Lec 4 Flashcards
The solvent poured through. It is in liquid or gas form. Also called eluent
Mobile phase
Solid base of powder packed into the column, where the mobile phase flows. Should be in solid or liquid form.
Stationary phase
The solid material which serves as the stationary phase in the adsorption chromatography.
Adsorbent
A solid material that supports the liquid film in partition chromatography.
Support
The process where mobile phase is allowed to flow over the adsorbent or support
Development
The analyte material that emerges from the chromatograph.
Eluate
Component molecules of the substance being separated. Also substances being dissolved.
Solutes
The process where the separated substances on the chromatogram are washed off the adsorbent.
Elution
It is to become separated into constituent parts. A technique developed to separate plant pigments into colored bands.
Resolution
The process of separation of a chemical mixture into different components based on their physical-chemical characteristics and interaction of molecules with the mobile and stationary phase through a support medium.
Chromatography
4 Physico-chemical properties used as a basis in the separation of soluble components in a solution
Differences in:
1. Adsorption to the medium
2. Relative solubilities of mobile & stationary phase
3. Ionic attraction to the sorbent
4. Penetration into a sorbent gel
Type of chromatography that includes:
Classical Adsorption chromatography & Ion exchange
Liquid-Solid
Type of chromatography that includes:
Classical Partition Paper Chromatography & Thin Layer Chromatography
Liquid-Liquid
Type of chromatography that includes:
Gas Liquid Chromatography
Gas-Liquid
In Adsorption:
• Mobile phase should be in ____ form.
• Stationary phase should be in ___ form.
• Mobile phase is in liquid or gas form
• Stationary phase is in solid or liquid form
In Partition:
• Mobile phase should be in ___ form
• Stationary phase should be in ___ form
• Mobile phase is in liquid or gas form
• Stationary phase is in liquid form
_____ Chromatography:
The affinity of the components of a mixture for the adsorbent and the solubility in the mobile phase result in the resolution of the mixture.
Adsorption Chromatography (principle)
The Physical forces that control Adsorption chromatography
Hydrogen bonding,
Electrostatic,
Dispersive interactions,
Van der Waal Forces
Properties of a Good adsorbent
• insoluble to the solvent used
• neither react with separated substance, nor catalyze their decomposition
• colorless adsorbent, if colored separated substance
____ Chromatography:
Separation of solutes is based on relative solubility of the organic nonpolar solvent and the aqueous polar solvent
Partition chromatography (principle)
• Organic molecules with unequal sharing of electrons
• hydrophilic
• partially soluble in water
Polar groups
• Organic molecules predominantly made of covalent bonds with equal sharing of electrons
• hydrophobic
Nonpolar groups
K = solute in stationary phase ÷ solute in mobile phase
Partition of Coefficient (formula)
The ratio of the solute’s concentration in the 2 liquids
Partition of Coefficient (K)
Normal Phase Partition
When the mobile phase is less polar than the stationary phase
A partition system when the mobile phase is more polar
Reverse Phase Partition
____ Partition chromatography uses ________ that are:
• chemically bonded to the support
• insoluble to the mobile phase
Modern Partition chromatography uses pseudo-liquid stationary phases that are…..??
The characteristics of a good support:
• retains stationary phase
• exposes a large surface area to the mobile phase
• mechanically stable
• impedes solvent flow
• has adsorptive properties
• inert to the separated substances
• one of the 3 most common solid support used
• Strong adsorptive properties
• retains 50% to 75% of water
Silica gel
• one of the 3 most common solid support used
• permits separation of large quantities of materials
Cellulose powder
Commercially available as kieselguhr or Celite
Diatomaceous Earth
• beads of cross-linked dextran
• granular, hydrophilic, water-insoluble, porous
Sephadex
• Agarose
• CANNOT stand pressure >800 psi
Sepharose
• Polyacrylamide
Bio Gel P
• most common gel type under Steric Exclusion
• resistant to pressure >800 psi
Water-silica-based product
• Mobile Phase is an aqueous solvent
• Stationary Phase is a gel
Steric Exclusion / Molecular Exclusion
It consists of a reversible exchange of ions between solid and liquid phases
Ion exchange chromatography
Column Chromatography:
3 supports used to retain the solid phase at the bottom of the column.
Glass wool plugs,
Filter paper,
Sintered glass disks
A Packing technique wherein the adsorbent is introduced into the column.
Dry packing
A packing technique wherein the adsorbent & solvent are poured into the column
Wet Packing
Descending Chromatography
The solvent moves by gravitational flow
Ascending chromatography
The solvent runs upwards by capillary action
Support medium for Paper Chromatography
• Whatman paper (a-cellulose of high purity)
• KIMTEC Chromatography paper
Paper Chromatography:
Methods used for Detection
- Color reactions
- Radioactivity scanning
- Fluorescence
- UV absorption
- Infrared absorption
It designated the degree of Monochromacity of a filter
Spectral band width
3 situations wherein you Calibrate the instrument (spectrophotometer)
Malfunction of instrument,
When newly installed,
Periodic calibration
A graph that shows the pattern of absorbances by a solute at different wavelengths.
Spectra Absorption graph
Spectra Absorption Graph (purpose)
To determine the highest absorbance at different wavelengths
Wavelength of choice. The wavelength which gives the highest absorption reading.
Analytical wavelength
5 ways to eliminate stray radiation
- Change the light source
- Verify wavelength calibration
- Seal light leaks
- Realigning instrument components
- Cleaning optical surfaces
The basic components of High Performance of Liquid Chromatography (HPLC)
Sample injection,
Pump,
Detector,
Recorder
It is used to identify compounds when compared with standard retention volumes that is run under identical conditions.
Retention volume
____ retention volume is when the solute has less affinity with the stationary phase
Low retention volume
___ retention volume is when the solute has the highest affinity with the stationary phase
Greatest retention volume
The time required for the sample to elute from the column
Retention Time
It has short retention time which means poor separation.
Early eluting compounds
It has long retention time which means decreased sensitivity.
Late eluting compounds
The substance’s affinity to the liquid stationary phase over the gaseous mobile phase
Gas chromatography (principle)