Separation of five different solutes by Gel exclusion
Chromatography and Determining the Elution
position/volume of the colorless Alkaline Phosphatase.
Elina Shrestha
Tuesday Lab session
Lab Partners: Victoire Ndong
Date: October 23rd 2007
Abstract:
Gel exclusion chromatography, also known as gel filtration chromatography, gel
permeation chromatography and molecular sieve chromatography, is a technique used to separate
molecules according to their sizes. The technique involves making up a packed column of inert
solid resin beads, consisting cross-linked strands of high molecular weight polysaccharide or
polyacrilamide. At the molecular level, these resin beads appear like sponges with holes that
from interconnected channels throughout the particle. A solution of different solutes can
therefore be separated into its individual solute solutions when added to the molecular sieve
column and the column buffer is allowed to flow through by gravity. The large sized molecules
are excluded from the pores and thus elute from the column quickly whereas the smaller solute
can be included by the resin bead and takes longer to elute off the column.1 In lab, we have used
Sephacryl S-100 resin column to separate five different solutes in a solution, namely the blue
dextran 2000(Mr 2,000,000), yellow dextran 20(Mr 20,000), vitamin B12 (Mr 1300, pink), plus
cytochrome C (Mr 12,400, orange) and alkaline phosphatase (Mr 86,000, colorless). A fraction
collector was set at 5 min/tube to collect ~20 fractions in total and the fraction’s average volume
measurements were taken the solution soon separated into its individual solute fractions, of
which blue, yellow, orange and pink fractions were recognized as blue dextran, yellow dextran,
vitamin B12 and cytochrome C respectively (starting from the first elution). The volume of
buffer required to elute each solute from the column (Ve) was calculated and was plotted against
the log of molecular wt of the solutes. It gave us a straight line with a negative slope. The Ve of
colorless alkaline phosphate was found to be 13.56ml from the plot. Whereas the elution position
of alkaline phosphatase was determined by enzymatic assay which required us to add BCIP and
NBT into aliquots of each fraction and look for the fraction that gave purple precipitate. The
peak fraction containing alkaline phosphatase was found to be in tube #7. From this, its Ve was
found to be 14ml.
Results:
The table below shows the relative molecular weights of the five different solutes in our
sample solution that were subjected to separation by gel chromatography and the calculation of
their log values and the volume of the column buffer required to elute each of them.
Solute
Molecular Weight
of solute
Log (Molecular wt.)
Fraction #
Elution Volume
Ve ( ml)
Blue dextran
2,000,000
6.3
5
5*2=10
alkaline
phosphatase
86,000
4.93
7
7*2=14
yellow
dextran
20,000
4.30
7
7*2=14
Cytochrome C 12,400
4.09
8
8*2=16
vitamin B12
1300
3.11
10
10*2=20
The Blue dextran was eluted off the column first followed by yellow dextran ,
Cytochrome C(orange) and vitamin B12(pink) detected by the visual examination of color.
The total space surrounding the gel particles in the packed column,Vo = Ve of blue
dextran= 10ml
Radius of the column, r = 0.75cm ; Height of the column, h= 17cm
Total volume occupied by the column,Vt= 3.14 r2h
= 30.03 ml
Figure 1. Determination of Elution volume for alkaline phosphatase by using the relation
between log of its molecular weight and Ve .
From the graph,
Ve of alkaline phosphatase was found to be 13.56ml.
The elution position of alkaline phosphatase was found to be in between the blue and
yellow fractions since the blue fraction gave light purple precipitate on addition of BCIP and
NBT whereas the darkest purple precipitate was seen in the yellow fraction. Thus, the peak
fraction containing the alkaline phosphate was the one in tube #7.
Discussion:
The experiment we performed using the gel fraction column helped us separate different
solutes that were dissolved in a solution. Separation was done by the size of the solute molecules.
The largest molecules were eluted from the bottom of the column first and the smallest the last.
This is evident from the results that the blue dextran is eluted from the column first, which has
the largest molecular weight out of all five solutes in the sample solution. Only then the other
smaller solute molecules were eluted size-wise. So, if the solute is sufficiently large in size such
that it is “excluded” from the pores, the volume of the column that is accessible to this very large
molecule is greatly reduced and it will elute from the column very quickly.1 However, for small
solutes, they can diffuse in and out of the pores and be “included” by the resin beads and hence
stay longer in the column. The plot of elution volume vs. log of molecular weight of the
molecule is a straight line with a negative slope of ~ -3 which also tells us that with decreasing
molecular size the volume of column buffer required to elute the solute will increase. Since
alkaline phosphatase is a colorless solute, its elution volume was determined by two methods,
one by plugging the log of its molecular weight into the equation obtained from the plot which
gave us 13.56 ml and the other by noting the peak fraction containing the alkaline phosphatase
that produced deep purple precipitate on addition of BCIP and NBT. The latter gave us the value
of Ve as 14ml. The molecular weight of alkaline phosphatase lies between those of blue dextran
and yellow dextran, so the alkaline phosphate was eluted from the column after blue dextran and
before yellow dextran as expected. This result serves as yet another evidence to the fact that gel
exclusion chromatography separates molecules by their size. Since all the solutes present in our
sample solution with their molecular weights ranging from 1300-2,000,000 were separated fairly
well, the Sephacryl S-100 has proved to be a suitable resin for separating these solutes. The
solutes can be collected in more pure form by setting the fraction collector such that it switches
the tube more frequently. This reduces the chance of mixing two solutes in one tube.
References
1. Biochemistry 311 Lab Manual Fall, 2007. Biochemistry Department, Mount Holyoke
College.