Applications Of Spec Flashcards
What things does spectroscopy help with
Enzyme assays
Blood oxygenation
Measuring turbidity
Raman microscopy
Forensics
What machine do enzyme assays use
What is a blank
What is measured
Uv/visible spec
Has all the stuff except the enzyme and we take a reading of it
Adding the enzyme then you take the measurement
What do you need to keep constant in enzyme assays
Ph , temp, ionic strength
What is an enzyme assay
How do you base it on absorbance
A measure of the enzyme activity in a solution, measurement is based on the amount is product formed or substrate used
In absorbance the product formed or substrate used absorbs light a a wavelength in either the uv or visible spectrum
What info do enzyme assays based on absorbance provide
They give the concentration of product or substrate which has to then get converted to amount
Give examples of two different enzyme assays
Measuring reaction of
Lactate dehydrogenase
Acid phosphotase
What is lactate dehydrogenase and what reaction does it do
An enzyme in glycolysis
Turns pyruvate into lactate using NADH
but pyruvate and lactate don’t absorb
We measure the reaction through disappearance of color from NADH which absorbs at 340nm (nad+ doesnt)
What is the enzyme acid phosphotase
Its used as a clinical marker for disease
Ex. Prostate acid phosphotase is used in reactions as an indicator of prostate cancer
What reaction do enzyme acid phosphotase do
It can help form things that give off colour so :
Removes phosphate (po3) group from things through use of water
Then it forms inorganic phosphate and an oh on the substrate to make p-nitrophenol
If something doesn’t give colour in enzyme assays, what can we do
Couple the substrate with something that does give off colour
Once the acid phosphotase makes p-nitrophenol, how can we stop it and why would we stop it
Since it’s an acid phosphotase we can add base (naOH) to make basic conditions and stop the reaction
Want to stop it because it gives us enough time to measure the absorbance of p-nitrophenolate
Once p-nitro phenol is formed and you’ve added base what then happens
The OH from the base reacts with p-nitrophenol to remove the H from its OH
This forms O- on the substrate and it is now p-nitrophenolate
p-nitrophenolate is yellow compound and absorbs at 405nm
Meaning the amount of p-nitrophenol formed can be measured though amount of p-nitrophenolate
What are higher throughout assays
What is a micro plate reader
Instead of doing one experiment at a time, you can do many
Instead of a single cuvette you have a bunch of wells that a bunch of samples can go into
What is a multichannel pipet
Instead of just one pipete tip with one sample you have multiple for different samples
Each one is set to the same volume and you can use thing to load into the micro plate reader wells
What is an oximeter
You put on your finger
It measures the saturation of hemoglobin in your blood with oxygen
What regions does hemoglobin absorb most and least
What about oxygenated hemoglobin
What is their isosbestic point
Red, far IR (FIR)
FIR, red
Absorbs the same amount at 800nm in the near IR region
What is an isosbestic point
The wavelength where the two NON INTERACTING species have the same absorption coefficient
And that value is not zero
If you have two noninteracting absorbing species how do you find their absorbance
The total absorbance is the sum of both species absorbances
What can the absorbances at an isosbestic point show us
The total concentration (sum) of the two species that are absorbing
Slide 11 equation for concentrations at isosbestic pint
Ok
How does an oximeter detect blood oxygenation
It has both IR and red light sources and a detector
Both lights go through the finger.
The red light is able to go through the oxygenated blood since it is not absorbed
The IR light is able to go through the deoxygenated blood since it is not absorbed
Then the detector see amounts of IR and red light going through , gets ratio of R/IR to see oxyblood/deoxyblood
What is turbidity and the sources of it
It’s an optical property where the light is scattered and absorbed instead of transmitted through the sample
Slit, microorganisms, plant fibres
What can turbidity be used for
Examining water clarity
Measuring effectiveness of processing water
Estimating the amount of bacteria in a sample
How cant turbidity tell us the amount of bacteria
Measure the turbidity of the bacteria as optical density
We have to measure the bacteria optical density between 520-700nm to only get bacteria optical density
This is because the tiny chemical bonds absorb in 200-280 range and we don’t want to measure these bio molecules
how can you tell the mass of a bacterial suspension
And what can this be useful for
What’s hard to quantify
By the turbidity (optical density) of the suspension
We can test new antibiotics in some samples and let them grow to a specific optical density
Then check for growth inhibition in those treated samples
Prokaryotes are cultured but hard to quantify
How has Raman spec changed to help more in spectroscopy
Used to be used to find scattering at a point in the sample
Now used to find Raman scattering over the entire 3D sample at diff depths of the sample
For that you can see a scattering map of the cell interior
What types of Raman microscopy are there and what do they do
Direct imaging : uses the characteristic wave number of a bio molecule in the cell
Ex. You can detect the cholesterol in the cell and see where it is in the cell
Hyper spectral imaging:
Collecting thousands of Raman spectra over a field of view of the sample
The resulting images show a bunch of different bio molecules in the cell (not just one) and their locations
How is spectroscopy used in forensics
They use mass spec , FTIR/RAMAN, or NMR (nuclear magnetic resonance spec)
In using FTIR, they can have a portable one for their field work
What can FTIR characterize in forensics
Solid and liquid (more materials and more rapid)
gasses or vapours (less material can be analyzed)
