly distinguish between the peaks after
What is column efficiency?
The plate height that are relative to the retention time and affect the band broadening
What is longitudinal diffusion?
- When it runs down the column and doesn’t stay in one tight band, it longitudinally diffuses to separate into a broad band
- The faster we run the column, the less our longitudinal diffusion, so it remains a tight band.
What is resistance to mass transfer?
- When the band broadens due to the resistance to diffusion of the molecule in the mobile and stationary phase
- DependIrs on diffusion co-efficient of compound in each phase, diameter and shape of stationary phase
- It’s parabolic flow profile: pressure driven flow
What is Eddy diffusion?
- When the mobile phase moves through the column which is packed with stationary phase
- The more the column is packed- the lower eddy diffusion
How do the parameters following increase or decrease column efficiency?
- Low flow rate
- Large particle size of stationary
- Irregularly shaped stationary phase
- Very fast flow rate
- Low flow rate- broadens peak due to longitudinal diffusion
- Large particle size of stationary phase- Increases eddy diffusion and mass transfer scales with particle size, this leads to peak broadening and decreases column efficiency
- Irregularly shaped stationary phase- Eddy diffusion decreases with increasing particle size and packing uniformity- irregular shaped will increase peak broadening- impacts on diffusion due to mass transfer
- Very fast flow rate- peak broadening due to resistance to mass transfer- diffusion in stationary phase
What does having a more efficient column mean?
Narrower peaks and better resolution
What does HPLC (high performance liquid chromatography) consist of?
Normal Phase:
- Polar stationary phase, non polar mobile phase
- Molecules elute in order of increasing polarity
Reverse Phase
- Non-polar stationary phase, polar mobile phase
- Molecules elute in order of decreasing polarity (most polar comes out first and least polar comes out last
- About 80% HPLC separations
Describe the normal phase in HPLC?
Consists of:
Stationary phase: Silica Gel with polar groups that are absorbed and retained
Mobile phase: hexane, dicloromethane, isopropanol, methanol (more polar mobile phase will elute compound more quickly)
What is elution?
The process of extracting one material from another by washing with a solvent
Describe the Reverse phase in HPLC?
Consists of:
Stationary phase: Octadecylsilane coated (ODS) Silica gel lipid groups adsorbed and retained
Mobile phase: Water, methanol, acetonitrile, tetrahydrofuran (the more lipophilic the mobile phase the faster elution of organic compounds)
Increasing in strength means increase in polarity
What do you have to consider when it comes to elution of neutral components?
Balance between polarity and lipophilicity of compound: H-bonding capacity
Example: Rapid elution of prednisoline and betamethasone
- Much longer elution of esters
What can you change for elution of ionisable compounds?
- Altering pH of the mobile phase can control solvent strength
- Works for weak electrolytes where solubility can be altered at working pH values in region of pKa
What are the two main types of HPLC detectors?
- One is responsive to physical and chemical properties of sample components- known as UV and fluorescence detectors
- Responsive to changes in properties of the mobile phase- known as refractive index detector
- non-specific and not as sensitive
- only used when other detectors like sugars cannot be used
What are the four types of HPLC detectors?
- UV absorption detector (most popular)
- fixed wavelength, variable wavelength with photo diode array
- mobile phase must not absorb UV - Fluorescence detector- selective and sensitive
- Refractive index detector
- Universal
- based on RI difference between solvent and solute
- Moderately sensitive
- very temperature sensitive - Mass Spectrometer
- structural information
What is HPLC-MS?
- An industry standard technique for the identification of chemicals in mixtures
- Very high sensitivity and specificity
