UHPLC TIP: Reducing the Column Temperature to Offset Frictional Heating Effects (Causing Poor Resolution)

HPLC column temperature is a critical variable that we adjust and optimize during method development. We use it as a variable during the method development process to improve solubility, optimize peak shape and increase resolution. Once established, it must be carefully controlled during the method analysis to provide reliable and reproducible analysis results. Change the column temperature and you may also change the results obtained. This is a fundamental method development tool and must not be forgotten.

If you are developing a new UHPLC method OR perhaps scaling an HPLC method to utilize 2.5 micron or smaller support particles, then you may observe a loss of resolution or poor peak shape in the new method. There are many reasons why this may occur, and the most common ones relate to not optimizing all of the method parameters correctly when scaling the method (e.g. dwell volume too large, flow cell volume too large, injection volume too large, sample rate too slow, flow rate not optimized, mobile phase composition changes not in scale with the gradient...). But there is another reason...

Resolution may be reduced or lost when all of the initial scaling and instrument set-up parameters are optimized. What is the most likely reason for this? In many cases the use of substantially higher flow rates (relative to linear flow rates) and the use of smaller diameter particles results in much higher backpressures (you may recall that if you halve the particle size, the backpressure increases 4x). The resulting backpressure might be 2, 3 or even 4 times higher than observed in the original method. While these higher backpressures were well within the operating parameters of the HPLC system used, the results obtained were poor. The possible cause? The much higher backpressure increased the amount of frictional heating inside the column, raising the actual analysis method temperature and changing the separation conditions. 

Pushing mobile phase (liquid) through a chromatography column generates heat and pressure. The heat generated increases the actual temperature of the column and reduces the viscosity of the fluid. In conventional columns (i.e. 4.6 x 150 mm, 5u) at 1.00 ml/min, this heating effect is minimal, but at much greater column pressures, > 400 bars, the frictional effects may be substantial. These types of very high pressures may be seen with methods which utilize columns containing the smallest particles (1.9 to 2.5 micron). Enough to change the temperature in the column by several degrees (e.g. >5 degrees C) and result in different method conditions. So, what can you do about this? The most direct way to address the problem is to run the same method at a lower temperature (perhaps decrease by 5 C to start with). This will slightly raise the backpressure (lower temperature equals higher viscosity), but it should cool the column and restore the original temperature conditions used. Additionally, we suggest that you always start column equilibration using a flow ramp to gradually increase the flow over time and reduce the overall heating effect and resulting "shock" placed on the column. An initial delay at equilibration may help reduce these effects (gradually ramp up to the regular flow rate and hold). You may need to try several temperatures and this may be easiest to do if your HPLC has a column compartment with heating and COOLING capabilities. Optimizing the temperature and internal pressures may increase the column lifetime and result in better overall data reproducibility.

HPLC Peak Tailing - Some of the Most Common Reasons For it

Three easy ways to minimize chromatography peak tailing:

(1) Tailing often results from using “Type – A” HPLC silica. Type-A silica often contains more acidic silanol groups and metal impurities than Type-B. To improve peak shape, use modern “Type – B” silicas which are of higher overall purity, have less metal contamination and feature minimal silanol ionization under higher pH conditions.

(2) Minimize ionic interactions and utilize a buffer or ion pairing agent (e.g. TFA 0.02%) in your mobile phase. Select a buffer that is at least 2.0 pH units away from your sample's pKa and use the smallest concentration or amount that gets the job done. For LC/MS or MS/MS applications, remember to only use volatile buffers and avoid the use of ion pairing agents unless absolutely necessary (and if used, use at the lowest possible concentration to avoid source contamination).

(3) Always use a freshly washed and equilibrated column. Is the column fouled or the inlet frit dirty? If the head of the column is fouled from sample overloading or from a failure to wash off strongly retained compounds from many runs (much more common problem), then your peak shape and reproducibility will suffer. Incorporate a washing step in between your analysis methods which utilizes a solvent which is stronger (in concentration) than your mobile phase to wash off any strongly retained material after each run. For example, if you normally end a method with an 80% concentration of ACN, utilize a separate wash method which has 95% ACN in it. Allow enough wash time for this work.

Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)

   Let's take a quick look at two different problems which you may encounter when operating an HPLC system. We start with the basic observation and then look at the most likely causes so we can begin the troubleshooting process and repair the problem. An automated HPLC system's flow path typically consists of: The Solvent Pickup Filters (in the mobile phase reservoirs); The Pump(s); AutoSampler; AutoInjector; Column and one or more Detectors.*You should have a good understanding of this flow path before you proceed to diagnose the problem(s).

 *A gradual increase of pressure for the same method over time is often due to column fouling or a dirty inlet frit (e.g. PTFE frit). This article specifically focuses on the causes of a sudden change, not a slow change over time.

   Sudden System Back Pressure Changes: We will assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the back pressure has changed from what is normally seen. The problem must lie within the flow path of the system.

   Excessive High Pressure: Typical reasons for this are:

1.      A fouled or plugged column;
2.      Wrong flow rate (higher than normal);
3.      Inlet frit/filter plugged or restricted;
4.      Plugged line;
5.      Wrong mobile phase composition.

   Large Drop in Pressure: Typical reasons for this are:

1.      A leak at a fitting, column or line (Number one reason);
2.      Wrong flow rate (lower than normal);
3.      Wrong mobile phase composition. 

• Start by checking the method parameters to insure that they have not changed (i.e. flow rate, mobile phase composition). Check for leaks or plugs. If the column is suspect, replace it with a zero dead volume union (ZDU) and restrictor and flush the system. Replace the column with a new one or wash the current column according to the column manufacturer's guidelines.

   Sudden Peak Shape Changes: We will again assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the peak shape of one or all of the peaks has changed from what is normally seen. *The key thing to keep in mind is that the change occurs all of a sudden, not because of poor initial method development.

   Typical reasons for this are:

1.      Tailing or Split Peaks: Sample overload, change in flow rate, mobile phase composition (e.g. composition or pH), void formation, dirty frit, injection solvent too strong or a fouled column.
2.      Fronting: Commonly seen when overloading sample on column.
3.      Ghost Peaks: Usually due to a contaminated mobile phase, contaminated sample vial or contaminated injector (e.g. rotor seal).
4.      Broad Peaks: Large sample injection volumes or extra column volume (bad connections with the system or tubing) are usually to blame. Try reducing the injection volume by a factor of 10 and see if the problem goes away. You may also want to wash the column as it may be fouled with sample.

   These are just two common problems we see when using HPLC systems. Note that a dirty or fouled column can cause many of these problems so take care of your columns and wash and test them regularly to insure they are in compliance. There are many other commonly seen problems besides these. If you would like to see a specific problem featured on this blog, then please send me a request.