Chromatographic techniques Flashcards
What are the stationary phases used in column chromatography?
Silica gel - most common
Alumina
Why is water never used as a solvent in column chromatography?
Because it is too polar
What is the elutropic series used for?
To identify which is the best solvent to use for separation
In column chromatography polar extracts come out first. True or false?
False - non-polar
Why do polar solvents come out first in column chromatography?
Because they have the least interaction with the matrix and more interaction with the solvent
Column chromatography is only used on a small-scale. True or false?
False - small and large
More usually, in column chromatography, the stationary phase and mobile phase are mixed together. True or false?
True
What is flash column chromatography?
When pressure is applied through using a pump or nitrogen gas, speeding up the process
In flash column chromatography, what influence does particle size have?
Small particles have a higher mesh values so need a pump to flow through mobile phase
In TLC, what is the stationary phase that is usually used?
Silica
What is the driving force in TLC?
Mobile phase moving through the stationery phase by capillary action
In TLC, what does separation depend on?
The polarity of the solute, solvent and stationary phase
TLC plates are treated with fluorescing dye and when placed under UV, the spots appear dark. Why is this?
Because the sample has aromatics and double bonds which will absorb UV light, making the spots appear dark, not green like rest of plate
What are the 5 ways in which TLC spots can be developed?
UV light (indirect)
Iodine - reversibly produces brown spots
Potassium permanganate - detection of sugars
Ninhydrin - pink spots for amines - used for gentamicin
Alkaline tetrazolium - blue spots with corticosteroids
What is TLC used for?
To select solvents for column chromatography Evaluating purity Identification of compounds Follow progress of a reaction Detect impurities BP qualitative test of pure substances
In gel filtration chromatograph, large molecules are separated from small ones. True or false?
True
Gel filtration can be used to remove salts from large molecules. True or false?
True
What are the common stationery phases used in gel filtration chromatography?
Dextran (sephadex)
Dextran-polyacrylamide (Sepharyl)
Agarose (Sepharose)
In gel filtration, what is meant by void volume?
The volume outside the gel matrix
In gel filtration, what is meant by elution volume?
Volume of buffer required to elute any given substance
Gel filtration can be used to desalt or purify a sample. True or false?
True
In ion exchange chromatography, what is separation based on?
Charge properties
Ion exchange chromatography is the most popular for proteins and peptides. True or false?
True
In which type of chromatography is the stationary phase usually cross-linked peptides, typically cellulose or agarose resins?
Ion exchange chromatography
The stationary phase in ion exchange chromatography has positive or negative functional groups. True or false?
True
In ion exchange chromatography, what happens in cation exchange?
positively charged molecules are attracted to a negatively charged solid support
In ion exchange chromatography, what happens in anion exchange?
negatively charged molecules are attracted to a positively charged solid support
In ion exchange chromatography, by using a linear salt gradient, molecules with the weakest ionic interactions start to elute from the column first. True or false?
True
In ion exchange chromatography, the pH of the mobile phase buffer must be between isoelectric point of the charged molecule and the pKa of the charged group on solid support. True or false?
True
In ion exchange chromatography, what factors affect eluting?
The size of the charge - divalent ions greater affinity than monovalent
Intensity of the charge - small monovalent ions show greater affinity than large monovalent
Conc of ions
What are the applications of ion exchange chromatography?
Isolation of metabolites from biological fluids
Neutral compounds separated by mixture of anionic and cationic resins, removing unwanted ions and replacing them with water
Charged compounds e.g. metals can be separated from unwanted constituents
