Chromatography Flashcards
What is chromatography used to do
To separate and identify the components in a mixture
What are retention times and Rf values used to do
Identify different substances
Characteristics of a stationary phase
Does not move
Normally a solid
Characteristics of a mobile phase
Does move
Normally a liquid or a gas
What does the mobile phase do
It flows through the stationary phase and carries the components of the mixture with it
Which solids are used in chromatography
Silicon (IV) oxide - silica
Aluminium oxide - alumina
How is separation achieved with a solid stationary phase
A solid stationary phase separates by relative adsorption.
The different components in the mixture have different affinities for the adsorbent and bind with different strengths to its surface.
If a substance has a stronger adsorption to the stationary phase, it will move more slowly
How is separation achieved with a liquid stationary phase
It separates by relative solubility.
The different components have different solubilities in the liquid stationary phase.
If a substance is more soluble in the stationary phase, it has a longer retention time
Thin layer chromatography (TLC)
A plate is coated with a solid and a solvent moves up the plate.
Can be used to separate and identify amino acids in a hydrolysed protein
What is the stationary and mobile phase in TLC
stationary phase - A solid adsorbent, usually silica (SiO2), that is highly polar
mobile phase - Liquid solvent
Describe the process of TLC
Use a pencil, draw a line across the TLC plate 1 cm from the bottom of the plate. This is the base line.
Use a capillary tube to put a small spot of the mixture on the base line of the TLC place and allow to dry.
Place the TLC plate in a beaker containing a suitable solvent, making sure the solvent is below the base line otherwise the solvent will just wash the sample off the plate.
Cover the beaker with a watch glass to prevent the solvent from evaporating
The solvent will rise up the TLC plate.
Leave the TLC plate in the beaker until the solvent is about 1cm below the top of the plate.
Remove the plate from the beaker and mark the position of the solvent front with a pencil line.
Dry the plate and examine the chromatogram.
Each separated substance appears as a spot on the TLC plate.
Rf =
Distance moved by a component / Distance moved by the solvent
Describe how to see colourless compounds in TLC
Use a locating agent such as iodine or ninhydrin.
How can substances in the mixture be identified
By comparing its Rf value with values for known reference compounds, recorded using the same solvent and adsorbent
Examples of adsorbents
Silica
Aluminum oxide
A student runs a second chromatogram on the protein sample, using a more polar solvent. Predict the effect, if any, on the Rf values of the amino acids. Explain your reasoning
Rf values would be larger.
Amino acids are more soluble in a polar solvent so would travel further up the plate
What are the limitations of TLC
Similar compounds have similar Rf values so will be difficult to distinguish
There will be no reference Rf values for comparison for unknown compounds
May be difficult to find a solvent that separates all the components in a mixture
What property determines whether compound A or B moves faster up the TLC plate
Its adsorption to the solid stationary phase
Adsorption vs affinity
Adsorption - How strongly a chemical sticks to the stationary phase
Affinity - How strongly attracted the chemical is to the stationary phase
They have a positive correlation
Gas chromatography
A column is packed with a solid or with a solid coated by a liquid, and a gas is passed through the column under pressure at high temperature
Stationary phase in gas chromatography
A liquid with a high boiling point adsorbed onto an inert solid support
Mobile phase in GC
an inert gas such as helium, argon or nitrogen
Retention time
The time between injection and detection of a component
How can an unknown compound be identified using its retention time
By comparing its retention time with values for known reference compounds.
What is gas chromatography used for
Separating and identifying volatile organic compounds present in a mixture
How does gas chromatography tell us how many components there are in a mixture
Through the number of peaks recorded.
It also tells us the abundance of each substance.
What does the area under each peak in GC represent
Used to determine the conc of components in the mixture as the area is proportional to the abundance
What does retention time in GC depend on
The stationary phase
The temperature of the column
The length of the column
The flow rate of the carrier gas
What happens to retention times when temperature and flow rate are greater
The retention times are shorter
Limitations of GC
Similar compounds will have similar retention times and so will be difficult to distinguish
There will be no reference retention times for comparisons for unknown compounds
What property determines which chemical moves faster through the capillary column in GC
Its solubility in the liquid stationary phase
A mixture containing propane and chloroethane is analysed using GC. Name the compound that moves through the column more slowly and why
Chloroethane as it is more polar than propane. Chloroethane has a greater affinity for the stationary phase so travels through the column more slowly and has a greater retention time.
Applications of GC
Measuring level of alcohol in blood samples from drivers
Detecting pesticides in river water
How is GC commonly used
A mass spectrometer is combined with GC to generate a mass spectra which can be analysed or compared with a spectral database by computer for positive identification of each GC component in the mixture.
Describe how GC-MS overcomes some of the limitations of GC
It produces a mass spectrum for each of the chemical separated on the GC column. These spectra are used to identify the compounds as each one has a unique mass spectrum.
Why is nitrogen rather than oxygen used as the carrier gas in GC
Nitrogen is unreactive but if oxygen was used ,the sample may get oxidised
Column chromatography
A column is packed with a solid and a solvent moves down the column
Describe the process of column chromatography
A glass tube is filled with the stationary phase usually silica or alumina in powder form to increase the surface area.
A filter is used to retain the solid in the tube.
Solvent is added to cover all the powder.
The mixture to be analysed is dissolved in a minimum of a solvent and added to the column.
A solvent or mixture of solvents is then run through the column.
The time for each component in the mixture to reach the end of the column is recorded (retention time)
Explain why cyclohexene has a shorter retention time than cyclohexanol
Cyclohexanol is more polar than than cyclohexene so cyclohexanol has a greater affinity for the stationary phase. Cyclohexene has a greater affinity for the mobile phase.
