Chromatography Flashcards
-is a technique in which the components of a mixture are separated based on differences in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase
Chromatography
Components of a mixture are carried through the stationary phase by the flow of a _______________, and separations are based on differences in ________________ among the mobile-phase components.
mobile phase
migration rates
-It is a phase that is fixed in place either in a column or on a planar surface
Stationary Phase
-It is the phase that moves over or through the stationary phase carrying with it the analyte mixture.
-It can be gas, liquid or a supercritical fluid
Mobile Phase
2 Classification of Chromatographic Methods
- Column Chromatography 2. Planar Chromatography
Type of chromatography
-Stationary phase is held in a narrow tube
-The mobile phase is forced through the tube under pressure or by gravity
Column Chromatography
2 types of Column Chromatography
- Gas Chromatography
- Liquid Chromatography
Type of Column Chromatography
-Used for the separation of steroids, barbiturates, blood alcohol and lipids
-If the molecule of interest is not volatile enough for direct injection, it is necessary to derivatized it into a more volatile form.
-Samples (urine or blood) are introduced into the GC column using a hypodermic syringe or an automated sampler.
-The specimens are vaporized and swept onto the column
-Useful for compounds that are naturally volatile or can be easily converted into a volatile form
-Flame ionization is used as a detector for GLC
-Elution order of volatiles is based on their boiling point
-Mobile Phase: Nitrogen, Helium, Hydrogen and Argon(inert gas)
Gas Chromatography
Column Chromatography
Two Types of Gas Chromatography
▪Gas Solid Chromatography
▪Gas Liquid Chromatography
Column Chromatography
Type of Gas Chromatography
-Measure differences in absorption at the solid phase surface
-the mobile phase is a gas, and the stationary phase is a solid that retains the analytes by physical adsorption
-permits the separation and determination of low-molecular-mass gases, such as air components, hydrogen sulfide, carbon monoxide, and nitrogen oxides -has limited application because of semipermanent retention of active or polar molecules and severe tailing of elution peaks
▪Gas Solid Chromatography
Column Chromatography
Type of Gas Chromatography
-Separation occurs by differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase
-the mobile phase is a gas, and the stationary phase is a liquid that is retained on the surface of an inert solid by adsorption or chemical bonding
-based on partitioning of the analyte between a gaseous mobile phase and a liquid phase immobilized on the surface of an inert solid packing or on the walls of capillary tubing
Gas Liquid Chromatography
4 Instruments for Gas – Liquid Chromatography
- Carrier Gas System
- Sample Injection System
- Column Configurations and Column Ovens
- Chromatographic Detectors
Instrument for Gas – Liquid Chromatography
-Mobile phase in gas chromatography
-Chemically inert -Helium is the most common mobile phase (argon, nitrogen, and hydrogen are also used)
-Pressure regulators, gauges, and flow meters are required to control the flow rate of the gas
-Flow rates in gas chromatography were regulated by controlling the gas pressure
Carrier Gas System
- is widely used to measure the desirable flow through the column.
-a soap film is formed in the path of the gas when a rubber bulb containing an aqueous solution of soap or detergent is squeezed -the time required for this film to move between two graduations on the burette is measured and converted to volumetric flow rate
Classical Soap Bubble
Instrument for Gas – Liquid Chromatography
-For high column efficiency, a suitably sized sample should be introduced as a “plug” of vapor.
-Slow injection or oversized samples cause band spreading and poor resolution.
-Calibrated microsyringes are used to inject liquid samples through a rubber or silicone diaphragm, or septum, into a heated sample port located at the head of the column.
-Sample port is usually kept at about 50oC greater than the boiling point of the least volatile component of the sample.
Sample Injection System
Instrument for Gas – Liquid Chromatography
Column Configuration and Column Ovens
Two Types of Columns
- Packed Columns (outdated)
- Capillary Columns (recently used)
-Chromatographic columns vary in length from less than __________ or more.
-They are constructed of __________,_____, fused silica, or Teflon.
2 m to 60 m
stainless steel, glass
-________ temperature is an important variable that must be controlled.
-________ is normally housed in a thermostat oven.
-Optimum ________ temperature depends on the boiling point of the sample and the degree of separation required
column
_____________________________ in gas chromatography is achieved by increasing the column temperature continuously or in steps during elution
TEMPERATURE PROGRAMMING
Chromatographic Detectors
- Adequate sensitivity
- Good Stability and Reproducibility
- A linear response to solute that extends over several orders of magnitude 4. A temperature range from room temperature to at least 400 oC
- Short response time that is independent of flow rate
- High Reliability and ease of use
- Similarity in response toward all solute
- Nondestructive of sample
Characteristics of an Ideal Detector
-Most widely used and generally applicable detector for gas chromatography -Effluent from the column is directed into a small air/hydrogen flame
Flame Ionization Detector (FID)
-One of the earliest detectors for gas chromatography
-Consist of an electrically heated source whose temperature at constant electric power depends on the thermal conductivity of the surrounding gas
Thermal Conductivity Detector (TCD)
-Most widely used detectors for environmental samples
-Selectively responds to halogen – containing organic compounds (pesticides and polychlorinated biphenyls)
Electron Capture Detector (ECD)
-One of the most powerful detectors for gas chromatography
-Based on the fragmentation and ionization of molecules using a suitable source of energy
-Before a compound can be detected and quantified by MS, it must be separated by GC
-It can also detect structural information and determination or molecular weight
Mass Spectrometry Detectors
-Gold standard for drug testing
-Uses an electron beam to split the drug emerging from the column into its component ions
-Quantitative measurement of drug can be performed by selective ion monitoring
Gas Chromatography – Mass Spectrometry (GC/MS)
- Can detect 20 inborn errors of metabolism from a single spot
Tandem Mass Spectroscopy (MS/MS)
Gas Chromatographic Columns and Stationary Phases (2)
Capillary Column Packed Columns
Gas Chromatographic Columns and Stationary Phases
-Also called open tubular columns
Capillary Column
Gas Chromatographic Columns and Stationary Phases
Capillary Column -Also called open tubular columns
Two basic Types:
- Wall – Coated Open Tubular (WCOT)
- Support – Coated Open Tubular (SCOT)
Gas Chromatographic Columns and Stationary Phases
Capillary Column -Also called open tubular columns
basic Type:
-Capillary tubes coated with a thin layer of the liquid stationary phase -Columns are made of stainless steal, aluminum. Copper, or plastic
Wall – Coated Open Tubular (WCOT)
Gas Chromatographic Columns and Stationary Phases
Capillary Column -Also called open tubular columns
basic Type:
-The inner surface of the capillary is lined with a thin film of a solid support material on which the liquid stationary phase is adsorbed
-This type of column holds several times as much stationary phase as does a wall-coated column and thus has a greater sample capacity
Support – Coated Open Tubular (SCOT)
Other Type of Capillary Column Fused
-Currently the most widely used GC columns.
-drawn from specially purified silica that contain minimal amounts of metal oxides
-these capillaries have much thinner walls than their glass counterparts
-. they are given added strength by an outside protective polyimide coating, which is applied as the capillary tubing is being drawn
– Silica Open Tubular Capillaries
Other Type of Capillary Column
-Modern packed columns are fabricated from glass or metal tubing.
-Typically 2 to 3 m long and have inside diameters of 2 to 4 mm.
-Tubes are densely packed with a uniform, finely divided packing material, or solid support, that is coated with a thin layer (0.05 to 1 micro meter) of the stationary liquid phase.
-Usually formed as coils with diameters of roughly 15 cm so that they can be conveniently placed in a temperature-controlled oven.
Packed Columns
Liquid Stationary Phase
Desirable Properties for the Immobilized Liquid Phase in Gas – Liquid Chromatography Column include: (4)
- Low Volatility
- Thermal Stability
- Chemical Inertness
- Solvent Characteristics
▪retention time for an analyte on a column depends on its _______________, which in turn is related to the chemical nature of the liquid stationary phase
distribution constant
to separate various sample components, their distribution constants must be sufficiently _______ to accomplish a clean separation
different
these constants must not be extremely large or extremely small because large distribution constants lead to prohibitively long retention times and small constants produce such short retention times that ___________ are incomplete
separations
- is applicable to species that are appreciably volatile and thermally stable at temperatures up to a few hundred degrees Celsius.
Gas-liquid chromatography
has been widely applied to the separation and determination of the components in a variety of sample types
- polydimethyl siloxane
- 5% (phenylmethyldimethyl) siloxane
- 50% (phenylmethyldimethyl) siloxane
- 50% poly(trifluoropropyldimethyl) siloxane
- polyethylene glycol
- 50% poly(cyanopropyldimethyl) siloxane
-Gas chromatography
-Widely used to establish the purity of organic compounds
-Contaminants are revealed by the appearance of additional peaks in the chromatogram
-Areas under these extraneous peaks provide rough estimates of the extent of contamination
-Useful for evaluating the effectiveness of purification procedures
Qualitative Analysis
-Based on comparison of either the height or the area of an analyte peak with that of one or more standards.
-If conditions are properly controlled, both parameters vary linearly with concentration.
-Peak area is independent of the broadening effects discussed earlier.
Quantitative Analysis
-Based on the distribution of solutes between a liquid mobile phase and a stationary phase.
-HPLC is the most widely used liquid chromatography.
Liquid Chromatography
-Used for the fractionation of drugs, hormones, lipids, carbohydrates and proteins.
-Uses pressure for fast separations, controlled temperature, in – line detectors and gradient elution technique.
-Reverse phase HPLC – mobile phase is more polar than stationary phase
HPLC (high Performance Liquid Chromatography)
Separation Mechanism:
▪Gel/Gel Permeation/ Gel Filtration/Size Exclusion/Molecular Sieve Chromatography
-Separates molecules based on differences in their size and shape -As solutes travel through the gel, large molecules remain in the mobile phase are eluted rapidly from the column
- for separation of enzymes, antibodies and proteins - Examples: DEXTRAN and AGAROSE
Hydrophilic Gel
Separation Mechanism:
▪Gel/Gel Permeation/ Gel Filtration/Size Exclusion/Molecular Sieve Chromatography
-Separates molecules based on differences in their size and shape -As solutes travel through the gel, large molecules remain in the mobile phase are eluted rapidly from the column
- for separation of triglyceride and fatty acid - Example: sephadex
b. Hydrophobic Gel (Gel Permeation)
-Separation of nucleic acid and proteins depends primarily on the sign and ionic charge density.
-For separation of amino acids, proteins and nucleic acids.
Ion Exchange Chromatography
-Separation is based on relative solubility in an organic (nonpolar) solvent and an aqueous (polar solvent)
-For separation of therapeutic drugs and their metabolites
Partition Chromatography (Liquid – Liquid Chromatography)
-Used to separate and prepare larger quantities of proteins and antibodies for study
-For separation of lipoproteins, Carbohydrates and glycated hemoglobin.
Affinity Chromatography
- Separation is based on the differences between the adsorption and desorption of solutes at the surface of a solid particle.
Adsorption Chromatography
- Rate of Diffusion
- Solubility of the Solute
- Nature of the Solvent
- Sample Volatility/solubility
- Distribution between 2 Liquid Phases
- Molecular Size
- Hydrophobicity of the molecule
- Ionic Attraction
- Differential Distribution between two Immiscible Liquids
10.Selective Separation of Substances 11.Differences in Adsorption and Desorption of Solutes
Bases of Separation
-based on adsorption of gaseous substances on solid surfaces
-distribution coefficients are generally much larger than those for gas-liquid chromatography
-useful for separating species that are not retained by gas-liquid columns, such as the components of air, hydrogen sulfide, carbon disulfide, nitrogen oxides, carbon monoxide, carbon dioxide, and the rare gases.
-performed with both packed and open tubular columns
-for open tubular column, , a thin layer of the adsorbent is affixed to the inner walls of the capillary called Porous Layer Open Tubular Columns (PLOT)
Gas – Solid Chromatography
