Agilent Community
Agilent Community
  • User
  • Site
  • Search the Community
  • User
Consumables
  • Technical Areas
Consumables
Wiki Best Practices for Making Good Connections
  • 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.

Best Practices for Making Good Connections

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

Properly fitted connections are key to optimizing separations.  This post illustrates the effects of poorly fit connections and suggests resources to help.

 

Many of us don’t spend much time thinking about the fittings used to plumb our LC, including connecting the column, but this seemingly small detail can cause major headaches if they aren’t correct. 

 

It’s especially important to pay attention to fittings when you’re switching back and forth between different vendor’s LCs, LC columns, or fittings.  Many vendors have a slightly different geometry to them, so taking an already-swaged fitting from one LC column to another may not result in a solid connection.

 


In the figure above, the middle image shows an example of the capillary being too long – sticking out too far past the ferrule.  This can lead to leaks as the fitting isn’t tight enough.  Sometimes these are obvious enough that they’ll trigger a leak sensor quickly and interrupt your run; other times they’re very slow leaks that are harder to pinpoint.  Especially with an organic mobile phase, the solvents may evaporate before triggering a leak sensor. 

 

The bottom image shows a case where the capillary doesn’t extend far enough past the ferrule, creating an empty space between the end of the capillary and the head of the column.  This dead-volume or mixing chamber will manifest as tailing, poor resolution, or generally poor peak shape.

 

Here we have a couple of examples collected by my colleague, Andy, of the effect this extra dead volume can have on separations.  In the first example of a single protein, the extra dead volume causes the tailing factor to increase from 0.901 to 1.093.

 

Uridine; AdvanceBio SEC 4.6 x 150 mm, 300 Å (p/n PL1580-3301); 150 mM phosphate buffer, pH 7.0; 0.35 mL/min

 

In this second example, with a mixture of peptides on a C18 column, the impact this peak broadening can have is readily apparent as minor peaks begin to disappear!

 

Peptide mix (Sigma H2016); AdvanceBio Peptide Mapping 2.1 x 150 mm (p/n 653750-902); water/acetonitrile gradient with 0.1% TFA; 0.2 mL/min

 

Agilent’s InfinityLab Quick Connect fittings are a simple way to ensure a good connection every time, as the fitting is spring-loaded to adjust to the needed capillary length.  If you’re creating connections yourself from loose capillaries, fittings, and ferrules, check out pages 43-46 of the LC columns catalog for detailed instructions with illustrations.

 

Speak soon!

Anne

 

Keywords: bio columns, liquid chromatography, best practices, tips and tricks, AdvanceBio blog

  • tips and tricks
  • Best practices
  • bio columns
  • advancebio blog
  • 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