HPLC Flashcards
What does HPLC stand for?
High performance liquid chromatography.
How does HPLC work?
It is essentially a column chromatography that has been automated with a pump.
What type of pump is used with HPLC? and what concerns are there with it.
The most efficient pump is a dual piston reciprocating pump, a downside to this pump is the fluctuating pressure and pulsating flow. Pulse dampeners can be used to mitigate these effects.
What is a gradient elution system (HPLC)?
This is where the HPLC mixes solvents from multiple reservoirs prior to entering the pump. This allows the mobile phase to be altered during the chromatography. It is a valuable technique to elute all of the components from a sample especially when the sample sticks harshly to the stationary phase. This also allows for similar components to be well resolved and allows for the separation of peaks in MS and spectroscopy.
What types of chromatography is gradient HPLC useful for?
All types of chromatography are routinely used except for size exclusion. Because the separation agent in size exclusion is the stationary phase filtering by size. Where as in other forms of Chromatography the mobile phase is responsible for the separation.
How do incorporate the sample into the flow of a HPLC instrument?
By using an Injector
What two phases does the injector work off? How do these work?
It uses a load phase and a inject phase.
The load phase requires an external loop to fill with the sample.
the inject phase then connects this external loop into the pathway to the column via the mobile phase.
What is an autosampler?
An autosampler allows for heaps of samples to measured by a computer in succession without the input of labour. This machine improves reproducibility and accuracy.
What is a HPLC column?
It is a specially designed column for used for HPLC, they are typically made from stainless steel tubing with two terminals that allows for connection the the injector and detector.
What is a HPLC precolumn?
This precolumn is made from the same packing material as the column itself, this allows for sample components that would otherwise irreversibly bind to the column to bind to the precolumn instead. This allows for the cheaper precolumn to be replaced instead of the expensive column.
What are analytical columns supports generally made from?
Porous Silica. Particle size and pore diameter are important characteristics of the silica.
What is the importance of particle size in a HPLC column?
The small the particle size the greater the surface area, which increases the efficiency of separation in the column. However the higher surface area decreases flow rate and subsequently a higher pressure is needed to push the flow through which is limited by the connectors.
What is the importance of the porous property in a HPLC column?
Half the volume of silica consists of pores which increase surface area. The smaller the pore diameter the greater the increase in surface area. This allows for more analyte to be tested at once allowing for minuscule components of a analyte matrix to be measured.
What do you need to consider with he detector?
The detector depends on the analyte you are detecting. and if it will actually record any data. You also need to consider detector sensitivity, linear range and solvent characteristics.
What is the most common type of chromatography put to use in a HPLC?
The most common type used is a reverse-phase column chromatography. This relies on the stationary phase of silica being derivitised with non-polar binding molecules and the mobile phase being a mixture of water and organic solvent. this causes non-polar analyte compounds to move slower through the column
