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

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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.

1 Master Layout Slide 412 Colorimetry Centrifugation 2 Slide 13-17 UV.Spectrophotometric analysis Slide 18-21 3 4 5 Slide 22-24 Laminar air flow .

mp4 .1 Step 1: T1: Colorimetry Scroll 2 Opening for cuvette display 3 4 5 Video File: Colorimeters.

like nothing but logarithm ratio of incident light (Io) passing through the incident light to reflected light is sample of concentration ‘c’. a path length “l” and coming out as epsilon constant and concentration of reflected light (I). the incident light. . Now animate the solution. travelling equal to the product of path length. reflected light and path length value.1 Step 2: T1: Colorimetry 2 3 4 5 Description of the action Audio Narration Absorbance of a sample that is Animate the above display. 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.

auto zero and absorbance buttons.1 Step 3: T1: Colorimetry Description of the action 2 3 4 5 Show a instrument labeled as “colorimeter” and draw it as shown in the figure. keep it on stand for 30min to attain the set wavelength. . Meanwhile prepare the dilution of samples. Audio Narration The instrument need to set to the required wavelength at first place before taking the reading. a opening and a display screen. Animate a scroll. Use of colorimetry is explained with the steps from IDD-47 quantitative and qualitative estimation of amino acidninhydrin. 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. for more information. Once the instrument is set for the wavelength.

1 Step 4: T1: Colorimetry 1 2 2 3 4 3 4 5 Video File: Colorimeters.mp4 .

now ask user to click “auto zero“ option and the display should show “0. instruct user to rinse the cuvette with water. auto zero the instrument and take the readings for all the sample tubes. let user clean the cuvette with tissue. Audio Narration Fill the cuvette with the blank solution and take the OD.00” reading then animate like removing the cuvette and pouring the solution out. place it in the opening and click on “Absorbance” the reading in the display should show ”0. .00”.1 Step 5: T1: Colorimetry Description of the action 2 3 4 5 After 30minutes. 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. (repeat the above step twice). Instruct the user to take 2000ul of the blank and add to the cuvette .

. 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. clean the edges of the cuvette with tissue.2”.6. pour the solution out and follow the same for the solutions in tubes “0. Let user take out the full volume from the tube with help of pipette and trasfer it to cuvette.2.0.4.8.unknown 1.1. remove the cuvette.0. Let user fill the cuvette with the solution from the tube labeled as “0.1 Step 6: )T1: Colorimetry Description of the action 2 3 4 5 Instruct the user clean the cuvette with blank. Keep it in the opening as instructed and press” absorbance” and note down the readings.3.

2 0.0 0.06 .05 0.6 0.03 0.01 0.02 Unknown 2 0.1 2 3 4 5 Step 7: T1: Colorimetry Sample OD at 570nm Blank 0.4 0.07 1.8 0.00 0.09 Unknown 1 0.04 Unknown 3 0.

1 2 3 4 5 Step 8: T1: Colorimetry .

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. The absorbing molecules must be uniformed distributed throughout solution.5mg. For colorimetry to work a reaction must be found which will produce a color that can be measured. 0. Choice of the wavelength is very important to get the better sensitivity and selectivity. animate like the user locating the point on y axis for “unknown 1 (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.3mg follow the same for other two unknowns and show the concentration as 0. .1 Step 9: T1: Colorimetry Description of the action 2 3 4 5 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.

1 Step 1: T2: centrifugation 2 3 rotor Centrifuge 4 5 Video File: Centrifuge.MTS and Centrifuge_part2.MTSc .

Now let user pick the rotor lid and close it by making clock-wise movement. time and temperature by regulating the nob.1 Step 1: T2: centrifugation Description of the action 2 3 4 5 The animator should draw a centrifuge instrument as shown in the figure. Now balance the tubes in the rotor. let user rotate in anti-clockwise to open the lid of the rotor. Now let user set the required parameters for speed. Please include the buttons like enter. Let user keep the lid on table. open a regulator nob along with display on the centrifuge. time and temperature. Let user take the centrifuge lid to its normal position and press to close it. animate the centrifuge lid opening on its own. set. Organelles separate when the density of the organelle equals the density of the sucrose gradient . Audio Narration Transfer the content into the centrifuge tube and perform a centrifugation at required speed. animate increase in speed from zero to the set point. along with temperature and count down time display. let user click on “START” button. Let user click on “OPEN” button. take out the tubes to centrifuged and place them in the opening. start. Once the parameters are set.

1 Step 2: T2: centrifugation r 2 A 3 B 4 http://www.com/watch?v=IhJNFGfsUus 5 .youtube.

relative centrifugal force.118 × 10 ^-5 ) R S^2 g.S . R -radius of the rotor in meters.speed of the centrifuge in revolutions per minute (RPM) r 3) Svedberg: Sedimentation rate: dr/dt = w^2r. and S .1 2 3 4 5 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.

Particle with high density will sediment faster to precipitate than the low density ones which are left out as supernatant (figure: A). The animator should draw a centrifuge as shown in the figure. If the particles are of varying density.1 Step : T2: centrifugation Description of the action 2 3 4 5 Instruct user to carry out centrifugation step. Meanwhile show the different color moving down and stopping at one point as in the previous slide. Relative centrifugal force takes the gravity into account during separation. 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. Now once centrifugation is going the animator must zoom in the instrument and show the rotor and tube rotating . This force along with the particle density and liquid density helps in the separation of the particles. than different layers are formed after centrifugation (figure: B). The sedimentation rate is expressed in terms of svedberg units .

autozero.MTS .flv and UV Spectrometer. absorbance 2 3 Lid that can be opened 4 5 Video File: UV vis spectroscopy.1 Step 1: ) T3:: UV-Visible spectrophotometer Display Options like number 0-9. set wavelength.

light source and sample holder and detector. Go through the IDD enzyme assay for more information. . 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. Audio Narration UV-Visible spectrophotometer has a monochromator.1 2 3 4 5 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.

1 Step 2: T3:: UV-Visible spectrophotometer 2 3 4 5 L .

. the law relates the absorbance and the concentration of the solution. In the equation L signifies the path length. 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. C concentration. The intensity of the light coming out of the cuvette decreases when the concentration of the substances in the cuvette increases. 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-Lambert’s law.1 Step 3: T3:: UV-Visible spectrophotometer Description of the action 2 3 4 5 Now show the figure as in slide (redraw) followed by the formula which is given in the slide.

1 Step 1: T4: Laminar air flow CLASS-2 cabinet 2 3 4 Video File: Laminar air flow_1 5 .

for handling most pathogenic microbes especially in maximum containment labs. this cabinet has HEPA filters for filtering the entering air and the exhaust air and provide personnel.97% of particles of size 0. environmental and product protection.for common usage in microbiological activities. 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. .1 Step2 : T4: Laminar air flow Description of the action 2 3 4 5 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. Air flow prevents the entry of any microbes from the environment. Class 3. This is only for environment protection Class 2. Class 1. The UV in the cabinet is used prior to the usage of cabinet to kill existing organism in cabinet. HEPA (High efficiency particulate air removes 99.7). The laminar air flow used in usage of laminar air flow (slide laboratories uses horizontal air flow type with the 6. The biosafety laminar flow and the types of cabinets is of 3 classes.for has HEPA cabinet filters that removes contaminants from the exhaust air .3micro meters. The filters used are of different types depending the mechanism of working of on the type of sample used.

Air flow prevents the entry of any microbes from the environment.later OFF the UV light. Let user “ON” UV light for 5min. .3micro meters. Audio Narration HEPA (High efficiency particulate air removes 99. zoom in the HEPA filter.1 Step2 : T4: Laminar air flow 2 3 4 5 Description of the action Animate to show the flow of air. Now let user On the “LIGHT” and ON the “AIR FLOW” and animate user doing the experiment on the laminar work bench. The UV in the cabinet is used prior to the usage of cabinet to kill existing organism in cabinet.97% of particles of size 0. blocking the microbes.

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 .

the intensity of the outcoming light a) b) c) d) Decreases Increases Remains same zero .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 .

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

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. .