Biomedical Engineering Reference
In-Depth Information
3 Chromatography
3.1 Thin-Layer Chromatography
Thin-layer chromatography (TLC) has passed its heyday as an an-
alytical procedure and has been surpassed by more sensitive and
automatic methods, but it still has some advantages in the analysis
of complex lipids. Despite its decrease in usefulness, because of the
simple realization and low-cost apparatus, some examples for TLC
are given in this chapter.
3.1.1 Identification of the N-terminal Amino Acid
in Polypeptides (TLC of Modified Amino Acids)
Prerequisite is the existence of a primary amino group at the N-
terminal end of a polypeptide, i.e., chemical or posttranslational
modifications of this amino group, e.g., by methylation or acety-
lation, prevents success. If the amino group is not protected or
the amino acid chain is not branched, this method suits well for
examination of the uniformity of a purified protein.
If spots of multiple labeled amino acids (e.g., lysine, cysteine,
histidine, tyrosine) are found, check whether the intensity of the
multiple labeled derivatives is in the same order as the mono-
labeled derivative. Only in that case could the determined amino
acid be N-terminal.
All chemicals and reagents must be of highest purity (“Sequence
Grade”); especially water must be at least freshly double distilled.
A
coupling reagent
:1.4mg DABITC (4-(dimethylamino)azoben-
Solutions/Reagents
zene-4
-isothiocyanate, M
r
282.4, recrystallized from acetone)
is dissolved in 1.0 ml acetone. The solution is divided into 40
µ
l
aliquots, and acetone is evaporated by nitrogen. The aliquots
are stable in a desiccator at room temperature for months.
For reaction freshly dissolve 1 aliquot (corresponding 56
µ
g)
µ
in 20
l of pyridine.
B
PITC (phenylisothiocyanate), undiluted, Sequence Grade
C
n-heptane/ethylacetate 2:1 (v/v)
D
40% TFA (trifluoroacetic acid) (v/v) in ddH
2
O
E
50% pyridine (v/v) in ddH
2
O
µ
F
diethylurea marker
:mix6
l diethylamine and ethanolamine
µ
each with 100
l Soln. D, flush with nitrogen and heat for 1 h
