TITLE: Analysis of monomeric aromatic compounds in alkaline lignin-rich liquors via UHPLC-DAD
AUTHORS: Sean P. Woodworth, Morgan A. Ingraham, Stefan J. Haugen, Kelsey J. Ramirez, Gregg T. Beckham, Davinia Salvachua

[DESCRIPTION]
An analysis method was developed to quantify aromatic compounds present in lignin-rich, alkaline pre-treated liquors produced from corn stover. This method employs ultra-high pressure liquid chromatography paired with diode array detection (UHPLC-DAD). A reverse phase Luna Omega PS C18 column from Phenomenex and a gradient were used to separate various aromatic compounds. 

[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 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. 

[STEPS]
SECTION: Preparation of standards
1. By weight, create individual 10 g/L stock standards of the following analytes using ethanol (EtOH) as a diluent:
Protocatechuic acid
Catechol
4-Hydroxybenzoic acid
p-Coumaric acid
Ferulic acid
Vanillyl alcohol
Vanillic acid
4-Hydroxybenzaldehyde
Vanillin
SECTION: Preparation of standards
2. Combine stock standards to make a 1 g/L aromatics mixed standard using EtOH as a diluent. A potential preparation can be made by adding 1 mL of each of the standards and 1 mL EtOH for an overall 10-fold dilution. 

Create a calibration curve with a minimum of 5 points using ultrapure water (18.2MΩ⋅cm)(UPW) as a diluent.


 



SECTION: Preparation of mobile phases
3. Mobile phase A consists of 10 mM phosphoric acid in UPW. This can be prepared by adding 0.67 mL of 85% (w/w) phosphoric acid per liter of UPW.

Mobile phase B was acetonitrile.
SECTION: Sample preparation
4. Confirm that the sample matrix is compatible with instrumentation. The calibration range for this method is between 1.0 µg/mL and 500 µg/mL. Any samples with analyte concentrations expected to be greater than 500 µg/mL should be diluted appropriately to minimize the potential for carryover. Filter all samples through 0.2 µm filtered prior to injection.
SECTION: UHPLC analysis
5. Analysis is completed on an Agilent 1290 UHPLC system utilizing the following parameters. 




 


 



Analysis was completed utilizing wavelengths 265 nm and 280 nm. Quantitation of these aromatic compounds can be accomplished on multiple wavelengths. Discretion should be used in choosing the quantitative wavelength that produces the optimal response. 
SECTION: Data analysis and quality control
6. Data analysis was completed using Agilent OpenLab CDS ChemStation Edition Rev. C.01.10

The following criteria are employed to ensure reproducibility and stability throughout the analysis:
All analyte calibration curves must have a  correlation coefficient (r2) of greater than or equal to 0.995. A quadratic or linear fit may be applied with the origin ignored. 
A calibration verification standard (CVS) should be analyzed every 20 or fewer samples. This is a point from the calibration curve re-analyzed throughout the instrument run to check for drift. An acceptable recovery range for this method is +/- 10% of the expected concentration. 
SECTION: Example chromatogram
7.