Transcript Spectrophotometry

Spectrophotometry
August 2011
SLCC/UVU STEP grant workshop
Spectrophotometry
• How much light of a particular wavelength is absorbed
by a sample?
Figure © David P. Goldenberg, University of Utah, 2003
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Absorbance vs. concentration
• Absorbance values above 2 tend to be unreliable
– best values: 0.1 < A < 1.0
Figure © David P. Goldenberg, University of Utah, 2003
The Beer-Lambert Law:
A=C·l·
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•
•
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A = absorbance
C = concentration
l = cuvette pathlength
 = extinction coefficient
– specific for a particular wavelength
– specific for a particular compound
The
electromagnetic
spectrum
http://www.rationalizing.us/blog/files/wavelength_figure.jpg
Different molecules have different
absorption spectra
http://dx.doi.org/10.1590/S0103-50532006000300006
UV and visible absorbance
often arises from…
• Coordinated metal ions
– Chlorophyll
– Heme
• Systems of conjugated double bonds
UV
absorbance
by aromatic
amino acids
…and nucleic acids!
Spectral overlap between
proteins and nucleic acids
Figure © David P. Goldenberg, University of Utah, 2003
Notes about absorbance
• Absorbance is unitless
• Absorbance is sometimes also referred to as
optical density (OD)
– often, the wavelength of light is denoted, e.g.
OD600 = absorbance at 600 nm = A600
The Beer-Lambert Law:
A=C·l·
• For double stranded DNA,
–  has been found to be 20 L/(g cm)
– C = 299 792 458 m / s or 3.0 x 108 m/s
– l = A rule of thumb in molecular biology, using
standard 1 cm cuvettes:
1 A260 unit for dsDNA = 50 ug/mL
• Use Beer’s Law to prove it
Practical points
• The cuvette must be transparent to light of the
wavelength of interest
– Glass or standard plastic ok for visible light (≥ 350 nm)
– Specialized cuvettes (quartz or TrUView) required for UV
light (~200-350 nm)
• Absorbances are measured relative to that for a
“blank” solution that contains everything except the
compound of interest
– If your compound is in water, blank with water
– If your compound is in a buffer, blank with the buffer
• Ensure cuvettes are placed in the proper orientation
Two types of specs in our lab
• SmartSpec Plus
• NanoDrop 1000
SmartSpec Plus
• uses cuvettes
– must be transparent to wavelength of interest
• built in programs estimate concentrations of
DNA, protein, cells, etc.
• relatively large sample volume (50-500 µL,
depending on type of cuvette)
NanoDrop
• Measures absorbance with sample volume as
low as 1 µL
Absorbance ratios
• Nucleic acid absorbs strongly at 260 nm
• Protein absorbs strongly at 280 nm
• Ratio of 260/280 allows comparison of amount of
nucleic acid vs. protein
– ratio of ~1.8 is generally accepted as“pure” for DNA
– ratio of ~2.0 is generally accepted as “pure” for RNA
– if the ratio is appreciably lower in either case, it may
indicate the presence of protein, phenol or other
contaminants that absorb strongly at or near 280 nm.
Absorbance ratios
• Nucleic acid absorbs strongly at 260 nm
• Carbohydrates, some reagents used in
purification absorb near 230 nm
• Expected 260/230 values are commonly in the
range of 2.0-2.2.
– If the ratio is appreciably lower than expected, it
may indicate the presence of contaminants which
absorb at 230 nm.