Chromatography

Chromatography

1. Definition

   • A process in which a chemical mixture is carried around over a stationary liquid or solid phase
   • Stationary Phase
     • A solid or liquid material that is either coated onto a thin layer or inert material such as plastic/ packed or coated
   • Mobile Phase
     • A gas or liquid that is allowed to flow over or through the stationary phase carrying the analyte with it
   • Polarity
     • Due to the uneven distribution of E in a molecule
       • O>N>F>Cl>Br>I
       • As you increase C:EN ratio the compound becomes less polar

2. Column Chromatography

   • the stationary phase is held in a tube and the mobile phase is forced through the column under pressure or gravity
   • Liquid
     • 4 TYPES
       • Adsorption
         • Stationary phase is a adsorbent solid
         • Polar groups on surface
         • molecules attracted to surface by dipole- dipole forces, hydrogen or weak VDW forces
       • Partition
         • Stationary phase is a liquid coated solid
           • liquid e.g. SiO2 / H2O
         • Normal Phase
           • stationary phase is polar
             • Silica
           • Mobile phase is non-polar
         • Reverse Phase
           • stationary phase is non-polar
           • ODS column
           • Mobile phase is Polar
       • Ion exchange
         • Stationary Phase has ionic bonded phase
         • stationary phase is a coatdd solid. it has cation and anions covalently bonded to it
           • Ions of opposite charge are bonded ionically
         • Separation occurs because, the smaller and highly charged an ion is, the stronger it will be retained in the column
       • Gel permeation
         • Stationary phase is a porous solid/ gel
         • Dependent on pour size
           • smaller molecules are allowed in the pours and therefore have to travel through the entire volume of the column
           • Larger molecules are excluded from the pours and therefore have less volume to travel through and elute more quickly .
           • Separation occurs according to the size of solutes
     • High performance liquid chromatography
       • overview
         • An extension of liquid chromatography, where particle size is smaller and the eluent is pumped around the column under pressure rather than relying on gravity
       • Column packing material
         • 1. Microspheres
           • used in ion exchange
           • Low mass transfer and High efficiency
         • 2. Porous microsphere
           • Good efficiency due to low mass transfer but have higher capacity
         • 3. Bonded phases
           • silica has different groups bonded on the surface
       • most HPLC use reverse phase
       • Nature of mobile phase
         • The more polar the mobile phase the more quickly it will elute a compound from silica gel column
           • Hexane<Dichromethane <isopropanol<methanol
         • The more lipophilic the mobile phase the more quickly it will elute a compound from a reverse ODS column
           • Water<methanol<acetonitrile<THF
       • Detector
         • Ideal detector
           • Rapid and reproducible response to solutes
           • Linear response
           • Stability of operation
         • Optical
           • Refractivity Index Monitors
           • UV/VIS detectors
             • Fixed wavelength detectors
             • Diode array Spectrometers
           • Florescence detector
         • Electrochemical
           • Conductivity dectector
             • Used in ION exchange chromatography
           • Redox electrochemical detectors
             • Useful for solutes that can oxidise and reduce
       • Structural Factors
         • balance between polarity and lipophilicity
         • The type of OH will indicate how well the OH will contribute to H-bonding
           • Tertiary OH group will be more sterically hindered than a primary OH and therefore form weaker H-bonds
       • Ionisable compounds
         • Control solvent strength of a mobile phase by changing pH, therefore you can alter pH to change retention times.
         • Carboxylic compounds are more polar than phenol compounds
         • if you had a basic drug that was eluting too fast from a reverse phase column
           • To increase retention time , make the mobile phase more basic
             • it will supress ionisation and become more
           • Acid + Base = Salt , more polar
             • This would decrease retention time (increase the elute to come out faster)
       • Help to aid with the identification of separate analytes
   • Gas Chromatography
     • Use compounds that reasonably volatile and heat stable
     • Gas-solid chromatography
       • Mobile phase = gas
       • stationary phase = stable adsorbent solid
         • Adsorbs vapours to various amounts according to their volatility and attraction to the solid .
     • Gas- liquid chromatography
       • Mobile phase= gas
       • Stationary phase = a liquid that is retained on the surface of an inert solid by adsorption or by chemical bonding
         • A wide range, almost all based on silica polymer
           • Methyl silicate, good for non-polar separations
           • Cyanopropyl phenyl methyl silicate, good for moderately polar compounds
           • Triflurosilicone , good for polar separations
       • volatility of the component at column temperature
         • As Carbon chain increases then BP increases
           • AS BP increases, compound becomes less volatile
       • The polarity (solubility) of the component in the stationary phase
       • compounds with the highest volatility and the lowest solubility come out first.
       • Column oven
         • Separation of volatile compounds in a mixture is dependent on temperature
           • The ability to increase the temperature can allow you to reduce time it takes to run the chromatogram and reduce band broadening in the later elute peaks
         • Types of columns
           • Packed column
           • Capillary column
       • Detectors
         • Response must be rapid and linear, preferably with a steep slope
         • measuring sensitivity
           • 1. Slope vs amount of sample graph
           • limit of detection
             • 95% confidence of a peak being an actual component= L.O.D.= MEAN + 2SD
               • 99%= Mean + 3SD
         • An ideal detector
           • sensitive
           • Linear response to solutes that extends over orders of magnitude
           • Temperature range to at least 400c
           • Quick response time
           • Has high reliability
           • highly predictable results
           • Should not destroy sample
         • Types
           • Thermal conductivity detector
           • Flames ionisation
             • most common
           • Electron capture
           • phosphorus/nitrogen
           • flame photometric for P + S compounds
           • Mass spectrum
         • Signals from the detectors can be recorded by a data recorder, in form of chart plotter or a computer
         • Derivatising molecules before they are run on the GC, can help molecules that are not volatile enough or they have strong affinity for stationary phase and cant be eluted from column
     • GCMS - allow the analyst to obtain structural information on the components in the mixture, and therefore identify components of the mixture
     • chromatogram
       • Definitions
         • t0= when compound is injected into the column
         • tm= when a compound has n o affinity to the stationary phase and comes out at the same time as mobile phase
           • This is also called dead time ( time for mobile phase to get through the column)
         • tR= the time taken for a solute that has some affinity to the stationary phase reaches detector after injection.
       • How good?
         • 1. Resolution
           • How well the peaks have separated
           • Greater the value of R , the better the resolution
             • R=1.5, GOOD BASE LINE SEPARATION
             • R=1 RESOLUTION NOT FULLY COMPLETE
             • R<1, SEPARATION NOT GOOD ENOUGH TO ALLOW THE ANALYSIS TO MAKE ANY QUANTITIVE ANALYSIS OF [ ]
           • Needs to use peak area quantitatively
           • R= 2(tr2-tr1)/ (wb1+wb2)
           • NO UNITS
           • How to improve
             • Increase column length
               • This is a disadvantage to efficacy as there is more time for diffusion, therefore broadening the bands
               • Increases theoretical plates (N)
             • Altering mobile and stationary phase
             • increasing efficacy and lead to narrow of peaks
               • E.g. low sample [ ]
         • 2. Efficacy
           • n
           • the broader/ thicker the peaks relative to its retention time, the less efficient the column is at eluting
           • Theoretical plates ( N )
             • where a single equilibrium occurs between the stationary and mobile phase
             • The more N, there are within a set length of a column, the better the efficiency the column
             • N=5.54X(tr/W0.5)^2
           • length of column is always in meters
           • Factors effecting efficiency of a column and causing band broadening in HPLC
             • molecules of a single compound (therefore having same properties) can take different lengths of time to travel through a column
               • the more time it takes for a analyte to travel through a column , the more the individual molecules with in the sample spread out and the broader the bands become.
                 • the more rapidly a peak broadens the less efficient the column is
             • 1. Multiple path effect
             • 2. Non-equilibrium mass transfer
               • When a molecule is more into on phase than another,
                 • There could be two molecules that are the same but because one molecule is more in one phase than the other then the two molecules will come out at different times.
               • Dead space= when molecules can't distinguish between the two phases.
             • 3. Sample size
               • Sample size has no effect on the ratio of solute in the two phases
                 • increase in [ ] of the solute should increase the [ ] in both phases proportionally
                 • if you increase the [ ] too much the S phase may become too saturated
                   • Leads to tail peaks
             • 4. Diffusion
               • Works to broaden peaks even when mobile phase is flowing
               • occurs mostly in mobile phase and is greater in gas than in liquid.
             • 5. Time column
               • The longer the compound is in the column , the more opportunity the molecules have had to diffuse
         • 3. Capacity Factor (K')
           • Void volume
             • Space between the stationary phase and the packaging
             • 0.7 X the volume of the packaging
             • knowing the rate at which the molecule flows, you can find out how long it takes for a molecule to pass through a void volume .
           • Is a measure of the degree with which a given compound partitions onto a given stationary phase from a given mobile phase.
             • the hight K' of a compound the more it retained on the column
               • The longer it takes to pass through the column
           • tr= (K' + Vo)/ Flow rate
       • Qualitative and Quantitative
         • Internal normalisation
           • USE: When equal amount of any compound will give the same peak area on a chromatograph
             • % of component = (area of component x peak/ total area of all peaks) X 100
             • This only works whenthe anayltes are very similar and the detector response is linear
           • Don't use as it can give in accurate data
         • Extrenal Sandardisation
           • A series of samples each containing various known amounts of the test substance are subjected to GC under identical conditions
           • A calibration curve of response area vs [ ]
           • Weakness
             • GC must be performed under the exact conditions
             • Accurate microliter volumes must be injected.
               • Very difficult in GC, manual injection is poor. (human error)
         • Internal Standard
           • Not needed in HPLC as it is quite reproducible if you can run an ESD curve on the same day as your sample to analyse
           • USE :Ointments and creams
             • Extraction procedure is not complete
           • added to the sample before it is extracted so any loses will affect both ISD and analyte
           • To overcome the problems associated with injecting in GC not being repeatable,
             • Add IS before injecting onto the GC.
               • Spiking
               • even if you inject different amounts the ratio of peak heights will always be the same
           • An IS must:
             • closely related to the assyed component
             • be completely resolved from both assay component and all other components of the mixture
             • ideally have a retention time close to that of the compound under analysis
             • used at [ ] similar to that youd expect to find in the sample under analysis