Chromatography Flashcards
What is the partition coefficient?
The equilibrium constant for a solute that is partial between two immiscible liquids
Columns in Chromatography?
Sample is flushed through the column stationary phase by mobile eluent. Movement of analyte down a column only occurs in mobile phase.
Chromatogram?
A plot of detector response vs retention time.
On a chromatogram when does the width of the band increase?
With time or distance
Adsorption
Solute equilibrates between mobile phase and surface of stationary phase. Adsorbed on surface of stationary phase
Ion- Exchange
Ions in mobile phase are attracted to counterions covalently attached to stationary phase. Anion exchange resin
Partition
Solute equilibrates between mobile phase and film of liquid attached to stationary phase. Bonded to surface of column
Size Exclusion
Large molecules are excluded
Affinity
Solute in mobile phase is attracted to specific groups covalently attached to stationary phase. All other molecules wash through
How is separation increased in the chromatogram?
Increasing band/peak separation or decreasing amount of spreading
Theoretical plate
Indication of efficiency of a column. Approximately the length of column required for one equilibration of solute between mobile and stationary phase.
Extraction vs Chromatography?
Extraction uses two immiscible liquids, chromatography uses stationary phase and mobile phase which are miscible
Van deemter equation!!
KNOW
Used to find the optimal flow rate for the best resolution!
H of Van deemter equation
Plate height
A of Van deemter equation
Multiple Flow Paths
The residence time in the column for molecules of the same species is variable. Some flow paths are longer than others.
B/Ux of Van deemter equation
Longitudinal Diffusion
Diffusional broadening of a band. Takes place along axis of column and while band is moving along column by flow of solvent. Species migrate from a more concentrated part of medium to a more dilute. The faster the less diffusional broadening.
Cux of Van deemter equation
Mass Transfer Coefficients
The finite time required for solute to equilibrate between mobile and stationary phases. Cs Rate of mas transfer through stationary phase. Cm is “ “ mobile phase
How can resolution be increased
as plate height is decreased
Peak area is proportional to…
Concentration
Chromatography applications
Qualitative and Quantitative
Qualitative: gives either retention time or position on the stationary phase
Quantitative: Peak height (amount of analyte present), Peak area (Shows concentration)
How is peak area affected?
Changes in retention time or column efficiency
Peak height is inversely related to…
Peak width
Chromatography advantages?
Cheap, fast, and simple
Gas vs Liquid chromatography
what is it
mobile phase
stationary phase
cost
analytes
Separation
Van D
Detectors
Look at table on desktop!
Gas Chromatography
Gas flow through a glass or metal column which separates compounds based on volatility and interaction with the liquid stationary phase
Open Tubular Column
Advantages and Disadvantages
Common in GC. Made of fused silica and plymides or support and protection.
Offers high resolution, short analysis time and greater sensitivity then packed columns.
has a less sample capacity
Three types of open tubular columns?
Wall Coated: Has a thick film of stationary liquid on inner walls
Support coated: Has solid particles coated with stationary liquid. Can handle larger samples but performance isn’t as good
Porous Layer: Solid particles are the active stationary phase
Why are narrow columns better and worse?
provide better resolution but require a higher pressure and have a lower sample capacity
Making Liquid Stationary phase
The choice is base on “like dissolves like” non-polar columns are the best for non-polar solutes (strong for strong ect..). Surface silanol groups are exposed and strongly retain polar compounds by adsorption. To prevent stationary phase bleed silanol is bonded to silica surface.
Column surface deactivated by silanization.
Column Bleeding?
Occurs at high temperatures where the stationary phase decomposes and the products elevate background signal and increase interference.
How to reduce column bleeding?
To reduce this use the thinnest stationary phase on the narrowest and shortest column
Temperature programming?
Temperature of column is raised during separation to increase analyte vapour pressure and decrease retention time.
Flame ionization detector
Advantages and Disadvantages
Effluent from column is directed into small flame which produces ions. The current produced by collecting charged carriers is measured.
highly sensitive and can measure a wide range of analytes.
slow and can be affected by interference
Electron capture detector
Advantages and Disadvantages
Selectively responds to halogen-containing compounds.
The sample eluate from column is passed over a radioactive beta emitter. An electron causes ionization of carrier gas and the production of an electron burst. The eluent passes through and electron capture detector.
highly sensitive and doesnt alter the sample
requires a special license to operate and several carrier gasses are needed
Olfactometry
Allows for identification of odour active compounds
Derivatization
Manipulating analyte to make its properties more optimal for separation and detection
