TITLE: Quantitation of aliphatic acids from lignin-derived streams by HPLC-RID
AUTHORS: Stefan J. Haugen, Alexander F. Benson, Sean P. Woodworth, David Brandner, Kelsey J. Ramirez, Gregg T. Beckham

[DESCRIPTION]
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. 

[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.

[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. 

[STEPS]
SECTION: Preparation of mobile phase and instrument equilibration
2. Instrument equilibration

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.
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.
Purge the reference cell of the refractive index detector (RID), leave reference cell purge valve open through the duration of the column equilibration.
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.
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. 
After the reference cell is closed, wait for the RID signal to stabilize before starting analysis.
SECTION: Preparation of mobile phase and instrument equilibration
1. 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).
 

SECTION: Preparation of standards
3. Standards


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.

 

SECTION: Preparation of standards
3.1. Calibration curve



 


SECTION: HPLC-RID analysis
6. Prepare an Agilent 1260 Infinity II LC System according to the following parameters for a total run time of 30 minutes. 
 

 

SECTION: Data analysis and quality control
7. Data analysis
Data analysis completed using Agilent OpenLab CDS, ChemStation Edition version A.01.11.138
SECTION: Example chromatogram
8. 
 

SECTION: Data analysis and quality control
7.1. Calibration curves
All compounds must have a correlation coefficient (r2) of 0.995 or greater using a linear calibration fit and ignore the origin.
SECTION: Data analysis and quality control
7.2. 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.
SECTION: Acidification procedure
5. 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).
SECTION: Acidification procedure
4. 
 
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]) 

SECTION: Acidification procedure
4.2. 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.
SECTION: Acidification procedure
4.1. Add acid at calculated volume to sample in labelled acidification microfuge tube and record sample volume used and vortex thoroughly.
SECTION: Acidification procedure
5.1. Samples that are expected to be over the linear range should be diluted to ensure accurate analysis and avoid carryover.