Materials and Reagents

1. Methanol (HPLC grade)
   
2. Pyridine reagent Plus >99% (Sigma-Aldrich, catalog number: P57506 )
   
3. H₂O (HPLC grade) (Merck KGaA)
   
4. Acetonitrile (HPLC grade)
   
5. HPLC solvent A (see Recipes)
   
6. HPLC solvent B (see Recipes)
   

Equipment

1. 0.5 ml polypropylene tubes
   
2. HPLC 2 ml glass vials and 200 μl glass inserts
   
3. HPLC system with an autosampler (Agilent, model: 1200 ) (Figure 1)
   
   
   Figure 1. The Agilent 1200 HPLC system equipped with an autosampler and two fluorescence detectors marked as FLD #1 and FLD #2
   
   
4. Two HPLC fluorescence detectors (Agilent, model: 1200 ) (see Note 1)
   The original Agilent 1200 detectors offer adequate sensitivity for emission wavelengths ranging 300-600 nm, however for longer wavelengths the sensitivity quickly falls almost to zero. This issue also applies to the newer 1260 version of the detector. As sensitivity to longer wavelengths is essential for detection of several heme/chlorophyll precursors, the original Agilent photomultiplier was replaced with the R928 photomultiplier tube (Hamamatsu, catalog number: R928) in both detectors. This modification significantly improved sensitivity within the entire wavelength range, and dramatically improved the sensitivity to wavelengths >600 nm. The replacement for the photomultiplier was provided by Agilent’s technical service.
   
5. Tabletop centrifuge (Eppendorf, MiniSpin plus)
   
6. Reverse phase column (ReproSil 100 C8-AB, 5-µm particle size, 4 x 250 mm) (Dr. Maisch, catalog number: r15.8b.s2504 )
   

Procedure