29 Chromatography and Spectroscopy

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what is chromatography used for

separating individual components from a mixture of substances

what do all forms of chromatography have

• a stationary phase

  • doesn’t move, is normally a solid/liquid supported on a solid

• a mobile phase

  • does move, normally a liquid or a gas

what is chromatography used for

Used in analysis of drugs, plastics, flavourings, air samples, and has applications in forensic science.

what is thin layer chromatography (TLC)

Quick and inexpensive analytical technique that indicates how many components are in a mixture

how does TLC work

• absorbent substance (usually silica)

  • In TLC the absorbent is the stationary phase.

  • The different components in the mixture have different affinities for the absorbent that bind with differing strengths to its surface.

  • Adsorption is the process by which the solid silica holds the different substances in the mixture to its surface.

  • Separation is achieved by the relative adsorptions of substances with the stationary phase.

how to carry out TLC

Rf equation

what is gas chromatography (GC) used for

• Useful for separating and identifying volatile organic compounds in a mixture.

GC schematic diagram

how does GC work

• The stationary phase is a high boiling liquid absorbed onto an inert solid support. The mobile phase in an inert carrier gas such as helium or neon.

• A small amount of the volatile mixture is injected into the apparatus (Called a gas chromatograph)

• The mobile carrier gas carries the components in the sample through the capillary column which contains the liquid stationary phase absorbed onto the solid support.

• The components slow down as they interact with the liquid stationary phase inside the column. The more soluble the component is in the liquid stationary phase, the slower it moves through the capillary column.

• The components of the mixture are separated depending on their solubility in the liquid stationary phase.

• The compounds in the mixture reach the detector at different times depending on their interactions with the stationary phase in the column.

• The compound retained in the column for the shortest time has the lowest retention time and is detected first.

  • The retention time is the time taken for each component to travel through the column.

what is the stationary phase in TLC

the absorbent

what is the process by which the solid silica holds the different substances in the mixture to its surface in TLC?

adsorption

how is separation achieved in TLC

• Separation is achieved by the relative adsorptions of substances with the stationary phase.

what is the the stationary phase in GC

• a high boiling liquid absorbed onto an inert solid support.

• column coated in thin layer of hydrocarbon liquid

• kept at constant temp

what is the mobile phase in GC

an inert carrier gas e.g. He or Ne

what happens to the components in GC as they interact w the liquid stationary phase inside the column

• they slow down

• the more soluble the component is in the liquid stationary phase, the slower it moves through the capillary column

what effects the speed with which the components move through the capillary column

the solubility in the liquid stationary phase

• relative interactions w mobile phase (carrier)

• compared to interactions w stationary phase (liquid hydrocarbon coating the column)

how are components of a mixture separated in GC

• depending on their solubility in the liquid stationary phase

• they reach the detector at diff times depending on their interactions w the stationary phase in the column

what is the retention time in GC

the time taken for each component to travel through the column to the detector

• The compound retained in the column for the shortest time has the lowest retention time and is detected first.

how can you determine the [components] in the sample in GC

using a gas chromatogram:

• the peak integrations (area under each peak) can be calculated to determine the [components] in the sample

• comparing the peak integrations w values obtained from standard solutions of the component

method for determining concentration of compounds

calibration curve

what effects retention time in GC

• temperature: (increased temp so gas moves faster)

• flow rate

• length of capillary tube/column

• liquid in stationary phase

• carrier gas

why would a component move very slowly down the liquid coating column/stationary phase

• if it’s very soluble

  • diff times for diff components to move through the column

  • time taken to emerge is related to the structure of component

what does a large peak on a chromotagram say about the component

• highest concentration (greatest area under peak)

how to calculate peak on gas chromatogram

½ base x height

if a peak has a high retention time on a gas chromatogram what does that say about the component

• longer retention time

• slow to emerge

• interacted more w/ stationary phase

advantages vs disadvantages of GC

advantages:

• can find the exact proportions using a calibration curve

• linked to mass spec: can identify compound straight away

disadvantages:

• (more expensive equipment)

• only for volatile organic compounds

• if you have an unknown compound and unknown retention time, it can’t be identified

• similar compounds w similar IMF will have similar retention times

uses of GC testing

• steroid testing in athletes

• F1 motor racing - fuel testing

advantages and disadvantages of TLC

advantages:

• cheap

• quick

• can change solvent for better separation

• same amount of sample

disadvantages:

• can’t do gas

• don’t know Rf value of compound

• don’t know concentration

• similar functional group → similar Rf values → difficult to find good solvent

test + observation for alkene

• add bromine water drop-wise

• bromine water decolourised from orange to colourless

test + observation for haloalkane

• + AgNO3 + ethanol

• warm to 50°C in water bath

  • Cl-: white ppt

  • Br-: cream ppt

  • I-: yellow ppt

test + observation for carbonyl

• add 2,4-DNP

• orange ppt

test + observation for aldehyde

• + Tollen’s reagent

• warm

• silver mirror

test + observation for primary and secondary alcohol and aldehyde

• add K2Cr2O7/H2SO4(aq)

• warm in water bath

• colour change from orange to green

(doesn’t work if XS K2Cr2O7/H2SO4(aq))

test + observation for c-acid

• + Na2CO3(aq)

• effervescence

how to identify a phenol

• add Br2

• room temp

• Br2 decolourises and white ppt forms