Spectrophotometry

August 2011SLCC/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 ·

• 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.