TLC and HPTLC MS-grade plates for mass spectrometry

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TLC and HPTLC MS-grade plates for mass spectrometry coupling

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

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.

With desorption-based techniques, the analyte 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).

With elution-based techniques, the analyte on the silica plate is dissolved in a solvent and transferred to the mass spectrometer in the liquid phase (see CAMAG interface).

BAElution-based techniques

Desorption-based techniques

2

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• 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.

analyte is transferred in the gas phase

capillary tipvaporizes the analyte

TLC / HPTLC plate

analyzes the sample

Mass spectrometer (DART)

DART ion source

1.3698820

3.27869586

5.201729269

7.002397974

9.403329779

[m/z]

[%]100

80

60

40

20

0

TLC / HPTLC plate

pressure controlledpiston lowers

analyte is transferred in the liquid phase

HPLC pump

delivers the solvent

Mass spectrometer

analyzes the sample

[m/z]

198.8 273.1 353.2

365.2

385.3

402.6

100

80

60

40

20

0

[%]

CAMAG interface Schema Result

HPTLC-DART-MS interface Schema Result

3

301.1

323.1

269.1

355.3413.3157.1 185.1

215.1

6

4

2

0100 200 300 400 500 600 700 [m/z]

[ Intensity x105 ]

413.3159.0185.1

6

4

2

0100 200 300 400 500 600 700 [m/z]

[ Intensity x105 ]

331.2 367.2 393.3 425.3 441.3413.3

469.4171.1150.1

195.1

229.2217.1

245.2273.2

306.3

6

4

2

0150 200 250 300 350 400 450 500 [m/z]

[ Intensity x105 ]

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 (Ord. No. 1.05642.0001) with mobile phase acetonitrile/water (95/5).

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).

This clearly demonstrates that MS-grade plates have very low background signal compared to standard HPTLC plates.

These experiments clearly demonstrate the performance of the NEW Merck Millipore MS-grade 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

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

Figure 1

Figure 2

Figure 3

4

200 250 300 350 400 450 500 550

60

80

100

40

20

0

384

362220.8

385.2226.8

[m/z]

O O

N

CH3

CH3

HPTLC Silica gel 60 F254 MS-grade glass plates Separation of UV filter in sun cream with spot identification by mass spectrometry

Application 1Chromatographic ConditionsPlate 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 cmMigration time 18 minChamber normal chamber without chamber saturationDetection Detection UV @ 254 nmIdentification TLC-MS Interface CAMAG/ESI (+) mode (electrospray ionization)

SampleStandards

Eusolex® 9020 (310 Da)

Eusolex® OCR

Eusolex® OCR (361 Da)

No. Volume Compounds Sample Concentration Transfer solvent1 1.0 µl Eusolex® 9020

Eusolex® OCRstandard 1 mg/ml ethanol

2 1.0 µl Eusolex® 9020Eusolex® OCR

suncream – isopropyl alcohol

5

0.4

0.6

0.8

0.2

0.0100 200 300 400 500 700600

605.5109325.2430

232.6539

303.2592

[m/z]

[ Intensity x105 ]

OHH

H

OH

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

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

HPTLC Silica gel 60 F254 MS-grade glass plates Separation of steroids with peak identification by mass spectrometry

No. Volume Compounds Sample Concentration Transfer solvent1 to 3 2.0 µl Hydrocortisone steroids mixture 1.2 mg/ml methanol

Reichstein' S 1.0 mg/mlMethyltestosterone 0.8 mg/ml

Chromatographic ConditionsPlate 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 cmMigration time 15 minChamber normal chamber without chamber saturationStaining noDetection Detection UV @ 254 nmIdentification TLC-MS Interface CAMAG/ESI (+) mode

Methyltestosterone (302 Da)

Reichstein' S (346 Da)

Hydrocortisone (362 Da)

Application 2

Application result

Figure 5

6

1.0

1.5

2.0

2.5

0.5

0.0100 200 300 400 500 600 700

304.3268

326.4076

372.3803400.4145

496.4649

287.2264

540.4974 633.4726 693.4971

239.1704

347.2529

195.1401

217.1246

119.0585

173.0946151.1105

[m/z]

[ Intensity x105 ]

1.0

1.5

2.0

0.5

0.0100 200 300 400 500 600 700

385.2325423.2535 553.8458 665.4619 725.4857

363.2486

195.1326 242.1339

303.2223

345.2364362.6486

[m/z]

[ Intensity x105 ]

OHH

HHO OH

OOH

OHH

HOH OH

O

Mass spectrum of Reichstein'S ( TLC-MS Interface CAMAG / ESI (+) mode)

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

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).

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

Ordering informationProduct name Comments Ord. No.TLC silica gel 60 F254 MS-grade 25 glass plates 20x20 cm

Elution- and desorption-based approach

1.00933.0001

HPTLC silica gel 60 F254 MS-grade 25 glass plates 20x10 cm

Elution- and desorption-based approach

1.00934.0001

HPTLC silica gel 60 RP18 F254s MS-grade 25 glass plates 20x10 cm

Elution- and desorption-based approach

1.51161.0001

HPTLC silica gel 60 F254 MS-grade for MALDI*20 aluminum foils 5x7.5 cm

Elution- and desorption-based approach

1.51160.0001

* only aluminum plates are suitable for MALDI

Summary

Figure 6

Figure 7

7

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