Lecture 2: Bioanalytical tools
- It is also called as molecular sieve chromatography or sieve exclusion chromatography.
- A column made up of porous beads is taken. Beads can be of agarose, dextran, sephadex, polyacrylamide etc. All these beads have a common property that they are porous in nature.
- These beads are available in various pore sizes; and the pore size of beads is selected based on the sample elements before the start of the experiment. Let us take a sample which has 5 components A, B, C, D and E of sizes 2, 4, 6, 8, and 10 µm. So, you can prepare the column using beads of size 2, 4, 6, and 8 µm.
- For the experiment, first of all, beads are added to the column. Then buffer is added to make a gel. This is known as column preparation and components of the column are known as stationary phase.
- After this, sample having A, B, C, D, and E is applied on top of the column.
- The sample components (can be proteins, amino acids, pigments, lipids etc.) are absorbed in the beads. Some components (whose size is larger than the beads, in this case, E) remain outside the beads, while others (A, B, C, and D) are absorbed by the beads. This is known as sample application.
- Sample distribution: It is important to understand that component, “A”, which is the smallest component, will enter all the beads whose pore size is larger than its size. On the other hand, component “B” whose size is 4 µm will move into beads of 4, 6 and 8 µm diameters, but will be excluded from the beads of 2 µm pore size. Similarly, component “C” will be excluded from beads of 2 and 4 µm and will enter on the beads of 6 and 8 µm. Component “D” will enter only into the beads of pore size 8 µm.
- Elution: To separate the sample components further, buffer is applied on top of the sample. This buffer is known as mobile phase. It pushes the sample components downwards, so that they can come out of the column.
- Please note that the largest components, i.e. E, will travel through the intercellular spaces of the gel, and will reach the end of the column first (Kd =0; because it has 0 concentration in stationary phase).
- On the other hand, the smaller components A, B, C, and D will be held within the porous beads of the column and will come out later on. Among all these, component “A” will travel through almost all the beads of the gel; therefore, it will be eluted out at the last (Kd = 1; because its concentration in stationary phase is highest). On the contrary, component “D” will travel only through beads of 6 µm diameter and will come out immediately after E.
Similar reasoning can be given for B and C.
- Larger molecule will be eluted first, followed by progressively decreasing sized molecules.
Importance of gel filtration chromatography
- This technique has been used to separate components of a mixture on the basis of size.
- Mainly, it has been used to determine molecular weight of unknown proteins. Known proteins of standard size are applied to the column first. Then their elution time (the time taken by them to come out of the column) is calculated.
A plot is constructed taking log (Mr) on the X-axis and elution time on Y-axis. A straight line graph will be obtained. From this, molecular weight of unknown sample can be calculated easily. Similar plot can be obtained using elution volume.
A mixture is having 5 components A, B, C, D and E of size 2, 4, 6, 8 and 10 µm. This mixture is applied to a column having beads of size, 2, 4, 6, and 8 µm. After this, mobile phase is applied. The value of Partition coefficients (Kd) of sample components in stationary and mobile phase will be in the ratio:
- a) E>D>C>B>A
- b) A>B>C>D>E
- c) D>C>B>A>E
- d) A>D>B>C>E
Correct Answer is b.
Concept of distribution coefficient in chromatography
Distribution coefficient = (concentration of component in stationary phase)/ (concentration of component in mobile phase).
It is represented by Kd (also called as partition coefficient).
E, will travel through the inter-cellular spaces of the gel, and will not be able to enter the gel. Kd =0; because it has 0 concentration in stationary phase.
On the contrary, “A” will travel through almost all the beads of the gel; therefore, it will be eluted out at the last. Kd = 1; because its concentration in stationary phase is highest.
Kd of B, C, and D will be in between 0 and 1. Kd of B, will be higher than C, because its concentration in gel will be higher than C and D. Same is the case for C and D.
All samples which are separated through chromatography have different components. Partition coefficient of sample components in a mixture is the basis of all chromatography experiments.
In all column chromatography, the component of the sample mixture having lowest Kd values will be eluted out first (because their concentration in stationary phase is lowest) and the component having highest Kd value will be eluted out later on (as explained above).
In paper chromatography, the component of the sample mixture which has low Kd will be distributed in stationary phase less. Such component will travel less and will have low Rf value. On the contrary, it is vice versa for sample component which has high Kd value.