Chem 347 Lecture 8-e Chrom

7/30/2019 Chem 347 Lecture 8-e Chrom

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Chem 347 Fall 2010 Prof. Rob Ronald

Chromatography. Chromatography, in its various forms, is a purification

and analytical technique that has the widestapplicability in organic chemistry. Virtually all stable molecules will survive one or more

types of chromatographic separation;

Depending upon the system used, the purity ofchromatographed compounds can be from modest tovery 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.


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Chromatography is a technique thatemploys the partitioning of a solutebetween a stationary phase (solid,or sometimes liquid), and a mobilephase (liquid, or gas).

Chromatography was discovered byMichael Tswett in the first part of the20th century; his first paper describingthe technique was published in 1906.

The technique was discovered in thecourse of investigating plant pigments;Tswett passed pentane solutions ofplant pigments through a bed ofpowdered sucrose and observed the

formation of colored bands hencethe name chromatography. At the time he speculated that it would

be possible to separate colorlesscompounds by the same technique,

but he never did it himself.

Powdered

Sucrose

Column

Tswetts Original

Experiment


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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.

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

Syringe (sample) Injector heater Oven Detector Recorder Output

Coiled

Column

Schematic for Gas Chromatography


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The sample is usually injected as a dilute solution in asolvent 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 gasstream immediately onto the column.

The column is contained in a thermostated variabletemperature oven that can be heated from roomtemperature to about 250 C.

There are usually provisions to program thetemperature 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.


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Various types of detectors are available for GasChromatography: 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 tothe effluent;

The TCD detector measures the changes in temperature of ahot filament that result of changes in composition of the gaspassing 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 ina region of high potential: the ionic products of combustionproduce a miniscule electric current.

FID detects compounds based on the carbon content and theeffluent is destroyed by burning in a hydrogen flame toproduce the ion current

the electron capture detector (ECD) detects halogensselectively and uses a radioactive source to generate theions.


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In the TCD mode, gas chromatography can also beused in a preparative mode to prepare small (10-

500mg) quantities of volatile compounds. In mostcases this older methodology has been superseded byliquid chromatographic methods.

Another type of detection that is universal and very

sensitive is to couple the gas chromatography withmass 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 themass spectrometer they can be scanned, and oftenthe components can be identified, by mass

spectroscopy.


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Packed columns for GC are typically stainless steel or glasscoils 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 withback pressures around 60psi.

Typical performance will have peak widths at half height around0.2-1 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 variousliquid phases such as silicone fluids (SE-30, SPB-1, etc.) orpolyethylene glycol waxes (Carbowaxes).

There are many types of packing materials and liquid phasesfor various types of separations.

A company such as Supelco is a good source for thesecolumns


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Capillary columns are long tubes of fused silicaproduced by the same processes used to make fiberoptics. 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 sensitivedetector (FID) has to be used.

The carrier gas pressure is adjusted to give a flow rate ofabout 20-30cm/sec down the column; this usually requires apressure of only 10-20psi.

Capillary columns give very high resolution with peak widthsat half height around 0.05-0.2min depending on the retention

time, which again is related to the column temperature.


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Liquid Chromatography TLC, LPLC, MPLC and HPLC These are all related forms of chromatography in which the

mobile phase is a liquid and the stationary phase is a poroussolid (sorbent).

In TLC(Thin Layer Chromatography) the sorbent is spread in a thinlayer, 0.25-2mm thick, on an inert, rigid surface (glass, plastic, oraluminum plates) and the liquid phase is allowed to travel through thesorbent by capillary action from a solvent pool in which the bottompart of the plate is dipped.

The samples to be analyzed are spotted near the bottom of theplate and travel upwards as they are carried along with thesolvent.

In LPLC(Low Pressure Liquid Chromatography FlashChromatography) the sorbent is packed into columns (usually glassor plastic) and the mobile phase is applied to the top of the columnand allowed to percolate through the sorbent under the influence ofgravity or a slight positive pressure of a gas (2-10 psi compressed airor 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.


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MPLC and HPLC differ mainly in plumbing and in the pressureapplied to the mobile phase (eluant) due to the different particlesizes 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 columnslimited to between 50-200psi. HPLC (High Pressure (sometimes called High Performance)

Liquid Chromatography) is the most sophisticated and employspumps and columns capable of delivering and withstandingpressures up to 8000psi. To resist these high pressures HPLC columns and fittings are mad

from stainless steel; the pumps have sapphire pistons, ruby checkvalves and sapphire valve seats.

Typically, HPLC is an analytical tool and uses various detectionmethods to achieve quantitative information on the separation of th

analyte


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The most common sorbentsfor chromatography arevarious forms of Silica Gel.

The silica gel used today is asynthetic material, but it isrelated to a natural materialcalled diatomaceous earth, amineral that consists of thesilicaceous skeletons of

microscopic diatoms. Because diatomaceous earth

comes from billions andbillions of identicalmicroorganisms the material

is very porous, and the poresizes are uniform. Diatomaceous earth is

sometimes used as in inertsupport for packed GCcolumns.

Example of the pore morphology of the diatom,

Thalassiosira weissflogii, Bars = 5 m, bottom

inset bar = 0.2 m (2000 ).


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Synthetic Silica Gel had been known in the 17th century as ascientific curiousity.

In World War I the use of chemical warfare agents led to Prof.Walter Patrick of John Hopkins U. to invent a process forproducing 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 gelatinousproduct.

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

During WWII Silica Gel was used as a desiccant to preservepenicillin, and as a catalyst for making high octane gasoline andsynthetic rubber.

After WWII the ready availability of silica gels led to their use asadsorbents in chromatography.


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The most common type of sorbent used forchromatography 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 isabout 430m2; that means that if all the surface area in ateaspoonful of silica gel could be spread out it would occupy thearea of a basketball court!

The silica gel is sieved to have an average particle size of 40-60m for column chromatography (LPCL and MPLC); althoughfor 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.

C P


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Ordinary synthetic silica gel is sometimes referred toas normal phase silica gel, or absorption silica gel.

In normal phase silica gel compounds are absorbed to thesurface based on their polarities because the surface of thesilica 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 polarfunctional groups with the Si-OH groups on the silicasurface.

The more polar a molecule is the more tightly it is held bythe silica gel surface.

More polar compounds require a more polar the solvent tobe released from the surface.

Non-polar compounds bind only weakly to silica and arereleased readily from the surface with non-polar or onlymoderately polar solvents.

Ch 347 F ll 2010 P f R b R ld


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When dealing with very polar compounds it is sometimesadvantageous 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-O-Alkyl groups, typically C18 chains (RP-18).



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While reversed phase sorbents are more expensivethan 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 mosttightly bound and the polar compounds are more mobile.

Solvent mixtures for reversed phase chromatography arebased on mixtures of water (or aqueous buffers) and watermiscible organic solvents such as acetonitrile, THF orMeOH.

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 thenon-polar components, which stick to the greasy surface.

HPLC mainly employs the use of RP type columns.



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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 readilyavailable and the art of making TLC plates is mostly lost.

TLC plates have a thin layer of silica gel dispersed evenly on aninert, 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 aninert binder (usually a form of hydrated silica, or hydratedCaSO4, but sometimes an organic polymer) to make the layersstable and durable.

Other sorbents such as cellulose, alumina (aluminum oxide) orreversed phase silica gel are also available.



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The layers are durable enough that they can be writtenon lightly with a soft pencil.

Typically the sorbent is impregnated with an inert

fluorescent material that has a large Stokes shift so thatwhen it absorbs in the ultraviolet region (254nm and/or366nm) it emits in the visible (yellow green or blue)region of the electromagnetic spectrum. (ZnS has beenused in the past, but the fluorophores in use now areproprietary compounds)

The fluorescent dye makes materials that have a UVchromophore (light absorbing functional group)absorbing UV light around 254nm (or 366nm) visible asdark 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 TLCplates under UV irradiation; however some compoundsgive 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), RR-

2MeArL1c-2b; and lane 3 is the remaining mother liquor solution.

Chem 347 Fall 2010 Prof Rob Ronald


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

For typical analytical work the sheets are cut into strips 1-5cmwide by 5-10cm long.

For analyzing the series of fractions produced from LPLC orMPLC 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.



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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 Pasteurpipette using a Bunsen burner; there are also spotting pipettes availablein the stockroom

(3) a clean, dry 10mL graduated cylinder for measuring out solventmixtures,

(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 (bystockroom personnel) and stored in desiccators for protection.

Handle the plates only by the edges, and do not touch the whitelayer; use tweezers or forceps to place the plates in the tanksand to remove them.

Take only one (1) plate at a time, and do not use awider plate than you need; cut the plates with

scissors if they are not the correct size.



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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 outlarge chunks, but only powder to insure you have a representative sample.

Dissolve the sample completely in about 1mL of a solvent that will dissolve itcompletely: 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 theplate.

Usually a spot at the origin 1-2mm in diameter is sufficient. It is important tonot overload the plate.



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The depth of the solvent in the tankshould 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.

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.



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Choosing the solvent This is a trial and error process.

Generally, the best results are usually obtained when usingbetween 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 reliableseparations.

Some Useful TLC solvent mixturesSolvent Mixtures* Comment

H2OMeOH Extremely polar solvent mixture (10-25%H2O), good for acids

MeOHEtOAc Very polar solvent mixture (10-25% MeOH)

MeOHCHCl3 Sometimes works better than MeOHCH2Cl2

MeOHCH2Cl2 Useful general TLC solvent for polar compounds (10-40%MeOH)

EtOAcCH2Cl2 Specialized solvent mixture, infrequently used

Et2OCH2Cl2 Specialized solvent mixture, infrequently used

EtOAcHexane Useful general TLC solvent (10-40% EtOAc)

Et2OHexane Useful when EtOAc - Hexane mixtures are too polar

Increasing

Polarity

CH2Cl2Hexane For only very non-polar compounds hydrocarbons

* The first solvent given is the minor component.



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If you need more than about 40% of the polar component it is usuallybetter to go to a more polar combination and to use a smallerproportion, 10-30%, of the somewhat more polar component.

Likewise, if you need less than 5% of the more polar component thenit is usually better to move to a less polar combination with a largerproportion of the somewhat less polar component.

If you have an acid it is sometime advantageous to add 1-2%HOAcor 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 todissolve 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 platedevelopments the solvent composition will have changed significantlyso that the separation results will have changed.

With alcohol samples it is advantageous to use solvent mixtures thatcontain MeOH because this minimizes tailing of the spots.



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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 ofsolvent in the tank (~5mm).

The table gives suggestions for various mixtures, but a good placeto 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 thesamples 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 solventand the samples on the plate in order to give the solvent front aspace to stabilize before the samples are encountered.

Close the lid and wait until the solvent front has risen to withinabout 5mm of the top of the plate.

Reach in with forceps and remove the plate.



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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 shouldbe no more than 5-6mm in diameter. You can only see spots under theUV lamp if the compound in the spot has a UV chromophore (absorbsUV 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 manycompounds 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-transfercomplexes with many compounds that appear as brown spots;

if amines are present the plate can be sprayed with a solution ofNinhydrin (1,2,3-indane-trione) and then heated amine containingmaterials give pink or purple spots.

Visualizations with CAN or Ninhydrin give permanent visible spots, butthe plates cannot be again visualized by UV. CAN and Ninhydrinreagents are available ask for help on these.

The spots generated by iodine vapor are transitory and fade within a fewminutes of removal from the I2 chamber.



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Recording and troubleshooting If you are using an electronic notebook you can take a

digital picture of your TLC plate and paste it into yourexperimental page.

I have a digital camera and we can take a picture ofyour plate I will send it to you by email.

If you are using a paper notebook you make a copy ofthe 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 youcan remember what it represents.

ChemDraw has a TLC plate drawing macro that you

can use to make a representation of your plate.



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The distance a spot travels on a plateis referred to as the Rf (Rf = b/a).

In lane 1 an optimal spot (shape andRf) is shown.

In lane 2 the Rf is good, but the spot istailing; in this case a combination witha hydrogen bonding solventcomponent (MeOH, AcOH, or NH4OH)could be useful.

In lane 3 a more polar combination isrequired.

In lane 4 the solvent is too polar; a lesspolar combination is required.

In lane 5 the Rf is good, but thecompound was spotted too heavily;

spot more lightly. In lane 6 the Rf is good, but the

compound was spotted too lightly; spotmore heavily.

In the cases of lanes 2-6 the TLCshould be repeated with the suggestedadjustments

1 2 3 654

a

b

Op

timalRfrange

0.3

to0.6

Chem 347 Lecture 8-e Chrom

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