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MES Flashcards

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1
Q

FLUORESCENCE SPECTROPHOTOMETRY is also called

A

Fluorometry

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2
Q

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.

A

chromophore; UV/visible radiation; longer

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3
Q

longer wavelength = _______ (lower/higher) energy

A

lower

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4
Q

in UV-Vis spectroscopy, we only need
________________ for them to absorb UV-Vis
radiation

A

chromophores

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5
Q

in fluorescence, you need a ___________ structure

A

rigid

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6
Q

What theory: promotion of electron to an excited state

A

Absorption

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7
Q

What theory: excited electron returns to the ground
electronic state by emitting a photon

A

EMISSION

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8
Q

emission arising from an “allowed”
transition that typically has a short lifetime between 1
ns and 10 ns

A

Fluorescence

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9
Q

T/F: does Fluorescence have short life time?

A

T

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10
Q

In Fluoroscence: transition from the excited
state to the ground state _____________ (change/does not change) in
electron spin

A

does not change

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11
Q

emission arises from a
“forbidden” transition that typically has a long lifetime
between 1 ms and 1 s

A

Phosphorescence

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12
Q

In Phosphorescence, there is ________ (a/no) change in electron spin

A

there is a change in electron spin

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13
Q

In phosphorescence, the decrease in energy is not only due to the
release of energy, but also because of the
__________________

A

change of electron spin (intersystem crossing)

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14
Q

change in spin → intersystem crossing →

A

release radiation

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15
Q

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

A

red shift or Stokes
shift

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16
Q

change in energy or shift in
longer wavelength

A

red shift or Stokes
shift

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17
Q

excitation light sources

A

xenon lamps, lasers

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18
Q

most common excitation light source, high intensity and
broad wavelength range (UV to NIR)

A

Xenon lamps

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19
Q

highest-intensity source

A

Lasers

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20
Q

Lasers are used in applications where _____________ (short/long) collection times
and __________ (big/small) amounts of sample are required

A

short ; small

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21
Q

a filter or a monochromator with a known peak
transmission wavelength and bandwidth

A

EXCITATION WAVELENGTH SELECTOR

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22
Q

enables fluorescence excitation spectra to be
resolved

A

EXCITATION WAVELENGTH SELECTOR

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23
Q

Separates the wavelength

A

EXCITATION WAVELENGTH SELECTOR

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24
Q

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

A

SAMPLING DEVICE

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25
cuvettes, microwell plates, microarrays, microscope slides, and flow systems
SAMPLING DEVICE
26
ensure that the emission wavelength region being detected does not overlap with the excitation wavelength profile
EMISSION WAVELENGTH SELECTOR
27
important for the rejection of stray light
EMISSION WAVELENGTH SELECTOR
28
filters, monochromators, and grating polychromators often are used for
for emission wavelength selection
29
a photomultiplier tube (PMT) or a charge-coupled device (CCD) array
DETECTOR
30
T/F: EXCITATION - EMISSION ORIENTATIONS depends on the nature of the sample
T
31
Angle used to measure dilute solutions and other transparent samples
right-angle or 0°/90° geometry
32
excitation light is at ______° with emitted light
90°
33
used to measure optically dense samples
front-face geometry
34
In front-face geometry, fluorescence is collected at an angle ___________
≤90°; although <90° is more common to use
35
example of front face geometry
epifluorescence geometry
36
excitation beam and collected fluorescence are both on the same side of the sample,
epifluorescence geometry
37
angle used in microscopy
0°/180° transmitting geometry
38
FACTORS THAT AFFECT QUANTITATION
intrument based, sample based
39
wavelengths, bandwidths, and detector intensity of the excitation beam linear intensity range of the detection system diffraction efficiency of gratings
Instrument based factors
40
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
41
instrument based factor that can change significantly with excitation wavelength or with time
intensity of the excitation beam
42
does not increase linearly with concentration because of significant absorption of the excitation beam and/or emission (reabsorption) by the sample
optically dense samples
43
optically dense samples do not increase linearly with concentration because of significant ________________ of the excitation beam and/or _____________ by the sample
absorption; emission
44
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
45
To fix opitcally dense samples we must ___________________
reduce the amount of fluorescence that reaches the detector
46
fluorescence intensity of a sample may ___________ (decrease/increase) with time of exposure to light because of photobleaching and photodegradation
decrease
47
fluorescence intensity of a sample may decrease with time of exposure to light because of ______________ and ____________
photobleaching and photodegradation
48
intensity might _____________ with longer analysis times
decrease
49
fluorescence intensity of fluorophores is temperature _____________ (dependent/independent)
dependent
50
can introduce peaks into the fluorescence spectrum - sample's solvent or matrix
Raman signal
51
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
52
T/F: proteins in general are also fluorescent, especially those that contains amino acids tyrosine and tryptophan
T
53
T/F: free drugs have higher fluorescence compared to bound drugs
T
54
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
55
Limitations of fluorescence
-only applies to a limited number of molecules. -subject to interference by UV-absorbing species,
56
According to Raman Spectroscopy, change in wavelength occurs because of the molecules that ________ light and not because of relaxation
deflect
57
change in wavelength occurs because of the molecules that deflect light and not because of relaxation
RAMAN SPECTROSCOPY
58
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
59
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
60
lasers are used to provide high-intensity radiation in the visible region, generally somewhere between ______________ and _________ (NIR lasers)
450 and 800 nm
61
T/F: lasers do not excite fluorescence
T
62
bands that absorbed weakly in middle -IR region will absorb strongly in the ________
Raman region
63
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
64
Raman strength
-requires very little sample preparation -Increasingly a readily available option
65
Raman limitations
-Not yet fully established -solvent may interfere
66
Instrument used in Raman
Laser
67
Two types of MES
Fluorescence, Raman
68
More modern type of MES
Raman
69
compound present in a material that is responsible for the color of the compound
Chromophore
70
T/F: compound with intense color have chromophore
T
71
Compounds with chromophores (do not need/ need) a light source to emit a color
do not need
72
rigid structure that gives glow characteristics to a material.
Fluorophore
73
Principle involved in Glow in the dark material.
Fluorophore
74
need a light source to be absorbed and the energy will be emitted as the “glow
Fluorophore
75
UV VIS, once absorbed, it will emit it immediately during the __________ state through a photon
excitation
76
have more wv that it can absorb; longer wv
MES
77
only one setting
UV:
78
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
79
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
80
T/F: Fluorescence will not come back since this is where it will emit the photon
False - it will come back
81
T/F: Fluorescence = can see nanoseconds
F
82
difference between the two peaks of intensity between excitation and emission
Stokes shift:
83
emit immediately after excitation
UV
84
MES has __________ resulting to its glow in the dark property
overlap
85
the energy at which is absorbed by the compound
Wv
86
In ________ vibration relaxation is not heard since compounds tested under UV does not contain a chromophore
UV
87
Is vibration relaxation heard in Chromophore
yes
88
has excitation and emission monochromator to resolve the Strokes shift
MES
89
Resolve where the excitation ended and emission started
MES
90
Signal is _________ in MES
accurate
91
light source used if compound is really hard to excite
Lasers
92
purpose: to separate the peaks of individual excitation to know how it can be quantified.
Excitation Wavelength Selector
93
to resolve, correct, or give clarity of the correct wv at which the compound was excited
Excitation Wavelength Selector
94
Light beams that reaches the detector without passing the sample
stray light
95
stray light appear as _______
false negative
96
resolve the emission wv so the overlap will be resolved
Emission Wavelength Selector (filters, monochromators, and grating polychromators)
97
right-angle excitation
0º
98
right-angle emission
90º
99
when the light source is placed at a different angle, it will (glow/not glow)
not glow
100
T/F: When (light source) titled to a particular geometry, it will then be able to excite, seen, emit photons
T
101
T/F: when fluorescence is collected form above, you cannot get anything from the light source
T
102
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
103
only one angle
microscope
104
T/F: In microscopy, you cannot see anything if viewed from another side.
T
105
T/F: Interferences can come from the instrument or the sample
T
106
if detector is placed in an incorrect geometry, there will be an ________
interference or the noise
107
Wrong wv: __________.
nothing will be absorbed
108

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