Ninhydrin Test

Definition of Ninhydrin Test

The ninhydrin test is a chemical test to detect the presence of amino acids. Ninhydrin test is a confirmatory test for protein after performing burnt test. It is optional test for biuret test. If this ninhydrin test observed positive then only you have to perform further tests for amino acids. This test involves the addition of ninhydrin reagent to the sample solution that results in the formation of deep blue color gives +ve test.

IUPAC name of Ninhydrin reagent is 2,2-dihydroxyindane-1,3-dione.

Ninhydrin test is generally used to detect the fingerprints over any surface because ninhydrin reagent reacts with dried fingerprints.

Why Ninhydrin Test?

  • As this test is confirmatory test so, ninhydrin test is used to detect the presence of amino acids and carbohydrates in the given sample.
  • To detect the presence of amines and specific amino groups in the sample solution.
  • To detect the exact quantity the amino acids present in the given sample.

Principle of Ninhydrin Test

This test is positive test for amino acids and proteins. The formation of a complex called Rheumann’s purple due to the condensation of two molecules of ninhydrin with one molecule of ammonia from amino acid is responsible for violet color. All amino acids give blue or purple color except proline and hydroxyproline, which give a yellow color under the same conditions. In amino acids like proline and hydroxyproline, this test yields turns yellow-orange in color.

Reaction of Ninhydrin Test

Requirements of Ninhydrin Test

Instruments and Materials

  • Test tubes
  • Test tube stand
  • Pipettes
  • Dropper
  • Hot Water Bath
  • Spectrophotometer

Chemicals and Reagents

  • 1 ml of test solution
  • 5 drops of Ninhydrin Reagent: 0.35g of ninhydrin in 100 ml ethanol

Procedure of Ninhydrin Test

  1. Take a dry and sterile test tube.
  2. Add 1 ml of the given sample solution into the test tube with the help of dropper.
  3. Add 5 drops of ninhydrin solution to test tube side by side.
  4. Boil the mixture for 2 minutes and cool.
  5. Note the change in color.
  6. If there is a deep blue or purple appearance then the presence of protein is confirmed.

Observation and Inferences of Ninhydrin Test

  • If there is appearance of a deep blue or purple then the presence of protein is confirmed. This test is confirmatory test only.
  • If there is appearance of yellow-orange color then it will be specific test for proline or hydroxyproline.

Results and Interpretations of Ninhydrin Test

  • When conducting the ninhydrin test, an instrumental process used to uncover the presence of amino acids in solutions, the color of the ensuing complex is a key indicator of whether protein or certain types of amino acids are present. This test acts as an important follow-up to preliminary protein tests, such as the biuret test.
  • The ninhydrin test employs a special reagent known as ninhydrin, also scientifically referred to as 2,2-dihydroxyindane-1,3-dione. The interaction between the ninhydrin and amino acids in the solution generates a deep, rich hue that ranges from an intense blue to a strong purple.
  • In a practical setting, like a high school chemistry lab, you would observe this color transformation in a test tube. Teachers often demonstrate this by adding the ninhydrin reagent to an amino acid solution, boiling the solution, and then closely observing the resultant color change. Seeing this process in action helps students visualize and understand the reaction that's occurring.
  • Interestingly, the same reaction occurs when ninhydrin comes into contact with dried, latent fingerprints, which contain traces of sweat that include amino acids. That's why ninhydrin is frequently used in forensics to detect fingerprints at crime scenes.
  • The presence of a vibrant purple-colored complex in the test tube is a positive result, signaling that proteins and amino acids exist in the sample solution. On the other hand, when proline and hydroxyproline—two special kinds of amino acids—are present, the color changes to a yellow-orange instead. This versatile color shift characteristic of the ninhydrin test makes it a valuable tool in chemistry.
  • Conducting the ninhydrin test requires various lab tools, including test tubes, a stand to hold the tubes, pipettes for precise measurements, a dropper for administering the ninhydrin, a hot water bath for boiling the solution, and a spectrophotometer to measure the intensity of the resulting color. All these pieces of equipment contribute to providing an accurate and reliable test result.
  • Following the ninhydrin test, if a purple hue is notably absent from the test tube, it suggests a negative result. This implies that in the tested sample, there were no proteins detected. The ninhydrin reagent, 2,2-dihydroxyindane-1,3-dione, didn't catalyze any reaction, establishing the absence of proteins in our tested substance.
  • Given the sensitive nature of ninhydrin in discerning proteins, it's crucial to consider that not all negative tests mean an absolute absence of amino acids. Keep in mind, Proline and Hydroxyproline, two specialized amino acids present in samples, trigger a yellow-orange hue instead of the regular blue/purple color. Thus, before making any definitive determinations, remember to watch for this distinctive coloration.
  • The lack of color change isn't just a negative test result; it also places importance on the tools and apparatus used during the procedure. Each component — the test tubes, pipettes, dropper, hot water bath, and the spectrophotometer — plays a significant role in ensuring the test's accuracy. Hence, careful handling of these elements is pivotal to avoiding any null reactions or anomalies.
  • Furthermore, if you're carrying out the ninhydrin test post a Biuret test, and you're met with a colorless outcome, this further strengthens the absence of proteins. Both tests act together to provide a broader and more precise understanding of the sample tested, minimizing any room for error.
  • In real-world applications, the ninhydrin test isn't confined to lab environments. It's frequently used in forensic science to detect fingerprints. The ninhydrin reacts with the trace amino acids present in our fingerprints. So, imagine, if this test renders your fingerprint undistinguished, it's like your unique touch has left no residue behind!
  • The fascinating ninhydrin test cleverly harnesses the power of chemistry to make the invisible, visible. You might not know it by its formal name, 2,2-dihydroxyindane-1,3-dione, but this highly reactive compound plays a crucial role in identifying the proteins hiding within daily objects - much like fingerprints left carelessly on a surface.
  • The meticulous setup required for the intense experiment is testament to the precision needed in chemistry. Required apparatus includes impartial test tubes lined up on their designated stand, just waiting to hold the answers to our questions. Precision-driven pipettes and droppers are along for the ride too, ensuring the exact delivery of the required reagents.
  • But not to forget that the hot water bath too has an important task at hand. It is not there for some casual soaking, rather for achieving the desired temperature to speed up the reactions. A spectrophotometer is the last piece of this scientific puzzle. Patiently standing by, it quantifies and records the light intensity changes that occur during the reaction.
  • As we dive into the specifics of the procedure, we delicately mix a secret sample believed to harbor the mystery proteins with the distinctive ninhydrin reagent. This mixture is then exposed to increased temperatures, causing interesting changes to the solution.
  • The magic happens if any amino acids are present. They react with the reagent to form a deep hue of blue or purple. This transformation, unfolds right in front of our eyes and confirms the presence of a protein. It is a sight to behold, as the sample solution goes from dull and boring to vibrant shades of mystical blues and purples!
  • But what about our special amino acids - proline and hydroxyproline? They are the out-of-the-box thinkers of this protein world. They don't conform to the color code followed by all their fellow amino acids. No, they stand out and make themselves noted by representing in an unexpected shade of yellow. Just like the inherently rebellious streak of teenagers, these two outcasts prefer to break the norm. Just as the yellow poppies in a field of purple flowers uniquely mark their presence, these amino acids display their distinct characteristics in this sea of blue or purple solutions.
  • While conducting the perceptive ninhydrin test, remember to not just consider the colors at the end of the rainbow, but also the radiant sun in the middle. A yellow-orange color marks the presence of the nonconformist proline or hydroxyproline, acting as a bright signal amidst the sea of blues and purples.

FAQs – Ninhydrin Test

1. What is the ninhydrin test for?

Ans – Ninhydrin test is used to detect the presence of protein and amino acid. This test is specific for amino acid proline and Hydroxyproline.

2. Which is the confirmatory test for protein?

Ans – Ninhydrin test is performed to detect the presence of protein. This test is confirmatory test for qualitative test protein.

3. What is the positive result of ninhydrin test?

Ans – There are 2 positive results in ninhydrin test: (1) All amino acids give deep blue or purple color as a positive result except Proline and Hydroxyproline. (2) Proline and Hydroxyproline give yellow color as a positive result.

4. Is ninhydrin test positive for all proteins?

Ans – No. Ninhydrin test is positive for all the amino except two amino acids. i.e. Proline and Hydroxyproline (because these are having free amino group).

5. Which is a better test for proteins biuret and ninhydrin?

Ans – Both the ninhydrin test and biuret test are confirmatory test for protein. But ninhydrin test is most accurate and easy test as compared to biuret test.

References and Sources

Further Readings

  1. 8 Qualitative Tests for Protein
  2. Acid Fast Staining
  3. Aseptic Transfer Technique
  4. Bacterial Flagella, Fimbriae and Pili
  5. Bacterial Growth and Nutrition
  6. Growth Curve of Bacteria
  7. Instruments Used in Microbiology Laboratory
  8. MacConkey agar
  9. McFarland Standards
  10. Monochrome Staining
  11. Negative Staining
  12. Preparation and Sterilization of Culture Media
  13. Serial Dilution in Microbiology
  14. Spread Plate Technique
  15. Streak Plate Technique