Module 12: Chromatography Flashcards
what is chromatography used for
separation, detection and quantitation of a complete mixture of analytes in a sample
What is separation based on
different physical interactions between individual components and the stationary phase of the system
2 phases
mobile and stationary
Mobile phase
carries the sample through the stationary phase
sample compounds that have a greater affinity for the mobile phase will prefer to
stay in it
causing them to travel faster than those samples that have a greater affinity to remain in the stationary phase
Ion exchange
solute mixtures are separated by the charge of ionic species
Ion exchanger
stationary phase
porour solid whose surface is covered with ionic sites (pos or neg)
2 types of Ion exchange
cation: stationary phase carries neg charge - selects cations from sample
anion: stationary phase carries pos charge - selects anions from sample
Steric exclusion
gel filtration/gel permeable
solute mixtures separated based on size and shape
stationary phase is porous and traps molecules inside pores
Adsorption
based on competition between the components in the sample and the mobile phase for adsoptive sites on the solid stationary phase
components stick to sites on surface of stationary phase “velcro”
Partition
separation is based on polarity
Normal or reversed
As sample moves through column with mobile phase, separation is obtained as the polar or non polar compounds interact with he two phases
normal partition
mobile phase is NONPOLAR
Stationary phase is POLAR
Reverse partition (more common)
mobile phase is POLAR
Stationary phase is NONPOLAR
Affinity
separation based on specific interaction between one kind of solute molecule and a second molecules that is immobilized on a stationary phase
Immobilizing molecule called a ligand
ligand
immobilizing molecule in affinity chromatography
may be an antibody, protein or specific resin group intended to bind a specific analyte
most selective type of chromatography used
Can be used to separate glycosylated hemoglobin (hba1c) from other hemoglobins
binds to boronate group of stationary phase
2 large groups of chromatography
plane and column
plane chromatography
paper
thin layer chromatography
paper chromatography
sheet of paper (filter paper) is used as the stationary phase
aliquot of sample near bottom of paper, placed in jar with small amount of liquid in the bottom.
Liquid mobile phase will migrate up paper by capillary action and pick up components of the sample that are more soluble in the mobile phase
Paper is visualized suing stains, iodine vapour or UV light
Rf
racing factor
distance a component migrated, compared with the distance the solvent front moves
rf = Distance leading edge of component moves / total distance solvent front moves
Each sample component Rf is compared with he Rf of standards to identify the component
Thin layer chromatography (TLC)
similar to paper chromatography
thin layer of sorbent is coated onto a glass or plastic plane (acts as stationary phase)
separated based on preferential solubility
Column chromatography
stationary phase is packed into a tube called a column
mobile phase carries sample through column
1) mobile phase reservoir (additional pump required for liquid system)
2) injector port with septum
3) column
4)detector
5)display output
High pressure liquid chromatography
Gas chromatography
High pressure liquid chromatography
liquid mobile phase is driven through the column using the pressure generated by a pump
As liquid passes through column, the ELUATE is collected at the end and passed through a detector (UV spec)
Used for glycosylated hemoglobins
Gas chromatography
Mobile phase is an inert gas Sample used must be volatile sample is injected, temp of infection port must be above the boiling point of the components Often used for alcohol analysis Often coupled with a mass spec
Types of detectors for Hight pressure liquid and gas chromatography
Flame Ionization Detector (FID) Most common
Thermal Conductivity Detectors
Electron Capture detectors (most sensitiv)
UV spec
Linear Diode Arrays- consist of a line of detection diodes arranged in a line. Each diode is dedicated to a few wavelengths. Advantage is that multiple different compounds can be detected at once
automated chromatography process
liquid sample injected through septum into the column using a needle and glass syringe
Each component elutes from the column at a different time and is recorded as an individual peak by detector
chromatogram
record of all separate components that have been detected
Each component is recognized by separate
retention time
the time it takes for each component peak to reach the detector after sample injection
Is used for the ID of each specific component
Area under peak is used to determine quantity of the individual components in a sample
Resolution
relative separation of 2 separate chromatographic peaks
Internal standard
structurally similar reference compound that is added in constant concentration to help account for system variations such as low them or flow rate
n-propanol used for gas chromatography
Errors: no peaks
leaky septum, detector not responding/not lit
Errors: ghost peaks
contaminated syringe
Not waiting for previously injected sample to elute
Poor injection technique
Errors: irregular shaped peaks
poor injection, overloading the column, poor column prep
Peaks with long tails
water contamination of column
