Chromotography And HPLC Flashcards
What is chromatography?
Separation of dissolved analytes depending on their relative attraction to various solid or liquid phases
One phase is fixed “stationary phase”
One phase moves “mobile phase”
Like attracts like
The composition of the stationary and mobile phases depend on the type of chromatography
- GC
- Size exclusion Chro
- TLC
- Ion exclusion chro
- LC
What are the mechanisms of separation?
Absorption
Uses electrostatic, hydrogen bonding or dispersive interactions between a molecule and solid support
Partition
Based on relative solubility; an analyte is in equilibrium between the two phases: A(mobile) A(stationary)
The equilibrium constant, k, is termed the partition coefficient;
Analyte (moles) in the stationary phase
————————————————— = k
Analyte (moles) in the mobile phase
Ion-exchange
Sign and magnitude of ionic charges; cations and anions
Stationary phase can be gels, charged resins, mobile phases include buffers and salts
Steric exclusion
Based on size; Stationary phase with pores e.g. polyacrylamide gels, sephadex and a buffer as the mobile phase.
Separation based on the size and shape of molecule - do they fit in pores?
Affinity
Based on bio-specific interactions e.g. antibody-antigen
What is Gas Chromatography?
Stationary phases; greases, gums and resins
Mobile phases; gases such as helium and nitrogen
Separation based on the relative affinities to the column material and gas
What is TLC?
Solid phase is silica or alumina
Mobile phase is a solvent mixture
What is strong vs weak ion exchange?
To elute from strong ion exchanger you need to replace the analyte with large amounts of salt with the same charge
To elute from a weak ion exchanger you can alter the pH, then the analytes are no longer bound
What is HPLC?
Stationary phases are many; usually silica based with or without functional groups
Mobile phases are usually an aq buffer and an org solvent
- can have a single mixed solvent and can gradient or isocratic elution
- Can have additives in depending on the application; buffers, salts, ion-pair reagents (half non-polar, half polar)
The pump forces the mobile phase into the column. If more than one pump is used, each with a different solvent, mixing of solvents can be achieved.
The chromatography column is usually stainless steel (high pressure <300 bar, ultra pressure <800 bar), containing a packing material inside, held in place by a frit (sieve).
Guard columns protect the more expensive analytical column from particulates. May have packing material inside or be just a filter.
Labelled with; type, length and diameter of column and particle size.
Can use the length and diameter to calculate the volume of the column:
Volume μL = (d/2)2 x π x L (mm)
Also UPLC – Ultra pressure (performance) liquid chromatography
Why do particles separate in normal phase HPLC?
In a non-polar mobile phase such as hexane and a polar stationary phase such as modified silicon.
Non polar species: like the mobile phase and elute first
Somewhat polar: like both phases so slow down within the mobile phase
Very polar: like the stationary phase so slow down the most
Why do particles separate in reverse phase HPLC?
In a polar mobile phase such as ethanol and a non-polar stationary phase such as silicon.
Non polar species: like the stationary phase and elute last
Somewhat polar: like both phases so slow down within the mobile phase
Very polar: like the mobile phase so travel the fastest
What are the principles:
The greater the concentration of organic substrate on the packed bed, the stronger the retention:
29%C > 20%C > 12%C
The longest alkyl bonded phase gives the greatest retention:
C18 > C8 > C4
Nonpolar groups on sample increase retention: toluene elutes after benzene
Polar functional groups on sample reduce retention: phenol elutes before benzene
What is back pressure?
Back pressure is the pressure generated as the mobile phase moves through the column
The smaller the particle, the greater the back pressure. Columns are most often silica particle based, very regular= higher back pressure.
Other factors include
- Flow rate (faster = higher pressure)
- Column dimensions (wider = lower pressure)
- Solvent composition (higher viscosity = higher pressure)
What are the features of the particles in the column?
HPLC particles are usually 2.5 – 15 μm
UPLC particles are smaller than this
Alternative types are available
They have a fused core
Particles can have functional groups added C18 C8 C4 Phenyl Amide CN PFP HILIC
The particles are very porous, like the pores of a sponge – 99% of chromatographic surface is inside the pores. Mobile phase must be allowed into the pore in order for chromatographic retention of the analyte to take place.
- 80–100 angstrom – analytical
- 120 angstrom – small peptides (1000 daltons)
- 300 angstrom - proteins (up to 700k daltons)
What does end-capping involve?
If you have only 8% C18 coverage, then the remaining silanol groups will ionically interact with analytes in the mobile phase. The solution is to cap them. This is a mechanism that results in covering of residual silanol groups, preventing ionic interaction with polar silanols. This also increases the pH stability of particle.
Silanols can be end-capped with different groups, e.g.
- aqueous groups can help to prevent pore de-wetting and enable more aqueous mobile phases to be used
- groups that bridge between functional attachments to increase robustness for e.g. pH
TMS is a commonly used group
What is the nature of the detector?
Detectors can be:
- UV-visible spectrometer
- Fluorescence detector
- Mass spectrometer
What is the plate model for chromatographic separation?
This assumes that the column contains a large number of separate layers or theoretical plates.
In each of these plates, there is equilibration of the analyte between the mobile and stationary phases.
The analyte moves through the column by transfer between mobile and stationary phase at each plate.
These plates do not actually exist:
Number of theoretical plates N =5.54 (t/W)2
- where t is retention time
- W is width of peak at half peak height
This is a measure of the efficiency of a column
The more theoretical plates, the more space for equilibrium or more space for separation making the column more efficient. But, shorter columns are more efficient compared to longer columns with the same number of plates.
Another way to express this is to look at the height equivalent to a theoretical plate (HETP)
HETP = Length of column / N
This is reported in millimeters, and the shorter the HETP, the more plates are ‘contained’ within a given length of column and the more efficient the column
What is the rate theory of chromatography?
The HETP is dependent on different properties of the column
These are described in the Van Deemter equation (describes the efficiency of the plate):
HETP = A + B/u + Cu
- Where A = Eddy Diffusion, B = Longitudinal diffusion (stat phase), C = Mass transfer (stat phase) and u = average velocity of the mobile phase
What are the principles of Eddy Diffusion?
Analyte molecules take different paths through the stationary phase at random.
This causes peak broadening as the different paths are different lengths.
The more regular the particle size and packing of the column, the less Eddy diffusion is observed.
Modern UPLC columns with very small particle sizes have much less Eddy diffusion than more traditional columns.
