Thin‐Layer Chromatography

C.L.F. Meyers1, D.J. Meyers1

1 Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Appendix 3D
DOI:  10.1002/0471142700.nca03ds34
Online Posting Date:  September, 2008
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Abstract

TLC is used extensively in nucleic acid chemistry to monitor the progress of chemical reactions, to assay fractions collected from a larger chromatographic separation (e.g., column chromatography), and to determine optimal conditions prior to column chromatography. This unit describes methods for spotting test compounds onto a TLC plate, developing the plate in a suitable solvent system, visualizing the results, and calculating the retention factor (Rf). Candidate compounds can be co‐spotted for identification without relying on Rf values. Curr. Protoc. Nucleic Acid Chem. 34:A.3D.1‐A.3D.13. © 2008 by John Wiley & Sons, Inc.

Keywords: thin‐layer chromatography; TLC; nucleosides; nucleotides; separation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Thin‐Layer Chromatography
  • Alternate Protocol 1: Co‐Spotting a TLC Plate
  • Support Protocol 1: Cutting TLC Plates
  • Support Protocol 2: Preparation of TLC Spotters
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Thin‐Layer Chromatography

  Materials
  • Test compound
  • Appropriate volatile solvent for test compound
  • Appropriate solvent system (eluent; see )
  • Reagent(s) for visualization (stain; see )
  • Solid iodine (for semi‐destructive visualization)
  • TLC plate with appropriate adsorbent (see ), cut to size (see protocol 3)
  • TLC spotter (see protocol 4)
  • Wide‐mouthed TLC chamber or beaker with lid (Fig. )
  • Filter paper
  • 100° to 140°C heat gun or hot plate (for destructive visualization)
  • Hand‐held UV light source, 254 or 366 nm (for nondestructive visualization)

Alternate Protocol 1: Co‐Spotting a TLC Plate

  • Unknown compound
  • Authentic compounds A and B
  • 2 × 5–cm TLC plate (see protocol 3)

Support Protocol 1: Cutting TLC Plates

  Materials
  • 10 × 20–cm TLC plates
  • Filter paper or paper towel
  • Ruler
  • Glass cutter (diamond cutters preferred) or scissors

Support Protocol 2: Preparation of TLC Spotters

  Materials
  • ∼1 × 100–mm open‐ended capillary tubes
  • Bunsen burner
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Figures

Videos

Literature Cited

   Sherma, J. and Fried, B. (eds.) 1996. Chromatographic Science Series, Vol. 71: Handbook of Thin‐Layer Chromatography. Marcel Dekker, New York.
   Steinberg, J., Cajigas, A., and Oliver, G. 1996. Nucleic acids and their derivatives. In Chromatographic Science Series, Vol. 71: Handbook of Thin‐Layer Chromatography (J. Sherma and B. Fried, eds.) pp. 921‐969. Marcel Dekker, New York.
   Touchstone, J. 1992. Practice of Thin Layer Chromatography, 3rd ed. John Wiley & Sons, New York.
   Zweig, G. and Sherma, J. 1972. CRC Handbook of Chromatography, Volumes I and II. CRC Press, Cleveland, Ohio.
Internet Resources
   http://www.ux1.eiu.edu/∼cfthb/research/handbook/TLCstains.htm
  Excellent reference for TLC stain recipes, the colors the stain produces, and functional groups sensitive to that particular stain.
   http://www.chemicalshift.com/chemistry/chemistry‐tips/tlc‐stains/
  This site contains TLC stain recipes.
   http://chem.chem.rochester.edu/∼nvd/tlcnotes.html
  Good reference for practical TLC use. Links to organic synthesis tips.
   http://chem.ps.uci.edu/∼srychnov/tlc_stain_recipes.htm
  This site is another good source containing TLC stain recipes.
   http://www.umich.edu/∼mssgroup/Group%20Business/TLCStains.pdf
  This site contains information on TLC stains and their uses.
   http://stoltz.caltech.edu/files/Techniques/TLCstains.pdf
  This site is another good source containing TLC stain recipes.
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