Isoelectric Point A Level Chemistry Ocr

[Breanna Mangels]

2Apply an electric current. When an electric current is applied across the gel, the charged amino acids in the sample will migrate through the gel towards the opposite charged cathode.

The R group of an amino acid can also accept or donate electrons depending on the functional groups in it. As a result, different amino acids have different isoelectric points.

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No! Amino acids are acids. They are also bases containing an amino group. The term amphoteric is often used to describe amino acids, meaning that they are capable of acting as both acids and bases.

If the pH is decreased to a low enough value (e.g. pH 1) then the carboxylate salt will be protonated to give the neutral carboxylic acid, and the molecule will have a net charge of +1.

For a typical amino acid, there will be a range of pH values where the positively charged form dominates, another where the neutral form dominates, and finally one where the negatively charged one dominates.

If only there were some formula we could use for figuring out the pH of points A and B on the graph above, where the acid and its conjugate base are present in equal concentration.

There is more to isoelectric point than just the calculation of pI values for individual amino acids! The same concepts also apply to peptides and proteins, each of which will have a pI value that is influenced by the characteristics of its side chains.

Suppose you put a single crystal of potassium manganate(VII) (potassium permanganate) onto some damp filter paper supported on a microscope slide. The crystal would dissolve in the water in the filter paper, and the deep purple colour would diffuse out to make a small circle around the original crystal.

Now let's modify this by connecting the filter paper into a simple electric circuit. This time, when you put the potassium manganate(VII) crystal onto the paper, the colour doesn't spread into a circle. Instead, the purple colour starts to move towards the positively charged crocodile clip.

There isn't anything very surprising about this. The colour of the potassium manganate(VII) is due to the manganate(VII) ions present. These are negatively charged, MnO4-, and move towards the positive electrode.

This movement of ions in an electric field is the basis of a separation technique known as electrophoresis. Let's apply this to the more interesting (and more complicated) case of amino acids.

Little troughs are made in the gel to hold the solutions being tested. In an exam, you may find that you have to interpret diagrams based on bits of paper or slabs of gel. It makes no difference whatsoever to what you would need to say.

At a particular pH, known as the isoelectric point, this is how the amino acid exists in solution. The amino acid won't move during electrophoresis, because the two charges cancel each other out, and there won't be any attraction either to the positive or the negative electrode.

Note: For many simple amino acids, the isoelectric point is at a pH of about 6. I have, however, come across the odd exam question which gives a diagram a bit like the one above, but describes the solution as being neutral. This is a bit careless! In fact, an amino acid with an isoelectric point of 6 would in fact move slightly towards the positive electrode at pH 7.

The ions have to find their way through the fibres in the paper or the pores in the gel. Smaller ions will travel faster than bigger ones. So, for example, the smaller aspartic acid will travel faster than the larger glutamic acid.

What do I mean by "smaller" or "larger"? This could be in terms of the masses of the ions, or their shapes. Ions with bulky groups (such as benzene or other rings) will travel more slowly than ones with, say, unbranched chains.

All of them will move to the negative electrode, but they will move at different rates because, for example, one of them carries 2+ charges whereas the others carry only 1+. And obviously they will be different sizes as well - both in mass and shape.

All of them will move to the positive electrode, but they will move at different rates because, for example, one of them carries 2- charges whereas the others carry only 1-. And obviously they will be different sizes as well - both in mass and shape.

The sodium dodecyl sulfate is a constituent of many detergents and cleaning products. It is also known as sodium lauryl sulfate. If a protein is treated with SDS and then heated, the protein is denatured.

That means that its secondary and tertiary structures are lost. It becomes covered in SDS molecules, and this turns the protein molecule into a long tube covered with negative charges on the outside. The negative charges come from the SDS molecules.

If these treated protein molecules undergo electrophoresis, all the molecules will move towards the positive electrode, but the smaller molecules will move through the gel faster than the bigger ones.

Obviously, protein molecules have to be stained in some way in order to find out how far they have travelled. The intensity of the colour is a measure of how much of any particular protein is in the mixture.

This technique is used to detect the presence or absence of proteins which might be an indicator of disease. For example, kidney disease can be recognised by the unusual presence of proteins in urine. Lots of diseases including liver disease and some cancers produce abnormal levels of some proteins in the blood.

Since amino acids, as well as peptides and proteins, incorporate both acidic and basic functional groups, the predominant molecular species present in an aqueous solution will depend on the pH of the solution. In order to determine the nature of the molecular and ionic species that are present in aqueous solutions at different pH's, we make use of the Henderson-Hasselbalch Equation, written below. Here, the pKa represents the acidity of a specific conjugate acid function (HA). When the pH of the solution equals pKa, the concentrations of HA and A(-) must be equal (log 1 = 0).

The titration curve for alanine in Figure \(\PageIndex2\) demonstrates this relationship. At a pH lower than 2, both the carboxylate and amine functions are protonated, so the alanine molecule has a net positive charge. At a pH greater than 10, the amine exists as a neutral base and the carboxyl as its conjugate base, so the alanine molecule has a net negative charge. At intermediate pH's the zwitterion concentration increases, and at a characteristic pH, called the isoelectric point (pI), the negatively and positively charged molecular species are present in equal concentration. This behavior is general for simple (difunctional) amino acids. Starting from a fully protonated state, the pKa's of the acidic functions range from 1.8 to 2.4 for -CO2H, and 8.8 to 9.7 for -NH3(+). The isoelectric points range from 5.5 to 6.2. Titration curves show the neutralization of these acids by added base, and the change in pH during the titration.

The distribution of charged species in a sample can be shown experimentally by observing the movement of solute molecules in an electric field, using the technique of electrophoresis (Figure \(\PageIndex2\)). For such experiments an ionic buffer solution is incorporated in a solid matrix layer, composed of paper or a crosslinked gelatin-like substance. A small amount of the amino acid, peptide or protein sample is placed near the center of the matrix strip and an electric potential is applied at the ends of the strip, as shown in the following diagram. The solid structure of the matrix retards the diffusion of the solute molecules, which will remain where they are inserted, unless acted upon by the electrostatic potential.

At pH 6.00 alanine and isoleucine exist on average as neutral zwitterionic molecules, and are not influenced by the electric field. Arginine is a basic amino acid. Both base functions exist as "onium" conjugate acids in the pH 6.00 matrix. The solute molecules of arginine therefore carry an excess positive charge, and they move toward the cathode. The two carboxyl functions in aspartic acid are both ionized at pH 6.00, and the negatively charged solute molecules move toward the anode in the electric field. Structures for all these species are shown to the right of the display.

It should be clear that the result of this experiment is critically dependent on the pH of the matrix buffer. If we were to repeat the electrophoresis of these compounds at a pH of 3.80, the aspartic acid would remain at its point of origin, and the other amino acids would move toward the cathode. Ignoring differences in molecular size and shape, the arginine would move twice as fast as the alanine and isoleucine because its solute molecules on average would carry a double positive charge.

Some amino acids have additional acidic or basic functions in their side chains. These compounds are listed in Table \(\PageIndex1\). A third pKa, representing the acidity or basicity of the extra function, is listed in the fourth column of the table. The pI's of these amino acids (last column) are often very different from those noted above for the simpler members. As expected, such compounds display three inflection points in their titration curves, illustrated by the titrations of arginine and aspartic acid (Figure\ (\PageIndex3\)). For each of these compounds four possible charged species are possible, one of which has no overall charge. Formulas for these species are written to the right of the titration curves, together with the pH at which each is expected to predominate. The very high pH required to remove the last acidic proton from arginine reflects the exceptionally high basicity of the guanidine moiety at the end of the side chain.

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