Mar 08, 2023
Carotenoid Pigment Analysis in Leaf Extracts by HPLC - UNFINISHED
This protocol is a draft, published without a DOI.
- Laurie Leonelli1,
- Lynn Doran2
- 1UIUC;
- 2Realizing Increased Photosynthetic Efficiency (RIPE)
Protocol Citation: Laurie Leonelli, Lynn Doran 2023. Carotenoid Pigment Analysis in Leaf Extracts by HPLC - UNFINISHED. protocols.io https://protocols.io/view/carotenoid-pigment-analysis-in-leaf-extracts-by-hp-cnc6vaze
Manuscript citation:
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.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 25, 2023
Last Modified: July 11, 2023
Protocol Integer ID: 75902
Keywords: HPLC, Chromatography, Carotenoids, Pigments, Plants
Funders Acknowledgement:
Realizing Increased Photosynthetic Efficiency (RIPE) that is funded by the Bill & Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the U.K. Foreign, Commonwealth & Development Office
Grant ID: OPP1172157
Abstract
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.
Materials
Equipment
- HPLC, as described in before you begin.
- Microcentrifuge
- Vortex, with attached tube rack
- Tube rack
- Micropipette, 20-200 ul
- (Optional) 11mm Stainless Steel Hand Operated Crimper with Grip, ThermoFisher 60180-CR11
***Crimp top vials are used in this protocol for cost savings. If screw cap vials are used, crimper is not necessary.
Consumables and Reagents
| A | B | C | D | E | F | |
| Item | Vendor | Part # | Size | ~ Amt/sample | Website | |
| Crimp caps with septa, Aluminium,Clear PTFE/Natural Red rubber, Silver, 11 mm | VWR | 46610-744A | Pack of 100 | 1 | https://us.vwr.com/store/product/37402009/vwr-crimp-caps-for-11-mm-crimp-top-vials | |
| 300 µl Amber Polypropylene Limited Volume Vial, 11mm Crimp/Snap Ring Top | JG Finneran Associates, Inc | 30111P-1232A | case of 1000 | 1 | https://www.novatech-usa.com/30111P-1232 | |
| Thermo Scientific™ Target2™ Nylon Syringe Filters | Fisher Scientific | 03-391-4B | Pack of 100 | 1 | https://www.fishersci.com/shop/products/target2-nylon-syringe-filters/033914B?matchedCatNo=F2504-2&searchHijack=true&searchTerm=F2504-2&searchType=RAPID | |
| AIR-TITE LUER SLIP SYRINGES, 1 mL (Low Dead Space) | Air-Tite Products | MS1 | Pack of 100 | 1 (Reusable) | https://www.air-tite-shop.com/p-16-air-tite-luer-slip-syringes.aspx?variantid=56 | |
| Acetone, HPLC Plus, for HPLC, GC, and residue analysis, ≥99.9% | Sigma-Aldrich | 650501-1L | 1L | 300 ul | https://www.sigmaaldrich.com/US/en/product/sigald/650501 | |
| Acetonitrile, HPLC Plus, ≥99.9% | Sigma-Aldrich | 34998-2.5L | 2.5L | ~40-60 mL | https://www.sigmaaldrich.com/US/en/product/sigald/34998 | |
| Ethyl acetate, HPLC Plus, for HPLC, GC, and residue analysis, 99.9% | Sigma-Aldrich | 650528-1L | 1L | ~20-40 mL | https://www.sigmaaldrich.com/US/en/product/sigald/650528 | |
| Methanol, suitable for HPLC, gradient grade, ≥99.9% | Sigma-Aldrich | 34885-1L-R | 1L | ~20-40 mL | https://www.sigmaaldrich.com/US/en/product/sigald/34885 | |
| Tris-HCl, Molecular Biology Grade (Tris-Hydrochloride) | Promega | H5121 | 100 g | <0.1g | https://www.promega.com/products/biochemicals-and-labware/biochemical-buffers-and-reagents/tris_hcl_-molecular-biology-grade-_tris_hydrochloride_/?catNum=H5121 | |
| Sodium Hydroxide (White Pellets) | Fisher BioReagents | BP359-500 | 500 g | <0.1g | https://www.fishersci.com/shop/products/sodium-hydroxide-white-pellets-fisher-bioreagents/BP359500 | |
| Gel-Loading Tips, 1-200μL | Fisherbrand | 02-707-138 | 960 tips | 2 | https://www.fishersci.com/shop/products/fisherbrand-gel-loading-tips-1-200-l-4/02707138 | |
| Micropipette tips, 20-200 ul, standard | Pipette Brand Dependent | |||||
| 1.5 mL Eppendorf Tubes | Fisherbrand | 02-682-002 | 500 | 1 | https://www.fishersci.com/shop/products/basix-microcentrifuge-tubes-standard-snap-caps/02682002#?keyword=02682002 |
To reuse 1 mL syringes:
To reuse steel grinding beads:
Safety warnings
Uses organic solvents.
- Familiarize yourself with safety data sheets for acetone, acetonitrile, ethyl acetate, and methanol before beginning analysis.
- Wear appropriate PPE.
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:
watersPIGCAR.amx Processing Method:
pigcarprocessed_v2.pmx 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.
Prepare HPLC
Prepare HPLC
Contact IGB GEGC Lab Manager to ensure the instrument is up to date on maintenance status, out of storage status, and in analysis mode.
Note
When the HPLC will not be used for >2 weeks, the lab manager will make sure the analytical column is rinsed with water and stored in 100% acetonitrile. The bypass column is used to fill the sample lines, gaskets, and stainless steel components with isopropanol. The lines need to be rinsed and prepared with analytical reagents prior to analysis. This is to be done by the lab manager.
Note
Due to the high concentration of Tris and the solvent changes from Tris to organic solvents, salt accumulation on the pre-column and column can occur. Coordinate with lab manager to rinse column with water at least once a week to minimize salt build up in the system and keep a low pump pressure.