Chromatography Flashcards
Types of gas chromatography (based on stationary phase)
Gas-solid
Gas-liquid
Define retention time, void time and adjusted retention time
Retention time, tR: the average time that is required for a particular chemical to pass through the column
Void time, tM: the time for a compound that is nonretained/that does not interact with the stationary phase
Adjusted retention time: retention time corrected for the void time (tR’ = tR-tM)
Explain how the efficiency of a chromatographic system is assessed.
Efficiency with regards to chromatography refers to separation performance. This is indicated by peak width (either at the base or at half-height). The narrower the peak, the more easily two peaks with similar interactions with the system can be separated
In chromatography, what is “k”?
The retention factor. The average time an analyte remains in the stationary phase, compared to its time spent in the mobile phase
What is the advantage of using retention factor to describe a compound’s retention in chromatography?
“k” is independent of flow rate and column size
It is directly related to the strength of interactions occurring between the compound and the stationary/mobile phases, as well as the relative amounts of stationary/mobile phase
With the aid of a diagram, demonstrate how efficiency and selectivity change the resolution of peaks in chromatography.
Increasing efficiency improves resolution by creating narrower peaks. Increasing selectivity improves resolution by increasing the differences in retention time/volume of different compounds.
What is column bleed? What problems are associated with it?
In gas-liquid chromatography, with each run, some of the liquid stationary phase may leave the column with the mobile phase. This is known as column bleed.
When column bleed occurs, it changes the amount of stationary phase present in the column, and therefore changes the ability of the system to retain chemicals. Also, as the liquid stationary phase elutes, it may actually produce signal that is picked up by the detector as high background/noise.
Advantages of HPLC over GC
Ability to work directly with liquid samples
Modern LC instruments have better resolution and lower limit of detection
Suited to automation
Wide range of separation mechanisms, stationary phases, solvents and detectors that can be employed
5 main types of LC based on separation mechanism
- Adsorption chromatography (AKA liquid-solid chromatography)
- Partition chromatography
a) Normal phased (polar stationary phase)
b) Reversed phase (non-polar stationary phase) - Ion exchange chromatography
- Size-exclusion chromatography
- Affinity chromatography
Define chromatography
A method of separation of analytes based on differential interaction between stationary and mobile phases
What is the difference between column and planar chromatography?
Column - support is the internal surface of the column or a material that is placed/packed into the column
Planar - support/stationary phase is present on a surface that is open and flat
Liquid chromatography hardware components
Mobile phase
Degasser - removes bubbles
Pump - constant, pulse free flow, programmable, if binary, can adjust proportions of mobile phase
Sample injector - injects sample, includes needle wash components
Column compartment
Temperature control - sample injector and column compartment
Detector - UV or MS
Computer hardware/software - to drive instrument parameters and collect/process instrument output
In HPLC, what is “response ratio”?
peak area (analyte) / peak area (internal standard). Used for quantification of analytes.
Detection methods in liquid chromatography
- UV absorbance
- Fluorescence
- Electrochemical detection
- Mass spectrometer
- Refractive index detector
Analyte(s) UV absorbance detection is useful for in chromatography
HbA1c
Analyte(s) Fluorescence detection is useful for in chromatography
Detectors used in gas chromatography
- Flame ionisation detector
- Nitrogen-phosphorous detector
- Mass spectrometer
Factors affecting chromatographic efficiency
Column length
Particle size of support/tube diameter
Uniformity of size/shape/packing of support
Flow rate/linear velocity
Temperature
Rate of solute diffusion
Mobile phase viscosity
Degree of compoud retention
Initial injection volume
Volume of connecting tubing, detector, system components other than the column
What is resolution?
The extent to which two peaks are separated in chromatography
What are theoretical plates?
Another expression of the resolution of a chromatographic column. Conceptually, the number of interactions of a chemical with the mobile and solid phases as it passes along the length of the column. Mathematically, retention time of the peak / std devn of the peak. Higher N, better resolution.
What is H/height equivalent of a theoretical plate?
Length of column/N. Smaller H, better resolution. Van Deemter equation can relate H to various band broadening factors such as flow rate and diffusion effects.
How is resolution calculated?
Retention time between two peaks / average width of the two peaks. Literally tells you how separated the peaks are. Resolution > 1.25 generally fit for most purposes.
What is peak capacity?
The number of separations that can be made during a single chromatographic separation.
General ways to improve peak resolution in chomatography
- Increase efficiency of the system (eg longer column)
- Increase overall degree of peak retention
- Increase selectivity of column for peak of one compound vs another
Forces affecting retention in adsorption chromatography
Electrostatic interactions
Hydrogen bonding
Dipole-dipole interactions
Dispersive interactions (ie van der Waals forces)
Principle underlying adsorption chromatography?
Analyte and mobile phase compete to adsorb to support.
What is elutropic strength?
The ability of a mobile phase to elute analyte off a support in adsorption chromatography. Mobile phases with high elutropic strength bind strongly to support, causing analyte to be displaced and elute off the column.
Types of supports in adsorption chromatography
- Polar acidic eg silica
- Polar basic eg alumina
- Non-polar eg polystyrene
Advantages of reversed phase chromatography over normal phased chromatography
- Polar mobile phase convenient for analysis and separation of chemicals in aqueous based samples like serum and urine
- Many applications in clinical chemistry and biomedical research as many organic compounds can be analysed
- Wide range of supports and stationary phases available for reversed-phase separations
How is the strength of a mobile phase described in partition chromatography?
By its solvent polarity index
How are pH adjustments used to separate compounds in chromatography?
Adjusting the pH to the pKa of a weak acid or base will render that analyte chargeless, making it less polar. This change in the analyte’s polarity changes whether it can be eluted off the column or not.
Isocratic vs gradient elution
Isocratic - no change in mobile phase during separation
Gradient elution - composition of mobile phase changes during separation eg by adjusting ratio of polar and less polar solvents
