Gel filtration

Student’s Name
Professor’s Name
Course
Date
The Goals of the Experiment
The experiment aims to separate a mixture containing blue dextran, DNP-serine, and horse, skeletal muscle myoglobin, using gel filtration chromatography in which the molecules are separated based on their sizes. The other purpose of the experiment is to determine the relative molecular weight of DNP-serine.
Principle of Test (Gel Filtration Chromatography)
The gel filtration chromatography that is also known as the size-exclusion chromatography seeks to separate the molecules based on their sizes. The stationary phase is made of the porous beads that have a well-defined pore size. The stationary phase has a fractionating range meaning that the molecules can be separated by different molecular ranges depending on their sizes. This experiment utilizes protein molecular mass ranges of between 1000 and 50000 as such 6-75 Sephadex is also applied. The molecules that are small in size can fit into the porous beads and can be said to be included. On the other hand, the more important molecules cannot fit into the dots and are excluded. The small molecules have access to the mobile phase and are eluted last.
Equations
The essential equations that form the basis of the experiment are:
Vt=Vo+ViWhere Vt is the total volume of the liquid in the column. Vo is the volume of the column void while Vi is the volume inside the gel. The equation is possible because the buffer in the gel filtration is seen as the partitioning outside and inside the gels.

To get the elution volume (Ve) the following equation is used where Kd is the partition coefficient. Elution volume is the volume between the injection of the solvent and its elution.
Ve=V0+(Kd×Vi)To obtain Kd, the above equation can be rearranged to give:
Kd=Ve-VoViSince Vt=Vo+Vi, Vi = Vt -Vo
Therefore Kd can be expressed as:
Kd=Ve-VoVt-VoInstrumentation
The instrumentation is made up of a set up that comprises of a gel filtration column, 150ml Erlenmeyer flask, stands, and beaker. The column acts to separate the mixture while the 150ml Erlenmeyer flask is a buffer reservoir. The flow of the buffer into the column is controlled using a paper clip. A spectrophotometer is used to measure the absorbance of the samples.