Liquid chromatography Flashcards
LO
- To understand the importance of (U)HPLC, HILIC and IC
- To understand how and why they work
- Be aware of the various modes of separation to help identify which might be best suited to a particular separation
- Be able to give practical examples of LC at work
What is liquid chromatography and what are the common forms of the MP, SP and separation systems?
- Separation technique for compound mixtures dissolved, or extracted into, liquids
* Mobile phase is a liquid
* Stationary phase is usually in a particle-packed column, or on a planar surface
* Separation system can be **preparative or analytical **(scales)
How does GC compare with LC?
- GC, interaction of compounds of interest with stationary phase only as mobile is inert (H2 and He)
How does LC interaction with the stationary and mobile phase differ to that of GC?
o More complex interactions to consider/ optimise
o Permits many modes of separation
o Flexibility
Different names for the MP depending on application
o TLC- ‘system solvent’, ‘developer’
o (U)HPLC- ‘mobile phase’, ‘eluent’
o IC- ‘eluent’
o GC- ‘carrier gas’
o CE- ‘buffer electrolyte’
Acronyms used
For NPLC whats the polarity of the MP and the SP.
Some examples
What order the solutes elute in
MP examples
- A non-polar MP
- A polar SP
- E.g., silica, alumina, amino, phenyl as polar/induced polarity groups
- Solutes elute in the order of increasing polarity (adsorption based)
- Mobile phases: hexane, dichloromethane, toluene
What different groups can attach to the Silica surface (Si-OH) in NPLC?
What are silanols?
A silanol is a functional group in silicon chemistry with the connectivity Si-O-H and is related to the hydroxy functional group (C-O-H)
With RPLC:
* Polarity of the MP and SP
* Order the solutes elute in
* SP examples
* MP examples
- A polar MP
non-polar SP - Solutes elute in order of increasing non-polarity (by adsorption/ partition)
- SP:
o E.g., silica bonded with C1-C18 non-polar chains
o E.g., polystyrene divinylbenzene (PS-DVB) - MP:
o E.g., methanol/ water
o Acetonitrile/ water
What is phase collapse in regard to RPLC?
Phase collapse is when >95% causes C18 chains to collapse. Adding amide groups adds polarity to stop this from happening (or other functional groups which are polar)
What is end-capping?
- Non-polar functional groups (e.g.,C18) can be bulky, and at manufacture can get ‘residual silanol’ groups, which make the SP more polar
- Cause ‘secondary interactions’, e.g., tailing peaks
- Want to get as many functional groups on Si as possible
- To use a secondary functionalisation with smaller non-polar group to reach residual Si to avoid 2˚ unwanted interactions
With ion chromatography:
* What does it separate ions based on?
* What used as an eluting species?
* What is used during gradient elutions and why?
* What is present on the SP surface?
* Examples
* Applications
- Separates ions based on their size and charge
- Uses a competing ion (e-) in the MP as an eluting species
- For gradient elution, incremental concentrations of this species are used to elute large mixtures
- SP surface is grafted with oppositely charge groups to electrostatically attract analyte ions
- Examples
o 20mM NaOH in H20 as an MP on alkanol quaternary ammonium as a SP (for anion separations)
o 20mM methansulfonic acid in H2O as MP on alkylsulphonate SP (for cations M+) - Applications:
o Inorganic anions (Cl-, NO3-,SO42-)
o Alkali, alkaline earth and transition metals
o AA,peptides and other charged biomolecules
Ion exchange equilibria
With HILIC:
* What is the polarity of the MP and SP?
* What is HILIC
* Order it elutes solutes
* examples
- Polar MP,
Polar zwitterionic SP - An intermediate between RPLC and NPLC where there is small % water in a predominantly polar organic water-miscible solvent
- Solutes elute in the order of increasing polarity
What are some MP/SP interactions?
How is the SP selected?
Tell me the following about mobile phases:
* What is it often mixtures of?
* Considerations when choosing the MP
* Common modifiers
* Common solvents
- Often mixtures of solvents and additives (‘modifiers’)
- Considerations
o Physical properties: high purity (especially for MS), no particulates, no gas, green, etc
o Strength: need to elute analytes, polarity (water vs hexane (NP vs RP), ion concentration (IC)
o** Selectivity:** interactions (change MP parameters e.g., acetate –> carbonate)
o Miscibility: components dissolved in different reservoirs must be miscible
Common modifiers:
* Buffers: maintain pH
** Acids/bases: **Ionise/ de-ionise analytes
** Ionic strength
* Ion-pair reagents **
o Association complexes between analyte/modifier, to aid retention in RP-LC
* **Amines: **reduce tailing for basic analytes in RP-LC
* Remember: must be volatile for MS
What are some types of stationary phases?
- **Columnar **(uses particles, monolith, and capillary scale column GC)
2.** Planar** (TLC)
What are some common solid substrates for TLC and what are they coated with?
What are some examples of MP used for TLC?
- Solid substrate (glass, plastic, Al) coated with SP (silica, alumina, polyamide)
- MP: toluene, methanol, acetonitrile (+buffers)
o Generally isocratic
What is the equation for calculating the retardation factor in TLC?
What are the types of TLC?
1D and 2D
What are some common detection methods for TLC?
o Visual (dyes)
o UV (drugs, light box)
o Fluorescence
o Radioactive labelling
o ‘Developers’,E.g.., ninhydrin, hexachloroplatinate
o Analysis of spots by LC-MS/GC-MS
o MALDI
What are the pros and cons to TLC?
*** Pros **
o Cheap
o Low volume MP
o Small sample volume
o Multi-dimension (2D)
* Cons
o Poor efficiency
o Non-confirmatory
o Limited for quantification
o Not easy to automate (HP-TLC)
Columnar chromatography:
* type of column
* Column material
* Preparative scale
* Analytical scale
-
Particle-packed, or monolithic column, rather than spread on a surface
o **Particles: **spherical particles, ideally with narrow size distribution (more common)
o Monolithic: single piece of material with flow-through pores - Columns typically stainless steel, though also fused silica, plastic, titanium, etc.
*** Preparative scale **
o Large-scale (up to tons of material)
- Purification, extraction, preparation of standards/ reagents, etc
*** Analytical scale **
o Small scale: typically, 30-250mm columns
o Analysis only- nL to µl injection volume
Compare HPLC vs (U)HPLC
HPLC vs (U)HPLC
o Small particles (HPLC 3-5 microns, UHPLC <2 microns)
o High-pressure (UHPLC very high pressure, specialist pumps)
o 4.6mm column id- ‘standard bore’
o 2.1mm i.d. or less- ‘narrow bone’
Draw the general features of the LC instrumentation
With the LC instrumentation there is a gradient pump and a degasser. Why are these present?
Mobile phase reservoir/ degasser
o Houses mobile phase(s) (100-1,000mL)
o Gradient pump- multiple reservoirs and ‘solvent selector’
o Degasser: used to remove air from liquids when they mix on-line
o (inert gas headspace for IC)
Whats good practice for preparing MPs?
o Separately measure components
o Mix and degas
o Add buffers/ pH adjust as needed
o Filter
o Rinse glassware (10-20mL)
o Fill reservoir and prime pump(s)
What does the gradient elution change?
Whats required for the analytical pump in the LC?
- Analytical pump- want to avoiding pumping in system
o Need pulseless flow (typically 0.05-5.00 mL/min)
- Capillary/ nano-flow «_space;micro-flow «_space;standard flow
o High pressure
o Corrosion-proof (typically stainless steel, PEEK with ruby/ sapphire/ titanium)
Why are there generally two-pump heads in LC?
One fills, whilst the other is emptying- continuous, pulseless flow)
Ideally small internal volume (‘dead’ volume, ‘delay’ volume)
Why is priming required in LC?
o Important step prior to analysi
o Open a tap on the outlet, to divert flow to waste
o MP(s) pumped from reservoirs at high flowrate for 5-10 mins (can do this as removed column)
o Check for air
How does an auto-injector/ auto-sampler work?
o Inject sample as discrete slug onto column
**o Uses flow-switching valve(s) **
o Reproducible sample volume injection- use sample ‘loops’
o Include loop/needle washes (carryover)
o Can include on-line sample preparation steps
Why are column ovens fitted?
o Columns fitted with 1/32” or 1/16” fitting, using a nut and ferrule
o Make sure all MP are primed through
o Ensure no leaks
Why are guard columns often used on columns?
- Increase col. lifetime
- Save money
- Can use for some separations (do at low pressure and quick if good enough for some separations)
- Generally, use 3-5 guard columns: 1 column
With the column over, what are the general temperature control ranges and the types of ovens used?
o Temperature control to within **+/- 0.5 to 1.0˚c **
o Pre-column MP heating ideal/useful
o Types of oven:
Fan oven
Water baths
Peltier ovens- uses semi-conductors
What is the van’t Hoff equation? What it is a useful parameter for and what can it affect?
Temperature and HPLC
o check Alters thermodynamics, hence interaction of SP and MP (Van’t Hoff equation: −ΔG∘=RTlogeKp))
∆G= Gibbs free energy
K= equilibrium constant
T= temperature
o Useful parameter to influence/adjust selectivity
o Can affect:
* Retention time
* Selectivity (peak resolution/ separation, and order)
* Peak shape
* Back-pressure
* Detector signal
MP pre-heating…
Column packing architectures
What is the chromatographic plate theory and its assumptions?
Chromatographic plate theory
What does efficiency, H,depend on? and when plotted what does it look like
The efficiency curve is comprised of three componenets, what does each of these components represent?
Tell me about Term A for efficiency curve
Tell me about Term B for efficiency curve
Tell me about Term C for efficiency curve
What are common LC detectors?
o UV/Vis
o Fluorescence
o Conductivity
o Evaporate light scattering
o Electrochemical
o Refractive index
o Mass spectrometry
What is peak fronting and peak tailing and the solutions for each?
What is peak splitting and the solution for it?
Black: gradient profile
* Over is RPC
* Start at 5% water to keep C18 ready to interact with amide
* Strength of elution is good for separation
Blue: sample
Red: pressure trace for injection
* Reproducible pressure trace
* Can show blockage(shoots up), leaks (drops off), bubble (not smooth trace), pressure trace looks different if acetonitrile used instead or if wrong way round
* Methanol less viscous than water which causes pressure drop
* Good diagnostic tool especially if peaks absent
