Bruker Daltonics
Application Note # MT-94
Direct Read-out of Thin Layer Chromatography (TLC)
using MALDI-TOF
Thin Layer Chromatography (TLC) is broadly established
to separate, characterize and quantify food and biological
ingredients/components. Recently interest in mass
spectrometric analysis of TLC-separated compounds has
increased significantly. Here we will introduce the direct
coupling of TLC with MALDI-TOF mass spectrometry:
TLC-MALDI; an adapter target for TLC plates and an
extension of the standard flexSoftware that turns the
MALDI into a molecular TLC readout platform. Also included
are the first results from lipid analysis.
Introduction
TLC separations remain indispensable for a number of
applications such as forensics, herbals, food, cosmetics
and clinical/industrial applications. Particular analytes
which prove difficult for HPLC separations, such as lipids,
are widely characterized by TLC. In addition, TLC remains
a quick and simple method to monitor the progress of
chemical reactions in the organic synthesis laboratory. For
many of these applications, the assignment or confirmation
of molecular structures to TLC-separated spots is
important, as RF (“ratio of fronts”) values alone are often
not highly reproducible and do not allow the unequivocal
assignment of a certain compound. However, currently
established TLC-MALDI protocols involve scratching the
chromatographic phase off the carrier and elution of the
analyte prior to spectroscopic or mass spectrometric
analysis. This process is not only time consuming and
carries the risk of loosing some compounds, but it also
relies on previously detected and manually marked spots.
Therefore, the structural analysis depends on the ability to
visualize the particular analyte by specific staining agents
or by unspecific fluorescence quenching. As staining
methods do not resolve individual compounds with similar
RF values, the achievable analytical resolution is typically
limited by the detection method. Here we introduce matrix-
assisted laser desorption/ionisation time-of-flight mass
spectrometry (MALDI-TOF MS) to directly read out TLC
traces reproducibly whilst maintaining the chromatographic
resolving power.
Workflow
The hyphenated TLC-MALDI approach requires the
uniform coverage of the TLC lanes with MALDI matrix as
a preparative step comparable with the effort of staining.
The mass spectrometric information is subsequently
read out automatically. MALDI plate movement along the
chromatographic lanes is driven by the standard movement
mechanics of the MALDI ion source without additional
robotic requirements. A chromatographic run is generated
for each lane. It can then be analyzed using TLC-MS
software, providing the extracted ion chromatograms
of individual analytes independent of dye staining and
at a selectable resolution which largely depends on the
chromatography itself.
 Fig. 1: MTP sized TLC-MALDI
Adapter Target (Order No
#255595) for 50x75 mm TLC
Silica gel (aluminum backed).
After TLC separation and matrix
coating they are ready for
MALDI analysis.

Experimental ImagePrep allows fully automated matrix preparation with

highest spatial resolution and reproducibility. A 100 mg/
TLC
ml solution of DHB in acetonitrile/water (1:1, v/v) was
All TLC-MALDI experiments were performed using either used for manual matrix application. 50 mg/ml DHB was
aluminum backed 50×75 mm TLC Silica gel 60 F254 plates, used for ImagePrep preparations. Generally, in contrast to
200 µm layer thickness (Merck, # 1.05549.0001) or 200×200 conventional MALDI, a larger excess of the matrix over the
mm TLC Silica gel 60 plates on aluminum backs, 200 μm analyte is needed if spectra are to be recorded directly from
layer thickness) (Merck, # 1.05553.0001) and developed in the TLC plate.
horizontal developing TLC chambers using CHCl3, ethanol,
TLC-MALDI software
water, triethylamine (35:35:7:35, v/v/v/v) as the solvent
system for lipids and phospholipids, whereas glycolipids
were separated under acidic conditions (CHCl3, CH3OH,
acetic acid (65:25:10, v/v/v)). Lipids were obtained by
extraction of cellular suspensions or biological tissues,
prepared and visualized by spraying with a solution of
primuline as described [1-3].

Coupling of TLC with MALDI-TOF MS

The 200×200 mm TLC plates were cut to 50×75 mm to fit

the TLC-MALDI Adapter target (Bruker, Order Nr. 255595);
50×75 mm TLC plates were unaltered (Fig. 1). MALDI
spectra were acquired from the matrix coated TLC plates
as described in [1]. Matrix was added either manually using
conventional dried droplet preparation on discrete primuline
stained spots (marked by pencil) or the entire TLC plate was
homogeneously coated with matrix (1-5 mg/cm2) for MALDI- Fig 2: TLC-MALDI setup dialog. The wizard driven software interface
imaging or TLC-scanning using the ImagePrep preparation allows for simple analysis setup with multiple chromatographic lanes
device (Bruker Daltonics) [4]. and defined step rasters along with or orthogonal to the chromato-
graphic axis.
MALDI-TOF MS

Selected Spots: All MALDI spectra from primuline spots Abbr. Lipid name MH +
were acquired on an Autoflex MALDI-TOF-MS with 50 Hz
nitrogen laser. The extraction voltage was 20 kV and gated
matrix suppression was applied to prevent the saturation PE Phosphatidylethanolamine 16:0/18:1 718.5
of the detector by matrix ions. All spectra were acquired
in reflector mode using delayed extraction as described in
[5]. Spectra were calibrated using a standard lipid mixture G Ganglioside 1261.8
desorbed from a standard DHB preparation next to the
spots of interest (positive ion mode). For negative ion mode,
PS Phosphatidylserine 18:0/20:4 834.6
the characteristic signals of the DHB matrix were used for
calibration [6].

MALDI: Imaging and scanning of lanes on TLC plates was PC Phosphatidylcholine 16:0/20:4 782.6
performed on an ultraflex III MALDI-TOF/TOF with 200 Hz
smartbeam laser in reflector mode. The extraction voltage
SM Sphingomyelin 16:0 703.6
was set to 25 kV and matrix suppression to m/z <200. Data
were acquired under Compass 1.2 (FC 3.0, FA 3.0).
LPC Lyso-Phosphatidylcholine 16:0 496.3
TLC-Imaging: A pixel raster of 400 μm × 400 μm spots was
defined with flexImaging 2.0 on the entire TLC lane area.
200 laser shots were accumulated for each pixel. False
PI Phosphatidylinositol 16:0/18:2 857.6
colors were assigned to each mass of interest and their
spatial distributions across the TLC plate were displayed as
a heat map. PA Phosphatidic acid 16:0/18:1 719.5

TLC-Scanning: The position of chromatographic lanes on

the TLC plate can be read directly from xy-scales on the
adapter target and entered into a new dedicated software
tool for TLC-MALDI (Fig. 2), which is available as a TLC-
MALDI software patch compatible with Compass 1.2 for
chromatographic readout. The Compass 1.3+ software
will incorporate TLC-MALDI functionality. The location and
dimensions of multiple TLC lanes can be defined, as can
raster dimensions (here 400 µm). The intuitive, easy-to-
handle TLC-MALDI software controls not only automatic
data acquisition but also provides a dedicated TLC data
viewer and post-processing tools.
Results
Erythrocyte membrane lipids
4×10 6 cells were extracted using CHCl3/ CH3OH/ H2O 2:1:1
(v/v/v) as described in more detail in [2]. The TLC plate was
analyzed using three different approaches as shown in Fig.
3. The traditional analysis used specific lipid staining by
primuline (Fig. 3A), which forms a non-covalent complex
with lipids, therefore, not interfering with subsequent
MALDI analysis. Three major spots consisting of PE, PC
and SM (see Tab. 1) were detectable using primuline and
a minor spot represented LPC. Detailed evaluation of the
TLC-MALDI imaging dataset revealed 3 more groups of
compounds (LPI, PI, PS) and permitted the differentiation
of lipids that could not be distinguished chromatographically
Tab. 1: Detected and characterized lipids from erythrocyte
membrane, stem cells and brain: abbreviation, name, structure
and typical protonated molecular ion (for several lipids, e.g., MNa+
are actually detected).
with the staining-scraping procedure: PE 18:0/20:4 vs. PE
16:0/18:1; LPI 22:5 vs. PI 18:0/18:2 (Figs. 3b, 4).
Detection of major lipids
Although not shown, all major lipids (phospholipids,
apolar lipids such as cholesterol, cholesteryl esters and
triacylglycerols) present in biological systems are easily
detectable using this approach. Our studies have also
provided evidence that even low abundance lipids (< 1%
of the total lipids) such as PI are easily detectable within a
single dataset providing a dynamic range of ~ 3 orders of
magnitude. Even mixtures of polyphosphoinositides (PI,
PIP, PIP 2) can be easily separated and analyzed (PI peak
labeled blue in Fig. 3 and present in Fig. 4 b). Glycolipids
require a more acidic solvent system in order to separate
them from the common and more abundant phospholipids
[7]. Although compounds such as gangliosides are
characterized by a much higher polarity resulting in reduced
detectability, they are easily detectable using this approach.
However, glycolipids are often negatively charged (due to
the presence of phosphate or sulphate groups), and require
negative mode analysis.
 Classical Staining vs. Imaging and Chromatographic Readout of TLC-MALDI Data

Mass Spectrum

A B C

Fig 3: A) Classical primuline

TLC Lane

MALDI Image

MALDI staining of separated lipids on a

Chromatogram TLC plate; B) TLC-MALDI imag-
ing analysis of identical TLC plate

Ion Cromatogram
Extracted
with matrix coating;
C) chromatographic TLC readout
as provided by the TLC-MALDI
software permitting direct
access to all molecular species
(m/z values on the x-axis) along
the chromatographic separation
on the y-axis. The colored circles
correspond to the compounds
m/z that are visualized in the TLC-
MALDI image.

Full readout in minutes

This representation of the data allows direct access to all
Although imaging readouts such as Fig. 3B appear attractive chromatographic mass peaks and a direct distinction can be
as an analysis format comparing directly and favorably with made between the peaks and background ions which are
classical stains (Fig. 3A), they require time consuming, represented by vertical streaks in the DataView. In addition,
manual data interrogation. Therefore, they do not appear distinguishing largely overlapping lipids is straight- forward
well suited for routine analysis purposes. However, as their mass separation adds another dimension not
chromatographic treatment of the TLC data from acquisition available with the mass image.
to visualization and analysis appears to be more efficient
Structure confirmation
and powerful, providing a full readout of the information
contained in the TLC-MALDI dataset (Fig. 3C). Acquisition After the analysis of the separated compounds, structure
times are reduced from hours to ~ 5 min/lane and the large confirmation typically requires the acquisition of MS/MS
data file size for an image is reduced to a fraction of the size spectra. The TLC-MALDI DataViewer facilitates manual
for the chromatogram. Therefore the TLC-MALDI software “pick a peak”, which permits the direct manual acquisition
has been designed to support the quick and simple of MS/MS spectra, in a few seconds, without the need to
definition of lanes in the visual interface for the acquisition find the proper spots (not shown).
of chromatographic traces. Up to 4 lanes can be defined per
TLC plate. After acquisition, the entire dataset is visualized
using a dedicated DataViewer (Fig. 3C). It provides direct
access to all separated compounds through a heat map of
all MS peaks along the chromatographic separation (y-axis).
For a cursor-selected position in the RF vs. m/z plane, the
extracted ion chromatogram is displayed on the right side
and the corresponding MS spectrum is shown on the top.
 TLC-MALDI Analysis of Erythrocyte Membrane Lipids
PE 18:0/20:4

PE 16:0/18:1
8 PE 18:0/20:4 (m/z = 812.5)
7 PE PE 16:0/18:1 (m/z = 762.5)
7
PC 16:0/20:4
6 Fragments of LPI 22:5
(m/z = 523.4 and 551.4)

PI 18:0/18:2
PC 16:0/18:1 (m/z = 885.6)
5
Fragment of PS 16:0/20:4
(m/z = 618.5)
SM 24:0 Fig. 4: Spectrum overview of
4
various lipids detected in posi-
Unknown (Impurity?)
SM 16:0 (m/z = 1157.7) tive ion mode from erythrocyte
3 6 PC 16:0/20:4 (m/z = 804.6) membrane. Spectra are num-
PC bered according to their elution
5 PC 16:0/18:1 ( m/z = 760.6)
LPC 18:0 order. TLC-MALDI imaging data
2 4 SM
SM 24:0 (m/z = 837.7)
(b) proved to be much more
3 SM 16:0 (m/z = 725.6)
sensitive than common staining
LPC 16:0 2 LPC LPC 18:0 (m/z = 524.3)
techniques (a: primuline stain).
1 1 LPC 16:0 (m/z = 496.3)
MS spectra obtained from (b)
500 550 600 650 700 750 800 850 (a) (b) provided detailed differences in
m/z
fatty acyl compositions (1-8).

Conclusion
TLC-MALDI runs on any Bruker Flex mass spectrometer Therefore, we developed software to automate the linear
equipped with a scoutMTP ion source under Compass 1.2 scanning of TLC traces with scan times in the 5 min range
and higher. The ImagePrep is equipped with TLC methods and use DataViewer to work with TLC-MALDI data in
to produce a uniform matrix coating with TLC separated chromatographic format, which drastically reduces manual
lipids. Previously, TLC-MALDI coupling was expected to evaluation times of the analysis to few minutes.
be problematic [8] because pieces of the stationary TLC All, hardware and software methods are available now for
phase might disintegrate and damage the MS. However, no the simple application of TLC-MALDI to routine analysis.
such events were observed even under routine laboratory Use of optimized matrix protocols with new matrices (such
conditions. It is also remarkable that under TLC-MALDI as 9-aminoacridine) are expected to further extend the
conditions, fragmentation of analytes was not significantly application range of this new and exciting method. TLC-
exaggerated in comparison with standard MALDI. MALDI is interesting in a great number of applications such
The new technology was applied to the analysis of lipids as oligosaccharides [10], food, cosmetics, herbals or for
from various sources such as mesenchymal stem cells [2], quick analytical support in the organic chemical synthesis
human erythrocytes [2] or chicken egg [1] extracts providing lab.
evidence that MALDI is a great readout platform for TLC
separations of lipids [9]. These analyses demonstrated
significantly improved detection limits compared to the
classical primuline staining approach. Imaging analysis
allowed the visualization of many hitherto undetected lipids
and to distinguish several compounds in broad primuline
spots. Although imaging mass spectrometry compares
favourably with primuline staining analytically, the relatively
long acquisition time (~1-2 h) and data evaluation process
associated with imaging (~1-2 h), calls for different
approaches in routine analysis.
Acknowledgements
We thank Michael Schulz, Merck KGaA, Darmstadt,
Germany, for providing various phases and plate formats for
protocol optimization.

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to change specifications without notice. © Bruker Daltonics 01-2009, # 262189

(MALDI-TOF) mass spectrometry in lipid and phospholipid

Bruker Daltonics is continually improving its products and reserves the right
research Prog. Lipid Res. 43, 449-488.
Bruker Daltonics, Bremen, Germany
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For research use only. Not for use in diagnostic procedures.

Keywords Instrumentation & Software

TLC-MALDI autoflex TOF/TOF
lipids ultraflex TOF/TOF
ImagePrep
flexSoftware 1.2
TLC-MALDI Adapter Target
# 255595

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