Transcript Quantitative and qualitative estimation of amino acids

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
> Prior Viewing – IDD-1. Extraction of bacterial protein, IDD-6. Extraction of serum
protein
> 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-ShareAlike 2.5 India license
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Learning objectives
After interacting with this learning object, the learner
will be able to:
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Define the presence of amino acid in the sample
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Identify the mechanism involved in the detection
3.
Operate the steps used in colorimeter
4.
Infer the law governing the colorimetric analysis
5.
Assess the troubleshooting steps involved in the
experiments.
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Definitions and Keywords
1. 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-dione), used to detect
ammonia in primary and secondary amines. During the reaction with amines ,
purple colour known as Ruhemann's purple is formed.
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3. Beer –Lambert’s 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
Ninhydrin reaction (Slide: 5-6)
Beer-Lambert’s Law (Slide:7-8)
Reagents for Ninhydrin test (Slide:9-12)
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Ninhydrin test (Slide:13-25)
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:
T1: Ninhydrin reaction
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Ninhydrin
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Amino acid
Amino acid
Ninhydrin
complex
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Step:
T1: Ninhydrin reaction
Description of the action
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Show a tab labeled as theory
behind the ninhydrin’s test and
colorimeteric absorbance.
Now first show the ninhydrin
structure followed by amino acid
and the product as in the slide.
Audio Narration
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:
T2: Beer-Lambert’s Law
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L
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Step :
T2: Beer-Lambert’s Law
Description of the action
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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
Audio Narration
Colorimeter works on the basis of
Beer-Lambert’s 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:
T3:Reagents for Ninhydrin test
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Description of the action
Audio Narration
(if any)
Show a measuring balance, with display,
ON, OFF and TARE/0 buttons on it. let
When measuing
user ON it, display reading as 0.000g, let
with paper, the
user picks up the paper from the rack,
weight of the
makes 1/10 of folding on the sides and
paper need to be
places it on the balance. Now the display tared from actual
reading changes to 0.003g. Instruct user
reading.
to TARE the reading. And animate to click
the tare button. Once user clicks it,
reading must show ”0”
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Step 1:
T3:Reagents for Ninhydrin test
2Amino acid standard
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Description of the action
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.
Audio Narration
Prepare stock
solution of amino
acid with the
concentration
1mg/ml
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Step 2:
T3:Reagents for Ninhydrin test
ninhydrin
Description of the action
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.
ethanol
Audio Narration
Prepare ninhydrin
in ethanol, prepare
the required volume
as required for the
experiment.
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Step 3:
T3:Reagents for Ninhydrin test
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ethanol
Description of the action
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%”.
Audio Narration
Prepare 50%
ethanol.
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Step 4:
T4: Ninhydrin test
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Step 4:
T4: Ninhydrin test
Description of the action
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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.
Audio Narration
Wash the boiling tubes, label it
accordingly for blank, 0.2 -1ml
and then unknown.
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Step 5:
T4: Ninhydrin test
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Step 5:
T4: Ninhydrin test
Description of the action
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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 as”0.4” 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.
Audio Narration
Prepare the working standard
in the concentration of 0.2mg1mg and unknown samples
taking in different volumes.
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Step 6:
T4: Ninhydrin test
Description of the action
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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
as”0.2” 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 unknown:1 , 3.8ml to
unknown:2 and 3.7 ml to
unknown:3 to make final volume to
4 ml in all the tubes.
Audio Narration
Make the volume to 4ml using
the distilled water in all the
tubes.
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Step 7:
T4: Ninhydrin test
Description of the action
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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.
Audio Narration
Add 1000ul of ninhydrin
reagent in all the tubes and
mix well for the reaction to take
place.
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Step 8:
T4: Ninhydrin test
Aluminum foil
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Step 8:
T4: Ninhydrin test
Description of the action
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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.
Audio Narration
Cover the tubes with aluminum
foil before a hot water
treatment.
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Step 9:
T4: Ninhydrin test
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Water bath
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tubes
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Step 9:
T4: Ninhydrin test
Description of the action
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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 100’C. 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.
Audio Narration
Place the tubes in the boiling
water for 15 minutes for the
reaction to occur.
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Step 10: T4: Ninhydrin test
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Step 11: T4: Ninhydrin test
Step
10
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and 11:
T4: Ninhydrin test
Description of the action
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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
tubes”0.2,0.4,0.6,0.8,1 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.
Audio Narration
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: T5: Colorimetric analysis
Scroll
opening
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display
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Step 12: T5: Colorimetric analysis
Description of the action
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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.
Audio Narration
Switch on the colorimeter and
set the wavelength to 570nm
to take the absorbance.
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Step 13: T5: Colorimetric analysis
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Step 13: T5: Colorimetric analysis
Description of the action
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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.
Audio Narration
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: 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: T5: Colorimetric analysis
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Step 16: T5: Colorimetric analysis
Description of the action
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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
Audio Narration
Plot the graph between OD at
570nm and the concentration
of the sample and extrapolate
the unknown OD value to find
the concentration.
Slide 56
Tab 01
Slide 7- Slide 98
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Tab 02
Tab 03
Slide
13-25
Tab 04
Slide
26-33
Tab 05
Tab 06
Tab 07
Name of the section/stage
Interactivity
area
Animation 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”
Button 01
Button 02
Instruction:
Button 03
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
APPENDIX 1
Questionnaire:
Question 1
Amino acid can be detected using
a)
b)
c)
d)
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
Absorbance can be taken using
a) Calorimetry
b) Colorimetry
c) Spectrometry
d) refractometry
APPENDIX 1
Questionnaire:
Question 4
Colorimetry works based on
a)
b)
c)
d)
Beers law
Lamberts Law
Beer-Lamberts Law
Raman spectrum
Question 5:
As the absorbance increases , the intensity of the outgoing light
a)
b)
c)
d)
Decreases
Increases
Remains same
zero
APPENDIX 2
Links for further reading

Reference websites:
http://www.youtube.com/watch?v=JdXbTWfOc18
&feature=related
Book
Biochemistry by Voet & Voet, 3rd edition
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.