Chromatography- CIE iGCSE Chemistry Notes - New Syllabus
Chromatography for iGCSE Chemistry Notes
Core Syllabus
- Describe how paper chromatography is used to separate mixtures of soluble coloured substances, using a suitable solvent
- Interpret simple chromatograms to identify:
(a) unknown substances by comparison with known substances
(b) pure and impure substances
Supplement Syllabus
- Describe how paper chromatography is used to separate mixtures of soluble colourless substances, using a suitable solvent and a locating agent
Knowledge of specific locating agents is not required - State and use the equation for Rf:
Rf = distance travelled by substance ÷ distance travelled by solvent
Paper chromatography to separate mixtures of soluble coloured substances
Paper chromatography to separate mixtures of soluble coloured substances
Principle
- Paper chromatography separates the components of a coloured mixture because each dye travels at a different speed along the paper
- Separation depends on two competing factors: how soluble each substance is in the mobile phase solvent and how strongly each substance is attracted to the stationary phase paper
Key terms
- Stationary phase: the chromatography paper (cellulose)
- Mobile phase: the solvent that moves up the paper by capillary action
- Solvent front: the furthest point reached by the solvent at the time you stop the run
Apparatus
- Chromatography paper or clean filter paper cut into a rectangle or strip
- Beaker or chromatography tank with a tight cover or watch glass
- Pencil and ruler for drawing the baseline and labels
- Capillary tube or fine pipette for spotting the sample
- Suitable solvent such as water for water-soluble inks or ethanol/propanone for less polar pigments
- Forceps for handling the paper and a drying rack or warm place for drying
Choosing a suitable solvent
- Select a solvent in which the coloured substances are at least partially soluble so they can move with the solvent
- Use water for food colourings and many marker inks when they are water-soluble
- Use organic solvents such as ethanol or propanone for plant pigments and less polar dyes
- If components do not separate, change the solvent or use a mixed solvent to alter solubility and separation
Preparation
- Draw a light pencil baseline about 1 cm from the bottom edge of the paper; pencil is graphite and will not dissolve
- Place a small concentrated spot of the coloured mixture on the baseline using a capillary tube; allow to dry and, if needed, re-spot to keep the spot small but intense
- Prepare the tank by pouring a shallow layer of solvent, about 0.5–1.0 cm deep; the solvent level must be below the baseline so the sample is not washed off
- Cover the tank to saturate the atmosphere with solvent vapour and improve separation
Running the chromatogram
- Hang or stand the paper so that its bottom edge is in the solvent but the baseline remains above the solvent level
- Keep the tank covered and avoid moving it while the solvent rises by capillary action
- Allow the solvent to travel up the paper until it is a few centimetres from the top edge
- Remove the paper and quickly mark the solvent front with pencil before the solvent evaporates
After the run
- Dry the chromatogram gently; the separated coloured spots represent different components of the mixture
- Label the spots and the origin for later interpretation and comparison
Why substances separate
- A component that is more soluble in the mobile phase moves further up the paper
- A component that is more strongly attracted to the stationary phase moves less and stays nearer the baseline
- Different balances between solubility and paper attraction produce distinct travel distances, giving separation
Good practice and common errors
- Use pencil for all lines and labels; ink would dissolve and interfere
- Keep the sample spot small to prevent streaking or overlapping bands
- Ensure the solvent level starts below the baseline; if not, the sample will dissolve into the solvent pool and be lost
- Keep the tank covered to prevent evaporation and uneven solvent fronts
- Do not touch the paper surface with fingers; oils can affect movement
Typical uses
- Separating food colourings into individual dyes
- Comparing inks from different pens
- Preliminary separation of plant pigments before further analysis
Example
A student runs chromatography on an unknown food dye along with three known dyes (A, B, C). The unknown produces a spot that travels the same distance from the baseline as dye B. What can the student conclude?
▶️Answer/Explanation
Explanation:
- If a spot from the unknown travels the same distance (same height and colour) as a reference dye, they are likely to be the same substance.
- To be more reliable, the \(R_f\) value should also be calculated and compared with the reference.
Final Answer: The unknown dye is likely to be the same as dye B, since they have the same chromatographic behaviour under identical conditions.
Separation of colourless substances using chromatography
Separation of colourless substances using chromatography
Why special treatment is needed
- Paper chromatography is often used to separate coloured substances such as dyes or inks
- However, many important substances (like amino acids, sugars, some organic compounds) are colourless and cannot be seen directly on the chromatogram
- To detect these colourless spots, a locating agent is used after the chromatogram is developed
Process of separating colourless substances
- The sample mixture is spotted onto chromatography paper at the baseline (in pencil)
- The paper is placed in a container with a suitable solvent (below the baseline)
- The solvent travels up the paper by capillary action, carrying the soluble colourless substances with it
- Each component travels at a different rate depending on its solubility in the solvent and its attraction to the paper
- Once the solvent has nearly reached the top, the chromatogram is removed, and the solvent front is marked
- At this stage, the substances are separated but invisible
Using a locating agent
- A locating agent is a chemical sprayed or applied onto the chromatogram to make the colourless substances visible
- For example, in amino acid chromatography, the chromatogram is sprayed with ninhydrin solution, which reacts with amino acids to produce purple or brown spots
- Different colourless substances may appear as different coloured or shaded spots depending on the reaction with the locating agent
- The positions of these visible spots can then be measured to calculate \( R_f \) values for identification
Example
A student wants to separate and identify amino acids in a mixture using paper chromatography. Since amino acids are colourless, how can the spots be made visible after running the chromatogram?
▶️Answer/Explanation
Explanation:
- Many substances like amino acids are colourless and cannot be seen directly on the chromatogram.
- After the chromatography run, the paper is treated with a locating agent (such as ninhydrin), which reacts with amino acids to produce coloured spots (usually purple).
- Alternatively, UV light can be used if the paper contains a fluorescent dye: the colourless spots appear as dark patches under UV.
Final Answer: The chromatogram is sprayed with ninhydrin (or viewed under UV light) to make the colourless amino acid spots visible, allowing their positions and \( R_f \) values to be determined.
Chromatograms
Chromatograms
(a) Identifying unknown substances by comparison with known substances
- Run the unknown sample and several known reference substances on the same chromatogram under identical conditions
- If a spot from the unknown travels the same distance as one of the reference spots (same height from the baseline and same colour), then they are likely to be the same substance
- More reliable identification can be done by calculating the \( R_f \) values of the spots and comparing with known reference \( R_f \) values
Example: If an unknown food dye produces a spot at the same distance as a known dye (say, Yellow 5), then that dye is present in the sample
(b) Distinguishing between pure and impure substances
- A pure substance will usually give a single spot on the chromatogram (assuming the solvent is appropriate for separation)
- An impure substance (a mixture) will separate into two or more spots, each representing a different component
- If multiple spots appear at different heights, it proves that the sample contains more than one soluble substance
Example: A black ink sample gives several coloured spots (blue, red, yellow) showing that the ink is a mixture of dyes, while a pure food colouring (E110) gives only one spot
Important points when interpreting chromatograms
- The solvent front must be marked in pencil before it dries to measure distances accurately
- Always compare spots from unknown and known substances run on the same paper, because the separation depends on the solvent and paper used
- Rf values provide a more precise comparison than just observing the height of spots
- If spots overlap or are smeared, results may not be reliable, so the experiment should be repeated with better technique or a different solvent
Example
Chromatography was performed on two samples: Sample X gave only one spot, while Sample Y gave three spots. What does this result show about each sample?
▶️Answer/Explanation
Explanation:
- A pure substance usually gives a single spot on the chromatogram.
- A mixture (impure substance) separates into two or more spots, each representing a component.
Final Answer: Sample X is pure (one substance), while Sample Y is a mixture of at least three different substances.
The \( R_f \) value
The \( R_f \) value
- The \( R_f \) value (retention factor) is a ratio used to identify substances separated by chromatography
- It is defined as the distance travelled by the substance (spot) divided by the distance travelled by the solvent front
Equation
\( R_f = \dfrac{\text{distance travelled by substance}}{\text{distance travelled by solvent}} \)
Key features of \( R_f \) values
- The value is always between 0 and 1 (because the substance cannot move further than the solvent front)
- Different substances usually have different \( R_f \) values under the same conditions, allowing identification
- For accurate comparison, both the unknown and known substances must be tested under the same solvent and same type of chromatography paper
- Using \( R_f \) values is more precise than simply comparing the height of spots on paper
Example
A student performs chromatography of a food dye. The solvent front travels 8.0 cm. The dye spot travels 6.0 cm from the baseline. Calculate the \( R_f \) value.
▶️Answer/Explanation
Distance travelled by spot = 6.0 cm
Distance travelled by solvent = 8.0 cm
\( R_f = \dfrac{6.0}{8.0} = 0.75 \)
So, the \( R_f \) value of the dye is 0.75. This can now be compared with reference \( R_f \) values of known dyes to identify the substance.
Important notes
- Rf values must always be measured from the same baseline (the pencil line where the sample was applied)
- The solvent front must be marked immediately before it evaporates, otherwise results will be inaccurate
- Factors such as solvent type, temperature, and paper type affect Rf values, so they must be controlled when comparing substances