Spectroscopy Flashcards
1
Q
Spectroscopy
A
- identify biological macromolecules
- main use: quantification of biological macromolecules
- how much protein and nucleic acids obtained
2
Q
Spectrophotometers
A
- Take light from polychromatic source and use a monochromator to select a narrow band width of light
- Measure relative intensity of light passing through a sample as compared to a control or blank
3
Q
- Take light from polychromatic source and use a monochromator to select a narrow band width of light
A
- light from light source separated into component wavelength by a diffraction gradient or prism.
4
Q
- Measure relative intensity of light passing through a sample as compared to a control or blank
A
- monochromatic light passed through a cuvette containing protein or nucleic acid being measured and then transmitted light is measured by a detector displayed on a spectrophotometer
- measure relative light intensity of beam before and after it passes through the test sample.
- light that comes through sample has to be compared to a control or blank that consists of the solution (water or buffer) the sample is in.
- ensures quantification of only the biological molecule of interest.
5
Q
Beer’s law
A
- the amount of light transmitted is inversely proportional to the concentration of molecules in a sample
- more molecules in a sample to absorb the light, the less that will move through the sample and be detected.
6
Q
Beer-Lambert Law
A
- there is a linear relationship between the absorbance and the concentration
A=Elc
A = measure of absorbance (no units) E = molar extinction coefficient or absorption coefficient (L/mol*cm) l = is the path length (cm) c = the concentration (mol/L) or (ug/mL)
7
Q
The molar extinction coefficient
A
is given as a constant and varies for each molecule.
8
Q
The light absorbed depends on:
A
- the wavelength
- the characteristics of the absorbing molecules
- the concentration of the absorbing molecules.
9
Q
Comparison of DNA and Protein Absorbance Spectra
A
- Nucleic acid absorbed at 260 nm
- Protein absorbed at 280 nm
10
Q
purity ratios
A
- indication of the purity of a DNA samples
- A260/A280
11
Q
a pure solution of DNA/RNA
A
- 1.8
- 2.0
the addition of protein will typically reduce these values.
12
Q
Extinction coefficient
A
ds-DNA = 0.02 ss-DNA = 0.025
13
Q
Proteins at 280
A
- The Protein A280 method is applicable to puried proteins that contain Trp, Tyr, or Phe residues
- Trp>Tyr>Phe
- helps to know AA composition of a protein when quantifying. Will have impact on extinction coefficient of your particular protein.
14
Q
Fluorescent protein spectra
A
- wavelengths of 400-800
15
Q
Each instrument has a linear range
A
- sensitivity of instruments in lab are important
- samples too concentrated or dilute will give an incorrect value on spec.