Victoire Ndong
Lab partner Elina Shrestha
Biochemistry 311 Tuesday lab
Instructor: Laurie Lentz-Marino
MOLECULAR SIEVE (GEL EXCLUSION) CHROMATOGRAPHY
ABSTRACT
The gel exclusion chromatography is a technique used to separate solutes that are present in a mixture.
A buffer flows in a column filled with gel particles and the solutes will elute depending on their size.
Large solutes elute faster because of their large size and the small solutes will elute slower.
In our experiment, we used Sephacryl S-100 as our column resin and we used it to separate the solutes in a mixture containing Blue dextran, alkaline phosphatase, yellow Dextran, cytochrome and vitamin B 12 . Using the graph of the elution volumes and the molecular weights, we tried to determine the elution volume of our unknown which was alkaline phosphatases. We checked the success of our experiment by taking tubes containing different solutes and testing for the presence of alkaline phosphatase.
RESULTS AND CALCULATIONS
First we have to calculate the total volume (V t) of the column. The measured height was 17cm;
Vt = (π) (.75) 2 (17) = 30.04 ml
The void volume (V 0 ) is the V e for Blue Dextran and it is equal to: 5x2=10ml. We used the tube numbers to calculate the V e of each substance using the equation V e = (Fraction #) x (2ml)
Table 1: Elution volume of the different solutes
Solute Fraction #
Blue Dextran
Alkaline phosphatase unknown
Yellow Dextran
Cytochrome
Vitamin B
5
7
8
10
Elution volume (v e )
10
14
16
20
We also made a graph of the elution volume versus the log of the M.W in order to determine the elution volume of alkaline phosphatase V e.
Table 2: Data for the different solutes
Solute Fraction Elution molecular weight Log of M.W
1

Victoire Ndong
Lab partner Elina Shrestha
Biochemistry 311 Tuesday lab
Instructor: Laurie Lentz-Marino
# volume (v e ) M.W
Blue Dextran
2000
Apase
Yellow dextran
20
Cytochrome
5 unknown
7
8
10
14
16
2000000
86000
20000
12400
6.301029996
4.934498451
4.301029996
4.093421685
Vitamin B12 10 20 1300 3.113943352
The table above allows us to draw the graph of the Ve versus the logarithm of the molecular weight.
Figure 1: Ve vs the logarithm of the molecular weight
y = -3,0114x + 28,408
R² = 0,9349
25
20
15
10
5
Ряд1
Линейная (Ряд1)
0
0 1 2 3 4
Log of M.W
5 6 7
Using the linear equation we can calculate the elution volume of alkaline phosphatase. Y= -
3.011x + 28.40 so let’s take x as the logarithm of the molecular weight of alkaline phosphatase.
We will have
Ve (Apase) = -3.011(4.93) +28.40 = 13.54 ml
DISCUSSION
This experiment was very straight forward and the results we got were very satisfying.
Different solutes when put in a resin column elute depending on their size. The largest solutes are the first to elute. That permits to separate the different solutes in a mixture and determine the solution’s composition. We were given 5 different solutes and each had a different
2

Victoire Ndong
Lab partner Elina Shrestha
Biochemistry 311 Tuesday lab
Instructor: Laurie Lentz-Marino molecular weight. The solutes run by gravity. Blue Dextran was the largest (M.W 2 millions) so it was the first to elute and Vitamin B12 (M.W 1300) was the last to elute and it had the biggest elution volume. Alkaline phosphatase elution volume was unknown. Using the graph of the Ve versus the log of M.W, we calculated it’s Ve since the flow rate should be constant. Our line was not straight meaning the data was not exactly perfect but we got an acceptable number: Ve
(Apase) = 13.54 ml. At the end of our elution, we tested the tubes to see which one contained alkaline phosphatase. When alkaline phosphate is present, we should see a purple precipitate after the addition of NBT and BCIP. We saw a purple precipitate on almost all our tubes, but the precipitate was bigger in the yellow Dextran and the Blue Dextran tubes. That means some of our alkaline phosphatase leaked into the first tube and the third tube. The reason for that may be that the resin was not very successful in separating all the solutes.
The bigger the resin gel particles are, the faster the flow rate and the lower the resolution and vice versa. If we were to choose another resin, we might have chosen one with finer gel particles. It would have given us a better separation of the solutes. We used Sephacryl S-100 and to improve our results we could have used Sephacryl S-150 maybe.
Our sources of errors were human errors, and systematic error. Our buffer reservoir had problems and we added sometimes too much or too little buffer.
REFERENCES
-Biochemistry 311 Lab manual, Fall 2007
3