3. Gel Permeation Chromatography

This type of chromatography is also called as "Molecular Sieve Chromatography" or "Molecular Exclusion Chromatography". When a gel having pores on its matrix is packed in the column and a heterogeneous mixture is pass through it, smaller ions enter the gel and come out of the column phase. The bigger molecules can not enter the gel, they move only with the mobile phase through the inner phases of the gel. In this process, a type of sieving of molecules occur.

Widely used gels are Agarose, Porous Glass, Sephadex, Polyacrylamide, Polystyrene and Silica Granules.

Gel Permeation chromatography can be carried out using the technique of column chromatography. (By determining the distribution coefficient, molecular weight of macro molecules can be determined).

Image Source: American laboratory.

After running a chromatogram, a fraction of macro molecules is collected and volume is known. From this value, distribution coefficient can be calculated.

The distribution coefficient (Kd) is referred to as the the distribution of solute particles between inner and outer solvent in a gel. The value of individual fraction (Ve) depends on the volume of outer solvent (Vo) and volume of inner solvent (Vi).

Ve = Vo+Kd(Vi)

Vi = (a)(Wr) [a = dry weight of the gel and Wr= water regain value.]

Ve = Vo + (Kd x aWr)

Distribution Coefficient (Kd) = (Ve - Vo) / (aWr).

To know the molecular weight of the fractioned compounds, a standard graph with Kd of standards plotted against their molecular weight.

Next Post: Ion-Exchange Chromatography

Comments

Statistics in Flow Cytometry Data and "MFI" values

The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity.. Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values. (Keep...

Nanodrop

Thermofisher, Nanodrop 1000 Nanodrop is a spectrophotometer which is designed to measure the concentration of nucleic acids at 260nm and Proteins at 280nm present in a very small minute volume. It is also capable of measuring the concentrations of dsDNA, RNA, ssRNA and purified protein. Required sample size is usually 1µL. Nanodrop instruments are usually full spectrum spectrophotometer, i.e., Ultra violet and visible range wavelengths (250nm to 750nm). Nanodrop is commonly used for the quantification of the DNA concentration and RNA concentration in a given sample. However there are many other applications which can be performed on this instrument. It works on the principle of Spectrophotometer or Sample Retention System.... Principle of Nanodrop: "The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of colum...

Protocol: Sample preparation for Cell Cycle Analysis by Flow Cytometry

Protocol: Preparation of Samples for Cell Cycle Analysis by Flow Cytometry: 1) Suspend approximately million cells (10^6) in 1mL of phosphate-buffered saline solution (1x). And with the help of pipette, gently aspirate about 7-8 times, to make the single cell suspension. 2) Take a centrifuge tube and add 4mL of 70% ethanol. (70% ethanol should be one ice before using). and add 1mL of cells in PBS to this tube. Keep the tubes on ice for minimum 2 hours at 4℃.. This step will fix the cells. ( cells can kept at 4 ℃ for a week). 3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet. 4) Add 5mL of cold 1x PBS to this pellet and suspend the cells using pipette. 5) Centrifuge the tubes at 300g for 5 minutes. and discard the supernatant. Again do not disturb the pellet. 6) if required, step 5 can be repeated for one more time. 7) Add 1mL of Propidium Iodide (PI...

BIOMAG

Search This Blog