ANALYTICAL METHODS AND INSTRMENTATION - PART 2 Flashcards
It measures the light emitted by a single atom burned in a flame
Flame Emission Photometry (FEP)
what is the principle behind the flame emission photometry
Excitation of electrons from lower to higher energy
what is the light source for the flame emission photometry
Flame (also serves as the cuvette)
what is the method used in flame emission photometry
Indirect internal Standard Method
internal standard used for flame emission photometry
Lithium/Cesium
these corrects variation in flame and atomizer characteristics
what is the purpose of Lithium/Cesium in flame emission photometry
corrects variations in flame and
atomizer characteristics
It is used for the measurement of excited ions (sodium and potassium)
Flame Emission Photometry (FEP)
in FEP, “ Flickering light indicates __.
changes in the fuel reading of the
instrument
Purpose of Flame in FES
Breaks the chemical bond to produce atoms
Source of energy absorbed by the atoms to enter an excited state
in FEP, it’s used to breaks up the solution into
finer droplets so that the atom will absorb heat energy from the flame and get excited
`ATOMIZER OR BURNER
interference filters as monochromator used in FLAME EMISSION PHOTOMETRY
NA, K , LITHIUM
what color do NA produced as an interference filter in FEP
transmit yellow light (589 nm)
what color do K produced as an interference filter in FEP
transmit violet light (767 nm)
what color do LITHIUM produced as an interference filter in FEP
transmit red light (761nm)
QUALITY CONTROL IN FES
referred internal standard; also acts as a radiation buffer
lithium
Reasons why lithium is preferred:
→ Its emission characteristics are similar to those of Na+ and K+
+ → Normally present as a trace element in human tissues and does
not present interferences in the determination
purpose of quality control in FEp
to achieve stability where there is fluctuations caused by
changes in fuel of air pressure which affects flame temperature and
rate of sample aspiration
It measures the light absorbed by atoms dissociated by heat in an unionized, unexcited, ground state
Atomic Absorption Spectrophotometry
(AAS)
WHAT IS THE PRINCIPLE OF AAS
Element is NOT EXCITED by merely dissociated from its chemical bond and
place in an unionized, unexcited, ground state.
what is the light source of atomic absorption spectophotometry
Hollow-cathode lamp
what are the interferences for atomic absorption spectrophotometry
chemical, matrix (differences in viscosity) and ionization
It is used for measurement of unexcited trace metals (calcium and magnesium)
atomic absorption spectophotometry
It is more sensitive than FEP; it is accurate, precise and very specific
Atomic Absorption Spectrophotometry
(AAS)
what is the internal standard used for atomic absorption spectrophotometry
Internal standard is not needed - changes in aspiration have little effect on the
number of ground state atom
what is the atomizer used for the atomic absorption spectophotometry
nebulizer/graphite furnace is used to convert ions to atoms
what is used in atomic absorption spectrophotometry to modulate the light source
chopper
in AAS;
is added to samples to form stable complexes with phosphate
lanthanum or strontium chloride
what is the principle behind the volumetric or titrimetric
The unknown sample is made to react with a
known solution in the presence of an indicator
examples of volumetric or titimetric
❑Schales and Schales method: chloride test
❑EDTA Titration Method: Calcium Test
For measuring abundant large particles (proteins) and bacterial
suspension
turbidimetry
what is the principle behind the turbidimetry
It determines the amount of LIGHT BLOCKED (reduction of
light) by a particulate matter in a TURBID SOLUTION
2 things that can affect the turbidimetry
specimen concentration
particle size
The measurement of reduction of light is due to particle formation.
TURBIDIMETRY
Solutions requiring quantitation by turbidimetry are measured
using____
visible photometers or visible spectrophotometers.
what are the uses of turbidimetry
- protein measurements (CSF and urine)
- to detect bacterial growth in broth cultures
- antimicrobial test (broth method)
- to detect clot formation
For measuring the amount of antigen-antibody complexes (proteins).
NEPHELOMETRY
what is the principle for NEPHELOMETRY
It determines the amount of scattered light by a particulate
matter suspended in a turbid solution
2 things that can affect nephelometry
Light scattering depends on WAVELENGTH and PARTICLE SIZE
photodetector used in nephelometry
photomultiplier tube
IN NPHELOMETRY,
Light scattered by particles is measured at an angle, typically ___
degrees to the beam incident on the cuvette.
15-90
Is the migration of charged particles in an electric field. it
separates proteins on the basis of their electric charge and
densities.
ELECTROPHORESIS
it separates protein on the basis f their electric charge densities
electrophoresis
example of buffer i electrophoresis
barbital pH 8.6
factors affecting the rate of migration in electrophoresis
net charge of the molecule
size and shape of the molecule
electric field strength
nature of the supporting medium
temperature of the operation
supporting media in electrophoresis which molecules are separated by molecular size
cellulose acetate
supporting media in electrophoresis which molecules are separated by electrical charges and does not bind protein
agarose gel
supporting media in electrophoresis which molecules are separated on the basis of charge and molecular size - combination of agarose gel and cellulose
polyacrylamide gel
polyacrylamide gel will separate proteins into how many fractions
20 fractions; used to study isoenzymes
this will determines the amount of current and the movement of the proteins for a fixed voltage
ionic strength of the buffer
explain the migration of proteins in high and low ionic strength buffer
inversely
high ionic buffer will cause less current and slower protein mobility
low ionic buffer will cause more current and faster protein mobility
relationship of electrophoretic mobility to netcharge, moleciular size, and viscosity of the medium
directly to net charge
inversely to molecular size and viscosity
alam mo na yan kung bakit, char, syempre kasi kapag net charge mas attracted papunta sa anode. Molecular naman mas malaki mas mabagal, as well as viscosity, mas malapot mas mahirap gumalaw
the larger the protein molecule, the most it interacts with the environment, creating a “__” effect which slows down the movement of the protein
solvent drag
what will happen to a particle without a netcharge during electrophoresis
it will not migrate and will remain on its position
stains for the visualization of the fractions/bands that is for CSF protein
coomasive blue
stains for the visualization of the fractions/bands that is very sensitive even to nanogram quantities of proteins
gold/silver stain
has a net charge that can be either
positive or negative depending on pH conditions
Amphoteric
is the movement of
buffer ions and solvent relative to the fixed support
Electroendosmosis/Endosmosis
is the migration of small charged
iontophoresis
is the migration of charged
macromolecules
Zone electrophoresis
Factors Affecting Rate of Migration:
- Net electric charge of the molecule
- Size and charge of the molecules
- Electric field strength
- Nature of the supporting medium
- Temperature of operation
what are the supportng media
Cellulose acetate
Agarose gel
Polyacrylamide Gel
a supporting media that separates by molecular size
cellulose acetate
a supporting media that separates by electrical charge; it does
not bind protein
Agarose gel
a supporting media that separates on the basis of
charge and molecular size; separates proteins into 20
fractions;
polyacrylamide gel
supporting media that is used to study isoenzymes
Polyacrylamide Gel
Stains for Visualization of Fractions
(Bands):
fats fats fats
O S F
OIL RED O
SUDAN BLACK
FAT RED 7B
Stains for Visualization of Fractions
(Bands):
protein
protein
AB
PS
Amido black
Ponceau s
Stains for Visualization of Fractions
(Bands):
CSF PROTEIN
Coomassie Blue
Stains for Visualization of Fractions
(Bands):
very sensitive even to nanogram
quantities of proteins
Gold/Silver stain
It measures the absorbance of stain - concentration of
the dye and protein fraction.
densitometry
It scans and quantitates electrophoretic pattern
Densitometry
at pH 8.6 WHAT’S THE DIRECTION OF GAMMA GLOBULINS in electrophoresis
to cathode, kahit negative pa yan. This process is called as ENDOSMOSIS
It separates molecules by migration through a pH gradient.
Isoelectric Focusing
It is ideal for separating proteins of identical sizes but with
different net charges.
Isoelectric Focusing - kasi pH gradient ang mobility nito. Kahit same pa ang size nila if di sila same ng pH, madidifferentiate pa rin natin ung mga molecules
In isoelectric focusing,
pH gradient is created by adding acid to the ___ of the electrolyte cell and adding base to the ___
anodic area; cathode area
in isoelectric focusing, Proteins move in the electric field until they reach a pH equal to their ___.
isoelectric point
what are thr supporting media for isoelectric focusing
agarose gel, polyacrylamide gel and
cellulose acetate
what are the advantages of isoelectric focusing
+ 1. The ability to resolve mixture of proteins.
+ 2. To detect isoenzymes of ACP, CK and ALP in serum.
+ 3. To identify genetic variants of proteins such as alpha-1 antityrpsin.
+ 4. To detect CSF oligoclonal banding
In this method, sample molecules are separated by electro-osmotic
flow (EOF)
Capillary Electrophoresis
It utilizes nanoliter quantities of specimens.
Capillary Electrophoresis
In capillary electrophoresis,
__ ions in the specimen emerge early at the capillary
outlet because the EOF and the ion movement are in the same
direction.
___ ions in the specimen move towards the capillary
outlet but at a slower rat
Positively charged; Negatively charged
what are the uses of the capillary electrophoresis
separation, quantitation and determination of molecular
weights of proteins and peptides; analysis of Protein products;
analysis of organic and inorganic substances and drugs
advanatge of capillary electrophoresis to routine electrophoresis
low sample volume (kasi nanometer lang ang need)
short around turnaround time - 10 mns lang
high resolving power
It involves separation of soluble components in a
solution by specific differences in physical-chemical
characteristics of the different constituents
CHROMATOGRAPHY
what are the bases of separation
+ 1. Rate of Diffusion
+ 2. Solubility of the solute
+ 3. Nature of the solvent
+ 4. Sample volatility/solubility
+ 5. Distribution between 2 liquid phases
+ 6. Molecular Size (molecular sieving)
+ 7. Hydrophobicity of the molecule
+ 8. Ionic attraction
+ 9. Differential distribution between two immiscible liquids
+ 10. Selective separation of substances
+ 11. Differences in adsorption and desorption of solutes
2 Forms of Chromatography
+A. Planar
+B. Column
a planar chromatography that is used for fractionation of sugar and amino acid
Paper chromatography
paper used for paper chromatography
Sorbent (stationary phase) - Whatman paper
It is a semiquantitative drug screening test
Thin Layer Chromatography (TLC)
a planar chromatography wherein the Sample components are identified by comparison with
standards on the same plate
Thin Layer Chromatography (TLC)
Extraction of the drug is pH ____ - which means the ph must be adjusted to reduce the solubility of the drug in the aqueous
phase.
(dependent, independent)
pH dependent
in the thin layer chromatography,
When all drug spots including the standards have migrated with
the solvent front, it is cause by ___
incorrect aqueous to nonaqueous
solvent mixture.
what are the samples we can use for thin layer chromatography
Biological samples such as blood, urine and gastric fluid can be used for the test
what is the sorbent used for thin layer chromatography
thin plastic plates impregnated with a layer of silica gel or alumina.
It is used for separation of steroids, barbiturates, blood, alcohol and
lipids.
Gas Chromatography (GC)
It is useful for compounds that are naturally volatile or can be easily
converted into a volatile form.
Gas Chromatography (GC)
in gas chromatography,
If the molecule of interest is not volatile enough for direct injection, it is
necessary to derivatize it into a more volatile form.
true or false
true
biological samples used in gas chromatography
urine and blood
urine and blood are introduced in the GC column using
hypodermic syringe or an automated sampler
In GC column, The specimens are _ and _onto the column.
vaporized; swept
what is the detector used in gas liquid chromatography GLC
Flame ionization
Separation occurs based on differences in absorption at
the solid phase surfaces
Gas Solid Chromatography (GSC)
Separation occurs by differences in solute partitioning
between the gaseous mobile phase and the liquid stationary
phase.
Gas Liquid Chromatography (GLC)
it is based on the fragmentation and ionization of molecules using a suitable source of energy
Mass Spectroscopy (MS)
it can also detect structural information and
determination of molecular weight.
Mass Spectroscopy (MS)
Before a compound can be detected and quantified by
mass spectroscopy, it must be separated by ___
Gas chromatography
It is the gold standard for drug testing
Gas Chromatography-Mass
Spectroscopy (GC-MS
It is also used for xenobiotics, anabolic steroids and pesticides.
Gas Chromatography-Mass
Spectroscopy (GC-MS)
in this method, quantitative measurement of drug can be performed
by selective ion monitoring
Gas Chromatography-Mass
Spectroscopy (GC-MS)
It uses an electron beam to split the drug emerging from the column into
its component ions - drugs are detected by means of the presence of
decomposition fragments which arise after degradation of the analytes.
Gas Chromatography-Mass
Spectroscopy (GC-MS)
In GC - MS,
The position of the parent molecule-lon and degradation products give
rise to fingerprint patterns which will provide the final identity of the drug
of interest
TRUE OR FALSE
TRUE
Every drug has its own fingerprint pattern which is compared to a
computer library of known fragmentation patters
true or false
true
- can detect 20 inborn errors of metabolism from a
single blood spot
Tandem mass spectroscopy (MS/MS)
It is based on the distribution of solutes between a
liquid mobile phase and a stationary phase.
Liquid Chromatography
_____is the most widely used liquid chromatography.
HPLC (High Performance Liquid
Chromatography)
It uses pressure for fast separations, controlled temperature, in
line detectors and gradient elution technique.
High Performance Liquid
Chromatography (HPLC)
what are the uses of High Performance Liquid
Chromatography (HPLC)
fractionation of drugs, hormones, lipids, carbohydrates
and proteins; separation and quantitation of various
hemoglobins associated with specific diseases (e.g.,
thalassemia); rapid HbA1c test (within 5 minutes)
In reverse phase HPLC, the mobile phase is ____ than
stationary phase.
more polar
It is for detecting nonvolatile substances in body fluids
Liquid Chromatography-Mass
Spectroscopy (LC-MS)
It is utilized to confirm positive results from screening of elicited
drugs - it is a complementary method to GC-MS
Liquid Chromatography-Mass
Spectroscopy (LC-MS)
It is also used in therapeutic drug monitoring, toxicology and
studies of drug metabolites
Liquid Chromatography-Mass
Spectroscopy (LC-MS
It requires interface methods to convert nonvolatile to volatile
compounds.
Liquid Chromatography-Mass
Spectroscopy (LC-MS)
whta are the interface methods in Liquid Chromatography-Mass
Spectroscopy (LC-MS)
Electrospray (ES) and Atmospheric Pressure
Chemical Ionization (APC
An ____ is used in HPLC and GC methods to
compensate for variation in extraction
internal standard
The mechanism in this type of chromatography is the
exchange of sample ions and mobile-phase ions with the
charged group of the stationary phase
Ion Exchange Chromatography
Is for separation of amino acids, proteins and nucleic
acids.
Ion Exchange Chromatography
Separation of nucleic acids and proteins depends primarily
on the charge and ionic charge density
Ion Exchange Chromatography
Separation of compounds is based on their partition
between a liquid mobile phase and a liquid stationary
phase coated on a solid support.
Partition Chromatography (Liquid-Liquid
Chromatography)
Is for separation of therapeutic drugs and their
metabolites.
Partition Chromatography (Liquid-Liquid
Chromatography)
It uses immobilized biochemical ligands as the
stationary phase to separate a few solutes from other
unretained solutes.
Affinity Chromatography
This type of separation uses the so-called lock-and-key
binding that is widely present in biologic systems.
Affinity Chromatography
affinity chromatogaphy ❑Is for separation of___
lipoproteins, carbohydrates and
glycated hemoglobins; antibodies.
Separation is based on the differences (competition)
between the adsorption and desorption of solutes at the
surface of a solid particle
Adsorption Chromatography (Liquid
Solid Chromatography)
in adsorption chromatography, LIQUID - SOLID
The compounds are adsorbed to a solid support such ___
as
silica or alumina
It measures the amount of light intensity present over a zero
background.
FLUOROMETRY /MOLECULAR LUMINESCENCE
what is the principle behind FLUOROMETRY /MOLECULAR LUMINESCENCE
It determines the amount of light emitted by a molecule
after excitation by electromagnetic radiation.
whta is the light detector used in FLUOROMETRY /MOLECULAR LUMINESCENC
Photomultiplier tube or phototube
monochromators used in fluorometry
filters, prisms, or grating
minerals or elements for which the FLUOROMETRY /MOLECULAR LUMINESCENCE is used
: porphyrins, magnesium, calcium and catecholamines
fluorometry or molecular luminescence,
It uses 2 monochromators____
(either filters, prisms or gratings) -
Is about 1000x more sensitive than spectrophotometer - emitted
radiation is measured directly
FLUOROMETRY /MOLECULAR LUMINESCENCE
fluorometry/molecular luminescence
It is affected by ___- pH and temperature changes, chemical
contaminants, UV light change
quenching
light source for fluorometry or molecular luminescence
Mercury arc lamp, Xenon lamp
FLUOROMETRY
to avoid potential interference from excitation signal, fluorescence measurements detect the emitted light at ___
right angle to the incident light
fluorescence and phosphorescence are both under LUMINESCENCE
what’s the difference
Fluorescence is the light emission from a single excited state
phosphorescence is the light emission from an excited triplet state
f- single
p- triplet
It differs from fluorescence and phosphorescence in that
the emission of light is created from a chemical or electrochemical reaction, and not from absorption of electromagnetic energy
CHEMILUMINESCENCE
what is the principle behind the CHEMILUMINESCENCE
The chemical reaction yields an electronically
excited compound that emits light as it is ground state, or that transfers its energy to another compound, which
then produces emission
what is the use of chemiluminecence
Immunoassays
photodetector used for CHEMILUMINESCENCE
Photomultiplier tube (luminometer
It Is more sensitive than fluorescence
CHEMILUMINESCENCE
chemiluminescence
In this method, no excitation radiation is required and no monochromators
are needed because the chemiluminescence arises from one species.
true or false
true
The excited products formed in the oxidation reaction produce
___ on return to the single state
chemiluminescence
chemiluminescence is a measurement of light against a dark backround since no excitation light is required
true or false
true
since chemiluminescence has no excitation light, which part of spectro is not used for this method
has no excitation light (light source)and monochromator
it is widely used nowadays due to its high sensitivity while even more sensitive than fluorometry
chemiluminescence
this method is best when differentiating two compounds having excitation reaction at the same wavelength but emit at different wavelengths
chemiluminescence
uses of chemiluminescence
autoantibody testing
measurement of hormones
drugs
vitamin
tumor markers
forensic analysis
microbial and infectious disease marker studies
toxicology
the light emission from a single excited state is called as
fluorescence
It is the measurement of the osmolality (KG)of an aqueous solution such as serum,
plasma, or urine.
OSMOMETRY
what is the principle behind the osmometry
It is based on measuring changes in the colligative properties of solutions
that occur owing to variations in particle concentration
osmotic particles in osmometry
glucose, urea nitrogen and sodium
Colligative properties of the solution
osmotic pressure, boiling point, freezing point,
and vapor pressure
As the osmolality of a solution increases the following reactions occur:
what will happen to the osmotic pressure, boiling point, freezing point, and the vapor pressure
osmotic pressure increases
boiling point is elevated
freezing point is depressed; and the
vapor pressure is also depressed
Is the most commonly used method for measuring the
changes in colligative properties of a solution
Freezing-point depression osmometry
It is based on the principle that addition of solute molecules
lowers the temperature at which a solution freezes.
Freezing-point depression osmometry
what is the freezing point of a 1.0 mOsm/kg solution
0.00186° C
what is the freezing point of a blood plasma that has an osmolality of 285 mOsm/k
-0.53° C.
The measurement of current or voltage
generated by the activity of a specific ion.
ELECTROCHEMISTRY TECHNIQUES
It is the measurement of electrical potential due to the activity of
free ions - change in voltage indicates activity of each analyte.
POTENTIOMETRY
It is also the measurement of differences in voltage (potential) at
a constant current.
POTENTIOMETRY
potentiometry It follows the ___ or which law
Nernst equation.
Concentration of ions in a solution can be calculated from the
measured potential difference between the two electrodes.
POTENTIOMETRY
what are the reference electrodes of potentiometry
calomel and silver-silver chloride
whata re the uses of potentiometry
blood pH and pCO2 tests
It is an electrochemical transducer capable of responding to one given
ion
Ion Selective Electrode (ISE)
It is very sensitive and selective for the ion it measures - it measures
the activity of one ion much more than other ions present in the sample
Ion Selective Electrode (ISE)
It is the measurement of the amount of electricity (in
coulombs) at a fixed potential.
Coulometry
It is an electrochemical titration in which the titrant is
electrochemically generated and the end point is detected by
amperometry
Coulometry
Coulometry follows which law
Faraday’s law
uses of coulometry
Chloride test in
csf, serum and sweat
interference in coulometry
bromide, cyanide and cysteine
It is the measurement of the current flow produced by an
oxidation-reaction
Amperometry
uses of Amperometry
pO2, glucose, chloride and peroxidase determination
→ It is the measurement of differences in current at a constant voltage
Polarographyt
it follows the ilkovic equation
Amperometry
The measurement of current after which a potential is
applied to an electrochemical cell
Voltammetry
It allows sample to be preconcentrated, thus utilizing
minimal analyte.
Voltammetry
___ voltametry - for lead and iron.
Anodic stripping
