Analytical Techniques Flashcards
Chromatography
A technique used for separating a mixture into components
Stationary Phase
Stays still and has an origin where the sample starts
Mobile Phase
Moves over the stationary phase and carries components of sample with it
Components move with the mobile phased according to how attracted they are due to IMF
Components with similar IMF to mobile phase travel further along stationary phase
Column Chromatography
Solid stationary phase is resin/gel beads packed into a column
Sample mixture placed at top of column, and solvent (mobile phase) passed through column and tap at bottom
Components pass through column at different rates according to attraction
Can be used for protein analysis
Size Exclusion (form of column chromatography)
Stationary phase has pores that catch smaller proteins and larger proteins leave column first
Ion Exchange (form of column chromatography)
Components of mixture are separated based on charge
Column has positive or negative beads
Hydrophobic interactions (form of column chromatography)
Hydrophobic portions or proteins have higher affinity with stationary phase and will take longer to pass through
Affinity (form of column chromatography)
Resin is coated in ligand (protein) which will bind with specific proteins of interest. Once sample has moved through column, resin is flushed with a chemical to detach protein
High Performance Liquid Chromatography (form of column chromatography)
A detector at bottom of column detects components passing through and produces peaks on a chromatogram
Time taken to pass through is retention time (Rt) - same components have same Rt
Electrophoresis
Uses a gel as a stationary phase and passes a current through it - positive and negative ends
Protein sample is coated in a substance so it has a negative charge
Gel is covered in a buffer solution containing ions that conduct the electric current
Samples loaded into wells at the end of the gel and current applied
Negatively charged proteins move towards positive end
Smaller proteins move faster
Mass Spectrometry
Used to determine the structure of a molecule and if certain elements are present
Ions are formed by colliding a beam of high energy electrons into a sample to lose an electron - it now has a positive charge. Positive ions from the sample are detected by a magnetic field based on mass charge ratio.
Fragmentation occurs when the beam breaks a covalent bond in the parent chain. Produces one part of the molecule with a positive charge and one neutral part that cannot be detected.
A common peak for an aldehyde is molecular ion -1
Molecular Ion
The peak with the largest m/z. Tells you the molecular mass of compound
Molecular ion peak is sometimes base peak as well.
Base Peak
Tallest peak produced by most abundant fragment ion
Isotope Patterns
Tiny peaks of +1 or +2 from major peaks due to isotopes in sample
Infrared Spectroscopy
Technique to determine the functional groups present in molecule
Based on molecules absorbing different frequencies of light depending on bond type, strength, length, atoms present
Transmittance is amount of radiation not absorbed by sample
Not possible to assign every peak in spectrum
