Have you ever looked at one of your chromatograms and thought: “Oh, I think there are two peaks overlapping?” Having a background in analytical chemistry and years in the lab, I know how it feels. You may have developed the ultimate LC method capable of separating your fifty-something analytes while simultaneously taking into account all HPLC and UHPLC guidelines, yet in the end, there are still overlapping peaks. If you fine-tune one part of the gradient, you just end up shifting others. Now you’re at your wit’s end and you have no time to re-develop the method from scratch. If this sounds familiar to you, read on.
No need to throw in the towel. There are technical solutions to help you out of the dilemma.
For the most challenging separations, one solution is to add more separation power by adding a second dimension to your standard one-dimension liquid chromatography using 2D-LC systems.
What is two-dimensional liquid chromatography?
What does it mean to add an extra dimension? One-dimensional means you have just one stationary phase to separate your peaks: This is pretty standard! Two-dimensional means you use the separation power of two stationary phases to increase your peak capacity.
In 2D-LC, discreet chromatogram sections of interest are collected from the effluent flow of the first dimension. These fractions are then analyzed using a second column with a different stationary phase chemistry. Therefore, each first dimension fraction is used to generate a second dimension chromatogram, enabling applications such as peak purity determination (shown below).
There are different ways to perform a 2D-LC separation. The main difference is the interface between the dimensions. Let’s see what possibilities we have and investigate the advantages and drawbacks of each.
- Offline or online fraction?
The fractionation of the first dimension can be made offline or online. Offline means the fractions are stored in fraction collector inside vessels and re-injected into the same or different LC for the second dimension analysis. For offline fractionation, high-resolution second dimension chromatography in a time-decoupled manner is possible for every single fraction collected in the first dimension. However, sample integrity might be affected because it leaves the flow path (e.g. analyte degradation under ambient conditions). Therefore, a thermostatting is a must, particularly for analysis of biopharmaceuticals.
With online 2D-LC the first dimension fractions are stored in certain interfaces and analyzed in near real-time. This approach is fast and easy to automate while assuring the sample integrity because the sample never leaves the flow path. However, the interface (details explained below in point 4) between the dimensions needs to be chosen thoughtfully to achieve the highest separation power possible.
- Fractionation Frequency
Besides the offline or online fractionation type, the fraction cutting frequency is a crucial parameter. When the entire 1D chromatogram is collected and analyzed, we are talking about comprehensive 2D-LC, also abbreviated as LCxLC. If certain sections are cut out of the 1D chromatogram for second dimension analysis, it is called a heart-cut 2D-LC. Depending on the number of fractions, it can be done as single or multiple heart cuts.
Like what you are learning?
- Loop or Trap?
In online 2D-LC there are different ways to store the fractions. They can be stored in sample loops for a fast fractionation and transfer to the second dimension. For large volume fractionation, analyte enrichment, or replacement of eluent fractions can be stored on a trap column.
- Which stationary phase to use?
This is a good question and there is no short answer because of the complexity and variety of applications. There is an interesting review article addressing this topic. In short, it always depends on the application details and analytical focus.
So, which way to go for your complex analyte mixture?
One option is to first deplete the matrix interfering with the analyte of interest. This approach, termed Online solid-phase extraction (SPE), might already solve the separation problem. One such configuration to trap, wash, and elute analytes of interest is the Thermo Scientific Online SPE HPLC and UHPLC System.
For fast peak purity verification, a simple combination of two complementary RP columns might do the job. The Thermo Scientific Vanquish Loop Heart-Cut 2D-LC System can be easily adapted for a variety of approaches including single or multiple loop heart-cutting and selective comprehensive 2D-LC (sLCxLC).
For deeper sample characterization, incompatible separation and detection methods may be needed. This requires the use of solvent modulation to reduce the eluent strength or desalt first-dimension buffered eluent for the second dimension. For example, sample desalting is required for mAb clone screening prior to accurate mass confirmation with mass spectrometry. Clones are first isolated on a Protein A column, then by reversed-phase analysis. Collecting such fractions on trap-columns is a good way to desalt even large peak volumes for making non-MS compatible UV methods MS compatible. This approach is facilitated by the Thermo Scientific Vanquish Trap Heart-Cut 2D-LC System.
Method development is another important consideration while installing a 2D-LC method in your lab. Usually, development starts with optimizing both dimensions separately from each other on single-channel LC flow paths. By using smart configuration of column switching valves and novel sampler technology with two separate injection needles and injection valves with the Thermo Scientific Vanquish Simple Switch 2D-LC system, it is even possible to do the individual method development in parallel on one dual LC. After optimization, you can simply switch and run 2D-LC without the need to replumb any fluidic connections. More details can be found in this technical note.
You’ve now seen various technical solutions for the analysis of complex samples. Now I am curious: What is your biggest challenge in analyzing difficult to separate analytes?
Let me know and drop me an email or leave a comment below.
- Technical Note: A pre-concentration and online solid phase extraction setup for the LC-MS analysis of therapeutic protein mixtures (TN72161)
- Technical Note: Complementary Dual LC as a convenient alternative to multiple heart-cut 2D-LC for samples of medium complexity (TN73184)
- HPLC and UHPLC system page