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Quantitative Estimation of Amino Acids by Ninhydrin
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Materials Required:

 

 

Reagents Required:

 

  1. Standard amino acid stock solution (150 micrograms of  Standard amino acid stock solution (150µg/ml)).
  2. 0.2M Acetate buffer (pH=5.5).
  3. 8% w/v of Ninhydrin reagent [ Preparation: Weigh 8g of ninhydrin and dissolve in 100ml of acetone].
  4. 50% v/v ethanol.
  5. Distilled water.

 

Apparatus and Glasswares Required:

 

 

  1. Test / Boiling tubes.
  2. Pipettes [glass / micropipette].
  3. Waterbath.
  4. Colorimeter.
     

 Procedure:

 

  1. Pipette out different volumes(0.1ml-1ml) of standard amino acid solution to the respective labelled test tubes.
  2. Add distilled water in all the test tubes to make up the volume to 4ml.
  3. Add 4ml of distilled water to the test tube labelled Blank.
  4. Now add 1ml of ninhydrin reagent to all the test tubes including the test tubes labelled 'blank' and 'unknown'.
  5. Mix the contents of the tubes by vortexing /shaking the tubes.
  6. Put a few marble chips in each tube.
  7. Cover the mouth of the tubes with aluminium foil.
  8. Place all the test tubes in boiling water bath for 15 minutes.
  9. Cool the test tubes in cold water and add 1ml of ethanol to each test tube and mix well.
  10. Now record the absorbance at 570 nm of each solution using a colorimeter.

 

How to set the colorimeter:

 

  1. Switch on the colorimeter and set the wavelength at which the absorbance is to be measured [this should be done about 30 min before taking the readings].
  2. Rinse the cuvette with the blank solution, drain off the solution by inverting the cuvette and touching its mouth on to the filter paper.
  3. Fill the cuvette with the blank solution up to the appropriate mark and place it in the hole provided on the colorimeter.
  4. Care should be taken that the cuvette is properly inserted into the hole.
  5. Read the OD value and tare the instrument to zero.
  6. Take out the cuvette and drain off the blank solution.
  7. Now measure the OD values from the lower to the higher concentration.

 

Differences Encountered In a Real Laboratory:



In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:

 

  1. Always wear lab coat and gloves when you are in the lab. When you enter the lab, switch on the exhaust fan and make sure that all the reagents required for the experiment are available. If it is not available, prepare the reagents using the components shown in the reagent preparation.
  2. Care should be taken while handling reagents like Ninhydrin reagent. This reagent is a strong oxidizing agent and should not be inhaled or spilled on hands or other body parts.  Accidental spill of this reagent will cause severe itching sensation. Wash the spilled area with cold water and inform the lab assistant immediately.
  3. Always check the water level in the water bath and if it is up to the level [nearly half the volume], switch on the water bath and adjust to the required temperature before proceeding with the experiment. Take care while using the water bath for the boiling step in the experiment.  Always  hold the test tube using a test tube holder.
  4. While operating the colorimeter, make sure that water droplets do not enter the slot of the colorimeter as it will damage the instrument. So wipe the cuvette using a lint free tissue paper before inserting it into the slot.
  5. Do not touch the part of the cuvette that gets inserted into the colorimetric slot  with hands and ensure that  the cuvette is free from water droplets.


     

 

 

 

 

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