Transcript Thin-Layer and Column Chromatography

Thin-Layer and
Column Chromatography:
A Practical Guide
Advanced Research Techniques Workshop
February 23rd 2011
Lynsey Cotterill
Outline
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
What is Chromatography?
Chromatography is a method of separating the individual components of a mixture
•
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
•
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
Stationary Phase
•
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)
•
each component will differ in their affinity for the stationary phase and will
repeatedly adsorb and desorb as they move along the stationary phase
•
components will also differ in their interaction with the mobile phase and so some
analytes will be carried farther than others
•
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?
polar surface of silica gel
non-polar compound
polar compound
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
Uses of Thin-Layer Chromatography

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
•
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
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
•
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
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
Choosing a Solvent System
Least Polar
Petroleum ether
Cyclohexane
Chloroform
Ethyl acetate
Hexane
Acetonitrile
Pentane
Isopropanol
Toluene
Ethanol
Diethyl ether
Acetone
Dichloromethane
Methanol
Most Polar
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
avoid touching the
plate with fingers!
~ 5cm
baseline should be
higher than solvent
depth!
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)
Rf values are only accurate to about 20%  it is best to compare
compounds on the same plate with a mixed spot!
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
Visualisation
Three general ways to visualise spots – can be used alone or in combination,
but should be carried out in the order shown:
1. UV light (λ = 254 and 365nm) to show any UV active spots
2. Iodine stain. Shake the plate in a sealed jar containing
an inch of sand and a few crystals of iodine. Good for
unsaturated compounds
3. 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
254 nm
365 nm
Visualisation
Method
Recipe
Used For
Vanillin
vanillin (6g) in ethanol (250ml) + c. H2SO4 Good general reagent, gives a
(2.5ml)
range of colours
PMA
phosphomolybdic acid (12g) in ethanol Good general reagent, gives
(250ml)
blue-green spots
Anisaldehyde
Anisaldehyde (6g) in ethanol (250ml) + c. Good general reagent, gives a
H2SO4 (2.5ml)
range of colours
Ceric sulphate
15% aq. H2SO4 saturated with ceric sulphate
Ninhydrin
Amines, amino acids, Boc
Ninhydrin (0.5g) in n-butanol (250ml) +
protected amino groups after
AcOH (2.5ml)
deprotection on TLC plate
DNP#
2,4-dinitrophenolhydrazine (12g) + c. H2SO4 Mainly aldehydes and ketones,
(60ml) + water (80ml) + ethanol (200ml)
gives orange spots
Potassium
Permanganate#
KMnO4 (3g) + K2CO3 (20g) + 5% aq. NaOH Mainly unsaturated compounds
(5ml) + water(200ml)
and alcohols, gives yellow spots
# - do not usually require heating
Fairly general, gives a range of
colours.
Visualisation
Visualisation
Circle the spots and calculate Rf (retention factor) values from the centre of the spot
Rf =
always quote Rf with
solvent system used!
distance travelled by compound
distance travelled by solvent
Rf = 0.70
Rf = 0.65
Rf = 0.40
Rf = 0.13
For column chromatography Rf of between 0.2 and 0.3 is ideal
Remember, the further up the plate spots travel, the more diffuse they become
Visualisation
Rf = 0.65
product - non-polar
Rf = 0.13
Rf = 0.24
Rf = 0.00
ethyl acetate – petrol 50:50
starting material - polar
Rf is too high = eluent is too polar
ethyl acetate – petrol 20:80
Rf is too low = eluent is too non-polar
For column chromatography Rf of between 0.2 and 0.3 is ideal
Troubleshooting
Some common problems encountered when carrying out thin-layer
chromatography:
•
Overloading of spots
•
Uneven elution
•
Acidic or Basic Compounds
•
Acid Sensitive Compounds
•
Decomposition
•
Poor separation
Problem 1 – Overloading
Problem:
Spots are too large, making it difficult to identify individual spots or
measure accurate Rf values
Possible 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 spots
Possible 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 values
Possible 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 compound
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 alumina TLC plates, these plates are basic in nature
(although the resolution is generally not as good)
Problem 5 - Decomposition
Problem:
Some compounds will decompose on silica. If this happens during column
chromatography, the yield of product could be greatly reduced or even lost
Possible 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
cut a square TLC plate and
spot the compound in the
bottom left hand corner
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 mixture
Possible 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
•
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
•
The absolute distance a compound runs up a TLC plate is variable –
always run comparisons on the same plate
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
•
Composition of fractions is monitored by TLC, UV absorption or fluorescence
•
Main advantages are speed of separation, relatively low cost and ability to
dispose of the used stationary phase
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
•
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
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
As a general guide:
Column
1cm
width
Sample
<100mg
mass
Pots:
2cm
3cm
100500mg
500mg-2g
4cm
5cm
2-5g 5-10g
choose appropriate size
•
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
1. Close tap and lightly plug with small
piece of cotton or glass wool
(nb. slow flow rate gives reduced
resolution, NOT improved separation!
2. Measure dry silica to around
18cm, then pour into a beaker
3. Add 1 inch of sand to give a flat
layer and add 2 inch of solvent mix
– check the tap for leaks!
4. Add enough solvent mix to beaker
of dry silica to form a pourable
slurry and add to column – tap to
remove bubbles
5. Compress the silica (tap open!)
using pressure until the solvent
reaches the surface of the silica
18cm
DO NOT LET SILICA DRY OUT!
Running a Column: Step by Step Guide
6. Dissolve sample in minimum amount
of solvent and add using pipette
around the edge of column - keep a
sample of crude for comparison!
7. Bring solvent level to the surface of
the silica and add 1 inch of sand to
protect the silica surface
8. 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
9. Check fractions by TLC - include
starting material and crude spot
10. Increase the polarity if necessary –
gradually!
Running a Column
•
Standard elution: when the components of a mixture run close together, a
single solvent system which gives the upper spot an Rf of 0.20.3 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)
Analysing Fractions
•
if using more than one plate, include a crude or comparison spot
?
•
when all compounds you are interested in have eluted and
you have identified which fractions they are, combine the
pots as appropriate
•
keep fractions that contain mixtures separate from those of
pure materials
Analysing Fractions
•
combined fractions can then be rotary evaporated to remove the solvent
•
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
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
Some common problems encountered when carrying out column
chromatography:
•
Insolubility of crude sample
•
Silica runs dry
•
Disappearing products
•
Acidic or Basic Compounds
•
Acid Sensitive Compounds
•
Poor separation
Problem 1 – Insolubility of Crude Sample
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 elution
Possible Solution: 
would be better to dry load sample:
1. dissolve sample in the minimum volume of volatile solvent (e.g.. DCM) in a rbf
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 out
Possible 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 4 – Acidic and Basic Compounds
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 plate
Possible 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 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 mixture
Possible 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
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
5.
finally, compounds can be reported as an inseparable mixture if necessary, and
often individual NMR peaks can be identified from mixed samples
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
•
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
•
Care must be taken when cleaning-up column as silica gel provides an
inhalable dust form for toxic chemicals
Acknowledgements
MJH Research Group
Dr. Michael J. Hall
Christopher Stephenson
Matthew 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
Dates, locations and titles to be confirmed
•
Sign-up sheet for each workshop will be available on the Postgraduate
Notice board two weeks in advance
•
PhD students: record attendance at each workshop in your e-portfolio
1 school PGRDP credit is available per workshop