MES Flashcards
FLUORESCENCE SPECTROPHOTOMETRY is also called
Fluorometry
Fluorescence Spectro Principle: Certain molecules, particularly those with a
____________ and a rigid structure (fluorophore),
can be excited by __________, and will then
emit the radiation absorbed at a _______ wavelength.
chromophore; UV/visible radiation; longer
longer wavelength = _______ (lower/higher) energy
lower
in UV-Vis spectroscopy, we only need
________________ for them to absorb UV-Vis
radiation
chromophores
in fluorescence, you need a ___________ structure
rigid
What theory: promotion of electron to an excited state
Absorption
What theory: excited electron returns to the ground
electronic state by emitting a photon
EMISSION
emission arising from an “allowed”
transition that typically has a short lifetime between 1
ns and 10 ns
Fluorescence
T/F: does Fluorescence have short life time?
T
In Fluoroscence: transition from the excited
state to the ground state _____________ (change/does not change) in
electron spin
does not change
emission arises from a
“forbidden” transition that typically has a long lifetime
between 1 ms and 1 s
Phosphorescence
In Phosphorescence, there is ________ (a/no) change in electron spin
there is a change in electron spin
In phosphorescence, the decrease in energy is not only due to the
release of energy, but also because of the
__________________
change of electron spin (intersystem crossing)
change in spin → intersystem crossing →
release radiation
a polyatomic fluorophore, once electronically
excited, experiences vibrational relaxation before
emitting a photon, causing a ______________ of the fluorescence spectrum relative to the
wavelength at which it was excited
red shift or Stokes
shift
change in energy or shift in
longer wavelength
red shift or Stokes
shift
excitation light sources
xenon lamps, lasers
most common excitation light source, high intensity and
broad wavelength range (UV to NIR)
Xenon lamps
highest-intensity source
Lasers
Lasers are used in applications where _____________ (short/long) collection times
and __________ (big/small) amounts of sample are required
short ; small
a filter or a monochromator with a known peak
transmission wavelength and bandwidth
EXCITATION WAVELENGTH SELECTOR
enables fluorescence excitation spectra to be
resolved
EXCITATION WAVELENGTH SELECTOR
Separates the wavelength
EXCITATION WAVELENGTH SELECTOR
includes all optics and other equipment needed to
deliver the excitation beam to the sample, collect
the emission from the sample, and hold the sample
in place
SAMPLING DEVICE
cuvettes, microwell plates, microarrays, microscope
slides, and flow systems
SAMPLING DEVICE
ensure that the emission wavelength region being
detected does not overlap with the excitation
wavelength profile
EMISSION WAVELENGTH SELECTOR
important for the rejection of stray light
EMISSION WAVELENGTH SELECTOR
filters, monochromators, and grating polychromators
often are used for
for emission wavelength selection
a photomultiplier tube (PMT) or a charge-coupled
device (CCD) array
DETECTOR
T/F: EXCITATION - EMISSION ORIENTATIONS depends on the nature of the sample
T
Angle used to measure dilute solutions and other
transparent samples
right-angle or 0°/90° geometry
excitation light is at ______° with emitted light
90°
used to measure optically dense samples
front-face geometry
In front-face geometry, fluorescence is collected at an angle ___________
≤90°; although <90° is more common to use
example of front face geometry
epifluorescence geometry
excitation
beam and collected fluorescence are both on the same side of the sample,
epifluorescence geometry
angle used in microscopy
0°/180° transmitting geometry
FACTORS THAT AFFECT QUANTITATION
intrument based, sample based
wavelengths, bandwidths, and detector
intensity of the excitation beam
linear intensity range of the detection system
diffraction efficiency of gratings
Instrument based factors
instrument based factor that can
introduce measurement uncertainty or bias that is
particularly significant when measured values are
compared between instruments
wavelengths, bandwidths, and detector
instrument based factor that can change
significantly with excitation wavelength or with time
intensity of the excitation beam
does not increase linearly
with concentration because of significant absorption
of the excitation beam and/or emission
(reabsorption) by the sample
optically dense samples
optically dense samples do not increase linearly
with concentration because of significant ________________
of the excitation beam and/or _____________ by the sample
absorption; emission
optically dense samples ____________ (increase / do not increase) linearly
with concentration because of significant absorption
of the excitation beam and/or emission
(reabsorption) by the sample
do not increase
To fix opitcally dense samples we must ___________________
reduce the amount of fluorescence that reaches
the detector
fluorescence intensity of a sample may ___________ (decrease/increase)
with time of exposure to light because of
photobleaching and photodegradation
decrease
fluorescence intensity of a sample may decrease
with time of exposure to light because of
______________ and ____________
photobleaching and photodegradation
intensity might _____________ with longer analysis
times
decrease
fluorescence intensity of fluorophores is
temperature _____________ (dependent/independent)
dependent
can introduce peaks into the
fluorescence spectrum - sample’s solvent or matrix
Raman signal
Applications of fluorescence
- analyzing fluorescent compounds (with
fluorophores)
-Determination of fluorescent drugs in low-dose
formulations in the presence of non-fluorescent
excipients.
-carrying out limit tests
-Useful for studying the binding of drugs
-Widely used in bioanalysis
T/F: proteins in general are also fluorescent, especially those that contains amino acids
tyrosine and tryptophan
T
T/F: free drugs have higher fluorescence compared
to bound drugs
T
Strenghts of fluorescence
-selective detection; quantify a strongly fluorescent compound in the
presence of a larger amount of non-fluorescent
material.
-used to monitor changes in complex
molecules
Limitations of fluorescence
-only applies to a limited number of
molecules.
-subject to interference by
UV-absorbing species,
According to Raman Spectroscopy, change in wavelength occurs because of the
molecules that ________ light and not because of
relaxation
deflect
change in wavelength occurs because of the
molecules that deflect light and not because of
relaxation
RAMAN SPECTROSCOPY
The Raman effect is analogous to fluorescence
except:
-not wavelength dependent
-not require the molecule to have a
chromophore
-Energy shift in cm-1 (wavenumber) is measured
instead of wavelength
T/F: In Raman Spectroscopy, the shifts measured correspond to the
wavenumbers of the bands present in the middle-IR
spectrum of the molecule.
T
lasers are used to provide high-intensity radiation in
the visible region, generally somewhere between
______________ and _________ (NIR lasers)
450 and 800 nm
T/F: lasers do not excite fluorescence
T
bands that absorbed weakly in middle -IR region
will absorb strongly in the ________
Raman region
Raman application
-for identifying complex samples,
-peptide pharmaceuticals can be
analyzed for changes in their three-dimensional
structure
-Provides additional fingerprint identity information
complementary to middle-IR spectroscopy
Raman strength
-requires very little sample preparation
-Increasingly a readily available option
Raman limitations
-Not yet fully established
-solvent may interfere
Instrument used in Raman
Laser
Two types of MES
Fluorescence, Raman
More modern type of MES
Raman
compound present in a material that is responsible for the color of the compound
Chromophore
T/F: compound with intense color have chromophore
T
Compounds with chromophores (do not need/ need) a light source to emit a color
do not need
rigid structure that gives glow characteristics to a material.
Fluorophore
Principle involved in Glow in the dark material.
Fluorophore
need a light source to be absorbed and the energy will be emitted as the “glow
Fluorophore
UV VIS, once absorbed, it will emit it immediately
during the __________ state through a photon
excitation
have more wv that it can absorb; longer wv
MES
only one setting
UV:
T/F: MES can absorb energy
at different wv so you can see the photon emission
at a longer time through the form of fluorescence or
glowing
T
Fluorescence absorb ________ wv therefore it cannot go back to ground state immediately but it will come back since this is where it will emit the photon
longer
T/F: Fluorescence will not come back since this is where it will emit the photon
False - it will come back
T/F: Fluorescence = can see nanoseconds
F
difference between the two peaks of
intensity between excitation and emission
Stokes shift:
emit immediately after excitation
UV
MES has __________ resulting to its glow in the dark property
overlap
the energy at which is
absorbed by the compound
Wv
In ________ vibration relaxation is not heard since compounds tested under UV does not contain a chromophore
UV
Is vibration relaxation heard in Chromophore
yes
has excitation and emission monochromator to resolve the Strokes shift
MES
Resolve where the excitation ended and emission started
MES
Signal is _________ in MES
accurate
light source used if compound is really hard to excite
Lasers
purpose: to separate the peaks of individual excitation
to know how it can be quantified.
Excitation Wavelength Selector
to resolve, correct, or give clarity of the correct wv at
which the compound was excited
Excitation Wavelength Selector
Light beams that reaches the detector without passing the sample
stray light
stray light appear as _______
false negative
resolve the emission wv so the overlap will be resolved
Emission Wavelength Selector (filters, monochromators, and grating
polychromators)
right-angle excitation
0º
right-angle emission
90º
when the light source is placed at a different angle, it will (glow/not glow)
not glow
T/F: When (light source) titled to a particular geometry, it will then be able to excite,
seen, emit photons
T
T/F: when fluorescence is collected form above, you cannot get anything from the
light source
T
Detector will not show a signal so must tilt the test instrumentation
at a particular orientation based on ________
the characteristic of the sample being
tested
only one angle
microscope
T/F: In microscopy, you cannot see anything if viewed from another side.
T
T/F: Interferences can come from the instrument or the sample
T
if detector is placed in an incorrect geometry, there will be an ________
interference or the noise
Wrong wv: __________.
nothing will be absorbed
