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: Thin-Layer Chromatography
  • Alternate Protocol: 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: Thin-Layer Chromatography

 Materials
  • Test compound
  • Appropriate volatile solvent for test compound
  • Appropriate solvent system (eluent; see Strategic Planning)
  • Reagent(s) for visualization (stain; see Strategic Planning)
  • Solid iodine (for semi-destructive visualization)
  • TLC plate with appropriate adsorbent (see Strategic Planning), cut to size (see Support Protocol 1)
  • TLC spotter (see Support Protocol 2)
  • Wide-mouthed TLC chamber or beaker with lid (Fig. A.3D.1)
  • 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: Co-Spotting a TLC Plate

 Additional Materials (also see Basic Protocol)
  • Unknown compound
  • Authentic compounds A and B
  • 2 × 5–cm TLC plate (see Support Protocol 1)

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

  •  FigureFigure A.3D.1 Diagram of a TLC developing chamber.
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  •  FigureFigure A.3D.2 Spotting a TLC plate. The baseline is ~1 cm from the bottom and spots are ~5 mm apart.
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  •  FigureFigure A.3D.3 Results of TLC. (A) The spot has a Rf of 0.0. For silica gel adsorbent, this means that the eluent is not polar enough. For C18 reversed-phase adsorbent, this means that the eluent is too polar (water content too high). (B) The spot has a Rf of 0.94. For silica gel adsorbent, this means that the eluent is too polar. For C18 reversed-phase adsorbent, this means that the eluent is not polar enough (organic content too high). (C) The mixture contains two compounds, as is apparent when the TLC plate is developed using a suitable eluent. The spots have Rf values of 0.29 and 0.67.
    View Image
  •  FigureFigure A.3D.4 Co-spotting a TLC plate (see Alternate Protocol). Lane (1) unknown compound. Lane (2) authentic compound A. Lane (3) authentic compound B. Lane (4) mixture of authentic compounds A and B and unknown compound. Notice that the unknown (lane 1) and authentic compound B (lane 3) have similar Rf values (0.44 and 0.40, respectively) when run side-by-side on the TLC plate. Upon closer examination of lane 4, it is shown that, in fact, the unknown and authentic compound B are identical.
    View Image
  •  FigureFigure A.3D.5 Preparation of TLC spotters.
    View Image

Videos

Literature Cited

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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