Separation of Colored Molecules Based on their Molecular - Lab Report Example

 SEPARATION OF COLORED MOLECULES BASED ON THEIR MOLECULAR WEIGHT Name of Student Institution affiliation Abstract The experiment was performed to use a chromatographic technique referred to as gel filtration chromatography to separate a mixture of blue dextran, hemoglobin and cytochrome c into individual components. Gel filtration column, 0.1 M phosphate buffer, gel, centrifuge tubes, measuring cylinder and pipette were used in the experiment. A specific procedure was followed with care and the mixture was able to be separated. From the results of the experiment, void volume, elution volumes of the components of the mixture and the molecular weights of hemoglobin and cytochrome c were determined. Introduction Chromatography is the method used in separating and analyzing complex mixtures. The separation process consists of a moving phase and a stationary phase. The mixture that is to be isolated is in the moving phase and it is made to pass through the immobile phase. Gel filtration is a procedure by which molecules are removed from a mixture based on their molecular shape and size. Stationary and moving phases in chromatography are determined by the chemical properties of the molecules to be separated. Apart from gel filtration chromatography other examples include: 1) Affinity chromatography-used in separating molecules that have an affinity for a particular macromolecule that is bonded on a solid support and a mobile phase. 2) Adsorption chromatography-used in separating low molecular weight molecules based on polar/non polar properties of the phases. 3) Ion exchange chromatography-used to separate charged molecules between a stationary resin and a liquid. In gel filtration chromatography, the stationary phase consists of beads that have pores which span a narrow range that determines the size of the macromolecules to be fractioned. Large molecules that are large to enter the pores of the beads are separated thus comes out of the column first. Examples of gels used in gel filtration are Dextran, Polyacrylamide and Agarose. When the column of Sephadex gel particles are packed: the volume of the solvent in the beads makes the internal volume (Vi) and that outside makes the external volume (Ve). The volume of the polymer forms the gel matrix volume (Vg). Bed volume is given by: Vt = Vg + Vi + Ve The elution volume of a molecular substance is the volume of solvent that is needed to move out of that substance. Its least amount is the void volume. Small molecules has higher volume available them than the void volume. Vl = Ve + Kd. Vi Where Kd is the coefficient of distribution and molecules have Kd between 0 and 1.The gel filtration column is divided into proteins of known molecular weight and then the Kd of each protein is calculated. The graph of Kd against log10 molecular weight is plotted and this graph allows the molecular weight of the unknown protein to be read from it. Procedure A mixture of haemoglobin, blue dextran and cytochrome was provided. One was required to have a 10 ml measuring cylinder and 20 centrifuge tubes that were numbered and calibrated to read 1.0 ml volume. The height of the column outlet and the reservoir were adjusted to give a Slow Flow Rate of about 0.5 ml per min. The buffer on top of the column was allowed to drain up to the level of the gel until the meniscus just disappeared. The buffer level was maintained throughout the procedure and the liquid that was drained of discarded. 0.3 ml - 0.5 ml of the mixture was pipetted onto the top of the gel while care was taken not to disturb the gel bellow. This solution was allowed to run onto the gel Using a disposable pipette, the top of the gel was washed with a few drops of the buffer to cater for spreading of the bands by back diffusion into the elution buffer when added. Collection of eluant into the 10 ml measuring cylinder was started at that point. Washing was repeated until the colored band just moved from the top of the gel bed. Very carefully, the column above the gel bed was filled with phosphate buffer so as not to disturb the surface of the gel. The running of the column was continued at a flow rate of about 1 drop per 4s. After the collection of the first 10 ml eluate in the measuring cylinder, 1.0 ml volumes were collected in the centrifuge tubes until visible color emerged from the column. The color and the intensity of each fraction was recorded using a ‘+’ scale for the intensity as shown in the table. Using the buffer, the 1.0 ml fractions and 1.0 ml eluate were diluted to a 4 ml volume. These diluted fractions were read on the Spectronic 20 spectrometer at 540nm and 650 nm while comparing with a dilute blank. Results Elution volumes: Blue dextran = 10 ml Haemoglobin = 11 ml Cytochrome c. = 12 ml Given blue dextran molecules are large and they cannot be excluded from the gel particles thus Elution volume of blue dextran = Void volume of the column. Void volume of the column is 10 ml. Total Volume Vt = (22/7*0.82 *2.1) cm3 Vt = 4.22 ml Internal volume = aWr and a = Vt/bed volume. Wr is the water regain (ml/g). a = (4.22/17.5) grams a= 0.24 grams and Wr = 10 ml/g Thus Vi = (0.24*10) ml Vi = 2.4 ml Kd = (Ve-Vo)/Vi Thus Kd for Haemoglobin = (11-10)/2.4 Kd for haemoglobin = 0.4 Kd for cytochrome c = (12-10)/2.4 Kd for cytochrome c = 0.8 From the calibration curve of log10 molecular weight against Kd, y = -1.8499x + 5.0876. Distribution coefficient corresponds to x and Log MWt corresponds to y thus; Log MWt = (-1.8499*0.4) + 5.0876 for hemoglobin and Log MWt = (-1.8499*0.8) + 5.0876 for cytochrome c. Log MWt for haemoglobin = 4.35 Log MWt for cytochrome c = 3.61 Estimated molecular weights Theoretical molecular weights Haemoglobin = 22.4 kDa Haemoglobin = 64.5 kDa Cytochrome c = 4.07 kDa Cytochrome c = 12 kDa Discussion The aim of the experiment was accomplished as the mixture of haemoglobin, cytochrome c and blue dextran was able to be separated. Also the molecular weights of cytochrome c and haemoglobin we determined experimentally and there values compared to the theoretical values. Blue dextran remained in the column as it has large molecules thus it could not be eluted. From this observation, the external volume of the gel filtration column was determined. The experimental values of molecular weights of haemoglobin and cytochrome varied from the values given in literature to errors that might have occurred during the performance of the procedure. Examples of the errors that might have occurred include poor measuring of the volumes, low flow rates to presence of an air gap in the column and the contamination of the apparatus accidentally. Bibliography Ucl.ac.uk,. 'Untitled Document'. N.p., 2015. Web. 3 May 2015. Mori, Sadao, and Howard G Barth. Size Exclusion Chromatography. Berlin: Springer, 1999. Print Striegel, Andre, Joseph J. Kirkland, and Donald D. Bly. Modern Size-Exclusion Liquid Chromatography: Practice of Gel Permeation And Gel Filtration Chromatography. 2nd ed. 2009. Print. Fischer, L. Gel Filtration Chromatography. Amsterdam: Elsevier/North-Holland Biomedical Press, 1980. Print. Walls, Dermot, and Sinéad T Loughran. Protein Chromatography. New York, N.Y.: Humana Press, 2011. Print. Work, T. S, E Work, and R. H Burdon. Laboratory Techniques In Biochemistry And Molecular Biology. Amsterdam, North-Holland Publishing Company: N.p., 1970. Print. Read More