Chromatography

1. Overview

Chromatography is a versatile analytical technique used to separate mixtures of soluble substances and identify the components present. It relies on the movement of a solvent (mobile phase) through a material like paper (stationary phase), separating substances based on their different solubilities and levels of attraction to the paper.

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Key Definitions

• Chromatography: An analytical method used to separate the components of a mixture based on their solubility in a solvent.
• Solvent: The liquid (e.g., water, $H_2O (l)$, or ethanol, $C_2H_5OH (l)$) that carries the substances up the paper.
• Solute: The substance that dissolves in the solvent to be separated.
• Chromatogram: The final piece of paper showing the separated spots after the process is complete.
• Baseline (Origin): The starting line drawn in pencil where the original mixture is placed.
• Solvent Front: The highest point the solvent reaches on the paper.
• Locating Agent: A chemical used to react with colorless substances to produce a colored product, making them visible.

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Core Content

How Paper Chromatography Works

1. A baseline is drawn in pencil near the bottom of the chromatography paper.
2. Small spots of the mixtures/substances are placed on this line.
3. The paper is placed in a beaker containing a small volume of solvent, ensuring the baseline is above the level of the solvent.
4. As the solvent travels up the paper, the substances in the mixture dissolve and move at different speeds.
5. The more soluble a substance is in the solvent, the further up the paper it will travel.

Interpreting Chromatograms

• Identification: An unknown substance can be identified by comparing it to a known reference substance. If two spots travel the same distance from the baseline in the same solvent, they are likely the same substance.
• Pure vs. Impure:
  • A pure substance will only produce one spot on the chromatogram.
  • A mixture (impure substance) will separate into two or more spots.

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Extended Content (Extended curriculum only)

Separating Colorless Substances

Some substances, such as amino acids or sugars, are colorless and cannot be seen on a chromatogram.

1. The chromatography is performed as usual.
2. The paper is dried and then sprayed with a locating agent (e.g., ninhydrin for amino acids).
3. The locating agent reacts with the colorless spots to turn them into colored spots, or the paper is viewed under Ultraviolet (UV) light.

The $R_f$ Value

The $R_f$ (retention factor) value is a ratio used to identify substances accurately. It is constant for a specific substance in a specific solvent.

Formula: $$R_f = \frac{\text{distance travelled by substance}}{\text{distance travelled by solvent}}$$

Worked Example: A spot of red dye moved $4.0 \text{ cm}$ from the baseline. The solvent front moved $10.0 \text{ cm}$ from the baseline. Calculate the $R_f$ value.

$$R_f = \frac{4.0 \text{ cm}}{10.0 \text{ cm}} = 0.4$$ (Note: $R_f$ values have no units and are always less than 1.0)

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Key Equations

Equation	Meaning	Units
$R_f = \frac{d_{substance}}{d_{solvent}}$	$R_f$ = Retention factor; $d$ = distance measured from the baseline	None (Ratio)

Note on Solvents: Common solvents used include:

• Water: $H_2O (l)$
• Ethanol: $C_2H_5OH (l)$
• Propanone: $CH_3COCH_3 (l)$

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Common Mistakes to Avoid

• ❌ Wrong: Drawing the baseline in ink.
• ✓ Right: Always use pencil for the baseline. Ink contains dyes that will dissolve in the solvent and interfere with the results.
• ❌ Wrong: Letting the solvent level start above the baseline.
• ✓ Right: The solvent level must be below the baseline, otherwise the samples will wash off into the solvent rather than moving up the paper.
• ❌ Wrong: Calculating an $R_f$ value greater than 1.
• ✓ Right: The substance can never travel further than the solvent; ensure the smaller number is divided by the larger number.

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Exam Tips

• Command Word - "State": If asked to "state" how many substances are in a mixture, simply count the number of vertical spots produced by that sample.
• Command Word - "Explain": If asked to explain why a substance didn't move, mention it is insoluble in that specific solvent.
• Measuring Distances: Always measure from the center of the spot to the baseline.
• Real-world Context: Be prepared for questions involving food coloring, ink analysis in forensics, or checking the purity of a synthesized drug in a laboratory.
• Typical Values: $R_f$ values are usually given to 2 decimal places (e.g., 0.65). If a spot stays on the baseline, its $R_f$ is 0.

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Exam-Style Questions

Practice these original exam-style questions to test your understanding. Each question mirrors the style, structure, and mark allocation of real Cambridge 0620 Theory papers.

Exam-Style Question 1 — Short Answer [5 marks]

Question:

A student uses paper chromatography to analyse the dyes present in three different coloured sweets: red, green, and blue. They also have samples of three known dyes, Dye 1, Dye 2, and Dye 3. The resulting chromatogram is shown below.

(a) State which dye is present in the red sweet. [1]

(b) Identify the dyes present in the green sweet. [2]

(c) Suggest why the blue sweet's dye spot does not match any of the known dyes. [1]

(d) State how the student could calculate the $R_f$ value for the dye in the red sweet. [1]

Worked Solution:

(a)

1. Dye 2 is present in the red sweet. The spot in the red sweet lane matches the spot for Dye 2.

How to earn full marks:

• State "Dye 2"

(b)

1. Dye 1 is present in the green sweet. The green sweet has a spot that matches Dye 1.
2. Dye 3 is present in the green sweet. The green sweet also has a spot that matches Dye 3.

How to earn full marks:

• State "Dye 1"
• State "Dye 3"

(c)

1. The blue sweet contains a dye that is not Dye 1, Dye 2, or Dye 3. The spot in the blue sweet lane does not match any of the known dyes.

How to earn full marks:

• Suggest that the blue sweet contains a dye that is different from the known dyes.

(d)

1. $R_f = \frac{\text{distance travelled by dye}}{\text{distance travelled by solvent}}$ This is the formula for the R_f value

How to earn full marks:

• State $R_f = \frac{\text{distance travelled by dye}}{\text{distance travelled by solvent}}$

Common Pitfall: When interpreting chromatograms, remember to carefully compare the positions of the spots in each lane. Don't just look at the number of spots; focus on which known dyes match the unknown samples.

Exam-Style Question 2 — Short Answer [6 marks]

Question:

A scientist is investigating the pigments present in spinach leaves using paper chromatography. They crush the spinach leaves and extract the pigments using a solvent.

(a) Describe how the scientist would set up the paper chromatography experiment to separate the pigments. [4]

(b) State two ways to ensure the accuracy of the experiment. [2]

Worked Solution:

(a)

1. Draw a pencil line near the bottom of the chromatography paper. The baseline must be drawn in pencil as ink would dissolve.
2. Spot the spinach extract onto the pencil line. The extract should be concentrated on the line.
3. Place the paper in a suitable solvent in a beaker, ensuring the solvent level is below the pencil line. The spots must not dissolve in the solvent before the experiment starts.
4. Allow the solvent to rise up the paper until it is near the top. Remove the paper and mark the solvent front with a pencil. The solvent front shows the maximum distance the solvent travelled.

How to earn full marks:

• Draw the baseline in pencil.
• Spot the spinach extract onto the baseline.
• Ensure the solvent level is below the baseline.
• Mark the solvent front.

(b)

1. Use a ruler to accurately measure the distance travelled by each pigment and the solvent. Accurate measurements are needed to calculate the Rf values.
2. Repeat the experiment multiple times and calculate the average $R_f$ values. Repeating the experiment reduces random errors.

How to earn full marks:

• Accurately measure the distance travelled by each pigment and the solvent.
• Repeat the experiment multiple times and calculate the average $R_f$ values.

Common Pitfall: Remember the practical details of setting up chromatography. Always use pencil for the baseline and solvent front, and make sure the initial spots are above the solvent level.

Exam-Style Question 3 — Extended Response [8 marks]

Question:

A forensic scientist uses paper chromatography to analyse the ink from a ransom note. They have four different pens (Pen A, Pen B, Pen C, and Pen D) that are suspected to have been used to write the note. The scientist performs chromatography on the ink from the ransom note and the inks from each of the pens. The results are shown below.

(a) State which pens were used to write the ransom note. Explain your answer. [3]

(b) Define the term '$R_f$ value'. [1]

(c) The solvent front travelled 8.0 cm. The lower spot in the ransom note travelled 3.2 cm. Calculate the $R_f$ value for this spot. [2]

(d) Suggest why the scientist used chromatography rather than simply comparing the colours of the inks. [2]

Worked Solution:

(a)

1. Pen A was used to write the ransom note because it contains one spot matching the lower spot in the ransom note. Pen A contains ink which matches one of the inks in the ransom note.
2. Pen D was used to write the ransom note because it contains one spot matching the upper spot in the ransom note. Pen D contains ink which matches one of the inks in the ransom note.
3. Pen B and Pen C were not solely used to write the ransom note because they contain spots which do not match the ransom note. Pens B and C contain inks which are not in the ransom note.

How to earn full marks:

• State that Pen A was used and give a reason.
• State that Pen D was used and give a reason.
• State that Pen B and Pen C were not used and give a reason.

(b)

1. The $R_f$ value is the ratio of the distance travelled by the substance to the distance travelled by the solvent. This is the definition of the Rf value.

How to earn full marks:

• State that the $R_f$ value is the ratio of distance travelled by substance to distance travelled by solvent.

(c)

1. $R_f = \frac{\text{distance travelled by dye}}{\text{distance travelled by solvent}}$ This is the formula for the Rf value.
2. $R_f = \frac{3.2 \text{ cm}}{8.0 \text{ cm}}$ Substitute the given values into the formula.
3. $R_f = \boxed{0.40}$ Calculate the Rf value.

How to earn full marks:

• State the formula for $R_f$ value.
• Correctly substitute the values into the formula.
• Calculate the correct $R_f$ value.

(d)

1. Different inks may appear to be the same colour but contain different dyes. Different inks can have the same visible colour due to a mixture of dyes.
2. Chromatography separates the different dyes in the ink, allowing for a more accurate comparison. Chromatography allows the comparison of individual dyes, not just the overall colour.

How to earn full marks:

• Suggest that inks that appear the same colour can contain different dyes.
• Explain that chromatography separates the dyes, allowing for a more accurate comparison.

Common Pitfall: When analysing chromatograms with multiple spots, remember that a match requires ALL spots of a known substance to be present in the unknown. If a pen has an extra dye not found in the ransom note, it wasn't used solely to write the note.

Exam-Style Question 4 — Extended Response [9 marks]

Question:

A student investigates the separation of amino acids using paper chromatography. Amino acids are colourless, so a locating agent is needed to visualise the spots.

(a) State why a locating agent is needed in this experiment. [1]

(b) Name a suitable locating agent for amino acids. [1]

(c) Describe the steps involved in using a locating agent after the chromatography paper has been developed. [3]

(d) The student performs two chromatography experiments using the same amino acid mixture, but with different solvents: Solvent X and Solvent Y. The table below shows the $R_f$ values obtained for three amino acids in each solvent.

Amino Acid	$R_f$ value in Solvent X	$R_f$ value in Solvent Y
Alanine	0.45	0.72
Glycine	0.28	0.55
Valine	0.63	0.88

Suggest which solvent, X or Y, would be better for separating this mixture of amino acids. Explain your answer. [4]

Worked Solution:

(a)

1. Amino acids are colourless and cannot be seen directly on the chromatography paper. Amino acids do not have a visible colour.

How to earn full marks:

• State that amino acids are colourless.

(b)

1. Ninhydrin is a suitable locating agent for amino acids. Ninhydrin reacts with amino acids to form a coloured compound.

How to earn full marks:

• State "Ninhydrin"

(c)

1. Allow the chromatography paper to dry completely. The solvent must be removed from the paper before applying the locating agent.
2. Spray the paper evenly with the locating agent. The locating agent must be evenly distributed for accurate results.
3. Heat the paper gently in a fume cupboard. Heating allows the locating agent to react with the amino acids.

How to earn full marks:

• Dry the paper.
• Spray the paper evenly with the locating agent.
• Heat the paper gently.

(d)

1. Solvent X is better at separating Glycine and Alanine as the difference in $R_f$ values is greater. ($|0.45 - 0.28| = 0.17$ for X, $|0.72 - 0.55| = 0.17$ for Y, so the difference is the same) The greater the difference in Rf values, the better the separation.
2. Solvent X is better at separating Alanine and Valine as the difference in $R_f$ values is greater. ($|0.63 - 0.45| = 0.18$ for X, $|0.88 - 0.72| = 0.16$ for Y) The greater the difference in Rf values, the better the separation.
3. Solvent X is better at separating Glycine and Valine as the difference in $R_f$ values is greater. ($|0.63 - 0.28| = 0.35$ for X, $|0.88 - 0.55| = 0.33$ for Y) The greater the difference in Rf values, the better the separation.
4. Overall, solvent X gives a better separation of the three amino acids as the $R_f$ values are more spread out. A more spread out range of Rf values is better for separation.

How to earn full marks:

• State solvent X is better.
• Calculate the difference in $R_f$ values for Alanine and Valine in solvent X.
• Calculate the difference in $R_f$ values for Alanine and Valine in solvent Y.
• Compare the difference in $R_f$ values between the two solvents and state why solvent X is better.

Common Pitfall: When comparing solvents for chromatography, focus on maximizing the differences in Rf values between the substances you're trying to separate. A solvent that spreads all the spots out evenly might not be as effective as one that creates larger gaps between specific substances.