Column chromatography

Column Chromatography:

In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column and the mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture.
The mixture to be analyzed by column chromatrography is applied to the top of the column. The liquid solvent (the eluent) is passed through the column by gravity or by the application of air pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as the solvent drips from the bottom of the column.
Column chromatography is separated into two categories, depending on how the solvent flows down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is called gravity column chromatography. If the solvent is forced down the column by positive air pressure, it is called flash chromatography, a "state of the art" method currently used in organic chemistry research laboratories The term "flash chromatography" was coined by Professor W. Clark Still because it can be done in a “flash."

The Adsorbent:

Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by the organic chemist for column chromatography. These adsorbents are sold in different mesh sizes, as indicated by a number on the bottle label: “silica gel 60” or “silica gel 230-400” are a couple examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process. If there are more holes per unit area, those holes are smaller, thus allowing only smaller silica particles go through the sieve. The relationship is: the larger the mesh size, the smaller the adsorbent particles.
Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for gravity chromatography. For example, 70–230 silica gel is used for gravity columns and 230–400 mesh for flash columns.
Alumina is used more frequently in column chromatography than it is in TLC. Alumina is quite sensitive to the amount of water which is bound to it: the higher its water content, the less polar sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity is designated as I, II, or III, with I being the most active. Alumina is usually purchased as activity I and deactivated with water before use according to specific procedures. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.
Silica gel and alumina are the only column chromatography adsorbents used in the CU organic chemistry teaching labs; please refer to the references for information on other column chromatography adsorbents.

The Solvent:

The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the successful application of column chromatography as a separation technique. TLC is generally used to determine the system for a column chromatography separation. The choice of a solvent for the elution of compounds by column chromatography is covered in the Chromatography Overview section.
Often a series of increasingly polar solvent systems are used to elute a column. A non-polar solvent is first used to elute a less-polar compound. Once the less-polar compound is off the column, a more-polar solvent is added to the column to elute the more-polar compound.

Interactions of the Compound and the Adsorbent:

Compounds interact with the silica or alumina largely due to polar interactions. These interactions are discussed in the section on TLC.

Analysis of Column Eluants:

If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. More commonly, the compounds to be isolated from column chromatography are colorless. In this case, small fractions of the eluent are collected sequentially in labeled tubes and the composition of each fractions is analyzed by thin layer chromatography. (Other methods of analysis are available; this is the most common method and the one used in the organic chemistry teaching labs.)

Procedures:

Columns for chromatography can be small or big, according to the amount of material which needs to be loaded onto the column. Pictured below are three glass columns, two of which are used in the organic chemistry teaching labs at CU.
The "column" on the far left in the photo is actually a Pasteur pipet. This size of column is suitable for 10-125 mg of material. The middle column is a 10 mL disposable glass pipet. You can load about a gram of material on this size column. The column on the right would be used for many grams of material.
Of the three columns pictured, only the column on the right is actually manufactured as a chromatography column. Note the stopcock at the bottom of the column. This is to control the flow of solvent through the column, important for gravity column chromatography applications.
The middle column is used for gravity column chromatography in a few of the chemistry majors' laboratory courses (chem 3361 and 3381). Note the piece of flexible tubing which has been added to the bottom of the column.To control the flow of solvent, a pinch clamp would be placed on the flexible tubing at the bottom.
The Pasteur pipet column is used for microscale gravity and microscale flash chromatrography procedures; these procedures (usually) do not require a means of control of gravity-induced solvent flow through the column.
Much larger chromatography columns are available than the one on the right. The size employed depends on the amount of material which needs to be separated. Large-scale flash columns look like this column but have a standard taper connection at the top so they can be connected to a source of pressurized air.
In the Organic Chemistry teaching labs at CU, the most frequently used column is the Pasteur pipet. They work well in microscale flash column chromatography procedures because a pipet bulb fits conveniently on top of them to serve as a source of pressurized air (when you press on the bulb!). Microscale procedures are used at CU Boulder whenever feasible to cut down on waste chemical production.
Here is a picture of a packed column of the type on the right

.
Procedure for Gravity Column Chromatography:

Gravity columns are not used in any of the non-major organic lab courses at CU Boulder (chem 3321/3341). The majors (chem 3361/3381) do use this type of chromatography. Gravity columns are a lot slower to run than microscale flash columns. They also are more difficult to set up or "pack" with adsorbent.

Procedure for Microscale Flash Column Chromatography:

Microscale flash chromatography is the method used almost solely in the organic chemistry teaching labs because it is both easy and environmentally friendly. The method is only limited by the fact that it can separate only small amounts of sample. It works best for 25 mg amounts, although we have pushed it to separate 125 mg mixtures if the TLC Rf's of the components of the mixture differ by at least 0.20.

Refrances:

1. http://en.wikipedia.org/wiki/Column_chromatography
2. http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html
3. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4TMYG2D
4. http://fa.wikipedia.org/wiki
5. http://orgchem.colorado.edu/hndbksupport/dist/distsetup/vacdist.html
6. http://fa.wikipedia.org/wiki/سانتریÙ

Thin Layer Chromatography ( TLC)

Introduction:

Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity and following the progress of a reaction. It also permits the optimization of the solvent system for a given separation problem. In comparison with column chromatography, it only requires small quantities of the compound (~ng) and is much faster as well.

Stationary Phase:

As stationary phase, a special finely ground matrix (silica gel, alumina, or similar material) is coated on a glass plate, a metal or a plastic film as a thin layer (~0.25 mm). In addition a binder like gypsum is mixed into the stationary phase to make it stick better to the slide. In many cases, a fluorescent powder is mixed into the stationary phase to simplify the visualization later on (e.g. bright green when you expose it to 254 nm UV light).

Preparing the Plate:

Do not touch the TLC plate on the side with the white surface. In order to obtain an imaginary start line, make two notches on each side of the TLC plate. You can also draw a thin line with pencil. Do not use pen. Why? The start line should be 0.5-1 cm from the bottom of the plate.

Capillary spotters:

Place a melting point capillary and in the dark blue part of the Bunsen burner flame. Hold it there until it softens and starts to sag. Quickly remove the capillary from the flame and pull on both ends to about 2-3 times its original length. If you pull the capillary inside the flame, you will have a "piece of art", but not a good spotter. Allow the capillary to cool down, and then break it in the middle. Make sure that you break off the closed end on one of them. Do not use gloves when you pull capillaries. You will have much better control without them!

Spotting the plate:

The thin end of the spotter is placed in the dilute solution; the solution will rise up in the capillary (capillary forces). Touch the plate briefly at the start line. Allow the solvent to evaporate and spot at the same place again. This way you will get a concentrated and small spot. Try to avoid spotting too much material, because this will deteriorate the quality of the separation considerably (‘tailing’). The spots should be far enough away from the edges and from each other as well. If possible, you should spot the compound or mixture together with the starting materials and possible intermediates on the plate. They will serve as internal reference since every TLC plate is slightly different.

Developing a Plate:

A TLC plate can be developed in a beaker or closed jar (see picture below). Place a small amount of solvent (= mobile phase) in the container. The solvent level has to be below the starting line of the TLC, otherwise the spots will dissolve away. The lower edge of the plate is then dipped in a solvent. The solvent (eluent) travels up the matrix by capillarity, moving the components of the samples at various rates because of their different degrees of interaction with the matrix (=stationary phase) and solubility in the developing solvent. Non-polar solvents will force non-polar compounds to the top of the plate, because the compounds dissolve well and do not interact with the polar stationary phase. Allow the solvent to travel up the plate until ~1 cm from the top. Take the plate out and mark the solvent front immediately. Do not allow the solvent to run over the edge of the plate. Next, let the solvent evaporate completely.
TLC chamber for development e.g. beacher with a lid or a closed jar
after ~5 min
after ~10 min
after drying

Visualization:

There are various techniques to visualize the compounds.1. Sulfuric acid/heat: destructive, leaves charred blots behind
2. Ceric stain: destructive, leaves a dark blue blot behind for polar compounds
3. Iodine: semi-destructive, iodine absorbs onto the spots, not permanent
4. UV light: non-destructive, long wavelength (background green, spots dark), short wavelength (plate dark, compounds glow), Do not look into the UV lamp!!!Circle the spots on the TLC plate to have a permanent record how far the compound traveled on the plate. Also draw a sketch of the developed plate in your lab notebook.

Analysis:

The components, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials. Measure the distance of the start line to the solvent front (=d). Then measure the distance of center of the spot to the start line (=a). Divide the distance the solvent moved by the distance the individual spot moved. The resulting ratio is called Rf-value. The value should be between 0.0 (spot did not moved from starting line) and 1.0 (spot moved with solvent front) and is unitless.
The Rf (=retardation factor) depends on the following parameters:
solvent system
absorbent (grain size, water content, thickness)
amount of material spotted
temperature
Due to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well.

Refrances:

1. http://en.wikipedia.org/wiki/Thin_layer_chromatography
2. http://orgchem.colorado.edu/hndbksupport/TLC/TLC.html
3. http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html
4. http://www.chem.ucla.edu/~bacher/General/30BL/tips/TLC1.html
5. http://www.files.chem.vt.edu/chem-ed/sep/tlc/tlc.html
6.http://www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/thin_layer_chrom.html
7. http://fa.wikipedia.org/wiki/الکتروفورز

Picrates:

Because tertiary amines are incapable of forming sustituted amides or ureas, the usual derivative is some type of salt Picrates, derivatives of picric acid (2,4,6-trinitophenol), are relatively easy to prepare.

A solution of 0.5 g of the amine in 10 mL of 95% ethanol is prepared. If all of the amine does not dissolve, filter the mixture.

The amine solution is added to 10 mL of a saturated solution of picric acid in 95% ethanol, which is contained in a 50-mL Erlenmeyer flask.
A boiling chip is added and the solution is heated to boiling on a steam bath.
Remove the flask from the heat source and allow the solution to cool slowly. The yellow picrate should precipitate.

Filter the crude product and crystallize it from 95% ethanol.

Picric acid combines with amines to yield molecular compounds (picrates), which usually possess characteristic melting points. Most picrates have the composition 1 mol amine : 1 mol picric acid. The picrates of the amines, particularly of the more basic ones, are generally more stable than the molecular complexes formed between picric acid and the hydrocarbons.

If the amine is soluble in water, mix it with a slight excess (about 25 per cent.) of a saturated solution of picric acid in water (the solubility in cold water is about 1 per cent.). If the amine is insoluble in water, dissolve it by the addition of 2-3 drops of dilute hydrochloric acid (1:1) for each 2-3 ml. of water, then add a slight excess of the reagent. If a heavy precipitate does not form immediately after the addition of the picric acid solution, allow the mixture to stand for some time and then shake vigorously. Filter off the precipitated picrate and recrystallize it from boiling water, alcohol or dilute alcohol, boiling 10 per cent. acetic acid, chloroform or, best, benzeneThe method described by Vogel may be a better procedure for heat sensitive amines, since the only heating step is after formation of the picrate and even then, by use of MeOH, temperatures could remain relatively low. Keep in mind that while he describes benzene as the a good choice during recrystalization, it has since been noted to be carcinogenic and is therefore probably not the best choice. The formation of a picrate does not in any way identify a compound as a tertiary amine, since they also form with hydrocarbons, aromatic hydrocarbons, some halogen derivatives and aromatic ethers. Picrates are simply a way of creating a derivative with a characteristic melting point, which is often used in melting point determinations by combination with a known compound (mixed melting points, M.m.p.), watching for depression of melting points.

Nickel Chloride, Carbon Disulfide, Ammonium Hydroxide Test

2oAmine

Procedure

Add 1 or 2 drops or 50 mg of unknown to 5 mL of water. If necessary, 1 or 2 drops of conc. HCl may be added to dissolve the amine. To 1 mL of nickel chloride in carbon disulfide reagent in a test tube, add 0.5 - 1 mL of conc. ammonium hydroxide, followed by 0.5 - 1 mL of amine solution.

Positive Test

2o amines- precipitate is a positive test.

Complications

Primary and tertiary amines with secondary amine impurities will yield a positive test.

AMIN1

AMIN2

AMIN3

Hinsberg Test



Procedure

To 0.3 mL or 300 mg of unknown in a test tube, add 5 mL of 10% NaOH solution and 0.4 mL of benzenesulfonyl chloride. Stopper the test tube, and shake the mixture vigorously. Test the solution to make sure that it is still alkaline. After all of the benzenesulfonyl chloride has reacted, cool the solution and separate the residue, if present, from the solution. Test the residue for solubility in 10% HCl solution. If no residue remains, then treat the solution with 10% HCl solution and observe whether a precipitate forms.

Positive Test

1o amines - dissolves in base and precipitates from acid is a positive test.
2o amines - precipitates from base and no change from acid is a positive test.
3o amines - precipitates from base and dissolves in acid is a positive test.

Complications

Amphoteric compounds give erroneous results.
Some sodium salts of benzenesulfonamides of primary amines are insoluble in the Hinsberg solution and may appear to be secondary amines.
Some tertiary amine hydrochloride salts are insoluble in dilute HCl and water and may also appear to be secondary amines

Hinsberg Test

1o Amine
2o Amine
3o Amine

Procedure
To 0.3 mL or 300 mg of unknown in a test tube, add 5 mL of 10% NaOH solution and 0.4 mL of benzenesulfonyl chloride. Stopper the test tube, and shake the mixture vigorously. Test the solution to make sure that it is still alkaline. After all of the benzenesulfonyl chloride has reacted, cool the solution and separate the residue, if present, from the solution. Test the residue for solubility in 10% HCl solution. If no residue remains, then treat the solution with 10% HCl solution and observe whether a precipitate forms.
Positive Test
1o amines - dissolves in base and precipitates from acid is a positive test.
2o amines - precipitates from base and no change from acid is a positive test.
3o amines - precipitates from base and dissolves in acid is a positive test.
Complications
Amphoteric compounds give erroneous results.
Some sodium salts of benzenesulfonamides of primary amines are insoluble in the Hinsberg solution and may appear to be secondary amines. Some tertiary amine hydrochloride salts are insoluble in dilute HCl and water and may also appear to be secondary amines

Nitrous Acid Test for amines

Procedure

Dissolve 0.5 mL or 0.5 g of unknown in 1.5 mL of conc. HCl diluted with 2.5 mL of water, and cool the solution to 0oC in a beaker of ice. Dissolve 0.5 g of sodium nitrite in 2.5 mL of water and add this solution dropwise, with shaking, to the cold solution of the amine hydrochloride. Continue the addition until the mixture gives a positive test for nitrous acid. The test is carried out by placing a drop of the solution on starch-iodide paper; a blue color indicates the presence of nitrous acid. If the test is positive, move 2 mL of the solution to another test tube, warm gently, and examine for evolution of gas.

Positive Test

1o aliphatic amines- rapid bubbling upon addition of sodium nitrite is a positive test.

1o aromatic amines- rapid bubbling after addition of sodium nitrite (with heating) is a positive test.

2o amines- pale yellow oil with no evolution of gas is a positive test.

3o aliphatic amines- immediate positive test for nitrous acid with no evolution of gas is a positive test.

3o aromatic amines- dark-orange solution or orange solid, when treated with base turns green is a positive test.

Complications

Compounds having a methylene group adjacent to a carbonyl group give a positive test.

Alkyl mercaptans yield red thionitroso compounds.

Nitrous acid will react with amides and phenols.

refrence

http://www.chemistry.ccsu.edu/

http://www.elmhurst.edu/

http://www.chemguide.co.uk/

http://www.redwop.com/download/ninhydrn.pdf

http://en.wikipedia.org/wiki/Ninhydrin

http://antoine.frostburg.edu/chem/senese/101/organic/faq/amino-acid-test.shtml

http://www.woelen.nl/chem

http://www.chemistry.ccsu.edu/glagovich/teaching/316/qualanal/tests/nitrousacid.html

http://www.chemguide.co.uk/organicprops/amines/nitrousacid.html#top

http://www.chemistry.ccsu.edu/glagovich/teaching/316/qualanal/tests/nitrousacid.html

http://www.chemguide.co.uk/organicprops/amines/nitrousacid.html#top

http://msds.chem.ox.ac.uk/BE/benzenesulfonyl_chloride.html

http://en.wikipedia.org/wiki/Ninhydrin

http://www.chemistry.ccsu.edu/glagovich/teaching/316/qualanal/tests/schotten.html

http://www.sciencelab.com/

Ninhydrin Test for amino acid

Procedure:

Add about 2 mg of the sample to 1 mL of a solution of 0.2 g of ninhydrin (1,2,3indanetrione monohydrate) in 50 mL of water. The test mixture is heated to boiling for 15-20 sec; This reaction is important not only because it is a qualitative test, but also because it is the source of the absorbing material that can be measured quantitatively by an automatic amino acid analyzer. This color reaction is also used to detect the presence and position of amino acids after paper chromatographic separation.

Positive Test:

A blue to blue-violet color is given by a-amino acids and constitutes a positive test.

Other colors (yellow, orange, red) are negative.

Complications:

Proline, hydroxyproline, and 2-, 3-, and 4-aminobenzoic acids fail to give a blue color but produce a yellow color instead.

Ammonium salts give a positive test.

Some amines, such as aniline, yield orange to red colors, which is a negative test.

Copper Complex Formation for amino acid

Procedure:

A small amount of the compound is dissolved in 1 mL of water. Two drops of 1 M copper(II) sulfate are added. If a blue color is not formed immediately, then heat the test tube in a hot water bath for 5 min.

Positive Test:

A moderate to deep blue color of a liquid or a dark blue solid is a positive test.

Complications:

Some a-amino acids are not very soluble in cold water.

However, these amino acids are soluble in hot water and will give a positive test when the solution is heated.

Aliphatic amines yield a blue precipitate.

Anilines give a brown or green color, but other aromatic amines produce a blue-purple color.

Schotten-Baumann Reaction for Carboxylic Acid

Procedure

A mixture of 0.20 g of the compound, 0.40 mL of absolute ethanol, and 0.20 mL of concentrated sulfuric acid is warmed over a steam bath for 2 min. The mixture is poured slowly into an evaporating dish containing 2 mL of saturated sodium bicarbonate solution. A second layer should be formed. Carefully smell the mixture. The presence of a sweet, fruity smell in the product, where no such smell existed in the original unknown, indicates that the original compound was a carboxylic acid and the acid was esterified. Large molecular weight carboxylic acids produce esters that are odorless.

Positive Test

Formation of an oily layer on top of the water is a positive test.

Complications

None.

Neutralization equivalent for carboxylic acids

Procedure:

Weigh 0.2 g (to three significant figures) of the unknown carboxylic acid, and place in a
125-mL Erlenmeyer flask. Dissolve the acid in about 50 mL of water or aqueous ethanol
(the acid need not dissolve completely, because it will dissolve as it is titrated). Titrate
the acid, using a standardized solution of NaOH of known molarity (in the range of 0.1000 M) and a phenolphthalein indicator (2 drops). Note the equivalent point (colorless to pink color).
Record the volume of NaOH used. Duplicate the run.

Calculate the neutralization equivalent (NE) from the equation:

NE = mg of carboxylic acid/molarity of NaOH x mL of NaOH used

The NE is identical to the equivalent weight of the carboxylic acid. If the acid has only
one carboxyl group, the NE and the molecular weight of the acid are identical. If the
acid has more than one carboxyl group, the NE equals the molecular weight of the acid
multiplied by the number of carboxyl groups, that is the equivalent weight. The NE can be used much like a derivative to identify a specific carboxylic acid.

Complication:

Many phenols are acidic enough to behave similarly to carboxylic acids. This is especially true of those substituted with electron-withdrawing groups at the ortho and para ring positions. These phenols, however, can be eliminated by the ferric chloride test or spectroscopy.