Lab Report on the Separation of Blue Dextran and Cobalt Chloride on Sephadex G25

Name of Student

Date of Submission

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.