Biomedical Engineering Reference
In-Depth Information
Formiate Gradient
Mark the start with a pencil on a PEI Cellulose sheet (dimensions as
in Protocol 3.1.2) and apply the samples with a microliter syringe
or a pipet.
DevelopthesheetwithH
2
O for about 1.5 h toaheightof16cm,
then dry it and continue development with Solvent 1 to a running
distance of 13 cm. Move the sheet in methanol for some minutes
and dry it again. Then do the same procedure with Solvent 1
,
followed by a third run with Solvent 1
.
Detect the phosphates as outlined in Protocol 3.1.2.
Lithium Chloride Gradient
If lithium chloride gradient is used, develop the sheet ascending
for 2 min with Solvent 2, and then for 6 min with Solvent 2
and last
up to 16 cm with Solvent 2
.
Depending on the nucleosides, which of the solvent systems
gives the best results should be verified.
3.1.4 Identification of Phosphates on TLC Plates
A
0.01% fluorescein sodium (w/v) in ethanol
Solutions/Reagents
B
Hanes' reagent: 0.5 g ammonium molybdate are dissolved in
5 ml H
2
O and supplemented with 2.5 ml of 25% HCl followed
by 2.5 ml of 70% perchloric acid. After cooling acetone is filled
up to 50 ml. The reagent is ready for use the next day and it is
stable for 2 - 3 weeks in the dark.
Identification by Phosphorescence
Phosphorescence is observed when the dry sheet is dipped in a con-
tainer with liquid nitrogen and illuminated by a UV lamp. Purine
derivatives phosphoresce light blue; pyrimidines are not visible.
Identification by Fluorescence Quenching
To detect the spots on the TLC plate spray it with Soln. A and dry
it. Illumination with a UV lamp (254 nm) shows the spots dark in
a greenish fluorescent surrounding. Indicate the spots with a pencil.
Identification by Molybdatophospho Complexes
Spray the dry plate with Soln. B and dry with air and heat in
a drying oven to 110
◦
C until blue spots of phosphoric acid esters,
e.g., phospholipids, appear.
References
Touchstone JC (1992) Practice of thin layer chromatography. 3rd. ed. Wiley,
New York
