Nov 26, 2025
Preparation and Analysis of Microbial Fermentation Samples using a Shimadzu Prominence-i LC-2030C 3D Liquid Chromatograph HPLC System
- Isaiah Richardson1,
- Bishal Dev Sharma1,
- Daniel G Olson1
- 1Dartmouth College Thayer School of Engineering
- Isaiah Richardson: PhD Student
- Bishal Dev Sharma: PhD Candidate
- Daniel G Olson: Principal Investigator of the Olson Lab
- Lynd/Olson Lab
Protocol Citation: Isaiah Richardson, Bishal Dev Sharma, Daniel G Olson 2025. Preparation and Analysis of Microbial Fermentation Samples using a Shimadzu Prominence-i LC-2030C 3D Liquid Chromatograph HPLC System. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrqbjblmk/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
Created: June 18, 2025
Last Modified: November 26, 2025
Protocol Integer ID: 220558
Keywords: HPLC Analysis, Microbial Fermentation Analysis, High Performance Liquid Chromatography, HPLC, Shimadzu HPLC System, Analyzing Samples on HPLC, Shimadzu HPLC Analysis, Shimadzu, analysis of microbial fermentation sample, microbial fermentation sample, metabolites present in microbial fermentation sample, standards for hplc analysis, hplc analysis, microbial fermentation, chromatograms of each sample, chromatogram, hplc instrument, hplc system, 3d hplc instrument, hplc, biological sample, metabolite, interest in biological sample, concentrations of compound, standard analytical approach, thermophilic microbe
Funders Acknowledgements:
US National Science Foundation
Grant ID: 2313152
Abstract
High Performance Liquid Chromatography (HPLC) is a standard analytical approach used across the field of biotechnology for quantifying the concentrations of compounds of interest in biological samples, such as samples from microbial fermentations. The purpose of this protocol is to provide users of the Shimadzu Prominence-i LC-2030C 3D Liquid Chromatograph HPLC system with a step-by-step method of how to: 1) prepare microbial fermentation samples and standards for HPLC analysis, 2) start and purge the HPLC system to initialize the instrument, 3) setup and start a batch run of samples, 4) analyze the chromatograms of each sample to quantify the metabolites present (in concentration units of mM), and 5) export the results to Microsoft Excel for subsequent data processing, analysis, and plotting. Although this protocol is specifically tailored for users of the Shimadzu Prominence-i LC-2030C 3D Liquid Chromatograph HPLC system for the quantification of metabolites present in microbial fermentation samples (i.e., from anaerobic, thermophilic microbes such as Clostridium thermocellum), the general workflow of this protocol and steps for using the LabSolutions CS software for purging the HPLC instrument, setting up and starting a batch file, and analyzing the results are broadly applicable to many users of the Shimadzu Prominence-i LC-2030C 3D HPLC instrument.
Materials
HPLC SYSTEM:
Shimadzu Prominence-i LC-2030C 3D (Liquid Chromatograph)
Link to Shimadzu website for i-Series Integrated HPLC and UHPLC Systems:
Shimadzu Prominence-i LC-2030C 3D Operation manual (PDF):
HPLC SYSTEM DETECTORS:
Shimadzu RID-20A (Refractive Index Detector [Detector B])
Shimadzu Photodiode Array (PDA [UV]) Detector (built into the Shimadzu Prominence-i LC-2030C 3D)
Shimadzu RF-20A XS (Prominence Fluorescence Detector) (used for quantifying amino acids)
ORGANIC ACIDS HPLC COLUMN:
BioRad Aminex HPX-87H HPLC Column
Catalog Number: 1250140; 300 x 7.8 mm, hydrogen form, 9 μm particle size, 8% cross linkage; pH range 1-3.
SHIMADZU HPLC SOFTWARE PROGRAM:
Shimadzu LabSolutions Client Server (CS) Software (Analysis Data System)
ADDITIONAL MATERIALS:
USA Scientific SealRite 2.0 mL Natural Microcentrifuge Tubes (500 tubes per 1 bag)
Catalog Number: 1620-2700; https://www.usascientific.com/seal-rite-2ml-tubes/p/Seal-Rite-2.0
Corning Costar Spin-X Centrifuge Tube Filters (100 per pack, 200 per case)
Product Number: 8169; Polypropylene tube; tube capacity: 2.0 mL; funnel capacity: 0.5 mL; 0.22 μm Nylon filter.
Chemglass Life Sciences HPLC Vials
Product Number: CV-1042-1232; Vial, Plastic Limited Volume, 0.5 mL, Polypropylene, 12 x 32 mm, 11 mm Snap Ring.
Chemglass Life Sciences HPLV Vial Caps
Product Number: CV-3570-B011; Chem Snap Cap, 11 mM, Blue with PTFE/Red Rubber Septum.
Fisherbrand Elite Pipettes
Catalog Number: 14-388-100F25. https://www.fishersci.com/shop/products/elite-pipette-kit-promo
Eppendorf Repeater E3x (Repeat Pipetter)
Eppendorf Combitips Advanced 1.0 mL (For the Eppendorf Repeater E3x)
Catalog Number: 0030089642. Biopur®, 1.0 mL, yellow, colorless tips. https://www.eppendorf.com/us-en/Products/Liquid-Handling/Pipette-Tips/Combitips-advanced-p-0030089642
Barnstead/Thermolyne 16700 Mixer Maxi-Mix 1 (Vortex)
Model Number: M16715; Serial Number: 1329040799136; Volts: 120, Amps: 0.4, Watts: 24, Hz: 60, Phase: 1.
Cole-Parmer Fisher Chemical Sulfuric Acid Solution (95-98%)
CAS Number: 7664-93-9; MPN: A300SI-212; F.W: 98.08; Certified ACS Plus Grade, Safe-Cote™ Glass Bottle, 2.5 L
Safety warnings
This experimental protocol requires users to work with both 100% and 10% w/v sulfuric acid (H2SO4) solutions. Handling and working with sulfuric acid at any concentration is very dangerous, as sulfuric acid even at diluted concentrations can severely burn and permanently damage human skin and eyes. Concentrated sulfuric acid can liquify (dissolve) clothing upon contact. Proper personal protective equipment (PPE), including an appropriately fitting laboratory coat, gloves, goggles, pants (no shorts), and closed toed shoes must be worn at all times while performing this protocol in the lab. 100% sulfuric acid solution should only be handled in a fume hood. Note that 10% and 100% sulfuric acid solutions are used in this protocol for the acidification of microbial fermentation samples, and the preparation of a dilute sulfuric acid mobile phase for the Shimadzu HPLC system, respectively.
Before start
Wear proper personal protective equipment (PPE), including an appropriately fitting laboratory coat, gloves, goggles, pants (no shorts), and closed toed shoes at all times while performing this protocol in the lab.
Introduction & Background
High Performance Liquid Chromatography (HPLC) is a standard analytical approach used across the field of biotechnology for quantifying the concentrations of compounds present in biological samples. A HPLC functions by pumping a biological sample through a column packed with a stationary phase (resin) under high pressure using a mobile phase solvent (i.e., dilute sulfuric acid or water). The different compounds present in a biological sample bind to and elute off of the stationary phase of the column at different retention times due to differences in their chemical structure and affinity for the stationary phase. A Refractive Index Detector (RID) and/or Photodiode Array (PDA) Detector subsequently detect the compounds present in the biological sample by measuring 1) the change in the refractive index of the mobile phase as the compounds elute, or 2) passing UV light through a flow cell containing the separated compounds, splitting the light into its spectrum with a diffraction grating, and measuring the absorbance at multiple wavelengths simultaneously using an array of photodiodes, respectively. Both the Refractive Index and Photodiode Array detectors generate a chromatogram for each biological sample, which contains peaks corresponding to the compounds present in the sample at their respective retention times. Analysis of the chromatograms enables the identification and quantification of the compounds present in each biological sample using standards and the height of each peak. Biological samples suitable for HPLC analysis can include both prokaryotic (i.e., Escherichia coli or Clostridium thermocellum etc.) or eukaryotic (i.e., Saccharomyces cerevisiae or Aspergillus fumigatus etc.) cultures or samples from cell-free systems.
The versatility of HPLC in quantifying different biological compounds arises primarily from the use of different columns, which can differ in 1) the stationary phase (resin) present inside the column, and 2) their physical dimensions (i.e., size of packing material, diameter, and length). An organic acids HPLC column is one of the most widely used columns for quantifying common biological metabolites (i.e., glucose, malate, pyruvate, succinate, lactate, formate, acetate, ethanol etc.). Although HPLC does not provide as high of a degree of resolution as mass spectrometry (MS), HPLC is a relatively straightforward analytical technique to learn and use for the identification and quantification of metabolites of interest in biological samples.
The purpose of this protocol is to provide users of the Shimadzu Prominence-i LC-2030C 3D Liquid Chromatograph HPLC system (such as in the Lynd and Olson Labs in the Thayer School of Engineering at Dartmouth College [including undergraduate and graduate students, post-doctoral researchers, collaborators, and visiting scholars etc.]) with a step-by-step method of how to: 1) prepare microbial fermentation samples and standards for HPLC analysis, 2) start and purge the HPLC system to initialize the instrument, 3) setup and start a batch run of samples, 4) analyze the chromatograms of each sample to quantify the metabolites present (in concentration units of mM), and 5) export the results to Microsoft Excel for subsequent data processing, analysis, and plotting. Please note that this protocol was developed to use with the specific HPLC system, column, detectors, and software detailed below:
High-Performance Liquid Chromatography (HPLC) System:
Shimadzu Prominence-i LC-2030C 3D Liquid Chromatograph
HPLC Column:
BioRad Aminex HPX-87H HPLC Column (Catalog Number: 1250140)
Column Details: 300 x 7.8 mm, hydrogen form, 9 μm particle size, 8% cross linkage, pH range 1-3
Detectors:
1. Shimadzu RID-20A (Refractive Index Detector [Detector B])
2. Shimadzu Photodiode Array (PDA) (UV) Detector
3. Shimadzu RF-20A XS (Prominence Fluorescence Detector)
Software:
Shimadzu LabSolutions CS (client server) software
Protocol Structure (Table of Contents)
This experimental protocol is structured into the following sections:
A. Microbial Fermentation Sampling and Preparation for Storage (Step 3 [3.1-3.10])
B. Preparation of Microbial Fermentation Samples for HPLC Analysis (Step 4 [4.1-4.6])
C. Preparation of Metabolite Standards for a HPLC Run (Step 5 [5.1-5.6])
D. Preparation of Dilute Sulfuric Acid Mobile Phase for a HPLC Run (Step 6 [6.1-6.8])
E. Starting the Shimadzu HPLC Instrument (Step 7 [7.1-7.8])
F. Switching Columns on the Shimadzu HPLC Instrument (Step 8 [8.1-8.18])
G. Purging the Shimadzu HPLC Instrument (Step 9 [9.1-9.8])
H. Setting up and Starting a Batch Run on the Shimadzu HPLC Instrument (Step 10 [10.1-10.25])
I. Editing the Batch File Table During an Active Batch Run of Samples (Step 11 [11.1-11.6])
J. Analyzing the HPLC Results After a Batch Run (Step 12 [12.1-12.24])
Microbial Fermentation Sampling and Preparation for Storage
This section of the protocol describes the steps required for 1) taking samples during an active microbial fermentation experiment and 2) preparing the samples for storage prior to HPLC analysis. Note that this section of the protocol (steps 3.1-3.10) can be repeated for as many timepoints and samples as desired throughout the course of a biological fermentation experiment.
At a desired timepoint during a microbial fermentation experiment, use a pipette to take a 1.0 mL sample from the microbial culture and transfer the sample to a new, labeled 2.0 mL microcentrifuge tube.
If necessary, a smaller volume than 1.0 mL can be taken for a fermentation sample. However, the fermentation sample volume should be at least 500 μL for each timepoint.
Centrifuge the sample for 5 minutes at 21,300 rcf (x g) (maximum speed) using a table top centrifuge. Ensure that the centrifuge is balanced prior to starting the centrifuge.
Remove the sample from the centrifuge and use a pipette to carefully extract 400 µL of supernatant without disturbing the cell pellet and transfer the supernatant to a new 2.0 mL microcentrifuge tube.
Use a repeat pipette to add 20 µL of 10% w/v sulfuric acid solution (sulfuric acid in MilliQ water at a final concentration of 10% w/v) to each of the 400 µL supernatant samples.
Despite being diluted, 10% sulfuric acid (H2SO4) should be handled with tremendous care and caution as sulfuric acid is a dangerous laboratory chemical. Ensure that proper PPE (laboratory coat, goggles, and gloves) is worn at all times when handling 10% sulfuric acid. If 10% sulfuric acid gets spilled on gloves, immediately remove and dispose of the gloves and thoroughly wash your hands for at least 5 minutes with soap and water. If 10% sulfuric acid gets spilled on clothing, it will stain and discolor the clothing. For safety reasons, the contaminated clothing must be thrown away inside the laboratory.
Vortex each acidified supernatant sample for 10-15 seconds to ensure that the dilute sulfuric acid is thoroughly mixed with the sample.
Centrifuge the acidified samples for 5 minutes at 21,300 rcf (x g) using a table-top centrifuge.
Following centrifugation, debris may be present at the bottom of the tube. This is expected especially for fermentations performed with high substrate concentrations (i.e., exceeding 50 g/L substrate).
Remove the samples from the centrifuge and carefully pour each acidified supernatant sample into a new, labeled Costar Spin-X Centrifuge Tube (spin filter tube).
Note that if there is residual, acidified supernatant remaining in the 2.0 mL microcentrifuge tube, do not force the supernatant into the spin-filter tube, just dispose of the microcentrifuge tube. This will help to prevent the filter in the spin filter tube from becoming clogged in the following step (Step 3.8).
Centrifuge the acidified supernatant samples in the spin filter tubes for 5 minutes at 21,300 rcf (x g).
During this time, the supernatant will pass through the filter into the bottom of the tube and leave any residual debris behind in the filter. This is a critical step as this helps to purify the supernatant sample and reduce the risk of the HPLC tubing and/or column from getting clogged by debris present in the sample.
Remove the acidified supernatant samples in the spin filter tubes from the centrifuge. Remove and dispose of the filter from each spin filter tube in a biohazard waste container. Close the spin filter tube caps and ensure that each cap is tightly sealed.
Store the acidified supernatant samples in the sealed spin filter tubes (filter removed) in a 4ºC freezer prior to running the samples on the HPLC. If planning to run the samples on the HPLC immediately following Step 3.9, proceed to Step 4.2.
The acidified supernatant samples can be stored in the 4ºC freezer for any time frame ranging from a few hours to approximately one month and still provide reliable metabolite quantification results on the HPLC.
Preparation of Microbial Fermentation Samples for HPLC Analysis
This section of the protocol describes the steps required for preparing acidified, fermentation supernatant samples in spin filter tubes (stored at 4ºC for up to one month) for HPLC analysis.
If applicable, remove the desired fermentation supernatant samples from long-term storage at 4ºC.
Vortex each sample for 10-15 seconds to thoroughly mix the acidified, supernatant sample in the spin filter tube (note that the filter should have been removed prior to long-term storage [Step 3.9]).
Use a pipette to carefully transfer 200 µL of acidified, filtered supernatant sample into a new, labeled HPLC vial and cap each HPLC vial.
Return the remaining volume of acidified, supernatant sample in the spin filter tubes (with the filter removed and cap sealed) to the 4ºC freezer until after the HPLC run and data analysis is finished.
This is a very important step, as HPLC samples sometimes have to be re-run due to an error during a HPLC run (i.e., if the run is stopped due to an instrument issue or power outage, the sample does not inject properly, or the presence of air bubble(s) in a sample interferes with the resulting chromatogram etc.).
Ensure that there are no air bubbles present in each HPLC vial. If an air bubble(s) is observed in a HPLC vial, tap the vial on a lab bench several times until the air bubble(s) pops and disappears. Repeat this step, (if necessary), until there are no air bubbles in all of the HPLC vials.
The presence of an air bubble(s) in a sample can clog the HPLC tubing and/or column, which may cause the pressure in the HPLC system to increase sharply and cause the run to stop prematurely. It can be very difficult and time-intensive to locate and remove air bubbles trapped inside HPLC tubing or a column.
Transfer all of the prepared HPLC vials into a sample tray. Place the sample tray in the sample tray drawer located at the front of the Shimadzu HPLC system and ensure that the draw is fully closed.
It is important to note and record the order in which the samples were placed in the sample tray (i.e., from vial position 1 to 54), as this information will be required for setting up a batch file table in Step 10 below.
Preparation of Metabolite Standards for a HPLC Run
This section of the protocol describes the steps required for preparing metabolite standards for a HPLC run so the concentration of the metabolites present in each sample run on the Shimadzu HPLC system can be accurately quantified (in concentration units of mM) during subsequent data analysis.
Refer to the 'HPLC Standard Recipe and Calculator.xlsx' Microsoft Excel spreadsheet file uploaded as an attachment to this protocol as a reference if you want to prepare your own standards from scratch.
Remove the pre-prepared, unacidified 1x (blue), 2x (mauve/pink) and 8x (white) metabolite reference standard boxes and the 60 mM (green), 30 mM (blue), and 7.5 mM (dark pink/red) Glucose standard boxes from long-term storage in the -20°C freezer. These metabolite reference standards historically have been pre-prepared by a research staff member in the Lynd Lab (Marybeth I. Maloney).
Remove one frozen 1x (blue sticker), 2x (mauve/pink sticker), and 8x (white sticker) reference standard 2.0 mL microcentrifuge tube and one frozen 60 mM (green line), 30 mM (blue line), and 7.5 mM (red line) Glucose standard 2.0 mL microcentrifuge tube from their respective boxes. Place these standards on the lab bench and allow them to thaw for 20-30 minutes until the standards are completely thawed. In the meantime, promptly return the reference standard boxes to their designated location in the -20°C freezer.
Vortex each thawed, reference standard microcentrifuge tube thoroughly for 10-15 seconds and use a pipette to transfer 400 µL of each reference standard to a new, labeled 2.0 mL microcentrifuge tube.
Use a repeat pipette to add 20 µL of 10% w/v sulfuric acid solution to each reference standard tube.
Vortex each acidified standard for 10-15 seconds and use a pipette to transfer 200 µL of each acidified reference standard to a new, labeled HPLC vial and cap each HPLC vial. Repeat this step twice such that there are two HPLC vials prepared for each standard (i.e., a 1x - 1 vial, and a 1x - 2 vial).
There should be twelve (12) total HPLC vials comprised of two replicates of each of the six standards (1x, 2x, 8x standards; 60 mM, 30 mM, 7.5 mM Glucose standards) acidified and ready to run on the HPLC.
Add the twelve (12) acidified reference standard HPLC vials to the sample tray containing the HPLC vials for the biological fermentation samples prepared above in Step 4 and close the sample tray drawer.
It is best practice to place one set of reference standards (comprised of 6 standards: 1x, 2x, 8x, 60 mM, 30 mM, 7.5 mM) at both the beginning and end of the set of experimental samples within the sample tray so a full set of standards run at both the beginning and end of the run on the Shimadzu HPLC system. Note the final order of the samples and standards in the sample tray as this will be needed for Step 10.
Preparation of Dilute Sulfuric Acid Mobile Phase for a HPLC Run
This section of the protocol describes the steps required for the preparation of the dilute sulfuric acid mobile phase (solvent) for a run on the Shimadzu HPLC System using the organic acids HPLC column.
Acquire a clean and dry 1.0 L glass bottle with an orange screw cap from the glassware storage cabinet.
If a 1.0 L glass bottle is not clean, thoroughly triple rinse a 1.0 L glass bottle and its corresponding orange, plastic cap at least three times (3x) using tap water, deionized (DI) water, and milliQ water in that order. Ensure that there is no residue remaining in the bottle or cap. Place the 1.0 L glass bottle in the 102°C oven overnight with the lid only slightly attached so the bottle can fully dry prior to use.
Using heat-resistant gloves, carefully remove the clean and dry 1.0 L glass bottle from the 102°C oven.
Fill the 1.0 L glass bottle with 1.0 L of milliQ water using the milliQ water station.
In the fume hood, use a pipette to carefully draw up and add 140 µL of 100% sulfuric acid solution to the 1.0 L of milliQ water. Pipette mix the solution a few times to ensure that all of the 100% sulfuric acid is out of the pipette tip. Dispose of the used pipette tip in the biohazard waste container.
Be sure to wear proper PPE at all times when handling 100% sulfuric acid (laboratory coat, goggles, and gloves). Never add water to concentrated sulfuric acid, as this is extremely dangerous and can cause a violent and potentially explosive chemical reaction inside the fume hood. Always add 140 µL of 100% sulfuric acid to 1.0 L of milliQ water in the fume hood in that order.
Tightly cap the 1.0 L glass bottle using the clean, orange screw-cap and throughly shake the bottle for a few seconds to mix the diluted sulfuric acid with the milliQ water.
Unscrew the red screw-cap of the large, 4.0 L glass bottle located to left of the Shimadzu HPLC system containing 'Eluent D' (labeled with a yellow piece of tape) and carefully pour the 1.0 L of dilute sulfuric acid solution into the 4.0 L glass bottle. This is the dilute sulfuric acid mobile phase labeled as 'Solvent D' that is used when running samples on the BioRad Aminex HPX-87H HPLC column. Repeat steps 6.4-6.7 as needed to ensure that the 'Eluent D' bottle is filled up to 4.0 L prior to each HPLC run.
Locate the 500 mL glass bottle labeled with a green piece of tape as '11/9/2022 Water' stored in the far back of the white container on the top of the Shimadzu HPLC system to the left of the Refractive Index Detector (RID). This bottle contains sterile-filtered milliQ water that is used to clean the autosampler needle prior to extracting and injecting each sample onto the organic acids HPLC column. Ensure that this bottle is filled with at least 400 mL of sterile-filtered milliQ water before starting a HPLC run.
Figure 1: Photographs correspond to Steps 6.7-6.8.
Starting the Shimadzu HPLC Instrument
This section of the protocol describes the steps required for starting the Shimadzu HPLC system prior to 1) switching columns (if necessary), 2) purging and initializing the instrument, and 3) creating and starting a batch file to run samples on the Shimadzu HPLC system.
Log onto the desktop computer located to the right of the Shimadzu HPLC system using the username and password. The Shimadzu LabSolutions CS software on this desktop computer is synched to this HPLC and is used to perform most operations on the Shimadzu HPLC system.
Detailed below is a brief visual overview of each of the components of the Shimadzu HPLC system:
Figure 2: Overview of the different components of the Shimadzu HPLC system.
Press the large circular power button located on the front of the Shimadzu HPLC system to the left of the touch screen interface to turn on the instrument. Press the power button located on the front left corner of the Refractive Index Detector (RID) to turn it on. It will take several seconds for both devices to turn on.
When prompted, enter the passcode on the touch screen of the Shimadzu HPLC instrument and click 'OK' on 1) the bottom left corner of the keyboard and 2) the bottom right corner of the 'Lock' dialog box.
Once the password is approved, the following display will appear on the touch screen on the front of the Shimadzu HPLC system. Please refer to the image below, which provides a brief description of each of the key parameters on the screen. It is important to familiarize yourself with each of the labeled parameters on the display screen prior to 1) purging the HPLC instrument and 2) starting a batch run with samples.
Figure 3: Overview of the different parameters and functions present on the 'home screen' of the touch screen display located on the front of the Shimadzu HPLC instrument.
Navigate back to the desktop computer. Double click on the LabSolutions CS software icon located on the far left side of the computer screen. A 'Login' dialog box may appear. Simply click 'Ok' to proceed.
After the LabSolutions CS software has opened, a 'LabSolutions Main (System Administrator)' screen will appear. Click on the large 'Instruments' tile located in the upper left corner of the screen. Next click on the blue-gray colored 'Shimadzu LC-2030' icon to connect the software with the Shimadzu HPLC instrument. A gray 'Connecting the Instrument...' box will appear while this process happens. Note that it may take several seconds for the next window to open upon clicking on the 'Shimadzu LC-2030' icon.
Upon connecting the LabSolutions CS software with the HPLC instrument, a 'Realtime Analysis (Shimadzu LC-2030-System Administrator)' window will appear. This window is the primary interface of the software for 1) purging the HPLC instrument and 2) setting up and starting a batch run with samples.
Figure 4: Screenshots correspond to Steps 7.6-7.8.
Switching Columns on the Shimadzu HPLC Instrument
This section of the protocol describes the steps required for switching columns on the Shimadzu HPLC instrument prior to selection of a method file and purging the Shimadzu HPLC instrument. Please note that this section of the protocol is only necessary to perform if a different mobile phase solvent (and most likely a different method file) was used during the prior HPLC run. This is a very important series of steps of the protocol to avoid contaminating and potentially damaging the stationary phase (resin) within the BioRad Aminex HPX-87H organic acids column prior to use.
No further steps are required on the LabSolutions CS software following Step 7.8 (see above) to switch columns on the Shimadzu HPLC instrument. With the instrument already turned on (from Step 7), direct your attention to the touch screen display located on the front of the Shimadzu HPLC instrument.
Press the blue 'lock' button located on the bottom right corner of the touch screen. This touch screen display has to be 'unlocked' to access most of the parameters and functions on this interface.
After pressing the blue lock button, enter the password when prompted. Note that this is the same password that was used in Step 7 above to turn on the HPLC instrument. Click 'Ok' after entering the password on the keyboard and click 'Ok' on the 'Lock' dialog box to submit the password for approval.
Figure 5: Photographs correspond to Steps 8.2-8.3.
Following password approval, all of the parameters and functions on the main display screen are now accessible and editable. Click on the portion of the touch screen that details a schematic of the valve positions numbered 1-6 (in different colors) for the different HPLC columns.
Navigate to and click on the 'Oven' tab located on the bottom of the display screen if it is not already open. Click on the 'Valve Position' box. Note that the 'Valve Position' is currently set to '2' for the organic acids column. Enter the number '6' on the keyboard and click 'Ok'. Valve position '6' is for the bypass.
The purpose of switching the valve position to the bypass is to flush any residual mobile phase solvent from the prior HPLC run that may be remaining in the tubing through the bypass and out of the instrument so it does not contaminate or disturb the stationary phase (resin) of the organic acids HPLC column.
Figure 6: Photographs correspond to Steps 8.4-8.5.
After completing Step 8.5 above, the 'Valve Position' box should be set to position '6'. Click 'Ok' in the bottom right corner of the 'Parameter Setting' box to return to the main display screen.
After switching the 'Valve Position' from position '2' (yellow column on schematic) to '6' (bypass) (orange column on schematic), this change should be reflected on the main display screen.
Figure 7: Photographs correspond to Steps 8.6-8.7.
With the 'Valve Position' switched to position '6' (bypass), navigate over to the solvent selection tile located on the bottom left corner of the main display screen. Check that the solvent tile is set to the desired mobile phase solvent (Solvent D - dilute sulfuric acid). If it is not set to Solvent D, click on the solvent selection tile and select 'D' in the 'Mobile Phase Port' dialog box and click 'Close'.
Figure 8: Photographs correspond to Step 8.8.
Click on the 'ISO' tile. 'ISO' stands for isocratic flow. In Isocratic flow, a constant mobile phase solvent composition is maintained for the duration of the run (this is what is desired for running samples on the organic acids HPLC column). Notice that if the 'ISO' tile is clicked, it will switch over to 'GRAD', which stands for gradient flow. In gradient flow, the composition of the mobile phase solvent (involving two or more mobile phases) changes over the duration of a sample run to achieve better separation of certain compounds. Click on the bottom left tile listing the percentage composition of solvents A, B, C and D. Click on the box next to each solvent letter in the 'Concentration' dialog box and use the keyboard to change the percentage composition of Solvent D to 100.0% and Solvents A, B, and C all to 0.0%.
Figure 9: Photographs correspond to Step 8.9.
With the valve position set to '6' and the mobile phase set to 100% of Solvent D (dilute sulfuric acid) as desired, navigate to the right side of the touch screen to the 'Lock' button, (which is currently unlocked).
Click on the 'lock' button to re-lock the parameters and functions on the main display screen. Note how the set of parameter tiles located within the dashed red box in the photograph below are grayed-out compared to earlier when the display screen was 'unlocked'. This is an important step as re-locking the main display screen prevents the method parameters from being changed accidentally prior to running 'pump' to flush out any residual solvent through the bypass (valve position '6').
Figure 10: Photograph corresponds to Step 8.11.
With the main display screen locked, navigate to the row of tiles located at the bottom of the main display screen and click 'Pump'. Allow 'Pump' to run for at least 10-15 minutes to allow any residual mobile phase solvent in the HPLC tubing from the prior run to flush through the bypass (valve position '6') and out of the instrument without disrupting the stationary phase (resin) of the organic acids HPLC column.
Figure 11: Photograph corresponds to Step 8.12.
After 10-15 minutes of running 'Pump', click on the 'Pump' button to turn it off. Note that the background color of the 'Pump' button will change from blue to gray, indicating that it is turned off.
With running 'Pump' finished, click on the 'Lock' button and enter the password to unlock the main display screen (in the same manner as before). We are going to change the 'Valve Position' from '6' (bypass) to position '2' (organic acids column) to prepare for a batch run. Click on the schematic of the valve positions (numbered 1-6) for the different HPLC columns on the main display screen.
Click on the 'Oven' tab located on the bottom of the display screen if it is not already open. Click on the 'Valve Position' box currently set to position '6' (bypass).
Use the keyboard located on the right side of the display screen to enter the number '2' to change the valve position from '6' (bypass) to position '2' (organic acids HPLC column) and click 'Ok'.
Figure 12: Photographs correspond to Step 8.16.
Note that the 'Valve Position' box has been updated to position '2' for the organic acids column. Click 'Ok' in the bottom right corner of the 'Parameter Setting' dialog box to return to the main display screen.
On the main display screen, check to ensure that the valve position has been updated to position '2' (organic acids column) (yellow column) on the schematic of the valve positions for the different HPLC columns. Click on the 'Lock' button to re-lock the parameters and functions on the main display screen.
Figure 13: Photographs correspond to Steps 8.17-8.18.
Purging the Shimadzu HPLC Instrument
This section of the protocol describes the steps required for purging the Shimadzu HPLC instrument prior to setting up a batch file for running samples on the HPLC. The purpose of this step is to initialize the HPLC instrument and prepare it for data analysis prior to starting a batch run with samples. Please note that this section of the protocol is required prior to starting any HPLC run unlike Step 8, which is only required when a different column and mobile phase solvent was used in the previous HPLC run.
Within the 'Realtime Analysis (Shimadzu LC-2030-System Administrator)' window open on the desktop computer, navigate to 'File' located in the upper left corner of the screen in the tool bar. Hover over 'File' and in the resulting drop down menu click on 'Open Method File...'
An 'Open Method File' dialog box will appear. Double click on 'Shimadzu HPLC data and methods'.
Double click on the 'Lynd Lab Methods' folder to access all of the method files for the HPLC instrument.
Figure 14: Screenshots correspond to Steps 9.1-9.3.
The 'Lynd Lab Methods' folder contains all of the existing method files for running the Shimadzu HPLC system. Double click on the 'h-column v2 IDR.lcm' method file for the organic acids HPLC column.
Upon opening the 'h-column v2 IDR.lcm' method file, a 'Method Editor (Instrument Parameters)' dialog box will appear. This is where method files can be edited if needed. The 'h-column v2 IDR.lcm' method file is already setup properly, so no changes need to be made to this method file. Click on the 'Download and Close' button in the bottom right corner of the 'Method Editor (Instrument Parameters)' dialog box.
Figure 15: Screenshot corresponds to Step 9.5.
Upon clicking 'Download and Close', the 'Method Editor (Instrument Parameters)' dialog box will close. Navigate to and click on the dark gray 'AutoPurge' button located below the chromatogram. Note that the 'AutoPurge' step includes purging flow lines, flushing the column, and equilibrating the instrument.
After clicking on 'AutoPurge...', a small 'Realtime Analysis' dialog box will appear in the center of the screen. Click 'Ok' to close the 'Realtime Analysis' dialog box and to start the 'AutoPurge' process.
Figure 16: Screenshots correspond to Steps 9.6-9.7.
A 'Purging...' box will appear during the 'AutoPurge' process, (which takes roughly 35-36 minutes). No further steps are required from the user to complete the auto purge process. The 'Purging...' box will close automatically when the 'AutoPurge' process is complete. When the 'AutoPurge' process is finished, the HPLC instrument is initialized and ready to set up a batch file for running samples.
Figure 17: Screenshot corresponds to Step 9.8.
Setting up and Starting a Batch Run on the Shimadzu HPLC Instrument
This section of the protocol describes the steps required for setting up a batch file and starting a batch run with samples on the Shimadzu HPLC instrument.
After the 'AutoPurge' process has finished, the Refractive Index Detector (RID) (Detector B) (black) and AD1 (pink) traces on the chromatogram should be stabilized (flat) with the AD1 (pink) trace at 0 mV and the Detector B (black) trace at slightly above 0 mV (usually at ~2.5 mV). It sometimes takes awhile for these traces to stabilize following the completion of the 'AutoPurge' process (up to ~ 1 hour). It is important to let both of these traces stabilize before starting a run on the HPLC. Once these traces have stabilized, the detectors are initialized and the instrument is ready to set up a batch file for a batch run.
Figure 18: Screenshot corresponds to Step 10.1.
To proceed with setting up a batch file for running a set of samples and standards on the Shimadzu HPLC instrument, navigate to and double click on the 'Documents' folder located on the bottom of the screen.
Figure 19: Screenshot corresponds to Step 10.2.
Upon opening the 'Documents' folder on the desktop, scroll down to the left side of the 'File Explorer' window and double click on the icon/folders in the following order:
1) 'OS (C:)' Icon
2) 'Shimadzu HPLC data and methods' Folder
3) 'Lynd Lab Data' Folder
4) Personal Data Folder (i.e., Isaiah Richardson)
Figure 20: Screenshots correspond to four sub-steps (1-4) within Step 10.3.
After opening an existing (or creating a new) 'personal data folder' to store all of your batch files (each HPLC run creates a batch file for each sample), right click within your personal data folder to create a new batch file folder for the HPLC run by clicking on 'New' (1) -> 'Folder' (2) and naming the new batch file folder (3) using the following format: year - month - day - initials - batch file name (omit hyphens except for the date). This particular naming format should be followed when naming all batch file folders.
Figure 21: Screenshots correspond to Step 10.4.
After creating and naming a new batch file folder for the HPLC run within your 'Personal Data Folder', navigate back to the 'Realtime Analysis (Shimadzu LC-2030-System Administrator)' window. With the HPLC system already purged and initialized for a batch run, navigate to the left side of the screen and click on the 'Main' tab (1) followed by the dark gray 'Batch Editor' icon (2). This will open a batch file table on the right side of the screen that can be edited to setup a batch run for a set of samples and standards (3).
Figure 22: Screenshots correspond to Step 10.5.
Right click on an empty cell in the batch file table to display a list of options. Click on 'Add Row' (1). This will open a 'Add Samples' dialog box. Type the total number of samples (samples + standards) for the HPLC run into the dialog box and click 'Ok' (2). An extra row should be added at the beginning and end of the batch file table for an 'Initialize' and a 'Shutdown' sample. This will expand the batch file table to your specified number of samples such that each row corresponds to a single sample injected on the HPLC (3).
Figure 23: Screenshots correspond to Step 10.6.
The best approach for setting up a batch file table for a batch run of samples on the Shimadzu HPLC system is to fill out the table column by column. You will notice that several of the columns are already filled out (i.e., 'Sample Type' and 'Method File'). No further action is required for these columns.
Start by filling out the leftmost 'Vial#' column in the batch file table. Write '-1' for the 'Vial#' for both the 'Initialize' and 'Shutdown' samples in the first and last rows of the table. A 'Vial#' of '-1' specifies to the HPLC instrument to not extract and inject a sample, which is desired, as this allows the mobile phase (dilute sulfuric acid) to flush through the organic acids column at both the beginning and end of the batch run. For all of the other rows in the 'Vial#' column, write a number in each row corresponding to the vial # specified on the sample tray where the HPLC samples and standards were placed in the instrument. The vial # specifies to the instrument which vial # in the tray to inject the needle into and extract sample from to run on the organic acids column. It is very important to double check the values entered in the 'Vial#' column, as an incorrect vial # can lead to: 1) an injection error and/or 2) a discrepancy between the order of sample vials in the tray and the batch file table, which can complicate subsequent data analysis. If all of the vials are in order within the same tray, right click on the second cell of the 'Vial#' column after entering a '1' and click 'Fill Series'. Ensure that the final 'Shutdown' row still specifies a '-1' as the 'Vial#'.
The next column to fill out in the batch file table is the 'Tray' column. In a somewhat similar manner to the 'Vial#' column, the 'Tray' column specifies the tray in which the sample vials are stored in the instrument. Collectively, the 'Vial#' and 'Tray' columns inform the instrument of the exact position of the vial for each sample in the batch run so the needle knows what sample to inject into, extract sample, and run on the organic acids HPLC column. Each tray can hold 54 sample vials. Unless a batch run has more than 54 samples, it is best practice to include all of the samples in order in the same tray. Right click on the first cell in the 'Tray' column and click 'Fill Down' to fill down the same tray # in all of the cells in the column.
The next column to fill out in the batch file table is the 'Sample Name' column. Enter a name for each sample in the 'Sample Name' column. It is good practice to give each sample a relatively detailed or specific name so it is easily remembered during subsequent data analysis. In a similar manner to the 'Vial#' column, it is very important to double check the entries in this column to ensure that the 'Sample Names' correspond precisely with the numbers in the 'Vial#' column. A simple mistake in either column can result in a discrepancy that can be extremely difficult to untangle during subsequent data analysis.
When running samples on the HPLC, it is best practice to run one set of standards (1x, 2x, 8x mixed standards + 60 mM, 30 mM, 7.5 mM Glucose standards = 6 standards total) at both the beginning and end of the batch run of samples (12 total standards). Thus, it is recommended to organize the sample vials for a batch run in the sample tray in this order and to enter this exact order in the 'Sample Name' column.
Be sure to enter 'Initialize' and 'Shutdown' in the 'Sample Name' column for the first and last rows of this column, corresponding to the '-1' value previously entered in the 'Vial#' column of the batch file table.
For the 'Sample ID' column, simple enter a '1' in the first cell of the column, right click on the cell and click on 'Fill Series'. This action will populate the column in numerical order. The purpose of this column is to specify the total number of samples in the batch run.
The final column to fill out in the batch file table is the 'Inj. Volume' column. The values in this column specify the injection volume (in units of μL) for each sample in the batch run. To maintain consistency between the samples and standards, it is best practice to set the injection volume for all of the samples and standards (including the 'Initialize' and 'Shutdown' samples) to 40 μL. Setting the same injection volume for both the samples and standards in a batch run enables direct comparison between the metabolite concentrations quantified in these samples without having to account for a dilution factor.
Due to pyruvate oversaturating the photodiode array (PDA) detector at an injection volume of 40 μL, (i.e., when zoomed in, the top of the pyruvate peak on the chromatogram will appear flat, indicating oversaturation), it is best practice to inject samples containing pyruvate at BOTH 4 μL and 40 uL so pyruvate can be accurately quantified using the chromatogram from the 4 μL injection, and the other metabolites in the sample can be accurately quantified using the chromatogram from the 40 μL injection.
With the columns specified in Steps 10.7-10.11 filled out in the batch file table, the initial phase of setting up the batch file table for a batch run of samples on the Shimadzu HPLC instrument is complete. At this stage of the process, the batch file table should look similar to the example batch file table detailed below:
Figure 24: Screenshot of an example batch file table, corresponding to Steps 10.7-10.12.
To finish setting up the batch file table, right click on the upper left corner of the batch file table in the blue 'Editor' box. A drop down menu of options will subsequently appear. Click on the 'Settings' option.
Within the 'Settings' dialog box, navigate to and click on the 'Shutdown' tab and check both the 1) 'Shutdown' and 2) 'Power Off after shutdown' boxes. Checking these boxes will turn off the HPLC instrument after the batch run of samples is finished. This is important for 1) not wasting the mobile phase and 2) ensuring that the mobile phase is not accidentally depleted (run out) after the run has finished.
Within the 'Settings' dialog box, navigate to and click on the 'Folder' tab. Within the 'Folder' tab, click on the tan folder icon next to the 'Data File' line. This will open a 'Browse for Folder' dialog box that allows you to navigate through the 'Documents' tab on the desktop computer.
Within the 'Browse for Folder' dialog box, navigate to and locate the batch file folder that was created within your 'Personal Data Folder' in Steps 10.2 - 10.4 above for the batch run and click on the folder. Click 'Ok' to exit the 'Browse for Folder' dialog box once the desired batch file folder is clicked on and highlighted in light blue (no further action is required). Note that at the completion of this step, the 'Data File' line is now populated with the path to the batch file folder. This specifies to the instrument to place the batch files created for each sample from the batch run into the specified batch file folder.
Still operating within the 'Folder' tab, click on the 'Use the same folder' box to uncheck it.
Still within the 'Folder' tab, click on the bright green folder icon to the right of the 'Method File' line.
In a similar manner to Step 10.16 above, use the 'Browse for Folder' dialog box to navigate through the 'Documents' folder on the desktop computer and locate and click on the 'Lynd Lab Methods' folder such that the folder is highlighted light blue. Click 'Ok' to exit the 'Browse for Folder' dialog box once the 'Lynd Lab Methods' folder is clicked on and highlighted in light blue.
With the necessary updates made within the 'Folder' tab, navigate to the 'Data File Name' tab within the 'Settings' dialog box. Within the 'Data File Name' tab, click on the 'Create filenames automatically with' box and click on the 'Add>>' and '<<Remove' toggle switches as needed to remove the 'Batch Start Date' item from the 'Selected Items' box and to add the 'Sample Name' item.
Click 'Ok' to exit the 'Settings' dialog box. Refer to the screenshots from the desktop computer below to assist in navigating Steps 10.13 - 10.20 described above:
Figure 25: Screenshots correspond to Steps 10.13-10.20.
With the batch file table setup complete, the next step is to save the batch file table. Navigate to 'File' in the upper left corner of the screen. Click on 'Save Batch File As...' in the dropdown menu. Within the 'Save Batch File As' dialog box, navigate through the 'Documents' folder to 'OS (C:)' -> 'Shimadzu HPLC data and methods' -> 'Lynd Lab Data' -> 'Personal Data Folder' (i.e., Isaiah Richardson) and locate and click on the batch file folder created previously in Steps 10.2-10.4 above within your 'Personal Data Folder'. This action specifies the batch file folder as the location for the software to store the batch files created for each sample in the batch run. After selecting the desired batch file folder, use the 'File name:' box to name the batch file using the same naming convention as for the batch file folder (for consistency).
Figure 26: Screenshots correspond to Step 10.22.
If not already completed earlier, carefully insert the sample tray(s) containing all of the samples (samples + standards ordered in the exact same manner as specified in the batch file table) for the batch run into its respective sample tray drawer at the front of the HPLC instrument. Note that tray positions 1 (front) and 2 (back) are correspond to the left (L) drawer and that tray positions 3 (front) and 4 (back) correspond to the right (R) drawer on the front of the HPLC instrument (refer to the labeled image in Step 7.2). Ensure that the sample tray(s) is set properly into its rack and that the drawers are fully closed. If the sample trays are properly inserted into their respective drawers, the HPLC instrument will make a small 'beep' noise.
With the HPLC vials for the batch run loaded into the HPLC instrument, click on the dark gray 'Queue Batch Run' tile located on the left side of the screen to start the batch run. Note that it may take several seconds for the batch run to start. Once the batch run is started, click on the 'Realtime Batch' icon on the left side of the screen to view both the batch file table and the chromatogram produced for each sample injected onto the organic acids column in real-time. Note that the row highlighted in blue in the batch file table under the 'Realtime Batch' icon specifies the sample or standard that is actively running on the HPLC instrument, while the white rows correspond to samples or standards that have finished running, and the yellow rows correspond to samples or standards that still have to run on the HPLC instrument.
Figure 27: Screenshots correspond to Step 10.24.
With the batch run of samples started, no further action on either the instrument or software is required from the user until after the batch run has finished and the Shimadzu HPLC instrument is turned off.
Editing the Batch File Table During an Active Batch Run of Samples
This section of the protocol describes the steps required for pausing an active batch run of samples on the Shimadzu HPLC Instrument to edit the batch file table if either 1) an error is identified in the table or 2) additional samples (either samples or standards) need to be added to the batch run.
Note: It is critically important to edit the batch file table while a sample is actively running on the HPLC column and to finish editing the table and save the changes before the instrument prepares to inject the next sample on the column. In other words, it is recommended to make and finalize any necessary edits to the batch file table before the 25 min time point within the 30 min run time per sample. While editing the batch file table, the RID and PDA detectors are still actively acquiring data for a sample.
To edit the batch file table during an active batch run, with the display set to 'Realtime Batch', navigate to the top tool bar and hover over the 'Batch' option. In the dropdown menu, click on 'Edit Table (Pause)'.
Within a few seconds (~5-10 seconds), a 'Pause' dialog box will appear, stating that, 'Data acquisition will be paused at line...' (the next row in the batch file table). Click 'Ok' to close out of this dialog box.
Make any necessary modifications, updates, or changes (i.e., add additional samples, correct a vial# entry, correct a sample name entry etc.) to the batch file table. This function allows you to edit the batch file table in the same manner as it was edited during the initial set up of the table. Double check that the changes made are correct. Do not make more edits to the table than necessary during an active batch run.
Once finished making changes to the batch file table, navigate back to and hover over the 'Batch' option located in the tool bar of options at the top of the screen and click on 'Edit Table (Pause)'.
A 'Realtime Analysis' dialog box will appear after a few seconds in the center of the screen. Click 'Yes' to save the changes made to the batch file table and to close out of the 'Realtime Analysis' dialog box.
Figure 28: Screenshots correspond to Steps 11.1-11.2 and 11.4-11.5.
After a few seconds (~5-10 seconds), the screen will return to the 'Realtime Batch' viewer that was displayed on the screen prior to making any edits to the batch file table. No further action is required. The batch run will proceed according to the changes made in the batch file table during Step 11.
Analyzing the HPLC Results After a Batch Run
This section of the protocol describes the steps required for analyzing and exporting the quantitative results from a batch run of samples using the organic acids column on the Shimadzu HPLC system.
Navigate to and double click on the LabSolutions CS software icon located on the far left side of the home screen of the desktop computer (in the same manner as in Step 7.6 above).
Once the LabSolutions CS software has opened, click on the large 'Postrun' tile located on the left side of the screen. Double click on the 'Browser' icon to open the document browser within the software for performing data analysis. Please note that it will take several seconds for the 'Browser' to open.
Navigate to the small, square folder icon located near the upper left corner of the screen. Click on this folder icon to open the 'Select Folder' dialog box as detailed in the screenshots below. Within the 'Select Folder' dialog box, click on 'OS (C:)' -> 'Shimadzu HPLC data and methods' -> 'Lynd Lab Data' -> 'Personal Data Folder' (i.e., Isaiah Richardson) (if necessary) and navigate to and click on your batch file folder containing all of the batch files for the samples and/or standards that you want to analyze.
Note the brief descriptions of the purpose of the different windows in the 'Browser' window below:
Figure 29: Screenshots correspond to Step 12.3.
Upon selecting the desired batch file folder in Step 12.3, all of the individual batch files (one batch file per sample run on the HPLC) will appear within the 'Filename' window on the far left side of the screen.
Click on the first batch file (sample) in the 'Filename' window. With the first batch file (sample) selected, hold down the 'Shift' key and scroll down to the bottom of the 'Filename' window to the last batch file (sample) and click on it. With all of the batch files (samples) selected (highlighted in blue), use the mouse to drag all of the files at once from the 'Filename' window into the 'Quantitative Results View' window located in the top center of the screen. Note that it may take several seconds to drag all the batch files into this window (a small dialog box will appear with a green progress bar denoting the progress of this step).
Note that the 'Initialize' and 'Shutdown' samples from the start and end of the batch run can be excluded from being dragged into the 'Quantitative Results View' window as they do not need to be analyzed.
With all of the desired batch files (samples) dragged into the 'Quantitative Results View' window, navigate to and hover over 'File' located in the upper left corner of the screen. In the resulting drop down menu, click on 'Open Method File'. A small 'Browser' dialog box will appear asking if you want to, 'Save current method file?' Click 'No' and the 'Browser' dialog box will close.
An 'Open Method File' dialog box will appear in the center of the screen. Click on 'Shimadzu HPLC data and methods' -> 'Lynd Lab Methods'. Within the 'Lynd Lab Methods' folder, double click on 'h-column v5 IDR.lcm' to open this HPLC method. Note that it may take several seconds for this method to open.
Upon opening the 'h-column v5 IDR.lcm' method file, a table of compounds (metabolites) that we are interested in quantifying the concentration of in each of our samples (in units of mM) will appear within the 'Compound' tab in the 'Method View - Compound Table' window located on the right side of the screen.
The key piece of information in this table is the 'Ret. Time' (retention time) for each of the compounds (metabolites). The retention time specifies the time at which each compound (metabolite) elutes off of the stationary phase (resin) of the organic acids column during the 30 minute run of each sample. This results in the formation of a peak for the metabolite on the chromatogram that can be used to quantify the concentration of that metabolite (in units of mM) in the sample based upon the height of the peak.
Navigate to the 'Quantitative Results View' window located in the top center of the screen. Use the horizontal sliding bar located on the bottom of this window to slide to the right within the table of batch files to the 'Level#' column. Set the 'Level#' to 1, 2, 3, 4, 5, 6 for the 1x, 2x, 8x, 60 mM Glucose, 30 mM Glucose, and 7.5 mM Glucose standards, respectively. All of the batch files for the samples (non-standards) should have a 'Level#' set to '0'. The specific 'Level#' for the standards arises from the concentration values set for these standards in the 'Conc. (1)' etc. columns located on the far right side of the table in the 'Method View - Compound Table' window under the 'Compound' tab.
Use the horizontal sliding bar located on the bottom of the 'Quantitative Results View' window to slide back to the left side of the table to the 'Sample Type' column. Click on the gray drop down arrow in the 'Sample Type' column for each of the standards and change the 'Sample Type' for the 1x, 2x, 8x, 60 mM Glucose, 30 mM Glucose, and 7.5 mM Glucose standards from 'Unknown' to 'Standard (Calc Point)'.
This is an important step as it sets the batch files for the standards as standards within the software and uses these specified standards to create a three-point standard curve for each compound (metabolite). The standard curve is used to quantify the concentration of the metabolites (units of mM) in each sample.
Figure 30: Screenshots correspond to Steps 12.5-12.10.
Note that after completing Step 12.10, a red three-point standard curve appears in the 'Calibration Curve/Spectrum View' window located in the bottom right corner of the screen for each compound (metabolite) when it is clicked on in the table of compounds (metabolites) under the 'Compound' tab in the 'Method View - Compound Table' window located in the upper right corner of the screen.
Note that there should not be standard curves for sulfuric acid or phosphate. These metabolites are present in each sample but do not need to be quantified as they are not relevant to this protocol.
Please refer to the PDF document attached to this protocol that provides an example of each of the standard curves for each of the metabolites within the table under the 'Compound' tab in the 'Method View - Compound Table' window for both the RID and PDA detectors. Also included in the PDF document are chromatograms for the 1x, 2x, 8x standards and the 60 mM, 30 mM, and 7.5 mM glucose standards.
With the standards specified and the standard curves created for each of the compounds (metabolites), slowly click through all of the standards and samples in the 'Quantitative Results View' window to analyze the chromatogram in the 'Chromatogram View' window for each of the samples. Note that the default detector for analyzing the chromatogram of each sample is Detector B (the Refractive Index Detector [RID]). Note that the RID Detector is used to quantify most metabolites, including cellobiose, glucose, malate, succinate, lactate, formate, acetate, MOPS (control), and ethanol. The detector type is specified in the top left corner of the screen below the main tool bar. The primary purpose of this step is to ensure that the software is accurately auto-integrating each of the metabolite peaks with respect to the baseline on the chromatogram for each of the samples. Note that all (or most) of the peaks in each sample are labeled with its metabolite name and specific retention time above the peak. If there is an unlabeled peak present on the chromatogram, that peak corresponds to a metabolite that is not listed in the table of compounds (metabolites) in the 'Method View - Compound Table' window. This does not mean that the unlabeled peak is not relevant, it may be very relevant depending upon the samples run.
Note that the table of compounds (metabolites) within the 'Method View - Compound Table' window can be edited by clicking on the 'Edit' box located in the top right corner of this window to add an additional row(s) for additional metabolites if desired. After editing the table within the 'Method View - Compound Table', click on the 'View' box located in the top right corner of this window to save the edits to the table.
Note that the integration for each peak is denoted by a red line under each peak flanked by a vertical red arrow pointing up (on the left side) and a vertical red arrow pointing down (on the right side) of each peak. It is very important to ensure that each peak is properly integrated in each sample (including both the samples and standards), as a bad integration of a peak (either above or below the baseline) will result in an inaccurate quantification of the concentration of that particular metabolite within a sample.
If a particular peak on the chromatogram of a sample or standard is poorly integrated by the software, navigate to and click on the icon of a blue, white, and red table with a hand located on the far right side of the tool bar at the top of the screen. This will open the 'Manual Integration Bar' dialog box that allows you to delete a poorly integrated peak and manually re-integrate it yourself. Detailed below is a description of a few of the most important icons in the 'Manual Integration Bar' dialog box for re-integrating a peak:
1. DELETE PEAK FUNCTION: Click on this icon to activate the ability to delete an existing integration of a peak or multiple peaks. Simply click on the desired peak (or peaks) to delete the existing integration.
2. MANUAL INTEGRATION FUNCTION: Click on this icon to activate the manual integration function. To use this function, click on the bottom left corner of the peak (along the baseline) that you want to re-integrate and drag the cursor to the right and click on the bottom right corner of the peak (along the baseline) to finish the integration. Note that the new integration line will change color from blue to red.
3. SPLIT PEAK FUNCTION: Click on this icon to activate the split peak function. To use this function, navigate to and click on the specific location between two merged peaks to split the merged peak into two separately integrated peaks. It can sometimes be difficult to determined where to accurately split two merged peaks. In these instances, best judgement has to be used.
Figure 31: Screenshot detailing the 'Manual Integration Bar' dialog box for Step 12.13.
After analyzing the chromatograms for all of the samples and standards using Detector B (the Refractive Index Detector [RID]), navigate to and hover over the 'File' option located in the upper left corner of the screen in the tool bar, and in the resulting drop down menu click 'Export Quantitative Results...'
A 'Export Quantitative Result (ASCII Conversion)' dialog box will appear in the middle of the screen. Under 'Items to Output', click the circle to the left of 'Summary output items' and ensure that the dropdown box located to the right of this option is set to 'Conc.' (concentration in units of mM). Click 'Ok' to close out of this dialog box. This action exports a table of concentration values (in units of mM) for all of the metabolites present in each sample and/or standard that was analyzed using Detector B (RID).
Note that under 'Summary output items', the 'Conc.' option can be changed in the dropdown box to export the 'Height' of the peaks (in units of mAu), among other options. Although this is not necessary for cellobiose, glucose, malate, pyruvate, succinate, lactate, formate, acetate, MOPS and ethanol (i.e., all metabolites included within the 1x, 2x, 8x or glucose standards), exporting the 'Height' of the peaks may be beneficial if you are interested in quantifying a specific metabolite that standards were not prepared for, but the starting concentration of the compound (i.e., a substrate) is known. This is not the ideal situation. It is always best to prepare your own standards for all the metabolites that you are interested in quantifying. Regardless, the concentration of the compound of interest can be quantified in units of mM from the height (mAu) by first converting the height (mAu) -> concentration (g/L) -> concentration (mM).
Figure 32: Screenshots correspond to Steps 12.14-12.15.
With the quantified results from Detector B (RID) copied in Step 12.15 above, minimize the 'Browser' window of the software. Navigate to and open Microsoft Excel on the desktop computer, and open a blank Excel workbook. Right click on a cell near the upper left corner of the workbook and paste the table of results into Excel. This table of results can be processed (if necessary), analyzed, and used to produce figures or plots from the data. It is recommended to 1) name the Excel file with the date, your initials, and the name of the fermentation in the same manner as the batch file folder and 2) to save the Excel file in the same batch file folder as the batch files from the batch run so it is easy to find and access in the future.
Note that the table of results exported from Detector B (RID) includes a separate column (from left to right) for the 'Data Filename', 'Sample Name', 'Sample ID', and each 'Compound (Metabolite)'. Note that each row is a different sample (or standard) and that the values are in concentration units of mM. During subsequent analysis, the 'Sample ID' column can be deleted, as well as the columns for 'sulfuric acid' and 'phosphate' as quantifying these compounds is not necessary within the scope of this protocol.
With the Detector B (RID) results exported and pasted into Microsoft Excel, navigate back to the 'Browser' window and in the gray drop down menu located at the top of the screen below the main tool bar, click on 'PDA'. This will switch the detector from RID to PDA. Recall that 'PDA' standards for the 'Photodiode Array'. This is the UV detector, and is useful for quantifying many of the same compounds as 'Detector B'. However, the primary metabolite that is quantified using the PDA detector is pyruvate.
Figure 33: Screenshot corresponds to Step 12.17.
Note that when the detector is switched from 'Detector B' to 'PDA', the table of compounds (metabolites) in the 'Method View - Compound Table' window switches to include: Pyruvate_320 (pyruvate quantified at 320 nm), Pyruvate (pyruvate quantified at 225 nm), Malate, Succinate, Lactate, Formate, Acetate, and Propionate. Note that several of these compounds were previously quantified using Detector B (RID). It is usually best practice to quantify Malate, Succinate, Lactate, Formate, and Acetate using Detector B (RID) instead of PDA (UV), but it is beneficial to examine the peaks for these metabolites using both detectors.
Note that within the 'h-column v5 IDR.lcm' method file, pyruvate is quantified at both 320 nm and 225 nm. The purpose of this is to ensure that pyruvate is being accurately quantified, as many more compounds tend to appear on the chromatogram from the PDA detector at a wavelength of 225 nm (that may merge with the pyruvate peak) than at a wavelength of 320 nm. If pyruvate was not used as a substrate in a fermentation, it is unlikely that a quantifiable amount of pyruvate will be present in the samples. However, for fermentations performed with pyruvate as a substrate, it is beneficial to quantify pyruvate at 320 nm and 225 nm using the PDA detector to ensure that pyruvate is accurately quantified.
In a similar manner to analyzing samples using Detector B (RID), carefully click through the chromatograms for all of the samples and standards in the 'Quantitative Results View' window and examine all of the metabolite peaks, ensuring that the software accurately integrated each of the peaks. If a peak was not integrated properly, use the 'Manual Integration Bar' to delete the poor integration and manually re-integrate the peak. Also be sure to examine the standard curves for all of the metabolites in the 'Method View - Compound Table' window to ensure that they appear as expected.
Note that the standard curve for pyruvate using the PDA detector is curved instead of linear (as for all of the other metabolites). If you are interested in quantifying pyruvate, it is very important to ensure that the concentration of pyruvate within each sample lies within the pyruvate standard curve. It is very important that the concentration of each metabolite in all samples lies within its linear standard curve (for both PDA and Detector B) to ensure that it is accurately quantified. However, the software does a much better job extrapolating the concentration of a metabolite that is above or below the concentration range of a linear standard curve than the concentration of pyruvate that is above or below the concentration range of the curved pyruvate standard curve. This is why it is important to inject all samples that contain pyruvate as a substrate at both 4 μL (to quantify pyruvate) and at 40 μL (to quantify all other metabolites) to ensure that: 1) the pyruvate concentration within the samples is within the concentration range of the curved pyruvate standard curve and 2) the pyruvate peak is not oversaturating the PDA (UV) detector.
After analyzing the chromatograms for all of the samples and standards using the Photodiode Array (PDA) (UV) detector, navigate to 'File' and in the drop down menu click 'Export Quantitative Results...'
Within the 'Export Quantitative Result (ASCII Conversion)' dialog box, select 'Summary output items' and ensure that the dropdown box located to the right of this option is set to 'Conc.' (concentration in units of mM). Once set, click 'Ok' to close out of this dialog box to export the PDA (UV) detector results.
Navigate back to the Microsoft Excel workbook opened earlier and right click in an open cell to paste the table of results from the PDA (UV) detector next to the Detector B (RID) results. Note that the table of PDA (UV) detector results is organized in the same manner as the Detector B (RID) results table with the values in concentration units of mM. The results are now ready to be processed, analyzed, and plotted.
Within Excel, it is recommended to manually include an additional column for 'Injection Volume (μL)' within the PDA (UV) detector results table to record the volume at which the samples were injected. This can be determined by referencing back to the batch file table that was setup to run the samples. This is particularly important for quantifying pyruvate, as the concentration of pyruvate within the samples injected at 4 μL likely fell within the concentration range of the curved pyruvate standard curve and the concentration of pyruvate within the samples injected at 40 μL likely oversaturated the PDA (UV) detector and fell far above the concentration range of the pyruvate standard curve. As a result, use the pyruvate concentration values from the samples injected at 4 μL in the analysis and account for this 10x dilution made by the HPLC instrument by multiplying these 4 μL pyruvate concentration values by a factor of 10.
Figure 34: Screenshots of example Detector B (RID) (left) and PDA (UV) (right) results exported from the LabSolutions CS software 'Browser' and pasted into Microsoft Excel for subsequent data analysis and processing. Note that these screenshots correspond to steps 12.16 and 12.22, respectively.
With the Detector B (RID) and PDA (UV) detector results for all of the samples and standards from the batch run analyzed and exported to Excel, navigate back to the 'Browser' and close this window. A 'Browser' dialog box will appear in the center of the screen asking if you want to, 'Save current method file?' Click 'No' and this will close out of the 'Browser' dialog box and the 'Browser' window.
No further steps are required for performing the data analysis within the LabSolutions CS software. It is recommended to copy and paste both the Detector B (RID) and PDA (UV) results tables into a new sheet within the same Excel workbook for 1) subsequent analysis and data processing and 2) to preserve the original, raw data tables exported from the LabSolutions CS software in the original Excel sheet. It is recommended to perform all of the data processing in Excel and to produce any figures or plots from the data using Python code in a Jupyter Notebook to streamline the figure development and design process.
Protocol references
1. Shimadzu. Shimadzu High Performance Liquid Chromatograph Prominence-i LC-2030 LC-2030C LC-2030C 3D Operation Guide. Shimadzu Corporation. (2013).
2. Shimadzu. LabSolutions Operators Guide. Shimadzu Corporation. (2015).