Instrumental Test 3
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retention time ( tr)
from the point of the injection to the time that the analyte is eluted form the column.
void time( tm)
The time that it take for n0n-retained species to get through the column and this usually shows up as a slight detection signal in the beginning of the elution.
Base width of the pek ( Wb)
This is in time units ( minutes, sec, etc,) and tells the width of the detected peak.
Two factors that determine solute separation.
- different retention
- narrow peak width
retention volume( Vr)
- the volume of mobile phase that it take to elute a peak off the column.
- void volume int he volume of mobile phase that it take to elute non-retained solute off the column
capacity factor (k')
- determined by the retention time and this is a universal measure
- IT IS INDEPENDENT OF COLUMN LENGTH AND FLOW RATE
Where is equilibrium achieved?
At the cent of the .eak?
k' values and what they mean
- k'<=1.0--> poor separation
- k> 30 --> slow separation
- k'=2-10, separation if optimum
The equilibrium constant for the distribution of A between the mobile phase and the stationary phase.
what happens when Kd increases ?
The solute's interaction with the stationary phase becomes more favorable and the retention time increases.
how does peak seperation relate to free energy?
peak sep, represents different changes in free energy
- N is dependent on the column length
- N is independent of the solute retention
Helped to distinguish small differences
plate height of height equivalent of theoretical plates ( HETP)
heigt of the theoretical plate that corresponds to the column length.
Why do band spread?
- eddy diffusion
- longitudinal diffusion
- stationary phase mass transfer
- Stagnant mobile phase mass transfer
- Mobile phase mass transfer
Eddy diffusion and MPMT
dependent on size and diffusion rate
- relate flow rate and linear velocity to H
- A represents eddy diffusion and MPMT
- B represents longitudinal diffusion
- C reps. SMPMS & SPMT
- H reps the total column hieght of a plate
- μ reps the linear velocity
Use to predict the effect of varying any of these parameters
separation factor( α)
α if greater than or equal to 1.1 is a goid separation.
- This is preferred over alpha since both retention (tr)and column efficiency are considered in defining peak separation.
- when the resolution is equal to or greater than 1.5 this represents baseline resolution/ complete separation of solutes
- RS=1 is adequate separation.
7 components of an HPLC
- precolumn filter,
- solvent reservoir and degassing system, sample injection system
- temperature control
what is the effect of particle size?
- plate height increases with increase in size of the particle
- small particles make the flow rate slow down and vice versa for large particles.
- Large particle have longer retention times, smaller N, and less pressure.
How is particle size related to efficency ?
Molecular ion peak
m/z that corresponds to the MW of the singly-charged molecules.
m/z less than the the M+ most intense m/z
Mass spectrum components
- inlet system
- ion source
- mass analyzer
- signal processor
hard ion source
- a lot of fragments
- less excess energy left in molecule.
Electron impact (EI)
- EI is a gas phase ion source and it is a hard source
- molecules are excited
- complex spectra
- helps with ID
- but poor determination of MW
- molecules must be thermally stable during vaporization
- molecules must be vaporized.
What causes there to be peaks with greater m/z than M+?
- Two things
- Isotopic peak and Collision Product Peak
what makes resorption and gas ion source different?
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