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Transcript
Titration curve of amino acids Science Background CHEMISTRY Acids and bases What does it mean for a solution to be acidic or basic? An acid is a substance that donates hydrogen ions. Because of this, when an acid is dissolved in water, the balance between hydrogen ions and hydroxide ions is shifted. Now there are more hydrogen ions than hydroxide ions in the solution. This kind of solution is acidic. A base is a substance that accepts hydrogen ions. When a base is dissolved in water, the balance between hydrogen ions and hydroxide ions shifts the opposite way. Because the base "soaks up" hydrogen ions, the result is a solution with more hydroxide ions than hydrogen ions. This kind of solution is basic (alkaline). An amphoteric substance is a substance that can both accept and donate hydrogen ions. It can react as both an acid and a base and will do so depending on the environment. The pH scale The pH scale was first used by the Danish biochemist Sören Sörensen in 1909 to create a more efficient notation for the enormous range of the H 3O+ concentrations confronted within his experiments. He defined pH as the negative logarithm (with base 10) of the hydronium ion concentration and in that way reduced an inconvenient number to a simple number. Henceforth we will write log instead of log10 𝑝𝐻 = −log([𝐻3 𝑂+ ]) [H3O+] is expressed in powers of 10: [𝐻3 𝑂+ ] = 10−𝑝𝐻 In neutral solution: [H3O+] = [OH-] = 10-7mol/L In acidic solution: [H3O+] In basic solution: [H3O+] < [OH-] > [OH-] pH = pOH =7. (pure water at 25°C) pH < 7. pH>7. Amino acids The name “amino acid” is derived from the general structure of molecules belonging to that group. They all contain an amino group (NH2) and an carboxylic acid group (COOH). Often, they are drawn as such. In reality, however, (solid) amino acids at relatively neutral pH values exist as zwitterions. This is a molecular ion with a net charge of zero. A clue for this can be found in the relatively high melting points of amino acids. These can not be explained using just Van der Waals forces and hydrogen bonds. It is the relatively strong ionic bonds between molecules that cause the higher melting points Titration curve of amino acids – Science background 1 The zwitterion is formed through an internal acid-base reaction. The amino group is a weak base and can deprotonate the carboxylic acid. The H+ (proton) shifts from the COOH group to the NH2 group, resulting in a negatively charged group (COO -) and a positively charged group (NH3+). This creates a molecular ion with a distinct positive and negative charge, though the molecule as a whole is still neutral. Most scientists use the zwitterion notation when discussing amino acids As a zwitterion, the amino acid can react as an acid and as a base. It is amphoteric. In alkaline (basic) environments, the NH3+ group will donate a proton, reverting to a NH2 group. This leaves the molecule with a net negative charge (on the COO - group). NH3+ reacts as a weak acid and has its own pKa value. In acid environments, the COO- group will accept a proton, reverting to a COOH group. This leaves the molecule with a net positive charge (on the NH3+ group). COO- reacts as a weak base and has its own pKb value. Table 1: pKa values for amino acids (via: http://www.cem.msu.edu/~cem252/sp97/ch24/ch24aa.html). pKb values can be deduced by assuming standard conditions where pKa + pKb = pKw pKz pKb pKz pKb R-groep COOH-groep COO--groep NH3+-groep NH2-groep Alanine 2.35 9.87 Arginine 2.01 9.04 12.48 Asparagine 2.02 8.80 Aspartic Acid 2.10 9.82 3.86 Cysteine 2.05 10.25 8.00 Glutamic Acid 2.10 9.47 4.07 Glutamine 2.17 9.13 Glycine 2.35 11.65 9.78 4.22 Histidine 1.77 9.18 6.10 Isoleucine 2.32 9.76 Leucine 2.33 9.74 Lysine 2.18 8.95 10.53 Methionine 2.28 9.21 Phenylalanine 2.58 9.24 Proline 2.00 10.60 Serine 2.21 9.15 Threonine 2.09 9.10 Tryptophan 2.38 9.39 Tyrosine 2.20 9.11 10.07 Valine 2.29 9.72 Amino Acid The acidic and basic groups that are present in the zwitterion (and every other form of the amino acid are weak acids and bases. Because of this, solution of amino acids have some limited buffering capabilities 2 CMA Learning and Teaching Resources Buffers A buffer is a mixture of a weak acid and its conjugated base (or vice versa). Such mixtures are capable of negating small changes in pH, thus keeping the pH of a solution more or less constant. During titrations of weak acids and bases, there is always a part of the curve in which the graph is almost horizontal. In those parts, some of the weak acid or base is converted to its conjugated partner, resulting in a mixture of both (a buffer). Adding more titrant has almost no effect on the pH. After adding more titrant, the ratio between the weak acid/base and its conjugated partner shift to favor one of them and the buffering capabilities of the mixture are lost. The curve increases or decreases significantly to reach an equivalence point in which all of the acid or base has been converted to the conjugated partner. Titration Titration is an analytical method in which a standard solution with the known concentration is used to determine the concentration of another solution. During titration the standard solution (also known as titrant) is slowly added to the solution of unknown concentration by means of a burette. The endpoint of the reaction can be observed by the colour change when using an indicator (for example phenolphthalein) or detected by pH measurement. At the end point an amount of standard solution has been added that just completely reacts with the solution titrated. The moles of standard solution can be calculated by multiplying the volume of standard solution used by its molarity. nstandard solution = Vstandard solution * cstandard solution The moles in the titrated solution of unknown concentration are then found using the coefficient in the chemical equation. Then, dividing the moles of the titrated solution by the volume of that solution gives the concentration of the titrated solution. ctitrated solution = ntitrated solution / Vtitrated solution. Step motor burette The step motor burette (titrator) is an actuator with which a liquid can be added to a solution automatically and relatively accurately. That way, a certain volume of a liquid can be added at constant speed, allowing for the creation of a graph in which you can plot pH versus volume added. This might also be achieved by using a drop counter and photo gate, but the step motor burette is much more convenient. More information about the function of the step motor burette, connecting it to an interface or tips about possible experiments can be found in the actuator’s manual. Titration curve of amino acids – Science background 3