Lecture 4 Flashcards
What is absorbtion
The molecules absorb energy (light) and transition the electrons to new states
Only certain wavelengths of light have the correct energy to transition an election to a higher state
When measuring absorbance what actually absorbs
The biomolecules
Solvent and other molecules
The cuvette
What type of cuvette is used for UV range
Visible range
Quartz (self masking so no stray light passes through the cuvette into the sample)
Normal clear plastic cuvette
What is a nano drop
A machine used to do absorbance reading of very small sample volumes
0.5-2microL of sample
How does the nano drop work
The detector is at the bottom light come in from the top
The sample drop goes directly on detector and the upper arm lowers to touch the drop
The path length can be adjusted so you get enough signal but don’t saturate the detector by easing and lowering the arm
In the nanodrop what do adhesion and cohesion do
Adhesion gives the sample a tug and makes it adhesive to the arm
Cohesion keeps the liquid in a column form
If the arm squishes the sample too much what happens
The sample is too concentrated on the detector
What is the absorbace at 205nm of something
At 280?
The peptide bond
Trp and tyr
What parts of the protein actually absorbs light (chromophores)
Peptide bonds, phe, tyr, trp, his, cystine, amides in asn and gln, carboxylates in asp and glu
C terminus
What other things absorb
Prosthetic groups (non protein part of the protein like a metal, heme)
DNA
What is the wavelength of absorption for a peptide bond
190-230
Peak at 205
Aromatic amino acids absorb at what wavelength
Which absorbs most
280
Trp, tyr and phenylalanine absorbs less
What is the wavelength of visible light
400-700nm
Beers law
A= ECL
What does the absorbance at 205nm tell us
Not dependent on amino acids composition just the peptide bond
The absorbace of the bonds is larger so we can use more dilute samples
What does delta A295 tell us
The absorbance of tyrosinate (a295 ph 12 - ph 7)
What is tyrosinate
Deprotonated form of tyrosine
What causes light scattering
Aggregates (from denatured protein)
Optical density equation
OD = A + scattering
What is the lambert beer law
A= -log (I/I0)
I is intensity measure at the detector, I0 is intensity from the lamp
If A=2 what is I/I0
If A=1 what is I/I0
0.01 , 1% of light is getting through (intensity out is 1%)
0.1, 10% of light is getting through
Why do we use air as a reference
To check that the cuvette doesn’t absorb in the wavelength range of interest
What are buffer you can use for measuring absorbance
Tris, phosphate, water
What is albumin
The most abundant protein in the blood
Carrier of fatty acids
What type of lamps do shimazdu specs have
visible and ultraviolet
What does a band pass of 1 nm mean if our light is 300nm
Means the light is actually from 299-301nm
What is the optimal range of absorbace
What happens if greater than the range
0.1-1
Dilute the sample if greater than 1
Once you change from uv to visible wavelength what do you do
Use the plastic cuvette
What do you clean quartz cuvette with
Uv -plastic
Water then ethanol
Methanol or water, or throw away
What are the errors with absorbace measure ments
Scratches on cuvette
Turbid samples
Absorabce not is 0.1-1 range
Using buffers that absorb
Not running a refence baseline (buffer vs buffer)
What are tips for measuring absorbance
Check if solution is turbid
Make sure enough solution in cuvette
Include the region where there’s no absorbance
What do you do for measuring difference spectra
If tyr vs tyros use quartz cuvette since 295nm
What is in the reference beam for a difference sorectrum
Put the ph7 solution in the reference because we’re cancelling the signals ofof the ph7 tyrosine and tyrosinate that are the same and only measuring tyrosinate
What are the advantages and disadvantages of measure spectra over single wavelength measurement
Advantages: can detect interference, can baseline , can identify chromophores
Disadvantage: the spectra takes more time to measure
What are the advantages and disadvantages of measure spectra over single wavelength measurement
Advantages: can detect interference, can baseline , can identify chromophores
Disadvantage: the spectra takes more time to measure
What can absorption spectra be used for
Measuring concentration
ID chromophores
get info on purity
Measure binding of ligands
