Dielectric Barrier Discharge Ionization in Characterization of Organic Compounds Separated on Thin-Layer Chromatography Plates
et al. (2014) Dielectric Barrier Discharge Ionization in Characterization of Organic Compounds
Separated on Thin-Layer Chromatography Plates. PLoS ONE 9(8): e106088. doi:10.1371/journal.pone.0106088
Dielectric Barrier Discharge Ionization in Characterization of Organic Compounds Separated on Thin-Layer Chromatography Plates
Micha Cegowski 0
Marek Smoluch 0
Micha Babij 0
Teodor Gotszalk 0
Jerzy Silberring 0
Grzegorz Schroeder 0
Andrew C. Gill, University of Edinburgh, United Kingdom
0 1 Department of Supramolecular Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Poznan, Poznan , Poland, 2 Department of Biochemistry and Neurobiology, Faculty of Materials Science and Ceramics, AGH-University of Science and Technology , Krakow , Poland , 3 Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology , Wroclaw , Poland , 4 Center for Polymer and Carbon Materials, Polish Academy of Sciences , Zabrze , Poland
A new method for on-spot detection and characterization of organic compounds resolved on thin layer chromatography (TLC) plates has been proposed. This method combines TLC with dielectric barrier discharge ionization (DBDI), which produces stable low-temperature plasma. At first, the compounds were separated on TLC plates and then their mass spectra were directly obtained with no additional sample preparation. To obtain good quality spectra the center of a particular TLC spot was heated from the bottom to increase volatility of the compound. MS/MS analyses were also performed to additionally characterize all analytes. The detection limit of proposed method was estimated to be 100 ng/spot of compound.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This work was supported by the Polish National Science Center (NCN: www.ncn.gov.pl; grant no. 2011/03/B/ST5/01573). GS received the funding. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Thin-layer chromatography (TLC) is a very simple,
costeffective and fast chromatographic technique allowing separation
of most chemical mixtures . Detection of separated compounds
relies mostly on their optical visualization using UV light or
appropriate reagents, such as Dragendorff reagent. The
information obtained by these means is, however very limited. For definite
identification of the compound, which has been visualized, it must
be compared with a standard that has been simultaneously eluted
on TLC. Coupling TLC and mass spectrometry (MS), which is a
very useful tool for structural analysis of organic compounds,
would make a simple and easy to operate technique. That is why
the application of mass spectrometry in characterization of
compounds separated on TLC plates has been a subject of
interest for many scientists [2,3] and recently some reviews on this
topic have been published [4,5,6,7]. The TLC-MS methods can
be divided into indirect sampling TLC-MS and direct sampling
Indirect sampling TLC-MS is of less importance because it
requires time-consuming processes, such as scratching of the spot
containing particular compound, followed by solvent extraction of
the adsorbed substance . The direct sampling TLC-MS
techniques allow for mass spectrometric analysis of compounds
directly from the surface of TLC plates. These methods can be
further divided into vacuum-based and ambient TLC-MS
Desorption/ionization methods that operate under vacuum
have been used to obtain mass spectra of compounds directly from
the surface of TLC plates. Several techniques such as fast atom
bombardment (FAB) [9,10], secondary ion mass spectrometry
(SIMS) , laser desorption (LDI) , matrix-assisted laser
desorption/ionization (MALDI) [13,14], surface-assisted laser
desorption/ionization (SALDI)  have been applied to
characterize compounds separated by TLC. These techniques however,
generate several problems: obtaining high vacuum in the ion
source considerably increases the time of analysis, volatile
compounds have poor sensitivity, MALDI matrices produce
interferences with mass spectra, poor reproducibility in
quantitative analysis, diffusion of analytes on TLC plates after applying
MALDI matrix solution .
Some of the presented problems were solved by coupling
ambient MS techniques with TLC. Hence, the TLC plates do not
need to be placed in a vacuum chamber for ionization, and the
analysis is much faster without the risk that volatile compounds
will desorb from the plate before MS analysis. Moreover, the TLC
plate size is not limited by the dimensions of a vacuum chamber.
Techniques, such as electrospray ionization (ESI) [17,18,19],
electrospray-assisted laser desorption ionization (ELDI) ,
desorption electrospray ionization (DESI) , laser desorption/
atmospheric pressure chemical ionization (LD-APCI) ,
atmospheric pressure matrix-assisted laser desorption/ionization
(APMALDI) [23,24] and direct analysis in real time (DART) [25,26]
have been adopted to generate mass spectra of compounds directly
from TLC plates.
Dielectric barrier discharge ionization (DBDI) produces
lowtemperature plasma by dielectric barrier discharges (DBD) .
DBD are obtained at ambient conditions and are formed between
two electrodes with a dielectric layer that separates them. The
presence of a dielectric layer limits the average current in the gas
space, which causes formation of the low-temperature plasma
containing a large number of high energy electrons [28,29]. DBD
plasma was used to produce mass spectra of compounds desorbed
from different surfaces , to detect nonvolatile chemicals
directly on various surfaces , utilized as an ion source for
liquid chromatography/mass spectrometry , and coupled on
line with TLC for mercury speciation .
Herein we report the coupling of DBD plasma source with
TLC. DBDI is capable of providing very fast and selective
ionization of each particular compound separated on TLC plate.
The ionized compounds can be characterized using MS and MS/
MS modes. Combination of these approaches can find many
applications, particularly in synthetic organic laboratories and
Materials and Methods
Synthesis of compounds 16, whose structures are presented in
Figure 1, as well as information about reagents used are given in
the Information S1.
The DBD plasma source consists of quartz capillaries (O.D.
1.5 mm and I.D. 0.8 mm). The electrodes of 2 mm width were
made of copper rings surrounding the capillary tube and the gap
between the inner edges was 5 mm. The grounded electrode was
placed 6 mm apart from the capillary end. The plasma was
operated with helium (99.996% purity) flow of 1 L/min, by
applying a voltage of 8 kV. Additional data describing DBD ion
source are given in details elsewhere . A Bruker Esquire 3000
quadrupole ion trap mass spectrometer (Bruker Daltonics,
Bremen, Germany) was used for all measurements. The typical
ESIMS source settings were found to be optimal also for the DBDI
source, with the exception of the mass spectrometer entrance glass
capillary voltage, where lower potential (1 kV) compared to the
standard ESI setting (4.5 kV) was used. The temperature of the
glass capillary was set to 200uC, the drying gas flow was
maintained at 3 L/min., and the nebulizer gas (N2) was not
applied. The scan range was set from 80 to 500 m/z. For MS/MS
experiments the isolation width was set to 2 m/z and the
fragmentation amplitude was in the range of 0.5 to 0.8 unit.
TLC preparation and detection
Compounds 16 were dissolved in dichloromethane to obtain
final concentration of 10 mg/mL. One mL sample solutions were
spotted on a TLC plate (Merck Millipore TLC silica gel 60 F254
aluminium sheets). The compounds were spotted in ester-alcohol
pairs on a single TLC plate. The plates with compounds 1 and 2
were developed with CH2Cl2/Et2O (1:1, v/v) solution, those with
3 and 4 were developed with CH2Cl2/Et2O (5:1, v/v), those with
5 and 6 were developed with CH2Cl2/Et2O (10:1, v/v). The plate
containing all six compounds was developed with CH2Cl2/Et2O
(10:1, v/v). For MS analysis, the spots were visualized on TLC
with UV light and marked with a pencil. The TLC plate was then
cut through the centers of all spots, resulting as a narrow strip, and
a heating element, of a diameter similar to the size of a spot, was
attached to the bottom of a particular spot. The strip was then
heated from the bottom and held manually so that the plasma
released from DBD ion source would emerge just above the
marked spot. The heating element consisted of resistance wire
which temperature was constantly increased in the range 25uC
400uC using manual control until the signal of particular analyte
was visible on mass spectra. Each analysis has been completed in
less than one minute. After each m/z measurement the heating
element was allowed to cool down. It was then attached to the
bottom of the next spot and the measurements were continued.
Figure 2 displays a schematic illustration of coupling DBDI to
TLC, whereas Figure 3 shows a photograph of experimental setup
used for TLC-MS analysis.
Results and Discussion
To examine the performance of DBDI in the on-spot detection
of compounds separated on TLC plates, solutions of six
compounds were deposited onto plates. The compounds were
applied in ester-alcohol pairs on a single TLC plate, because these
pairs are resolved properly on TLC and alcohols are products of
ester reduction. The spectra obtained from three TLC plates with
the six compounds are presented in Figure 4. Each spectrum
represents the major ion corresponding to the protonated
molecule with little or no background ions. The most intense
signals belonging to contaminants are observed at m/z 279.1 and
205.1 and derive from di-n-butyl phthalate (DBP) , which is
commonly used as a plasticizer in plastics, from which DBD ion
source has been manufactured. Particularly, the tube used to
transport helium from gas cylinder to DBD ion source, is made of
polyvinyl chloride (PVC) for which DBP is used as a plasticizer.
We therefore, believe that the possible source of contamination is
desorption of DBP by helium stream. An advantage of DBDI
technique is its ability to produce intact (usually protonated)
species with little or no fragmentation what substantially simplifies
identification of the compounds separated on TLC.
To show that good quality mass spectra can be generated even
when compounds are poorly resolved on TLC, all six compounds
were deposited and separated on a single TLC plate. The resulting
mass spectra of all six compounds are presented in the
Information S1. Each spectrum obtained represents the major
ion corresponding to the protonated molecule of a particular
compound with only small traces of other compounds resolved on
The structures of the analytes were confirmed by MS/MS
analysis of all compounds. The exemplary MS/MS spectrum of
the signal at m/z 190.1 (assigned to protonated compound 4) is
presented in Figure 5. Only one daughter ion ([4+H H2O]+ =
m/z 172.1) appeared in the mass spectrum. MS/MS spectra of all
compounds examined are presented in the Information S1.
The detection limits of compounds deposited on TLC plates
analyzed by DBD ion source were estimated using the solutions
containing different concentrations of compounds 16. Each
solution was deposited onto TLC plate and after air-drying the
Figure 4. Photographs of TLC plates (visualized in UV light) with corresponding mass spectra obtained from particular spots. The
spots were assigned to the label of particular compound. Left panel represents the mass spectra of compounds with higher Rf value (esters).
plate was heated as described in the Methods section and
introduced into the gas stream. The best result was obtained for
compound 3 where 100 ng/spot was detected. However, in the
spectrum obtained (presented in Figure 6) the most intense signal
comes from DBP contamination. Nevertheless, this result is
sufficient for TLC-MS analysis because the amount of 100 ng/
spot of compound 3 could not be seen under the UV light,
therefore this amount is far less than needed for common TLC
analysis. It is worth noting, that this is only semi-quantitative
analysis because the ion intensity of an analyte desorbed from
TLC plate varies depending on the thickness and composition of
silica gel, temperature, diffusion coefficient of the analyte on the
In this paper we demonstrated the use of DBDI in conjunction
with TLC separations. This technique appeared to be effective for
direct analysis of compounds that can be separated by thin layer
chromatography. The coupling of DBDI and TLC has many
advantages, such as ionization under ambient conditions, low-cost
of a DBD ion source, easy and fast analysis of volatile and
semivolatile compounds, unlimited dimensions of TLC plate and fast
sample switching. These advantages make DBDI a robust and
convenient mass spectrometric technique, which allows for fast
screening of TLC plates that are run every day in many
Figure S1 Photograph of TLC plate (visualized under
UV light), on which all six compounds have been
separated, combined with mass spectra obtained from
respective spots. The spots and the mass spectra were assigned
to the number labeling of particular compound.
Figure S2 MS/MS spectra of ions at: a) m/z 155.2
(assigned to protonated compound 1); b) m/z 113.3
(assigned to protonated compound 2); c) m/z 232.1
Conceived and designed the experiments: MC MS JS GS. Performed the
experiments: MC MS. Analyzed the data: MC MS JS GS. Contributed
reagents/materials/analysis tools: MC MS MB TG JS GS. Contributed to
the writing of the manuscript: MC JS GS.
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