CHROMATOGRAPHY, MASS SPECTROPHOTOMETRY AND LUMINESCENCE Flashcards
1
Q
is an analytical technique commonly used for
separating a mixture of chemical substances into its
individual components, so that the individual
components can be thoroughly analyzed
A
Chromatography
2
Q
→ could be Gas or liquid
→ Solvent moving through the column
→ Carries the sample
A
Mobile phase or carrier
3
Q
→ Solid or liquid
→ Where the mobile phase flows
→ it does not move and stay fixed inside the column
A
Stationary phase or adsorbent
4
Q
→ holds the stationary phase
→ it is where the sample moves
A
Column
5
Q
→ separated components
A
● Eluate
6
Q
→ the process of washing out a compound through a
column using a suitable solvent
A
● Elution
7
Q
→ Mixture whose individual components have to be
separated and analyzed.
A
● Analyte
8
Q
The time it takes for a compound or analyte to elute
A
Retention time or factor
9
Q
● Chromatographic techniques may be classified
according to their mobile phase:
A
→ Gas Chromatography
→ Liquid Chromatography
10
Q
separating compounds based primarily on their
volatility.
A
Gas Chromatography
11
Q
easily evaporated at normal temperatures
● is useful for compounds that are naturally volatile or
can be easily converted into a volatile form
A
● Volatility
12
Q
based on partition
A
Gas-Liquid Chromatography:
13
Q
based on adsorption
A
Gas-Solid Chromatography:
14
Q
is the emission of light by a substance
A
Luminescence
15
Q
Give me the three types of luminescence
A
Fluorescence
Phosphorescence
Chemiluniscence
16
Q
a beam of light is incident on certain substances, they emit visible light or radiation
A
fluorescence
17
Q
atom or molecule that fluoresces
A
fluorophore
18
Q
quick disappearance of fluorescence
A
quenching
19
Q
give me the 5 basic components of fluorometry
A
light source
excitation
cuvette
emission
photodetector
20
Q
delayed fluorescence
A
phosophorescence
21
Q
when light radiation is incident on a certain substances they emit light continuously even after the incident light is cut off
A
phosphorescence
22
Q
transition form a singlet to triplet state
A
intersystem crossing
23
Q
opposite spin direction
A
Single state
24
Q
same spin direction
A
triplet state
25
is a production of light from a chemical reaction
chemiluminescence
26
→ Packed columns or capillary columns
columns
27
very packed beads column, porous layer or
conventional inside the hollow portion of the
column.
It can be used on Gas-liquid and Gas-Solid
Chromatography
packed columns
28
coating (the red and blue) on the side of the
column, no particles have been added.
▪ Can only be used when using Gas-liquid
chromatography.
capillary or open column
29
two types of detector
thermal conductivity detector
Flame ionization detector
30
contain wires (filaments) that change electrical
resistance with change in temperature.
Thermal Conductivity detector
31
measures the unknown resistance values on
TCD.
▪ used for calibration on different instruments.
wheatstone bridge
32
carrier gas
helium
33
→ more sensitive than TC detectors.
→ uses small hydrogen flame (needed) and needs to
have a collector electrode.
Flame ionization detector
34
has mobile phase: (Carrier gas)
helium
argon
nitrogen
HAN
35
to measure analyte in a gas stream attached the
GC system
▪ universally employed where the flame is
commonly generated with hydrogen and air.
Flame Ionization Detector
36
introduce a sample into the inlets.
▪ manual insertion is possible.
Auto-Sampler
37
attached to the column head and provide the
means to introduce a sample into a continuous
flow of carrier gas
common inlet or injector
38
less interaction, travels faster and diluted
out first.
Components with less affinity of stationary
phase
39
more interaction, travels slower and diluted
later.
Components with more affinity to the stationary
phase
40
based on detention of ions formed during
combustion of organic compounds in a flame,
which is generated by hydrogen and air.
flame ionization detector
41
(where flame
produces)
Positive Electrode
42
(positioned
above the flame)
(positioned
above the flame)
43
Retention time
→ X axis
44
Responses
→ Y axis
45
Uses lower temperatures for separation achieving
better separation of thermolabile compounds (unstable
when heated and readily destroyed by heat)
Liquid Chromatography
46
→ liquid-solid chromatography
Absorption
47
acidic polar
silica gel),
48
basic polar
alumina),
49
nonpolar
charcoal)
50
involved attractive forces like Hydrogen bonding
and Van der waals interaction
→ Absorption
51
has Si-OH functional group also known as silanol,
Silica
52
less interaction
release first
53
More interaction
release last
54
Liquid-Liquid Chromatography
Partition
55
→ separation of substances according to their solubility
in an organic/non-polar solvent and in an
aqueous/polar solvent
Partition
56
uses paper to facilitate
Paper Chromatography
57
the distance moved by your solvent from its
starting point.
Solvent front
58
→ Variation of L-S chromatography
→ A.k.a. size exclusion chromatography
Steric exclusion
59
→ use of a resin (stationary solid phase) for covalent
attachment of anions or cations onto it
Ion-Exchange
60
positively charge
cation
61
negatively charge
anion
62
→ most selective type of chromatography employed
→ utilizes the specific interaction between one kind of
solute molecule and a second molecule that is
immobilized on a stationary phase
Affinity
63
Example that can be used as matrix:
− Agarose
− Polyacrylamide
− Polystyrene
− Cellulose
− Silica
64
is attached to a spacer (-CH2-), where it
prevents nonspecific interaction of the ligand with
the matrix itself
Matrix
65
NAD dependent dehydrogenase, kinase
AMP
66
NADP dependent dehydrogenase
ADP
67
Calmodulin binding enzymes
Calmodulin
68
Biotin containing enzymes
Avidin
69
Protein with histidine tag
Ni+2
70
Purification of rRNA
Lysine
71
● Variant of column chromatography
● A thin layer of sorbent, such as alumina, silica gel,
cellulose or cross-linked dextran, is uniformly coated on
a glass or plastic plate
● Most commonly used as a semiquantitative screening
test
THIN-LAYER CHROMATOGRAPHY
72
Uses pressure for fast separations, controlled
temperature, in-line detectors and gradient elution
techniques
● Highest sensitivity
B. HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY
73
Forces the mobile phase through the column at a
much greater velocity
Pump
74
→ Long stainless steel
→ Silica gel – most common
Columns
75
Can be used to introduce the sample into the path of
the mobile phase that carries it into the column
Sample injectors
76
detect absorbances of visible
or UV light
Spectrophotometers
77
used for spectral comparisons
and compound identification and purity and for drug
analysis in urine
Photodiode array
78
measures current produced when the analyte of
interest is oxidized or reduced at some fixed
potential set between a pair of electrodes
Amperometric or electrochemical detector
79
Used to record detector signal versus the time
mobile phase passed through the instrument,
starting from the time of sample injection
The graph is called
recorder - chromatogram
80
based on fragmentation and ionization of molecules
using a suitable source of energy
MASS SPECTROMETRY
81
→ method that requires a source of electrons in the form
of filament to which an electric potential is being
applied
Electron Ionization
82
direct electrical current and radiofrequency voltages
of selected magnitudes are applied to two pairs of
metallic rods
Quadrupole mass spectrometer
83
Three electrodes, in a ring shape and two end caps,
produce ions in the cavity until selectively ejected to
the ion detector
Ion-trap mass spectrometer
84
link three quadrupoles in series
Triple quad
85
used to scan across a preset m/z range and
select an ion of interest
Q1
86
functions as a collision cell
Q2
87
serves to analyze the product ions
generated in Q2 (full product ion scan or selected
reaction monitoring)
Q3
88
chemiluminescence Reactions are oxidation reactions of
luminol, acridinium
esters and dioxetanes
89
is a physical phenomenon that results from
the interaction of light with particles in solution
Light scattering
90
Measures the amount of light scattered in a particulate
suspension at 90deg angle
NEPHELOMETRY
91
the instrument of nephelometry is called
Nephelometer or Nephelometric
Turbidimeter
92
Factors that affect Scattered Light
Particle size
● Concentration of particles
● Molecular weight particles
● Wavelength dependence
93
→ wavelength of light > particle size
→ light symmetrically scattered around the particle
Rayleigh
94
→ wavelength of light < particle size
→ light scatters backward but appears forward due to
destruction out of phase background scatter
Mie
95
wavelength of light = particle size
→ more forward light scatter
→ antigen-antibody reactions
● Rayleigh Debye
96
● Measures the amount of light blocked in a particulate
suspension
TURBIDIMETRY
97
● Separating compounds based primarily on their volatility
● Is useful for compounds that are naturally volatile or can be
easily converted into a volatile form
A. GAS CHROMATOGRAPHY
98
Gas-Liquid Chromatography:
based on partition
99
Gas-Solid Chromatography:
based on adsorption
100
packed columns (GLC/GSC) or capillary columns (GLC)
Columns
101
→ monitor the eluate as it leaves the column
→ produce an electronic signal proportional to the
concentration of each separated component
Detectors
102
used for spectral comparisons and compound
identification and purity and for drug analysis in urine
Photodiode array
103
measures current produced when the analyte of interest
is oxidized or reduced at some fixed potential set
between a pair of electrodes
Amperometric or electrochemical detector
104
▪ best and most widely used
▪ high reproducibility
▪ used at high pressures
loop injector
105
Basic Components of Mass Spectrometry
● Sample inlet
● Ionization source
● Mass analyzer
● Ion detector
