Digitally enhanced thin layer chromatography: Further development and some applications in isotopic chemistry

Article abstract of DOI:10.1002/jlcr.3052

Improvements to thin layer chromatography (TLC) analysis can be made easily and cheaply by the application of digital colour photography and image analysis. The combined technique, digitally enhanced TLC (DE-TLC), is applicable to the accurate quantificat

Full text of DOI:10.1002/jlcr.3052

Research Article
Received 16 February 2013,
Revised 22 March 2013,
Accepted 22 March 2013
Published online 23 July 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3052
Digitally enhanced thin layer chromatography:
further development and some applications in
isotopic chemistry
Daniel P. Manthorpe and William J. S. Lockley*
Improvements to thin layer chromatography (TLC) analysis can be made easily and cheaply by the application of digital colour
photography and image analysis. The combined technique, digitally enhanced TLC (DE-TLC), is applicable to the accurate
quantification of analytes in mixtures, to reaction monitoring and to other typical uses of TLC. Examples are given of the
2
application of digitally enhanced TLC to: the deuteromethylations of theophylline to [methylꢀ H
3
]caffeine and of umbelliferone
2
to [ H ]7-methoxycoumarin; the selection of tertiary amine bases in deuterodechlorination reactions; stoichiometry
3
optimisation in the borodeuteride reduction of quinizarin (1,4-dihydroxyanthraquinone) and to the assessment of xanthophyll
yields in Lepidium sativum seedlings grown in deuterated media.
2
2
Keywords: digitally enhanced TLC; DE-TLC; deuteromethylation; Lepidium sativum carotenoids; methylꢀ H
3 3
]caffeine; [ H ]7-
2
methoxycoumarin; [9,10ꢀ H
2
]1,4-anthraquinone; umbelliferone; quinizarin
Introduction
Moreover, the compounds or mixtures under investigation can
be subjected to a very wide range of detection modes including
fluorescence, fluorescence-quenching, chemical derivatisation,
dye absorbtion, colour reactions and so on. Hence, the DE-TLC
procedure is applicable to compounds that are inappropriate
analytes for HPLC or HPLC-MS.
Thin layer chromatography (TLC) is still of great utility to
chemists. However, it is normally considered as a qualitative
technique. In contrast, quantitative TLC is usually carried out
using high performance TLC plates in conjunction with
expensive automated spotting apparatus and with multi-
wavelength plate scanners costing tens of thousands of dollars.
This paper describes how digital photography and public
domain image processing software can be employed to enhance
both the qualitative and quantitative uses of ordinary TLC.
The current work constitutes an extension of, and a substantial
2
Discussion
The methodology used for DE-TLC is simple, employing ordinary
TLC tanks and plates. The improvements described in this paper
arise from four primary sources; (a) the use of quantitative
spotting via a syringe, microcap or micropipettor; (b) data
acquisition via digital colour photography; (c) quantification via
improvement upon, a previous study of digitally enhanced TLC
1
(
DE-TLC). This previous study illustrated the utility of digital
enhancement of TLC but was dependent on a bespoke data
processing program (TLC-Analyzer), which was suitable for some
types of DE-TLC analysis but limited in its ability to analyse parallel
data. Unfortunately, this program is now outdated and will not
operate on many modern computer systems. Hence, a re-
evaluation and updating of the technique is overdue. This is
particularly the case in view of the easy accessibility of digital
cameras and of public domain image processing programs. When
3
IMAGEJ image processing software (NIH, USA) ; and (d) application
of a simple mathematical transform to convert the raw data to
linear form. Essentially, the TLC plate is run, photographed
(usually under visible or 254/360 nm ultraviolet light), the image
quantified and the data is linearised via the transform. These
stages are discussed in more detail below.
combined together, these developments have enabled a new Quantitative thin layer chromatography procedure
approach to DE-TLC quantification and data manipulation, which
Throughout this work, manual spotting via fixed volume
is described herein. This new approach has proved, practical,
microcaps (0.5–5 μl capacity) has been used and the spotting
versatile and inexpensive. As such, DE-TLC continues to be an
volumes for each analysis have been kept constant. Microcaps
economical procedure that is well suited to laboratories with a
limited budget or to those laboratories where the potential
contamination of expensive commercial high performance TLC
systems currently precludes the use of quantitative TLC techniques. Department of Chemistry, Faculty of Engineering and Physical Sciences,
University of Surrey, Guildford, GU2 7XH, UK
It should be noted that DE-TLC maintains and extends all the
advantages of TLC as an analytical method. It allows parallel
analyses, all components of the mixture under analysis are
*
Correspondence to: William J. S. Lockley, Department of Chemistry, Faculty of
Engineering and Physical Sciences, University of Surrey, Guildford, GU2 7XH, UK.
visible and a variety of separation mechanisms can be employed. E-mail: w.lockley@surrey.ac.uk
J. Label Compd. Radiopharm 2013, 56 544–552
Copyright © 2013 John Wiley & Sons, Ltd.

D. P. Manthorpe and W. J. S. Lockley
were chosen because they are cheap and disposable but have a
very high degree of dispensing precision. In our hands, similar
analytical precision could be obtained using a fixed volume glass
gas-liquid chromatography syringe or a good quality low volume
micropettor set to a fixed volume (1–10 μl ). The best data were
obtained when the analyte was spotted in a solvent of lower
polarity than the TLC mobile phase and when approximately
circular spots were obtained. That said, good quality data were
still obtainable when both these criteria could not be fully
met. For quantitative analysis, duplicate sets of standards were
prepared by spotting fixed volumes of a series of standard
concentrations of the analyte. The test samples or quality
control samples were placed between the two sets of standard
sample spots. All the spots were allowed to dry before
development. Typically, a 20 × 10 cm plate was used in
landscape orientation with a development distance of 10 cm.
The TLC tank was lined with filter paper and was thoroughly
equilibrated with the eluent prior to chromatography.
Visualisation of the analytes
For coloured compounds, no specific technique of visualisation
was necessary because the digital camera could record the spots
directly and selection of the appropriate colour domain was
straightforward. For compounds with chromophores that
crossed 254 nm, visualisation was by fluorescence quenching
using F254 TLC plates. In this case, the pictures were taken under
2
54 nm illumination from a 4 W ultraviolet lamp. For fluorescent
Figure 1. Camera and light source arrangement for fluorescence or fluorescence-
quenching assay.
compounds, the fluorescence was recorded under 4 W ‘black-
light’ illumination at 360 nm. In the case of compounds detected
by iodine adsorbtion, the TLC plates were allowed to stand in an
atmosphere of iodine vapour until the spots were clearly visible or the area of the plate that is of interest, fills about 80% of the
against an essentially white background and photographed frame. This allows for any non-linearity (vignetting) towards the
promptly. No other visualisation techniques were evaluated edge of the photograph, which can be experienced with
2+
quantitatively in the current work, but colour detection of Ni
inexpensive cameras. The ultraviolet lamps used in this study had
by dimethylglyoxime spray reagent worked well. Hence, in fluorescent tubes of ca. 10 cm length, and the lamp was arranged
principle, the method should work with any TLC visualisation such that the tubes were parallel to the line of spots on the TLC
2
technique or development reagent.
plate to maximise the uniformity of the illumination.
Digital photography and thin layer chromatography plate
illumination
Image processing and spot quantification
Image processing was carried out using several public domain
3
No special camera is required for the DE-TLC technique. The data in packages. In our hands, the IMAGEJ software available from the
this paper were obtained using a 10.1 megapixel camera. It should US National Institute of Health proved the simplest, most
be noted, however, that for good quantification, the photography straightforward and most versatile, although other packages
of the plates does require very even lighting. For coloured analytes could be utilised. For IMAGEJ, the photographic image may be
or iodine-developed plates, this is best achieved in outside input in joint photographic expert group (JPEG) or tagged image
daylight, although other lighting can be used provided that the file format (TIFF) format. The spot image can be optimised
lamp is at a significant (effectively infinite) distance from the plate (differentiated from the background or from interfering
and that no nearby surfaces are present to act as reflectors to substances) by selection of the best colour domain (red, green
reduce the evenness of the illumination. In the case of ultraviolet or blue). Options are available within the program to invert the
adsorbing or fluorescent compounds, siting the plate at a image, to subtract background and so on. The best option to
significant distance (ca. 1.5 m) from the ultraviolet lamp and using select depends on the image. For example, an image from a
a manual exposure of several seconds at apertures around F8 TLC plate that involves fluorescence quenching by the analyte
worked well. The International Organization of Standardization/ or detection by colour or by colour reactions will usually require
American Standards Association (ISO/ASA) setting (if this was selection of the appropriate colour domain and the image will
selectable) was left on auto to allow the camera to assess the best need to be inverted such that the analyte pixels have large
sensitivity for the photograph. The best settings for a particular positive values against the background pixels, which will ideally
camera and experimental set-up were determined by bracketing have small values. Conversely, fluorescent analytes will require
the exposures around the initial values above. A typical the appropriate selection of the colour domain but will not
experimental arrangement is shown in Figure 1. The zoom require inversion as the background will be dark and the
function of the camera is used to ensure that the TLC plate image, fluorescent spots will be bright.
J. Label Compd. Radiopharm 2013, 56 544–552
Copyright © 2013 John Wiley & Sons, Ltd.
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D. P. Manthorpe and W. J. S. Lockley
Quantification of the spots via IMAGEJ can utilise various The ‘camera factor’ is a measure of the change in the pixel
approaches; two of which are simple and effective. These use response when the camera exposure is varied by a factor of 2.
different approaches to the selection and quantification of the The factor may be determined experimentally as the change in
spots. The spots may be selected, either individually by using integrated intensity of a fixed area of the camera image which
an ellipse selection tool or as a batch by selection of a channel is obtained upon doubling the camera exposure. Conversely,
across or along the plate using a sub-routine designed for the the factor can be varied, using the equation in the previous texts,
quantification of gels. Either procedure can yield good results so as to linearise the data as judged by the linear regression
depending on the actual plate under analysis and they are often coefficient of the standard line obtained. In our hands, both
equivalent in terms of accuracy and precision. In the case of the methods work well and yield the same or very similar values
gel sub-routine, the quantification is by integration of peaks for the camera factor. Once the transform is carried out, the
derived by the subroutine from the strip of spots selected. In resulting TLC data is linearised. Thus, the normalised data from
the case of individual spot selection, each spot is selected an umbelliferone analysis (Figure 2a), which shows a typical
manually and a list of integrated densities compiled by the standard curve with a negative second order coefficient, yields
program. This integrated density, sometimes referred to as ‘spot a linear standard curve after the transformation is carried out
volume ’, is essentially a mean pixel value multiplied by the (Figure 2b). The transformation approach described in the
number of pixels in the spot area selected. This selection of previous texts enables the rapid and accurate analysis of many
individual spots generally functions well and is the best option samples once the transformation has been incorporated into a
for plate images that have a high background or where the spots spreadsheet such as Microsoft Excel. However, if only a few
are not circular. Although background subtraction can be carried determinations are to be made, then simply reading the
out digitally by a subroutine in IMAGEJ, in our hands, we found unknown concentrations from a plotted standard curve is
that background subtraction was usually best achieved by straightforward and requires no mathematical or computational
quantification of equivalent blank areas of the plate near to the
analyte spots, for example, a row of blank areas above, below
or to the side of the spots. Keeping the area of the selection a)
ellipse constant at that of the largest spot removes subjectivity
in the analysis if this approach is used.
2
2
1
1
50
Integrated
Spot Density
Normalised
Data linearisation
00 0-255
Neither TLC nor photography are inherently linear techniques. In
the case of TLC, the spot shape and analyte density within the
spot can vary. Hence, it is essential to work within a reasonable
concentration range. At the low end of the standard curve, the
method is often limited by sensitivity and/or background;
whereas at the top of the range, the limitations are usually spot
shape and saturation. However, provided that a reasonable
range is selected, the resulting standard curve can be linear.
More generally, however, it follows a second order polynomial
curve. Typically, a selection of a 10-fold to 20-fold concentration
range proves most suitable for high quality analysis.
50
00
50
Concentration (mg/ml).
1.5
0
0
0.5
1
2
The raw data from the charge-coupled device detector of
digital cameras is subjected to a significant amount of
automated processing before the data is recorded as a joint
photographic expert group or TIFF file. This manipulation
enables the final file to have the correct intensities as judged
by the human eye. Unfortunately, the eye does not respond to
changes in light intensity in a linear fashion. Moreover, the
absorbtion of light by the analyte on a TLC plate should follow
an approximation to Beer's law, and hence the raw data from
quantification of spot integrated intensity needs mathematical
transformation in order to yield the linear standard curves
desirable for an analytical method. Because several factors are
involved, there is no straightforward method to linearise the
data. The procedure utilised in this work to achieve the
b)
"
Optical Density"
12
10
8
6
y = 5.8279x + 0.6462
R² = 0.9981
4
1
transformation is a modification of the method of Hess in which
an ‘optical density’ parameter is calculated from the determined
2
spot integrated density such that:
Concentration (mg/ml).
1.5
0
0
ðnormalised spot integrated density=camera factorÞ
0
Optical density ¼ 2
0
0.5
1
2
where the spot integrated density is normalised such that the
integrated density of the most intense spot (i.e. that of the
Figure 2. a. Normalised data from an umbelliferone DE-TLC analysis prior to
transformation. b. ‘Optical density’ transformed (linearised) data from an umbelliferone
highest concentration spot) has a value of 255 (for 8 bit images). DE-TLC analysis.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 544–552

D. P. Manthorpe and W. J. S. Lockley
expertise. Indeed, if a simple quantification is required, such as progress of the deuteromethylation reactions (Scheme 1) of
that for the measurement of mass for specific radioactivity theophylline and umbelliferone to yield trideuterated internal
determination, then simply using a linear interpolation from standards for the assay of caffeine and 7-methoxycoumarin by
standard samples set at multiples of 0.75-fold and 1.5-fold, the gas chromatography–mass spectrometry (GC-MS). Monitoring
expected concentration will yield reasonable results due to the for reaction completion ensured that good yields of the internal
limited curvature over this narrow twofold range.
standards were obtained by running the reactions at room
temperature and monitoring by DE-TLC until the reaction was
complete. The results of the monitoring are shown in Figures 3a
and 3b.
Results and discussion
Another example is the use of the technique to determine the
reaction stoichiometry for the borodeuteride reduction of
Determination of accuracy and precision
Table 1 shows the quality of analytical data for nine analytes quinizarin to yield labelled 1,4-anthraquinone. In this case, a
across 11 unoptimised methods using various detection
techniques. Accuracy and precision data were determined for
D
all the analytes from the analysis of triplicate high and low
concentration quality control samples against duplicate standard
curves covering the standard range and were surprisingly good.
Overall, a mean accuracy of 99 ± 6% and mean coefficient of
variation (CV) of 4.1 ± 2.9% were obtained across all the methods.
To provide some practical examples of the use of the
technique, several studies from the isotopic chemistry area are
described in the succeeding texts. They illustrate the ways in
which DE-TLC can enhance both the quantitative, semi-
quantitative and qualitative uses of the TLC technique.
O
N
D
D
O
N
H
N
CD3
I
N
N
N
N
K CO
N
2
3
O
O
D
MeOH
CD3
K CO
I
D
D
2
3
O
O
O
HO
O
O
Acetone
The first two examples are the uses of DE-TLC for monitoring
reactions. In these cases, the technique is used to monitor the
Scheme 1. Deuteromethylations of theophylline and umbelliferone.
Table 1. Accuracy and precision data for nine analytes across 11 methods using digitally enhanced thin layer chromatography
with various detection techniques
Domain
(detection)
Concentration
range
Regression
coefficient
QC CVs(%)
high, low QC
Accuracies(%)
high, low QC
Analyte
Adsorbent eluent
Quinizarin
Silica gel
Toluene
Silica gel
Blue
(Orange colour)
Green
(Magenta colour) 0.5-4 μl
Green
0.1-2.0 mg/ml
0.1-2.0 mg/ml
0.5–4 μl
0.9954
0.9954
0.9993
0.9994
0.9962
0.9895
2.55, 1.23
2.7, 2.24
4.2, 3.1
4.0, 3.2
5.1, 3.0
3.5, 4.6
95, 106
92, 105
87, 101
92, 105
100, 102
100, 99
1
,4-Diamino-
anthraquinone
Theophylline
CH Cl2
2
Silica gel F254
EtOAc/iPrOH/H O
0.2–2.0 mg/ml
0.2–2.0 mg/ml
(254 nm)
2
10:7:6 v/v/v.
Caffeine
Silica gel F254
EtOAc/iPrOH/H O
Green
(254 nm)
0.2–2.0 mg/ml
0.2–2.0 mg/ml
0.9892
0.9897
2.5, 3.0
2.0, 3.8
101, 110
100, 102
2
10:7:6 v/v/v.
Umbelliferone
Umbelliferone
Umbelliferone
Acetanilide
Silica gel F254
Green
25% EtOAc/CH Cl2 (254 nm)
0.2–2.0 mg/ml
0.1–2.0 mg/ml
0.9902
0.9988
5.6, 12.8
4.2, 0.91
98, 97
100, 99
2
Silica gel F254
Green
25% EtOAc/CH Cl2 (360 nm)
2
2
2
Silica gel F254
Blue
(Iodine vapour.)
Green
0.1–2.0 mg/ml
0.125-1.0 mg/ml
6.25-100 μg/ml
0.9839
0.9918
0.9969
3.9.9.2
1.9, 1.4
6.9, 3.2
99,92
2
5% EtOAc/CH
Silica gel F254
5% EtOAc/CH
Silica gel F254
EtOAc/iPrOH/H
0:7:6 v/v/v.
Cl
2
95, 98
98, 101
2
Cl2 (254 nm)
Green
Riboflavin
2
O
(360 nm)
1
Nicotinamide
Silica gel F254
EtOAc/iPrOH/H
Green
(254 nm)
0.1–2.0 mg/ml
0.1–2.0 mg/ml
0.9957
0.9808
8.0, 3.4
89,94
2
O
10:7:6 v/v/v.
2
′-Chloro-
Silica gel F254
25% EtOAc/CH
Green
(254 nm)
11.9, 1.9
117,95
acetanilide
2
Cl
2
J. Label Compd. Radiopharm 2013, 56 544–552
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D. P. Manthorpe and W. J. S. Lockley
a)
yielded the information required to set a suitable stoichiometric
ratio (Figure 4).
100
A similar use of the technique was in the optimisation of the
deuterodechlorination of 2'-chloroacetanilide by deuterium gas,
%
9
0
0
0
0
0
0
0
0
0
2
8
7
6
5
4
3
2
1
catalysed by 5% palladium on carbon, to yield [2'ꢀ H]acetanilide
4
(
Scheme 3). Here, extensive studies have been carried out into
the role played by the tertiary amine base, which is normally
5
included in the reaction. Both DE-TLC and quantitative nuclear
Theophylline
Caffeine
magnetic resonance (NMR) methods were utilised to determine
the yield of product in the presence of various tertiary amine
bases (Figure 5) or in the presence of a single base at different
times (Figure 6). There was a reasonable correlation between
the DE-TLC determinations and the quantitative NMR method
(Figure 7), which was based upon the integration of the methyl
Time (h)
group resonances of the starting material and product in the
proton NMR of the reaction mixture. However, the latter method
was not validated and would clearly be susceptible to any
interferences arising from minor undetected and/or unresolved
by-products. Hence, the source of the observed differences
cannot be definitively ascribed to one or other of the methods.
The aforementioned examples are included to show the ability
of DE-TLC to carry out many determinations in parallel.
The selectivity conferred upon the DE-TLC technique by the
ability to analyse TLC plates in the three different colour domains
is shown clearly by some studies from the field of carotenoid
chemistry. Figure 8 shows how the carotenoids, chlorophylls
and pheophytin present in an aged acetone extract of cress
seedlings are differentiated by the selection of the appropriate
colour domain.
In a further carotenoid study designed to investigate the
preparation of poly-deuterated carotenoids for HPLC-MS
analysis, the reduction in carotene and xanthophyll yields
occasioned by growing cress seedlings on a 1:1 mixture of water
and deuterium oxide was easily quantified by DE-TLC analysis of
a simple saponified extract (Figure 9). In this case, there were
clear reductions in all the xanthophyls and carotenes; and in
the case of two uncharacterised xanthophylls, the reductions
were very marked (Figure 10).
0
0
50
100 150 200 250 300 350 400 450 500
b)
100
%
9
0
0
0
0
0
0
0
0
0
8
7
6
5
4
3
2
1
%
Umbelliferone
%7-MeO-Coumarin
Time (h)
0
0
200
400
600
800
1000
1200
1400
1600
Figure 3. a. Monitoring of the deuteromethylation of theophylline to yield
Although the aforementioned studies were carried out as
inexpensively as possible, some improvements to the technique
might well be made for only small increases in cost. Thus the use
of the JPEG file format was selected as it is the commonest file
2
[
methylꢀ H
3
]caffeine. b. Monitoring of the deuteromethylation of umbelliferone
2
to yield [methylꢀ H
3
]7-methoxycoumarin.
semi-stable intermediate is formed, which is converted to the format used in inexpensive cameras. This format only has an 8
product by acid catalysed hydrolysis and dehydration bit data depth (i.e. a maximum range of 0–255 per pixel per
(Scheme 2). Analysis of the reaction mixtures by DE-TLC allowed colour domain). Moreover, JPEG data are compressed, with the
the optimum stoichiometry to be determined prior to a large potential for some data loss, especially if files are saved several
scale reaction. Because all the components present were times. Nevertheless, JPEG files are very economical in the
coloured, analysis required no specific detection method and amount of memory used. The use of TIFF or raw files would avoid
was straightforward. Of course, in the absence of known the possibility of data loss during the processing of the image
extinction values for all the analytes, the method allowed only and additionally could lead to a better dynamic range because
a semi-quantitative determination of concentrations but still the file format supports a greater data depth (typically 8,12,16
R
B
O
O
OH
OH
O
O
O
O
D
D
O
O
D
D
+
NaBD4
MeOH
H
B
R
Scheme 2. Borodeuteride reduction of quinizarin.
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 544–552

D. P. Manthorpe and W. J. S. Lockley
1
1
1
4.00
2.00
0.00
"Optical density"
8
6
4
2
0
.00
.00
.00
.00
Quinizarin
BD4-Complex
1,4-Anthraquinone
.00
0
.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Mol. ratio NaBD4/quinizarin
Figure 4. ‘Optical density’ of labelled 1,4-anthraquinone versus reaction stoichiometry.
O
O
O
1
00
90
80
2
'-Chloroacetanilide (DE-TLC)
HN
HN
HN
[2'-2H]Acetanilide (Q-NMR)
2'-2H]Acetanilide (DE-TLC)
Cl
D2
D
H
[
+
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
Pd on carbon
%
Yield
Scheme 3. Deuterodechlorination of 2'-chloroacetanilide.
Time (Minute)
0.0
50.0
100.0
150.0
200.0
Figure 6. Percentage yield of labelled acetanilide against time (minutes) determined
by DE-TLC or nuclear magnetic resonance.
60.0
Figure 5. DE-TLC analysis of the efficiency of 11 tertiary amine bases in the
deuterodechlorination of 2'-chloroacetanilide. The bottom row of spots is the
labelled acetanilide product and the top row (higher Rf) is the 2'-chloroacetanilide
starting material. The extreme right bottom spot is an acetanilide standard.
Q-NMR
Method
50.0
y = 0.9979x-3.3338
R² = 0.982
4
3
2
0.0
0.0
0.0
or 24 bits). However, not all cameras can support these formats
and the data files are considerably larger placing greater
demands on the storage capacity of the camera.
Another possible improvement would be the addition of a
cut-off filter to the ultraviolet light source to remove light other
than the desired wavelength. Only one of the light sources used
in this study (Mineral Light UVGL-25, (UVP Inc, San Gabriel,
California, USA)) was equipped with such a filter, which should
reduce the background light on the image and hence could
improve contrast, sensitivity and selectivity.
10.0
DE-TLC
Method
0.0
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
Experimental
The TLC plates used throughout were Machery-Nagel aluminium-backed
Figure 7. Correlation between DE-TLC and an unvalidated quantitative nuclear
silica gel plates of thickness 0.1 mm containing a fluorescent indicator
magnetic resonance method for the determination of labelled acetanilide.
J. Label Compd. Radiopharm 2013, 56 544–552
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D. P. Manthorpe and W. J. S. Lockley
that could be excited at 254 nm and were obtained from Fisher Scientific,
(
Loughborough, UK).
The camera used was a Panasonic Lumix FZ28 model (Amazon.co.uk,
Slough, Berkshire, SL1 1QP), athough similar results were obtained with
other cameras.
The ultraviolet lamps used for 254 nm illumination were a Hygeniclean
4
W UV-C sterilising wand (UK Import & Export Ltd, Braithwell S66 7RL, UK
or a 4 W Mineral Light UVGL-25, (UVP Inc, San Gabriel, California, USA).
The latter was also used for fluorescence illumination at 360 nm. An
alternative light source constructed from commercial 8 W 254 nm and
3
2
60 nm T5 300 mm screened fluorescent tubes was also evaluated, at
m distance from the TLC plate, and this arrangement also proved
suitable for DE-TLC.
The IMAGEJ program was available free of charge from the National
Institute of Health (NIH), USA and was downloaded from the website at
http://rsbweb.nih.gov/ij.
The spotting of solutions was carried out using Drummond microcaps
(
Sigma-Aldrich Company Ltd, Dorset, England), although similar results
were obtained using a 5 μl Hamilton syringe from the same supplier or
an Eppendorf Reference variable volume pipette (1-10 μl), set at 5 μl
Figure 8. DE-TLC analysis of an aged acetone extract of cress (Lepidium sativum)
seedlings in the various colour domains. Carotenes at highest Rf values.
(
Fisher Scientific, Loughborough, UK).
Deuterium oxide, sodium borodeuteride, 2'-chloroacetanilide,
acetanilide, caffeine, umbelliferone, theophylline and β-carotene were
obtained from Sigma Aldrich Company Ltd, Dorset, England). Standard
®
lutein was obtained by hydrolysis of Xangold (Lifeplan Products Ltd,
Lutterworth, Leics LE17 4ND) using sodium hydroxide in MeOH and
was purified by TLC and stored atꢀ20°. Deuteromethyl iodide was
obtained from Cambridge Isotope Laboratories (UK agents CK Gas
Products Ltd. Hook, Hampshire, RG27 7GR). All other reagents, chemicals
and solvents were obtained from recognised chemical suppliers and
used without purification.
Typical assay procedures
Determination of accuracy and precision values
A TLC plate (20 × 10 cm in landscape format) was spotted with
aliquots (typically 5μl) of the standard and quality control samples.
Under these conditions, 18 spots can be accommodated per plate.
Generally, the data in Table 1 were generated using six standard
Figure 9. Variation of carotenoid recovery from cress (Lepidium sativum) seedlings concentrations of analyte spotted in duplicate (12 spots) covering
grown using H
plate (highest Rf value).
2
O or 50%D
2
O (= D
2
O/H
2
O 1:1 v/v). Carotenes are at the top of the
a 10-fold or 20-fold concentration ranges. Two quality control test
samples with concentrations near the upper and lower ends of the
assay standard curve were also spotted in triplicate (6 spots)
making the 18 spots in total. The transformed data from all the
standard spots were utilised to construct a standard line and the
concentrations of the quality control samples were determined
using the transformed data from the quality control samples and
the equation of the standard line. Analysis of the resulting
concentration data enabled the determination of accuracy and
precision for the low and high quality control samples.
Reaction monitoring and optimisation
2
[
methylꢀ H ]caffeine from theophylline
3
To theophylline (184 mg,1.02 mmol) dissolved in MeOH (16 ml)
and potassium carbonate (207 mg, 1.5 mmol) contained in a
2
0 ml screw-top glass vial complete with stirrer, was added
2
[
H
3
]methyl iodide (128 μl, 2.0 mmol) and the reaction kept at
room temperature and monitored by DE-TLC. For this procedure,
0 μl aliquots of the reaction mixture were evaporated and
5
dissolved in water (1 ml). Aliquots (5 μl) of the resulting solution
were analysed by DE-TLC using silica gel F254 eluted with ethyl
acetate/propan-2-ol/water: 10:7:6 v/v/v). After the reaction was
completed, the MeOH was evaporated and the resulting white
Figure 10. Relative yields of carotenoids from cress grown on a mixture of H
and D O (1:1 v/v) in comparison with cress grown on H O.
2
O
2
2
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 544–552

D. P. Manthorpe and W. J. S. Lockley
solid was triturated three times with dichloromethane (3 ml each acetone (0.5 ml). Aliquots of the resulting solution (5 μl) were
time). The filtered extracts were combined and evaporated to analysed by DE-TLC (silica gel F254, dichloromethane/toluene
2
yield [methylꢀ H
3
]caffeine (186 mg, 92% based on theophylline, 1:1 v/v) at 0.5, 1.0 and 1.5 h. The analyses were identical at all
4
6% based on CD I which was used in 100% excess). The the time-points showing that the reaction was rapid. Analysis
3
compound was pure by TLC (silica gel F254, EtOAc/iPrOH/H20 of the samples from the five reactions at 1.0 h were used to
0:7:6 v/v/v). The yield would have been 95% if the material construct Figure 4, which showed that a mol-ratio of ca. 2.5 or
1
utilised for DE-TLC monitoring was taken into account. The greater would be required to achieve a high yield of the desired
2
isolated material was characterised by GC-MS (m/z (%): 197 product. The [9,10ꢀ H ]1,4-anthraquinone product had m/z 210
2
(100%), 168 (5%), 112 (40%), 85 (14%), 70 (11%), 58(16%) with (10%),182 (24%),154 (75%),128 (69%), 99 (4%) 77(16%) and δ
no detectable peak at the unlabelled molecular ion of 194) and (500 MHz, CDCl3) 7.05 (2H), 7.68 (2H), 8.10 (2H) and 8.6 (small
1
2
by H-NMR, δ (500 MHz, CDCl ) 3.41(3H), 3.59 (3H), 4.00 (absent, residual traces) ppm. The H-NMR (CHCl ) showed only a single
3
1
3
3
<
0.06%), 7.51 (1H) ppm and C-NMR (δ,CDCl , 27.9, 29.7, 33.6 resonance at δ 8.59 ppm, the expected position of labelling at
3
(small multiplet only), 107.6, 141.4, 148.7, 151.7, 155.5 ppm. The the nine and ten positions. The corresponding unlabelled
deuterated batch assayed as 97.8% caffeine by GC-MS against material had the same retention time as the labelled compound,
authentic unlabelled caffeine (Sigma Aldrich) by comparison of and the NMR and MS spectra were essentially identical with the
6
the intensities of the 197 and 194 ions of the labelled and literature mass spectrum , and the labelled compound showed
unlabelled caffeine.
only the differences associated with labelling in the nine and
ten positions.
2
[
methylꢀ H ]7-methoxycoumarin from umbelliferone
3
Efficiency of tertiary amine bases in facilitating the
deuterodehalogenation of 2'-chloroacetanilide with
deuterium gas over palladium
Potassium carbonate (236 mg, 1.71 mmol) was weighed into a
0 ml flask and umbelliferone (162 mg, 1.00 mmol) in acetone
5ml) was added. Deuteromethyl iodide (200μl, 3.15 mmol) was
1
(
then added, and the reaction stirred slowly with monitoring To 2'-chloroacetanilide (84.8 mg, 0.500 mmol) in tetrahydrofuran
by DE-TLC (5μl aliquots analysed using silica gel solution (2 ml) was added 5% 2.0 mmol, palladium supported on
F /
254
dichloromethane) until the reaction was entirely complete. The carbon (Aldrich item 27,670–7, 30 mg) and the chosen base
solvent was removed under a stream of nitrogen, and the residue (0.72 mmol) added. The reaction suspension was sealed in
was triturated with three 2 ml portions of dichloromethane. The 10 ml capacity reaction tube, evacuated under vacuum then
combined dichloromethane extracts were filtered and the filter flushed twice with deuterium. Under these conditions, ca.
cake also was washed with dichloromethane (2ml). Removal 0.36 mmol of deuterium gas was present. The reaction was then
2
of the dichloromethane under a stream of nitrogen yielded [ H
3
]
stirred rapidly for 180 min. Under these conditions, the substrate
7-methoxycoumarin as a very pale yellow solid (180.2 mg, 100% was in excess over the deuterium gas and hence the maximum
based on umbelliferone, 31% based on CD I which was used in possible yield of acetanilide was 72%. After the reaction, the
3
ca. threefold excess to speed the reaction). TLC, as catalyst was removed by filtration and the solvent removed
aforementioned, showed only a single spot (Rf = 0.35) with no under a stream of nitrogen. The crude product was dissolved
detectable umbelliferone (Rf = 0.1) and no other components. in dichloromethane (2 ml), washed, to remove base, with 2 N
GC-MS similarly showed only a single peak. Crystallisation from HCl, (2 ml) then with water (2 ml). The dichloromethane was
dichloromethane (1.5ml) by slow addition of hexane (6× 1 ml) dried and evaporated to yield the crude reaction product for
yielded diamond-shaped crystals (140.1mg, 78% overall based evaluation by DE-TLC, NMR and GC-MS. Portions (2 mg) of the
on umbelliferone) along with material isolated from the mother isolated material from reactions carried out with each base were
liquors (38.8mg), which still showed no other compounds present separately dissolved in dichloromethane (2 ml) and aliquots of
by TLC. The material was characterised by GC-MS (m/z (%): 179 the solutions (5 μl) spotted for DE-TLC comparison of the extent
(
100%), 151 (80%), 133 (94%), 105 (10%), 77 (19), 51 (14%) and of reaction. A standard sample of acetanilide at 1 mg/ml in
1
by NMR H-NMR, δ (500 MHz, CDCl ), 3.88 (not detectable), 6.25, dichloromethane was also spotted.
3
(
d,1H), 6.86–6.82 (complex multiplet, 2H),7.37(d,1H), 7.64 (d, 1H)
1
3
ppm and C-NMR (δ, CDCl3), 55.8 (not detectable),100.9, 112.6,
13.1, 128.8, 143.4, 156.0, 161.2,162.9 ppm.
Growth of Lepidium sativum seedlings on deuterated
medium and extraction of carotenes and xanthophylls
1
Into the bottom of each of four screw-top glass tubes (height
Determination of the optimum stoichiometry for the sodium
8
cm and diameter 1.5 cm) was placed a pad of sufficient surgical
borodeuteride reduction of quinizarin to yield labelled
2
gauze to reach to 1.5 cm above the base. Aliquots of water (2 ml
each) was added to two of the vials and a mixture of water and
[
9,10ꢀ H ]1,4-anthraquinone
2
Sodium borodeuteride (42 mg, 1.00 mmol) was dissolved in deuterium oxide (1:1 v/v, 2 ml each) was added to the other two
MeOH (2 ml) containing sodium hydroxide (1.2 μl of 10% w/v vials. To each of the vials was added 15 seeds of Lepidium
solution in MeOH) to ensure that the solution was alkaline, thus sativum, such that the seeds were spread over the surface of
stabilising the borodeuteride. Aliquots of this solution (sufficient the gauze and the vials were capped. The seedlings were then
to achieve a final molar ratio of sodium borodeuteride to allowed to germinate. Germination at 10 days was 26/30 for
quinizarin of 0.3125, 0.625, 1.25 and 5.0) were added to five the 1:1 deuterium oxide/water vials and 29/30 for the water only
reactions vials each containing quinizarin (25 mg, 0.104 mmol) vials. The seedlings were separated, blotted dry using filter
dissolved in sufficient MeOH to ensure that the final volume in paper, and the cotyledons were removed with scissors (yields,
each reaction was 2 ml. Aliquots (50 μl) from each reaction were 301 mg for the deuterium oxide/water group and 270 mg for
treated with hydrochloric acid (2 N, 100 μl) and dissolved in the water only group). To each group of cotyledons was added
J. Label Compd. Radiopharm 2013, 56 544–552
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

D. P. Manthorpe and W. J. S. Lockley
1
0% w/v sodium hydroxide in MeOH (2 ml) and dichloromethane chemical analysis. DE-TLC is particularly appropriate for
(
2 ml) and the vials stirred in the dark for 18 h to destroy the laboratories without access to expensive quantitative TLC
chlorophyll and to hydrolyse the xanthophyll esters. Water equipment or to those laboratories where contamination issues
4 ml) was added to each of the hydrolysis reactions and the precludes the use of such equipment.
(
dichloromethane layer separated. The aqueous layer was
washed with dichloromethane (1 ml) and the combined
dichloromethane extracts were washed three times with water
Conflict of Interest
(
3 × 5 ml) the dichloromethane layer was then dried by passing The authors did not report any conflict of interest.
through a pad of cellulose tissue, washing through with a further
ml of dichloromethane, and evaporated to dryness at room
1
References
temperature in the dark. The resulting extracts were each
dissolved in dichloromethane (0.5 ml) and the carotene and
[
1] A. V. I. Hess, J. Chem. Educ. 2007, 84, 842.
xanthophyll contents compared by DE-TLC (silica gel F254, [2] R. Johnsson, G. Träff, M. Sundén, U. Ellervik, Evaluation Of quantitative
thin layer chromatography using staining reagents, J. Chromatogr. A
acetone/hexane 2:3 v/v or ethyl acetate/dichloromethane 1:1v/v)
by spotting equal fixed aliquots (15 μl).
2
007, 1164, 298-305.
[
3] The excellent ImageJ image processing software. http://rsbweb.nih.
gov/ij
[4] a) W. J. S. Lockley, D. J. Wilkinson, J. R. Jones. J. Label. Compd.
Radiopharm. 2006, 49, 401-402 and references therein;
b) W. J. S. Lockley, D. D. Jenkins, D. P. Manthorpe, J. Label. Compd.
Radiopharm. 2013, 56(2), 50-63.
Conclusions
The combination of digital photography with image processing
and data transformation has enabled the easy and cost effective
use of TLC as a quantitative technique for assay or as a semi-
quantitative technique for reaction monitoring. The DE-TLC
approach improves both the sensitivity and selectivity of TLC
analysis and will enable the technique to play a greater role in
[
5] Y. Monguchi, A. Kume, K. Hattori, T. Maegawa, H. Sajiki, Tetrahedron
006, 62, 7926–7933.
2
[
6] The Spectral Database for Organic Compounds: Advanced Industrial
Science and Technology, Japan. http://sdbs.riodb.aist.go.jp [25 Jan 2013],
spectrum 15066.
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 544–552