TITLE: Carotenoid Pigment Analysis in Leaf Extracts by HPLC - UNFINISHED
AUTHORS: Laurie Leonelli, Lynn   Doran

[DESCRIPTION]
Pigment extraction and carotenoid analysis by high performance liquid chromatography (HPLC) from leaf tissue.


Additional References:
García‐Plazaola, J. I., & Becerril, J. M. (1999). A rapid high‐performance liquid chromatography method to measure lipophilic antioxidants in stressed plants: simultaneous determination of carotenoids and tocopherols.Phytochemical Analysis,10(6), 307-313.
Leonelli, L. (2022). Chapter Twenty - An in vivo plant platform to assess genes encoding native and synthetic enzymes for carotenoid biosynthesis. Methods in Enzymology, 671, 489-509. https://doi.org/10.1016/bs.mie.2022.03.005.
Meuller-Moule, P., Conklin, P. L., & Niyogi, K. K. (2002). Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo. Plant Physiology, 128(3), 970–977. https://doi.org/10.1104/pp.010924.

[BEFORE_START]
Collect tissue into tubes and place directly into liquid nitrogen. Grind under liquid nitrogen using tissuelyzer or mortar and pestle.  This protocol assumes ~3X #7 cork borer leaf discs (ID 12mm) were collected per sample.

This protocol assumes that HPLC hardware, software, and method has already been properly configured for this analysis.  

Protocol as presented is run on an HPLC (Agilent 1290 Infinity II) configured with an XSelect HSS C18 5 um VanGuard Cartridge 3.9 mm x 5 mm pre-column (Waters, 186007856)  and a Spherisorb 5 um ODS1 4.6 mm × 250-mm cartridge column (PSS830615, Waters, Milford, MA) at 30°C (1290 MCT isothermal column oven, Agilent G7116B) and a vial auto-sampler (1290 Vialsampler, Agilent G7129B). A quaternary pump (Agilent 1290 Infinity II) provides a linear gradient from 100% (v/v) solvent A (acetonitrile:methanol:0.1MTris-HCl, pH 8.0; 84:2:14 [v/v]) to 100% (v/v) solvent B (methanol:ethyl acetate, 68:32 [v/v]) for 15 min, followed by 3 min of solvent B at a solvent flow rate of 1.2 mL min−1 to a A445with a reference at 550 nm by a diode array detector (1260 DAD WR, Agilent G7115A).  

Software screenshots or example chromatograms are taken from OpenLab CDS Acquisition ver.  2.5 with Agilent Chemstation Integrator enabled in processing methods.

Analytical Method:
  

Processing Method:
   
NOTE: 
Sample and hardware performance can vary.  Processing method is provided as a TEMPLATE ONLY and will need to be adjusted for each use case.
This processing method does not contain calibration standards.  UIUC GEGC uses external software.  Calibration standards can be added to this processing method for direct analysis in OpenLab.


HPLC maintenance and storage status is maintained by the IGB GEGC Lab Supervisor.  ALWAYS contact the lab supervisor before beginning analysis on the instrument.  Often it may be in a storage state and will need to be changed to the appropriate chemicals and columns for analysis.

Communicate OFTEN with the lab supervisor when performing analysis.  
Immediately notify lab supervisor of any large changes in pump pressure or if pump pressure exceeds 200 bar.
Notify lab supervisor if instrument provides any errors pertaining to any equipment (i.e. auto-sampler, pump, DAD, etc).
Notify lab supervisor if there are any unexplained leaks on the counter or equipment.
Notify lab supervisor if waste container has greater than 8L of waste and you are not familiar with the hazardous waste disposal protocols.
Notify lab supervisor weekly if you are completed or if you plan to continue analysis on Monday.  Communicate with lab supervisor again on Monday to see if weekly HPLC maintenance has been completed.  Do not start weekly analysis until lab supervisor has confirmed that required maintenance has been completed.  Lab supervisor typically tries to complete maintenance on Friday afternoons to avoid salt buildup in the instrument when it is idle over the weekend.


[STEPS]
SECTION: Prepare HPLC
1. Contact IGB GEGC Lab Manager to ensure the instrument is up to date on maintenance status, out of storage status, and in analysis mode.