Basic Instrumentation

Handling the instruments form the basis of the

practical knowledge and learning its mechanism of
working ensures the proper handling and
significance of its usage
Related LOs: Environment and health safety issues
> Prior Viewing - IDD-1. Extraction of bacterial protein, IDD-6. Extraction of serum
protein
> Future Viewing IDD-11. Protein quantification

Course Name: Basic Instrumentation

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

1.

2.

3
3.

After interacting with this Learning Object, the

learner will be able to:
Operate the steps involved in the instruments
Analyse the theory and the mechanism of working for
different instruments
Assess the troubleshooting steps involved in the
experiments.

Master Layout
Slide 412

Colorimetry
Centrifugation

Slide
13-17

UV- Spectrophotometric analysis Slide

18-21

Slide
22-24

Laminar air flow

Step 1:

T1: Colorimetry

Scroll

Opening for
cuvette
display

3
4

Video File: Colorimeters.mp4

Step 2:

T1: Colorimetry

3
4

Description of the action

Audio Narration

Absorbance of a sample that is

Animate the above display, like
nothing but logarithm ratio of
incident light (Io) passing through the incident light to reflected light is
sample of concentration c, travelling equal to the product of path length,
a path length l and coming out as
epsilon constant and concentration of
reflected light (I). Now animate
the solution. Form the above
putting these parameters in the
equation one can calculate the
equation above for user click to
concentration of the solution given
explain accordingly.
the incident light, reflected light and
path length value.

Step 3:

T1: Colorimetry

Description of the action

3
4

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 buttons.
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
stand for 30 minutes.
Use of colorimetry is explained
with the steps from IDD-47
quantitative and qualitative
estimation of amino acidninhydrin, for more information.

Audio Narration

The instrument need to set to

the required wavelength at first
place before taking the
reading. Once the instrument is
set for the wavelength, keep it
on stand for 30min to attain the
set wavelength. Meanwhile
prepare the dilution of
samples.

Step 4:

T1: Colorimetry

4
3

Video File: Colorimeters.mp4

Step 5:

T1: Colorimetry

Description of the action

3
4

After 30minutes, instruct user to

rinse the cuvette with water. let user
set the pipette to 1000ul to take out
blank solution to add into the cuvette
and show like the user inverting the
cuvette and pouring out the solution
into a beaker. Instruct the user to
take 2000ul of the blank and add to
the cuvette , (repeat the above step
twice), let user clean the cuvette with
tissue, place it in the opening and
click on Absorbance the reading in
the display should show 0.00. now
ask user to click auto zero option
and the display should show 0.00
reading then animate like removing
the cuvette and pouring the solution
out.

Audio Narration

Fill the cuvette with the blank

solution and take the OD,
auto zero the instrument and
take the readings for all the
sample tubes.

Step 6:

)T1: Colorimetry

Description of the action

3
4

Instruct the user clean the cuvette

with blank. Let user fill the cuvette with
the solution from the tube labeled as
0.2. Let user take out the full volume
from the tube with help of pipette and
trasfer it to cuvette, clean the edges of
the cuvette with tissue. Keep it in the
opening as instructed and press
absorbance and note down the
readings, remove the cuvette, pour
the solution out and follow the same
for the solutions in tubes
0.4,0.6,0.8,1,unknown 1,2,3.
Show the values as in next slides and
the graph

Audio Narration

Before taking reading for each

sample the cuvette need to be
rinsed with blank.

3
4

Step 7:

T1: Colorimetry

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

Unknown 1

0.02

Unknown 2

0.04

Unknown 3

0.06

3
4

Step 8:

T1: Colorimetry

Step 9:

T1: Colorimetry

Description of the action

3
4

Instruct the user to plot the graph as

OD in y axis and the concentration in
x axis and show like a straight line is
drawn (red line) once this is done,
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 blue line touches
the red line drag the blue 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. For
colorimetry to work a reaction
must be found which will
produce a color that can be
measured. The absorbing
molecules must be uniformed
distributed throughout
solution. Choice of the
wavelength is very important
to get the better sensitivity
and selectivity.

Step 1:

T2: centrifugation

3
rotor

Centrifuge

Video File: Centrifuge.MTS and Centrifuge_part2.MTSc

Step 1:

T2: centrifugation

Description of the action

3
4

The animator should draw a centrifuge

instrument as shown in the figure. Please
include the buttons like enter, set, start,
open a regulator nob along with display on
the centrifuge.
Let user click on OPEN button, animate
the centrifuge lid opening on its own, let
user rotate in anti-clockwise to open the lid
of the rotor. Let user keep the lid on table,
take out the tubes to centrifuged and place
them in the opening. Now balance the
tubes in the rotor. Now let user pick the
rotor lid and close it by making clock-wise
movement. Let user take the centrifuge lid
to its normal position and press to close it.
Now let user set the required parameters
for speed, time and temperature by
regulating the nob. Once the parameters
are set, let user click on START button,
animate increase in speed from zero to the
set point, along with temperature and
count down time display.

Audio Narration

Transfer the content into the

centrifuge tube and perform
a centrifugation at required
speed, time and
temperature.
Organelles separate when
the density of the organelle
equals the density of the
sucrose gradient

Step 2:

T2: centrifugation

2
A

4
http://www.youtube.com/watch?v=IhJNFGfsUus

3
4

Step :

T2: centrifugation

1) sedimentation coefficient
S = 1/ w^2 r dr/dt
where w = angular velocity of the rotor in radians/sec
r = the distance between the particle and the centre of rotation (m)
dr/dt = the rate of movement of the particle (m/sec)
2)RCF
g = (1.118 10 ^-5 ) R S^2
g- relative centrifugal force, R -radius of the rotor in meters, and S - speed
of the centrifuge in revolutions per minute (RPM)
r

3) Svedberg:
Sedimentation rate: dr/dt = w^2r.S

Step :

T2: centrifugation

Description of the action

3
4

Instruct user to carry out

centrifugation step. The animator
should draw a centrifuge as shown
in the figure. Now once
centrifugation is going the animator
must zoom in the instrument and
show the rotor and tube rotating .
Meanwhile show the different color
moving down and stopping at one
point as in the previous slide. And
show the formulas and the
governing relations in the animation
along with the audio narration

Audio Narration

Centrifugation works on the basis of

the centrifugal force which acts away
from the center. Relative centrifugal
force takes the gravity into account
during separation.
This force along with the particle
density and liquid density helps in
the separation of the particles.
Particle with high density will
sediment faster to precipitate than
the low density ones which are left
out as supernatant (figure: A). If the
particles are of varying density, than
different layers are formed after
centrifugation (figure: B).
The sedimentation rate is expressed
in terms of svedberg units

Step 1:

)
T3::
UV-Visible spectrophotometer

Display
Options like number 0-9,
set wavelength, autozero,
absorbance

Lid that can be

opened

Video File: UV vis spectroscopy.flv and UV Spectrometer.MTS

3
4

Step 1:

T3:: UV-Visible spectrophotometer

Description of the action

Animate the instrument as in figure

and redraw the instruments with the
specification mentioned in the figure
and zoom the instrument and show a
schematic as shown in the figure
with the labeling but redraw
completely.
Go through the IDD enzyme assay
for more information.

Audio Narration

UV-Visible spectrophotometer has

a monochromator, light source and
sample holder and detector, Light
from the source are converted to a
monochromatic light of particular
wavelength and allow it pass
through the sample and amount of
light that emerges is detected by a
detector. The wavelength used in
the specific for the sample to be
measured.

Step 2:

T3:: UV-Visible spectrophotometer

3
4

Step 3:

T3:: UV-Visible spectrophotometer

Description of the action

3
4

Now show the figure as in slide

(redraw) followed by the formula
which is given in the slide, audio
narration should take place
simultaneously show the animation
of air flow in the direction shown in
the figure

Audio Narration

UV-Visible spectrophotometer 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.

Step 1:

T4: Laminar air flow

CLASS-2
cabinet

3
4
Video File: Laminar air flow_1

Step2 :

T4: Laminar air flow

Description of the action

3
4

Audio Narration

Laminar air hood is used to maintain the aseptic

Go through the IDD for the
condition that can be used for microbiological
bacterial extraction to know the activities. The laminar air flow used in
usage of laminar air flow (slide laboratories uses horizontal air flow type with the
6,7), let the user click on the
air flow direction is facing towards the user and
laminar flow in the figure to know the velocity of air flow is maintained constant.
The filters used are of different types depending
the mechanism of working of
on the type of sample used. The biosafety
laminar flow and the types of
cabinets is of 3 classes. Class 1- for has HEPA
cabinet

filters that removes contaminants from the

exhaust air . This is only for environment
protection Class 2- for common usage in
microbiological activities, this cabinet has HEPA
filters for filtering the entering air and the exhaust
air and provide personnel, environmental and
product protection. Class 3- for handling most
pathogenic microbes especially in maximum
containment labs. HEPA (High efficiency
particulate air removes 99.97% of particles of
size 0.3micro meters. The UV in the cabinet is
used prior to the usage of cabinet to kill existing
organism in cabinet. Air flow prevents the entry of
any microbes from the environment.

Step2 :

T4: Laminar air flow

3
4

Description of the action

Animate to show the flow of air,

zoom in the HEPA filter, blocking
the microbes. Let user ON UV
light for 5min,later OFF the UV
light. Now let user On the
LIGHT and ON the AIR
FLOW and animate user doing
the experiment on the laminar
work bench.

Audio Narration
HEPA (High efficiency particulate air removes
99.97% of particles of size 0.3micro meters. The
UV in the cabinet is used prior to the usage of
cabinet to kill existing organism in cabinet. Air
flow prevents the entry of any microbes from the
environment.

Slide 4- Slide
13-17
12
Tab01

Slide
18-21

Tab 02

Slide
22-24

Tab 03

Tab 04

Tab 05

Tab 06

Tab 07

Name of the section/stage

Animation area

Interaction1: Animator should frame a question if you require a aseptic

condition for working with bacteria which instrument you will prefer
a)Centrifuge
b)Clean room
c)Laminar air flow
d)UV-Visible spectrometry

Interactivity
area

Button 01
Button 02

Instruction: if the user selects laminar air flow show the animation involving
laminar air flow

Button 03

Interaction 2: In slide-17: give user un-know/any sample and instruct to find

the exact wavelength for the sample determination?
Instruction: let user start taking the absorbance of the sample at all the
wavelength, compare the readings and conclude the wavelength for higher
absorbance.

Instructions/ Working area

Credits

APPENDIX 1

Questionnaire:
Question 1
Absorbance can be taken using
a) Calorimetry
b) Spectroscopy
c) spectrophotometry
d) Refractometry
Question 2
UV-Visible spectrophotometer works based on
a)
b)
c)
d)

Beers law
Lamberts Law
Beer-Lamberts Law
Raman spectrum

Question 3:
As the absorbance increases , the intensity of the outcoming light
a)
b)
c)
d)

Decreases
Increases
Remains same
zero

APPENDIX 2

Linkswebsites:
for further
Reference

reading

1. http://www.stanford.edu/dept/EHS/prod/researchlab/bio/docs/typ
es_biosafety_cabinets.pdf
2. http://www.nuaire.com/download/brochure/airegard_laminar_airfl
ow_products.pdf
3. http://www.structuralchemistry.org/teaching/downloads/scm08_1
0.pdf
4. http://www.fondriest.com/pdf/thermo_colorimeter_theory.pdf
5. Video for pipette: http://www.labtricks.com/2010/01/11/how-touse-a-pipette/
6. Video for buffer preparation:
http://www.labtricks.com/2010/01/01/how-to-make-and-phbuffers/

APPENDIX 3

Summary
The instruments discussed are routinely used in the proteomics
experiment. Each and every step of the instrument as a principle
behind, which when followed properly will yield better result.