I am trying to repeat an experiment conducted by FDA on Malachite Green (MG).

According to this paper,

Prepare a series of LC calibrants by aliquoting into individual 15 mL graduated 
glass or disposable polypropylene centrifuge tubes, 50 µL, 100 µL, and 200 µL
of the 0.1 µg/mL MG2 solution and 50 µL and 100 µL of the 1.0 µg/mL MG1 solution.
Dilute each to 5.0mL with (1:1 vol) acetonitrile/ammonium acetate buffer
(mobile phase A), vortex mix or stopper and invert to mix thoroughly. 
Based on a 5 g sample weight, these solutions will generate standards with
concentrations of 1, 2, 4, 10 and 20 ng/g of MG. 

If I am not wrong, Malachite Green used in this paper is from the salt, Malachite Green Oxalate.

  • Is Acetonitrile used here as a pH buffer?
  • What is the role of Ammonium acetate?

If I plan on using Raman Spectroscopy to detect MG in my samples, shouldn't I be using the pure forms of MG as my reference signal? If so, wouldn't the addition of these two chemicals affect my readings?


The pdf document originally linked in this post (see edits) is no longer available. The most similar reference identified (available here via the Internet Wayback Machine) is:

  1. Wendy C. Andersen, José E. Roybal, Sherri B. Turnipseed. Determination of Malachite Green and Leucomalachite Green in Salmon with In-Situ Oxidation and Liquid Chromatography with Visible Detection. Laboratory Information Bulletin (LIB) 4334: Malachite Green and Leucomalachite Green in Salmon. Volume 20, No. 11, November 2004.

Since they're using ACN/ammonium acetate to elute from solid phase extraction cartridges and that's what they're injecting into their LC analysis, they probably want to use the same mixture to avoid problems with unmatched matrices with their standards. The ammonium acetate is the pH buffer. Malachite green has different protonation states at different pH, so it's important to set the pH properly for elution from the SPE and LC analysis. MG is fairly soluble in water, but still has a lot of organic functionality, so adding an organic solvent would help elute from the SPE column and keep the analyte from being too strongly retained by the LC stationary phase.

As for raman analysis, water has rather weak raman scattering, so it's not usually a problem. Acetonitrile has one very strong peak at 2942 $\mathrm{cm^{-1}}$ and a few moderate peaks, so it might be an issue. Since you're not doing LC, you can likely prepare standards in buffer only. Unfortunately, ammonium acetate is also raman-active, but if you're not doing LC, you might be able to do without as well. Fortunately, one of the great powers of IR and raman spectroscopies is that there is often enough bandwidth to find one or two non-overlapping peaks in a complex mixture and use that for quantitation, so it may still work as written. They're both also very amenable to chemometric techniques like partial least squares when measuring complex samples.

The big problem is going to be sensitivity if you're interested in the concentrations they use in the paper.. Normal raman is not that sensitive and the smallest standard is only ~3 nM, so it's probably going to be hopeless. These guys do surface-enhaced resonance raman and are able to get to about 1 nM, and the raman setup's not actually that hard since MG has a huge absorbance at 620 nm, right where HeNe lasers emit. Unfortunately, MG doesn't fluoresce much in solution or it would be very easy to measure.

  • $\begingroup$ Thanks for the detailed answer. It is very informative. Can I just prepare my standards in methanol without the buffer? And further dilute it into various ppb solutions also in methanol? Since methanol evaporates fast, this allows it to dry quick on the SERS substrate. What do you think? $\endgroup$
    – Tinker
    Jun 2 '15 at 21:46
  • $\begingroup$ They use acetonitrile in that paper, but I don't see why you couldn't use methanol, you'll just need to be more careful with your standards as it evaporates very quickly. You may also want to use the chloride salt of MG rather than oxalate, to avoid complicating your spectrum. $\endgroup$ Jun 2 '15 at 22:38

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