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retention time ( tr)
from the point of the injection to the time that the analyte is eluted form the column.
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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.
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Base width of the pek ( Wb)
This is in time units ( minutes, sec, etc,) and tells the width of the detected peak.
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Two factors that determine solute separation.
- different retention
- narrow peak width
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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
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capacity factor (k')
- determined by the retention time and this is a universal measure
- IT IS INDEPENDENT OF COLUMN LENGTH AND FLOW RATE
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Where is equilibrium achieved?
At the cent of the .eak?
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k' values and what they mean
- k'<=1.0--> poor separation
- k> 30 --> slow separation
- k'=2-10, separation if optimum
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KD
The equilibrium constant for the distribution of A between the mobile phase and the stationary phase.
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what happens when Kd increases ?
The solute's interaction with the stationary phase becomes more favorable and the retention time increases.
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how does peak seperation relate to free energy?
peak sep, represents different changes in free energy
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N
- N is dependent on the column length
- N is independent of the solute retention
Helped to distinguish small differences
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plate height of height equivalent of theoretical plates ( HETP)
heigt of the theoretical plate that corresponds to the column length.
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Why do band spread?
- eddy diffusion
- longitudinal diffusion
- stationary phase mass transfer
- Stagnant mobile phase mass transfer
- Mobile phase mass transfer
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Eddy diffusion and MPMT
dependent on size and diffusion rate
-
Van Deemter
- relate flow rate and linear velocity to H
- H=A+B/μ+Cμ
- 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
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Van deemter
Use to predict the effect of varying any of these parameters
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separation factor( α)
α if greater than or equal to 1.1 is a goid separation.
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resolution (Rs)
- 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.
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7 components of an HPLC
- Pump,
- precolumn filter,
- solvent reservoir and degassing system, sample injection system
- columns
- temperature control
- detector
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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.
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How is particle size related to efficency ?
Indirect relationship
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Molecular ion peak
m/z that corresponds to the MW of the singly-charged molecules.
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Fragment peak
m/z less than the the M+ most intense m/z
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Mass spectrum components
- inlet system
- ion source
- mass analyzer
- detector
- signal processor
- readout
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hard ion source
- a lot of fragments
- less excess energy left in molecule.
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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.
- sensitive
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What causes there to be peaks with greater m/z than M+?
- Two things
- Isotopic peak and Collision Product Peak
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what makes resorption and gas ion source different?
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