Thin Layer Chromatography

Li Cai1

1 Division of Mathematics and Science, University of South Carolina Salkehatchie, Walterboro, South Carolina
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 6.3
DOI:  10.1002/9780470089941.et0603s08
Online Posting Date:  February, 2014
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Thin layer chromatography (TLC) is a quick, sensitive, and inexpensive technique used to determine the number of components in a mixture, verify the identity and purity of a compound, monitor the progress of a reaction, determine the solvent composition for preparative separations, and analyze the fractions obtained from column chromatography. This unit is mainly aimed at novice experimenters, describing in detail the strategies and principal steps for performing a TLC experiment, with illustrations of the relevant instruments, as well as approaches for obtaining and understanding results. Valuable practical tips and troubleshooting solutions are also provided throughout the unit. Curr. Protoc. Essential Lab. Tech. 8:6.3.1‐6.3.18. © 2014 by John Wiley & Sons, Inc.

Keywords: chromatography; TLC; separation; silica gel; organic solvents; staining

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Table of Contents

  • Overview and Principles
  • Strategic Questions
  • Strategic Planning
  • Safety Considerations
  • Protocol
  • Basic Protocol 1: Principal Steps of TLC and Required Instrumentation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Principal Steps of TLC and Required Instrumentation

  • Organic sample solution: the sample for TLC can be dissolved in any compatible solvent because the solvent used to dissolve the sample will be completely dried out after the sample is spotted on the TLC (avoid high‐boiling‐point solvents which would make it difficult to dry the sample after spotting and cause the TLC look like a smear; see Troubleshooting)
  • Developing solvents (CH 2Cl 2, hexanes, ethyl acetate, methanol, etc.)
  • Iodine (I 2; also see Table 6.3.1)
  • TLC staining reagents (Table 6.3.1)
  • TLC plates (aluminum, glass, or plastic; e.g., EMD Millipore, SORBTECH, Sigma‐Aldrich)
  • Guillotine paper trimmer
  • Diamond‐tipped glass cutter
  • Glass Pasteur pipets for capillary spotters
  • TLC chamber (CAMAG, cat. no. 022.5255; Sigma‐Aldrich, cat no. Z126195 or Z243906) or small wide‐mouth flat‐bottom glass jar/bottle with a lid
  • Tweezers
  • Heat gun (optional)
  • UV lamp
  • Iodine chamber: a screw‐top glass jar with a well‐fitting lid can be used as the vaporization chamber (see Table 6.3.1 for preparation details)
  • Filter paper
  • TLC spray cabinet equipped with radial fan (CAMAG;
  • Sprayer (see Fig. )
  • Hot plate or TLC plate heater
Table 6.3.1   MaterialsFrequently Used TLC Stains

Name Application Preparation
Iodine (I 2) Temporary stain; insert the TLC plate into the chamber and remove it after it develops a light brown color over the entire plate To a glass bottle with cap (bottle size depends on how much stain you prepare) add 100 g of silica and 5 to 7 g of iodine crystals (no liquid). Close the cap and shake many times so that iodine is dispersed over the silica.
p‐Anisaldehyde Carbohydrates; heating required to stain the plate; various colors Dissolve 18 ml of p‐anisaldehyde in 540 ml 95% ethanol and cool the solution in an ice/water bath. Mix 30 ml of 97% H 2SO 4 and 6 ml of acetic acid. Cautiously add the acid mixture to the prechilled ethanol solution dropwise at 0°C with vigorous stirring, without splashing. Store the resulting colorless solution in a − 20°C freezer before use.
Bromocresol green Carboxylic acids yield yellow‐green spots on blue background; no heating required Dissolve bromocresol green (0.08 g) in ethanol (200 ml) to get a clear colorless solution. Slowly add 0.1 N NaOH dropwise until blue color just appears in the solution.
CAM Universal stain; heating required to stain the plate; yields dark blue spots on light background Slowly add conc. H 2SO 4 (80 ml) to water (720 ml) under stirring followed by ammonium molybdate (40 g) and ceric ammonium sulfate (1.6 g). Stir the resulting mixture to get a clear solution.
Cerium(IV) sulfate [Ce(SO 4) 2] General staining, very effective for alkaloids; should be sprayed on to the plate (not dipped) and then heated for the stain to appear as black spots on yellow‐white background 15% aqueous sulfuric acid saturated with cerium (IV) sulfate
Chromic acid General staining; yields black spots To a cold (0°C) solution of sulfuric acid (100 ml, 20% v/v aq.), slowly add potassium chromate (2.5 g). Warm the resulting clear bright red/orange solution to room temperature and use directly.
2,4‐DNP Mainly for aldehydes and ketones; yields orange spots, no heating required Dissolve 2,4‐dinitrophenylhydrazine (6 g) in 95% ethanol (100 ml) and add water (40 ml). Stir the resulting mixture to get a clear solution, slowly add conc. H 2SO 4 (60 ml), and stir to get a clear solution.
Dragendorff reagent Unreactive amines (e.g., carbamate protected amines), alkaloids; yields orange spots, no heating required Solution A: 1.7 g basic bismuth nitrate in 100 ml water/acetic acid (4:1). Solution B: 40 g potassium iodide in 100 ml water. Mix reagents together as follows: 5 ml A + 5 ml B + 20 ml acetic acid + 70 ml water. Spray plates; orange spots develop. Spots intensify if sprayed later with HCl or 50% water‐phosphoric acid.
Ehrlich's reagent Amines, indole derivatives, antibiotics, steroids; mild heating (lower temperature and shorter heating time; remove the heat source before the background color obscures the spots) required to stain the plate Dissolve p‐dimethylaminobenzaldehyde (1.0 g) in 75 ml of methanol and add 50 ml of conc. HCl.
Ferric chloride spray Phenols Dissolve ferric (III) chloride (1 g) in a mixture of methanol (50 ml) and deionized water (50 ml). Stir the above mixture to get a homogenous solution.
Iodoplatinate (PIP) Alkaloids Dissolve hexachloroplatinate (0.5 g) and potassium iodide (10 g) in deionized water (295 ml). To the above mixture add conc. HCl (27 ml). Stir the mixture 4 hr at 0°C.
Morin hydrate General stain; fluorescently active Dissolve morin hydrate (100 mg) in methanol (100 g) and stir to get a clear solution
Ninhydrin Excellent stain for amines, amino acids, and aminosugars Dissolve ninhydrin (1.5 g) in n‐butanol (100 ml) and then add glacial acetic acid (3 ml). Ethanol can be used in place of butanol.
Potassium permanganate (KMnO 4) Mainly for unsaturated compounds and alcohols; alkenes/alkynes/aromatics usually stain without heating while other oxidizable groups require heating; yields yellow spots on purple background Dissolve KMnO 4 (1.5 g) and K 2CO 3 (10 g) in deionized water (200 ml). To this add 10% NaOH (1.25 ml) and stir to get a clear solution. It will take some time for the solution to clear.
Phosphomolybdic acid (PMA) Good general reagent; heating required to stain the plate, yields blue–dark green spots Dissolve 12 g phosphomolybdic acid in 250 ml ethanol.
Sulfuric acid Heating required to stain the plate; permanent charred spots are produced 5% sulfuric acid in methanol
Vanillin Good general reagent; heating required to stain the plate, yields a range of colors To a cold (0°C) clear colorless solution of vanillin (15 g) in absolute ethanol (250 ml), slowly add sulfuric acid (2.5 ml). Warm the resulting clear solution to room temperature and use directly. Store the excess in a refrigerator.

 Chemical reactions on the TLC plate after separation, which help find and characterize a substance, are almost unlimited and have not been fully investigated. Some frequently used staining reagents, and their application and preparation, are summarized in this table (adapted from OChemOnline Some TLC derivatization reagents (ready‐to‐use) are readily available from major suppliers such as Sigma‐Aldrich (‐chromatography/analytical‐products.html?TablePage=8659122) and Machery‐Nagel (‐‐US/Default.aspx). Although in most cases we talk about dipping solutions, the same reagents are often applicable for spraying TLC plates.
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Literature Cited

  Cai, L., Li, Q., Ren, B., Yang, Z., Zhang, L., and Zhang, L.‐H. 2007. Synthesis of aminodisaccharide‐nucleoside conjugates for RNA binding. Tetrahedron 63:8135‐8144.
  Cai, L., Guan, W., Kitaoka, M., Shen, J., Xia, C., Chen, W., and Wang, P.G. 2009. A chemoenzymatic route to N‐acetylglucosamine‐1‐phosphate analogues: Substrate specificity investigations of N‐acetylhexosamine 1‐kinase. Chem. Commun. (Camb.) 20:2944‐2946.
  Ettre, L.S. and Kalász, H. 2001. The story of thin‐layer chromatography. LCGC 19:712‐721.
  Gardner, H.W. 1968. Preparative isolation of monogalactosyl and digalactosyl diglycerides by thin‐layer chromatography. J. Lipid Res. 9:139‐141.
  Hahn‐Deinstrop, E. 2006. Applied Thin‐Layer Chromatography, 2nd ed. Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.
  Halpaap, H. and Ripphahn, J. 1976. Entwicklung, Daten und Ergebnisse der Hochleistungs‐Dünnschicht‐Chromatographie (HPTLC). Kontakte (Merck) 3:16‐34.
  Kim, Y.J. 1998. A simple streaking device for preparative thin layer chromatography. J. Chem. Ed. 75:640.
  Waksmundzka‐Hajnos, M., Petruczynik, A., Hajnos, M.L., Tuzimski, T., Hawryl, A., and Bogucka‐Kocka, A. 2006. Two‐dimensional thin‐layer chromatography of selected coumarins. J. Chromatogr. Sci. 44:510‐517.
  Zhao, G., Guan, W., Cai, L., and Wang, P.G. 2010. Enzymatic route to preparative‐scale synthesis of UDPGlcNAc/GalNAc, their analogues and GDP‐fucose. Nat. Protoc. 5:636‐646.
Internet Resources‐‐en/KATEN200001‐4TLC‐www.pdf
  MACHEREY‐NAGEL: Basic Principles of TLC and product catalog.'s_for_techniques/TLC.pdf
  TLC student experiments and the principles of separation.
  Grace TLC catalog with brief introduction to TLC and solvent system selection.
  Useful TLC stains with illustrations of selected stained TLC plates.‐chromatography/analytical‐products.html?TablePage=8659122
  Commercial TLC derivatization reagents from Sigma‐Aldrich. Some of these reagents can be easily prepared according to Table .‐‐US/Default.aspx
  More TLC detection reagents from MACHEREY‐NAGEL with preparation and application guide.‐ms_bioluminenscence/tlc_visualizer.cfm
  CAMAG TLC Catalog with detailed introduction to the TLC visualizer.
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