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Flashcards in Spectroscopy Deck (17):
1

Spectroscopy

- identify biological macromolecules
- main use: quantification of biological macromolecules
- how much protein and nucleic acids obtained

2

Spectrophotometers

1. Take light from polychromatic source and use a monochromator to select a narrow band width of light
2. Measure relative intensity of light passing through a sample as compared to a control or blank

3

1. Take light from polychromatic source and use a monochromator to select a narrow band width of light

- light from light source separated into component wavelength by a diffraction gradient or prism.

4

2. Measure relative intensity of light passing through a sample as compared to a control or blank

- 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

Beer's law

- 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

Beer-Lambert Law

- 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

The molar extinction coefficient

is given as a constant and varies for each molecule.

8

The light absorbed depends on:

- the wavelength
- the characteristics of the absorbing molecules
- the concentration of the absorbing molecules.

9

Comparison of DNA and Protein Absorbance Spectra

- Nucleic acid absorbed at 260 nm
- Protein absorbed at 280 nm

10

purity ratios

- indication of the purity of a DNA samples
- A260/A280

11

a pure solution of DNA/RNA

- 1.8
- 2.0

the addition of protein will typically reduce these values.

12

Extinction coefficient

ds-DNA = 0.02
ss-DNA = 0.025

13

Proteins at 280

- 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

Fluorescent protein spectra

- wavelengths of 400-800

15

Each instrument has a linear range

- sensitivity of instruments in lab are important
- samples too concentrated or dilute will give an incorrect value on spec.

16

if too concentrated

- dilute first then measure
- multiply by dilution factor once you have your reading.

17

Nanodrop

- use a small amount of sample (~1 mL) to quantify
- most traditional specs require 1 mL or 100 mL
- normally connected to a computer that does calculation for you.