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TLC-Scanning for Direct

Quantitation of Natural Waxes
a a
Ruben Martínez & Patrick Moyna
a
Farmacognosia y Praductos Naturales Facultad
de Química , Av. Gral. Flores, 2124, Montevideo,
Uruguay
Published online: 23 Oct 2006.

To cite this article: Ruben Martínez & Patrick Moyna (1990) TLC-Scanning for Direct
Quantitation of Natural Waxes, Journal of Liquid Chromatography, 13:10, 1959-1965,
DOI: 10.1080/01483919008049004

To link to this article: http://dx.doi.org/10.1080/01483919008049004

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 JOURNAL OF LIQUID CHROMATOGRAPHY, 13(10), 1959-1965 (1990)

TLC-SCANNING FOR DIRECT

Q U A N T l T A T l O N OF
NATURAL W A X E S
RUBEN M A R T ~ N E ZA N D PATRICK MOYNA
Farmacognosin y Prodnctos Naturales
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Facultad rle Quiniica

Av. Gral. Flores 2124
Aloiitevideo, Urugiiny

ABSTRACT

H2S04/char6-ing is used a s visualization method for quanti-

tative TLC scanning of waxes. The appropriate technical
details are described.

I NTRODUC T I O N

The range of components usually present in waxes ( 1 ) makes

their direct quantitation difficult. This has been achieved
by quantitative elution in Column Chromatography (CC) (2) or
by combined Thin Layer Chromatography-Gas Liquid Chromatog-
raphy (TLC-GLC) (3). For a limited group o f components direct
GLC of the wax has been tried (4). TLC scanning techniques
seem particularly appropriate for this problem (5). Four
stages of the TLC technique have t o be controlled for ade-
quate quantitation ( 5 ) : plates with even surfaces,proper

1959

Copyright 0 1990 by Marcel DeMter, Inc.

 1960 MARTINEZ A N D M O Y N A

application of samples; adequat'a s o vent mixtures; good

visualization methods. For working on waxes even labor-
atory-prepdred plates give adequate results. As sample
spotting is simultaneous. it is simple to control the ap-
plication stage ( 1 , 3 , 6 ) . Slight variations of the solvent
mixtures used in wax analysis will give good separations
for most fractions present. This leaves visualization as
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the main difficulty: most wax constituents lack chromo-

phores and do not fluoresce. With the exception of the
more general visualization reagents, few cover well the
wide range of wax components.
Given the available information on H2S04/charring ( 6 . 7 1 ,
the method was selected as worth assaying as a visualization
procedure with quantitative capability.

MATERIALS AND METHODS

Plates were prepared with Si1ic:a-gel G (E. Merck, Nr. 7 7 3 0 ) .

All solvents were analytical grade (Mallinckrodt, USA).
~ , c 3 0 ) and hydrocarbons (c24,c26) were
Pure acids ( c ~ ~ , C ,C26,
from Aldrich (USA).
Pure alcohols (CI6, CI8, Cz0) were from Fluka (Switzerland).
Triterpenes and esters were p w p a r e d from natural samples
by CC ( 3 , 8 , 9 ) .
Wax sarnplps were prepared from Colletia paradoxa by the meth-
ods described (8).
A TLC Scanner (Shirnadzu C S - 9 0 0 0 ) was used with the following

parameters:
Photo mode: ABS REF PKF filter: 50
Zero set mode: STORED Drift line: 10.00
Scan mode: ZIG-ZAG Minimum width: 5.0
Swing width: 10.0 mm Minimum ares: 1000
Oelta "Y": 0.20 mm
 QUANTITATION OF NATURAL WAXES 1961

*
.8
41
.7

.6
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a
V
r: .5
rn
n
L
0
lo .4
n
fu
.3

.2

FIGURE 1 - INFLUENCE OF OVEN TIME

RESULTS AN0 DISCUSSION

The usual charring conditions (H2S04:H20 ( 1 : l ) ; 2OO0C oven

temperature) (6) were used t o establish the basic operation
parameters. The influence o f the amount of charring agent
and oven time were determined and are shown in Figures 1

and 2 . It could be established that on these conditions a

maximum response could be obtained after 2 minutes, indepen-
dently of the amount o f charring agent applied when measur-
ing below 450 nm. Over 450 nm the maximum could only be ap-
proached with increased time.
 1962 MARTINEZ A N D MOYNA

60C

50C

40C

aJ
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v)
c
0
a 30C
v)
aJ
L

2oc

1o c

milligrams charring agent per cm2

FIGURE 2 - INFLUENCE OF AMOUNT OF CHARRING AGENT

(*) Response is expressed in "Area" values of the
TLC-scanner f o r a sample of 30 mg of acids.

The operating conditions thus established (10 min; 2 O O 0 C

oven temperature; 4 g charring tigent per 20 x 20 cm plate)
were used t o scan t h e absorbance-450 nmlconcentration ratios
for pure wax components. The results are shown in Figure 3.
It can be observed that there i s a concentr-ation over which
the results are non-1inear.These "saturation" thresholds are
different for the different fractions and the slope under
 QUANTITATION OF NATURAL WAXES 1963

1500

400
300
200
100
000

aJ 900
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In
e
0 800
a
In
a, 700
L
esters
600
500
400
300
200
- hydrocarbons

100
0
0 40 80 120 160 200
micrograms o f sample
FIGURE 3 - RESPONSE FOR DIFFERENT COMPONENTS
(*) R e s p o n s e is e x p r e s s e d in " A r e a "
v a l u e s o f t h e TLC-scanner.

Table 1 - Linear Range and Correlation Data for Wax Components

Component Linear range Ratio (Acids = 1)

(micrograms per plate)
Linear Acids 20 - 100 1 .oo
Linear Alcohols 20 - 100 0.89
Esters
Hydrocarbons
40
40
-- 120
120
0.18
0.10
Triterpenes
Taraxerol 1 - 5 4.66
Oleanoic Ac. 2 -
10 2.84
Taraxerone 10 50- 1.74
 1964 MARTINEZ A N D MOYNA

Table 2 - Composition of Colletia paradoxa

by TLC-GC (a)(*) by TLC-scanning
Hydrocarbons 22 % 22 %
Ketones 18 % 16 %
Free Acids 14 % 15 %
free Alcohols ( * * I 13 % 17 %
Esters 10 % 8 %
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(*) The TLC-GC values are corrected for the polar compounds present in
the original sample (8).
(**) "Free Alcohols" is expressed directly in taraxerol and needs further
refinement. It does not take into account that it is a mixture of 3 %
alkanols and 97 % triterpenic alcohols (lupeol, gerrnanicol, taraxerol.
2:l:l).

this limit is specific for each component. The correlation

data are chown in Table 1.
The method was tried on the war o f Colletia paradoxa and
the results were compared agairist those obtained by other
quantitation approaches (8). lhis wax was selected as
previously analysed samples were available and also for
its broad range of components. The results are shown in
Table 2.
The TLC scanning results are directly related to the o r ginal
wax composition by the correlat,ion data, while other me hods
tend to discard the more polar fractions that cannot be eluted
from CC or be observed by GLC.
The correlation is good, that for the "Free alcohols" fraction
being the lowest. In this wax, this fraction is 97 % terpenic
alcohols and only 3 % alkanols s o its quantitation needs fur-
ther refinement.
 QUANTITATION OF NATURAL WAXES 1965

ACKNOWLEDGEMENTS

The authors acknowledge support from the PEDECIBA (PNUD-

URU D02/84) and o f the CEC Scientific Directorate, which
made the work possible.

BIBLIOGRAPHY
Downloaded by [Michigan State University] at 19:14 09 February 2015

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