3.3.16 Chromatography Flashcards
What are the two phases in chromatography?
- Mobile phase - where molecules can move. This is always a liquid or gas
- Stationary phase - where the molecules can’t move. This muct be a solid, or a liquid on a solid support
What are the basic principles of chromatography?
Chromatography is used to separate chemical in a mixture, once they’ve been separated they can be identified.
- The mobile phase moves up throughor over the stationary phase
- The distance each substance moves up the plate depends on it’s solubility in the mobile phase and it’s retention by the stationary phase
- Components that are more soluble in the mobile phase will travel further up the plate
- It’s these differences in solubility and retention by the stationary phase that separate out the different substances
Give the method for thin layer chromatography
- Draw a line in pencil near the bottom of the TLC plate (the baseline) and put a very small drop of each mixture to be separated on the line
- Allow the spots on the plate to dry
- Place the plate in a beaker with a small volume of solvent (this is the mobile phase). The solvent must be bellow the baseline or it will dissolve the sample away
- The solvent will start to move up the plate. As it moves, the solvent carries the substances in the mixture with it - some chemical will be carried faster than others and therefore will travel further up the plate
- Leave the beaker until the solvent has moved almost to the top of the plate. The remove the plate from the beaker. Before it evaporates, use a pencil to mark the solvent front
- Place the plate in a fume cupboard and leave to dry - the fume cupboard prevents toxic or flamable fumes from escaping into the room
- The result is called a chromatogram, the position of the spots can be used to identify the chemical
What is a solvent front?
How far the solvent travelled up the plate
What is the stationary phase in TLC
In thin-layer chromatography (TLC), the stationary phase is a thin layer of silica (silicon dioxide) or alumina (aluminium dioxide) fixed on a glass or metal plate
Explain how to show colourless chemical on a TLC plate using UV light
TLC plates have a fluorecent dye added to the silica or alumina layer that glows when UV light shines on it. You can put the plate under a UV lamp and draw aroud the dark patches to show where the spots of chemical are.
Explain how to show a colourless chemical on a TLC plate using iodine vapour
Expose the chromatogram to iodine vapour (leave the plate in a sealed jar with a couple of iodine crystals). Iodine vapour acts as a locating agent - it sticks to the chemicals on the plate and they’ll show up as brown/ purple spots.
Explain how the postion of the spots on a TLC plate can identify the substances present
- To find the number or substances present in a mixture, count the number of spots that from on that plate
- To find out what each chemical is, the Rf value can be calculated, the formula is:
Rf = distance travelled by spot/ distance travelled by solvent
- Rf values aren’t dependent on how big the plate is or how far the solvent travels - they’e properties of the chemicals in the mixture and so can be used to identify those chemicals
- This means that the Rf found can be compared to a table of standard Rf values to identify the substance
- However, if the composition of the TLC plate, the solvent or temperature change (even slightly), the Rf value will change
- Instead if you suspect a chemical is present then it can be put on the baseline of the same plate, if the two spots have the same Rf value they are the same chemical
Explain how column chromatography separates solutions
Column chromatography is normally used for purifying an organic product.
- It involves packing a glass column with a slurry of an absorbant material such as aluminium oxide, coated with water. This is the stationary phase
- The mixture to be separated is added to the top of the column and allowed to drain down into the slurry. A solvent is then run slowly and continually throught the column. This solvent is the mobile phase
- As the mixture is washed through the column, it’s components separate out according to how soluble they are in the mobile phase and how strongly they are adsorbed onto the stationary phase (retention)
- Each component will spend some time adsorbed onto the stationary phase and some time dissolved in the mobile phase. The more soluble each component is in the mobile phase, the quicker it’ll pass through the column
Explain how gas chromatography is used to separate mixtures of volatile liquids
- If you’ve got a mixture of volatile liquids (ones that turn to gasses easily), then gas chromatography is the way to separate them so they can be identified
- The stationary phase is a solid or a solid coated by a viscous liquid, such as an oil, packed into a long tube. The tube is then coiled to save space and built into an oven. The mobile phase is an unreactive carrier gas such as nitrogen
- Each component takes a different amount of time from being injected into the tube to being recorded at the other end, this is retention time
How do you read a gas chromatography chromatogram?
- Each seperate substance will have a unique retention time - so you can use the retention time to identify the components of the mixture, a known sample must be run under the same conditions to use as a comparison
- The area under each peak on a G.C. chromatogram tells you the amount of each component that’s present in the mixture
What may gas chromatography be used for?
Finding the level of alcohol in blood or urine - the results are accurate enough to be used as evidence in court
It’s also used to find the proportions of various esters in oils used in paints - this lets the picture restorers know exaclty what paint was used origionally
Explain how mass spectrometry can be combined with gas chromatography
- Mass spectrometry is a technique used to identify substances from their mass/ charge ratio. It’s very good at identifying unknown compounds, but would give confusing results from a mixture of substances
- Gas chromatograpghy is good at separating a mixture into it’s individual components
- Therefore by combining the two techniques you get a very useful anylitical tool
The sample is separated by gas chromatography but instead of going to a detector, the separated components are fed into a mass spectrometer. The spectrometer produces a mass spectrum that can be used to identify each one and show what the origional sample consisted of
- The advantage of this method over normal G.C. is that the separated components can be positively identified, which is imposible from a chromatogram alone
- Computers can also be used to match up the mass spectrum for each component of the mixture against the database, so the whole process can be automated
