Lecture 6: Liquid Chromatography Flashcards
What is chromatography?
Physical separation method where the components to be separated are distributed between two phases – stationary and mobile (moves in definite direction).
Describe the phases in HPLC
liquid mobile phase
solid stationary phase
What is the relationship between analytes, the stationary phase and the column and what does this lead to?
analytes that like the stationary phase spend longer on column and separate from analytes that prefer mobile phase
Describe a C18 column
highly non-polar
18 carbon chain
What is the role of bead pore size in separation?
reduce pressure and facilitate solvent transfer through column.
What is significant about the column length and separation?
longer = more solvent and therefore, more separation space.
What does sorbent mean?
stationary phase
Describe the interaction in normal phase
vPOLAR (silica) stationary phase i.e. silanol (Si-OH) groups
Describe sample loading in normal phase
vNONPOLAR organic mobile phase (hexane, ethylacetate)
Describe elution in normal phase
more polar solvent to compete for interaction (can be by adsorption to stationary phase itself)
Elution order = least to most polar
what is reversed phase used for?
to separate analytes of a range of polarities (inc. drugs) and desalt/remove vPOLAR interferences
Describe interaction in reversed phase
NONPOLAR phase, i.e. C-8 (more retention)
Describe sample loading in reversed phase
use polar mobile phase (water and MeOH/ACN)
> 95% water can cause stationary phase to collapse (= reduced separation efficiency as reduced SA)
Describe elution in reversed phase
increased (polar) organic solvent (ACN)
What are the stationary phases/separation modes (adsorption)?
normal phase (NP)
reversed phase (RP)
ion exchange (IEX)
ion pair (IP)
Affinity chromatography
Describe sample loading in ion exchange
low ionic strength solvent (e.g. aqueous buffer at correct pH).
Describe how ion exchange differs from other stationary phase/modes
- Strong or weak exchangers = strong is over broad pH range, weak = narrow pH range.
- pH gradient instead of organic solvent gradient
Describe interaction in ion exchange
anionic or cationic groups (e.g. sulfonic acid or tertiary amine) interact with opposing charge of target analyte.
Describe elution in ion exchange
increase ionic strength/change pH to compete for charge site.
Describe ion pair
IP reagent (e.g. Alkylsulphonates, tetra-alkylammonium surfactant) at low concentration (0.1-0.3%) in mobile phase binds to RP stationary phase, providing charge site to retain ionisable, oppositely charged analytes
Once column and tubing used with ion pair, it will not behave as before as not all IP will be removed during wash
What are the practical considerations in ion pair?
o Charge and hydrophobicity of IP reagent and analyte
o Concentration of IP reagent (strength of interaction)
o Type and concentration of organic modified (solvent) in mobile phase
o Ionic strength of analytes (presence of salt, Na+, K+)
Describe the interaction in ion pair
specific, reversible interaction with a ligand on stationary phase, based on analyte structure or function
Describe sample application in affinity chromatography
o Use buffer at physiological pH of 7-7.4 and ionic strength for proteins to enable binding at optimised flow rate and reduce analyte loss (too high = reduce binding if weak interaction between analyte and ligand)
What is the stationary phase/separation modes for porosity
Size exclusion chromatography (SEC)
Describe SEC
No interaction = separation via cross-sectional area and shape in solution (i.e. hydrodynamic volume)
Gel permeation chromatography (GPC) = organic solvent e.g. polymer analysis.
Gel-filtration chromatography (GFC) = aqueous solvent e.g. biopolymer separations (proteins/oligonucleotides).
Describe the interaction in SEC
- No interaction = separation via cross-sectional area and shape in solution (i.e. hydrodynamic volume)
What is Gel permeation chromatography (GPC)
organic solvent e.g. polymer analysis.
What is Gel-filtration chromatography (GFC)?
aqueous solvent e.g. biopolymer separations (proteins/oligonucleotides).
What are the practical considerations for LC?
cut off size
void volume
column volume
Describe cut-off size as a practical consideration of LC
approximate max size of molecule that can enter pore (use MW due to impracticality of calc. hydrodynamic volume)
Describe void volume as a practical consideration of LC
volume of vessel unoccupied by stationary phase (molecule larger than pore (cut off size) – elutes in void volume)
Describe column volume as a practical consideration of LC
volume of separation vessel accessible by solvent (small molecules like salts elute in column volume so can desalt samples) (determine prior to use along with void and elution (retention) volume/time)
What are the flow considerations?
Mobile phase flow and analyte elution:
1. Isocratic = same conditions
2. Gradient = vary conditions throughout – increase chance of separation
Why is flow rate key and what does it depend on?
key for good separation and depends on column dimensions and packing
Purpose of analytical LC
To identify and quantify a substance (typically <ug of material)
Aim for the outcome of analytical LC
well resolved symmetrical (Gaussian) peaks that are repeatable and reproducible.
How does analytical LC work?
- Sample injected on column in low amounts and volumes vs stationary phase (typically < 100000th)
How would you mitigate the disadvantages of nanoLC?
use column switching
what are the advantages of nanoLC?
less solvent consumption
lower i.d. and flow rate so higher sensitivity as analyte is eluted in less volume
reduced column packing size = narrower peak width so increased separation efficiency
nano LC on chips = simultaneous separations as no. of columns on chips
use nanospray chips which limits contamination between samples
Describe UHPLC - how is it different to normal LC, what is the aim, and the output?
- Faster, higher-er pressure separations
- Aim: good separation efficiency and resolution in minimum time
- Speed of separation is assessed according to number of peaks showing baseline separation/unit time (peak capacity).
- Separations can involve different morphology of sorbent.
what are the disadvantages of nanoLC?
higher back pressures
lower flow rates so need specialist pump and a pre-column flow split
extra band broadening so minimise dead volume - ensure correctly installed connections and fittings with minimum tubing lengths
