Hptlc ms

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HIGH PERFORMANCE THIN
LAYER CHROMATOGRAPHY
MASS SPECTROSCOPY
HPTLC-MS

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PRESENTED BY:
Ashutosh Agarwal
1st Year M. Pharm
Department of Quality Assurance
SSR COLLEGE OF PHARMACY
UNDER THE GUIDANCE OF:
Dr. Sonal Desai
Associate Professor
Department of Quality Assurance
SSR COLLEGE OF PHARMACY
Self Introduction

Table Of Content
Introduction
History
Principle of HPTLC
Principle of Mass Spectroscopy
Steps for Performing HPTLC-MS
HPTLC-MS Interface
Application of HPTLC-MS
Case Study
References
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INTRODUCTION
• Chromatography is an non destructive procedure for resolving a multi-component mixture of trace
minor or major constituent into its individual fractions.
• Chromatography can be applied both quantitatively and qualitatively, but it is primarily a
separation technique.
• HPTLC-MS is an hyphenated form of HPTLC.
• High performance thin layer chromatography is an automated, simple, robust rapid, and efficient
tool in quantitative analysis of compounds.
• HPTLC is an analytical technique based on TLC, but with enhancements intended to increase the
resolution of the compounds to be separated. Whereas HPTLC- MS coupling allows for
verification of the chemical structure of a compound with its molecular mass.
4

HISTORY
• MIKHAIL TSVET was the first person who coined the term ‘thin layer
chromatography’ in 1903.
• HPTLC was first introduced by R.E. KAISER in 1955.
• In 2013, the concept of HPTLC-MS was developed to enhance the
identification of the compound
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PRINCIPLE of HPTLC
• HPTLC is based on an simple principle which is separation of the compound from sample, by the
phenomena of adsorption.
• Adsorption takes place between the polar substructures of solutes and the various adsorptive centers of the
sorbent. Solutes with high adsorption capacity bind more strongly to the sorbent, resulting in enhanced
retention, whereas solutes with lower adsorption strength elute more easily.
• It includes the interaction of compound to be separated with the stationary phase and mobile phase.
• The stationary phase is a HPTLC glass plate or aluminum sheet coated with a uniform thin layer (typically
200 micron) of porous particles (2 – 10μm) with an average particle size of 5μm. The layer typically
consists of silica gel with a pore size of 60 Angstroms, a polymeric binder and a so called fluorescence
indicator (F254). The standard format of the plate is 20×10 cm
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Principle of Mass Spectroscopy
• Mass spectrometry (MS) is commonly regarded as an instrumental technique for
separation of electrically charged species in the gas phase.
• The charged species (ions) are produced in the ion source. In some cases, the ion
source also assists the transfer of solid-phase or liquid-phase analytes into the gas
phase.
• The gas-phase ions subsequently are transferred into the mass analyzer. The mass
analyser sorts the ions—in space or time—according to the mass-to-charge ratios
(m/z)
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Schematic diagram of mass spectroscopy

MASS SPECTROSOPY
Types Of Interfaces used in HPTL-MS
• Electron Spray Ionization
• Matrix Associated Laser Desorption Ionisation Technique (MALDI)
• Atmospheric Pressure Chemical Ionization(APCI)
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Electron Spray Ionization
• ESI provides the softest ionization method available, which means, it can be
used for highly polar, least volatile or thermally unstable substances
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Steps of HPTLC-MS
Sample
preparation
Selection of
chromatographic
layer
Plates Pre-washing Conditioning
Sample
Application
Pre-Conditioning
Mobile Phase
Chromatographic
development
Detection of spot
Scanning and
Document
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Sample Preparation and Application
• A good solvent system is one that moves all components of the mixture off the
baseline but does not put anything on the solvent front.
• The peaks of interest should be resolved between Rf 0.15 and 0.85.
• Pharmaceutical preparation with sufficiently high concentration of analyte is simply
dissolved in a suitable solvent that will completely solubilize the analyte and leave
excipients undissolved to yield a test solution that can be directly applied on HPTLC
plate.
• Solvent used for dissolving the sample can be ethanol, methanol, chloroform N-
hexane etc.
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Stationary phase
• HPTLC can be regarded as the most advanced form of modern TLC.
• It uses HPTLC plates featuring small particles with a narrow size distribution. As a result, homogenous
layers with a smooth surface can be obtained.
• HPTLC uses smaller plates (10 × 10 or 10 × 20 cm) with significantly decreased development distance
(typically 6 cm) and analysis time (7–20 min).
• HPTLC plates provide improved resolution, higher detection sensitivity, and improved in situ quantification
and are used for industrial pharmaceutical densitometric quantitative analysis.
• Normal phase adsorption TLC on silica gel with a less polar mobile phase, such as chloroform– methanol,
has been used for more than 90% of reported analysis of pharmaceuticals and drugs.
• Lipophilic C-18, C-8, C-2; phenyl chemically-modified silica gel phases; and hydrocarbon- impregnated
silica gel plates developed with a more polar aqueous mobile phase, such as methanol–water or dioxane–
water, are used for reversed-phase TLC.
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Plate size
• 20 × 20 cm
• 10 × 20 cm
• 5 × 10 cm
• 5 × 7.5 cm
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HPTLC-MS Plates
• The techniques for coupling TLC with mass spectrometry can be
divided into elution-based, or desorption-based.
• Both approaches are offline, and are performed after the separation
is completed and the plate dried.
• Sample transfer to the mass spectrometer is fast and typically takes
less than one minute.
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• Elution-based TLC-MS
The analyte on the silica plate is dissolved in a solvent and transferred
to the mass spectrometer in the liquid phase.
• Desorption-based TLC-MS
The analyte is vaporized from the silica, and transferred to the mass
spectrometer in the gas phase. Vaporization techniques include gas
beam, ion bombardment, and MALDI (matrix-assisted laser
desorption/ionization).
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HPTLC-MS Plates

Features and Benefits
• Enhanced sensitivity
• Extremely low background signal
• Trace analysis in nanogram range
• Flexible choice of mobile phases
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HPTLC-MS Plates

Pre- Washing
• Plates need to be washed to be remove water vapors or volatile impurities .
The plates are cleaned by the methanol, chloroform : methanol (1:1),
ammonia solution 1%.
Conditioning
• The pre washed plates are placed in oven at 120 for 15-20 min. This process
is known as conditioning.
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Sample Application
The selection of sample application technique and device to be used depends
on:
• Sample volume
• No. of sample to be applies
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Sample application
• The sample should be completely transferred to the layer.
• Micro syringes are preferred if automatic application devices are not
available.
• Volume recommended for HPTLC - 0.5-5μlSample spotting should not be
excess or not low
• Problem from overloading can be overcome by applying the sample as band.
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Sample applicators used for spotting are:
• Capillary tube
• Micro bulb pipette
• Micro syringe
• Automatic sample applicator
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A. CAMAG Nanomat : Samples applied in the form of spots. The volume is
controlled by disposable platinum iridium of glass capillary which has
volume of 0.1-0.2μl
B. CAMAG Linomat : Automated sample application device. Sample is
loaded in micro syringe (Hamilton Syringe) 1ul capacity. Sample can apply
either as spot or band by programming the instrument with parameters like
spotting, volume ,band length etc.
C. CAMAG automatic TLC sampler III : Applies sample as spot or bands
automatically from the rack of sample vials
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Automatic applicators

Pre- Conditioning
• For low Polarity mobile phase there is no need of chamber saturation.
However saturation is needed for highly polar mobile phase.
• Time required for the saturation depends on the mobile phase.
• If plates are introduced into the unsaturated chamber, during the course of
development, the solvent evaporates from the plate mainly at the solvent
front and it results in increased Rf values.
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Mobile Phase
• The selection of mobile phase is based on adsorbent material used as stationary
phase and physical and chemical properties of analyte.
• General mobile-phase systems that are used based on their diverse selectivity
properties are diethyl ether, methylene chloride, and chloroform combined
individually or together with hexane as the strength-adjusting solvent for normal-
phase TLC.
• Methanol, acetonitrile, and tetrahydrofuran mixed with water for strength adjustment
in reversed-phase TLC.
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• Volumes smaller than 1 ml are measured with a suitable micropipette.
• Volumes up to 20 ml are measured with a graduated volumetric pipette of suitable
size.
• Volumes larger than 20 ml are measured with a graduated cylinder of appropriate
size.
• To minimize volume errors, developing solvents are prepared in a volume that is
sufficient for one working day.
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Mobile Phase

Generally used Mobile phase in detection of some chemical compounds
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Mobile Phase

Development of Chambers
1. Twin trough chambers
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• Rectangular Chambers
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• Automatic Developing Chambers
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Development
• Ascending, descending, horizontal
• Plates are spotted with sample and air dried andplaced in the developing
chambers
• After the development plate is removed from chamber and mobile phase is
removed under fume cup-board to avoid contamination of laboratory
atmosphere
• The plates should be always laid horizontally because when mobile phase
evaporates the separated components will migrate evenly to the surface
where it can be easily detected
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Drying
• Drying of chromatogram should be done in vacuum desiccators with
protection from heat and light
• If hand dryer is used there may be chances of getting contamination of
plates, evaporation of essential volatile oils if any present in the spot or
compounds sensitive to oxygen may get destroyed due to the rise in
temperature
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HPTLC-MS Interface
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Elution based HPTLC MS
TLC -MALDI -MS TLC –DART -MS
LESA

• LESAT technology was originally developed to investigate tissue slices, but
it can analyze almost any surface with its nano-robotic ESI source- and that
includes TLC plates.
• The Tri Versa® NanoMate (Advion) automatically works its way across the
plate, taking a fresh pipette tip to analyze each "zone," which practically
eliminates carry over.
• The robot picks up a pipette tip, draws extraction. solvent from a reservoir,
moves to the zone of interest, allows a small droplet of solvent to mix with
the sample spot for a preset time, and draws up the mixture before nano
spray injection into any high-end MS system.
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Liquid Extraction Surface Analysis TLC MS

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• Bruker Daltonics introduced an adapter that allows us to directly insert your
TIC plate into a MALDI instrument.
• The fully automated measurement process allows an entire plate to be
scanned and produces a visual representation of separations.
• However, the data evaluation software enables so called MALDI
chromatograms that plot molecular mass against TLC position, producing a
two dimensional view; analytes that overlap on the TLC plate are separated
by mass and shown in a different color.
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TLC MALDI-MS

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Dipping protocol optimized for protein and peptide
measurement on HPTLC Si60 F254 MS-grade for
MALDI:
250 mg/ml DHB in 90% ACN0.1% TFA/10%
ultrapure H₂O0.1% TFA with 10 mM (NH,), HPO
Matrix application: three times dipping
TLC MALDI-MS

• Rapid and contamination-free elution of selected zones
• Plug & play installation
• Compatible with any LC-MS system
• Confirmation of known substances within a minute Highly effective
backwashing function prevents the elution path from becoming blocked
• Easy handling ensures accurate and reproducible plate positioning Low
solvent consumption
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Elution based TLC-MS

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Eluents for positive ionization:
Acetonitrile/ Water 95/5+ 0.1% formic acid
Acetonitrile/ Water 50/50+ 0.1% formic acid
Methanol/ Water 80/20+0.1% formic acid
Methanol / Water 50/50+ 0.1% formic acid
Solvent grade: hypergrade for LC – MS
possible adducts:
[M+H], [M+Na], [M+K], [M+MeOH+H], [M+ACN+H]*

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Eluents for negative ionization
Acetonitrile / Water 95/5 + 10 mM ammonium formate / acetate buffer
Acetonitrile / Water 50/50+ 10 mM ammonium formate / acetate buffer
Methanol / Water 80/20 + 10 mM ammonium formate / acetate buffer
Methanol / Water 50/50+10 mM ammonium formate lacetate buffer
Solvent grade: hypergrade for LC-MS
possible adducts:
[M-H], [M-2H+Na], [M+HCO₂]

• Measurement procedure including background measurement
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STEP TIME DISCRIPTION
1 5min Rinsing and system blank
2 1-2 min First plate blank
3 2 min Rinsing and system blank
4 1-2 min Second plate blank
6 1-2 min Substance measurement
8 result Substance measurement and subtract plate blank

Comparison of coupling techniques
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METHODS CHARACTERISTIC FEATURES
Elution based TLC-MA Interface • Connectable to any LC-MS system
• Most type of YLC layers can be used
• Extraction into vials possible
TLC-MALDI Adapter • High degree of automation
• Scan mode (imaging)
• Large mass range
Liquid extraction surface
analysis(LESA)
• High degree of automation
• High sensitivity by nano spray
• Hydrophobic layers
TLC-DESI-MS • Scan mode
• Fast measurement

Applications of HPTLC-MS
• TLC-MS of proteins and peptides
• TLC-MALDI-MS of small molecules
• TLC-MS of dirty samples
 UV filters in sun screen
 Paracetamol in different formulations
 Caffeine in energy drinks
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SRNO. COMPOUND SAMPLE HPTLC HPTLC-MS REFERENCE
SAMPLE
PREPARATION
STATIONARY
PHASE
MOBILE PHASE IONIZATION
TECHNIQUE
MASS ANALYSER MOBILE PHASE FLOW
RATE
1 Acetylcholinesterase
Inhibitors from
Galbanum†
17 g DCM extract
of galbanum
HPTLC silica gel
60 F254 plates
gradient elution with
hexane and chloroform
(20–100%)
atmospheric
pressure chemical
ionization (APCI)
quadrupole mass
spectrometer
Methanol 0.4 mL/min
for 2 min
1
2 Cyclanthera Pedata
leaves, fruits, and
dietary supplement
250 mg of
powdered
lyophilized
samples in 5 mL of
80% aqueous
methanol
10 cm HPTLC
silica gel and C18
RP plates
methanol-water (7:3, v/v);
Ethyl acetate-formic acid-
acetic acid-water
(30:1.5:1.5:3, v/v); ethyl
acetate-water-formic acid
(17:3:2, v/v);
electrospray
ionization (ESI)
ion source
- 70% (v/v)
aqueous
methanol solution
0.2 mL /min 2
3 Hedera helix Extract of Hedera
helix in methanol
- ethyl acetate: methanol:
water: acetic acid
(20:5:4:1, v/v/v/v)
ESI ion source - chloroform and
methanol (1:2)
0.25 mL/min 3
4 harmane and Glu-
P-1
0.2mg/ml of harmane
and Glu-P-1 in
methanol containing
1% aq ammonia
silica gel 60 F254
HPTLC plates
10 mL diethyl ether–
methanol 49:1 (v/v)
electrospray
ionization (ESI)
ion source
- 95% MeOH, 5%
10 mM formate
buffer,
0.1 mL/ min 4
5 Genista saharae
Coss. & Dur
10g of Genista
saharae
extracted in
methanol
silica gel 60 F254
plates 200 × 100
mm
ethyl acetate-formic acid-
acetic acid-water
100:11:11:26, v/v/v/v and
dichloromethane-
methanol 95:5, v/v
electrospray
ionization (ESI)
ion source
triple quadrupole
mass spectrometer.
methanol 0.2 mL
min−1
5
6 Isopropyl thioxanthone
(ITX) in milk, yoghurt
and fat
4 mL milk or 4 g
yoghurt + r, 25 μL
DTX solution (8.0 μg
mL−1 acetonitrile)
silica gel 60 or
RP18 HPTLC
plates
toluene and n-hexane
(4:1, v/v); acetonitrile and
water (9:1, v/v)
electrospray
ionization (ESI)
ion source
Quadrupole methanol and
ammonium
formate
buffer(95:5)
0.1 mL
min−1
6
7 Isopropyl thioxanthone
(ITX) in milk, yoghurt
4 mL milk or 4 g
yoghurt + r, 25 μL
DTX solution (8.0 μg
silica gel 60 or
RP18 HPTLC
plates
toluene and n-hexane
(4:1, v/v); acetonitrile and
water (9:1, v/v)
DART-MS - Helium 1L/min 6

63
SRNO. COMPOUND SAMPLE HPTLC HPTLC-MS REFERENCE
SAMPLE
PREPARATION
STATIONARY
PHASE
MOBILE PHASE IONIZATION
TECHNIQUE
MASS
ANALYSER
MOBILE PHASE FLOW RATE
8 Bergenin from Mallotus
philippinensis
Methanol extract of
Bergenin
10 × 20 cm HPTLC
glass plates
coated with 0.25-
mm layers of silica
gel Si 60F254 (
ethyl acetate–methanol–acetic
acid–formic acid (8:1:0.5:0.5%
v/v),
electrospray
ionization (ESI)
ion source
- methanol 8 L/min 7
9 tryptic protein digests protein in 25 mM
ammonium
bicarbonate buffer.
And trypsin was
added such that the
trypsin: protein ratio
was 1:100.
Silica gel 60 F254s
plates
2-butanol/pyridine/acetic acid/
water (30:20:6:24, v/v/v/v)
DESI-MS - Nitrogen gas 1.7 l/min 8
10 benzodiazepines
in urine samples
1 mL of drug-free
urine spiked with
stock solution of
standards to make a
concentration of
10 µg/mL
silica gel G 60
F254
chloroform–glacial acetic acid
(9:1, v/v)
electrospray
ionization (ESI)
ion source
- Methanol (with
0.1% ammonium
hydroxide)
0.5 mL/min 9
11 caffeine, ergotamine,
and metamizol in a
solid pharmaceutical
formulation
1 mg ergotamine
tartrate, 100 mg
caffeine, and 300 mg
+ 40 mL methanol–
water 7:3 (v/v).+
10ml of methanol–
water 7:3 (v/v).
silica gel 60 F254 ethyl acetate–methanol–
ammonia 90:15:1 (v/v/v)
electrospray
ionization (ESI)
ion source
single-
quadrupole MS
methanol and
formate buffer
(10 mmol/L, pH
4.0) 19:1 (v/v)
0.1 mL/min 10

CASE STUDY
HPTLC-MS of Flavonoids
 Stationary phase:
20 cm×10 cm glass backed HPTLC silica gel 60 F254 and HPTLC silica gel 60 plates .
 Mobile phase:
1st methanol–formic acid (10:1, v/v), 2nd methanol
64

 Preparation of standard solutions:
Standards of flavonoids and phenolic acids were dissolved in methanol in
concentrations of 0.1 mg mL-1
 Preparation of sample test solutions :
500 mg of all pulverized materials (roasted coffee, rose hip, hibiscus, sage and
rosemary) were separately dispersed in 5 mL of 80% aqueous ethanol
65
CASE STUDY

 HPTLC
• Separations were performed on HPTLC plates.
• Standard solutions of flavonoids and phenolic acids as well as STSs were
applied on the plates by an Automatic TLC Sampler as 8 mm bands, 8 mm
from the bottom of the plate.
• The developed plates were dried in a stream of warm air for 3 min
66
CASE STUDY

 HPTLC-MS
• Chromatographic zones for further TLC–MS analyses were marked under
illumination at 366 nm, to ensure the appropriate positioning of the plate under the
oval elution head (4 mm×2 mm) of the TLC–MS interface, which was used for
elution of the compounds from the plates into the mass spectrometer
• A flow rate of 70%(aq) methanol, which was used as the eluent, was 0.2 mL min−1
• Heated electrospray ionization (HESI) in the negative ion mode was applied for
ionization of the compounds.
67

• Chromatograms of Flavonoids on HPTLC
68
CASE STUDY

• Mass Spectra of Flavonoids by HPTLC-MS
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CASE STUDY

Proper Understanding
Through
70
• https://www.youtube.com/watch?v=gTN6fcTrHGM

1. Adhami, H.-R., Scherer, U., Kaehlig, H., Hettich, T., Schlotterbeck, G., Reich, E., & Krenn, L. (2013). Combination of
Bioautography with HPTLC-MS/NMR: A Fast Identification of Acetylcholinesterase Inhibitors from Galbanum†.
Phytochemical Analysis, 24(4), 395–400. doi:10.1002/pca.2422
2. Orsini, F., Vovk, I., Glavnik, V., Jug, U., & Corradini, D. (2019). HPTLC, HPTLC-MS/MS and HPTLC-DPPH methods for
analyses of flavonoids and their antioxidant activity in Cyclanthera pedata leaves, fruits and dietary supplement. Journal of
Liquid Chromatography & Related Technologies, 1–12. doi:10.1080/10826076.2019.1585630
3. Shawky, E., & El Sohafy, S. M. (2019). Untargeted and targeted chemical profiling for efficacy-directed discrimination of
Hedera helix L. subspecies using HPTLC- image analysis and HPTLC/MS. Industrial Crops and Products,
111980. doi:10.1016/j.indcrop.2019.111980
4. Morlock, G., & Jautz, U. (2008). Comparison of two different plunger geometries for HPTLC-MS coupling via an extractor-
based interface. Journal of Planar Chromatography – Modern TLC, 21(5), 367–371. doi:10.1556/jpc.21.2008.5.9
71
REFERENCES

6. Morlock, G., & Schwack, W. (2006). Determination of isopropylthioxanthone (ITX) in milk, yoghurt and fat by HPTLC-FLD,
HPTLC-ESI/MS and HPTLC-DART/MS. Analytical and Bioanalytical Chemistry, 385(3), 586–595. doi:10.1007/s00216-006-
0430-5
7. Haribabu, K., Ajitha, M., Ramesh, B., Babu, K., & Rao, J. (2012). Quantification of bergenin fromMallotus philippinensisby
HPTLC-MS and study on different extraction methods. Journal of Planar Chromatography – Modern TLC, 25(5), 445–
449. doi:10.1556/jpc.25.2012.5.10
8. Pasilis, S. P., Kertesz, V., Van Berkel, G. J., Schulz, M., & Schorcht, S. (2008). HPTLC/DESI-MS imaging of tryptic protein
digests separated in two dimensions. Journal of Mass Spectrometry, 43(12), 1627–1635. doi:10.1002/jms.1431
9. Choudhary, P., Bansal, S., & Verma, K. L. (2020). HPTLC–MS method for the determination of benzodiazepines in urine
samples. JPC – Journal of Planar Chromatography – Modern TLC. doi:10.1007/s00764-020-00053-w
10. Aranda, M., & Morlock, G. (2007). Simultaneous Determination of Caffeine, Ergotamine, and Metamizol in Solid
Pharmaceutical Formulation by HPTLC-UV-FLD with Mass Confirmation by Online HPTLC-ESI-MS. Journal of
Chromatographic Science, 45(5), 251–255. doi:10.1093/chromsci/45.5.251
72
REFERENCES

REFERENCES
11. Attimarad, M., Mueen Ahmed, K. K., Aldhubaib, B. E., & Harsha, S. (2011). High-performance thin layer chromatography: A
powerful analytical technique in pharmaceutical drug discovery. Pharmaceutical Methods, 2(2), 71–75. doi:10.4103/2229-
4708.84436
12. Arshad Hala; 2012; HPTLC Instrumentation: An Overview; articles@pharmatutor.org
13. Urban, P. L. (2016). Quantitative mass spectrometry: an overview. Philosophical Transactions of the Royal Society A:
Mathematical, Physical and Engineering Sciences, 374(2079), 20150382. doi:10.1098/rsta.2015.0382
14. Urška Jug, Vesna Glavnik, Eva Kranjc & Irena Vovk (2018) HPTLC–densitometric and HPTLC–MS methods for analysis of
flavonoids, Journal of Liquid Chromatography & Related Technologies, 41:6, 329-341, DOI: 10.1080/10826076.2018.1448690
15. www.merckmillipore.com
16. www.slideshare.com
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