HPTLC


What is High-Performance Thin Layer Chromatography?

High-Performance Thin Layer ChromatographyHPTLC (high-performance thin layer chromatography) is a sophisticated form of TLC, which provides superior separation efficiency. The HPTLC concept includes validated methods for qualitative and quantitative analysis, and fulfills all quality requirements for use in fully regulated environments.

HPTLC vs. TLC

The process steps of HPTLC are identical to classical TLC. The main difference between them is in the characteristics of the separation plate. HPTLC plates are based on optimized silica gel 60 with a significantly smaller particle size than used for classical TLC. This allows a higher packing density and a smoother surface. Hence, sample diffusion is reduced, resulting in compact bands or spots. Furthermore, the smaller particle size and thinner layer significantly increase detection sensitivity and analysis speed.

Features of HPTLC versus classical TLC
HPTLC
Classical TLC
Mean particle size 5 - 6 µm 10 - 12 µm Comparison of the particle size distribution of TLC and HPTLC plates
Particle size distribution 4 - 8 µm 5 - 20 µm
Layer thickness 200 µm (100 µm) 250 µm
Plate height 12 µm 30 µm
Typical migration distance 3 - 6 cm 10 - 15 cm
Typical separation time 3 - 20 min 20 - 200 min
Number of samples per plate < 36 (72) < 10
Sample volume 0.1 - 0.5 µl 1 - 5 µl
Detection limits: absorption 100 - 500 pg 1 - 5 ng
Detection limits: fluorescence 5 - 10 pg 50 - 100 pg

 The separations below demonstrate the advantages of HPTLC over TLC.

Comparison of classical TLC
versus HPTLC plates
Sample 1. N-alpha-dansyl-L-aspargine
2. alpha-dansyl-L-arginine
3. Dansyl-L-cysteic acid
4. N-Danysl-L-serine
5. Dansyl-glycine
6. N-N-Didansyl-L-tyrosine
A. TLC
 
B. HPTLC
 
Mobile phase Ethyl acetate/methanol/propianic acid (20/10/3)
Detection UV 366
Sample volume 4 µm 0.3 µm
Migration distance 10 cm 5 cm
Analysis time 42 min 13 min 45 sec
- - -

Comparison of the separation of dansyl amino acids under identical conditions. The comparison clearly demonstrates that the HPTLC plate delivers sharper zones with shorter migration distances and faster analysis times. In addition the HPTLC plate allows these separation of twice the number of samples simultaneously.

Classical TLC silica gel 60 plate HPTLC silica gel 60 plate

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HPTLC Benefits

  • Faster analysis, only 3 to 20 minutes for optimal separation
  • 5 to 10 times better detection sensitivity than classical TLC
  • Highly reproducible, sharp bands for quantitative analysis
  • Easy coupling with bioassays, thus particularly beneficial for effect-directed analysis
  • Defined zones can be absorbed by mass spectrometry (MS) after evaluation, hence no need to record every run including matrix and background

HPTLC Plates

HPTLC AMD Plates
  • Developed for automated multiple development (AMD)
  • Extra thin layers of 100 μm
  • Best suited for qualitative and quantitative detection of pesticides
HPTLC Premium Purity Plates
  • Specially designed for demanding pharmacopoeia applications
  • Wrapped in aluminum foil to prevent deposition of plasticizers from standard wrapping material, which could result in unknown extra zones when using solvent systems of medium polarity, e.g. toluene/ethyl acetate (95/5)
LiChrospher® HPTLC Plates with Spherical Particles
  • Optimized for high-throughput analysis of complex samples
  • Based on spherical silica gel 60 with a particle size of 7 μm and narrow particle size distribution
  • Excellent selectivity similar to standard HPTLC plates, yet even higher performance and speed with improved detection limits

HPTLC Application Fields

HPTLC plates offer superior separation performance for quantitative evaluation of highly complex samples in:

  • Pharmaceutical quality control
  • Analysis of medicinal plants and herbs
  • Analysis of pesticide mixtures

HPTLC Application Examples

Comparison of detection limits on HPTLC LiChrospher® Si 60 F254s plates and normal HPTLC Si 60 F254 plates (Detection Limits, UV 254 nm, ng/spot).

Substance
Visually
Spectrophotometrically
Silica gel 60 F254
LiChrospher® Si 60 F254s
Silica gel 60 F254
LiChrospher® Si 60 F254s
Ascorbic acid 100 100 100 25
Cortisone 50 25 25 10
Atrazine 50 25 10 5
Prometryne 25 10 10 5
Theophylline 50 25 25 10
o-Aminophenol 50 25 25 5
m-Aminophenol 10 5 10 5
p-Aminophenol >100 50 50 25

Scanning electron pictures of the cross-section

(A) classical silica TLC plate (B) high performance silica HPTLC plate(C) HPTLC LiChrospher® plate

Pesticide separation
Sample 1. Hexazinone Conventional HPTLC Si 60 plateHPTLC LiChrospher® Si 60 plate
2. Metoxuron
3. Monuron
4. Aldicarb
5. Azinphos-methyl
6. Prometryn
7. Pyridate
8. Trifluralin
Sample volume 50 nl
Mobile phase Petroleum benzene 40 - 60°C / acetone 70/80
Detection 5 - 10pg

Recommended HPTLC Tutorials

CAMAG HPTLC Application Tutorial

HPTLC Demonstration

Recommended HPTLC Literature

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