TLC and HPTLC MS-grade plates
for mass spectrometry
EMD Millipore Corp. is a subsidiary of Merck KGaA, Darmstadt, Germany
TLC and HPTLC MS-grade plates
for mass spectrometry coupling
2
Simple thin layer chromatography (TLC) is the most widely used technique in planar
chromatography, whereas high performance TLC (HPTLC) is considered to be the most
efficient and powerful technique. In HPTLC, the silica used has a smaller particle size
(4 – 8 µm) and a narrower particle distribution.
In 1969, Prof. R.E. Kaiser reported the coupling of thin-layer chromatography with mass
spectrometry (MS) for the first time. TLC spots were heated and desorbed into a gas
stream in front of the source of a mass spectrometer. Later H.J. Issaq demonstrated the
use of multi-dimensional TLC coupled to MS, where separated zones were eluted from
the TLC plate with methanol and introduced into the MS using the Eluchrom interface
(CAMAG special products). Since then, many TLC-MS publications have been made,
in particular in the last 3 – 4 years as interest has strongly grown.
Today, coupling TLC plates to Mass Spectrometry is a new field of high interest, which will
contribute strongly to the progress of planar chromatography, today and in the future.
The techniques
The techniques for coupling TLC with
mass spectrometry can be divided into:
A
B
ith elution-based techniques, the analyte on the
W
silica plate is dissolved in a solvent and transferred
to the mass spectrometer in the liquid phase (see
CAMAG interface).
Elution-based techniques
Both approaches are offline, and both are
performed after the separation is finished
and the plate is dried. The sample transfer to
the MS is fast and typically takes less than
one minute.
ith desorption-based techniques, the analyte
W
is vaporized from the silica and transferred to
the MS in the gas phase. Vaporization techniques
include gas beam, ion bombardment and MALDI
(matrix assisted laser desorption / ionisation) or
DART (direct analysis in real time).
Desorption-based techniques
Key benefits of TLC-MS are:
• Mass spectra are obtained quickly by direct sample access on the TLC plate at room temperature –
high quality spectra are obtained with low background signal.
• T argeted recording of mass spectra on zones or lines of interest is performed after the TLC chromatogram
has been developed, thus providing high efficiency.
•O
ne particular advantage of TLC-MS and HPTLC-MS is the flexibility in choosing mobile phases for
a separation. By contrast, with standard LC-MS coupling using HPLC, some mobile phases cannot
be used (e.g. inorganic buffers).
High efficiency / high resolution
• TLC-MS and HPTLC-MS plates have less impurity in silica gel matrix compared to standard products
• T he selectivity of MS-grade plates is the same as EMD Millipore standard TLC and HPTLC plates
• T he detection limit of HPTLC-MS-grade plates is in the lower nanogram range
3
Compatible with different TLC-MS techniques
• Elution-based techniques
•D
esorption-based techniques
Separation performance
• The separation performance of the new products is equivalent to the standard TLC/HPTLC plates,
so that the method with standard TLC plates can be directly transferred to MS-grade plates.
Cleanness
• The important difference between MS-grade plates and standard EMD Millipore plates is that
the new MS-grade products are much cleaner (more sensitive, reduced background signals).
• T LC/HPTLC MS-grade plates are packed in aluminum foil to maintain cleanness and prevent
contamination.
HPLC
pump
Mass
spectrometer
delivers the solvent
analyzes the sample
[%]
100
365.2
80
60
pressure controlled
piston lowers
analyte is transferred
in the liquid phase
40
385.3
20
0
TLC/HPTLC plate
CAMAG interface
Schema
DART
ion source
Mass spectrometer
(DART)
capillary tip
vaporizes
the analyte
analyzes the sample
Schema
353.2
402.6
[m/z]
Result
[%]
100
9.40
3329779
80
7.00
5.20
2397974
1729269
60
analyte is transferred
in the gas phase
TLC/HPTLC plate
HPTLC-DART-MS interface
198.8
273.1
3.27
869586
40
20
0
Result
1.36
98820
[m/z]
Comparison of HPTLC MS-grade glass plates
with EMD Millipore standard HPTLC glass plates
under same chromatographic conditions
The following experimental results demonstrate the enhanced sensitivity of TLC-MS-grade plates:
MS background signal measurement using a standard HPTLC silica gel 60 F254 glass plate
(VWR Cat. No. EM1.05642.0001) with mobile phase acetonitrile/water (95/5).
4
Figure 1
[ Intensity x105 ]
269.1
6
215.1
4
323.1
301.1
2
413.3
157.1 185.1
0
100
355.3
200
300
400
500
600
[m/z]
700
MS background signal measurement using an MS-grade HPTLC silica gel 60 F254 glass plate
(VWR Cat. No. 10755-322) with mobile phase acetonitrile/water (95/5).
Figure 2
[ Intensity x105 ]
6
4
2
185.1
0
413.3
159.0
100
200
300
400
500
600
[m/z]
700
This clearly demonstrates that MS-grade plates have very low background signal compared to
standard HPTLC plates.
Trace measurement of caffeine [sample: 20 ng caffeine (MH+) 195.1] on a HPTLC silica gel 60 F254
MS-grade glass plate (VWR Cat. No. 10755-322) with mobile phase acetonitrile/water (95/5) +
0.1 % formic acid
Figure 3
[ Intensity x105 ]
195.1
6
4
2
217.1
150.1
0
150
229.2 245.2
171.1
200
250
273.2
306.3
331.2
300
367.2
350
393.3
400
413.3
425.3 441.3
450
469.4
500 [m/z]
ESI-MS mass spectrum of caffeine, measured from a 20 nanogram TLC spot
These experiments clearly demonstrate the performance of the NEW EMD Millipore MS-grade
HPTLC plates.
HPTLC Silica gel 60 F254 MS-grade glass plates
Separation of UV filter in sun cream
with spot identification by mass spectrometry
Application 1
Chromatographic Conditions
Plate
HPTLC Silica gel 60 F254 MS-grade glass plate (VWR Cat. No. 10755-322)
Sample preparation
1 g sun cream in 10 ml isopropyl alcohol at room temperature for 15 min
and filtration through a 0.45 μm syringe filter (PTFE Millipore)
Mobile Phase
Migration distance
Migration time
Chamber
Detection
Identification
Standards
toluene / n-heptane (6/4, v/v)
5 cm
18 min
normal chamber without chamber saturation
Detection UV @ 254 nm
TLC-MS Interface CAMAG/ESI (+) mode (electrospray ionization)
Sample
100
CH3
O
80
O
N
60
Eusolex® OCR
40
385.2
226.8
20
362
220.8
0
CH3
384
200
250
300
350
400
450
500
550
Eusolex® 9020 (310 Da)
Eusolex® OCR (361 Da)
No.
1
Volume
1.0 µl
Compounds
Eusolex® 9020
Eusolex® OCR
Sample
standard
Concentration
1 mg/ml
Transfer solvent
ethanol
2
1.0 µl
Eusolex® 9020
Eusolex® OCR
suncream
–
isopropyl alcohol
[m/z]
5
HPTLC Silica gel 60 F254 MS-grade glass plates
Separation of steroids with peak identification
by mass spectrometry
6
Application 2
Chromatographic Conditions
Plate
HPTLC Silica gel 60 F254 MS-grade glass plate
(VWR Cat. No. 10755-322)
Mobile Phase
Migration distance
Migration time
Chamber
Staining
Detection
Identification
petroleum benzene / acetone (8/2, v/v)
5 cm
15 min
normal chamber without chamber saturation
no
Detection UV @ 254 nm
TLC-MS Interface CAMAG/ESI (+) mode
Methyltestosterone (302 Da)
Reichstein’S (346 Da)
Hydrocortisone (362 Da)
Application result
No.
Volume
1 to 3 2.0 µl
Compounds
Hydrocortisone
Reichstein’ S
Methyltestosterone
Sample
steroids mixture
Concentration
1.2 mg/ml
1.0 mg/ml
0.8 mg/ml
Transfer solvent
methanol
See corresponding mass spectrums of Methyltestosterone (figure 5), Reichstein’ S (figure 6) and
Hydrocortisone (figure 7).
Figure 5
[ Intensity x105 ]
303.2592
OH
H
0.8
H
O
0.6
H
0.4
0.2
325.2430
232.6539
0.0
100
200
300
605.5109
400
Mass spectrum of Methyltestosterone (TLC-MS Interface CAMAG / ESI (+) mode)
500
600
700
[m/z]
HPTLC Silica gel 60 F254 MS-grade glass plates
Separation of steroids | Summary | Ordering information
[ Intensity x105 ]
Figure 6
347.2529
O
2.5
2.0
195.1401
1.0
0.5
173.0946
151.1105
119.0585
0.0
100
H
O
1.5
287.2264
326.4076
239.1704
304.3268
200
OH
OH
H
217.1246
H
400.4145
372.3803
300
633.4726
496.4649 540.4974
400
500
693.4971
600
7
[m/z]
700
Mass spectrum of Reichstein’ S ( TLC-MS Interface CAMAG / ESI (+) mode)
Figure 7
[ Intensity x105 ]
OH
363.2486
O
OH
HO
2.0
H
1.5
H
O
303.2223
H
1.0
0.5
0.0
100
345.2364 385.2325
423.2535
362.6486
195.1326 242.1339
200
300
400
665.4619
553.8458
500
600
725.4857
700
[m/z]
Mass spectrum of Hydrocortisone (TLC-MS Interface CAMAG / ESI (+) mode)
Summary
The separation efficiency and selectivity of the new MS-grade plates is equivalent to the standard
TLC/HPTLC plates; the only difference is that the new products are much cleaner than the standard plates.
This gives higher sensitivity and reduced background signals, allowing trace analysis with mass
spectrometry detection in the lower nanogram range (see figure 3).
Ordering information
Product name
TLC silica gel 60 F254 MS-grade
25 glass plates 20 x 20 cm
Comments
Elution- and desorption-based
approach
VWR Cat. No.
10755-320
HPTLC silica gel 60 F254 MS-grade
25 glass plates 20 x 10 cm
Elution- and desorption-based
approach
10755-322
HPTLC silica gel 60 RP18 F254s MS-grade
25 glass plates 20 x 10 cm
Elution- and desorption-based
approach
10755-328
HPTLC silica gel 60 F254 MS-grade for MALDI* Elution- and desorption-based
20 aluminum foils 5 x 7.5 cm
approach
10755-326
* only aluminum plates are suitable for MALDI
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be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own
responsibility for checking the suitability of our products for the envisaged purpose.
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EMD Millipore, the M logo and Eusolex are registered trademarks of
Merck KGaA, Darmstadt, Germany. W.288730 06/2016 LAm-16-12910
© 2016 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.
0816 Lit No.131710W