Thin-Layer Chromatography

72 3 Chemical Methods of Detection
Table 15 (Continued)
Substances Method, reagent and end products References
Steroid ketones
Aldehydes, ketones
Carvone, menthone,
acetophenone etc
Phenolic aldehydes
Chloro-, hydroxy-, and
methoxybenzaldehyde
derivatives
p-Benzoquinone derivatives
o-, m-, p-chloro-, 2,4- and
3,4-dichlorobenzoic acids
3.1.7 Dansylation
Apply the sample solution followed by GIRARD'S [81]
reagent (0 1 % tnmethylacetyl hydrazide in 10%
acetic acid) and allow to react for 15 h in an atmosphere
of acetic acid Then dry at 80 °C for 10 mm
and after cooling chromatograph the hydrazones
that have been formed
Apply sample solution and moisten with 2 N 2,4- [14]
dinitrophenylhydrazine in acetic acid After reacting,
dry and chromatograph the 2,4-DNPH derivatives
Apply an acidic solution of 2,4-dinitrophenyl- [82]
hydrazine, 4-nitrophenylhydrazine or 2,4-dinitrophenylsemicarbazide
onto the previously applied
sample solution Aliphatic and aromatic hydrazones
and carbazones can be differentiated by
their colors
Apply sample solution and then acidic 2,4-dimtro- [15]
phenylhydrazine solution, allow to react, dry and
develop
Denvatize with 2,4-dinitrophenylhydrazine solu- [83]
tion in hydrochloric acid Heat to 80 °C, cool and
chromatograph
Denvatization with 2,4-dinitrophenylhydrazine [84]
solution in hydrochloric acid Heat to 80 °C, cool
and chromatograph
Apply p-bromophenacyl esters of the substances, [85]
followed by 0 5% 2,4-dinitrophenylhydrazine in
2 mol/1 hydrochloric acid and allow to react for
10 to 15 min in the desiccator
Prechromatographic dansylation has the advantage that chromatography separates
excess reagent and also the fluorescent by-products (e g dansyl hydroxide)
from the reaction products of the substances to be determined In the case of
postchromatographic dansylation the whole of the plate background fluoresces
blue, so that in situ analysis is made more difficult
3 1 In Situ Prechromatographic Denvatization 73
Primary and secondary amines, amino acids and phenols react In the case of longwavelength
UV light (A = 365 nm) the DANS amides fluoresce yellow-green, while
amines that have reacted at a phenolic OH group have an intense yellow to yelloworange
fluorescence The detection limit for DANS amides is ca 10" 10 mol [86]
Acids can also be converted to fluorescent dansyl denvatives The reaction of C8
to C24 fatty acids with dansyl semipiperazide or semicadavende provides an
excellent example (Fig 34) [87] Odd-numbered and unsaturated fatty acids [88]
and propionic, sorbic and benzoic acid [89] can be detected in the same manner
Fig. 34 Fig. 35
Fig. 34: Chromatogram of various even- and odd-numbered fatty acids after in situ denvatiza-
Uon with dansyl semicadavende The separations are with increasing Rf — track 1 C-24 to
C 16 track 2 C-24 to C-6, track 3 C-20 to C-12, track 4 C-20 to C-ll, track 5 C 19 to
C 11 track 6 C-24 to C-16
Fig. 35: Fluorescence scan of the dansyl semicadavende denvatives of 1 behenic acid
2 erucic acid, 3 steanc acid, 4 oleic acid, 5 linoleic acid, 6 hnolenic acid