Chem 347 Fall 2010

Prof. Rob Ronald

Chromatography.
Chromatography, in its various forms, is a purification and analytical technique that has the widest applicability in organic chemistry. Virtually all stable molecules will survive one or more types of chromatographic separation; Depending upon the system used, the purity of chromatographed compounds can be from modest to very high even to analytical purity. There are many forms of chromatography, but this semester we will use just three of the types:
GLPC (Gas Liquid Phase Chromatography); used in GC/MS TLC (Thin Layer Chromatography) LPLC (Low Pressure Liquid Chromatography) sometimes called Flash Chromatography.

Chem 347 Fall 2010

Prof. Rob Ronald

Chromatography is a technique that employs the partitioning of a solute between a stationary phase (solid, or sometimes liquid), and a mobile phase (liquid, or gas). Chromatography was discovered by Michael Tswett in the first part of the 20th century; his first paper describing the technique was published in 1906. The technique was discovered in the course of investigating plant pigments; Tswett passed pentane solutions of plant pigments through a bed of powdered sucrose and observed the formation of colored bands hence the name chromatography. At the time he speculated that it would be possible to separate colorless compounds by the same technique, but he never did it himself.

Tswetts Original Experiment

Powdered Sucrose Column

Chem 347 Fall 2010

Prof. Rob Ronald

GLPC (Gas Liquid Phase Chromatography)

In this form of chromatography the mobile phase is a gas and the stationary phase is a viscous liquid absorbed in a thin layer on: the surface of an inert material packed in a tube (packed columns) on the walls of a long narrow capillary (WCOT Wall Coated Open Tube) also referred to as Capillary GC. The columns are typically heated in an oven, and the sample is vaporized in a hot zone prior to injection into the column. The mobile phase is typically He or H2, which is referred to as the carrier gas.
Schematic for Gas Chromatography

Coiled Column

Syringe (sample) Injector heater

Oven

Detector

Recorder

Output

We will use GLPC to analyze the product from our Et3N distillation experiment, in the form of GC-MS.

Chem 347 Fall 2010

Prof. Rob Ronald

The sample is usually injected as a dilute solution in a solvent such as CH2Cl2 using a 1L or 10L syringe; the top of the column is sealed with a silicone rubber septum through which the sample is injected.
The injection port is typically heated to 200-250 C so the sample is instantly vaporized and swept by the carrier gas stream immediately onto the column.

The column is contained in a thermostated variable temperature oven that can be heated from room temperature to about 250 C. There are usually provisions to program the temperature of the oven during the chromatographic run: temperature programmed GC. The oven can also be kept at constant temperature: isothermal GC. Usually the average oven temperatures for GC are in the range of 75-150 C.

 Various types of detectors are available for Gas Chromatography:

Chem 347 Fall 2010

Prof. Rob Ronald

the most universal detector is the thermal conductivity detector (TCD) this detector has relatively low sensitivity, but detects almost all compounds and is non-destructive to the effluent;
The TCD detector measures the changes in temperature of a hot filament that result of changes in composition of the gas passing over it what is actually measured are changes in resistance of the filament

the flame ionization detector (FID) is much more sensitive,

The effluent from the column is burned in a hydrogen flame in a region of high potential: the ionic products of combustion produce a miniscule electric current. FID detects compounds based on the carbon content and the effluent is destroyed by burning in a hydrogen flame to produce the ion current

the electron capture detector (ECD) detects halogens selectively and uses a radioactive source to generate the ions.

Chem 347 Fall 2010

Prof. Rob Ronald

In the TCD mode, gas chromatography can also be used in a preparative mode to prepare small (10500mg) quantities of volatile compounds. In most cases this older methodology has been superseded by liquid chromatographic methods. Another type of detection that is universal and very sensitive is to couple the gas chromatography with mass spectroscopy (GC-MS).
In this combination the peak detection is accomplished by measuring the total ion current in the mass spectrometer;

As the peaks emerge from the column and enter the mass spectrometer they can be scanned, and often the components can be identified, by mass spectroscopy.

Chem 347 Fall 2010

Prof. Rob Ronald

Packed columns for GC are typically stainless steel or glass coils 2-5mm in i.d. and are typically 1-2m in length.
The columns are usually mounted in the oven supported only by fittings at either end. Gas flows for packed columns are typically 20-60mL/min with back pressures around 60psi. Typical performance will have peak widths at half height around 0.2-1 min depending upon the retention time, which is related to the column temperature

The packing material is typically a porous substance such as diatomaceous earth that has been impregnated with various liquid phases such as silicone fluids (SE-30, SPB-1, etc.) or polyethylene glycol waxes (Carbowaxes). There are many types of packing materials and liquid phases for various types of separations. A company such as Supelco is a good source for these columns

Chem 347 Fall 2010

Prof. Rob Ronald

Capillary columns are long tubes of fused silica produced by the same processes used to make fiber optics.
A typical capillary column is 15 to 30m in length with a bore of 0.25mm. The walls of the tube are coated with a thin layer, 0.25m, of the liquid phase; these are often referred to as WCOT (wall coated open tubes). The columns can handle only a few nanograms of sample so an injection splitter is employed, and a very sensitive detector (FID) has to be used. The carrier gas pressure is adjusted to give a flow rate of about 20-30cm/sec down the column; this usually requires a pressure of only 10-20psi. Capillary columns give very high resolution with peak widths at half height around 0.05-0.2min depending on the retention time, which again is related to the column temperature.

Chem 347 Fall 2010

Prof. Rob Ronald

These are all related forms of chromatography in which the mobile phase is a liquid and the stationary phase is a porous solid (sorbent). In TLC (Thin Layer Chromatography) the sorbent is spread in a thin

Liquid Chromatography TLC, LPLC, MPLC and HPLC

layer, 0.25-2mm thick, on an inert, rigid surface (glass, plastic, or aluminum plates) and the liquid phase is allowed to travel through the sorbent by capillary action from a solvent pool in which the bottom part of the plate is dipped. The samples to be analyzed are spotted near the bottom of the plate and travel upwards as they are carried along with the solvent. In LPLC (Low Pressure Liquid Chromatography Flash Chromatography) the sorbent is packed into columns (usually glass or plastic) and the mobile phase is applied to the top of the column and allowed to percolate through the sorbent under the influence of gravity or a slight positive pressure of a gas (2-10 psi compressed air or nitrogen). The samples are applied to the top of the column and then eluted out the bottom.

In Chem 347 we will concentrate on TLC and LPLC.

Chem 347 Fall 2010

Prof. Rob Ronald

MPLC and HPLC differ mainly in plumbing and in the pressure applied to the mobile phase (eluant) due to the different particle sizes used to pack the columns
MPLC uses particles typically 15-30m in diameter HPLC uses particles typically 5-10m in diameter Both of these techniques rely on mechanical pumps to force the eluant through the column.

MPLC (Medium Pressure Liquid Chromatography) is a preparative technique and employs glass, or plastic columns limited to between 50-200psi. HPLC (High Pressure (sometimes called High Performance) Liquid Chromatography) is the most sophisticated and employs pumps and columns capable of delivering and withstanding pressures up to 8000psi.

To resist these high pressures HPLC columns and fittings are made from stainless steel; the pumps have sapphire pistons, ruby check valves and sapphire valve seats. Typically, HPLC is an analytical tool and uses various detection methods to achieve quantitative information on the separation of the analyte

Chem 347 Fall 2010

Prof. Rob Ronald

The most common sorbents for chromatography are various forms of Silica Gel. The silica gel used today is a synthetic material, but it is related to a natural material called diatomaceous earth, a mineral that consists of the silicaceous skeletons of microscopic diatoms. Because diatomaceous earth comes from billions and billions of identical microorganisms the material is very porous, and the pore sizes are uniform. Diatomaceous earth is sometimes used as in inert support for packed GC columns.

Example of the pore morphology of the diatom, Thalassiosira weissflogii, Bars = 5 m, bottom inset bar = 0.2 m (2000 ).

Chem 347 Fall 2010

Prof. Rob Ronald

Synthetic Silica Gel had been known in the 17th century as a scientific curiousity. In World War I the use of chemical warfare agents led to Prof. Walter Patrick of John Hopkins U. to invent a process for producing Silica Gel to be used as an adsorbent for gas masks.
Synthetic Silica Gel is made by acidifying solution of sodium silicate (water glass) and then washing and drying the gelatinous product.

Some Silica Gels can have surface areas as high as 800m2/g and have pore sizes as small as 24.

During WWII Silica Gel was used as a desiccant to preserve penicillin, and as a catalyst for making high octane gasoline and synthetic rubber. After WWII the ready availability of silica gels led to their use as adsorbents in chromatography.

Chem 347 Fall 2010

Prof. Rob Ronald

The most common type of sorbent used for chromatography in organic chemistry is Silica Gel 60 (sometimes also called Kieselgel 60).
The number 60 refers to the average size of the pores - 60. Other pore sizes are available. The surface area of Silica Gel 60 averages about 450m2/g.
To put this in perspective: a level teaspoon of silica gel weighs about a gram; and the area of a NBA/NCAA basketball court is about 430m2; that means that if all the surface area in a teaspoonful of silica gel could be spread out it would occupy the area of a basketball court!

The silica gel is sieved to have an average particle size of 4060m for column chromatography (LPCL and MPLC); although for TLC the particle size range is usually somewhat broader (10-200m) For HPLC columns the particle sizes are 5-10m, which is one of the reasons that such high pressures are required.

Chem 347 Fall 2010

Prof. Rob Ronald

Ordinary synthetic silica gel is sometimes referred to as normal phase silica gel, or absorption silica gel.
In normal phase silica gel compounds are absorbed to the surface based on their polarities because the surface of the silica has many exposed silanol residues (S-OH groups) Polar interactions of the solute with the surface are dominant The binding is principally due to interactions of polar functional groups with the Si-OH groups on the silica surface. The more polar a molecule is the more tightly it is held by the silica gel surface. More polar compounds require a more polar the solvent to be released from the surface. Non-polar compounds bind only weakly to silica and are released readily from the surface with non-polar or only moderately polar solvents.

Chem 347 Fall 2010

Prof. Rob Ronald

When dealing with very polar compounds it is sometimes advantageous to use Reversed Phase Silica Gel (RP).
This type of silica gel is chemically modified by attaching hydrocarbon chains to the Si-OH groups converting them to Si-OAlkyl groups, typically C18 chains (RP-18).

Chem 347 Fall 2010

Prof. Rob Ronald

While reversed phase sorbents are more expensive than normal phase silica they are very useful and are available for TLC, LC, and HPLC.
Many types of groups can be attached in place of C18. In this type of silica non-polar compounds are the most tightly bound and the polar compounds are more mobile. Solvent mixtures for reversed phase chromatography are based on mixtures of water (or aqueous buffers) and water miscible organic solvents such as acetonitrile, THF or MeOH. The order of elution is the opposite from normal phase silica the more polar compounds move faster, and it takes increasing amounts of the organic phase to mobilize the non-polar components, which stick to the greasy surface.

HPLC mainly employs the use of RP type columns.

Chem 347 Fall 2010

Prof. Rob Ronald

TLC (Thin Layer Chromatography)

The technique of TLC was invented by Egon Stahl shortly after the end of WWII.
Since that time it has become one of the most useful and used analytical techniques in chemistry. In the beginning researchers had to make their own layers, but in the 1960s commercially manufactured plates became readily available and the art of making TLC plates is mostly lost.

TLC plates have a thin layer of silica gel dispersed evenly on an inert, rigid backing (glass, plastic or aluminum).
For analytical TLC plates the layer is 0.25mm thick; and for preparative TLC plates the layers can be up to 2mm thick.

The sorbent, typically silica gel 60 particles, is applied with an inert binder (usually a form of hydrated silica, or hydrated CaSO4, but sometimes an organic polymer) to make the layers stable and durable. Other sorbents such as cellulose, alumina (aluminum oxide) or reversed phase silica gel are also available.

Chem 347 Fall 2010

Prof. Rob Ronald

The layers are durable enough that they can be written on lightly with a soft pencil. Typically the sorbent is impregnated with an inert fluorescent material that has a large Stokes shift so that when it absorbs in the ultraviolet region (254nm and/or 366nm) it emits in the visible (yellow green or blue) region of the electromagnetic spectrum. (ZnS has been used in the past, but the fluorophores in use now are proprietary compounds) The fluorescent dye makes materials that have a UV chromophore (light absorbing functional group) absorbing UV light around 254nm (or 366nm) visible as dark spots against a glowing green (or blue) background. In Chem 347 all of the unknowns have a UV chromophore and will be visible on fluorescent TLC plates under UV irradiation; however some compounds give darker, more easily seen spots than others.
Legend: the picture at the right, above is a TLC chromatogram of the results of the purification of the crude reaction product; the plate was developed with 8%MeOH in 20%EtOAc-hexane, and visualized in the UV. Lane 1 is the major crystalline product (1st crop), RR-2MeArL1c-2a; lane 2 is the recrystallized mother liquor (2nd crop), RR2MeArL1c-2b; and lane 3 is the remaining mother liquor solution.

Chem 347 Fall 2010

Prof. Rob Ronald

TLC plates come as 20cm x 20cm sheets that are usually cut up to the desired sizes as needed.
Precut plates in other sizes can be purchased

For typical analytical work the sheets are cut into strips 1-5cm wide by 5-10cm long. For analyzing the series of fractions produced from LPLC or MPLC separations wider plates can be used.

Legend. The picture above is the UV visualization of the chromatograms of the chromatographic separation showing the individual fractions; the plates were developed 1x with the same solvent used for eluting the column.

Chem 347 Fall 2010

Prof. Rob Ronald

Protocols for running TLC analyses

To do TLC you will need:
(1) a small screw-capped jar that is at least 3-4cm in diameter and about 10cm deep for a TLC tank, suitable jars are available; (2) spotting pipettes; these can be made by drawing out a Pasteur pipette using a Bunsen burner; there are also spotting pipettes available in the stockroom (3) a clean, dry 10mL graduated cylinder for measuring out solvent mixtures, (4) forceps or tweezers to place and remove the plates from the tank; (5) and also a sharpened, soft lead pencil for marking the surface of the plates.

TLC plates (Silica Gel 60) for Chem 347 are pre-cut (by stockroom personnel) and stored in desiccators for protection. Handle the plates only by the edges, and do not touch the white layer; use tweezers or forceps to place the plates in the tanks and to remove them.

Take only one (1) plate at a time, and do not use a wider plate than you need; cut the plates with scissors if they are not the correct size.

Chem 347 Fall 2010

Prof. Rob Ronald

Applying the Sample to the TLC Plate.

To run TLC on your unknown, weigh out about 5-10mg of your unknown mixture into a small vial or test tube, making sure that you do not take out large chunks, but only powder to insure you have a representative sample. Dissolve the sample completely in about 1mL of a solvent that will dissolve it completely: CH2Cl2 or CHCl3 are good choices. Label the vial or tube and keep it closed with a cork so the solvent does not all evaporate. If the solvent evaporates it can be replaced with fresh solvent. Mark dots on your plate according to the diagram (on the next slide) with a pencil; do not draw a line for spotting, it only gets confusing later. You may mark each lane with a number or symbol so you can keep track of what you spotted. Take a spotting pipette and touch it to the stock solution of your unknown drawing up a column of liquid 1-2 cm high. Touch the tip of the spotting pipette to one of the dots on your plate to apply the sample. It is a good idea the first few times you do this to use the UV lamp while applying the sample so that you can see how much you are putting on the plate. Usually a spot at the origin 1-2mm in diameter is sufficient. It is important to not overload the plate.

Chem 347 Fall 2010

Prof. Rob Ronald

Allow the solvent to migrate nearly to the top of the plate.

Keep the spots at least 5-6mm apart from each other and 5-6mm away from the edge of the plate.

Mark where the plate is to be spotted with a pencil at least 1cm above the end of the plate.

Use the UV lamp when spotting the samples so that you get an adequate concentration of the sample without spotting too heavily.

The depth of the solvent in the tank should be no more than 5-6mm; there should be at least 5mm from the top of the solvent to where the solvent front encounters the samples.

Chem 347 Fall 2010

Prof. Rob Ronald

Choosing the solvent

This is a trial and error process.
Some Useful TLC solvent mixtures Solvent Mixtures* Comment H2O MeOH Extremely polar solvent mixture (10-25%H2O), good for acids MeOH EtOAc Very polar solvent mixture (10-25% MeOH) MeOH CHCl3 Sometimes works better than MeOH CH2Cl2 MeOH CH2Cl2 Useful general TLC solvent for polar compounds (10-40%MeOH) EtOAc CH2Cl2 Specialized solvent mixture, infrequently used Et2O CH2Cl2 Specialized solvent mixture, infrequently used EtOAc Hexane Useful general TLC solvent (10-40% EtOAc) Et2O Hexane Useful when EtOAc - Hexane mixtures are too polar CH2Cl2 Hexane For only very non-polar compounds hydrocarbons

* The first solvent given is the minor component.

Generally, the best results are usually obtained when using between 5% and 30% of the more polar solvent in a mixture. Using less than 5% of the more polar solvent or more than 50% of the more polar solvent does not usually produce reliable separations.

Increasing Polarity

Chem 347 Fall 2010

Prof. Rob Ronald

If you need more than about 40% of the polar component it is usually better to go to a more polar combination and to use a smaller proportion, 10-30%, of the somewhat more polar component. Likewise, if you need less than 5% of the more polar component then it is usually better to move to a less polar combination with a larger proportion of the somewhat less polar component. If you have an acid it is sometime advantageous to add 1-2%HOAc or aq. HCl to the solvent mixture to minimize the tailing of the spots. If you add aq. HCl the whole solvent mixture has to be able to dissolve the aqueous component. For an amine or 1-2% aq. NH3 or Et3N can be added. When using low amounts of these polar additives it is important to change the solvent in the tank frequently since these compounds are strongly adsorbed by the silica and usually after a few plate developments the solvent composition will have changed significantly so that the separation results will have changed. With alcohol samples it is advantageous to use solvent mixtures that contain MeOH because this minimizes tailing of the spots.

Chem 347 Fall 2010

Prof. Rob Ronald

Developing the plate

Use a 10mL graduated cylinder to measure out 5-6mL of a solvent mixture; this amount of solvent usually gives a good depth of solvent in the tank (~5mm). The table gives suggestions for various mixtures, but a good place to start is 20%EtOAc-hexane (1mL of EtOAc and 4mL of hexane). Pour the solvent mixture into the TLC tank (developing chamber), close the lid and swirl the solvent to completely mix it and to equilibrate the tank to the solvent. Pick up your spotted plate with forceps and place it in the tank so that it is nearly vertical; be sure that the end of the plate where the samples are spotted is dipped into the solvent mixture. The depth of the solvent in the tank should be about 5mm and there should be about 5mm space between the top of the solvent and the samples on the plate in order to give the solvent front a space to stabilize before the samples are encountered. Close the lid and wait until the solvent front has risen to within about 5mm of the top of the plate. Reach in with forceps and remove the plate.

Chem 347 Fall 2010

Prof. Rob Ronald

Visualizing the separation

After the plate is developed it can be held under a UV lamp. The plate will fluoresce green and compounds will appear as dark spots. If you have spotted the correct amount of sample the major spots should be no more than 5-6mm in diameter. You can only see spots under the UV lamp if the compound in the spot has a UV chromophore (absorbs UV light). There are other methods to visualize TLC plates: the plate can be sprayed with a solution of ceric ammonium nitrate (CAN) in dil. H2SO4 and then heated to 150-200 C many compounds will char to give brown spots; the plate can be placed in a jar containing a small amount of iodine (I2) iodine from iodine vapor forms colored charge-transfer complexes with many compounds that appear as brown spots; if amines are present the plate can be sprayed with a solution of Ninhydrin (1,2,3-indane-trione) and then heated amine containing materials give pink or purple spots. Visualizations with CAN or Ninhydrin give permanent visible spots, but the plates cannot be again visualized by UV. CAN and Ninhydrin reagents are available ask for help on these. The spots generated by iodine vapor are transitory and fade within a few minutes of removal from the I2 chamber.

Chem 347 Fall 2010

Prof. Rob Ronald

If you are using an electronic notebook you can take a digital picture of your TLC plate and paste it into your experimental page. I have a digital camera and we can take a picture of your plate I will send it to you by email. If you are using a paper notebook you make a copy of the picture and paste it into your notebook You could draw a representation of your plate in your notebook. In all cases be sure to annotate the picture so that you can remember what it represents. ChemDraw has a TLC plate drawing macro that you can use to make a representation of your plate.

Recording and troubleshooting

Chem 347 Fall 2010

Prof. Rob Ronald

The distance a spot travels on a plate is referred to as the Rf (Rf = b/a). In lane 1 an optimal spot (shape and Rf) is shown. In lane 2 the Rf is good, but the spot is tailing; in this case a combination with a hydrogen bonding solvent component (MeOH, AcOH, or NH4OH) could be useful. In lane 3 a more polar combination is required. In lane 4 the solvent is too polar; a less polar combination is required. In lane 5 the Rf is good, but the compound was spotted too heavily; spot more lightly. In lane 6 the Rf is good, but the compound was spotted too lightly; spot more heavily. In the cases of lanes 2-6 the TLC should be repeated with the suggested adjustments.

1 2 3 4 5 6

Optimal Rf range 0.3 to 0.6