Thin layer chromatography
Chromatography in which the stationary phase is a thin layer of adsorbent gel coated on a glass or plastic plate. The sample is applied in a small spot at the base of the plate, and the plate is made to stand on edge in a solvent. As the solvent rises in the gel by capillary action, the sample components move at different rates and are separated into different spots. The plate is removed from the solvent, dried, and stained to visualize the components.
In this lesson, we will learn about paper and thin layer chromatography, both of which are used in forensic science applications.
Chromatography is a separation technique, and this will be demonstrated using food colouring.
Using a pencil, draw a line at the bottom of a strip of chromatography paper.
Dab a small spot of red food colouring on the pencil line, and repeat with green and blue food colouring.
Place this in a beaker with water, ensuring that the water level does not touch the spots.
As the water is absorbed, it separates the multiple components of the food colouring.
We have now created a chromatogram.
The chromatography paper is called the stationary phase as it “stays put”.
Water is called the mobile phase as it “moves” up the chromatography paper.
Here is the key idea – a component that is more soluble in water travels a greater distance up the chromatography paper, and a less soluble component travels a shorter distance.
The retention factor, or Rf value, is a measure of the distance that a separated component has travelled compared to the distance that the solvent, in our example, water, has travelled.
Rf = distance travelled by a component / distance travelled by the solvent
These distances can be measured using a ruler.
[Is it possible to demonstrate this in the video?]
Rf = distance travelled by a component / distance travelled by the solvent
Distance travelled by solvent = 6cm
Distance travelled by component A = 4cm
Distance travelled by component B = 2cm
Rf for component A = 4cm / 6cm = 0.67
Rf for component A = 2cm / 6cm = 0.33
What does the Rf value tell you about the solubility of a separated component in the mobile phase solvent?
Please pause the video to think about this.
A larger Rf value means that the component is more soluble in the solvent.
A smaller Rf value means that the component is less soluble in the solvent.
Thin layer chromatography, or TLC, works in exactly the same manner.
Instead of chromatography paper, a glass plate with a thin layer of silica gel or any other adsorbent material is used.
This thin layer of adsorbent material is the stationary phase.
Any suitable solvent may be used as the mobile phase.
If the separated components are colourless, the dried chromatogram may be sprayed with ninhydrin, which can turn some components purple, or the chromatogram may be viewed under UV light.
Ninhydrin is be used to detect fingerprints from a crime scene.
Both chromatography methods can be used to identify and compare samples of drugs, inks, lipsticks, saliva, and explosives, just to name a few.
In conclusion, paper and thin layer chromatography are used to separate components of a mixture based on their solubility.
The relative solubility of separated components can be determined by calculating their Rf values.
More soluble components have a larger Rf value, and less soluble components have a smaller Rf value.