Qualitative analysis in biochemistry is a foundational technique for identifying the presence of specific biomolecules, such as proteins, nucleic acids, carbohydrates, and lipids, within a sample.
Test tubes (5 mL capacity)
Pipettes (1 mL and 5 mL)
Test tube rack
Water bath (set to 60°C)
Gloves, safety goggles, and lab coat (for safety)
Vortex mixer or manual shaker
Fume hood (for handling nitric acid)
Tongs or test tube holder
Concentrated nitric acid (HNO₃, 65–70%)
Sodium hydroxide solution (NaOH, 2M)
Solid sodium hydroxide
Solid copper(II) sulfate (CuSO₄·5H₂O)
Solid sodium potassium tartrate (Rochelle salt)
Lead acetate solution: 1% w/v lead(II) acetate trihydrate (Pb(CH₃COO)₂·3H₂O) in distilled water
Distilled water
Xanthoproteic Reaction
The xanthoproteic method is a classical qualitative test used to detect proteins or amino acids with aromatic side chains, such as tyrosine and tryptophan, in a biochemical sample. The test relies on the reaction of concentrated nitric acid with the aromatic rings, forming a yellow nitro-derivative compound. Upon neutralization with a strong base like sodium hydroxide, the color intensifies to an orange hue, indicating a positive result. This method is widely used in biochemistry education and research to confirm the presence of aromatic amino acids in protein samples.
You are provided with:
Six samples
The staff listed above
Your brain and hands
The courage to dive into unknown
Tasks:
Find the samples containing proteins or amino acids with aromatic side chains
Write down observations
Put on gloves, safety goggles, and a lab coat. Perform the experiment in a fume hood due to the use of concentrated nitric acid, which is corrosive and produces toxic fumes
In a clean test tubes, add 1 mL of each test sample separately
Carefully add 0.5 mL of concentrated nitric acid to the each tube using a pipette. Gently mix by swirling the tube
Place the test tube in a water bath set to 60°C for 2–3 minutes. Observe for the formation of a yellow color, indicating the nitration of aromatic rings
Remove the test tube from the water bath and allow it to cool to room temperature (approximately 2 minutes)
Slowly add 1 mL of 2M sodium hydroxide (NaOH) to the cooled mixture. Mix gently by swirling the tube. Observe any color change
Note the final color of the solution. A shift from yellow to orange confirms the presence of aromatic amino acids
Interpretation of Results
Positive Result: A yellow color after adding nitric acid, turning to orange upon addition of NaOH, indicates the presence of aromatic amino acids (e.g., tyrosine, tryptophan) in the sample.
Negative Result: No color change (solution remains colorless or pale) suggests the absence of aromatic amino acids.
Notes: False positives may occur if the sample contains other compounds that react with nitric acid. Ensure the sample is free of contaminants. The test is not specific to proteins; it detects any aromatic ring-containing compounds.
Safety Notes
Concentrated nitric acid is highly corrosive and can cause severe burns. Handle with care and use in a fume hood.
Sodium hydroxide is a strong base and can cause skin irritation. Avoid contact with skin and eyes.
Dispose of chemical waste according to local regulations.
Biuret Test
The Biuret test is based on the reaction of peptide bonds (–CO–NH–) in proteins or polypeptides with copper(II) ions (Cu²⁺) in an alkaline solution. Under these conditions, the peptide bonds coordinate with Cu²⁺ to form a violet or purple-colored complex. The intensity of the color is proportional to the number of peptide bonds, indicating the presence of proteins (typically requiring at least two peptide bonds, i.e., tripeptides or longer).
You are provided with:
Albumin solution, glucose solution and distilled water
The staff listed above
Your brain and hands
The courage to dive into unknown
Place 1–2 mL of each sample in a clean test tube.
Prepare a biuret reagent
Weigh 30 g of sodium hydroxide (NaOH)
Dissolve the NaOH in approximately 500 mL of distilled water in a 1 L beaker. Use a stirring rod or magnetic stirrer to mix gently
Allow the solution to cool to room temperature
Weigh 6 g of sodium tartrate (Rochelle salt)
Add the tartrate to the cooled NaOH solution and stir until completely dissolved. This stabilizes Cu²⁺ ions in the alkaline environment
Weigh 1.5 g of copper(II) sulfate pentahydrate (CuSO₄·5H₂O)
Dissolve the CuSO₄·5H₂O in approximately 50 mL of distilled water in a separate small beaker to ensure it is fully dissolved.
Slowly add the copper sulfate solution to the NaOH/tartrate mixture while stirring continuously. The solution should remain clear and blue, indicating stable Cu²⁺ ions
Transfer the solution to a 1 L volumetric flask and add distilled water to bring the total volume to 1 L
Mix thoroughly to ensure homogeneity
Add 1–2 mL of biuret reagent to the test tube (use equal or double the volume of the sample for clear color development).
Gently mix the contents by swirling the test tube or using a vortex mixer for 5–10 seconds.
Note: No heating is typically required, as the reaction occurs at room temperature.
Allow the mixture to stand for 5–10 minutes at room temperature to ensure full color development. A violet or purple color indicates the presence of peptide bonds (proteins or polypeptides)
Observe and note the color change (violet, purple, or no change) after incubation.
Compare the colours of different samples
Interpretation of Results
Positive Result: A violet or purple color indicates the presence of proteins or polypeptides with at least two peptide bonds (e.g., tripeptides or longer)
Negative Result: No color change (solution remains blue, the color of CuSO₄) indicates the absence of peptide bonds or insufficient protein concentration
Notes: The test is specific to peptide bonds, so free amino acids or dipeptides do not react
Safety Notes
Sodium hydroxide is highly corrosive and can cause severe burns. Handle with care and avoid contact with skin and eyes.
Copper(II) sulfate is toxic so do not dispose it in the sewage
Dispose of chemical waste according to local regulations
Xanthoproteic Reaction
The Lead Acetate Test involves the reaction of free sulfhydryl groups (-SH) in cysteine with lead(II) ions (Pb²⁺) from lead acetate in a strongly alkaline environment (provided by sodium hydroxide). The sulfhydryl groups react with lead ions to form lead sulfide (PbS), a black or dark brown precipitate. The test is specific for free -SH groups and does not detect disulfide bonds (-S-S-) unless they are first reduced. Heating enhances the reaction by promoting the release of sulfide ions from cystein.
You are provided with:
1% albumin solution
The staff listed above
Your brain and hands
The strong desire to go home
Place 1 mL of the sample in a clean, heat-resistant test tube.
Optional (for disulfide bonds): To detect cysteine residues in disulfide bonds (-S-S-), pre-treat the sample with 100 µL of 0.1 M DTT or 2-mercaptoethanol for 30 minutes at room temperature to reduce disulfide bonds to free -SH groups.
In a fume hood, add 1 mL of 2 M sodium hydroxide (NaOH) to the test tube to create a strongly alkaline environment
Gently mix by swirling the test tube
Note: The solution may become cloudy due to protein denaturation, which is normal
Add 0.5 mL of 1% lead acetate solution to the test tube
Mix gently by swirling or vortexing for 5–10 seconds
Place the test tube in a water bath set to 80–100°C (or heat gently in a boiling water bath using a Bunsen burner) for 2–5 minutes
Observe the black or dark brown precipitate (lead sulfide) forms if free sulfhydryl groups (cysteine) are present
Compare with positive and negative controls to confirm the presence of cysteine
Control tests:
Positive control: Test a known cysteine-containing sample (e.g., 0.1% L-cysteine solution or reduced albumin) to confirm the formation of a black/dark brown precipitate.
Negative control: Test a sample lacking free sulfhydryl groups (e.g., distilled water or glycine solution) to ensure no precipitate forms.
Blank: Test NaOH and lead acetate alone (without sample) to rule out reagent-related precipitation.
Interpretation of Results
Positive Result: A black or dark brown precipitate indicates the presence of free sulfhydryl groups, confirming cysteine residues in the protein or peptide.
Negative Result: No precipitate (solution remains clear or slightly cloudy from protein denaturation) indicates the absence of free sulfhydryl groups. This may occur if:
The protein lacks cysteine
Cysteine residues are involved in disulfide bonds (test with a reducing agent to confirm)
The sample concentration is too low
False Positives: Other sulfur-containing compounds (e.g., methionine, thiols like glutathione) may produce a weak precipitate, though less intense than cysteine. Use controls to validate protein-specific results
Safety Notes
Sodium hydroxide is highly corrosive and can cause severe burns. Handle with care and avoid contact with skin and eyes.
Do not pour lead-containing waste down the drain due to environmental toxicity
Dispose of chemical waste according to local regulations
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