CHEM 411L Instrumental Analysis Laboratory Revision 2.1 Fluorescence Quenching of Human Serum Albumin by Caffeine In this laboratory exercise we will examine the fluorescence of Human Serum Albumin (HSA) and its quenching by Caffeine. In molecular fluorescence spectroscopy an analyte is stimulated by excitation photons and then responds by fluorescing; emitting longer wavelength photons. The emitted photons are detected by a spectrometer, generating a signal that can be analyzed. It would be expected that the response signal should be linearly proportional to the analyte concentration. And this is generally true over a wide range of analyte concentrations. However, due to limitations in the technique, non-linearities do set in at higher analyte concentrations. Additionally, other species present in the analyte's solution matrix can quench the analyte's fluorescence signal. In the present case, HSA's fluorescence signal is quenched by the presence of Caffeine. Both static and dynamic quenching processes are observed for this system. Although quenching diminishes an analyte's fluorescence signal, for the present case, we can use quenching data to determine binding parameters for the interaction of Caffeine with HSA. Luminescence involves emission of photons from excited atoms or molecules. Fluorescence and Phosphorescence, both luminescent processes, involve emission of photons from systems that have been excited by absorption of photons. In molecular Fluorescence Spectroscopy, an analyte molecule first absorbs a photon (excitation, E excite ) that leaves the analyte in an electronically and vibrationally excited state. At this point, the molecule rapidly looses excess vibrational energy by non-radiatively relaxing to the ground vibrational level of the excited electronic state. This occurs because energy is transfered to solvent molecules as the analyte molecule jostles against them. This relaxation process is very efficient and very rapid. Now, the molecule can fluoresce (E relax ). Or, the molecule can undergo a non-radiative transition (Internal Conversion) to the ground state. Molecules undergoing Internal Conversion transit to the Ground State without emitting radiation.
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CHEM 411L
Instrumental Analysis Laboratory
Revision 2.1
Fluorescence Quenching of Human Serum Albumin by Caffeine
In this laboratory exercise we will examine the fluorescence of Human Serum Albumin (HSA)
and its quenching by Caffeine. In molecular fluorescence spectroscopy an analyte is stimulated
by excitation photons and then responds by fluorescing; emitting longer wavelength photons.
The emitted photons are detected by a spectrometer, generating a signal that can be analyzed. It
would be expected that the response signal should be linearly proportional to the analyte
concentration. And this is generally true over a wide range of analyte concentrations. However,
due to limitations in the technique, non-linearities do set in at higher analyte concentrations.
Additionally, other species present in the analyte's solution matrix can quench the analyte's
fluorescence signal. In the present case, HSA's fluorescence signal is quenched by the presence
of Caffeine. Both static and dynamic quenching processes are observed for this system.
Although quenching diminishes an analyte's fluorescence signal, for the present case, we can use
quenching data to determine binding parameters for the interaction of Caffeine with HSA.
Luminescence involves emission of photons from excited atoms or molecules. Fluorescence and
Phosphorescence, both luminescent processes, involve emission of photons from systems that
have been excited by absorption of photons. In molecular Fluorescence Spectroscopy, an analyte
molecule first absorbs a photon (excitation, Eexcite) that leaves the analyte in an electronically
and vibrationally excited state.
At this point, the molecule rapidly looses excess vibrational energy by non-radiatively relaxing
to the ground vibrational level of the excited electronic state. This occurs because energy is
transfered to solvent molecules as the analyte molecule jostles against them. This relaxation
process is very efficient and very rapid. Now, the molecule can fluoresce (Erelax). Or, the
molecule can undergo a non-radiative transition (Internal Conversion) to the ground state.
Molecules undergoing Internal Conversion transit to the Ground State without emitting radiation.
P a g e | 2
This is an efficient relaxation process when higher vibrational states of the Ground Electronic
State overlap with lower vibrational states of the Excited Electronic State.
Because of non-radiative relaxation in the electronically excited state, excitation energy is
always greater than relaxation energy.
Eexcite > Erelax (Eq. 1)
Since the energy of photons involved in these transitions is inversely related to the photons'
wavelength:
Ephoton = h c / (Eq. 2)
(c is the speed of light and h is Planck’s constant), the wavelength of an exciting photon is
always shorter than that of a photon emitted during relaxation:
excite < relax (Eq. 3)
Fluorescence spectra can be measured using a Spectrofluorometer. Light from a source is
dispersed and an excitation wavelength is selected using a monochrometer. The excitation
radiation impinges upon the sample, which then begins to fluoresce. Fluorescent radiation is
itself dispersed by a monochrometer and the spectrum is measured using an appropriate detector.
In an actual spectrofluorometer, the dispersing element is usually a diffraction grating. In
simpler fluorometers, wavelength selection is accomplished using filters.
P a g e | 3
The Flourescent Intensity (F) of an analyte solution will be proportional to the radiant Power
absorbed by the sample (Po-P):
F = K’ (Po – P) (Eq. 4)
Inserting Beer’s Law:
P/Po = 10-bc
(Eq. 5)
and expanding the exponential term, gives us:
F = K’ Po {2.3bc – (2.3bc)2/2 - ...} (Eq. 6)
Provided the sample Absorbance is relatively low, we can truncate the expansion after the first
order terms:
F = 2.3 K’ Po bc (Eq. 7)
When Po is constant, we see the Fluorescent Intensity is proportional to the Concentration of the
analyte:
F = K c (Eq. 8)
This, then, provides a method for quantifying the amount of analyte in a system based on
fluorescence measurements.
A few words of caution. If the concentration of the analyte is high enough, higher order
expansion terms become important and the relationship between F and c is no longer linear.
And, if the concentration becomes very high, the system begins to absorb its own emitted
radiation, causing a decrease in fluorescence intensity and as a result severe non-linearities set in.
Fluorescence spectroscopy is much more sensitive than corresponding Absorbance spectroscopic
techniques. This is because light emitted against a dark background (fluorescence) is much
easier to detect than a slight dimming of intensity against a light background (absorbance).
However, fluorescence techniques are severely limited by the number of analytes that actually
fluoresce. Most systems shed their excitation energy via radiationless pathways. Structurally,
molecules that possess unsubstituted aromatic rings or other structurally rigid elements have a
propensity for fluorescing. Fused-ring heterocycles also fluoresce nicely.
In our case, we will be examining the fluorescence of Human Serum Albumin (HSA). Albumins
constitute a family of globular proteins commonly found in blood serum. They are involved in
the transport of fatty acids, bind cations and buffer the pH of their solution. HSA is a monomeric
67 kDalton protein consisting of 585 amino acid residues and comprises ~50% of all plasma protein.