Analytical Techniques 2 Flashcards
____ involves the separation of soluble components in a solution by specific differences.
Chromatography
____ refers to the group of techniques used to separate complex mixtures.
Chromatography
Chromatography refers to the group of techniques used to separate complex mixtures on the basis of different physical interaction between ____.
Individual compounds and stationary phase of the system
2 Forms of Chromatography
- Planar
- Column
Techniques under Planar Chromatography
- Paper
- Thin Layer
Techniques under Column Chromatography
- Gas
- Liquid
____ determines the fraction of sugar and amino acid.
Paper Chromatography
Paper chromatography measures the fraction of ____.
sugar and amino acid
In paper chromatography, the sorbent ____ is used.
Whatman paper
____ is used for drug testing.
Thin Layer Chromatography (TLC)
TLC is used for ____.
drug testing
____ is a semi-quantitative screening test.
Drug test
In ____, each drug has a characteristic Rf value and it must match the Rf value with the standard.
Thin Layer Chromatography (TLC)
Rf value stands for ____.
Retention Factor
____ is a relative distance of migration from the point of the application.
Retention factor (Rf)
In TLC, the extraction of the drug is ____.
pH dependent
Sorbet for TLC
Thin plastic plates impregnated with a layer of silica gel or alumina
Kinds of Gas Chromatography
- Gas solid
- Gas liquid
- Mass Spectrometry
GC-MS
MS/MS
Kinds of Liquid Chromatography
High Performance Liquid Chromatography (HPLC)
____ has a gaseous mobile phase and a liquid stationary phase.
Gas Liquid Chromatography
____ is used for the separation of steroids, barbiturates, blood, alcohol, and lipids.
Gas Chromatography
Gas chromatography is used for the separation of ___.
- Steroids
- Barbiturates
- Blood
- Alcohol
- Lipids
____ is useful for compounds that are naturally volatile or can be easily converted into a volatile form.
Gas Chromatography
What is the indicator (detector) used in Gas chromatography?
Flame ionization
In gas chromatography, flame ionization is used as ____.
indicator (detector)
Gas Chromatography
The elution order of volatile is based on their ____.
boiling point
Gas Chromatography
Mobile Phase
- Nitrogen
- Helium
- Hydrogen
- Argon
Gas Chromatography
Volatile substances mixed in the sample
- Heptane
- Acetone
Components of Gas Chromatography
- Carrier gas
- Flow controller
- Sample injector
- Column
- Thermostatic oven
- Detector
- Waste
____ is based on the fragmentation and ionization of molecules using suitable source of energy.
Mass Spectrometry (MS)
Mass Spectrometry (MS) is based on the ____ of molecules using suitable source of energy.
fragmentation and ionization
Mass Spectrometry
The substance should be first separated by ____.
Gas Chromatography
In ____, the substance must be separated first by gas chromatography.
Mass Spectrometry
____ can also detect structural information and determination of molecular weight.
Mass Spectrometry
Mass spectrometry can also detect ____ and determination of ____.
structural information; molecular weight
____ is the gold standard for drug testing.
GC-MS
____ uses an electron beam to split the drug.
GC-MS
____ is used for xenobiotics, anabolic steroids, and pesticides.
GC-MS
____ can detect 20 in-born errors of metabolism from a single blood spot.
Tandem Mass Spectroscopy (MS/MS)
____ 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.
High Performance Liquid Chromatography (HPLC)
____ uses pressure for fast separations, controlled temperature, in-line detectors and gradient elution technique.
High Performance Liquid Chromatography (HPLC)
____ is used for fraction of drugs, hormones, lipids, carbohydrates, and proteins.
High Performance Liquid Chromatography (HPLC)
____ is used for the separation and quantitation of various hemoglobins associated with specific diseases; rapid HbA1c.
High Performance Liquid Chromatography (HPLC)
5 Separation Mechanisms used in Liquid Chromatography
- Gel permeation/filtration
- Ion exchange
- Partition
- Affinity
- Adsorption
____ separates molecules based on differences in their size and shape.
Gel Permeation/Gel Filtration
Gel Permeation/Filtration separates molecules based on differences in their ____.
size and shape
____ is used for the separation of enzymes, antibodies, and proteins.
Hydrophilic Gel (Gel Filtration)
____ is also known as Gel Filtration.
Hydrophilic Gel
Hydrophilic gel makes use of ____.
- Dextran
- Agarose
____ is used for the separation of triglycerides (TAG) and fatty acids (FA).
Hydrophobic Gel (Gel Permeation)
____ is also known as Gel Permeation.
Hydrophobic Gel
Hydrophobic gel makes use of ____.
Sephadex
____ is the exchange of sample ions and mobile ions with charged group of the stationary phase.
Ion Exchange Chromatography
____ resembles gas chromatography.
Ion Exchange Chromatography
____ is used for the separation of amino acids, proteins, and nucleic acids.
Ion Exchange Chromatography
In ____, the separation of nucleic acids and proteins depends primarily on the size, shape, and ionic charge density.
Ion Exchange Chromatography
In ion exchange chromatography, separation of nucleic acids and proteins depends primarily on the ____.
size, shape, and ionic charge density
Liquid to liquid chromatography
Partition Chromatography
In ____, separation compounds are based on their partition between liquid mobile phase and a liquid stationary phase coated on a solid support.
Partition Chromatography
____ is used for the separation of therapeutic drugs and metabolites.
Partition Chromatography
____ uses immobilized biochemical ligands as the stationary phase.
Affinity Chromatography
Affinity chromatography uses ____ as the stationary phase.
immobilized biochemical ligands
____ uses the so-called lock and key binding for separation of substances.
Affinity Chromatography
____ is used for the separation of LPP, CHO, and glycated Hgb.
Affinity Chromatography
____ is used to separate and prepare larger quantities of proteins and antibodies for study.
Affinity Chromatography
Liquid to solid chromatography
Adsorption Chromatography
In ____, separation is based on the differences between adsorption and desorption of solute at the surface of solid particles.
Adsorption Chromatography
In adsorption chromatography, the compounds are adsorbed to a solid support such as ____.
silica or alumina
Fluorometry is also known as ____.
Molecular Luminescence Spectrophotometry
____ measures the amount of light intensity present over a zero background.
Fluorometry or Molecular Luminescence Spectrophotometry
____ is almost similar to colorimetry.
Fluorometry or Molecular Luminescence Spectrophotometry
Principle: It determines the amount of light emitted by a molecule after excitation by electromagnetic radiation.
Fluorometry or Molecular Luminescence Spectrophotometry
Fluorometry or Molecular Luminescence Spectrophotometry
Light source
- Mercury arc
- Xenon lamp
- Wavelength (365-366 nm)
Fluorometry or Molecular Luminescence Spectrophotometry
Light detectors
Photomultiplier tube or phototube
Fluorometry or Molecular Luminescence Spectrophotometry uses how many monochromators?
2
Fluorometry or Molecular Luminescence Spectrophotometry
The ____ selects wavelength that is best absorbed by the solution to be measured.
Primary filter
Fluorometry or Molecular Luminescence Spectrophotometry
____ prevents incident light from striking the photodetector.
Secondary filter
Fluorometry or Molecular Luminescence Spectrophotometry is ____ more sensitive than spectrophotometer.
1000x
____ is affected by quenching, pH and temperature changes, chemical contaminants and UV light changes.
Fluorometry or Molecular Luminescence Spectrophotometry
Uses: measurement of porphyrins, magnesium, calcium and cathecolamine.
Fluorometry or Molecular Luminescence Spectrophotometry
How is chemiluminescence different from fluoroscence and phosphofluorescence?
The emission of light is created from a chemical or electrochemical reaction rather than from absorption of electrochemical energy.
In ____, the emission of light is created from a chemical or electrochemical reaction rather than from absorption of electrochemical energy.
Chemiluminescence
In ____, no excitation radiation is needed and no monochromators are needed.
Chemiluminescence
____ is more sensitive than fluoroscence.
Chemiluminescence
In ____, the chemical reaction yields an electronically excited compound that emits light as it returns to its ground state, or that transfers its energy to another compound, which then produces emission.
Chemiluminescence
____ involves the oxidation of an organic compound by an oxidant.
Chemiluminescence
Chemiluminescence
Examples of organic compound
- Dioxetane
- Luminol
- Acridinium ester
Chemiluminescence
Examples of oxidants
- Hypochlorite
- Hydrogen peroxide
- Oxygen
Chemiluminescence is used for ____.
immunoassays
____ is used for immunoassays.
Chemiluminescence
Photodector for Chemiluminescence
Photomultiplier Tube (PMT (luminator)
____ is the measurement of the osmolality of an aqueous solution such as serum, plasma, and urine.
Osmometry
____ is based on measuring changes in the colligative properties of solutions.
Osmometry
Osmotic Particles
- Glucose
- Urea
- Nitrogen
- Sodium
____ is the most commonly used method for measuring the changes in colligative properties.
Freezing Point Depression Osmometry
____ is the measurement of current or voltage generated by the activity of specific ions.
Electrochemistry Techniques
____ is used for the measurement of blood gas, blood pH, electrolytes, glucose, urea, ionized Calcium, lead and Chloride.
Electrochemistry Techniques
Electrochemistry Techniques
- Potentiometry
- Coulometry
- Amperometry
- Voltammetry
Potentiometry
Ion Selective Electrode
Amperometry
Polarography
____ measures the electrical potential due to the activity of free ions.
Potentiometry
____ is the measurement of differences in voltage at a constant current.
Potentiometry
____ follows the Nernst Equation.
Potentiometry
Potentiometry follows the ____ equation.
Nernst
Potentiometry
Reference Electrodes
- Saturated calomel
- Silver-silver chloride
Uses of Potentiometry
pH and pCO2
____ measures the activity of one ion much more than the other ions present in the sample.
Ion Selective Electrode
Ion selectivity depends on the ____.
membrane or barrier composition
Ion Selective Electrode
Interferences
Excess protein
2 Types of Ion Selective Electrode (ISE)
- Direct
- Indirect
2 Types of Ion Selective Electrode (ISE)
without sample dilution
Direct ISE
2 Types of Ion Selective Electrode (ISE)
with sample dilution
Indirect ISE
Ion Selective Electrode
Glass aluminum silicate is used for ____.
Na
Ion Selective Electrode
Valinomycin gel is used for ____.
K
Ion Selective Electrode
Organic liquid membrane ion exchanger is used for ____.
Ca and Li
pH Electrodes
- Indicator
- Reference
pH Electrode
Silver wire coated with AgCl, immersed into an internal solution of 0.1 mmol/HCl, and placed into a tube containing a special glass membrane tip.
Indicator electrode
pH Electrode
Calomel is in direct contact with metallic mercury in an electrolyte solution of potassium chloride.
Reference electrode
Other parts of pH electrode
- Liquid junction
- Read-out meter
3 Major ISE Types
- Inert metal
- Metal
- Membrane
3 Types of ISE
____ is in contact with a redox couple.
Inert Metal Electrode
3 Types of ISE
____ participate in a redox reaction.
Metal Electrodes
____ is used for the measurement of the electricity at a fixed potential.
Coloumetry
____ is an electrochemical titration in which the titrant is electrochemically generated.
Coulometry
In ____, the endpoint is detected by amperometry.
Coulometry
____ follows the Faraday’s Law.
Coulometry
Coulometry follows the ____ law.
Faraday’s
Use of Coulometry
Chloride test
Interferences in Coulometry
- Bromide
- Cyanide
- Cysteine
____ is the measurement of the current flow produced by oxidation reaction.
Amperometry
____ is the measurement of differences in current at a constant voltage.
Polarography
____ follows Ilkovic equation.
Polarography
Polarography follows the ____ equation.
Ilkovic
____ is the measurement of the current after which a potential is applied to an electrochemical cell.
Voltammetry
____ allows sample to be preconcentrated thus utilizing minimal analyte.
Voltammetry
____ is used for lead and iron testing.
Anodic Stripping Voltammetry
Anodic stripping voltammetry is used for ____.
lead and iron testing