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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: 1.4 mg DABITC (4-(dimethylamino)azoben- Solutions/Reagents
zene-4 -isothiocyanate, Mr 282.4, recrystallized from acetone)
is dissolved in 1.0 ml acetone. The solution is divided into 40 µl
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 µg)
in 20 µl of pyridine.
B PITC (phenylisothiocyanate), undiluted, Sequence Grade
C n-heptane/ethylacetate 2:1 (v/v)
D 40% TFA (trifluoroacetic acid) (v/v) in ddH2 O
E 50% pyridine (v/v) in ddH2 O
F diethylurea marker: mix 6 µl diethylamine and ethanolamine
each with 100 µl Soln. D, flush with nitrogen and heat for 1 h
84 3 Chromatography
at 55 ◦ C. Dry in vacuum, dissolve in 1 ml ethanol, aliquot to

100 µl portions and store at −20 ◦ C
butylchloride or butylacetate
TFA, anhydrous
HCl, concentrated
Solvents for TLC 1 acetic acid/H2 O 1:2 (v/v)
2 toluene/n-hexane/acetic acid 2:1:1 (v/v/v)
This protocol gives a survey of the method. For details and special
tips see Choli-Papadopoulou et al. (1997).
Derivatization, cleavage, and conversion

Dry a desalted sample of the protein or polypeptide (0.5–5 nMoles)
over solid KOH in vacuum. Add 20 µl of Soln. E and flush with
nitrogen. Add the described aliquot of A in pyridine (20 µl). Flush
the tube with nitrogen, and then close it and heat it to 52 ◦ C for 20–
30 min. Add 2 µl reagent B, flush again with nitrogen and incubate
the closed tube for further 20 min at 52 ◦ C.
Remove uncoupled reagents by extraction with Soln. C. For this
purpose, add 200 µl of Soln. C to the reaction mixture, centrifuge,
carefully withdraw the supernatant and repeat extraction twice.
Discharge the supernatants and dry the aqueous phase in vacuum.
To remove residual traces of water, add 10 µl of ethanol to the tube
and dry again.
Add 20 µl of anhydrous TFA to the dry sample in a nitrogen
atmosphere. The solution changes to red. Heat the closed tube to
55 ◦ C for 10 min, and then blow off the TFA in a hood.
Wet the residue with 30 µl of H2 O and extract the solution
twice with 30 µl butylchloride or n-butylacetate. Separate phases
by centrifugation and collect the upper organic layer into a new
tube.
Evaporate the organic solvent with nitrogen, and then add 10 µl
of Soln. D. Flush the tube with nitrogen, close it, and incubate it for
30 min at 55 ◦ C. Then dry the solution in vacuum.
Chromatography and identification of N-terminal amino acid

Dissolve the dry residue of the conversion step in 5 µl of ethanol.
Spot 0.5–1 µl of this solution together with the same amount of
marker Soln. E onto the start point of a 25 × 25 mm polyamide TLC
sheet, about 3 mm distant from an edge.
Cover a chromatographic chamber, with at least 30 mm diam-
eter, using filter paper and equilibrate with Solvent 1. After equi-
libration, pour off the solvent and fill up new solvent to a hight of
2 mm.
Develop the sheet for 4–5 min and remove it when the solvent
front is about 2 mm from the top of the sheet. Remove the sheet
and dry it with a cold fan.

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3 Chromatography

3.1 Thin-Layer Chromatography

Thin-layer chromatography (TLC) has passed its heyday as an an-

3.1.1 Identification of the N-terminal Amino Acid

Prerequisite is the existence of a primary amino group at the N-

at 55 ◦ C. Dry in vacuum, dissolve in 1 ml ethanol, aliquot to

Derivatization, cleavage, and conversion

Chromatography and identification of N-terminal amino acid

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