Flashcards in Medicine - Chromatographic Techniques Deck (31):
Uses of chromatography
Common features of chromatography
All have a stationary phase (solid or liquid supported by a solid)
And a mobile phase (liquid or gas)
The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates dependent on their attraction to the mobile phase and the stationary phase
What is meant by development when referring to chromatographic techniques?
What is the solute referred to as?
What is the mobile phase referred to as?
Describe the process of column chromatography in 5 steps
1 - load the stationary phase material into the column
2 - Equilibrate column stationary phase with the mobile phase
3 - Load sample in as small a volume as possible
4 - Add more mobile phase to the column
5 - Collect sample fractions from the column
What are the two possibilities of substance the column can be loaded with in terms of mobile and stationary phases? (In column chromatography)
The column is either loaded and filled with mobile phase, which is then flushed through the column,
More usually, loaded with a slurry of stationary phase and mobile phase together (care taken to avoid bubbles)
When is sand used in column chromatography?
Sand is used if cotton wool is used in place of a sinter disc
Describe how molecules of analyte are separated in column chromatography in terms of stationary and mobile phases
Molecule with high affinity for the stationary phase will take longer to travel through the column.
Thus, molecules of different polarities separate out
Stuff to monitor in normal phase chromatography and how to monitor it
What one factor determines the type of chromatography it is?
The type of solid/stationary phase determines the type of chromatography
Most common stationary phase in normal phase chromatography? Give particle sizes
Silica gel (Si-OH) is mostly commonly used
-40-63um particle size
-high surface area (~800m2/g)
Alumina is less commonly used
50-200um particle size
Describe how use of column chromatography as a purification tool would happen in 4 steps
1) Collect fractions
2) Determine component of interest by methods such as colour, TLC, UV, fluorescence
3) Pool fractions
4) Column can then be washed with eluting solvent and re-used or discarded
Use of chromatography in laboratories and in industry?
Used to separate an purify compounds on scale from mg to kg
What is the stationary phase in TLC?
Usually silica, but alumina and cellulose are used too
What does TLC stand for?
Thin layer chromatography
What is the driving force in TLC?
Mobile phase moves through stationary phase by capillary action
What is the elutropic series?
A scale of compounds starting at high polarity (water, acetic acid) down to low polarity (cyclohexane, n-hexane)
What is flash column chromatography used (a lot of the time) for?
In flash column chromatography, what affects solvent flow? Discuss
Particle size affects solvent flow
70-230 silicia gel for gravity columns
Smaller particles (higher mesh values) need a pump to flow the mobile phase
Describe the instrumentation in flash column chromatography
Pump (causes fast elution, can use gas instead)
Detector (fraction collector)
All of this is in a closed system
Elution expected in order of organic classes for flash column chromatography?
Increasing polarity (more slow)
Acids and bases
Name 7 uses of TLC
-Quality control and purity evaluation
-synthetic chemistry for identifying compounds
-To select a solvent system for column chromatography
-To follow the progress of a reaction
-To follow purification process
-Basic identity to check on pharmaceutical raw materials
-Detect impurities in pharmaceutical raw materials and formulations
What interactions go on in TLC and normal phase chromatography?
Weak polar interactions i.e.
Name the specific stationary phase used sometimes in TLC to detect things with no colour
F254 = fluorescent reagent (shine UV light and it looks green)
Name some ways you would detect colourless components when doing TLC
-chemical treatment (dip, spray) e.g. Ninhydrin
-chemical vapour e.g. Iodine
TLC colour for UV light (indirect)
Dark spot on yellow/green background
TLC colour for iodine vapour
Spraying with starch makes it permanent
In TLC, what does potassium permanganate detect?
Sugars and sugar-like molecules
What colour spot does Ninhydrin cause in TLC? What is it used to detect?
Purple/pink spot with amines
Used for gentamycin