Oct 16, 2024

Public workspaceQuantitation of aliphatic acids from lignin-derived streams by HPLC-RID

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxrn2gx1/v1
  • 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA
  • NREL
    Tech. support email: ftlb_analysis@nrel.gov
Alexander F. Benson
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzxrn2gx1/v1
Protocol Citation: Stefan J. Haugen, Alexander F. Benson, Sean P. Woodworth, David Brandner, Kelsey J. Ramirez, Gregg T. Beckham 2024. Quantitation of aliphatic acids from lignin-derived streams by HPLC-RID. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxrn2gx1/v1
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: January 10, 2024
Last Modified: October 16, 2024
Protocol Integer ID: 93350
Keywords: lignin-derived streams, aliphatic acids, HPLC-RID, oxalic, malic, malonic, succinic, glycolic, lactic, formic, acetic, propionic
Funders Acknowledgement:
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 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 analytical method was developed using high performance liquid chromatography with refractive index detection (HPLC-RID) to quantify the concentrations of aliphatic acids. The analytes quantified were oxalic acid, malic acid, malonic acid, succinic acid, glycolic acid, lactic acid, formic acid, acetic acid, and propionic acid in samples from lignin-derived streams. This method utilized an ion exclusion column to provide chromatographic separation across a 30 minute run-time.
Guidelines
This protocol utilizes an high pressure liquid chromatography equipped with a refractive index detector (HPLC-RID) 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
Reagents:

ReagentSulfuric Acid 10N SolutionFisher ScientificCatalog #SA200-1
ReagentSulfuric Acid, 72% (w/w)Ricca Chemical CompanyCatalog #R8191600-1A

Standards:

ReagentAliphatics Mix 9 components Absolute StandardsCatalog #67191
Download Aliphatics Mix 9 components COA.pdfAliphatics Mix 9 components COA.pdf

The Aliphatics Mix 9 components standard contains the following compounds.
  1. Oxalic acid
  2. Malic acid
  3. Malonic acid
  4. Succinic acid
  5. Glycolic acid
  6. Lactic acid
  7. Formic acid
  8. Acetic acid
  9. Propionic acid

Consumables:

Syringe filters for aqueous matrices
Equipment
syringe filters, 13mm nylon membrane
NAME
syringe filter
TYPE
Cytiva
BRAND
4550
SKU
https://www.cytivalifesciences.com/en/us/shop/lab-filtration/syringe-filters-non-sterile/nylon-non-sterile-syringe-filters/acrodisc-syringe-filters-with-nylon-membrane-p-36371
LINK
13mm
SPECIFICATIONS

Equipment:
Equipment
Agilent 1260 Infinity II LC System
NAME
HPLC System
TYPE
Agilent
BRAND
Agilent 1260 Infinity II LC System
SKU
https://www.agilent.com/en/product/liquid-chromatography/hplc-systems/analytical-hplc-systems/1260-infinity-ii-lc-system
LINK
G7111B Quat Pump, G7167A 1260 Multisampler, G7116A 1260 MCT, G7117C DAD HS, G7162A 1260 RID
SPECIFICATIONS
Column:

Analytical Column


Equipment
Rezex ROA-Organic Acids H+ (8%)
NAME
LC Column
TYPE
Phenomenex
BRAND
00H-0138-K0
SKU
https://www.phenomenex.com
LINK
300 x 7.8 mm
SPECIFICATIONS
Guard Column

Equipment
Rezex ROA-Organic Acid H+ (8%)
NAME
LC Guard Column
TYPE
phenomenex
BRAND
03B-0138-K0
SKU
https://www.phenomenex.com/
LINK
50 x 7.8 mm
SPECIFICATIONS


Safety warnings
All chemicals used for this procedure are hazardous. Read the Safety Data Sheet (SDS) for all chemicals and follow all applicable chemical handling and waste disposal procedures. Manufacturer specific SDS information can be found by following the CAS numbers of compounds in 'Materials' list.

Sulfuric acid can cause serious chemical burns. See SDS for additional information:
https://beta-static.fishersci.com/content/dam/fishersci/en_US/documents/programs/education/regulatory-documents/sds/chemicals/chemicals-s/S25899.pdf
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 phase and instrument equilibration
Preparation of mobile phase and instrument equilibration
Mobile phase preparation

  • To make 0.02 N sulfuric acid (0.01 M), dilute 2.0 mL of 10 N sulfuric acid into 1.0 L of 18.2MΩ⋅cm ultrapure water (UPW).
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 is not identical to the original mobile phase. This method uses roughly 15.0 mL of mobile phase per injection. Calculate how much mobile phase is needed before beginning analysis.

Instrument equilibration

  1. Purge the high performance liquid chromatography (HPLC) instrument with 0.02 N sulfuric acid mobile phase made in step 1. Ensure the instrument is purged through the entire flow path including the detector, before the analytical column is installed.
  2. Install the Rezex ROA-Organic Acid LC guard column and the Rezex ROA-Organic Acid analytical column to the system. Begin equilibrating the column at a low flow rate of 0.1 mL/min while the column approaches compartment temperature.
  3. Purge the reference cell of the refractive index detector (RID), leave reference cell purge valve open through the duration of the column equilibration.
  4. At intervals of at least 10 minutes, increase the flow by 0.2 mL/min until you reach the method flow of 0.5 mL/min.
  5. Once the system is at method flow rate and the column compartment and the RID reach analysis temperature, close the reference cell purge valve. This typically takes around 30 minutes after method flow rate is reached. A longer purge of the reference cell is not detrimental to the system.
  6. After the reference cell is closed, wait for the RID signal to stabilize before starting analysis.
Preparation of standards
Preparation of standards
Standards


  1. Remove aliphatics mix ampule (listed in 'Materials' section) from freezer and allow it to come to room temperature, vortex, and transfer contents of ampule into a 2 mL amber HPLC vial.
2. Using this 5 g/L stock mix, follow the table below to create a calibration curve.

Note
Standards should be kept in a freezer capable of maintaining a temperature of -20°C.

Calibration curve



Example calibration curve preparation (click to enlarge)


Acidification procedure
Acidification procedure

Note
For alkaline samples or samples containing high molecular weight lignin the following sample acidification should be performed prior to analysis to avoid column fouling. If samples don't meet these criteria, proceed to step 5 below.
An addition of concentrated acid in excess is needed to lower sample pH and remove components that are harmful to the HPLC guard column and analytical column. This allows for better chromatographic results and subsequently better quantitation of aliphatic acids. Acidification is performed by adding 72% sulfuric acid to samples at a proportion of ~3.5% of the total sample volume.

Define volume of sample to be acidified, noting that filtration will retain approximately 30 µL of liquid. Use the equation below to calculate the volume of acid to be added to each sample respectively. Example: 1.5 mL of sample requires 52.2 µL of 72% sulfuric acid.

(sample volume[µL]) * (0.035) = (amount of 72% sulfuric acid to be added [µL])

Add acid at calculated volume to sample in labelled acidification microfuge tube and record sample volume used and vortex thoroughly.
Affix filtration system (0.2 µm nylon) to luer lock syringe. Add sample to syringe, carefully and slowly plunge acidified sample through filter to avoid bursting filter membrane into labelled HPLC vial. Samples are now ready for instrument analysis.
Skip step 5 if acidification procedure was performed. Samples must be filtered through a 0.2 µm or smaller filter prior to injection on the HPLC ('Materials' section includes part numbers for filters).
Samples that are expected to be over the linear range should be diluted to ensure accurate analysis and avoid carryover.
HPLC-RID analysis
HPLC-RID analysis
Prepare an Agilent 1260 Infinity II LC System according to the following parameters for a total run time of 30 minutes.
Defined HPLC parameters (click to enlarge)

Defined HPLC-RID parameters (click to enlarge)

Data analysis and quality control
Data analysis and quality control
Data analysis
  • Data analysis completed using Agilent OpenLab CDS, ChemStation Edition version A.01.11.138
Calibration curves
  • All compounds must have a correlation coefficient (r2) of 0.995 or greater using a linear calibration fit and ignore the origin.
Calibration verification standard (CVS)
  • A CVS is a standard from the calibration curve that is re-analyzed 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 chromatogram
Example chromatogram

Example chromatogram including elution order of 'Aliphatics Mix 9 components' located in 'Materials'. (click to enlarge)