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• Someone has stolen Dr Kemp’s badminton racquet just hours before he has another important match with a year 11 student. The thief left a ransom note behind. How could Dr Kemp use chromatography to find the culprit?
So, Who is the Thief?
The thief’s ink Mrs McDonald’s
ink
Mr Middleton’s Ink
Mr Spoor’s Ink
That’s right. It was me, Mr Middleton who stole Dr Kemp’s racquet and
I’d do it again!
I’m just so sick of hearing how good at
badminton he is!
Learning OutcomesContent• Describe and compare paper and thin-layer chromatography• Be able to calculate and define Rf values
• Describe the procedure for gas chromatography• Interpret gc printouts.
ProcessCarry out thin layer chromatography on some samples and measure their Rf values
BenefitIt is important to know the differences in the methods of chromatography so that choices can be made about which one to use.
Different types of chromatography
Paper
TLCGC
Not used much
Used a lotCheap, quick, requires
Only small amount of solventUsed a lot
Expensive, quick, requires a skilled operator, very
precise.
In paper chromatography what is the stationary phase and what is the mobile
phase?
TLC is used a lot Why?
What does aqueous and non aqueous mean?
What does mobile and stationary phase mean?
Thin-Layer Chromatograpy (TLC)
• TLC is used a lot in analytical laboratories at room temperature
• A plate of glass is coated with silica (silicon dioxide) or alumina (aluminium oxide)
• Spots are put on the plate about 1-2 cm from the end and the plate placed in a small amount of solvent. Different substances travel different distances.
• When the solvent has run, spots can be identified using a UV lamp or a locating agent
• Spots can be compared to a reference material (a known substance) to check for purity.
Analysing Chromatograms
Rf = Distance moved by chemical
Distance moved by solvent
= y/x
Rf = retardation factor
Larger the Rf the further the spot moves up the plate!
Rf values can be compared to known values but only for the same solvent system!
X
Y
Spot compounds ref A, then compounds B and C on your TLC plates.
Run the TLC plates in a mobile phase of Ethyl acetate/hexane 40:60
Take the TLC plate out and mark your solvent front
Allow to dry and check spot positions under UV light and mark spots with a pencil.
Draw a sketch of your plate in your book and work out the Rf values of your spots.
Half of you….take your TLC plates to an iodine tank and place in the tank until all the spots have been visualised.
The other half visualise your TLC plates with ninhydrin (I’ll spray on the ninhydrin), then heat the TLC plates
Activity
Learning OutcomesContent• Describe and compare paper and thin-layer chromatography
• Be able to calculate and define Rf values
• Describe the procedure for gas chromatography• Interpret gc printouts.
ProcessCarry out thin layer chromatography on some samples and measure their Rf values
BenefitIt is important to know the differences in the methods of chromatography so that choices can be made about which one to use.
How could I get a better separation between my spots if they overlay?
• Change my mobile phase (solvent system)
•Develop the TLC plate for longer•Use a different UV wave length
How could I see my spots better?
Can you think of a way that TLC could be used in a quantitative way?
Measure the intensities of the spots against a known concentration
Demonstrate
Gas chromatography (GC)Very sensitive techniqueGood separationQuantitative
Gas – mobile phaseOften He (carrier gas)
Sample injection port.Only a very small sample is needed.Often in liquid form
The heat of the oven vaporises The sample in the column at 400 ⁰C
Column in oven – stationary phasePacked with a thin film of powered solid. Some columns are 25m long and only 0.25mm in diameter.
1, The column is at temperature2, The gas is turned on3, The sample is injected4, The sample passes through the column 5, The chemicals separate6, The separation is detected by a computer 7, A chromatograph of the sample is obtained
Learning OutcomesContent• Describe and compare paper and thin-layer chromatography
• Be able to calculate and define Rf values
• Describe the procedure for gas chromatography • Interpret gc printouts.
ProcessCarry out thin layer chromatography on some samples and measure their Rf values
BenefitIt is important to know the differences in the methods of chromatography so that choices can be made about which one to use.
GC chromatograph
Retention time
Peak
inte
nsi
ty
Separated compounds
What affect does the size of the hydrocarbon in this chromatograph have on the retention time?
Learning OutcomesContent• Describe and compare paper and thin-layer chromatography
• Be able to calculate and define Rf values
• Describe the procedure for gas chromatography • Interpret gc printouts.
ProcessCarry out thin layer chromatography on some samples and measure their Rf values
BenefitIt is important to know the differences in the methods of chromatography so that choices can be made about which one to use.
Review – Complete the table
Paper TLC GC
Temperature
Stationary Phase
Mobile Phase
Review – Complete the table
Paper TLC GC
Temperature Room temp Room Temp 400 ⁰C
Stationary Phase
Paper Silica or alumina
Lining of column
Mobile Phase Liquid Liquid Gas (He, Ar, N2)