Thin-Layer and Column Chromatography:

A Practical Guide

Advanced Research Techniques Workshop February 23rd 2011

Lynsey Cotterill

What is Chromatography?• Stationary phase• Mobile phase

Thin-Layer Chromatography• Preparing a tank• Choosing a solvent• Preparing a plate• Visualisation techniques• Troubleshooting

Column Chromatography• Before you begin• Preparing a column• Running a column• Analysis• Clean-up• Troubleshooting

Outline

What is Chromatography?

Chromatography is a method of separating the individual components of a mixture

• thin-layer and column chromatography are the most

commonly used examples

of analytical and preparative chromatography, and involve the

separation of components of a mixture between a solid

stationary phase and a liquid mobile phase

• chromatography may be analytical or preparative:

- analytical chromatography is used to

identify and measure the relative

proportions of components in a mixture

-

preparative chromatography separates components of a mixture

for further use

Stationary Phase

OR

RHC(CH3)2

F2C

SiO

O

O

SiO

SiO

Si

OH

O

OH

O

O

O

SiO

SiO

SiO

Si

HH

Silica Gel

• silica gel (SiO2) has hydroxyl groups at the surface of the

particles

• the surface of silica gel is highly polar

• polar functionality can bind in two ways:- through hydrogen bonds

- through dipole-dipole interactions

• more polar compounds will have greater interactions with the

stationary phase, and so will move slower along it

Mobile Phase or Eluent (organic solvents)

• as the mobile phase moves past silica gel it transports analytes past the particles of the stationary phase

• an equilibrium is established between the molecules of analyte that are adsorbed to the silica (stationary phase) and those which are in solution (mobile phase)

• components will also differ in their interaction with the mobile phase and so some analytes will be carried farther than others

• each component will differ in their affinity for the stationary phase and will repeatedly adsorb and desorb as they move along the stationary phase

• the result is that more polar analytes that have a high affinity for the silica will move much slower along the polar stationary phase, while non-polar analytes will have very little interaction with the stationary phase and so will move very quickly

What is Chromatography?

non-polar compound polar compound

polar surface of silica gel

• TLC plates consist of an adsorbent, usually silica gel, mixed with a small amount of an inert binder, ZnS and water to form a slurry. This slurry is then

spread on plates and activated in an oven

Introduction to Thin-Layer Chromatography

• TLC is an analytical chromatography technique which is quick, cheap and very easy, using plates coated in a stationary phase placed in a tank of eluent

monitor the progress of a reaction

identify compounds present in a given

substance

determine the purity of a sample

identify if compound is likely to decompose

on silica gel

identify suitable separation conditions for

column

chromatography

Uses of Thin-Layer Chromatography

Preparing a Tank

• a clean, dry lipless beaker (100mL) covered with a watch-glass

is ideal

• eluting solvent(s) should be 0.5cm deep (lower than plate

baseline!)

• filter paper can be used to saturate the tank atmosphere with

solvent vapour

Choosing a Solvent System

Common solvent combinations:

Diethyl ether / petroleum ether 50:50 general organic compounds

Ethyl acetate / petroleum ether 50:50 more polar organic compounds

Ethanol / diethyl ether 5:95 very polar organic compounds

Methanol / dichloromethane 2:98 sometimes useful when others fail

• eluent (mobile phase) which will give the best separation is by

experiment

• for most organic molecules, a good standard starting point is a

50:50 mixture of diethyl ether and petrol

• most solvent systems consist of a non-polar solvent with a

more polar solvent

Choosing a Solvent System

Least Polar

Petroleum ether Chloroform

Cyclohexane Ethyl acetate

Hexane Acetonitrile

Pentane Isopropanol

Toluene Ethanol

Diethyl ether Acetone

Dichloromethane Methanol

Most Polar

CRSM MIX

Preparing a Plate

1. Cut plate carefully using guillotine or scalpel – not scissors!

• allow 0.5cm between each spot and from the edge

2. Lightly mark baseline and lanes with pencil

3. Compare starting material(s) and product, including a mixed spot

4. Dissolve 1-3 mg of material in 1mL of a volatile solvent (1-2% solution)

• Can often spot directly from reaction mixture (may require mini-work

up) or from NMR sample – dilution may be necessary

baseline should be higher than solvent

depth!

~ 5cm

avoid touching the plate with fingers!

Preparing a Plate

5. Apply spot lightly to plate using a small capillary tube or micropipette

• spot should be as small as possible (1 – 2mm in diameter)

6. Place TLC plate in the tank and allow solvent to creep up plate to 0.5cm from the top, then remove it and mark the solvent front with a pencil

Rf values are only accurate to about 20% it is best to compare compounds on the same plate with a mixed spot!

Three general ways to visualise spots – can be used alone or in combination,

but should be carried out in the order shown:

Visualisation

1. UV light (λ = 254 and 365nm) to show any UV active spots

254 nm 365 nm

2. Iodine stain. Shake the plate in a sealed jar containing an inch of sand and a few crystals of iodine. Good for unsaturated compounds3. Treat with a chemically activating reagent, and then heat the plate

• let TLC eluent evaporate, immerse the plate as completely as possible

in the stain and quickly remove

• allow the excess stain to run off onto paper towel

• heat carefully on a hot plate or with a heat gun – in a fume hood!

This method is irreversible and so should always be carried out last

Visualisation

Method Recipe Used For

Vanillin vanillin (6g) in ethanol (250ml) + c. H2SO4 (2.5ml)

Good general reagent, gives a range of colours

PMA phosphomolybdic acid (12g) in ethanol (250ml)

Good general reagent, gives blue-green spots

Anisaldehyde Anisaldehyde (6g) in ethanol (250ml) + c. H2SO4 (2.5ml)

Good general reagent, gives a range of colours

Ceric sulphate 15% aq. H2SO4 saturated with ceric sulphate

Fairly general, gives a range of colours.

Ninhydrin Ninhydrin (0.5g) in n-butanol (250ml) + AcOH (2.5ml)

Amines, amino acids, Boc protected amino groups after deprotection on TLC plate

DNP# 2,4-dinitrophenolhydrazine (12g) + c. H2SO4 (60ml) + water (80ml) + ethanol (200ml)

Mainly aldehydes and ketones, gives orange spots

Potassium Permanganate#

KMnO4 (3g) + K2CO3 (20g) + 5% aq. NaOH (5ml) + water(200ml)

Mainly unsaturated compounds and alcohols, gives yellow spots

# - do not usually require heating

Visualisation

Visualisation

Circle the spots and calculate Rf (retention factor) values from the centre of the spot

Rf = distance travelled by compound distance travelled by solvent

CRSM MIX

Rf = 0.70

Rf = 0.40

For column chromatography Rf of between 0.2 and 0.3 is ideal

Rf = 0.13

Rf = 0.65

Remember, the further up the plate spots travel, the more diffuse they become

always quote Rf with solvent system used!

Visualisation

Rf = 0.13

Rf = 0.65

ethyl acetate – petrol 50:50

OMe

O

N

N

product - non-polar

OMe

O

N

NH

starting material - polar ethyl acetate – petrol 20:80

Rf = 0.24

Rf = 0.00

Rf is too high = eluent is too polar Rf is too low = eluent is too non-polar

For column chromatography Rf of between 0.2 and 0.3 is ideal

Troubleshooting

• Overloading of spots

• Uneven elution

• Acidic or Basic Compounds

• Acid Sensitive Compounds

• Decomposition

• Poor separation

Some common problems encountered when carrying out thin-layer

chromatography:

Problem 1 – Overloading

Problem: Spots are too large, making it difficult to identify

individual spots or

measure accurate Rf valuesPossible Solution: the concentration of sample may be too high –

dilute with

further solvent and keep spots as small as possible (1-2mm)

before dilution after dilution

Problem 2 – Uneven elution

Problem: TLC plate is damaged and solvent runs unevenly, leading to

uneven spotsPossible Solution: damaged silica may be removed by very

carefully trimming

the edges of the

plate using scissors (held at 45o)

Problem 3 – Acidic or Basic Compounds

Problem: Acidic or basic compounds will often streak up a TLC

plate, making it

difficult to identify individual spots and measure Rf valuesPossible Solution: for acidic compounds: add a small amount of

carboxylic acid to

the solvent mixture (e.g.

0.1% acetic acid) for basic compounds: add a small amount of amine to the solvent mixture (e.g. 0.1%

triethylamine)

Problem 4 – Acid Sensitive Compounds

Problem: Silica on TLC plates is slightly acidic. Compounds sensitive

to acid may

decompose on TLC plate, leading to additional spots and loss of compoundPossible Solution: add a small amount of amine to the solvent

mixture to

neutralise the acidic sites on silica (usually 0.1-0.5% ammonia

or triethylamine)

try alumina TLC plates, these plates are basic in

nature

(although the resolution is generally not as good)

Problem 5 - Decomposition

cut a square TLC plate and

spot the compound in the

bottom left hand corner

Problem: Some compounds will decompose on silica. If this happens

during column

chromatography, the yield of product could be greatly reduced or even lostPossible Solution: if you suspect this is happening, or that your

product may

be labile on

silica, you can check for decomposition by

running a 2D TLC plate

Problem 5 - Decomposition

elute

turn the plate

and elute

again

no decomposition decomposition

Problem 6 – Poor separation

Problem: Two spots run closely together and do not give good

separation in the

chosen solvent mixturePossible Solution: the degree of separation of two compounds

depends on the

solvent mixture in

which they are run – try an alternative

solvent system

if there isn’t an ideal

solvent mix,

choose the best available and

try to purify by

column chromatography.

Often the spots will appear in

different pots with

minimal overlap

if separation still cannot

be achieved,

speak to your supervisor as there

are many

other methods of purification!

Summary

• Thin-Layer Chromatography is a fast, easy and cheap analytical technique with a wide range of uses

• Analytes are carried by a mobile phase across a stationary phase and are adsorbing and desorbing in equilibrium• Stationary phase (silica gel) is highly polar so more polar compounds will have greater interactions and will move slower across it and so are

eluted slower

• The absolute distance a compound runs up a TLC plate is variable – always run comparisons on the same plate

• Non-polar compounds have very little affinity for silica gel and will have more interactions with solvent, moving quickly across the silica and

eluting quickly

Introduction to Column Chromatography

• Column chromatography is a preparative chromatography

technique which is used to purify sufficient quantities of a

substance for further use, rather than analysis

• Stationary phase is held within a glass tube and components are

carried down the stationary phase at different speeds to

the bottom, where they are

collected in fractions

• Main advantages are speed of separation, relatively low cost

and ability to dispose of the used

stationary phase

• Composition of fractions is monitored by TLC, UV absorption or fluorescence

• Flash chromatography – introduced in 1978 by Still, Khan and

Mitra1, has provided

chemists with a fast and simple technique of separating materials of

similar polarities

- rapid

- carried out under pressure to drive

compound through and decrease band

dispersion

Column Chromatography

• Gravity chromatography – the traditional method which employs

very long columns filled with silica, usually

running under gravity

- very slow elution rate

- leads to band dispersion, which

reduces the resolution and leads to a large

number of mixed fractions

1. Still, W. C.; Khan, M.; Mitra, A.; J. Org. Chem., 1978, 43, 2923

Before you Begin

• Crude NMR – ensure you have analysed the crude by NMR to

enable you to compare with the product fractions

• Identify a suitable solvent system by TLC which gives:

- good separation of the component

spots

- Rf of 0.2-0.3 for the highest spot

you wish to collect (there are often

irrelevant impurities or residual starting materials

which are either very polar

or very non-polar and these can largely be ignored)

• Large volumes of solvent are used in column chromatography,

take care to avoid breathing in vapours or exposing them to

sparks Remember: silica dust is very toxic if inhaled,

always handle in a fume cupboard!

Preparing a Column: Equipment

• Choose your equipment in relation to mass of crude sample!

Column Size: maintain silica depth of 18cm and

vary the width of column

Pots: choose appropriate size

Column width 1cm 2cm 3cm 4cm 5cm

Sample mass <100mg 100-

500mg 500mg-2g 2-5g 5-10g

As a general guide:

• It is a myth that collecting smaller pots leads to less mixtures – the mixture will

just appear in more tubes!• collecting larger pots will reduce the time spent running a column

Preparing a Column: Equipment

Other equipment:

• tall clamp stand

• clamp and ring

• measuring cylinder

• powder funnel

• beaker

• conical flask

• TLC tank

• TLC plates

Running a Column: Step by Step Guide

18cm

DO NOT LET SILICA DRY OUT!

Close tap and lightly plug with small piece of cotton or glass wool(nb. slow flow rate gives reduced resolution, NOT improved separation!

1.

Measure dry silica to around 18cm, then pour into a beaker

2.

Add 1 inch of sand to give a flat layer and add 2 inch of solvent mix – check the tap for leaks!

3.

Add enough solvent mix to beaker of dry silica to form a pourable slurry and add to column – tap to remove bubbles

4.

Compress the silica (tap open!) using pressure until the solvent reaches the surface of the silica

5.

Running a Column: Step by Step Guide

Dissolve sample in minimum amount of solvent and add using pipette around the edge of column - keep a sample of crude for comparison!

6.

Bring solvent level to the surface of the silica and add 1 inch of sand to protect the silica surface

7.

Top up with solvent mix, tap to remove bubbles, apply pressure and begin collecting fractions. Solvent should run rather than drip – slow flow rate causes reduced resolution

8.

Check fractions by TLC - include starting material and crude spot

9.

Increase the polarity if necessary – gradually!

10.

• Standard elution: when the components of a mixture run close together, a

single solvent system

which gives the upper spot an Rf of 0.2-

0.3 will be effective

• Gradient elution: when the spots are a long way apart, increasing the polarity

of the solvent mixture will save a lot of time and solvent –

must be very careful!

1. begin running column with solvent system which gives highest product

spot an Rf of 0.2-0.3

2. when TLC analysis indicates this component is almost off, increase the

solvent polarity to that which gives the second highest spot an Rf of 0.3

3. continue this process until all spots are off the column

• Flushing: it is sometimes necessary to flush remaining compounds

from the column using a large volume of polar solvent

(usually ethyl acetate)

Running a Column

Analysing Fractions

NO

Br

Br

• when all compounds you are interested in have eluted and you have identified which fractions they are, combine the pots as appropriate

N N

O

O

SO O

NMeMe

NHOBr

Br

N N

O

O

SO O

NMeMe

• if using more than one plate, include a crude or comparison spot

?

• keep fractions that contain mixtures separate from those of pure materials

Analysing Fractions

• occasionally crystals will form in the pots if they are left to evaporate slightly in the fume hood

while running the column the following morning

• combined fractions can then be rotary evaporated to remove the solvent

Clean-up

• to clean-up the column, allow silica gel to dry by forcing solvent out with pressure, or using a low vacuum• once the silica is completely dry, it will pour easily from column

• silica should be emptied into a suitable solid waste container inside a fume hood!

Caution! Silica gel dust impregnated with toxic chemicals is very easily inhaled, take extra care

when emptying column!

Troubleshooting

• Insolubility of crude sample

• Silica runs dry

• Acidic or Basic Compounds

• Acid Sensitive Compounds

• Disappearing products

• Poor separation

Some common problems encountered when carrying out column

chromatography:

Problem 1 – Insolubility of Crude Sample

1. dissolve sample in the minimum volume of volatile solvent (e.g.. DCM) in a rbf

Problem: When loading column, sample is insoluble in a small

volume of solvent, or

is only soluble in highly polar solvents which would affect column elutionPossible Solution: would be better to dry load sample:

2. add a mass of dry silica equivalent to the mass of (dry) crude sample

3. evaporate the solvent to dryness so that the impregnated silica is free flowing in the flask

4. add the dry, impregnated silica to the top of the pre-packed column (leave an inch of solvent above the pre-packed silica to protect it when adding sample, or use sand) and tap to remove any air bubbles

Caution! Silica gel dust impregnated with toxic chemicals is very easily inhaled, take extra care when dry loading!

Problem 2 – Silica Runs Dry

Problem: The solvent falls below the level of the sand and the

silica dries outPossible Solution: top-up the solvent as soon

as possible!

It is likely that ‘cracks’ will

appear in the

silica gel due to the

presence of air. These

cracks reduce the resolution

and results

are unlikely to be as good as

they could

have been.

Problem 3 – Product ‘disappears’ on column

Problem: After running numerous pots, product is still not visible

by TLC

Possible Solution: pots may have been too large and

concentration is too

weak to show product by TLC, either spot multiple times or

allow pots to concentrate

and retry TLC

product may have

become ‘stuck’

on the column, try gradient

elution or flushing

with a large volume of

more polar solvent

(usually ethyl

acetate)

product may have

decomposed on

the silica, check for this on TLC

Problem: Acidic or basic compounds will often streak through a

column making it

difficult to isolate individual compounds, and this should have been

evident on the TLC

platePossible Solution: for acidic compounds: add a small amount of

carboxylic acid to

the solvent mixture (e.g..

0.1% acetic acid)

for basic compounds: add a small amount of

amine to the

solvent

mixture (e.g.. 0.1% triethylamine)

Problem 4 – Acidic and Basic Compounds

Problem 5 – Acid Sensitive Compounds

Problem: Silica gel is slightly acidic. Compounds sensitive to acid

may decompose

on silica during column, and this should have been evident from TLC plate

Possible Solution: add a small amount of amine to the solvent

mixture to

neutralise the acidic sites on silica (usually 0.1-0.5% ammonia

or triethylamine)

try using alumina as the stationary phase as it is

basic in nature

Problem 6 – Poor Separation

Problem: No solvent mixture gives effective separation on silica gel,

if columned

the spots co-elute to give a mixturePossible Solution: the Rf values of some compounds are just too

similar to

separate. However, before giving up, try the following:1. try using alumina TLC plates, the Rf values will be

different and separation may be better

2. speak to your supervisor about the other methods of

chromatography available -

Normal-phase: Silica, Alumina (acidic, basic, neutral), Celite

-

Reverse-phase: C18, C8, cyano and phenyl bonded silica

- Size-

exclusion gel: Sephadex, LH20

- Ion-

exchange: Agarose, Cellulose

-

Partition, Affinity... the list goes on!

Problem 6 – Poor Separation

5. finally, compounds can be reported as an inseparable mixture if necessary, and often individual NMR peaks can be identified from mixed samples

3. consider if it may be possible to use recrystallisation or distillation to separate compounds

4. occasionally it’s easier to take the mixture through to the next synthetic step in the synthesis and separate afterwards

Summary

• Column Chromatography is a rapid and reliable preparative technique

• Solvent system which gives an Rf of 0.2-0.3 for the highest product spot is required initially

• Once analytes have been added to silica gel, column flow must not be stopped and silica gel must not be allowed to dry out

• Care must be taken when cleaning-up column as silica gel provides an inhalable dust form for toxic chemicals

• Fractions are eluted from the bottom of the column and collected in a series of pots. Fractions are then analysed by TLC, always including

comparison spots

Acknowledgements

MJH Research Group

Dr. Michael J. Hall

Christopher StephensonMatthew Dunn

LJH Research Group

Manuel Abelairas Edesa

The Postgraduate Committee

Other Workshops in the Series

Workshop Date

Introduction to Mestre-Nova to analyse NMR data 23rd March 2011

Air Sensitive Techniques 1: Using Schlenk Lines 20th April 2011

Effective Work-ups and Quenching of Reactions 18th May 2011

Recrystallisation and Growing Samples for X-Ray 15th June 2011

Industry Techniques 1: Flash Chromatography 13th July 2011

Air Sensitive Techniques 2: Using a Glove box 10th August 2011

Industry Techniques 2: Microwave Reactions 7th September 2011

• Sign-up sheet for each workshop will be available on the Postgraduate Notice board two weeks in advance

Dates, locations and titles to be confirmed

• PhD students: record attendance at each workshop in your e-portfolio 1 school PGRDP credit is available per workshop