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159.Column Chromotography

159.Column Chromotography

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Column Chromatography
Number 159
In Factsheet 156, paper chromatography and thin layer chromatography were considered as well as some general points aboutchromatography. In this Factsheet, column chromatography (CC), gas chromatography (GC) and high performance liquid chromatography(HPLC) will be explained. In fact, these three techniques are essentially all types of 
since the stationary phase is contained in a“column”.The rates at which the solutes move through a column depends on the balance between two factors:(i) their solubility in the mobile phase and, (ii) their retention on the stationary phase.The distribution process between mobile and stationary phases is
 partition (“sharing”)
when the stationary phase (SP) is a liquid but
adsorption (sticking”)
when the SP is a solid.
Column Chromatography
TypeColumnGCHPLCColumn arrangementVertical as gravity takes the liquid eluent (the mobile phase) through the column.Column is so long that it is made into a spiral to conserve space.Any but heavily re-inforced because the eluent (the mobile phase) is forced throughunder high pressure at up to 40000kPa.Length/bore of column/cm20 – 100 / 1 – 5500 – 1300 / 0.2 – 15 to 30 / 0.1 – 0.9
concentrated solution of the sample, (A + B)uniform settled slurry of solid adsorbent in glasscolumnglass wool plugtap closedfurther solvent (eluent)is addedbands (A and B) areseparated componentsBAThe pattern of separatedbands are called a
)tap opencollected eluenttap closedsolute B dissolved insolvent (eluent)(1) Start(2) After several minutes(3) After further time
A typical procedure is to make slurry of the solid adsorbent (e.g. alumina (Al
) or silica (SiO
)) with a liquid such as water. This is pouredinto the glass column and allowed to settle. In the column, the wet solid adsorbent must be uniformly distributed – i.e. no air pockets orchannels.The sample mixture to be separated is dissolved in a second solvent (the eluent and mobile phase) to produce a concentrated solution.This solution is poured on to the top of the column. (See diagram 1).The tap is opened and the components begin to separate into “bands” (each of which is
a pure compound derived from theoriginal sample) as the eluent is allowed to trickle slowly through the column. The column is continually topped up with fresh solvent(eluent) at the top of the column at the same rate as it is “eluted” from the bottom. This is continued until all of the bands separate (seediagram 2) and each is eluted from the bottom of the column into separate collection vessels (as shown for component B in diagram 3).
Chem Factsheet
159. Column Chromatography
NoteThe progress, separation and collection of the bands is easilyachieved if the components are coloured (e.g. a mixture of plant pigments) but use of automatic detection devices suchas infra-red sensors, may be needed if the components arecolourless (e.g. a mixture of pharmaceutical drugs).Note In the above example the water adsorbed on the solid’ssurface is the stationary phase, so partitioning of the solutesbetween the water and the sovent has taken place. Soluteswith a tendency to dissolve more in water than the solventmove through the column more slowly.Note In the simple example illustrated, to elute component A afterB has been collected, an alternative solvent could be usedin which A is more soluble. This allows A to travel throughthe column more quickly.NoteEach band
may not 
be a pure compound because it is possiblefor molecules (especially those with closely relatedstructures) to have similar tendencies to partition betweenthe mobile and stationary phases. This can only beestablished by analysis (e.g. IR spectroscopy) of each elutedband. However, in practice, the solvent will be varied andtested until one is found which
give a definite resolution(separation) of the components in the sample.
Advantages of Column Chromatography over ThinLayer Chromatography
1.Column chromatography can separate much larger quantities omixtures.2.Column chromatography allows the separated components tobe readily obtained by evaporation of the solvent after they areseparately removed from the column. Thus columnchromatography may be used to purify compounds made in thelaboratory.Column, paper, and thin-layer chromatography are most suitablefor the separation of solids and non-volatile liquids in solution.NoteCC is essentially a
technique, whereas paperand thin-layer chromatography can be used to measure theR
values (see FS 156) of the components and hence
Gas Chromatography (GC)
Gas-solid chromatography (gsc) & gas-liquid chromatography (glc)GC is a sensitive technique best suited for the separation of gasesand volatile liquids or solids that have been vaporised.In “gsc” the stationary phase (SP) is a fine inert porous solid (e.g.Al
) packed into a column,
(adsorption column)
, and the mobilephase (MP) is a stream of ‘carrier gas’. 
The analysis of mixtures of gases is carried out by gsc.In “glc” the SP is a thin liquid film (e.g. silicon oil) on the surface of a fine inert solid support (e.g. fine glass beads) packed into a column,
(partition column)
, and the MP is a stream of ‘carrier gas’ (e.g.nitrogen).
The analysis of mixtures of volatile liquids and / or solids iscarried out by glc.
carrier gasflow controllersample injectorcolumncolumn ovenusually 50-250
Cchromatogramdetectormeasures thethermal conductivityof the gaseouscomponentwasteInjection: 0.1 - 10
l for gases and volatile liquids, 1
lfor volatile solids (i.e. in concentrated solution) by amicrosyringe, with a self-sealing cap.
The chromatogram (signal from the detector against retentiontime / min) shows a series of peaks. Each peak represents asingle compound from the mixture. The times taken for differentcompounds to travel through the apparatus (their "retentiontimes") are reproducible providing all conditions on the columnare identical. Hence a substance can be identified providedthe retention time has been measured for a pure sample underidentical conditions.For similar compounds (e.g. hydrocarbons) the areas underthe peaks give the relative quantities of the compoundspresent. (Actual concentrations can be determined bycomparing areas with those for pure substances of knownconcentrations.)An inert carrier gas is passed at a controlled rate through athermostatically controlled column. Before the column, a very smallsample is injected into the gas stream. The sample is transportedthrough the column in the gas stream and, as it travels, itscomponents are separated as a result of their different tendenciesto be adsorbed or partitioned.On leaving the column, the separated components pass
in turn
through a detector (e.g. infra-red or combustion) which measuresthe retention time and the % composition (after calibration) of each.The
produced is a plot of the “signal from thedetector” against retention time.Compounds with lower boiling points than the column temperaturewill tend to spend all of their time in the gas phase and so have ashorter retention times.Compounds which are readily soluble in the liquid SP or areadsorbed strongly by the solid SP, spend less time in the gas phase,making the retention times longer.The higher the temperature of the column the more the moleculeswill tend to be in the gas phase and so, for all components, theretention times will be shorter. Too high a temperature means theresolution of the components will be poor – there will be little spacebetween the peaks on the chromatogram. Too low a temperaturemeans it takes a very long time to get all components through thecolumn.
Apparatus for Gas Chromatography

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