TLC and HPTLC MS-grade plates

for mass spectrometry

Merck Millipore is a division of

 TLC and HPTLC MS-grade plates
for mass spectrometry coupling

Simple thin layer chromatography (TLC) is the most widely used technique in planar
2
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

A
The techniques for coupling TLC with
mass spectrometry can be divided into:  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

B
Both approaches are offline, and both are  ith desorption-based techniques, the analyte
W
performed after the separation is finished is vaporized from the silica and transferred to
and the plate is dried. The sample transfer to the MS in the gas phase. Vaporization techniques
the MS is fast and typically takes less than include gas beam, ion bombardment and MALDI
one minute. (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.
• Targeted recording of mass spectra on zones or lines of interest is performed after the TLC chromatogram
has been developed, thus providing high efficiency.
• One 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 3
• The selectivity of MS-grade plates is the same as Merck Millipore standard TLC and HPTLC plates
• The detection limit of HPTLC-MS-grade plates is in the lower nanogram range

Compatible with different TLC-MS techniques

• Elution-based techniques
• Desorption-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 Merck Millipore plates is that
the new MS-grade products are much cleaner (more sensitive, reduced background signals).
• TLC/HPTLC MS-grade plates are packed in aluminum foil to maintain cleanness and prevent
contamination.

[%]
HPLC Mass 365.2
100
pump spectrometer
80
delivers the solvent analyzes the sample
60
pressure controlled analyte is transferred
40
piston lowers in the liquid phase 385.3
20
198.8 273.1 353.2 402.6
0
TLC / HPTLC plate [m/z]

CAMAG interface Schema Result

[%] 9.40
DART Mass spectrometer 3329779
100
ion source (DART)
capillary tip analyzes the sample 80 7.00
5.20 2397974
vaporizes 1729269
60
the analyte
analyte is transferred 3.27
40 869586
in the gas phase
20 1.36
98820
0
TLC / HPTLC plate [m/z]

HPTLC-DART-MS interface Schema Result

 Comparison of HPTLC MS-grade glass plates
with Merck 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
4 (Ord. No. 1.05642.0001) with mobile phase acetonitrile/water (95/5).
Figure 1 [ Intensity x105 ]
269.1
6

4 215.1 323.1

2 301.1
157.1 185.1 413.3
355.3
0
100 200 300 400 500 600 700 [m/z]

MS background signal measurement using an MS-grade HPTLC silica gel 60 F254 glass plate
(Ord. No. 1.00934.0001) with mobile phase acetonitrile/water (95/5).
Figure 2 [ Intensity x105 ]

2
185.1
159.0 413.3
0
100 200 300 400 500 600 700 [m/z]

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 (Ord. No. 1.00934.0001) with mobile phase acetonitrile/water (95/5) + 0.1%
formic acid
Figure 3 [ Intensity x105 ]
195.1
6

2
217.1
413.3
229.2 245.2 273.2
150.1 171.1 306.3 331.2 367.2 393.3 425.3 441.3 469.4
0
150 200 250 300 350 400 450 500 [m/z]

ESI-MS mass spectrum of caffeine, measured from a 20 nanogram TLC spot

These experiments clearly demonstrate the performance of the NEW Merck 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 5
Plate HPTLC Silica gel 60 F254 MS-grade glass plate (Ord. No. 1.00934.0001)
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 toluene / n-heptane (6/4, v/v)
Migration distance 5 cm
Migration time 18 min
Chamber normal chamber without chamber saturation
Detection Detection UV @ 254 nm
Identification TLC-MS Interface CAMAG/ESI (+) mode (electrospray ionization)

Standards Sample
100
CH3
O O CH3
80
384
N
60

40
Eusolex® OCR

226.8 385.2
20
220.8 362

0
200 250 300 350 400 450 500 550 [m/z]

Eusolex® 9020 (310 Da)

Eusolex® OCR (361 Da)

No. Volume Compounds Sample Concentration Transfer solvent

1 1.0 µl Eusolex® 9020 standard 1 mg/ml ethanol
Eusolex® OCR
2 1.0 µl Eusolex® 9020 suncream – isopropyl alcohol
Eusolex® OCR
 HPTLC Silica gel 60 F254 MS-grade glass plates
Separation of steroids with peak identification
by mass spectrometry

Application 2
6 Chromatographic Conditions
Plate HPTLC Silica gel 60 F254 MS-grade glass plate
(Ord. No. 1.00934.0001)
Mobile Phase petroleum benzene / acetone (8/2, v/v)
Migration distance 5 cm
Migration time 15 min
Chamber normal chamber without chamber saturation
Staining no
Detection Detection UV @ 254 nm
Identification TLC-MS Interface CAMAG/ESI (+) mode

Methyltestosterone (302 Da)

Reichstein' S (346 Da)

Hydrocortisone (362 Da)

Application result No. Volume Compounds Sample Concentration Transfer solvent

1 to 3 2.0 µl Hydrocortisone steroids mixture 1.2 mg/ml methanol
Reichstein' S 1.0 mg/ml
Methyltestosterone 0.8 mg/ml

See corresponding mass spectrums of Methyltestosterone (figure 5), Reichstein' S (figure 6) and
Hydrocortisone (figure 7).

Figure 5 [ Intensity x105 ]

303.2592
OH

0.8 H

H H
0.6 O

0.4

0.2
325.2430
232.6539 605.5109
0.0
100 200 300 400 500 600 700 [m/z]

Mass spectrum of Methyltestosterone (TLC-MS Interface CAMAG / ESI (+) mode)

 HPTLC Silica gel 60 F254 MS-grade glass plates
Separation of steroids | Summary | Ordering information

[ Intensity x105 ] Figure 6

347.2529
O
2.5
OH
OH
217.1246 H
2.0
195.1401 H H
1.5 O

1.0 287.2264
326.4076 400.4145
0.5 173.0946 239.1704 372.3803
151.1105 304.3268
119.0585 496.4649 540.4974 633.4726 693.4971
0.0
100 200 300 400 500 600 700 [m/z] 7
Mass spectrum of Reichstein'S ( TLC-MS Interface CAMAG / ESI (+) mode)

[ Intensity x105 ]
Figure 7
363.2486 OH
O
2.0 HO OH
H
1.5 H H
303.2223 O
1.0

0.5
362.6486 345.2364 385.2325
423.2535
195.1326 242.1339 553.8458 665.4619 725.4857
0.0
100 200 300 400 500 600 700 [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 Comments Ord. No.
TLC silica gel 60 F254 MS-grade Elution- and desorption-based 1.00933.0001
25 glass plates 20x20 cm approach
HPTLC silica gel 60 F254 MS-grade Elution- and desorption-based 1.00934.0001
25 glass plates 20x10 cm approach
HPTLC silica gel 60 RP18 F254s MS-grade Elution- and desorption-based 1.51161.0001
25 glass plates 20x10 cm approach
HPTLC silica gel 60 F254 MS-grade for MALDI* Elution- and desorption-based 1.51160.0001
20 aluminum foils 5x7.5 cm approach
* 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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and our products contact:

Merck KGaA
08/13

64271 Darmstadt, Germany

E-mail: [email protected]
W288130

www.merckmillipore.com/chromatography
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