Module 3d: Chromatography Flashcards
Refers to the group of techniques used to separate complex mixtures on the basis of different physical and chemical interactions between the compounds and the stationary phase.
Chromatography
Four basic components in any chromatographic technique:
- stationary phase
- mobile phase
- column
- eluate
Component that carries the complex mixture (sample)
mobile phase
component which can either be solid or liquid through which the mobile phase flows
stationary phase
component that holds the stationary phase
column
The separated components
eluent
5 separation mechanisms of chromatography
- ion-exchange
- partition
- adsorption
- size exclusion
- affinity mechanisms
Separation mechanism that is based on the differences or magnitude of their ionic charge.
ion-exchange chromatography
An exchangable ion found in close proximity with the fixed charge to maintain electrochemical neutrality.
counterion
Ion exchange chromatography used primarily for strongly acidic groups
Cation-exchange particles
acids have high H+ that can exchange with negatively charged stationary phase
Ion exchange chromatography used primarily for strongly basic quarternary amines with positive charges.
anion exchange packings
basic solutions have less H+ and adhere to negatively charged stationary phase
Chromatography involving separation of solutes based on their differential distribution of between two immiscible liquids
partition chromatography
partition chromatography separates solutes based on
the difference in solubility of the solute molecules between the stationary and mobile phase
Partition chromatography is classified into
gas liquid chromatography
liquid liquid chromatography
Liquid liquid chromatography is further classified into:
normal phase
reverse phase
Normal phase LLC has
stationary - polar
mobile - non- polar
Reverse phase LLC has
stationary - non polar
mobile - polar
Version of partition chromatography wherein analytes elute in order of increasing polarity
hydrophilic interaction chromatography
Separation technique based on the differences between the adsorption and desorption of solutes at the surface of a solid.
Adsorptiom chromatography
adsorption chromatography is also known as
liquid- solid chromatography
In adsorption chromatography, the molecules that are most soluble in the mobile phase move the ___________
fastest
Separation method based on the molecular sizes and shape
Size exclusion/ steric exclusion chromatography
In size exclusion chromatography, the molecules that remain entirely in the mobile phase are large or small??
large
Process where hydrophilic gel isused in separation of enzymes, antibodies and proteins.
Gel filtration
example of hydrophilic gels
dextran
agarose
Process where hydrophobic gel is used to separate triglycerides and fatty acids
Gel permeation
example of hydrophobic gels
sephadex
separation method that uses the the unique and specific biological interaction of the analyte and ligand
affinity chromatography
Two forms of chromatography
planar
column
two types of planar chromatography
- paper chromatography (PC)
2. thin layer chromatography (TLC)
type of paper commonly used in paper chromatography
Whatman paper
separation mechanism in paper chromatography
normal phase partition
non polar mobile phase, polar stationary phase
Applications of papaer chromatography
fractionation of sugar and amino acid
Thinl layer chromatography setup is consist of
thin layer of silica gel coated on glass plate
separation method of thin layer chromatography
Any mode depending on the sorbent or thin layer and solvent applied
Migration of solutes in PC and TLC is expressed as
Retention Factor (Rf) value
solute migration/ solvent migration
Two categories of column chromatography
gas chromatography
liquid chromatography
Column chromatography which usually provides higher resolution due to much longer columns and higher linear flow rate
Gas chromatography
Column chromatography of choice for volatile components, including analytes of lower molecular weight.
gas chromtography
column chromatography usually used for larger molecules like peptides, proteins, nucleic acids, and large polymers.
liquid chromatography
Setup of Gas chromatography
mobile phase: gas
statiomary phase: nonvolatile liquid bonded to the surface of capillary
examples of carrier gases (typically inert)
N, He, Ar
In gas chromatography, molecules that are ______ spend more time in the gas phase
more volatile with higher pressure
two types of gas chromatography
Gas solid chromatography
gas liquid chromatography
Basic components of a gas chromatograph
- carrier gas with flow controller
- injector or inlet
- chromatographic column
- column oven
- detector
- computer
Two types of chromatographic column in gas chromatograph
packed column
capillary column
These columns can be filled with uncoated support particles.
packed column
carrier gas most usually used for packed columns
nitrogen
Chromatographic column which whose fused silica tubular columns are wall-coated with a thin film of liquid phase. They are very efficient but have low sample capacities.
Capillary column
carrier gas usually used for capillary column
high purity hydrogen or helium
Most commonly used detector for gas chromatography clinical analysis because of its simplicity, reliability, versatility, sensitivity and ease of operation.
flame ionization detector
Column chromatography that is based on the distribution of solutes between a liquid mobile phase and a stationary phase
liquid chromatography
most widely used form of LC where particles of small diameter are employed as the stationary phase support
High performance liquid chromatography
Basic components of a liquid chromatograph
- solvent reservoir
- pumps
- injector
- chromatographic column
- column oven
- online detector
- computer
Identify type of chromatography:
Important tool for monitoring drugs and their metabolites.
LC
Identify type of chromatography:
Identify and quantify such drugs as alcohol, used in forensics to compare fibers found on a victim
GC
Identify type of chromatography:
Detecting pesticide or insecticide residues in food, also used in forensics to analyze the
dye composition of fibers
TLC
Identify type of chromatography:
Separating amino acids and anions, RNA, fingerprinting, separating and testing histamines, antibiotics
PC
Used to analyze metal ions and organic compounds in solutions using liquids which
may incorporate hydrophilic, insoluble molecules.
LC
Used to analyze volatile gases where Helium is used to move the gaseous mixture through a
column of absorbent material
GC
Simple and rapid method to check the purity of the organic compound using an absorbent material on flat glass plates
TLC
Most common type of chromatography which uses capillary action to pull the solutes up
PC
Considered as the gold standard for drug testing
GC- MS
Used for xenobiotics, anabolic steroids and pesticides.
GC-MS
Drugs are detected through the presence of
decomposition fragments
Has great potential for measuring low-level and mixed-polarity analytes such as vitamin D, testosterone, and immunosuppressant drugs
LC- MS