Lab Report on the Separation of Blue Dextran and Cobalt Chloride on Sephadex G25
Lab Report on the Separation of Blue Dextran and Cobalt Chloride on Sephadex G25
Name of Student
Date of Submission
Lab Report on the Separation of Blue Dextran and Cobalt Chloride on Sephadex G25
Results
The radius, height of bed and the bed volume of the column
The formula for the volume of a right cylinder is 3.142*r*r*h
Where r is the radius of the column and h is the height which are both expressed in cm
For the Econo column which is clamped in an upright position the radius is the diameter divided by 2
Which is 0.7 /2 = 0.35
The height is 20 cm
Therefore, the volume is 3.142 * 0.35*0.35*20 = 7.6979 cm3
2. The plots for the elution profiles is as shown below
The graph below shows the elution profile of blue dextran. The absorbance versus fraction number using the wavelength data is the first loading
Sample ID Description 619.6
512.9
Sample1920 Blue Dextran Fraction 1 0.0361 0.0356
Sample1921 Fraction 2 0.0121 0.0125
Sample1922 Fraction 3 0.1563 0.0389
Sample1923 Fraction 4 1.1281 0.2408
Sample1924 Fraction 5 0.2651 0.0570
Sample1925 Fraction 6 0.0416 0.0096
Sample1926 Fraction 7 0.0121 0.0051
The Graph above is a graph of absorbance of blue dextran fraction against the fraction number
619.6 512.9
Sample1927 Cobalt Chloride Fraction 1 0.0010 0.0008
Sample1928 Fraction 1 0.0013 0.0012
Sample1929 Fraction 2 0.0010 0.0008
Sample1930 Fraction 3 0.0029 0.0026
Sample1931 Fraction 4 0.0022 0.0038
Sample1932 Fraction 5 0.0095 0.1363
Sample1933 Fraction 6 0.0282 0.4087
Sample1934 Fraction 7 0.0155 0.2063
Sample1935 Fraction 8 0.0024 0.0206
Sample1936 Fraction 9 0.0014 0.0025
Sample1937 Fraction 10 0.0019 0.0015
The graph above is a plot of absorbance against the fraction number.
The graph for absorbance versus fraction number for the mixture
Sample ID Description 619.6 512.9
Sample1938 Mixture Fraction 1 -0.0004 -0.0006
Sample1939 Fraction 2 -0.0004 -0.0006
Sample1940 Fraction 3 -0.0004 -0.0002
Sample1941 Fraction 4 0.1345 0.0290
Sample1942 Fraction 5 0.2255 0.0487
Sample1943 Fraction 6 0.0312 0.0446
Sample1944 Fraction 7 0.0447 0.5608
Sample1945 Fraction 8 0.0574 0.8250
Sample1946 Fraction 9 0.0230 0.3293
Sample1947 Fraction 10 0.0022 0.0257
Sample1948 Fraction 11 0.0007 0.0039
3. The elution volume for Blue dextran
Ve = Vo + KdVi
Where Ve is the elution volume
Vo is the volume external to the medium
Vi is the volume included within the gel
Ve = 0.25 + 1(.5)
Ve = 0.25 + 1.5
Ve = 1.75ml
The elution volume for cobalt chloride
Ve = Vo + KdVi
Ve = 1 + 1(1.5)
Ve = 1 + 1.5
Ve = 2.5ml
4. For blue dextran the mass is `0.0042g
0.4moles/1 * 1 mole * 1L/1000ml = 0.004 moles
Cobalt Chloride = 237.93g
0.004 moles * 237.93g = 0.952g (Expected) Cobalt Chloride
Actual Cobalt Chloride is 0.9683g
The Cobalt peak maxima is (512.85nm, 1.5373A)
The Blue dextran maxima is (619.6nm, 0.32954A)
Discussion
The results appear not to be consistent despite the fact that there may be some errors. This may be because the samples stick to the column. The inconsistency is due to errors in calibration. The gel filtration columns should be calibrated regularly as the elution volumes change with time. Since the lab experiment occurred under limited timeframe, cleaning was intense. My results were not consistent since I accidently mixed up the chemicals in the laboratory. The degree of separation was consistent.
The main method of making the separation more effective is by ensuring the purification is done perfectly. Unlike other chromatography methods, the strength of gel chromatography comes in through the interaction of the according to shapes and sizes. Two samples which differentiate with respect to the molecular size come up due to differences in the size. If the shape counteracts the size difference the separation may be ineffective (Liu et al,.).
Reference
Liu, H., Nishide, D., Tanaka, T., & Kataura, H. (2011). Large-scale single-chirality separation of single-wall carbon nanotubes by simple gel chromatography. Nature communications, 2, 309.
