chromatography Flashcards
chromatography
technique used to separate and analyse the substances present in a mixture. it can be used to analyse numerous organic and inorganic substances.
eg: contaminants in water, toxic gases in air, additives and impurities in food, drugs in blood
chromatography only works for small amounts of different substances because the solvent can only carry small amount of the mixture with it.
how it works
has stationary phase (solid/liquid), and a mobile phase (liquid/gas). mobile phase moves up stationary phase due to capillary action.
as components of the mixture are swept upwards over the stationary phase by the solvent, they undergo a continual process of adsorption onto the solid stationary phase, followed by desorption and dissolving into the mobile phase. the ability of component molecules to adsorb depend on the polarity of stationary phase and molecules. attraction between component and solvent molecules depend on the polarity as well.
components
chemicals in a mixture
chromatogram
output of a chromatography procedure
in TLC and paper: patterns of bands or spots formed on a plate or on the paper
in HPLC: graph produced
rate of movement depends on…
how strongly the component adsorbs onto the stationary phase
how readily the component dissolves in the mobile phase
paper and TLC
qualitative analysis
paper: the stationary phase is high quality absorbent paper
TLC: the stationary phase is a thin layer of a fine powder (aluminium oxide) spread on a glass or plastic plate
small spot of solution of sample is placed on one end of chromatography paper/plate, spot is called the origin. the paper/plate is placed in a container with the solvent. origin is a bit above the solvent level, so that components can be transported up the paper/plate and not be dissolved in the solvent.
as the solvent rises up the paper/plate, the component of each sample separate depending on their attraction tot the stationary phase and their solubility in the solvent
Identifying components of a mixture
- include standards of known chemicals on the same chromatogram as the unknown sample and comparing the resulting positions of the unknown components with those of the known samples
- calculate the retardation factor of the sample and comparing these with the values of known samples
RF= distance component travelled/ distance solvent travelled
*from the origin
component most strongly adsorbed to the stationary phase moves the shortest distance and has lower RF value
solvent front
describe the movement of the solvent during chromatography. it is visible as the wet moving edge of the solvent as the solvent travels up the stationary phase
RF
each component has a characteristic RF value for the conditions under which the chromatogram was obtained
thing that change RF value:
changes in temp
type of stationary phase
amount of solvent vapour around paper/plate
type of solvent
colourless compounds
many organic compounds fluoresce and appear blue when viewed under UV light
chromatogram can be sprayed with a chemical that reacts to form coloured/fluorescent compounds
paper v TLC
paper: cheap little preparation more efficient for polar and water soluble compounds easy to handle and store
TLC:
detects smaller amounts
better separation for less polar compounds
corrosive materials can be used
wide range of stationary phases can be used
only use a give a guide to identify the chemical, since for. a particular combination of stationary phase and mobile phase, many different chemicals may have similar RF values.
column chromatography
the solid stationary phase is packed into a glass column. the sample is applied carefully to the top of the packed column, and a solvent which acts as a mobile phase is dripping slowly onto the column from a reservoir above
a tap at the bottom of the column allows the solvent (eluent) to leave the column at the same rate it enters at the other end
paper and TLC summary
components undergo a continual process of adsorption to stationary phase and desorption back into mobile phase
components undergo adsorption and desorption to different degrees depending on the strength of their attraction to the stationary and mobile phases. thus components separate as they move past the stationary phase
high performance liquid chromatography
based on column chromatography
allows sensitive analysis of a wide range of mixtures
separation and identification of complex mixtures of similar compounds (contaminants soluble in water, drugs in blood, hydrocarbons in oil samples, presence and concentration of dioxins; insecticide, pesticides and oil spills in water)
particles in the solid used in HPLC column are 10-20 times more smaller than column chromatography, allows more frequents adsorption and desorption of components giving better separation
small particle size in HPLC creates a considerable resistance to flow of mobile phase, so solvent is pumped through column in high pressure
range of solids can be used in HPLC, some with chemicals specially bonded to their surfaces to improve the separation of particular classes of compounds
components are detected by passing the eluent stream through Uv light beam. many organic components absorb UV light, so when an organic compound passes in front of the beam of light, a reduced signal is picked by a detector. amount of light received by detector is recorded on a chart that moves slowly at a constant speed or sent to a computer. resulting trace Is called a chromatogram. each component forms one peak in the chromatogram.
can separate compounds with relative molecular mass 1000 or even more
Gas Chromatography
most sensitive technique
limited to components that can be readily vaporised without decomposing (relative molecular mass <300)
analysis of trace contaminants, or tiny amounts of very potent components (drugs in urine)
mobile phase is an inert gas (nitrogen), as a carrier gas
small amount of sample is injected into the top of the column through an injection port. the port is heated to vaporise the sample which is swept into the column by the carrier gas
the column is a series of loops of glass/metal (length=2-3m, diameter=4mm), it is heated. the column is packed with a porous solid coated with an ester/liquid hydrocarbon with a high BP; or packed with and absorbent solid such as silica gel/ alumina. this is the stationary phase
components repeatedly interact with the stationary phase and are swept forward by the carrier gas. components that adsorb least strongly to the stationary phase are swept out first by the gas. as components emerge from the end of the column, they are sensed by the detector.
