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 (Vt) of the column. The measured height
was 17cm;

Vt = () (.75)2 (17) = 30.04 ml

The void volume (V0) is the Ve for Blue Dextran and it is equal to: 5x2=10ml.
We used the tube numbers to calculate the Ve of each substance using the
equation Ve = (Fraction #) x (2ml)

Table 1: Elution volume of the different solutes

Solute Fraction # Elution volume (ve)

Blue Dextran 5 10
Alkaline
phosphatas
e unknown
Yellow
Dextran 7 14
Cytochrome 8 16
Vitamin B 10 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 Ve.

1
Victoire Ndong
Lab partner Elina Shrestha
Biochemistry 311 Tuesday lab
Instructor: Laurie Lentz-Marino

Table 2: Data for the different solutes

Elution
Fracti volume molecular
Solute on # (ve) weight M.W Log of M.W
Blue Dextran
2000 5 10 2000000 6.301029996
unkno
Apase wn 86000 4.934498451
Yellow
dextran 20 7 14 20000 4.301029996
Cytochrome 8 16 12400 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

Ve vs Log M.W
25

20
f(x) = - 3.01x + 28.41
15 R = 0.93

Elution volume (Ve) Linear ()

10

0
2.5 3 3.5 4 4.5 5 5.5 6 6.5

Log of M.W

Using the linear equation we can calculate the elution volume of alkaline
phosphatase. Y= -3.011x + 28.40 so lets 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

2
Victoire Ndong
Lab partner Elina Shrestha
Biochemistry 311 Tuesday lab
Instructor: Laurie Lentz-Marino

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 solutions
composition. We were given 5 different solutes and each had a different
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 its 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