is spectrophotometry? | Basic Theory
| BLANKS | Basic
Procedures | Beer-Lambert Law |
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) while other
substances absorb at shorter wavelengths (i.e., ultraviolet =
UV) or at longer wavelengths (infrared = IR). 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. In addition,
the bulbs used to generate the light come in a variety of wavelength
ranges. For example, you can use bulbs which emit light in only
the blue range, or only in the red. For general purpose work,
we utilize broad range bulbs which allow absorbance to
be measured over the entire visible light range.
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Transmittance and Absorbance
is defined as the fraction of incident light which is transmitted,
ie, 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
or optical density, is a logarithmic function of T
and is expressed as:
A = log10
(1/T) = log10 (Io/I)
that absorbance has no units. The shorthand for absorbance is
Axxx, where xxx is the wavelength at which made the
So, for example, at 100% transmittance, A = log 1.0
= 0. At 50% transmittance, A = log (1/0.5) = 0.30. Your 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).
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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
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D. How to
Operate the Spec 20:
- Zero the Spec 20 to infinite absorbance
and a BLANK
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 (Fig. 2).
Figure 2. Wavelength control.
Figure 1. ON/OFF knob and adjuster
for zeroing the spec to infinite absorbance.
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, you will measure only the absorbance
due to the substance in question.
no tube in the holder, adjust the meter needle to read infinite
absorbance (= 0% transmittance) using the left
front knob (= power switch).
a Kimwipe, wipe off/polish the outside of the BLANK cuvette to
remove greasy finger smudges etc. (Fig. 3; you might want to
wear gloves). If not etched in to the cuvette, use a wax pencil
or Sharpie to make a small vertical mark at the top of each cuvette
for alignment in the sample holder (Fig. 4).
the sample holder trapdoor and insert the cuvette such that the
line on the cuvette lines up with the line on the sample holder
(Fig. 5). Close the lid.
Figure 5. Loading the blank.
the right front knob (Fig. 6), adjust the meter
needle to read absorbance = 0.0 (= 100 % transmittance).
This step is called setting the "full scale".
Figure 6. Use right knob to set
full scale against the blank.
is now calibrated to this BLANK. If your experiment involves
multiple reaction tube formulations, each one may need its own
blank and the Spec 20 must be rezeroed for each.
- Measuring Absorbance or Transmittance
on the Spec 20
BLANK and insert cuvette containing your sample. Close lid.
the absorbance (lower scale) OR transmittance (upper scale) as
appropriate for your sample (Fig. 7).
Figure 7. Meter for reading percent
transmittance (upper scale) or absorbance (lower scale).
for subsequent samples which use the same BLANK. (SEE
NOTE: When taking several measurements at the same wavelength
over a short time period, you do not need to reblank for each.
Over longer times, however, the unit may drift and recalibration
to the BLANK will be necessary. IF, however, you change the wavelength,
you must re-zero the instrument. If you are taking readings
over an extended period or sharing the instrument, re-zero for
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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
it is clear that concentration can be
determined from absorbance alone. Absorption coefficients for
biological molecules can be determined experimentally or can
be found in the literature.