Rapid Separation of Rhodamines

September 1, 2009

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2009, Volume 0, Issue 0

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

This application note describes a rapid UHPLC method for the separation of Sulforhodamine 101 (Texas Red® ) and its three water-soluble derivatives using a column packed with Thermo Scientific Hypersil GOLD™ 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, and also as fluorescent tracers in histochemistry (1). Due to their polycyclic aromatic structures, rhodamines are hydrophobic, which makes them incompatible for biological applications, so the ability to synthesize water-soluble 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 been developed for the separation of Sulforhodamine 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 column packed with Hypersil GOLD 1.9 μm particles.

Experimental Conditions

LC System: Thermo Scientific Accela™ High Speed LC

Column: Hypersil GOLD, 1.9 μm, 50 × 2.1 mm

Part Number: 25002-052130

Mobile Phase: A: 0.1M Triethylammonium acetate (TEAA), pH 7; B: Acetonitrile

Temperature: 30 °C

Flow Rate: 0.55 mL/min

Injection Volume: 1 μL

UV Detection: 595 nm

Std. Solutions: Alexa Fluor 594 (Isomer I): 1.09 mg in 2.09 g MeCN + 6.79 g water; Alexa Fluor 594 (Isomer II): 0.10 mg in 1.15 g MeCN + 3.81 g water; Texas Red water soluble (TR-WS): 0.25 mg in 1.17 g MeCN + 4.97 g water; Sulforhodamine 101 (Texas Red): 0.25 mg in 1.29 g MeCN + 6.16 g water

Results and Discussion

The use of a short Hypersil GOLD column packed with 1.9 μm particles allows for rapid analysis of the rhodamines. Using the UHPLC method described above, the separation of Sulforhodamine 101 (Texas Red) and its three derivatives can be achieved in under two 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

Further optimization of the gradient could potentially lead to a greater ability to separate the ortho-and para-isomers of Texas Red water soluble (TR-WS).

Acknowledgements

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

References

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

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