Spectrophotometry
Exp. 1: Spectrophotometry
【Objective】
1. To learn and understand the concepts and principles of spectrophotometric assay,
plotting standard curve and its application and the calculating results of the
standard compare
2. To become familiar with the commonly used spectroscopic methods for protein
quantification.
3. To become able to operate spectrophotometer and to make measurements.
【Principle】
Suppose you are looking at two solutions of the same substance, one is of a
darker color than the other. Your common sense tells you that the darker color
solution has more substances there. In other words, as the color of solution
becomes darker and darker, the concentration of the substance in this solution
becomes higher and higher. This is a basic principle of spectrophotometric assay:
the intensity of color is a measure of the amount of a material in solution.
The quantitative measurement of colorless materials is usually achieved based
on the principle that they will be converted to colored substances in certain
chemical or biological reactions.
1. The relationship of substance color and the light it absorbs
Complementary color
When two colors are mixed in appropriate proportions, a white color will be
produced. Thus, these two colors are called complementary color.
The color we see in a sample solution is due to the selective absorption of
certain wavelengths of visible light and transmittance of the remaining light. If the
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Spectrophotometry
sample absorbs all wavelengths in the visible region, it will appear black; if it
absorbs none of them, it will appear white or colorless. We see the various colors
when particular wavelengths of radiant energy strike our eyes.
Assuming that we shine a beam of white light at lactoflavin, it absorbs blue
light. Since the blue component of the white light gets absorbed by the substance,
the light that is transmitted is mostly yellow, the complementary color of blue. This
yellow light reaches our eyes, and we “see” the substance as a yellow colored
substance.
The visible range is only a very small part of the electromagnetic spectrum.
Ultraviolet and infrared spectrophotometric methods are suitable for many
colorless substances that absorb strongly in the UV or IR spectral regions.
2. Lambert- Beer Law
When monochromatic light (light of a specific wavelength) passes through a
color solution, there is usually a quantitative relationship between the solute
concentration and the intensity of the transmitted light, that is,
I0
I
Where:
I0 = the intensity of incident light
I = the intensity of the transmitted light
C = the concentration of the solute being measured
L = the path length, i.e. the length of the radiation path
through the sample
C
L
Transmittance (T) is defined as the ratio of the amount of the transmitted light
to the amount of the incident.
Transmittance =
I
I0
=
intensity of incident light
intensity of the transmitted light
Absorbance (A) is defined as the negative logarithm of the transmittance, and
you will note that absorbance and transmittance bear an inverse relationship.
Absorbance = - log T = - log I/I0
The absorbance of a solution is proportional to the number of absorbing
molecules, that is the concentration (C), and the distance (L) that the light passes
through.
A = KCL
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Spectrophotometry
This relationship is called the Lambert-Beer law.
K, the proportionality constant, goes by any names: extinction or absorption
coefficient or absorptivity constant. This is a characteristic of each solute. It is
dependent on the wavelength of light and on the solvent conditions.
The unit for the extinction coefficient depends on the units used to express the
concentration and the path length. A molar extinction coefficient (expressed in ε)
has units of M-1cm-1. Sometimes, the proportionality constant is given as the
absorbance of a 1% w/v solution in a cell with a path length of 1 cm. In this case
the constant in the Beer-Lambert Equation becomes E1%.
3. The calculation of the concentration of unknown substance
(1) Standard comparison method
In practice, the concentration of a solute in a sample with unknown
concentration or named as unknown sample (u) can be determined directly by
comparing the A of the unknown sample to the A of a standard solution whose
concentration is known or named as standard sample(s) providing that such
compounds obey the Lambert-Beer Law and all conditions under which standards
and unknowns are prepared should be kept identical.
Standard sample
As=Ks×Cs×Ls
Unknown sample
Au=Ku×Cu×Lu
Since the L, the path length and the extinction coefficient will be constant; that
is, Ls=Lu and Ks=Ku, thus,
As/Au=Cs/Cu, Cu=Au/As×Cs
(2) Standard curve method
According to the Lambert - Beer Law, there exists a linear relationship between
absorbance and concentration of a solute when all conditions under which
standards and unknowns are prepared should be kept identical. So a plot of
absorbance vs. concentration of absorbing solute yields a straight line passing
through the origin.
This is usually done by preparing a series of standard solutions, each with a
known concentration of a given compound, measuring their absorbance values and
plotting absorbance vs. concentration to construct a curve. The concentration of the
unknown sample can be located by drawing a straight line from point of
absorbance of the unknown until it intersects with concentration curve, and then
draw perpendicularly to the X-axis to identify the concentration of your unknown
sample.
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Spectrophotometry
The Lambert-Beer law implies that when concentration is equal to zero (C = 0),
absorbance must also be zero (A = 0). In other words, the standard curve must pass
through the origin.
4. General operation procedure
（1） Parameter selection
1) Choice of Wavelength
The plot of absorbance of a sample versus
wavelengths is called the absorption spectrum. The plot
below gives the absorption spectrum of potassium
permanganate (KMnO4). You will see that the absorbance
changes with wavelengths.
Theoretically we could choose any wavelength for
quantitative estimations of concentration. However, the
magnitude of the absorbance is important, especially
when you are trying to detect very small amounts of
material. For this reason we generally select the
wavelength of maximum absorbance for a given sample and use it in our
absorbance measurements.
2) Choice of Absorbance
It is strongly recommended to measure absorbance in the range 0.05-1.0 for
the following reasons. 1. When you are trying to detect very small amounts of
material, the magnitude of the absorbance is important. 2. When the absorption
band has a “flat” top, the rate of change in absorbance with wavelength is smaller
than that on the rising and falling shoulder of the peaks.
3) Blank reference solution (blank)
Since transmittance is a relative measurement, the light transmitted by the
sample should be compared to the light transmitted by a "reference" solution
(Blank). The reference solution is generally the solvent in which the colored
compound you are interested in is dissolved. A reference is necessary because the
solvent itself might absorb some light at the wavelength you are using and you
must correct for that absorbance.
We assume that the blank transmits 100% of the light entering it that is the
scale is set to read zero absorbance. Now you can use the full scale of the
spectrophotometer.
（2）Instrument components
The instrument used in spectrophotometic assay is called spectrophotometer.
All spectrophotometers have the following fundamental parts: a source of light, a
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Spectrophotometry
prism or grating to separate the light into narrow wavelength regions, a device for
holding the sample, and a photoelectric cell for measuring light intensity. The
“Spectronic 722” is a conventional instrument for spectroscopic measurement.
Your laboratory instructor will demonstrate how to use it.
（3）Operation for Spectronic 722
1)
2)
3)
4)
Turn on the instrument and allow it to warm up for at least 15 minutes.
Set the desired wavelength with the wavelength control.
Place the blank cell and the sample cell into the cell holder.
With the cover opened, adjust the readout to 0% T (transmittance) using zero
control (with blank in the light path) on the %T scale. Then close the cover
to adjust T to 100% using 100% control (or zero absorbance).
5) Turn to the absorbance scale by mode select button.
6) The instrument is now correctly calibrated. Measure absorbance of the
samples.
【Note】
1. Always handle the cuvettes in the top and bottom edges. The cuvettes have two
pairs of side surfaces. One pair is clear and used for light transmittance, and the
other pair is cloudy and for easier manipulation. Never touch the clear surfaces,
except to clean them with kimwipes.
2. Fill the cuvettes with the sample solution to the three-quarter volumes and then
wipe up the surface using kimwipes. Place the cuvette into the sample holder
with the clear surface aligned up with the light source.
5.
Quantization of Protein
(1) The Kjeldahl method of nitrogen analysis
Protein is a biomacromolecule made of nitrogen-containing amino acids.
When making protein determination, the measured quantity of nitrogen is
converted to the protein content using an appropriate numerical factor. For meat
samples, this factor is 6.25 since meat protein is approximately 16% nitrogen.
P = %N × 6.25
(2) UV Absorbance (280 nm)
Measure the absorbance of protein solutions at 280 nm. The absorbance
around 280 nm is primarily due to the aromatic amino acid side chains. Tryptophan
is the most important absorbant; tyrosine makes a small absorption. Other amino
acids are known to be transparent at this wavelength.
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Spectrophotometry
(3) Colorimetric determination (Visible Spectrophotometry)
Protein samples often consist of a complex mixture of many different proteins.
The quantitative determination of the protein content is usually achieved based on
the principle that certain reactions will convert functional groups of proteins to
dye-forming reagents. The intensity of the dye correlates directly with the
concentration of the reacting groups, and can be measured exactly.
【Experiment】
Method 1: Folin method (Lowry protein assay)
【Principle】
Under alkaline conditions, the divalent copper ion can form a complex with
peptide bonds and the copper ion is reduced to a monovalent ion. The monovalent
copper ion and the groups of tyrosine, tryptophan, and cysteine react with Folin
reagent (phospho-molybdic-phosphotungstic reagent) to produce a blue color
product. The intensity of the blue correlates directly with the concentration of the
reacting groups and can be measured exactly.
【Reagents】
1. Lowry stock reagent: Prepare immediately before use by mixing the following
three stock solutions A, B, and C in the proportion 100:1:1 (v:v:v), respectively.
Solution A: 2% (w/v) Na2CO3 in distilled water.
Solution B: 1% (w/v) CuSO4·5H2O in distilled water.
Solution C: 2% (w/v) sodium potassium tartrate in distilled water.
2. 0.9 % NaCl
3. Folin′s reagent (Folin reagent)
4. Standard bovine serum albumin (BSA) solution (0.1%＝1mg/ml)
【Procedure】
1. Add the following reagents in the order listed to the masked tubes.
Volume: ml
1
2
3
4
5
6(B)
BSA
0.1
0.2
0.3
0.4
0.5
－
0.9% NaCl
0.9
0.8
0.7
0.6
0.5
1.0
Lowry stock reagent
5.0
5.0
5.0
5.0
5.0
5.0
2.
3.
4.
Incubate for 20 minutes at room temperature.
Add 0.5ml of Folin′s reagent, mix well with Vortex.
Incubate for 30 minutes at room temperature.
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Spectrophotometry
5.
6.
Read at 650 nm against the blank solution.
Plot absorbance vs. concentration to construct a standard curve.
Method 2: Biuret method
【Principle】
Under alkaline conditions substances containing two or more peptide bonds
form a purple complex with copper salts in the Biuret reagent. The intensity of
purple correlates directly with the concentration of the reacting groups and can be
measured exactly.
【Reagents】
1. Biuret reagent
2. 0.9% NaCl
3. Standard serum (7%)
4. Unknown serum
【Procedure】
1. Add the following reagents in the order listed to the masked tubes.
Volume: ml
ml
2.
3.
4.
1 (u)
2 (s)
3 (B)
unknown serum
0.1
－
－
standard serum
－
0.1
－
0.9% NaCl
－
－
0.1
Biuret reagent
5.0
5.0
5.0
Incubate for 30 minutes at room temperature, mix well with Vortex.
Read at OD readings at 540 nm against the blank solution.
The protein concentration is determined using the following formulation below
Cu = Au/As×Cs
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