Aug 09, 2024
Version 3
Muconic acid isomers and aromatic compounds analyzed by UHPLC-DAD V.3
- Sean Woodworth1,
- Stefan Haugen1,
- William E. Michener1,2,
- Kelsey Ramirez1,2,
- Gregg T. Beckham1,2
- 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA;
- 2Agile BioFoundry, Emeryville, CA, USA
- National Laboratory of the Rockies (NLR)Tech. support email: [email protected]
Protocol Citation: Sean Woodworth, Stefan Haugen, William E. Michener, Kelsey Ramirez, Gregg T. Beckham 2024. Muconic acid isomers and aromatic compounds analyzed by UHPLC-DAD. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjjxyvk5/v3Version created by Kelsey J. Ramirez
Manuscript citation:
Black, Brenna A., William E. Michener, Courtney E. Payne, and Gregg T. Beckham. 2019.
Determination of cis,cis- and cis,trans-Muconic Acid from Biological Conversion. Golden,
CO: National Renewable Energy Laboratory. NREL/TP-5100-74473.
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: Working
We use this protocol and it's working
Created: August 09, 2024
Last Modified: August 09, 2024
Protocol Integer ID: 104998
Keywords: muconic acid, aromatic acids, isomers, c18, gradient, UHPLC, DAD, lignin, lignin aromatics, lignin-derived aromatics, lignin-derived aldehydes, lignin-derived carboxylic acids, BEH, quantitation of muconic acid isomer, muconic acid isomer, aromatic compounds by ultra high pressure liquid chromatography, muconic acid, muconic isomer, various aromatic analyte, ultra high pressure liquid chromatography, additional aromatic analyte, chromatographic separation, aromatic compound, compound, uhplc
Funders Acknowledgements:
This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. This work was performed as part of the Agile BioFoundry (ABF) and was supported by Office of Energy Efficiency and Renewable Energy (EERE) and Bioenergy Technologies Office (BETO).
Grant ID: DE-AC36-08GO28308
Disclaimer
This protocol is for research purposes only.
Abstract
An analysis method was developed to allow for quantitation of muconic acid isomers (cis, cis and cis, trans) and aromatic compounds by ultra high pressure liquid chromatography paired with diode array detection (UHPLC-DAD). This was achieved by reproducible preparation of c,c and c,t isomers of muconic acid and chromatographic separation using a mobile phase gradient to separate various aromatic analytes and muconic isomers on a UHPLC reverse phase analytical column.
In version 3 of this protocol a different C18 column option is used with the same gradient and mobile phases. This change was made due to inconsistancies in product specifications and QC with the previous column choice. Additional aromatic analytes were also added to the analytes quantified.
Guidelines
This protocol utilizes an ultra-high pressure liquid chromatography diode array detection (UHPLC-DAD) system manufactured by Agilent Technologies as referenced in 'Materials'. A similar chromatography and detection system can be utilized; however, some parameter nomenclature may deviate depending on the manufacturer.
Materials
Standards:
cis cis-Muconic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #15992
Aromatics Mix Large Rev. 1Absolute StandardsCatalog #67185
HydroquinoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #H17902
L-PhenylalanineMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5482
Caffeic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0625
trans-Cinnamic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #C80857
Reagents:
Sodium hydroxide 10N ACS reagent grade (≥30% w/w)Fisher ScientificCatalog # SS255-1
Formic acid 98 % pureThermo ScientificCatalog # AC147932500
Acetonitrile OptimaFisher ScientificCatalog # A996SK
Equipment:
vials for isomer preparation-
Equipment
40mL Amber Borosilicate Glass Vials
NAME
Vials
TYPE
Environmental Sampling Supply
BRAND
0040-0400-QC
SKU
LINK
40mL volume / Open-Top / Polypro with 0.125" Septa
SPECIFICATIONS
syringe filters-
Equipment
Syringe Filters, 13 mm PTFE
NAME
syringe filter
TYPE
Cytiva
BRAND
2400
SKU
LINK
13mm
SPECIFICATIONS
Equipment
syringe filters, 13mm nylon membrane
NAME
syringe filter
TYPE
Cytiva
BRAND
4550
SKU
LINK
13mm
SPECIFICATIONS
guard column-
Equipment
ACQUITY UPLC Guard column
NAME
VanGuard Pre-column BEH
TYPE
Waters Corporation
BRAND
186003975
SKU
LINK
ACQUITY UPLC BEH C18 VanGuard Pre-column, 130Å, 1.7 µm, 2.1 mm X 5 mm, 3/pk
SPECIFICATIONS
analytical column-
Equipment
ACQUITY UPLC Analytical Column
NAME
ACQUITY UPLC BEH Analytical Column
TYPE
Waters Corporation
BRAND
186002350
SKU
LINK
ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 mm X 50 mm, 1/pk
SPECIFICATIONS
UHPLC-DAD system-
Equipment
1290 Infinity UHPLC
NAME
Ultra-high performance liquid chromatography system
TYPE
Agilent Technologies
BRAND
1290 Infinity UHPLC
SKU
LINK
Safety warnings
All chemicals used for this procedure are hazardous. Read the Safety Data Sheet (SDS) for each chemical listed and follow all applicable chemical handling and waste disposal procedures. Manufacturer specific SDS information can be found by following the catalog numbers of compounds in 'Materials' list.
Before start
All solvents and chemicals used are listed in the ‘Materials’ section. These are excluded from in-line references to maintain clarity and keep the steps concise.
Preparation of Mobile Phases
Mobile Phases
- To make the aqueous 0.2% formic acid, dilute 2.0 mL of formic acid into 1.0 L of 18.2MΩ⋅cm ultrapure water (UPW). Volumetric preparation is optimal.
- Acetonitrile is used as the organic mobile phase.
Note
It is advised to prepare sufficient mobile phase for the entire analysis to reduce the need to add additional mobile phase during an active sequence. Adding mobile phase during an active sequence may cause retention time shifting if the new mobile phase varies in pH or acid concentration. This method uses 5.6 mL of mobile phase per injection, calculate how much mobile phase is needed before beginning analysis.
Preparation of Standards
Standards
This procedure for standard preparation is previously documented in our work published as the National Renewable Energy Laboratory (NREL) Laboratory Analytical Procedure (LAP) 'Determination of cis,cis-and cis,trans-Muconic Acid from Biological Conversion' (https://www.nrel.gov/docs/fy19osti/74473.pdf). Preparation of both cis,cis- and cis,trans-muconic acid isomer standard solutions are outlined in LAP sections 10.1.3 and 10.1.4 and below in protocol step 2.1. Both standard working solutions of c,c and c,t isomers of muconic acid are prepared at 1 g/L concentrations in 0.05% v/v sodium hydroxide solution (preparation of sodium hydroxide is outlined in the referenced LAP, section 10.1.2 and below in step 2.1). Each muconic acid isomer is to be prepared in separate calibration curves in subsequent steps, the isomers should not be combined due to the tendency for c,c to irreversibly isomerize to c,t-muconic over time at room temperature.
The LAP uses the following acronyms:
c,c-muconic acid (ccMA)
c,t-muconic acid (ctMA)
10.1.2 from LAP
Prepare a sodium hydroxide solution (0.05% v/v) for standard preparation. Prepared by adding 66 μL 10N sodium hydroxide with an air displacement pipette to 39.934 mL of UPW measured using a repeater pipette. This solution may be scaled if necessary.
10.1.3 from LAP
Prepare the ccMA stock standard by weighing 40.0 ± 0.5 mg of the ccMA standard into a 40 mL amber vial and record the weight of the standard to the nearest 0.1 mg. Add an appropriate volume of 0.05% v/v sodium hydroxide solution using a repeater pipette to make a final concentration of exactly 1.0 mg/mL solution and mix well (vigorous shaking periodically over approximately 1 hour to allow for solubilization). The muconic acid should be completely dissolved before use otherwise the concentration of the standard will be unknown. Record the date of preparation, concentration, and any other pertinent information on the vial and store sealed at 4 °C for up to 4 months (stability study ongoing).
10.1.4 from LAP
Prepare the ctMA stock standard by preheating a water bath to 60 ± 3 °C. Weigh exactly 40.0 mg of the ccMA standard into a 40 mL amber vial (vial REQUIRED as ordered per Step 7.2.1; vial variation will lead to heat transfer difference and the reaction time will either lead to incomplete ctMA formation or lactone formation). Record the weight of the standard to the nearest 0.1 mg. Add 39.934 mL UPW or similar using a repeater pipette and mix well. Record the concentration of the standard, date of preparation, and any other pertinent information on the vial. Seal the standard with compatible vial top and place into the water bath so that the liquid in the vial is completely submerged for 2 hours. Shake every 15 minutes (use personal protective equipment as necessary). After 2 hours, immediately add 66 μL of 10 N sodium hydroxide using an air displacement pipette and mix. Store the sealed vial at 4 °C for up to 4 months (stability study ongoing).
Additionally, aromatic analytes compatible with this method are listed in the 'Materials' section.
Preparation of aromatic analyte standard stocks should be performed in a compatible solvent for compound solubility. A 1 g/L concentration ampule of Aromatics Mix Large Rev.1 (detailed in 'Materials') is used and diluted in UPW to reach relevant calibration curve concentrations.
Calibration Curve
Example calibration curve preparation (click to enlarge)
Note
Reporting limits and linear ranges may vary and should be determined for each instrument and analyte individually. The standard ranges provided by the manufacturer are suggested starting amounts and may change depending on detector response.
Preparation of Samples
Samples
- All samples containing muconic acid require a minimum 5x dilution scheme (4:1 diluent to sample ratio) for reliable quantitation of isomers due to matrix effects causing chromatographic issues. Compounds included in the aromatic mix do not require dilution without the presence of muconic acid.
- Samples must be filtered through a 0.2 µm or smaller filter prior to injection on the UHPLC.
- Samples expected to be over the linear range of the instrument should be further diluted to be within the calibration range to ensure accurate analysis and avoid carryover.
UHPLC-DAD Analysis
Method Specifications
Analysis of muconic acid isomers and aromatics is performed using an Agilent 1290 series ultra high performance liquid chromatography (UHPLC) system with a diode array detector (DAD). Complete method parameters are in the tables below.
Defined UHPLC parameters (click to enlarge)
Defined UHPLC-DAD parameters (click to enlarge)
Use the analytical column listed here, as well as associated guard column phase (with associated holder) listed in 'Materials'.
Equipment
ACQUITY UPLC Analytical Column
NAME
ACQUITY UPLC BEH Analytical Column
TYPE
Waters Corporation
BRAND
186002350
SKU
LINK
ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 mm X 50 mm, 1/pk
SPECIFICATIONS
Note
Muconic acid isomers are quantified using the 265 nm wavelength and aromatics on 265 nm, 280 nm, and 310 nm. Varying wavelength signals can be used for different aromatic compounds and should be chosen at the discretion of the analyst.
Analytical Quality Control
Multiple strategies are utilized when performing this analysis to ensure instrument stability and reproducibility.
Calibration Curves
A minimum of 5 standard levels should be used.
All compounds must have a correlation coefficient (r2) of 0.995 or greater using a linear calibration fit and ignoring the origin.
Calibration Verification Standards (CVS)
A calibration verification standard (CVS) is a standard from the calibration curve that is re-injected every 20 or fewer samples to ensure instrument drift remains within the determined acceptance criteria. Acceptable CVS recoveries for this analysis are within 10% of the expected amount. Acceptance criteria may differ between instruments and should be determined experimentally.
Example Chromatography
Data Reporting
Muconic acid should be reported as a sum of the two isomers. While isomerization from c,c-muconic to c,t-muconic is irreversible, environmental conditions (decreased pH, exposure to heat, etc.) may cause further isomerization of c,c-muconic acid to c,t-muconic acid. This will cause a change in the ratios of these isomers but the total muconic acid concentration will remain constant. Discrepancies in data are avoided by reporting the total of c,c-muconic acid plus c,t-muconic acid as a sum of the isomers.
Protocol references
Black, Brenna A., William E. Michener, Courtney E. Payne, and Gregg T. Beckham. 2019.
Determination of cis,cis- and cis,trans-Muconic Acid from Biological Conversion. Golden,
CO: National Renewable Energy Laboratory. NREL/TP-5100-74473.