chromatography Flashcards
explain what chromatography is
method of separating components in a mixture that are distributed between a mobile phase and stationary phase, based on their interaction with the stationary phase.
give some examples of POLAR stationary phases
- silica
- alumina
- paper
example of a non-polar stationary phase
charcoal
explain partition chromatography
- both phases are liquid
- ## a liquid stationary phase is bonded onto silica support
why is chromatography needed in pharmacy
- drug design and manufactures
- quality assurance
-clinical trials
list some quality issues with chromatography in pharmaceuticals
- quality of actives (purity, identity, content)
- quality of excipients (raw materials)
- cleanliness of manufacturing plant.
- stability testing. including prediction of degradation pathways.
draw out a diagram of a gas chromatography instrument
COMPONENTS:
- gas cylinder on the left with regulator on top
- autosampler/injector
- inlet
- two columns with a modulator inbetween
- the gc oven
- detector feeding info to a computer
briefly explain the process of gas chromatography.
- a sample mixture heated 50degree C higher than its BP
- top of column is lower than heating block to encourage concentration condensation
- mixture carried through column by a continuous flow of inert gas.
what are GC columns packed with?
solid particles or a solid support coated with a liquid either by adsorption or chemically bonded to it
difference between capillary and packed columns?
capillary much smaller diameter but a lot longer.
3 types of capillary columns?
- wall coated open tubular(WCOT)
- Support-coated open tubular(SCOT)
- porous-layer open tubular (plot)
what effect does mcreynolds constant have on the stationary phase?
higher the constant, the more polar the stationary phase
why is derivatization used in GC
- increases volatility, decreases polarity of analytes
- decreases thermal degradation of analytes
- improves detector response
- improves separation and decreases band broadening
- improve extraction efficiency
what are some disadvantages to derivatization in gc
- derivatizing agent can be difficult to remove and interferes with analysis
- can cause unintended chemical changes to analyte
- increases analysis time
- can cause extra response
what are the three types of derivatization in gc
- alkylation
- acylation
- silylation
how do you calculate resolution
difference of retention divided by mean of peak base widths
explain plate theory
says that there are theoretical plates along a stationary phase. the more plates the more efficient the chromatography column is
over what digit, does resolution be considered ‘valid’
over 1.5
what three principles are considered for Rate theory
- eddy diffusion (A term)
- longitudinal molecular diffusion(B term)
- resistance to mass transfer in the liquid stationary phase (C term)
draw out a diagram of a liquid chromatography apparatus
- mobile phase reservoir
- leading to pump beside injection valve.
- column followed by detector
- detector leads to waste and data recorder
what force is required for LC
very high pressure
what is the difference between normal phase and reverse phase HPLC
- normal has a polar stationary phase(silica) and less polar mobile phase
- reverse phase is, you guessed it, the reverse.
reverse would use water or methanol as mobile.
90% of time reverse phase is used
suggest how you would manipulate the separation of HPLC
- change the composition of mobile phase (increase water content, increase organic modifier.)
- change polarity of the column
- heat the column
- derivatisation
why is HPLC detection useful
- used for quantitative and qualitative analyses.
- analyzing peaks on a chromatogram
- finding the identity of analyte(s) within each chromatogram peak. (detector selectivity)
list three types of detectors and rank their selectivity
- mass spectral detection (MSD), the greatest selectivity
- UV & Fluorescence detectors, much better selectivity.
- Refractive index detectors. (RID) low selectivity
how would you modify a system to enhance detector selectivity
derivitisation of analytes
explain what a chromophore is
region in a molecule where the energy difference between 2 different molecular orbitals falls within the UV or visible spectrum
explain pre-column derivatisation and why its done
- analyte/sample undergoes derivatisation before separation on column
- any reaction conditions can be made (time, temp, reagents)
- however it might alter chromatographic properties like resolution or elution order of analytes
explain post column derivatisation and possible disadvantages
- occurs on-line between column and detector so doesnt affect resolution
*disadvantages are: - might need to alter HPLC system to introduce reaction chambers
- reaction conditions employed must be kept moderate
- reaction must complete within 30 secs
explain the layout of a CZE setup
- capillary with tube diameter less than 1mm
- filled with a running buffer
- one end has a ‘destination vial’
- other end has a source vial with an anode
give a brief description of Electro Osmotic Force. ie. buffer, silica, interactions, layer
- capillary is made of fused silica so have Si-OH at inner surface
- running buffer above Ph 3 has OH minus ions
- these interact causing this eqn through de-protonation:
Si-OH + -OH -> SiO- + H2O - SiO- groups attract postive cations ie Na+ from buffer.
- this then forms a fixed layer held through non-covalent interactions, but is negative.
- a 2nd layer of cations form on fixed layer, which is deemed the mobile phase.
why is CZE used as a form of chromatography
- an electrically driven system is uniformly distributed along the capillary.
- thus there is no pressure drop and velocity of flow is uniform.
- this cause a lot less band broadening when viewed after detection
