Normal Phase Chromatography Flashcards
What is normal phase chromatography?
Separation mode that utilises a polar stationary phase with a non polar mobile phase - the sample components are retained on the stationary phase through interaction of polar functional groups on the solute with polar sites on the stationary phase surface
Interactions in normal phase chromatography?
Dipole-dipole, dipole-induced dipole, hydrogen bonding, pi couples bonding
How do adsorption strengths increase in the following order?
These leave first (lowest interactions with non polar analytes):
saturated hydrocarbons < olefins < aromatic ~ organic halogen compounds < sulphides < ethers < nitro compounds < esters ~ aldehydes ~ ketones < alcohols ~ amines < sulphides < sulphides < amides < carboxylic acids These leave last (stronger interactions longest retention times leave last)
Stationary phases in normal phase HPLC?
Silica
Silica functionalist with either diol, cyanopropanol or amino groups (less polar functional groups)
Mobile phases in normal phase HPLC?
Generally consist of mixtures of organic miscible solvents (eg hexane/acetonitrile or hexane/dichloromethane) two types of solvent of weak and strong eluting strength to facilitate interactions
Order of increasing polarity and increasing elution power?
n-Hexane Isopropyl ether Ethyl acetate Tetrahydrofuran Acetonitrile Methanol
Deactivators (moderators) in normal phase HPLC?
A substance added to the mobile phase to deactivate the most highly adsorptive centres of the stationary phase such as water (water in non polar mobile phase has high affinity towards polar surface of silica) and alcohols (methanol, ethanol, isopropanol)
What is reversed phase chromatography?
Separation mode that utilises a non polar stationary phase with a polar mobile phase
Interactions in reversed phase chromatography?
The retention mechanism is complex and could be best described a combination of partition and adsorption - aim to have one type of interaction Van der Waals C18 carbon type of interaction
How do retention of analytes decrease in the following order?
Highest logKow values higher affinity to stationary phase longer retention time last to be eluted:
aliphatics > induced dipoles (eg CCl4) > permanent dipole (eg CHCl3) > weak Lewis bases (ethers, aldehydes, ketones), > strong Lewis bases (amines) > weak Lewis acids (alcohols, phenols) > strong Lewis acids (carboxylic acids)
Lowest logKow values eluted first
When are analyses retained better?
Analytes are better retained by the reversed phase surface the less water soluble (ie the more non polar) they are
Stationary materials in reversed phase HPLC?
Bonded phase silica stationary phases (stability pH: 2-8)
Polymeric reversed phase stationary phases (stability pH: 2 -12)
Mobile phases in reversed phase HPLC?
Generally consist of mixtures of water or aqueous buffer solutions with various water miscible organic solvents (water or methanol first choice, if retention time is too long move down the list to stronger eluting solvents such as isopropanol)
Solvents in order of increasing elution power and decreasing polarity?
Methanol Acetonitrile Ethanol Isopropanol Propan-1-ol Dioxane Tetrahydrofuran
How to remove interaction in the separation of basic compounds in reversed phase HPLC?
To avoid interaction of basic molecules with silanol groups acidic fluent should be used (pH of mobile phase < pHpzc of surface silanol groups ), to remove interactions remove charge by changing pH decrease pH to lower pic of silica, won’t have negative charge on silica so won’t have interaction with basic molecules but silica doesn’t like hitting low pH range, hence why second choice of polymeric materials - could increase pH to very high values to remove charge on basic molecules, strong base to block silanol group
What is ion suppression?
To separare bases, neutral or alkaline mobile phases has to be used (because in acidic pH basic molecules carry positive charge and are not retained in the column) - ion suppression
How to separate basic compounds in reversed phase HPLC?
To separate bases in reversed phase HPLC another base (alklyamine or alkaline buffer) can be added to block silanol groups
What is ion pair formation?
Organic acid (counter ion) can be added to form ion pairs with basic molecules - ion pair formation (add another acidic and basic molecule to mobile phase to interact with analyte of interest to form ion pairs and hence no charge)
How can chromatographic separation of charged analytes be achieved in reversed phase ion pair HPLC?
Ion suppression (adjustment of the mobile phase pH to result in a non ionised analyte) Ion pair formation (addition of ionic compounds to the mobile phase to promote the formation of ion pairs with charged analytes)
Ion pair formation basic analytes?
sample+ + counter ion- —> [sample+counter ion-]pair
Ion pair formation acidic analytes?
sample- + counter ion+ —> [sample-counter ion+]pair
How can quaternary alklyamines be used as a counter ion in reversed phase ion pair HPLC?
Eg tetramethyl, tetrabutylammonium, suitable for strong and weak organic acids
How can aryl and arylsuphonates be used as a counter ion reversed phase ion pair HPLC?
Eg methane and heptanesulphonate, suitbale for strong and weak organic bases
Advantages of reversed phase ion pair HPLC?
Separation of mixtures of acids, bases and neutral compounds and also amphoteric molecules
Selectivity can be influenced by the choice of the counter ions
Disadvantages of reversed phase ion pair HPLC?
Reduction of the sensitivity of detection
When should reversed phase ion pair HPLC be used?
Reversed phase ion pair HPLC should be used when other methods (reversed phase and ion suppression modes) have failed or when the sample contain both non ionic and ionic compounds
