Zwitterion and isoelectric point: Definition, Separation of amino acid, TLC.

 Zwitterion and isoelectric point

Hello everyone today will discuss the zwitterion of amino acids. We are already discussing what amino acids? What is the amino acid structure with an amino acids list?

Amino acids are given an Amino acid chart, which follows amino acid 20 total.

We are discussing a total of 20 amino acids code (Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asp, Glu, Lys, Arg, His, Asn, Glu, ), and amino acids example of essential amino acids are  Isoleucine, Leucine, Tryptophan, Lysine, Methionine, Histidine, Phenylalanine, Threonine, and Valine. 

Non-essential amino acid

The remaining 10 amino acids are non-essential amino acids. Which are made on the body naturally. Not requirement any supplement, non-essential amino acids are automatically generated in the human body    

Table of the contents

1. Definition of amino acid

2. What is the zwitterion definition?

3. How to zwitterion form?

4. Zwitterion of Lysine

5. Zwitterion structure of glycine

6. What is the zwitterion formation of Histidine?

7. Isoelectric point definition

8. What is the isoelectric point

9. How to calculate isoelectric point

10. Isoelectric point graph of the titration of the glycine and determination of the isoelectric point

11. How to determine the isoelectric point of the solution

12.  Arrange the isoelectric point of the amino acid: Val, Ary, Asp

13. Separation of amino acid

14. Separation of the amino acids gel chromatography

15. Separation of amino acids using paper chromatography and Thin Layer chromatography.

16. Ion exchange chromatography

17. Frequently asked question

18. How do you calculate the isoelectric pH of a protein?

19. Conclusion

Definition of Amino acid

We are already discussing the definition of amino acids is. Amino acids contain one amino group and one acidic group, and another one group( R) variable, which is attached with alpha carbon. This R group variation made 20 amino acids. That is the Amino acid definition. 

What is the Zwitterion definition?

Now today we will discuss what is zwitterion and isoelectric points. First of all, we are discussing the zwitterion of amino acids. We know that amino acids are molecules that have two parts: one is the amino (NH3+) part, another is the acidic part( COO-). They exist +ve charge and -ve charge respectively.

Clearly, We can see that one same molecule has two different types of charge present. These types of molecules are called a zwitterion. A zwitterion is highly soluble in water. 

Zwitterion salts are highly soluble due to the Dipole moment of the zwitterion molecule amino acid.

How to zwitterion form?

Zwitterion of lysine

First of all understanding zwitterion formation of amino acids we see the one example of amino acid to zwitterion formation.

In this case, zwitterion lysine present amino group (-NH2) is electron pulling (-I effect) due to Nitrogen atom. Nitrogen atoms have electronegativity effects and lone pairs. Where carbonyl group has a -I effect, which causes the carbonyl group to have H+ easily released, and the NH2 group which easily accepts it. That means nitrogen lone pairs easily donate H+ ions.

That's why the nitrogen group is on the longer chain side, which is more More electron density, so more Easily electrons are shared with H + ions. 

 

Zwitterion structure of glycine

Similarly, In the zwitterion chemistry formation of a neutral potion of the glycine amino acid. In glycine, the same group is present that is why we see Gly as an achiral compound or one of the achiral amino acids. 

First of all, we can see that in the presence of water glycine is a stable molecule, this glycine is an aliphatic amino acid. 

Now,

What is the zwitterion formation of Histidine?

In case Histidine has an imidazole ring, in-ring nitrogen does not participate in zwitterion formation. Because Ring nitrogen is sp2 hybridized, less electron density compared to another chain amino group nitrogen. On the other hand chain, amino acids have sp3 hybridized.

We know that the order of the basic character is sp3 > sp2 > sp Hybridization, and that the main chain amino group nitrogen has a more basic character. Chain amino groups easily donate the lone pair electron compared to another ring nitrogen lone pair.  

Isoelectric point definition

The isoelectric point is at a point zwitterion charge Neutral, no charge is formed. At the condition of solution pH value is increased H+ < OH-, solution basicity is increased PI < pH.  Where PI is known as an isoelectric point. In the solution Neutral zwitterion amino acid -ve Charge part (-COO-) movement toward Anode( +ve charge) plate, and Neutralization of negative charge by positive charge cathode. That is why the overall solution of the charge is positive.   

Similarly

On the other hand, at the condition of the solution pH value is increased, H+ > OH-, solution acidity is increased PI > pH. In case the isoelectric point value is greater than the pH value. In this solution, zwitterion movement toward the -ve charged cathode. That mains zwitterion +ve part ( NH3+)  movement toward -ve charged cathode. 

What is the isoelectric point?

The pH at which the molecule is electrically neutral is called isoelectric point(PI).

• It is the pH at which no net charge forms.

• It is the pH at which the condition of amino acids exactly balances the amount of negative charge and possessive charge.

How to calculate isoelectric point

A conjugate acid released the H+ iron and formation of dipole iron. We have written that k1 is equal to dipolar ion concentration into H+  ion concentration divided by the concentration of conjugate acid. Where k1 is the equilibrium constant.

Similarly, we have to write that a dipolar ion is converted into a conjugate base and relies on the proton. Now, k2 is equal to the concentration of the conjugate base into the concentration of H+ ion divided by the concentration of dipolar ions. Where k2 is the equilibrium constant.

 

At the isoelectric point concentration of conjugate acid is equal to the concentration of conjugate base.

In isoelectric point, electrophoresis steps determine the pH value of the equilibrium of the solution. Which is the one type of isoelectric point in the solution of the amino acid. 

Isoelectric point graph of the titration of the glycine and determination of the isoelectric point

Titration of amino acid

In this curve, we can see that at isoelectric point 5.97, the solution pH is 7. For any Pka value change corresponding to their isoelectric point is variable. When the pka value is increased corresponding isoelectric point is increased. When pka values decrease than their isoelectric point decreases. 

Now, 

How to determine the isoelectric point of the solution

The summation of all Pka values divided by 2 is equal to the isoelectric point of the solution. 

 

Q1. Arrange the isoelectric point of the amino acid: Val, Arg, Asp

Ans: The increasing order of isoelectric points of the given amino acid is Arg>Val>Asp. The Isoelectric point of the Acidic amino acids is less compared to neutral and basic amino acids. And the isoelectric point of the basic amino acids is greater than neutral and acidic amino acids. Now in case, Valine is a neutral amino acid, Arg is a basic amino acid and Aspartic acid is an acidic amino acid. Clearly, we can see that the isoelectric point of the Arg is much higher than Valine and Aspartic acid. And Aspartic acid is much more acidic compared to another, therefore the isoelectric point of the Asp is much less compared to Val and Arg.

Separation of amino acid

We follow a different path for the separation of amino acids. This process is very useful for peptide bond breaking and the separation of amino acids.

1. Gel electrophoresis method 

2. Ion exchange chromatography

3. Paper chromatography

4. Thin-layer chromatography(TLC)

Separation of the amino acids gel chromatography

In-gel chromatography using electric current separated the mixture of the amino acids. In this method, positive charge amino acids are moved towards the negative electrode and on the other hand, the negative charge carries amino acids is moved towards the positive electrode. In this case positive amino acid mains, amino acids carry a positive zwitterion form. 

Using this method mixture of the amino acids, all carry different types of charge therefore separation of amino acids using electrical electrodes. 

In this electrophoresis method, the medium of the cell is agoras gel. 

Separation of amino acids using paper chromatography and Thin layer chromatography

  

Ion exchange chromatography

Mainly ion-exchange chromatography has two types one is cation exchange chromatography and another is anion exchange chromatography. This method uses resin, which has a positive charge or negative charge. They bond with the amino acid side chain and block the whole amino acids, then they are separated by column chromatography. Amino acid mixtures separated by the side chain group have a positive charge in case the resin negative side attacks on this amino acid positive side chain. And block which amino acid is retained by column. The relatively non-polar amino acid first eluted then polar amino acid eluted sequentially.   

Frequently asked question

Q1. How do you calculate the isoelectric pH of a protein?

Proteins are made with amino acid-binding so we can easily find the isoelectric point of the protein. First of all, check what type of amino acids are bound with protein, then we can easily find the protein terminal group. Then the terminal group pka value is found in the given table chart of the amino acids. Greater pka value summation divided by 2 is equal to the isoelectric pH of the protein.

Conclusion

Today we are discussing what is the zwitterion of amino acids and the isoelectric point of the amino acids. We are hoping for this type of chem_article. These amino acids are from the bioorganic part of organic chemistry. We are referenced in the organic book, Clayden, Greenies, Warren, and Wothers Organic Chemistry.