Presentation on theme: "TLC and Column Chromatography Workshop"— Presentation transcript:

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Thin-Layer and Column Chromatography: A Practical Guide Advanced Research Techniques Workshop February 23rd 2011 Lynsey Cotterill



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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



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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



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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



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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



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What is Chromatography? Solvent polar surface of silica gel Solvent non-polar compound polar compound



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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 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



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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



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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 more polar organic compounds Ethanol / diethyl ether 5:95 very polar organic compounds Methanol / dichloromethane 2:98 sometimes useful when others fail



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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



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Preparing a Plate Cut plate carefully using guillotine or scalpel – not scissors! allow 0.5cm between each spot and from the edge Lightly mark baseline and lanes with pencil Compare starting material(s) and product, including a mixed spot 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!



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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



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Visualisation Three general ways to visualise spots – can be used alone or in combination, but should be carried out in the order shown: 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 254 nm 365 nm 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



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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) 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



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Visualisation



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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 always quote Rf with solvent system used! Rf = 0.65 Rf = 0.70 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



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Visualisation Rf = 0.65 product - non-polar Rf = 0.24 Rf = 0.13 Rf = 0.00 ethyl acetate – petrol 50:50 ethyl acetate – petrol 20:80 starting material - polar 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



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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



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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



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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)



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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)



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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 % ammonia or triethylamine)  try alumina TLC plates, these plates are basic in nature (although the resolution is generally not as good)



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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



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Problem 5 - Decomposition elute turn the plate and elute again no decomposition decomposition



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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!



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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



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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



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



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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 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!



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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 width 1cm 2cm 3cm 4cm 5cm Sample mass <100mg mg 500mg-2g 2-5g 5-10g Pots: 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



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Preparing a Column: Equipment Other equipment: tall clamp stand clamp and ring measuring cylinder powder funnel beaker conical flask TLC tank TLC plates



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Running a Column: Step by Step Guide 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. 18cm Add enough solvent mix to beaker of dry silica to form a pourable slurry and add to column – tap to remove bubbles 4. DO NOT LET SILICA DRY OUT! Compress the silica (tap open!) using pressure until the solvent reaches the surface of the silica 5.



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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.



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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 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! begin running column with solvent system which gives highest product spot an Rf of 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)



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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



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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



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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!



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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



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



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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.



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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



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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)



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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 % ammonia or triethylamine)  try using alumina as the stationary phase as it is basic in nature



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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!



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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



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Summary Column Chromatography is a rapid and reliable preparative technique Solvent system which gives an Rf of 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



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Acknowledgements MJH Research Group Dr. Michael J. Hall Christopher Stephenson Matthew Dunn LJH Research Group Manuel Abelairas Edesa The Postgraduate Committee



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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

