Lecture 3: Bioanalytical tools
- This is the most common method for separation of proteins from a mixture. This is so because first of all, the cost of matrix (stationary phase) used in this method is low. Second, it gives high resolution.
- Principle: Many biological molecules like proteins, amino acids etc. have charge (either positive or negative). This property of having charge can be exploited for separation purpose.
- Matrix preparation: the matrix contains stationary phase, which is an ion exchanger. This can be anion or cation exchanger. A cation exchanger binds to positively charged cations, therefore it has negative charge. It is also called as acidic ion exchanger. An anion exchanger binds to negatively charged anions and therefore has positive charge; also called basic ion exchanger.
- Choice of ion-exchanger: It totally depends on the charge of the sample protein to be separated. Charge depends on isoelectric point. You will get isoelectric point of all proteins on the internet.
Now, suppose, isoelectric point of a protein is not known, then it is calculated via isoelectric focusing (technique discussed later) or it is estimated from the sequence. How??
Qs. Decode the given protein sequence: “WATER”. What is the overall charge present on this protein.
- 0 (neutral)
Charge on Glu is -1; and charge on Arg is +1. Try, Ala and Thr is neutral, with charge 0, so, net charge on this protein sequence “WATER” is 0.
Check out the charge on WATERR (Ans is +1).
Types of ion exchangers
- Weak ion exchanger- ionized over a narrow pH range e.g. carboxylate, diethyl-ammonium.
- Strong ion exchanger – ionized in almost all pH e.g. sulphonate, quaternary ammonium.
Please note that an ion exchanger/matrix should be charged for ion exchange chromatography to occur. A charged matrix will be able to bind to the sample; and thus aid in separation. The choice of ion exchanger depends on the charge of analyte, which is further dependent on isoelectric point (as stated earlier).
What is Iso-electric point and which ion exchanger should be used when?
It is that pH at which net charge on a protein is 0. Analytes for ion-exchange chromatography may be stable below or above isoelectric point.
Above isoelectric point, OH– will be more. These OH– will bind to NH3+ of amino acid. So, net charge on the protein will be negative. So, above isoelectric point anion exchanger which exchanges anions should be used.
Let us take the case of enzyme pepsin. Its isoelectric point is 1.0. It means that net charge on pepsin at pH1 = 0. So, if it is kept in pH 2 (i.e. above 1), its charge will be negative; i.e. it will become an anion. As said before, anions are separated via anion exchanger. Now, if you have a mixture containing many enzymes, out of which one is pepsin, then you should load this mixture on an anion exchanger for separation of pepsin. Please note one pH above or below can be employed, therefore pH 2 is used. This small change in pH ensures that the sample component which we have to separate is charged, and binding has taken place.
Now, let us take the case of acid phosphatase, which has isoelectric point of 7.6. Above 7.6 it will have –ve charge and below 7.6 it will have +ve charge. You can load sample containing acid phosphatase at pH 8.6 on an anion exchanger and at pH 6.6 on a cation exchanger.
So, sample loading totally occurs on the basis of isoelectric point i.e. pI.
Sample volume: In isocratic elution, sample volume is generally 1-5% of bed volume. But in gradient elution, sample volume is not important at all. You can apply very dilute sample also.
Sample pH: pH of sample used should be either one pH below or one pH above the isoelectric point.
Elution in ion exchange chromatography
In ion-exchange chromatography, elution occurs on the basis of ionic strength and pH.
- Ionic strength = ½ ∑(concentrations of ions * charge ^2)
For e.g. ionic strength of 1M (NH4)2SO4 = (concentration of ammonium * square of charge on ammonium) + (concentration of sulphate * square of charge on sulphate)
= ½ [2*(+1)2 + 1*(-2)2] = ½ * (1+ 4) = ½ * 5 = 3M (Two molecules of ammonium are there, so, ammonium concentration becomes 2M)
Qs. Calculate the ionic strength of 1M NaCl.
Qs. Why a solution of higher ionic strength is employed for elution in ion-exchange chromatography?
For elution always a solution of higher ionic strength is employed. This is so because an eluent with higher ionic strength will bind more strongly to the matrix in comparison to an eluent with low ionic strength. When the eluent binds to the matrix, the sample/analytes becomes free/neutral and comes in the inter-molecular space.
Two types of elution are there:
A. Gradient elution– In this the composition of mobile phase is changed continuously. First, mobile phase of 50 mM ionic strength is used, then second time the ionic strength should be greater than 50mM, say 100mM, 150mM, 200 mM and so on.
It can be a continuous or discontinuous gradient. Continuous gradient is like the one given above, i.e., 50, 100, 150mM….. and so on (with a constant difference in ionic strength each time). Discontinuous gradient is when difference between every successive ionic strength is not the same, say 50, 80, 100mM… etc.
2. pH of eluent:
In case of anion exchanger, pH gradient decreases and ionic gradient increases. Take the e.g. of above enzyme mixture “X” containing pepsin given above. 50mM solution “X” is first of all applied to the bed at pH 2. For elution, 100mM buffer of pH 1 is applied. At pH1, pepsin will have charge 0 and will be separated from the matrix, and eluted out. 100mM Ionic strength further aids the process; because it allows binding of pH1 buffer more strongly.
For cation exchanger, pH and ionic gradient always increases. Here, eluent of higher pH will make bound sample neutral.
B. Isocratic elution: If composition of mobile phase remains constant, then it will be called isocratic elution. If is generally employed in case of gel filtration chromatography. In ion-exchange chromatography, gradient elution is preferred.
Qs. What benefit does low ionic strength of initial sample and eluent provide in ion-exchange chromatography?
Initially sample as well as eluent of low ionic strength is used because large numbers of unwanted charged particles are also present in the sample. These will also bind to the matrix. If you use a solution of low ionic strength, then minimum number of contaminants will bind.
In subsequent elution, solvents/buffers of successively higher ionic strength is used. By this time, greater part of the sample has already been separated out. Higher ionic strength now facilitates maximum number of contaminants to be left in the bed.