Chromatography 1 Flashcards
What is chromatography ?
Involves the physical separation of analytes based on their differential migration in a mobile phase as they move along a stationery phase.
What is the mobile phase?
Can be a gas, liquid or some sort of fluid
What is the stationery phase?
Immiscible to the mobile phase
Fixed in place in a column or on a solid surface.
What is column chromatography ?
Stationery phase is held in a narrow tube through which the mobile phase is forced under pressure or by gravity
What is planar chromatography ?
Stationery phase is supported on a flat surface and the mobile phase moves through the stationery phase by capillary action or by gravity
What is thin layer chromatography and what can it be used for?
Fast and simple analytical technique used to determine or monitor :
Number of components in a mixture
Identity of two substances
Progress of a reaction
Effectiveness of purification
TLC involves spotting a dilute solution of sample on one end of sheet that is coated with silica gel or alumina, known as the stationery adsorbent phase. The sheet is placed upright in a jar with a small amount of solvent. The components then travel up different rates depending on the interactions with the mobile phase and stationery phases.
How do u calculate Rf value ?
Distance travelled by component / Distance travelled by solvent front
How does column chromatography work?
The mobile phase is either a gas or a liquid
The stationery phase is usually a viscous liquid bonded to the inside of a tube or onto the surface of solid particles
What is the partition coefficient ?
K= Cs / Cm
Cs - concentration in the stationery phase
Cm - concentration in the mobile phase
The larger the K value the more a molecule interacts with the column and the longer it is retained
What is high pressure liquid chromatography ? (HPLC)
Characterised by the use of high pressure to push a mobile phase solution through a column containing a stationery phase allowing separation of complex mixtures with high resolution.
What does a HPLC consist of ?
Pump System - mobile phase pressures up to 6000 psi
Injection system - to introduce small samples
Solvents
Chromatographic column
Detector
Computer for control and display
What is the difference between isocratic elution and gradient elution ?
Gradient elution and isocratic are two common methods used in liquid chromatography. Isocratic elution involves using a constant mobile phase composition throughout the entire chromatographic run. This means that the solvent composition remains unchanged, resulting in a constant elution strength. On the other hand, gradient elution involves changing the mobile phase composition over time. This allows for a varying elution strength, which can be beneficial for separating complex mixtures. Gradient elution is often used when there is a need to separate compounds with different polarities or when dealing with complex samples. Isocratic elution, on the other hand, is simpler and more straightforward, making it suitable for separating compounds with similar polarities.
HPLC in pharmaceuticals
To control drug stability
Quantity of drug determination
Drug monitoring
Purity check
Toxicology
Ultra Performance liquid chromatography
Resolution in HPLC can be increased by decreasing particle size in the stationery phase.
As particle size decreases the optimum flow rate to reach max column efficiency increases
HPLC works with pressures up to 6000 psi
UPLC requires pressures up to 15000 psi.
