Applications Of Spec

Question 1

What things does spectroscopy help with

Answer 1

Enzyme assays

Blood oxygenation

Measuring turbidity

Raman microscopy

Forensics

Question 2

What machine do enzyme assays use

What is a blank

What is measured

Answer 2

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

Question 3

What do you need to keep constant in enzyme assays

Answer 3

Ph , temp, ionic strength

Question 4

What is an enzyme assay

How do you base it on absorbance

Answer 4

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

Question 5

What info do enzyme assays based on absorbance provide

Answer 5

They give the concentration of product or substrate which has to then get converted to amount

Question 6

Give examples of two different enzyme assays

Answer 6

Measuring reaction of

Lactate dehydrogenase

Acid phosphotase

Question 7

What is lactate dehydrogenase and what reaction does it do

Answer 7

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)

Question 8

What is the enzyme acid phosphotase

Answer 8

Its used as a clinical marker for disease

Ex. Prostate acid phosphotase is used in reactions as an indicator of prostate cancer

Question 9

What reaction do enzyme acid phosphotase do

Answer 9

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

Question 10

If something doesn’t give colour in enzyme assays, what can we do

Answer 10

Couple the substrate with something that does give off colour

Question 11

Once the acid phosphotase makes p-nitrophenol, how can we stop it and why would we stop it

Answer 11

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

Question 12

Once p-nitro phenol is formed and you’ve added base what then happens

Answer 12

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

Question 13

What are higher throughout assays

What is a micro plate reader

Answer 13

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

Question 14

What is a multichannel pipet

Answer 14

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

Question 15

What is an oximeter

Answer 15

You put on your finger

It measures the saturation of hemoglobin in your blood with oxygen

Question 16

What regions does hemoglobin absorb most and least

What about oxygenated hemoglobin

What is their isosbestic point

Answer 16

Red, far IR (FIR)

FIR, red

Absorbs the same amount at 800nm in the near IR region

Question 17

What is an isosbestic point

Answer 17

The wavelength where the two NON INTERACTING species have the same absorption coefficient

And that value is not zero

Question 18

If you have two noninteracting absorbing species how do you find their absorbance

Answer 18

The total absorbance is the sum of both species absorbances

Question 19

What can the absorbances at an isosbestic point show us

Answer 19

The total concentration (sum) of the two species that are absorbing

Question 20

Slide 11 equation for concentrations at isosbestic pint

Answer 20

Ok

Question 21

How does an oximeter detect blood oxygenation

Answer 21

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

Question 22

What is turbidity and the sources of it

Answer 22

It’s an optical property where the light is scattered and absorbed instead of transmitted through the sample

Slit, microorganisms, plant fibres

Question 23

What can turbidity be used for

Answer 23

Examining water clarity

Measuring effectiveness of processing water

Estimating the amount of bacteria in a sample

Question 24

How cant turbidity tell us the amount of bacteria

Answer 24

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

Question 25

how can you tell the mass of a bacterial suspension

And what can this be useful for

What’s hard to quantify

Answer 25

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

Question 26

How has Raman spec changed to help more in spectroscopy

Answer 26

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

Question 27

What types of Raman microscopy are there and what do they do

Answer 27

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

Question 28

How is spectroscopy used in forensics

Answer 28

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

Question 29

What can FTIR characterize in forensics

Answer 29

Solid and liquid (more materials and more rapid)

gasses or vapours (less material can be analyzed)