Aug 22, 2024

Public workspaceAnalysis of ethylene glycol and 1,4-butanediol by HPLC-RID

 Forked from Analysis of sugars, small organic acids, and alcohols by HPLC-RID
DOI
dx.doi.org/10.17504/protocols.io.6qpvr8wrplmk/v1
  • 1Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA;
  • 2Agile BioFoundry, Emeryville, CA, USA
  • NREL
    Tech. support email: ftlb_analysis@nrel.gov
Alexander F. Benson
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr8wrplmk/v1
Protocol Citation: Hannah M. Alt, Alexander F. Benson, Stefan J. Haugen, Morgan A. Ingraham, William E. Michener, Sean P. Woodworth, Kelsey J. Ramirez, Gregg T. Beckham 2024. Analysis of ethylene glycol and 1,4-butanediol by HPLC-RID. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8wrplmk/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: August 22, 2024
Last Modified: August 22, 2024
Protocol Integer ID: 106282
Keywords: formic acid, acetic acid, propionic acid, butyric acid, lactic acid, glucose, xylose, arabinose, glycerol, carboxylic acids, 87H, HPLC, refractive index detector, RID, monomeric sugars
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 performed as part of the BOTTLE™ Consortium and was supported by Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (BETO), and Advanced Materials and Manufacturing Technologies Office (AMMTO).
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 concentration of ethylene glycol and 1,4-butanediol in aqueous samples. This list is non-exhaustive as this isocratic method with an acid modified mobile phase is compatible with analysis of many alcohols, small acids, and sugars. This method utilizes a Bio-Rad Aminex HPX-87H Ion Exclusion Column to provide chromatographic separation.

Guidelines
This protocol utilizes an high pressure liquid chromatography refractive index detector (HPLC-RID) system manufactured by Agilent Technologies as referenced in 'Materials'. A similar HPLC-RID system can be utilized; however, some parameter nomenclature may deviate depending on the manufacturer.
Materials

Reagents:

Reagent10N Sulfuric AcidFisher ScientificCatalog #SA200-1

Standards:

ReagentEthylene glycolToronto Research Chemicals IncCatalog #E890140-1mL

Reagent1,4-butanediolMerck MilliporeSigma (Sigma-Aldrich)Catalog #493732-1L



Materials:

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
Syringe filters for organic 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
Instrumentation:
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 Multisampler G7116A 1260 MCT G7117C 1260 DAD HS G7162A 1260 RID
SPECIFICATIONS

Column:

Analytical Column
Equipment
HPX-87H Column
NAME
Column
TYPE
Bio-Rad
BRAND
125-0140
SKU
https://www.bio-rad.com/en-us/sku/1250140-aminex-hpx-87h-column?ID=1250140&WT.mc_id=170208017160&WT.srch=1&WT.knsh_id=_kenshoo_clickid_&gclid=EAIaIQobChMI5vbXvujS9wIVTxPUAR2GPgz2EAAYAiAAEgIuV_D_BwE
LINK
Guard Column
Equipment
Micro-Guard Cation H Cartridge
NAME
Guard Column
TYPE
Bio-Rad
BRAND
1250129
SKU
https://www.bio-rad.com/en-us/sku/1250129-micro-guard-cation-h-refill-cartridges?ID=1250129
LINK

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 phases

  1. To make 0.01 N sulfuric acid (0.005 M), dilute 1.0 mL of 10 N sulfuric acid into 1.0 L of 18.2MΩ⋅cm ultrapure water (UPW). Volumetric preparation of this mobile phase will yield the most reproducible chromatography. See note below.

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 18.0 mL of mobile phase per injection. Calculate how much mobile phase is needed before beginning analysis to prepare enough for the entire analysis.
Instrument equilibration

  1. Purge instrument with 0.01 N sulfuric acid solution made in the previous step. Be certain the instrument is purged through the entire flow path including the detector, before the analytical column is plumbed in. The Aminex 87H column is sensitive to solvents, for example, a higher amount of methanol will damage the column. The purge step is needed to remove all solvents/mobile phases from the analytical system. (See Bio-Rad's 'Instruction Manual' for Aminex resin-based columns for solvent compatibility and installation details).
  2. Add the guard column and analytical column to the system and begin equilibrating the column at a low flow rate of 0.2 mL/min while the column reaches analysis temperature.
  3. During the column equilibration, begin purging the reference cell of the refractive index detector (RID), this process will continue through the final equilibration of the column.
  4. At intervals of at least 10 minutes, increase the flow by 0.2 mL/min until you reach the method flow of 0.6 mL/min.
  5. Once the column is up to method flow and both the column compartment and the RID are at method temperature and stable, the RID reference cell can be closed. This typically takes around 30 minutes after method flow is reached. A longer purge of the reference cell is not detrimental.
  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. Prepare a 10 mg/mL ethylene glycol stock standard by weighing 50 mg of the ethylene glycol raw material into a 20 mL amber vial. Record the weight of the raw material to the nearest 0.1 mg. Add 5 mL of UPW to amber vial and vortex thoroughly until raw material is dissolved.
  2. Repeat step 1 in order to create a 10mg/mL 1,4-butanediol stock standard.
  3. Make separate calibration curves for ethylene glycol and 1,4-butanediol by following the calibration table below.

example calibration curve preparation (click to enlarge)

Note
Reporting limits and linear ranges may vary and should be determined for each instrument individually. Stock standards can be made at varying concentrations that allow for quantification of samples within a desired range.

Preparation of samples
Preparation of samples
Samples
  • 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 to use depending on matrix composition)
  • Samples expected to be over the linear range of the instrument should be diluted to ensure accurate analysis and avoid carryover.

HPLC-RID analysis
HPLC-RID analysis
Method Specifications

Analysis is performed using an Agilent 1200 Series High Performance Liquid Chromatography (HPLC) system. An isocratic concentration of 0.01N sulfuric acid through an Aminex HPX-87H column (300 x 8.7 mm, 9 μm particle size) is used to achieve separation at a flow rate of 0.6 mL/min. Quantitation is determined using refractive index detection (RID). Column and RID are both held constant at 55 °C and each sample and standard is injected at a volume of 6.0 μL.


Retention time of analytes is dependent on the configuration of the HPLC and will vary from instrument to instrument. Retention time markers for each analyte should be run individually to assess the total required run time of the analysis based on elution. Additional analytes not listed in this protocol may be compatible with these instrument parameters. This re-emphasizes the need to run single analyte retention time markers to prevent co-eluting peaks. The method run time is 27 minutes.

Note
Injection volume can be increased to 20 μL to obtain a lower reporting limit if necessary. This has the possibility to reduce the upper limit of quantitation due to signal saturation.

Analytical Quality Control
Analytical Quality Control
Multiple strategies are utilized when performing this analysis to ensure instrument stability and reproducibility.
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 Standards (CVS)

A calibration verification standard (CVS) is a level provided by the manufacturer 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 Chromatography
Example Chromatography


Example chromatogram including elution order of ethylene glycol and 1,4-butanediol. (click to enlarge)