|
|
|
A. Introduction
Spectrophotometry is a technique used to measure how much radiant energy a substance absorbs at varying wavelengths of light. Pigments such as chlorophyll and other colored materials (i. e., dyes) absorb energy in the visible light range (380-760 nm). Do not use the Spec 20 for other substances which absorb at shorter wavelengths (i.e., ultraviolet = UV) or at longer wavelengths (infrared = IR) The instrument is not capable of a wider absorption range. The other spectrometers and the Spectronic 21 are used to make measurements in the UV or IR regions.
By measuring the Absorption Spectrum of a substance, i.e. all the wavelengths at which it absorbs, it is possible to identify it or at least place it in a particular class of compounds. The wavelength at which peak absorption occurs, the absorption maximum, is very useful when trying to identify an unknown. By creating and measuring a series of standards, it is possible to quantify the amount or concentration of a substance in a sample. For example, in their pure form, the nucleic acids can be quantified by absorption measurements in the UV range.
Wavelength is selected by adjusting a prism within the instrument such that only a narrow range of light wavelengths are directed through the sample.
B. Theory: Transmittance and Absorbance
Transmittance (T) is defined as the fraction of incident light which is transmitted, i. e., passes through, a sample. Thus, T = I/Io, where Io equals the intensity of light which strikes the sample and I is the intensity of light after passing through the sample. Transmittance is usually expressed as a percentage:
%T = (I/Io) x 100
Absorbance (A), or optical density, is a logarithmic function of T and is expressed as:
A = log10 (1/T) = log10 (Io/I)
Note that absorbance has no units.
So, for example, at 100% transmittance, A = log 1.0 = 0. At 50% transmittance, A = log (1/0.5) = 0.30. The Spec 20 has two scales, one calibrated from 0 to 100% Transmittance and the other as Absorbance, ranging from infinity to 0. Note that the highest calibrated unit of absorbance is 2.0. Spectral data are usually plotted as absorbance (Y-axis) vs. wavelength or concentration (X-axis).
C. BLANKS
In order to measure the absorbance of a particular substance in a reaction mixture, it is necessary to first "zero out" the spectrophotometer such that only the absorbance of the substance of interest is measured. This is done with a blank - a cuvette which contains all the carrier solvents EXCEPT the substance of interest. A separate BLANK is needed for every unique reaction mixture.
Formulation of the BLANK depends on the particular experiment and may involve use of dyes or other colorimetric indicators.
D. How to Operate the Spec 20:
Zero the Spec
20 to infinite absorbance and a BLANK
1. Turn on the Spec 20 and allow it to warm up for 5 - 10 minutes (left front knob). Set wavelength using the dial on top of the Spec 20.
2. Prepare a BLANK cuvette by adding all solvents EXCEPT the substance to be measured. (Refer to your experimental protocol).
A BLANK is
used to calibrate the Spec 20 so that any absorbance attributable
to the solvent and/or glass cuvette can be compensated. By zeroing
the Spec 20 to the blank, an instrumental subtraction will measure
only the absorbance due to the substance in question.
3. With no sample tube in the holder, adjust the meter needle to read infinite absorbance (i. e. 0% transmittance) using the left front knob (i.e. power switch).
4. Using a Kimwipe, wipe off/polish the outside of the BLANK cuvette to remove greasy finger smudges etc. (Wear gloves).
Using a wax pencil or Sharpie, make a small
vertical mark at the top of each cuvette for alignment in the
sample holder. 
5. Raise the sample cover door and insert the cuvette such that the line on the cuvette lines up with the line on the sample holder, close the lid.
6. Using the right front knob, adjust the meter needle to read 0.00 absorbance (i. e. 100 % transmittance). This step is called setting the "full scale".
The spectrophotometer is now calibrated to this BLANK. If the experiment involves multiple reaction tube formulations, each one will need its own blank and the Spec 20 must be re-zeroed for each.
Measuring Absorbance
or Transmittance on the Spec 20
7. Remove BLANK and insert cuvette containing the sample. Close lid.
8. Read the absorbance (lower scale) OR transmittance
(upper scale) as appropriate for your sample.
9. Repeat for subsequent samples which use the same BLANK. (SEE NOTE BELOW)
NOTE: When taking several measurements at the same wavelength over a short time period, no need to re-blank for each. Over longer times, however, the unit may drift and recalibration to the BLANK will be necessary. If, however, the wavelength is changed, then re-zero the instrument. If readings are taken over an extended period or sharing the instrument, re-zero for each measurement.
E. Beer-Lambert Law
The Beer-Lambert law describes an important relationship that exists between absorbance (A) and two sample parameters - solute concentration (c) and length of the light path (l). Simply put, the law states that absorbance, A, is directly proportional to c and l, and is represented by the following equation:
A = µcl
where, µ is a constant, the absorbance coefficient.
In biological research, concentration (c) is usually expressed on a mass/volume basis, e.g., ug/ml, length of light path (l) in centimeters (usually l=1 cm), and µ, the absorbance coefficient, is also expressed on the basis of weight. For practical purposes, the light path is the interior diameter of the cuvette and is the same for all samples. Therefore, a plot of absorbance vs. concentration yields a straight line with slope µ. Such a curve using known concentrations of a pure substance is called a standard curve. A standard curve is then useful for determining the concentration of the same substance in solutions of unknown concentration. By algebraic rearrangement of the above equation to,
c = A / µl
Concentration can be determined from absorbance alone. Absorption coefficients for biological molecules can be determined experimentally or can be found in the literature.