Professional Documents
Culture Documents
Figure 1. Reaction of dansyl chloride with terminal amino groups and identification by TLC.
Page 2 of 13
Consult Materials Safety Data Sheet (MSDS) for all reagents used in this procedure for detailed
safety information
Solutions are stabilized with acids or bases, which are potentially corrosive. Wear appropriate eye
protection, gloves and a lab coat at all times.
Perform all procedures in the fume hood.
After incubation precipitate the excess bicarbonate by adding 1.6 mL of acetone and mix. Make
sure you condition your micropipette for using acetone.
5.
6.
Except for the unknown dipeptide, aspirate the liquid layer with a Pasteur pipette to a new set of
microfuge tubes.
7.
Label your tubes with tape and leave them open in the fume hood to allow the solvent to dry for the
next lab session.
8.
For the unknown dipeptide, aspirate the liquid layer into a vial from your locker and dry it down
using a gentle stream of air while placing the vial in a beaker of hot tap water.
Page 3 of 13
Once the unknown dipeptide is dry add 0.2 mL of 6 M HCl (careful, strong acid) to the residue
and mix using gentle vortexing until dissolved.
10. Transfer this solution to an ampule labelled with tape, Parafilm and submit to the TA. The ampule
will be sealed by a glass blow torch for you, and then be placed in an oven at 110C for 18 hours.
The samples will be returned to you in the following lab period for analysis. How does acid
hydrolysis occur?
Would the above method be adequate for longer polypeptides, such as proteins?
Disposal
Rinse the microfuge tubes with water to suspend the precipitate and dispose the solution into the
organic waste container. Once clean, the tubes can go in the garbage.
Discard used plastic pipette tips in the garbage.
Discard used Pasteur pipettes in the broken glass container.
Vt = V0 + Vi + Vinert
The substances being chromatographed (or separated) are called the solutes.
(1)
In column
chromatography the mixture of solutes is initially applied to the top of the column in a small volume of
solvent.
Elution is achieved by continually applying pure solvent. The various solutes then move
Page 5 of 13
coefficient, K, as the equilibrium ratio of the concentration of the solute in the two phases (K=Cs/Cm,
where Cs and Cm are the concentrations of the solute in the stationary and mobile phases,
respectively). It should be intuitively obvious that if a mixture contains 2 solutes with different Ks, then
theoretically one of these solutes will move faster in the mobile phase than the other. The solute with
the higher K value will spend more time in the stationary phase and will be eluted later.
The relative mobility of the solute can be defined as:
R=
V0
V0 + KVi
(2)
Because the column parameters Vo and Vi are constant for a given column, the relative mobility will
increase as K decreases.
The ability of a solute to partition between a mobile and stationary phase does need not be based
only on preferential solubility. Any physical method of retaining one solute preferentially in one phase
will also work. In ion exchange chromatography, the solute is held preferentially in the stationary phase
(or resin) because of electrostatic interaction with the resin.
stationary resin itself also serves to bind the solute, but not through electrostatic interactions. But in all
cases, the same principle applies i.e. differential partitioning or distribution between mobile and
stationary phases, the latter being the liquid, a liquid-solid matrix or a solid support.
Calculating the relative polarity of the mobile phase is made possible by the Snyder Polarity
Scale/Index (P'), which is a measure of the ability of the solvent to interact with the following polar test
solutes: ethanol, dioxane, and nitromethane. Solubility measurements of each of the test solutes in a
given solvent are used to determine adjusted partition coefficients, kg', where kg' is the solute
distribution coefficient corrected for the molecular weight of solvent and solute. The polarity index (P') is
Page 6 of 13
% + % + + %
100
Rf =
(3)
Rf will be constant for a particular solute in a defined solvent and will vary between 0 (no migration from
origin) to 1 (migration along with solvent front).
Cellulose Chromatography
The principle of TLC using cellulose is basically the same as that of TLC using silica. In this case,
the inert support is the cellulose matrix, which is very polar. As the solvent permeates the cellulose, it
separates into a water-rich phase, which binds to the cellulose, and the organic solvent or non-polar
rich phase that is mobile. Solutes that are more soluble in the aqueous layer have lower Rf values.
Amino acids can be separated by cellulose chromatography generally because of their differences
in polarity. For example, glutamic acid will be more soluble in the water-rich phase than isoleucine.
Page 7 of 13
Consult Materials Safety Data Sheet (MSDS) for all reagents used in this procedure for detailed
safety information
Solutions are stabilized with acids or bases, which are potentially corrosive. Wear appropriate eye
protection, gloves and a lab coat at all times.
Do not inhale loose silica powder from the TLC plates.
TLC tanks will contain solvent that emit strong fumes; do not move the tanks from the fume hoods.
Allow wet TLC plates to dry in the fume hood.
Perform all procedures in the fume hood.
DNS-Glu
DNS-
DNS-
DNS-
control
Phe
Peptide
DNS-Gly
DNS-
DNS-
Peptide
Ala
DNS
5. When you are ready to spot the samples, reconstitute them by adding 100 microL of acetone and
mix completely by vortexing.
6. Dip a capillary tube into one of your dansylated amino acid samples and apply ONE spot of no more
than 2-3 mm in diameter to one of the pencil dots. Multiple dabs will give you big spots that will
smear and you will NOT get good results. Repeat with the rest of the dansylated standards. To save
glassware you should use the other end of the capillary tube to spot another sample. If you want to
practice, try spotting acetone only at the top of the TLC plate above the solvent front.
7. Place the plate in a pre-equilibrated tank containing the solvent (chloroform/methanol/acetic acid,
95:10:1). Let it run until the solvent front has reached 1 cm from the top of the plate. Calculate the P
of this solvent mixture.
8. Remove the plate and immediately mark the solvent front. Allow the plates to completely dry in the
fume hood (1-2 minutes) before viewing under ultraviolet light.
9. Record the Rf values in the table provided at the end of this procedure. Also note the colours of all
spots.
B. Cellulose TLC for C-terminal determination
1. Three groups will share a 20 cm x 10 cm pre-coated cellulose TLC plastic plates. Handle the plates
as little as possible and only at the very edges with gloved hands.
2. Plan and organize such that each group will spot their third of the plate, which is in a landscape
orientation, as follows with the peptides spotted in duplicate:
Glu
Peptide
Ala
Phe
Peptide
Gly
3. Glutamic acid, alanine, glycine and phenylalanine are supplied at concentrations of 0.1 mM.
4. Dip a capillary tube into one of the samples and apply ONE spot of no more than 2-3 mm in
diameter to one of the pencil dots. Multiple dabs will give you big spots that will smear and you will
Page 9 of 13
Page 10 of 13
DNS-Glu
DNS-Peptide
DNS-Phe
DNS-Peptide
DNS-Ala
DNS
1.00-0.90
0.89-0.80
0.79-0.70
0.69-0.60
0.59-0.50
0.49-0.40
0.39-0.30
0.29-0.20
0.19-0.10
0.09-0.00
Cellulose TLC
Rf value
Glu
Peptide
Ala
Phe
Peptide
Lys
1.00-0.90
0.89-0.80
0.79-0.70
0.69-0.60
0.59-0.50
0.49-0.40
0.39-0.30
0.29-0.20
0.19-0.10
0.09-0.00
Page 11 of 13
Lab report
In addition to what is written in the syllabus, the following guide provides a few more details for this
particular report.
Introduction
For the last paragraph, what do you expect to see on your silica and TLC plates? Think of where the
standards will appear on the plate in terms of polarity.
Results
Provide labelled pictures of both silica and polyamide TLC plates. Identify ALL spots on the TLCs.
Fill out the following table and include it in your results section. Note which dipeptide you analysed.
N-Terminal amino acid
Dipeptide #:
Rf of dipeptide
Rf of matching
standard
Rf of matching
standard
Dipeptide
structure and
name
Discussion
Recall the purpose of the experiment and what techniques are being used to address it.
Look at your TLCs and Rf tables and start to match up and identify spots. Are the spots appearing
where you expect them to in terms of polarity?
If the experiment did not work, suggest how you would change the experiment.
Conclude with restating the purpose and the techniques used to answer it. Identify your unknowns and
state if the purpose was answered or not. If not, how would you change the experiment?
Appendix
References
Calculations
Sample calculation of Rf values
Page 12 of 13
Print out the Rf values tables and include them in this section
Methods: state that the lab manual was followed, provide its reference, and include any deviations
from the procedure here.
Copies of flow charts
Copies of observations and raw data
Page 13 of 13