Lab Partner Elina Shrestha

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 B12. 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 = (π) (.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 phosphatase 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.

Table 2: Data for the different solutes

Fraction Elution molecular weight Solute # volume (ve) M.W Log of M.W

1 Victoire Ndong Lab partner Elina Shrestha Biochemistry 311 Tuesday lab Instructor: Laurie Lentz-Marino Blue Dextran 2000 5 10 2000000 6.301029996 Apase unknown 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

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 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.

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

-Biochemistry 311 Lab manual, Fall 2007