L10. OPTICAL METHODS OF ANALYSIS.

DETERMINATION OF PROTEINS
USING THE BRADFORD METHOD

This category of analyzing methods is based on the optical properties of the analyzed
system, manifested during its excitation, respectively the interaction with radiant energy of all
wavelengths.
The differentiation between the optical methods is also done following the spectral
domain in which the measurements are done. In this respect, there are: absorption
spectroscopy in ultraviolet (UV), visible (VIS), infrared (IR) or X spectroscopy (absorption
or emission), etc.
From experimental point of view, another classification criteria refers to the
modalities of observation of the leaving signal. On this respect there are: visual methods (the
observation is done with the eye – less used nowadays) and instrumental methods that use
different transductors.
The optical analytical methods are used for the determination of concentrations in the
clinical laboratory, the elucidation of the reaction mechanisms, kinetic studies, discovery of
the structural aspects of compounds in biochemistry researches.

Spectrophotometry

Absorption Spectra
Measurements of the absorbance of light by substances in solution are widely used in
biochemistry because many compounds of interest absorb light in the ultraviolet, visible, or
near infrared domain. Spectrophotometry is the quantitative measurement and study of the
electromagnetic spectra – including visible light, UV, and near-infrared - by absorption of
matter.
The absorbance of light is characterized by two parameters, the wavelength of
maximum absorption and the extent of absorption (extinction coefficient).

Spectral domains
Wave length
Spectral domains
Cm Other used units
3
10 radio
102 1m
10
1 1 cm microwave
-1
10 1 mm microwave
10-2 100 µm IR
10 µm
-3
10 IR
-4
10 1 µm
400 - 760 nm visible
10-5 UV
10-6 10 nm UV (vid)
100 Å
10-7 1 nm X ray
10-8 1Å
-9
10 Gamma ray
10-10 0,01 Å
Visible domain with wavelength characteristics for some colors
Color Peak wavelength (nm)
Blue 470
Cyan 525
Green 560
Yellow 585
Orange 600
Red 645
Deep red 700

Apparatus
Measurements of the absorbance of light are made using a photometer, an instrument
for the direct measurement of light intensities. There are two basic types, a filter photometer
and a spectrophotometer. The former uses filters for the isolation of relatively broad
bandwidths and the latter uses a monochromator, composed of prisms or diffraction gratings,
for the isolation of very narrow bandwidths. The basic parts of a spectrophotometer are a
light source (often an incandescent bulb for the visible wavelengths, or a deuterium arc lamp
in the ultraviolet), a holder for the sample, a monochromator to separate the different
wavelengths of light, and a detector. The detector is typically a photodiode. Photodiodes are
used with monochromators, which filter the light so that only light of a single wavelength
reaches the detector.

Source Monochromator Sample Detector Recorder

Features of an absorption spectrophotometer

Source + Sample Monochromator Detector Recorder

Features of an emission spectrophotometer

The cuvettes for the samples must be made out of substances transparent for the
domain of analysis and their optical length must be reproducible or very exact.

The laws of radiation absorption

The absorbance of light by a solution is described by the Lambert-Beer Law:

log10I0/I = Elc

where I0 = intensity of the incident light

I = intensity of the transmitted light
c = concentration of the absorbing substance
l = length of the light path through the solution
Ɛ = extinction (absorption) coefficient

The quantity log10(I0/I) is known as the absorbance (optical density or O.D. in older
terminology). Thus the Lambert-Beer Law can be rewritten as:
A = Ɛlc where A = absorbance (no units)
 As mentioned, the extinction coefficient is a key parameter of absorption; it may be
defined as the absorbance of a solution having unit concentration and measured with a unit
light path.
The measurement units of the extinction coefficient can be many, but they must be
always related to the units of the concentration and the length of the light path, so that Ɛlc is a
dimensionless quantity. This must be the case since absorbance, a logarithmic function of the
ratio of light intensities, has no measurement units.
In spectrophotometric assays, first a standard curve (or calibration curve) is
constructed by measuring the absorbance of a series of solutions containing varying amounts
of the compound to be assayed. Once the curve has been constructed, the concentration of an
unknown solution may be determined from the absorbance. It is also possible to determine
the concentration of an unknown sample from the absorbance of one standard. In either case,
it is important that measurements are made in the linear part of the standard curve.

Absorption measurements applied in quantitative determinations

The concentration of substances could be determined based on measurements of the

light absorbance by substances in solution especially in the visible domain (color reactions)
in several situations:
The analyzed substance has a characteristic color;
The analyzed substance leads to a colored compound which absorbs light;
The analyzed substance, reacting with a colored compound, modifies that color.

For the analysis of a given compound by measurements of light absorbance:

The obtained absorbance (the color) depends only on the analyzed compound;
The color must be stable in time;
The color development is independent of the ambient conditions;
The Lambert-Beer law could be applied;
The measurements of the light absorbance should be made at maximum of absorption
or other specific λ.

The following methods can be used:

Standard curve method;
The method of reporting to a single standard;
The molar extinction coefficient.

Standard curve method

The method uses 5 or more standard solutions (solutions with known concentrations).
The standards and the analyzed compound are participating to a specific color reaction and all
of them are processed in a similar way. After the colored reaction is finished, the absorbance
of each solution (standards and sample) is measured with a spectrophotometer zeroing it after
a blank solution. The blank solution contains all reagents used for the developing of color
except the analyzed compound. By zeroing the spectrophotometer using the blank, the total
absorbance of these reagents is optically cancelled and uncounted in the total absorbance of
the following samples. The absorbance of each standard is graphically represented versus to
the concentration values (Fig. 1). The graphic should be a line, as it is demonstrated by the
Lambert-Beer law. The unknown concentration is determined based on the graphic
representation and corresponds to the absorbance which was read for the given sample.
 2

A
1

0
10 20 30 40 50
C(µg)
Figure 1. The standard curve representation

Reporting to a single standard method

From all standard solutions from the above experiment you chose a single standard
solution with the known concentration Cs and the absorbance Es (for example tube 3). For
the sample with unknown concentration Cx and the absorbance measured by
spectrophotometer Ex, the next calculation could be done:
Cs................Es
Cx................Ex

Cx= (Ex/Es) x Cs

If the analyzed solution has a dilution coefficient D then:

EP
Cx = ⋅ CS ⋅ D
ES

Reporting to the extinction coefficient

This modality is used only in the situation when a standard is not available in order to
obtain a standard curve. On the other hand, it is known that the Lambert-Beer law is strictly
respected only in the investigated domain of concentration. In this case, very correct
calibrated pipettes are used to measure the volumes of reagents; also cuvettes with a very
well known layer thickness are used (b). The absorption (E) measurement is done at a well
defined wavelength. This way of calculation is used for the determination of enzyme activity
(see optical test) or to establish the sample concentration for which standards are not
available.

Experimental part: Determination of proteins using the Bradford method

Principle of determination:
Proteins react with the Bradford reagent (Coomassie Brilliant Blue G250) in an acidic
medium and form a blue colored compound, whose absorbance of radiation at 595 nm is
proportional with the protein concentration in the sample.
For the calibration curve, 5 solutions of BSA (bovine serum albumine) must be
obtained, using a stock solution of 2 mg/ml concentration. The diluted solutions must be
prepared at a volume of 1 ml, in Eppendorf vials, at the following concentrations:
P1 = 0.2 mg/ml
P2 = 0.4 mg/ml
P3 = 0.6 mg/ml
 P4 = 0.8 mg/ml
P5 = 1.0 mg/ml

After the diluted solutions were obtained, the color reactions are assessed according to
the working protocol:
For the BSA diluted solutions:
Reagent, µl
Diluted BSA sample 50
Bradford reagent 1500

For the unknown sample:

Reagent, µl
Unknown sample 50
Bradford reagent 1500

For the blank:

Reagent, µl
Distilled water 50
Bradford reagent 1500

Homogenize the samples, and incubate at room temperature for 20 minutes. Read the
absorbance of the diluted solutions and unknown sample against the blank at 595 nm.
With the obtained values, draw the calibration curve (A vs concentration) and then
find the concentration of the unknown sample.

Normal values: Serum proteins: 6.2-8.0 g/100 ml.

Pathological values
Pathological variations of total serum proteins have a minor clinical significance due
to their low incidence and low specificity.
Hyperproteinemia is defined by the increase of total serum proteins more than
8 g/100 ml. It appears as relative in hemoconcentration after dehydration states (nausea,
diarrhea), and real by high increase of one or many of the immune system components
(multiple myeloma, collagen diseases, leukemia) or the apparition in excess of some
pathologic proteins.
Hypoproteinemia means the decrease of total serum proteins under 6.2 g/100 ml and
is always associated with a negative N balance. It appears in excessive renal loss of proteins,
hepatic diseases which lead to the decrease of protein synthesis, in immunodeficiency
syndrome - here the low synthesis of the proteins being an effect of the illness, due to the
lower quantity of antibodies which are produced - and also in burns, traumatisms of different
kinds, as an effect of accelerated protein catabolism. Clinically, the protein diet deficiency
(malnutrition, protein malabsorption) produces “hungry edema”, with the decrease of
albumins and osmotic pressure. Immunity decrease, anemia, osteoporosis, gastro-intestinal
troubles appear to these patients.