Agilent Community
Agilent Community
  • User
  • Site
  • Search the Community
  • User
Consumables
  • Technical Areas
Consumables
Wiki Pass the Salt, Please – Mobile Phase Preparation for HIC
  • Announcements
  • Forum
  • Files
  • Wiki
  • More
  • Cancel
  • Consumables
  • A Beginner’s Guide to Hydrophobic Interaction Chromatography
  • A simple tip to protect your columns
  • A Tip for Preparing Robust and Consistent Mobile Phases
  • Achieve accurate and repeatable gas flow meter measurements
  • AdvanceBio Columns Blog Series
  • Agilent 123 Meter Sodium probe maintenance
  • Agilent Collection of Columns, Supplies, and Standards Resources
  • Analysis of microplastics in the environment
  • Analyzing ADCs by HIC
  • Avoiding downtime in the lab: top tips for GC/MS success
  • Best Practices for Aqueous Mobile Phases
  • Best Practices for Making Good Connections
  • Bio LC Column User Guides
  • Bursting Tubing and Columns (GC and HPLC)
  • Calculate the Maximum Allowable Working Pressure for Tubing
  • Calculating Column Volume
  • Cannabis Potency Testing: a Reliable, Cost-Effective Method
  • Carbohydrate Analyses in LC
  • Checking Your Intuition - Sub 2 µm vs Superficially Porous
  • Choosing the right GC Injection Technique
  • Choosing the right pore size for size exclusion chromatography
  • Columns, Supplies, and Standards Knowledgebase
  • Consumables Applications and Workflows
  • Consumables Recommended Supplies Lists for Agilent.com
  • Custom Product Request
  • Extreme Makeover – Derivatizations in Chromatography – Part 1 GC
  • Extreme Makeover – Derivatizations in Chromatography – Part 2 LC
  • Fake It Until You Make It: When BioInert Isn’t an Option
  • Flipping Amino Acid Analysis on Its Head
  • Glycans at a glance:  Analyzing therapeutic glycoproteins
  • Handle and Care of Syringes
  • Help! My Peaks Look Strange - Fronting and Tailing in GC
  • Help! My Peaks Look Strange - Saddle Points - LC/GC Troubleshooting
  • How do I select a Split/Splitless liner?
  • Hydrophobic Interaction Chromatography of Proteins and mAbs
  • Importance of Silica Particle Strength for Sub-2 µm SEC Columns
  • KB: Ferrules recommended for GC self-tightening column nut
  • LC and LC/MS Columns - USP Designations
  • LC Column User Guides
  • LC Method Translation - the Dwell Volume
  • Minimize spectroscopy workflow disruptions
  • Minimizing Metals for Best HILIC Results
  • More than just a drink: Analyzing the elemental composition of beer
  • Multi-Attribute Methods – Peptide Mapping Part IV
  • Must See Webinars
  • Nomenclature of CFC's/Freons/Halons/Coolants
  • Oligonucleotide Analysis - Unexpected Details Matter
  • Optimizing Bonding Chemistry for Sub-2 µm SEC Particles
  • Pass the Salt, Please – Mobile Phase Preparation for HIC
  • Pesticides and their stability during GC analyses
  • Pre-Columns - the forgotten art of using retention gaps
  • Problematic polar analytes? Hello HILIC…
  • Protecting your laboratory productivity
  • Recommended Reading
  • Sample Prep Pointers - Peptide Mapping Part I
  • Save your results with sample filtration
  • Simplified cone inspection with the new Agilent LED measuring magnifier
  • Software - Supported Method Development - The Scanview Application
  • Software tool for the ADM Flow Meter (G6691A)
  • Stay Safe: A Win-Win for Solvent Storage
  • Streamline your sample processing
  • The importance of chemical composition for vial performance
  • Tips & Tricks for Amino Acid Analysis – Part I
  • Tips & Tricks for Amino Acid Analysis – Part II
  • Tips & Tricks for Amino Acid Analysis – Part III
  • Tips & Tricks for Amino Acid Analysis – Part IV
  • Tips for Smooth Sailing with HIC
  • Troubleshooting HPLC autosamplers
  • Troubleshooting HPLC degassers
  • Troubleshooting Sequence Coverage – Peptide Mapping Part III
  • UltiMetal Plus Flexible Metal Ferrule
  • UV, MS, TFA, and Formic Acid – What to use? Peptide Mapping Part II
  • What are the typical % Gain or EHT values for hollow cathode lamps?
  • You Need Lamps or Chemical Standards for Atomic Absorption Single-Element Analyses?
Still Need Help?

Post your question in our User Forum or Contact Support.

Pass the Salt, Please – Mobile Phase Preparation for HIC

Created by anne_blackwell anne_blackwell over 2 years ago | Last modified by Agilent Agilent over 2 years ago

Hi again. One of the first tasks for any separation experiment is mobile phase preparation. So that’s where my colleague Sandeep is going to take the lead to describe some unique features of mobile phase preparation for hydrophobic interaction chromatography.

*********************************************************

In the last blog post, Greg introduced us to hydrophobic interaction chromatography (HIC) and its increasing demand as an analytical tool for characterizing monoclonal antibody (mAb) variants and antibody drug conjugates (ADCs). We also learned that in HIC separations the mobile phase contains a salting-out agent, which at high concentration retains the protein through increased hydrophobic interaction between the protein and stationary phase. This post will focus on preparing high salt mobile phases and buffer conditions needed for HIC separations.

Salts are employed in HIC to increase or decrease analyte binding. The ability of a salt to promote hydrophobic interaction depends on the ionic species present and its concentration. Based on the Hofmeister series shown in Figure 1, sodium chloride (NaCl) is going to be considerably less effective than ammonium sulfate ((NH4)2SO4) in protein binding to HIC stationary phase. In practice, NaCl solution needs to be around four times as concentrated as (NH4)2SO4 to achieve the same effect. The most commonly used salting out agents in HIC are ammonium sulfate, sodium sulfate, and ammonium acetate. We typically recommend about 1.5–2 M ammonium sulfate as a starting point. If the retention time of the analyte is too long, decrease the salt concentration of the starting mobile phase.

 Figure 1. Hofmesiter series showing effect of some anions and cations on protein precipitation

 

The high salt concentrations used in HIC separation can be detected in the UV range and are observed as baseline drift, as shown in Figure 2. LC grade reagents and solvents are recommended to avoid any background signal from reagent impurities. In HIC separations, it is important to collect a blank gradient run before the sample. Baseline drift in the sample can then be minimized by subtracting the blank chromatogram to improve reproducibility.

 

Figure 2. Differences in baseline are observed depending on grade of Ammonium sulfate

 

SALT

The type of salt, the concentration of salt in the starting mobile phase, addition of organic solvent, and pH are all important parameters to consider in HIC. The starting salt concentration can affect the resolution as shown in Figure 3. In this case, peaks elute early and are poorly resolved with a low starting salt concentration. Better retention and resolution were observed when the mobile phase salt concentration was increased.

 

 

Figure 3. Effect of salt concentration on HIC separation

 

ORGANIC SOLVENT

Addition of organic solvent may improve the resolution especially for large hydrophobic proteins such as monoclonal antibodies and ADCs. The most commonly used organic solvents are isopropanol and acetonitrile. The effect of adding acetonitrile (ACN) to mobile phase is demonstrated with the NIST mAb sample in Figure 4 – improved peak shape and recovery are observed.

 

Figure 4. Effect of organic solvent in HIC mobile phase

 

MOBILE PHASE PREPARATION

For the Agilent AdvanceBio HIC column, use these conditions as starting point and further adjust the starting salt concentration if needed.

For less hydrophobic samples:
Mobile phase A, 2 M ammonium sulfate, 50–100 mM sodium phosphate, pH 7.0.
Mobile phase B, 50–100 mM sodium phosphate, pH 7.0.

For more hydrophobic samples such as ADCs:
Mobile phase A, 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 / isopropanol (95:5 v/v).
Mobile phase B, 50 mM sodium phosphate, pH 7.0 / isopropanol (80:20 v/v).

Ammonium sulfate at 2 M readily dissolves into solution within a couple minutes. Proper precaution needs to be taken while working with organic solvent in high salt mobile phase since too much organic solvent can lead to precipitation. For the AdvanceBio HIC column, we recommend use of isopropanol and acetonitrile organic solvents (up to 40 %) to ensure no precipitation on the column. Always filter mobile phases using a 0.22 µm filter. (Here’s a reminder what happens with poor aqueous mobile phase preparation).

Such high salt concentrations can cause issues with some LC instruments. It is therefore recommended that you use a fully bio-inert LC, ideally with a quaternary pump so the additional channels can be used for flushing. It is not advisable to leave either the HIC column or the LC instrument in 2 M ammonium sulfate solution, instead flush the column into mobile phase containing low concentration buffer after use. For more details on method development and for proper column care (including long term storage), please see the following documents.

Agilent AdvanceBio HIC Column Quick Start Guide
Agilent AdvanceBio HIC Column User Guide

SAMPLE PREPARATION

To ensure proper binding of the sample, dissolve the sample in starting mobile phase. The effect of sample matrix on peak shape is demonstrated in Figure 5. When the sample is dissolved in DI water, the early eluting protein peaks (peak 1 and 2) are broader and fronting is observed compared to the sample that is dissolved in 0.5× mobile phase A. Often, directly dissolving the sample in high salt concentration mobile phase causes precipitation of the protein. Therefore, we recommend the following procedure. If the sample is solid, dissolve the sample in water or appropriate buffer, then dilute the sample two-fold with the starting mobile phase. If your sample is already in a solution, simply dilute the sample with the starting mobile phase. Make sure that the sample does not precipitate!

Figure 5. Effect of sample matrix

 

*****************************************************

Thanks, Sandeep! Over the next few weeks, I’ll be back with my colleague, Andy, who will walk us through some of the other method parameters for HIC analyses. Talk to you soon!

Anne

Keywords: Bio columns, liquid chromatography, hydrophobic interaction chromatography, HIC, LC mobile phase, AdvanceBio blog

  • hic
  • lc mobile phase
  • bio columns
  • advancebio blog
  • hydrophobic interaction chromatography
  • Biopharma LC Columns & Consumables
  • liquid chromatography
  • Share
  • History
  • More
  • Cancel
Anonymous
Related

Agilent Community Feedback

Agilent Community Feedback

×
We are glad this was helpful! We are sorry this was not helpful. If you still need assistance please create a community post or contact support. To help us improve, please provide any additional feedback. For full details of how we will treat your information, please view our privacy policy.
Submit Cancel
Submit Cancel
Recommended
Privacy Statement
Terms of Use
Contact Us
Site Help