High-Performance Liquid Chromatography Flashcards
where does HPLC depend
nature of stationary phase
liquid-solid extraction
Adsorption chromatography
liquid-liquid extraction
Partition chromatography
based on the distribution of analyte between the mobile pase and stationary phase
separation principle
analyte
sample
mobile phase
eluent
stationary phase
packing material in column
how are compounds identified
difference in their peak’s retention time with prior injected reference standards
helps in quantifying specific compounds
chromatogram and related data
group of organic liquids used in HPLC
solvents
generates a flow of eluent from the solvent reservoir into the system
pump
- liquid that dissolves the target compound
- mixture of solvent can be used (polar and non-polar)
mobile phase
designed to remove gases from liquid such as the mobile pase in HPLC
degasser
main cause of noise and cycling in the baseline, and poor compressibility of the eluent
Gases
allows the introduction of a precise sample volume onto the column
injector
used in chromatography for separation of chemical compounds
HPLC column
- made up of an adsorbent material that has very small particle size
- particle size are kept uniform to obtain better performance
- part of column that interacts with the target compound
stationary phase
designed to allow to keep your columns at a constant temperature or utilize a series of temperature increases and constant hold temperatures
column heater
constant temperature
isothermal
temperature is raised from below (IC) to above (SEC) the CAP temperature if the sorption of the polymer solute to the stationary phase is a typical, exothermic process
temperature gradient elution
- monitor the compounds to quantify and identify the compounds separated in the column
- called as “eyes” for HPLC
detector
examples of detector in HPLC
- UV detector
- IR detector
- refractive index detector
- mass spectrometer
translates the signal generated by the detector into a graph called a chromatogram
recorder
safely collects all the mobile phase and sample components after they pass through the HPLC system
waste reservoir
Two common types of HPLC
- Normal phase HPLC
- Reverse phase HPLC
- utilizes a polar stationary phase (usually silica) and nonpolar (nonaqueaous) eluting solvents
- used to separate hydrophobic compounds and matrices
normal phase HPLC
what is usually used in polar stationary phase
silica
normal phase HPLC is used to separate what?
hydrophobic compounds and matrices
stationary phase is non-polar and the mobile phase is polar
reversed phase HPLC
example of application of HPLC
profile analysis of tissue homogenates in cervical cancer
