Rapid Separation of Rhodamines

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2008, Volume 0, Issue 0

This application note describes a rapid U-HPLC method for the separation of sulphorhodamine 101 (Texas Red®) and its three water-soluble derivatives using a Hypersil GOLD™ column packed with 1.9 μm particles.

Monica Dolci, Thermo Fisher Scientific

This application note describes a rapid U-HPLC method for the separation of sulphorhodamine 101 (Texas Red®) and its three water-soluble derivatives using a Hypersil GOLD™ column packed with 1.9 μm particles.

Rhodamines are a family of 3,6-di(substituted amino)-9-benzoate derivatives of xanthene. Fluorescent molecules from the rhodamine family are widely used in bio-analysis.They are used as dyes and indicators for various metals; they are also used as fluorescent tracers in histochemistry (1). Due to their polycyclic aromatic structures, rhodamines are rather hydrophobic, which makes them incompatible for biological applications, so the ability to synthesize hydrosoluble analogues is an important consideration for biocompatibility.

In order to compare the polarity of such water-soluble rhodamine derivatives with the starting molecules, it is important to have in place a sound analytical methodology. This application note describes a fast LC method that has been developed for the separation of sulphorhodamine 101 (Texas Red®) and its three derivatives: Alexa Fluor® 594 (Isomer I), Alexa Fluor® 594 (Isomer II), and Texas Red® water soluble (TR-WS) using a Hypersil GOLDTM column packed with 1.9 μm particles.

Experimental Conditions

Results

The use of a short (50 mm) column packed with 1.9 μm particles allows for rapid analysis of the rhodamines. Using the UHPLC method described above, the separation of sulphorhodamine 101 (Texas Red®) and its three derivatives can be achieved in under 2 min (Figure 1). The low dwell volume of the Accela pump means that the gradient is delivered onto the column in a short time and the column can be quickly re-equilibrated. The total analysis time is 3 min sample to sample.

Figure 1

The narrow, symmetrical peaks produced by this method enabled separation of the ortho- and para- isomers of Texas Red® water soluble (TR-WS). Further optimization of the gradient could potentially lead to a greater ability to separate these two isomers.

Conclusions

A fast LC method has been developed using a short (50 mm) column packed with 1.9 μm particles which allows for the separation of sulphorhodamine 101 (Texas Red®) and its three derivatives in a total analysis time of 3 min sample to sample.

References

(1) http://www.online-medical-dictionary.org.

Acknowledgements

This rapid separation was kindly initiated by the provision of existing methodology using a Hypersil GOLD 150 × 2.1 mm, 5 μm column. Thank you to: <IRCOF> Equipe de Chimie Bio-Organique, UMR 6014 CNRS, INSA de Rouen et Université de Rouen 76130 Mont Saint Aignan, France.

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