WKU Bio 114 Online Spectroscopy Lab

- Protein Concentration and Spectroscopy
Spectroscopy is the measurement of the wavelength and intensity of absorption of near-ultraviolet (UV) and visible (vis) light by a sample. UV-vis spectroscopy is usually applied to molecules and inorganic ions or complexes in solution. The UV-vis spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements. The concentration of a substance in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert Law.

The Beer-Lambert law (or Beer's law) states the linear relationship between absorbance and concentration of an absorbing substance. The general Beer-Lambert law is usually written as:

A = a() * b * c

where A is the measured absorbance, a() is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration.

When working in concentration units, the Beer-Lambert law is written as
A = X * b * c; where X is the wavelength-dependent absorptivity coefficient

Instrumentation - Spectrophotometers ("Spec's") measure the amount of transmitted or absorbed light. We will be using various types of the "Spec 20". The light source for a Spec 20 is usually a tungsten lamp that produces light in the visible range. The wavelengths of the light sources are selected with a wavelength separator such as a prism or grating monochromator. Spectra are obtained by scanning the wavelength separator and quantitative measurements can be made from a spectrum or at a single wavelength.

The Spec 20's can measure either Transmittance or Absorbance. Absorbance is the light removed (absorbed) as it passes thru a solution. Transmittance is the light that makes it thru the solution. These, naturally, are inversely proportional. If very little light is absorbed by a solution, then a lot of light will be transmitted thru the solution. We will concern ourselves with Absorbance in all our measurements.

Preparing your Spec for use - As we said above, there are several variations of Spec 20s, below is one example. Move your mouse over the dials to see what each does, then carefully follow the instructions. Remember that 0.0 Absorbance = 100% Transmittance and that infinite Absorbance (9999.9999) = 0% Transmittance.