Nile red

[wheels]

Does anyone have an analytical method for nile red using a c8 or c18 column and a uv/vis detector? I've looked on the net but can't seem to find one anywhere. Thanks for your help!

[JMB]

Wheels,

Here is a reference,

Simple liquid-chromatographic method for Nile Red quantification in cell culture in spite of photobleaching.

Lamprecht A and Benoit JP, J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Apr 25;787(2):415-9.

JMB

[wheels]

Thanks for that. I had already tracked down that reference but it's only available for hard cash and, much as I love my job, I'm not inclined to pay my hard earned dosh straight back in the form of journal subscriptions.
If no one can help then I'll just have a play with it and bung a very dilute sample down the c8 on a gentle ETOH gradient and see what happens!

[Kostas Petritis]

Wheels,

Here is the basic information from that reference. I think you can extrapolate from here...

Separation was carried out on a reversed-phase RP-18 column (LiChrospher© 100, Merck, Darmstadt, Germany). The mobile phase was kept as simple as possible which consisted of an isocratic eluent of methanol-water 93:7. The flow-rate was maintained at 1.0 ml/min. The effluents were monitored for their UV-Vis spectra of Nile Red and were recorded between 200 and 700 nm. For quantification purposes, Nile Red was detected by absorption at 559 nm, samples of 50 ml being injected into the column. All chromatographic analyses were performed at room temperature (25 °C).

[tom jupille]

samples of 50 ml being injected into the column.

I hope you meant "50 μL" .

[wheels]

Thanks for the information guys that'll do nicely! Just one small question though - what sort of Nile Red concentrations were being detected?

[Kostas Petritis]

Tom, yes it was 50 uL.

Wheels, you may find below the LOD concentrations and the linearity range tested...


The limit of detection (LOD), defined as the lowest concentration of Nile Red which can be detected with a signal-to-noise ratio greater than 7:1, was established by serial dilutions with decreasing concentrations of Nile Red. The LOD was 9 ug/l for Nile Red.

Nile Red-free DMEM buffer solutions were spiked with the appropriate volume of the working solutions to contain Nile Red at concentrations of 3, 15, 50, 200, 800 and 3500 ug/l

[wheels]

Fantastic, thanks. Have tested it out and it works a treat.