The gold standard of any chromatographic technique, such as HPLC, is to achieve symmetrical Gaussian peaks for all analytes fully separated from one another. Achieving this peak perfection makes quantifying your analytes easy and gives you confidence that a neighboring peak does not affect your peak of interest.
But getting needle-sharp peaks isn’t always straightforward. Many factors can distort your peak shapes – things like your column packing technology, flow rate, mobile phase, sample prep, HPLC instrument design, temperature fluctuations, extra-column volume and dead volume.
Dead volume in particular is something you need to understand and learn how to work around because these volumes vary by manufacturer make and model, which can affect both your method development and method transfer processes.
In this blog post, we will explore what causes dead volume and how you help improve your separation efficiency by fixing the issues.
What is dead volume?
Dead volume is essentially the areas within an HPLC system your analytes could get trapped and reside in, harming the chromatographic efficiency of your separation. This volume comes from multiple components and encompasses voids found in your fittings/connections, column and instrument design.
Why is dead volume bad for your analysis?
Dead volume is intrinsically tied to separation efficiency, which has a huge impact on your peak width and peak shape. And consequentially, changes in peak shape can lower your resolution and alter retention times. Here’s the main ways dead volume can impact your analysis:
1. Broadens peaks
Dead volume can broaden peaks, lower peak resolution and decrease the separation between your closely eluting compounds. This change makes quantifying the analytes difficult and thus reduces your confidence in the data set.
2. Reduces sensitivity
Broader peaks can lead to decreased sensitivity. That’s because as peaks broaden, the peak height subsequently drops, making it more challenging for you to distinguish the analyte signal from the baseline.
3. Compliance/quality assurance issues
Many methodologies have strict limits regarding these variables and results that fall out of these restrictions will cause System Suitability Test (SST) failures, potentially failing the analysis batch. Investigating and repeating a test could cost you thousands of dollars.
This is a good point in this post to note that, if SST failures concern you, this case study shows how to reduce SST failures and how that equates to huge savings.
4. Incompatible method transfers
Changes in dead volume between two HPLC instruments can lead to retention time reproducibility issues, hampering your efforts to easily transfer methods from one system to another.
Also, if your originating HPLC system has significant dead volume, you’re more likely to see sample matrix/mobile phase mismatch issues when moving a method to a system with less dead volume.
This mismatch happens because the dead volume on the originating system allows the sample matrix and mobile to fully mix and disperse before entering the column, so running this method on a new system could cause abnormalities in the peak shape and retention time. You can see a specific example about eluent mismatches in TN74138.
What causes dead volume and how can you fix the problem?
Because dead volume is intrinsic to your HPLC system and components, you can’t fully eliminate the sources. But there are things you can do help fix the problem.
1. Select columns compatible with your analytical conditions and handle with proper care
If your column is damaged/misused, inappropriate for the analysis or of poor quality, voids can form within the packing beads.
- Ensure you’re using an appropriate column within the set limits (staying within the mobile phase/flow rate/pH/temperature restrictions). But also handling it carefully, not letting it dry out, dropping it on the flow etc., all of which can introduce voids into the column packing. Need help finding an appropriate column? Our column selection tool can help.
2. Opt for LC specific tubing/fittings that are built to minimize dead volume
Using incorrect or poor-quality tubing and fittings creates additional volume in the system where your analytes could get trapped.
- Fittings that support virtually zero dead volume, like our universal tool-free Thermo Scientific Viper Fingertight Fittings, can help improve your separation efficiency.
3. Research the HPLC instrument flow path, method transfer capabilities and troubleshooting features before you buy
A poorly designed flow path creates unnecessary extra volume (using long/wide-bore tubing) or has an inefficient injection system design, the instrument will inherently have high dead volume
- When choosing an HPLC system, you need to consider the design:
- Is there a logical flow path that, by design, reduces dead volume?
- Is the system easy to maintain and troubleshoot?
- Is there a built-in health-check functionality that allows you to identify sources of dead volume?
- Does it have the capability to mimic other systems including the dead volume characteristics to enable seamless method transfer without affecting system performance?
The great news is the Thermo Scientific Vanquish HPLC & UHPLC Systems incorporate all these intelligent design considerations.
Are you new to HPLC and want to learn more?
Visit our Learning Center to learn more about HPLC, covering all the fundamentals: from learning how HPLC works and the applications, all the way to developing and transferring your methods.