Chromatography Flashcards
What is chromatography?
A method of separating soluble substances by their partition between two phases
What two things are needed for all forms of chromatography?
Stationary and mobile phase
Name the three main types of chromatography
Thin layer chromatography
Column
Gas
What are the mobile and stationary phases in TLC?
Mobile - solvent
Stationary - SiO2 or Al2O3 on a glass or plastic plate
Is the TLC plate polar or non-polar?
Polar
Outline the method of doing TLC
- Prepare TLC plate and draw pencil line as baseline
- Use capillary tube to dot samples onto the plate
- Put plate into the solvent with the baseline above the solvent
- Put on a lid and allow to run for half an hour
- Draw line on solvent front and measure it’s distance from baseline
- Identify the spots and measure their distance from BL
- Calculate Rf values and compare them to standard runs
Why do you hold the TLC plate with gloves?
So it doesnt get contaminated
Why do you use a pencil to draw the lines in TLC?
Bc it’s insoluble and pen contains substances which would run up the plate and would affect the results
Why don’t you submerge the line in the solvent in TLC?
So that the samples and the line don’t dissolve
If the spots in TLC are colourless, how do you identify them?
UV light
Ninhydrin - developing agent
Iodine - stains it brown
Potassium manganate
How does TLC separate compounds?
Adsorption
How does column chromatography separate compounds?
Adsorption
What are the stationary and mobile phases in column chromatography?
Stationary phase - silica or alumina
Mobile phase - non-polar solvent e.g. hexane
Define retention time
The amount of time that a substance remains in the column
What does assayed mean?
Analysis
Summarise how column chromatography works
- SP poured into column and air bubbles removed
- Mobile phase then added and equilibrated
- Sample dissolved in a small bit of solvent poured through
- Sample follows mobile phase through column and substances begin to separate
- Separated compounds colllected in fractions as they elute from the column
If a substance is eluted later in column chromatography, what can be said about its polarity?
It’s polar because it has a greater affinity for the stationary phase so it adsorbs to the stationary phase more
How does separation occur in gas-liquid chromatography?
Partition
What are the stationary and mobile phases in GC?
Stationary - non-volatile liquid such as a waxy polymer
Mobile - Inert gas
Name potential mobile phases for GC?
Argon, nitrogen and helium
What size is the column in GC?
1-4m long and about 4mm wide
Explain the process of gas chromatography
- Sample injected using a needle into carrier gas and heated
- Gaseous sample swept through column
- Column is in an oven and heated to temp lower than the gas
- Some gases condense into stationary phase, others are carried through in the gas phase
- When eluted, it can be detected by mass spec or by flame ionisation detector
In GC, are the less volatile substances retained for a longer or shorter time and why?
Longer bc they can condense onto or dissolve into the stationary phase
In GC, are the more volatile substances retained for a longer or shorter time and why?
Shorter because they stay in the gas phase and pass straight through
In GC, what is the temperature of the column oven?
50-250 degrees so that it’s slightly lower than the injector oven so some gases can condense and be retained
How is the information from the gas chromatogram analysed?
A graph of retention time against relative intensity is plotted. Relatve amount of each substance can be found and compared with standards under the same conditions
Put through a mass spec
What does the area under the peak in GC tell?
The relative amount of each substance
What three things may happen to a molecule in GC?
Condenses onto the stationary phase
Dissolves into the stationary phase
Stays in the gas phase
How can GC and MS be used to analyse a mixture of tripeptides?
GC separates the substances
Mass spec has a distinctive fragmentation pattern
Compare m/z values to database
Explain how you could use a gas-liquid chromatogram to determine the percentage composition of each component in a mixture
Chromatogram has retention time against relative intensity
Find area under each peak
Find total area
Do area under peak/total area x100
What is partition chromatography?
Separation when the stationary phase is liquid (substance can partition between the phases). Distribution between SP and MP
What is adsorption chromatography?
When the stationary phase is solid e.g. TLC
What things happen to a molecule being separated in GC chromatography?
Stays as a gas
Condenses to a liquid onto the stationary phase
Dissolves in the liquid on the SP
What factors does the retention time depend on?
temperature of the column
boiling point
solubility in liquid phase
Why do different amino acids have different Rf values?
Different polarities
Different retention by SP or diff solubility in the MP
