SPECTROPHOTOMETRY PART 2 Flashcards
1
Q
- not routinely done
A
SPECIAL
2
Q
excitation: unstable
A
FLAME PHOTOMETRY
3
Q
ground state: emit (emission) radiant energy
A
FLAME PHOTOMETRY
4
Q
heating: dissociation of atoms
A
FLAME PHOTOMETRY
ATOMIC ABSORPTION SPECTROPHOTOMETRY
5
Q
Detection of trace elements
A
ATOMIC ABSORPTION SPECTROPHOTOMETRY
6
Q
- in combination with proteins
A
mercury
7
Q
: absorb (absorption) radiant energy
A
hollow cathode lamp
8
Q
- allowed to pass through; converted to electrical signal
A
transmitted light
9
Q
Light Source: FEP
A
Flame
10
Q
Light Source: AAS
A
Hollow Cathode Lamp
11
Q
State of atoms: FEP
A
Excited
12
Q
State of atoms: AAS
A
Ground State
13
Q
Energy measured: FEP
A
Thermal
14
Q
Energy measured: AAS
A
Radiant
15
Q
Basis of measurement: FEP
A
Light emission
16
Q
Basis of measurement: AAS
A
Light absorbance
17
Q
Sensitivity: FEP
A
Lesser
18
Q
Sensitivity: AAS
A
Greater
19
Q
- failure of the flame to dissociate the sample
A
- CHEMICAL INTERFERENCE
20
Q
*unique trace elements in CHEMICAL INTERFERENCE
A
- Lanthanum
- Strontium
21
Q
*calcium phosphate remedy:
A
displacement principle
22
Q
- excitation instead of dissociation
A
- IONIZATION INTERFERENCE
23
Q
*absorbs radiant energy at ground state = EXCITATION instead
A
- IONIZATION INTERFERENCE
24
Q
*cause: HIGH TEMPERATURE
A
- IONIZATION INTERFERENCE
25
- interference on the absorption by evaporation of solvents or formation of solids
26
3. MATRIX INTERFERENCE
*examples:
1.
2. Use of organic compounds
27
USE OF FLAMELESS AAS
carbon rod or graphite furnace
28
heated until dried/charred
FLAMELESS AAS
29
FLAMELESS AAS: dissociation of atoms
atomized
30
more sensitive
FLAMELESS AAS
31
permits determination of trace metals in small samples
FLAMELESS AAS
32
Atoms absorb light of a particular wavelength and emit light of a longer wavelength (lower energy)
FLOURESCENCE SPECTROPHOTOMETRY
33
USE OF FLOURESCENCE SPECTROPHOTOMETRY
Porphyrins, hormones, amino acids, vitamins, cathecolamines
34
FLOURESCENCE SPECTROPHOTOMETRY COMPONENTS
Energy: mercury or xenon arc lamp, slit 1 degree monochromator, quartz cuvettes, 2 degree monochromator, detector, readout device
35
1. Xenon
2. Entrance slit
3. Primary monochromator
4. Secondary filter
FLOURESCENCE SPECTROPHOTOMETRY
36
*absorption causes excitation
*light emission
*filtered by 2nd filter
*PHOTOMULTIPLIER TUBE
FLOURESCENCE SPECTROPHOTOMETRY
37
- has the ability to magnify the radiant energy converted to electical energy
*PHOTOMULTIPLIER TUBE
38
- excitation filter (intends to isolate the specific wavelength of light: short wavelength, high intensity/energy)
Primary monochromator
39
- an energy emission that occurs when certain compounds absorbs electromagnetic radiation become excited and then return to an energy levelnthat is usually slightly hugher than their original level
FLOURESENCE
40
- emitted energy is equal to or lower than the absorbed energy
PHOSPHORESCENCE
41
absorbs short wavelength, high energy, great intensity
excitation (unstable)
ground state
emit radiant energy
XENON LAMP
42
measurement of light blocked by a suspension of particulate matter
TURBIDIMETRY
43
measurement of the reduction in light transmission caused by particle formation
TURBIDIMETRY
44
protein determination
TURBIDIMETRY
45
TURBIDIMETRY PRECIPITATION AGENTS
(turbid solution)
Sulfosalicylic acid and Trichloroacetic acid
46
Factors affecting turbidimetry and nephelometry:
Size and number of particles
The depth of the tube
Cross-sectional area of each particle
47
- measure turbidity of samples to detect bacterial growth in broth cultures
MICROBIOLOGY ANALYZERS
48
- measure the antibiotic sensitivity from such cultures
MICROBIOLOGY ANALYZERS
49
- detect clot formation in sample cuvets
COAGULATION ANALYZERS
50
- quantify protein concentration in biological fluids such as urine and CSF
CLINICAL CHEMISTRY ANALYZERS
51
measurement of light scattered by small particles at an angle to the beam incident on the cuvet
NEPHELOMETRY
52
more specific than turbidimetry
NEPHELOMETRY
53
immunoglobulins, complement, immune complexes
NEPHELOMETRY
54
When light passes from one medium into another, the light beam changes its direction at the boundary surface if its speed in the second medium is different from the first
REFRACTOMETRY
55
Refractivity depends:
wavelength of the incident light
temperature
nature of the liquid medium
concentration of the solute dissolved in the medium
56
Determines osmolality based on freezing point depression
Component parts
57
Component parts: OSMOMETRY
Cooling bath, thermistor probe, stirring wire, galvanometer
58
OSMOMETRY USE:
Serum and urine ormolality
59
compounds are separated based on differences in affinity/attraction between a mobile phase and a stationary phase
CHROMATOGRAPHY
60
– solid support (coated or uncoated)
Stationary phase
61
not treated with any substance
Stationary phase
62
CHROMATOGRAPHY
General types:
Adsorption Chromatography
Partition Chromatography
63
CHROMATOGRAPHY
Kinds of Chromatography
1. Paper chromatography
2. Thin Layer Chromatography
3. Ion-Exchange Chromatography
4. Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion
5. Gas Chromatography
6. High Performance Liquid Chromatography
64
- molecules adhere on the surface of the uncoated solid support (non-mobile)
Adsorption Chromatography
65
- solid support is coated with a film of water or non-volatile organic liquid
Partition Chromatography
66
Partition Chromatography Examples:
TLC, GLC
67
– flowing gas or liquid (eluent or carrier fluid)
Mobile phase
68
coated/moistened with either water or organic solvent
Mobile phase
69
Rf
retardation factor
70
ratio of the distance of movement by a compound to the distance of the solvent front
Rf (retardation factor)
71
Rf =
a/B
72
= distance travelled by compound from origin to front of spot
a
73
= distance travelled by solvent
B
74
- coated stationary phase
PARTITION
75
- compact spots easier to detect
Thin Layer Chromatography
76
- used for screening of drugs of abuse
Thin Layer Chromatography
77
- Separation is based on electrical charge
Ion-Exchange Chromatography
78
– capture anions
- Anion exchangers
79
– capture cations
- Cation exchangers
80
- Separation is based on differences in molecular size
Gel Filtration
81
- confirmation and quantitation of drugs
Gas Chromatography
82
* Some substances dissolve more in the immobile film of wax or oil-like material on a solid support medium while others in the surrounding steam of flowing gas
Gas Chromatography
83
- selective adsorption with the application of pressure
High Performance Liquid Chromatography
84
- for high MW compounds
High Performance Liquid Chromatography
85
immobile phase:
1. Paper chromatography
cellulose paper (coated with H2O)
86
immobile phase:
2. Thin Layer Chromatography
H2O (bound to silica)
87
immobile phase:
3. Ion-Exchange Chromatography
H2O (bound to silica)
88
immobile phase:
4. Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion
a. Polyacrylamide (plastic)
b. Sephadex (cross-linked polysaccharide)
c. Porous beads
89
immobile phase:
5. Gas Chromatography
- silica-coated (organic liquid) or uncoated
- diatomaceous earth (silica) coated with a non-volatile organic liquid
90
mobile phase:
1. Paper chromatography
organic solvent
91
mobile phase:
2. Thin Layer Chromatography
organic solvent
92
mobile phase:
3. Ion-Exchange Chromatography
water
93
mobile phase:
4. Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion
flowing water
94
mobile phase:
5. Gas Chromatography
Inert carrier gas (Helium or Nitrogen)
a. Gas – Liquid Chromatography (GLC) – based on partition
b. Gas – Solid Chromatography (GSL) – based on adsorption
95
– base on partition
– based on adsorption
a. Gas – Liquid Chromatography (GLC)
b. Gas – Solid Chromatography (GSL)
96
– polystyrene beads
Synthetic resins
97
* The stationary phase contains fixed charged group (constant) and a mobile charge with an opposite charge (exchangeable)
Ion-Exchange Chromatography
98
separation of a substance in a pure form and then determining its dry weight
GRAVIMETRIC METHOD
99
Example: Total Lipid determination
GRAVIMETRIC METHOD
100
detection of light energy scattered or reflected toward a detector that is not in the direct path of the transmitted light (90° to the incident beam)
NEPHELOMETRY
101
The factors affecting turbidimetric measurements are the same factors affecting
NEPHELOMETRY
102
It is more specific than turbidimetry
NEPHELOMETRY
103
measurement of the amount of light blocked by a particulate matter suspended in solution (180° to the incident beam)
TURBIDIMETRY
104
Factors affecting turbidimetry:
Size and number of particles
The depth of the tube
Cross-sectional area of each particle
105
Types of Radiation in Scintillation Counters:
Alpha
Beta
Gamma
106
Exists in two forms Scintillation Counters:
soft and hard gamma
107
Types of Scintillation Counters:
Solid Scintillation Counter
Liquid Scintillation Counter
108
– measures gamma radiation using thallium activated NaI crystal as scintillator and PM tube as detector with preamplifier circuit
Solid Scintillation Counter
109
– measures beta radiation using liquid flour as scintillator
Liquid Scintillation Counter
110
– positively charged particles; resemble the nucleus of helium atom with a mass of 4
Have very litenergytle
Alpha
111
– resembles an electron with both negative (β-) and positive (β+) charges but essentially no mass
Exists in two forms: soft and hard beta
Beta
112
– a form of electromagnetic energy with no mass, only energy
Gamma
113
SEPARATION OF SUBSTANCES
I. PRECIPITATION
II. ULTRAFILTRATION and DIALYSIS
III. CENTRIFUGATION
114
• Separation is based on solubility
PRECIPITATION
115
• The precipitate is studied by:
a. Turbidimetric method
b. Chemical reaction (after being dissolved)
c. Gravimetric method
116
- separates dissolved materials
ULTRAFILTRATION
117
- removes particulate matter
ULTRAFILTRATION
118
- movement through a semi-permeable membrane driven by a force or pulled through a vacuum
ULTRAFILTRATION
119
Cellulose esters
Cellulose acetate
Polyamide
Polyvinyl chloride
Sheets, disks or hair thin fibers, conical
ULTRAFILTRATION
120
- For desalting, fractionation of protein solutions and the preparation of pff
ULTRAFILTRATION
121
180° to the incident beam
TURBIDIMETRY
122
90° to the incident beam
NEPHELOMETRY
