Quantitative and qualitative estimation of amino acids-Ninhydrin test

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Quantitative and qualitative estimation of amino
acids-Ninhydrin test
Amino acid determination is required for the
quantitative and qualitative determination of the
specific amino acid or all the amino acid.
Specific reagents have been used for such
experimental analysis and this amino acid
reaction forms the basics behind the protein
sequencing studies.

• ?

• Related LOs Amino acid properties
• gt Prior Viewing IDD-1. Extraction of
  bacterial protein, IDD-6. Extraction of serum
  protein
• gt Future Viewing IDD-17. SDS-PAGE, IDD-33.
  Western blot assay
• Course Name Quantitative and qualitative
  analysis of amino acids-Ninhydrin test
• Level(UG/PG) UG
• Author(s) Dinesh Raghu, Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

The contents in this ppt are licensed under
Creative Commons Attribution-NonCommercial-ShareAl
ike 2.5 India license
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Learning objectives
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• After interacting with this learning object, the
  learner will be able to
• Define the presence of amino acid in the sample
• Identify the mechanism involved in the detection
• Operate the steps used in colorimeter
• Infer the law governing the colorimetric analysis
• Assess the troubleshooting steps involved in the
  experiments.

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Definitions and Keywords
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• Amino acid The basic monomeric unit of
  polypeptides and proteins. There are twenty
  standard amino acids with different structures
  and properties that can be combined in multiple
  ways to make up the wide range of proteins known
  to us. Each amino acid is also specified by a
  three-letter and single letter code.
• 2. Ninhydrin Ninhydrin (2,2-Dihydroxyindane-1,3-d
  ione), used to detect ammonia in primary and
  secondary amines. During the reaction with amines
  , purple colour known as Ruhemann's purple is
  formed.
• 3. Beer Lamberts law the law relates the
  absorbance and the concentration of the solution
  .
• In the equation L signifies the path length, C
  concentration, E(epsilon) absorption coefficient
  and Io and I corresponds to the intensity of
  light before entering the solution and the
  intensity after coming out of the solution. The
  intensity of the light coming out of the cuvette
  decreases when the concentration of the
  substances in the cuvette increases. This is the
  law that governs the colorimetric analysis

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Master Layout
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Ninhydrin reaction (Slide 5-6)
Beer-Lamberts Law (Slide7-8)
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Reagents for Ninhydrin test (Slide9-12)
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Ninhydrin test (Slide13-25)
Colorimetric analysis (Slide 26-32)
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Please animate the layout with figures, by raking
them from each steps.
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Step
1
T1 Ninhydrin reaction
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Amino acid Ninhydrin complex
Ninhydrin
Amino acid
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Step
1
T1 Ninhydrin reaction
Show a tab labeled as theory behind the
ninhydrins test and colorimeteric
absorbance. Now first show the ninhydrin
structure followed by amino acid and the product
as in the slide.
Alpha amino acid reacts with the excess of the
ninhydrin reagent to give the purple color, the
intensity of the color signifies the
concentration of the amino acid, more the color
intensity signifies the presence of excess amino
acid.
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Step
1
T2 Beer-Lamberts Law
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L
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Step
1
T2 Beer-Lamberts Law
Now show the figure as in slide above (redraw)
followed by the formula which is given in the
slide , audio narration should take place
simultaneously
Colorimeter works on the basis of Beer-Lamberts
law, the law relates the absorbance and the
concentration of the solution. In the equation L
signifies the path length, C concentration,
E(epsilon) absorption coefficient and Io and I
corresponds to the intensity of light before
entering the solution and the intensity after
coming out of the solution. The intensity of the
light coming out of the cuvette decreases when
the concentration of the substances in the
cuvette increases.
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Step 1
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T3Reagents for Ninhydrin test
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Audio Narration (if any)?
Description of the action
Show a measuring balance, with display, ON, OFF
and TARE/0 buttons on it. let user ON it, display
reading as 0.000g, let user picks up the paper
from the rack, makes 1/10 of folding on the sides
and places it on the balance. Now the display
reading changes to 0.003g. Instruct user to TARE
the reading. And animate to click the tare
button. Once user clicks it, reading must show
0
When measuing with paper, the weight of the paper
need to be tared from actual reading.
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10
Step 1
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T3Reagents for Ninhydrin test
Amino acid standard
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Audio Narration
Description of the action
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Let user pick up amino acid standard bottle,
spatula, pipette from the rack and keeps it on
the table next to balance. Instruct user to
weigh 10mg of amino acid let user tare the
balance, user should click on the amino acid
bottle, uncap it, with help of spatula weigh the
required amount on a paper over the balance.
Display a gradual increase in reading with
quantity addition. if the gram exceeds user
should remove some quantity or if it less add the
quantity to get the exact required amount. After
weighing transfer the quantity to beaker. Now
show a beaker labeled as water and the user
should click on the pipette to set at 1000ul to
take water in the pipette and add to the tube
with amino acid standard ( repeat 5 times) and
show like mixing by shaking and add water 4 times
to make the final volume to10ml.
Prepare stock solution of amino acid with the
concentration 1mg/ml
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Step 2
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T3Reagents for Ninhydrin test
ninhydrin
2
ethanol
Audio Narration
Description of the action
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Let user pick up ninhydrin bottle, spatula,
pipette from the rack and keeps it on the table
next to balance. Instruct user to weigh 0.2g of
ninhydrin let user tare the balance, user should
click on the ninhydrin bottle, uncap it, with
help of spatula weigh the required amount on a
paper over the balance. Display a gradual
increase in reading with quantity addition. if
the gram exceeds user should remove some quantity
or if it less add the quantity to get the exact
required amount. After weighing transfer the
quantity to beaker. Now show a beaker labeled as
ethanol, user should click on the measuring
cylinder, animate like pouring ethanol to the
cylinder till the volume reaches to100ml and add
to the beaker containing weighed ninhydrin, show
like user shaking the beaker to mix the content
and show the clear solution as in figure.
Prepare ninhydrin in ethanol, prepare the
required volume as required for the experiment.
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Step 3
1
T3Reagents for Ninhydrin test
water
2
ethanol
Audio Narration
Description of the action
3
Prepare 50 ethanol.
Let user pick up the bottle labeled as ethanol
and animate like pouring the ethanol in measuring
cylinder till the volume reaches 25ml (zoom in to
show the volume in cylinder). Now instruct the
user to click on water to take it and add to the
ethanol in cylinder till the volume reaches 50ml
later transfer in a bottle labeled as ethanol
50.
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Step 4
1
T4 Ninhydrin test
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Step 4
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T4 Ninhydrin test
Now instruct the user to take the boiling tubes
as shown in figure and animate like user washing
and drying the tubes. Now instruct the user to
take marker and label the tubes as shown in
previous slide and place it on the stand as
shown. But animate 3 more tubes labeled as
unknown 1,2,3.
Wash the boiling tubes, label it accordingly for
blank, 0.2 -1ml and then unknown.
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Step 5
1
T4 Ninhydrin test
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Step 5
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T4 Ninhydrin test
Animate like user taking the pipette setting the
value to 200ul to take out amino acid to pipette
out 200ul into the tube labeled as 0.2, again
instruct the user to set the pipette to 400ul and
pipette out the amino acid in to the tube labeled
as0.4 follow the same instruction by setting
the pipette to 600ul, 800ul and 1000ul , pipette
out the amino acid standard and adding to the
tube labeled as 0.6,0.8, 1.0. Now show a tube
labeled as unknown (sample) and instruct user to
take the pipette set to 100ul to take the unknown
add to the tube labelled as unknown 1, follow the
same by setting pipette to 200,300ul and adding
to tubes unknown 2,3. events must happen when the
user clicks on pipette.
Prepare the working standard in the concentration
of 0.2mg-1mg and unknown samples taking in
different volumes.
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Step 6
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T4 Ninhydrin test
Animate like the user taking the glass pipette or
5ml pipette and taking the distilled water beaker
and pipette out 4ml when user clicks on it and
adding to the tube labeled as blank, again
instruct the user to take 3.8ml of water in
pipette and add to the tube labeled as0.2
follow the same instruction by taking the water
in the pipette to 3.6ml, 3.4ml, 3.2ml and 3ml
adding to the tube labeled as 0.4,0.6,0.8,1.0 to
make final volume 4 ml. Now show a tube labeled
as unknown and instruct the user to take water in
the pipette to 3.9ml and add to unknown1 , 3.8ml
to unknown2 and 3.7 ml to unknown3 to make
final volume to 4 ml in all the tubes.
Make the volume to 4ml using the distilled water
in all the tubes.
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Step 7
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T4 Ninhydrin test
Animate like the user taking the pipette setting
the value to 1000ul to take the ninhydrin
solution and pipetting out 1000ul in each tubes.
Animate each time user taking 1000ul using
pipette and adding to the tubes. animate like the
user taking the tubes and mixing by gently
shaking it.
Add 1000ul of ninhydrin reagent in all the tubes
and mix well for the reaction to take place.
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Step 8
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T4 Ninhydrin test
Aluminum foil
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Step 8
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T4 Ninhydrin test
Show aluminum foil, user must click on it, show
like user tearing it in small pieces and covering
all the tubes as shown in the figure.
Cover the tubes with aluminum foil before a hot
water treatment.
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Step 9
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T4 Ninhydrin test
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Water bath
tubes
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Step 9
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T4 Ninhydrin test
Show instrument labeled as water bath. Redraw the
image, show the buttons like start, set and stop
with red light up and down arrow
button. Instruct user to click on bottle labeled
as distilled water and animate like pouring in
the water bath, now instruct the user to click
start and then set the temperature by pressing
the up arrows to 100C. After 15mins (animate a
clock) show like the water boiling. Now instruct
user to take the tubes covered with foil to keep
it in the water bath as shown in the figure
animate a clock for 15minutes.
Place the tubes in the boiling water for 15
minutes for the reaction to occur.
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Step 10
1
T4 Ninhydrin test
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Step 11
1
T4 Ninhydrin test
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Step 10 and 11
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T4 Ninhydrin test
After 15 minutes show like taking the tubes to
keep in the cold water in the beaker as shown in
the figure. Animate the colors as in
figure. Animate like the user taking the pipette
setting the value to 1000ul and taking the 50
ethanol bottle and pipette out 1000ul when user
clicks on it and adding to the tube labeled as
blank, again animate like pipetting 1000ul and
adding to the tubes0.2,0.4,0.6,0.8,1 unknown .
Animate each time the user taking 1000ul using
pipette and adding to the tubes. animate like the
user taking the tubes and mixing by gently
shaking it. After sometime show the color
intensity from blank (left to right) should
increase as in the tubes like shown in figure.
Place the tubes in cold water and allow it to
cool down. Add 1 ml of 50 ethanol in all the
tubes.
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Step 12
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T5 Colorimetric analysis
Scroll
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opening
3
display
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Step 12
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T5 Colorimetric analysis
Show a instrument labeled as colorimeter and
draw it as shown in the figure. Animate a scroll,
a opening and a display screen, auto zero and
absorbance Instruct the user to click on start
in the instrument and the user should move the
scroll so that the wavelength is set to 570nm as
shown in the image and allow it to stand for
30minutes.
Switch on the colorimeter and set the wavelength
to 570nm to take the absorbance.
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Step 13
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T5 Colorimetric analysis
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Step 13
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T5 Colorimetric analysis
After 30 minutes instruct user to take the
cuvette (as in figure), set the pipette to 1000ul
to draw the blank solution into the cuvette and
show like the user inverting the cuvette and
pouring out the solution in a beaker, again
instruct user to take 1000ul of the blank and add
to the cuvette, (repeat it once more to have the
volume of 2000ul in the cuvette) clean the
cunette with tissue on sides and place the
cuvette in the opening, and click on absorbance
the reading in the instrument should show 0.00.
now ask the user to click auto zero and the
display should show 0.00 and then animate like
removing the cuvette and pouring the solution
out. Follow the same steps for other tubes also.
Show the values as in next slides.
Rinse the cuvette with the blank and discard it.
Fill the cuvette with the blank solution and take
the OD, auto zero the instrument and take the
readings for all the other tubes.
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Step 15
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T5 Colorimetric analysis
Sample OD at 570nm
Blank 0.00
0.2 0.01
0.4 0.03
0.6 0.05
0.8 0.07
1.0 0.09
unknown1 0.02
Unknown 2 0.04
Unknown 3 0.06
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Step 16
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T5 Colorimetric analysis
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Step 16
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T5 Colorimetric analysis
Instruct the user to plot the graph as OD in y
axis and the concentration in x axis to show a
straight line drawn (red line) meeting the
standard points with absorbance. animate like the
user locating the point on y axis for unknown 1
(0.02) and drawing a line towards the red line
and when the line touches the red line drag the
line down to find the concentration as 0.3mg
follow the same for other two unknowns and show
the concentration as 0.5mg,0.7 mg
Plot the graph between OD at 570nm and the
concentration of the sample and extrapolate the
unknown OD value to find the concentration.
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Slide 13-25
Slide 26-33
Slide 5-6
Slide 7-8
Slide 9-12
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• Animation area

Interactivity area
Slide 28- 31 Show like the user touching the
sides of the cuvette and doing the experiment and
taking the reading ,show the blank absorbance
value as 0.10 Instruction Now instruct the
user to clean the sides of the cuvette using the
tissue and taking the reading , show the blank
absorbance as 0.01
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1

• Question 1
• Amino acid can be detected using
• Chloroform
• Ethanol
• Acetone
• Ninhydrin
• Question 2
• Amines when react with Ninhydrin gives
• a) Red color
• b) Blue clor
• c) Orange color
• d) Purple color
• Question 3

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Questionnaire
APPENDIX 1

• Question 4
• Colorimetry works based on
• Beers law
• Lamberts Law
• Beer-Lamberts Law
• Raman spectrum
• Question 5
• As the absorbance increases , the intensity of
  the outgoing light
• Decreases
• Increases
• Remains same
• zero

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APPENDIX 2
Links for further reading

• Reference websites
• http//www.youtube.com/watch?vJdXbTWfOc18feature
  related
• Book
• Biochemistry by Voet Voet, 3rd edition

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APPENDIX 3
Summary
The method mostly involves the quantitative and
qualitative estimation of amino acid using the
Ninhydrin test. Steps involved are preparation of
standards, ninhydrin and ethanol, preparing
working standards, addition of water, ninhydrin
followed by incubation and addition of ethanol to
see the color, followed by colorimetric analysis.
Depending on the color intensity developed, one
can make out the presence and absence of the
amino acid.