Reading-
1.  Switzer - Chapter 4 Introduction, 4.2 ion exchange chromatography, 4.5 TLC, 4.11 amino acid
2. There are several good web sites which provide useful information about amino acids. There are also some excellent study guides about properties and behavior. Not all web sites are good.  I have reviewed the information contained in the following sites for accuracy.

Acid-base chemistry and structure of amino acids:
http://www.dentistry.leeds.ac.uk/biochem/thcme/amino-acids.html

Amino acid chirality: http://chemistry.about.com/library/weekly/blaminochiral.htm

The chemical and structural properties of amino acid side chains:
http://www.mcb.ucdavis.edu/courses/bis102/AAProp.html
explore the links within this site for the properties of the side chains

Titrations of amino acids - take some time experimenting with the plot options on this site.  See if you can correlate structure with the titration plots.
http://cti.itc.virginia.edu/~cmg/Demo/markPka/markPkaInstr.html

Prelab Questions (completed in notebook prior to class):
1. Predict the dominant ionization state and total charge of the amino acids used in this laboratory at pH=1,7 and 11.
2. Using the Henderson-Hasselbach equation, prove the relationships denoted with an asterisk(*) in the introduction are true.
3.  Given the ion exchange procedure in this experiment what order of elution would you predict for a mixture of the amino acids, lys, arg and gly?

In the Laboratory Exercise Outline:

1. Determine the Rf values for a series of amino acids.
2. Separate a mixture of amino acids using ion exchange chromatography.
3. Identify the column eluant using TLC.

Amino Acids:
Amino acids are the building blocks of proteins. Proteins are biomolecules essential to the function of living systems. The amino acids also serve as the monomers used to create polymeric proteins. Amino acids all have the same basic backbone which contains a carboxylic acid and an amide attached by a tetrahedral methylene carbon. Each amino acid is defined by the side chain or R group pendant to the methylene carbon.

Twenty different R groups occur naturally which provides twenty different natural amino acids. Individually, amino acids play important roles in metabolism and signaling (especially as neurotransmitters). A key chemical property of every amino acids is that it has at least one acidic (carboxylic acid) and one basic (amide) functional group (some amino acids have more than one titratable group, if the R group exhibits acidic or basic properties). Side chains may also be protonated or deprotonated dependant upon the pKa of the ionizable group. Amino acids can be classified based on their side chains into acidic, basic, neutral (polar and non-polar). Since an amino acid has both an acid (carboxylic acid) and a base (amine) within the same molecule, at neutral (or near neutral) pH an ionic form called a Zwitter ion is the predominant species. Amino acids are ampholytes.
 

Each titratable group on an amino acid molecule has a characteristic pKa value. Acids and bases do not coexist in water in high concentrations simultaneously. Water acts as a proton transfer reagent to form the Zwitter ion (The most stable form at pH = 7) from the amino acid. The pH of an aqueous solution of amino acid will change the predominant form of the amino acid present. This is dependant upon the pKa's of the carboxylic acid, the amine and the side chain if acidic or basic moieties are present. All amino acids are polyprotic acids. That means that there is more than one site capable of protonation/deprotonation. The carboxylic acid and the amide functional group are both capable of acid/base behavior. Additional acid/base behavior may be observed in the side chains. By titrating an unknown amino acid sample, one can often identify the compound from the shape of the titration curve and the experimentally determined pKa values.Knowing the pKa of the amine and the carboxylic acid will allow you to gauge which amino acid species is present. The following relationships are helpful and can be derived from the Henderson Hasselbach equation.
 

*When pH=pKa, a 50/50 mixture of protonated and deprotonated forms exist.

*At pH 1 unit <pKa  an ~ 90/10 mixture of protonated/deprotonated exists.

*At pH 2 units <pKa  a > 99/1 mixture of protonated/deprotonated exists.
 

Thin Layer Chromatography
As each of you have completed general and organic chemistry, it is assumed that you are familiar with TLC. Thin-layer chromatography consists of a stationary phase immobilized on a glass or plastic plate, and an organic solvent. The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. The constituents of a sample can be identified by simultaneously running standards with the unknown. The bottom edge of the plate is placed in a solvent reservoir, and the solvent moves up the plate by capillary action. When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir. The different components in the mixture move up the plate at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. Please review the general principles, information on plate and developing chamber preparation and calculation of an Rf value.  We will be using filter paper as our stationary phase.
 

Amino Acid Detection
Since amino acids do not absorb light in the visible region they are not colored an cannot be seen with the "naked" human eye. In this case, like with many organic compounds, visualization reagents must be used to identify the compound. Amino acids react with a dye called ninhydrin to form a highly conjugated aromatic derivative which absorbs light in the visible portion of the spectrum. The ninhydrin amino acid derivative is purple and easily visible.

Ion Exchange Chromatography
Ion exchange chromatography separates substances on the basis of their charge. There are two general classes of ion exchange media or resins; anion-exchange media, which have positively charge groups attached to the media, and bind to anionic (negatively charged) compounds, and cation-exchange media, which have negatively charge groups attached to the media, and  bind to cationic compounds. Dowex-50 is a cation-exchange resin, i.e., it has covalently attached sulfonic acid groups which, at pH 3, are deprotonated and charged-balanced by associated sodium ions. Proteins with significant regions of opposing charge will bind to this column material by ionic attraction, displacing the sodium ions. In separation of amino acids, peptides or proteins, the initial analyte binding is usually done in a solution of low ionic strength  where the sample, can displace the sodium ions, and bind to the column material. In this experiment we will begin at low pH and proceed to higher pH to change the ionic state of the amino acids and thus their affinities for the resin.   In purification of a single protein or peptide from a mixture, a solution containing the desired analyte is passed through a column of ion exchange media, and the eluent checked to ensure that the desired component of mixture has bound to the column. The column is then washed with 2 - 5 column volumes of low ionic strength buffer (initial buffer) to remove any unbound material which has simply adhered (not bound) to the column. In our case we desire to separate all species in the mixture so we will collect the initial column washing as it may contain an analyte of interest.  After the washing point, the analytes bound to the column can be selectively released by increasing the salt concentration or changing the pH. In a salt gradient, the salt competes with the analytes for the charge groups on the column, and at a salt concentration characteristic for each analyte, the analyte is eluted. In this experiment, a mixture of amino acids are selectively eluted with three different buffers, 0.1M Na citrate, pH 3, 0.1 M Na citrate, pH6, and 0.1M CAPS pH 11. Competition of the analytes with Na+ ions will change as the pH, and thus the charge of the amino acid, changes.

1. Mixture Purification

Colum Preparation: Prepare a Dowex 50 column by loading the column approximately 2/3 full with the resin in buffer  (0.1 M citrate pH 3) and allowing the excess buffer to drain to just above the column head. A minimal volume of solvent should remain but do not allow the resin to run dry at any point!!!
Note - You should have about 10 ml of resin for the separation- your colum may be less  or more than 2/3 full depending on its diameter.

Sample Loading: Load approximately 0.5 mL of your sample mixture onto the column.

Elution: After the sample has eluted onto the column (do not allow the resin to run dry at any point!!!) add eluant I (0.1M citrate pH 3) in 5 ml increments to the column head. Collect 1 mL fractions, checking the fractions for the presence of amino acids using the ninhydrin reagent and chromatographic paper strips (do not allow the resin to run dry at any point!!!) .When amino acids have ceased to elute, change the buffer to eluant II (0.1 M citrate, pH 6) and repeat the elution as for eluant I. When eluant II fails to provide amino acids, change the buffer to eluant III (0.1M CAPS, pH 11). If the addition of two column volumes of any buffer fails to produce any amino acids, discontinue use of that buffer and proceed to the next buffer.
Note:  Just because you have added pH 6 buffer to the top of the column this does not mean that the entire column is immediately pH 6!  Think about it!

2. Establishing Standard Rfs-. Use gloves at all times your hands have amino acids in them.    Solvents will be provided.  Developing chambers (beakers) will be provided. Place about 1 cm of water saturated phenol in the bottem of a beaker.  in the center of the beaker place a 50 ml beaker with 20 ml of 0.3% ammoniumn hydroxide.  Let the chromatogram develope for appoximately 3 hours.   Visualization will be accomplished by spraying the chromatogram with ninhydrin and heating it gently with a hair dryer or placement in an oven.  (Spray and dry in the hood. Wear gloves. Ninhydin is toxic and your fingers contain amino acids!!!!!).  Run Standards and unknowns on the same sheet of paper

3. Identification:

Using the standards, identify the amino acids in your collected fractions.

Post laboratory Questions (answered in your notebook)
1. Why doesn't proline react with ninhydrin?
2. Why is it important to calculate an Rf value (as opposed to simple visual comparison)?
3. Given your data what other identifications could you have made? In other words, what is your second and third guess?

Data analysis/Reporting Results - see the syllabus for the report format. (your data analysis should also be in your notebook)

1. Make sure to include your unknown number and all Rf data for standards and unknowns.  Organize your data in a table; make sure to provide a figure caption and labeling
2. Identify the fraction number for each amino acid and the eluant buffer. Organize your data in a table; make sure to provide a figure caption and labeling
3. In your report make sure to discuss why your identified amino acids eluted using a particular buffer.
4. If any discrepencies are present explain to the best of your ability using data.