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HPLC (Basic principles (Pump (Generates eluent flow from solvent to system…
HPLC
Basic principles
Pump
Generates eluent flow from solvent to system at constant pressure
Injector
Allows repeated injection at scheduled timing.
Column
To separate analytes in a sample
made up from PEEK
Detector
To observe the obtained separation
By measuring differences in composition of eluent
Degasser
Detect gases in system
Helium purging
Removes 80%
Vacuum degassing
Removes 60%
Sonication
Removes 30%
Recorder
Data processor of electronic signal
Effects of Resolution
Retention
Measure retention of analyte to eluate (Rt)
Solevent must eluted first then followed by compound
Shorter Rt is better
tR1-tR0/tR0
Effeciency
The higher the peak, the nore efficient
Particle size affect plate count affect efficiency
Use longer column
allow compound interact longer with stationary phase
Change particle size
plate count increase
Use different stationary phase
Selective
Ability of system to chemically distinguish between sample component
tR2-tR0/tR2-tR0
Change composition of mobile phase
Change pH of eluent contains ionise group
Type of HLPC
Normal phase
Stationary phase
polar
Mobile phase
non-polar
Non-polar compound eluted faster
minimal interaction with Stationary phase
short retention time
Reverse phase
Stationary phase
non-polar
Mobile phase
polar
Polar comppund eluted faster
minimal interaction with Stationary phase
short retention time
Type of analysis
Paper chromatography
Qualification only
HPLC
Qualification
Identify Rt with the sample
Retention time
1 peak
Quantification
Different concentration of same standard
Many peak
AUC
Tye of elution
Isocratic
Mobile phase
One type of solvent
Mixture of solvent
Composition of mobile.phase remains constant
Gradient
Mobile phase
Change the ratio of polar to non-polar compound