Chromatographic Techniques Flashcards
What is Chromatography
Separation method to isolate, identify and/or quantify compound of interest (analyte)
Remove other compounds that could interfere with analyte
What are the chromatographic techniques classified by
Type of chromatographic bed - planar or column
Type of mobile gas - gas or liquid
Type of separation mechanism - size, polarity etc.
What are the phases of chromatography
Mobile Phase (MP) and Stationary Phase (SP)
How do you work out the Partition Coefficient of an analyte (also known as Equilibrium constant K)
Conc of analyte (A) in Stationary Phase / Conc of analyte (A) in Mobile Phase
What is Retention time (Tr)
Time from sample injection to elution from the column
What does a higher K value or Partition Coefficient represent
A higher affinity in the Stationary Phase
What does graphical column capacity and peak size mean
Column capacity - Maximum amount of analyte in the sample
Peak size - Greater area underneath a peak means more analyte in the sample
What causes fronting and tailing peaks
fronting peak - caused by overloading column with analyte mixture
Tailing peak - caused by analyte retention (hydroxyl group)
What are the different types of Liquid chromatography techniques
Ion-exchange
Size-exclusion
Affinity
Explain Ion-exchange liquid chromatography
(What the SP consists of and how it works, How analytes are removed from the SP, what this technique separates)
Separates analytes based on electrical charge.
SP: resin with either positively charged (anion-exchange) or negatively charged (cation-exchange) functional groups.
Different analytes have different affinities for SP, depending on their charge.
Bound analytes removed from SP by altering pH of MP or salt concentration.
Separates: amino acids, peptides, proteins & small nucleotides.
Explain size-exclusion liquid chromatography
(What the SP consists of, what the MP consists of, what this techniques separates)
Also called: Gel permeation chromatography, gel filtration chromatography & molecular sieve chromatography.
Separates analytes based on physical size.
SP chemically inert (inactive) porous beads e.g. dextran (Pore sizes vary)
MP aqueous or organic solvent-based.
Smaller molecules enter bead pores = delays passage down column; larger molecules elute first (removed or blocked)
Separates: proteins (in aqueous solution).
Explain affinity Chromatography
Separates analytes based on interaction between: Antibody & antigen
Enzyme & substrate
SP is gel matrix (e.g. agarose) to which ligand is covalently bound (specific shape for desired filtrate)
Ligand binds selectively to target analyte; other (unwanted) analytes pass through column.
Target analyte washed from column by altering MP composition
Separates: nucleic acids, proteins
e.g. antibodies.
Very specific/selective
(google it rat if it don’t make sense)
What is HPLC and what does it consists of (SP and MP)
High Performance (or pressure) Liquid Chromatography
Column filled with porous beads with SP compound chemically bound to them
MP containing the sample mixture pumped through the column at high pressure
Interaction through either polar / non-polar interactions as well as either any method previously described (ion-exchange, size-exclusion, affinity)
Normal phase HPLC vs reverse-phase HPLC
Normal phase HPLC - polar-polar interaction
Reverse-phase HPLC - non-polar - non-polar interaction
What are the advantages of HPLC
faster elution times due to higher pressure
better resolution (separation) due to small stationary phase particles
HPLC particle size, pressure
UPLC (ultra performance Liquid chromatography) particle size, pressure
HPLC particle size = 2-50 micrometres
pressure = 50 - 350 bar
UPLC particle size = 1.7 micrometres
pressure = 1500 bar
Explain Normal phase HPLC
Separates analytes based on polar interaction with polar SP
MP starts as non-polar, non-aqueous
Analytes dissolve in non-polar MP but sufficiently polar enough to interact with polar SP
Most polar molecules elute last
Explain reverse phase HPLC
Most frequently-used phase in HPLC
Separates analytes based on non-polar hydrophobic interaction with non-polar SP
MP moderately polar; mixture of water & organic solvents
Strength of analyte bond to SP proportional to analyte hydrophobicity
More hydrophobic molecules elute last from column
MP that remains constant in composition: isocratic elution
What equipment is used in HPLC ancillary equipment
Liquid handling pump: Must provide a constant flow of MP at constant (high) pressure. Also mixes solvents → elution gradient.
Detectors:
UV-Vis spectrophotometer (non-destructive).
Mass spectrometer (destructive)
Fraction collectors: Automated system of tubes collecting eluent leaving column. Can also be done manually.
Uses for HPLC
Manufacturing:
Pharmaceuticals (analytical and preparative).
Legal/Forensic:
Illegal drugs/doping in urine.
Research:
Very wide range of applications in chemistry, biochemistry and pharmacology.
Medical:
Nutrients e.g. vitamins in blood serum.
