Retention Time Fluctuations - Part 2

HPLC Troubleshooting Course

10 - Retention Time Fluctuations - Part 2

11 - Column Lifespan - Part 1

12 - Column Lifespan - Part 2

13 - Detector Issues

14 - Flow Line Leakage

15 - Course Summary

Last week, we discussed the influence of temperature and fluctuating flow rates on retention time. This week, we will look at the influence of eluents on retention time. If the composition or density changes, it quickly leads to retention time fluctuations.

1. Inadequate Re-Equilibration in the Gradient

At the beginning of each measurement, it is important to ensure that the column is in equilibrium. When switching between two different mobile phases, such as when replacing the storage solution, the column must be equilibrated with sufficient column volumes (typically 10 column volumes for reversed phase chromatography). This also applies to gradient methods between measurements. Typically, the measurement methods have a rinsing step with a high proportion of organic solvent at the end. This must be followed by a sufficiently long equilibration step that corresponds to the initial conditions of the method.

If the equilibration is too short, it can lead to retention time fluctuations or, as shown in the example above, progressively shorter retention times from measurement to measurement. In this case, the column is no longer in equilibrium.

2. Change in Eluent Composition

Great attention should be paid to the mobile phase. Together with the column, the eluents are crucial for separation and stable measurements. Therefore, store your eluents in suitable storage bottles and seal them with appropriate tight-fitting caps. Do not leave the eluents in the sun or in the direct draft of an air conditioner.

Depending on the composition of the eluent, a certain amount of organic solvent can quickly evaporate, changing the composition of the eluent. The influence of an unsealed eluent bottle was tested with a mixture of water and acetonitrile. The more acetonitrile evaporates, the lower the elution strength, and the longer the retention times.

3. Preparation of Eluents

Care in handling eluents should start with their preparation. You should develop precise procedures to ensure consistent quality of the eluents. A good example is the preparation of a 50% methanol solution in water. This solution can be prepared in different ways, and depending on which employee prepares it, different results can occur.

Water/Methanol = 1/1 (v/v)
Measure 500 ml of water and 500 ml of methanol separately and mix them together
The total volume created is not 1 L, but 0.94 L

50% (v/v) Methanol Water Solution
Add 500 ml of methanol to a 1L volumetric flask and fill up to 1 L with water
The total volume created is 1L, with a higher proportion of water when compared with the previous example

In the first case, 500 ml of water is mixed with 500 ml of methanol. The result is 0.94 L of a 1/1 solution in terms of volume. In the second case, 500 ml of methanol is placed in a 1 L volumetric flask and filled up to 1 L with water. In this case, you get 1 L of a 50% methanol solution based on volume.

These two solutions have different elution strengths, as seen in the following example with a phosphate buffer and acetonitrile. The retention times of the individual analytes fluctuate significantly and, in the worst case, can fall outside the identification time window.

In addition to the reasons listed here, there are many other reasons that can cause retention time fluctuations. These can include insufficient buffer capacity, poor online mixing, a defective or dried-out column, or accumulating contaminants.

If you are dealing with fluctuating retention times, it is recommended to first check the temperature control of the column and then check the pump and the eluents. For a quick test, replace the eluents with new ones and check the retention times with your references. Using the instrumentation to create these kinds of mixtures will ensure maximum accuracy.

Things to consider daily:

Susceptible Conditions:

Reversed-phase eluent is 100% water
Extremely acidic or alkaline eluents in silica columns

Additional Equilibration Time for:

Normal-phase chromatography
Reverse-phase chromatography with ion-pair reagents

General:

Keep a column diary
Document maintenance
Develop procedures for the preparation of eluents
Store storage bottles sealed and safely
Pay attention to sufficient equilibration time

The upcoming two course units will focus on the separation column and its lifespan.

Your Shimadzu LC Team

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