Chromatography – In General
• Separation of compounds based on the polarity of the compounds being separated • Two potential phases for a compound to exist in: mobile (liquid or gas) and stationary • Partitioning of compounds between mobile phase and stationary phase occurs – some
move more in mobile phase, some “stick” more on stationary phase, resulting in compounds moving at different rates which can then be separated
1. Mobile Phase – Solvent System
• The polarity of a solvent is defined as its ability of the solvent to dissolve increasingly polar organic compounds.
• The more polar a solvent is, the larger the numbers of compounds that can dissolve into the solvent, starting with non-polar, then a little polar, more polar and finally (for very polar solvents) totally polar compounds!
• Compounds dissolved into the solvent spend more time moving in the mobile phase in chromatography.
For Chromatography, you must remember “Polar Dissolves More,” not “like dissolves like”.
Some Common Solvents: (listed by increasing polarity)
• Petroleum ether (fake ether, C5 hydrocarbons) (non-polar) • Ligroin (C6 hydrocarbons) (non-polar, higher BP)
• Diethyl ether (real ether, CH3CH2OCH2CH3) (slightly polar) • Dichloromethane (CH2Cl2) (polar)
• Ethyl acetate (ester, CH3C(O)OCH2CH3) (pretty polar) • Methanol (alcohol, CH3OH) (really polar)
• Acetic acid (carboxylic acid, CH3C(O)OH) (extremely polar) And of course – mixtures of any of the above can be used.
Consider the following example: Compound A is a non-polar compound and compound B is a polar compound.
Add Petroleum ether to the mixture. Which will dissolve?
Non-Polar A, because a non-polar solvent can only dissolve non-polar compounds.
What if you added methanol instead? Which will dissolve?
Both! The more polar the solvent, the more compounds it can dissolve, starting with the non-polar ones and increasingly dissolving more polar ones.
Keep in mind that the more polar the solvent the more the compounds’ will dissolve into the mobile phase. This means the compounds will move further through the stationary phase.
2. Stationary Phase – Alumina (Al2O3) or Silica (SiO2) on a solid support
• The more polar the compound, the stronger the compound adheres (“sticks”) to the adsorbent.
Functional Groups (listed by increasing polarity): Alkanes (nonpolar, non-stick)
Alkenes and aromatics (still pretty nonpolar) Ethers (little more polar)
Esters (even more polar)
Ketones and aldehydes (yes, holding on tighter) Amines (reaching the polar end of the options)
Alcohols (even more polar than amines – better at H bonding) Carboxylic Acids (the most polar, holding on like its super-glued!) Partitioning Effect:
Generally, polar compounds spend more time on the stationary phase (“stuck”) and non-polar compounds spend more time in the mobile phase (moving along with the solvent). The goal is to use a solvent system that moves ONLY the least polar compound, until it is completely removed from the system…
…THEN use a more polar solvent to move the next compound… …and so on, until all components are separated out one at a time…
Just remember that non-polar compounds move in ALL solvents but polar compounds need more polar solvents in order to enter the mobile phase and be moved through the stationary phase.
Column Chromatography:
• Used for separation (purification) of organic compounds (solids or liquids) • Size of separation can range from milligrams to kilograms
Packing of the Column: -demo-
• Must remove air bubbles – pockets of air allow compounds to travel in the mobile phase faster through air pockets resulting in separation that is not uniform
• Column must be assembled in a vertical fashion, so bands will travel downwards in an even and uniform fashion for the best separation
• Crooked bands tend to stay overlapped slightly
• Sample must be loaded in the most concentrated method
o need the narrowest band possible for best separation
o wide bands tend to stay wide (or get wider) and thus stay overlapped
• Again, you may use a single solvent or you may change solvents during column by
allowing solvent level to drain to the top of the sand in column, THEN refill with new solvent.
• Use gravity to push your solvent through or you can use gas pressure to move the mobile phase quicker through the stationary phase (“flash column chromatography”)
Sand Sand alumina or silica gel Sand Sand alumina or silica gel Sand Sand alumina or silica gel Not Separated
• Collect fractions containing the bands and evaporate the solvent.
• Any crystals “growing” on the bottom of the column should be rinsed into the appropriate vial.
Take a look at the following columns. Determine which compound is the less polar compound.
a. b. c.
Thin-Layer Chromatography (TLC): • Used to identify compounds (like GC) • Used to check the purity of a compound • Used to check a reaction’s progress
Only need micrograms of material to do this process.
Truly, the only major difference between column chromatography and TLC is the direction of the flow of the mobile phase!
Similarities between Column Chromatography and TLC: • Mobile Phase: Same Solvents
• Stationary Phase: Same Alumina or Silica applied on some solid support • Same partitioning effect based on compound polarity
Process Summary: -demo-
1. Spotting the Plate (application of the compounds)
• TLC plate is marked with a pencil (not pen) to show point of origin (where compound is applied). This point of origin must be about 1 cm above the bottom of the plate
(above the level of the solvent used as the mobile phase). This avoids having the mobile phase wash the compounds off. Don’t spot too close to the end of the plate either…
• The microcapillary tube uses capillary action to pull liquid into tube and then capillary action will release the liquid onto the adsorbent by gentley tapping the microcapillary tube to the TLC plate.
2. Developing the Plate (movement of eluent through adsorbent)
• Developing Chamber – glass container, such as beaker or jar, with a cover.
• Mobile phase uses capillary action to “climb” the plate in a vertical fashion, thus often a “wick” is added to the chamber to provide saturated atmosphere throughout, so the mobile phase does not evaporate away as it climbs the plate. • Must be sure the plate does not touch any wick or the side of the chamber as the
plate develops. This would cause the mobile phase to move in a sideways fashion also, ruining the “lanes” of travel by the compounds and preventing any identification. • Plate should always be monitored – watch the “solvent front” – and removed prior to
the solvent exceeding the distance of the plate. MARK the distance the solvent has traveled when removing!
• Probably a good idea to pour out old solvent from first TLC plate and add in fresh solvent before doing second TLC plate or your solvent system (1:1) may not still be 1:1 – which will skew your results.
What will happen to your peaks if your solvent system evaporates and becomes, for instance, more polar?
3. Visualization
• Typically organic compounds are colorless and not seen by the human eye.
• To visualize, you may use iodine, a UV lamp, or one of many organic stains. Circle the spot with a pencil. Record the appearance of the spot on the plate.
• In today’s experiment, ferrocene and acetylferrocene are colored compounds so you will be able to see without any stain or UV light.
Plate 1: Plate 2:
x x x x x
Draw each TLC plate into your notebook LIFE-SIZED. Trace around the plate and transfer all details to your drawings – origin, shapes/sizes/colors of spots, solvent front distance. No thumbnail sketches please!
4. Calculations (For EACH spot):
Rf value (Retardation Factor): Relative amount the spot traveled when compared to the solvent.
The Rf value is calculated by dividing the distance the spot travels by the distance the solvent travels (from the origin to the solvent front), as shown below:
All measurements should be done in metric units (cm or mm, not inches). The Rf value will always be between 0 and 1 and is NOT expressed as a percentage and has no units. What problem occurs if you forget to draw the solvent line when you
remove your TLC plate from its developing chamber?
Rf values are commonly used for identification purposes.
• the same compound with always have the same Rf value under the same conditions (i.e. same solvent system).
• Apply standard compounds on the same TLC plate as an unknown compound and run the compounds side-by-side to compare. (Your Plate #1)
• Keep in mind that the spots for the same compounds will always have the same appearance, as well as the same Rf value.
Y
X
Rf = X/Y
Same Compound or Not?
no solvent front
TLC can also be used for checking the purity of a compound. A pure compound will always appear as a single spot (because it is a single compound). (Your Plate #2)
If the Rf value is too low (<0.2) or too high (>0.8), try a different solvent system. You may be using a system that is too polar or too non-polar to differentiate how many compounds (spots) you are seeing.
Pure or Not? Can you even tell…?
Probably the most popular reason for doing TLC: Following the Progress of a Reaction: Use TLC to monitor the status of a reaction’s progress. Always spot the beginning starting material on the same plate as the reaction mixture. If the starting material is still present, you will see starting material (same appearance, same Rf value) as a spot for the reaction mixture.
If the starting material is completely gone (i.e. Reaction is Finished!), then you should not see any spot that corresponds to starting material in the reaction mixture.
Consider the following questions: Which compound is the most polar? Which compound is the least polar?
non-polar solvent polar solvent O OH LiAlH4 x x sm rxn T = 0 min x x sm rxn T = 15 min x x sm rxn T = 30 min x x sm rxn T = 45 min x x sm rxn T = 60 min A B C
In what order would these compounds come out of a column?
One more time:
Which compound is the most polar? Which compound is the least polar?
In what order would these compounds come out of a column?
What would this TLC plate look like, if it was developed in an even more polar solvent?
How about a less polar solvent?
What problem is associated with determining the purity of the following spot?
Today in Lab:
Each pair of students must separate ferrocene and acetyl ferrocene using column chromatography.
Each pair of students need to run one TLC plate with the two known compounds on it and a sample of the mixture in order to identify which of the spots is which compound. You will need to measure to obtain Rf values for all four of those spots. (Four separate calculations)
You need to run a second TLC plate on the contents of Vial 1 and Vial 2 from your column separation. This is to check the purity of those separated compounds. You will also need to measure to obtain Rf values for both of those spots, even though Rf has nothing to do with purity.
Upon completing the second TLC plate, see your instructor about where to place the two vials so they may evaporate off solvent to form crystals for next weeks MP analysis.
