Retention Time Fluctuations - Part 1
HPLC Troubleshooting Course
6 - Changes in Peak Shape - Part 2
9 - Retention Time Fluctuations - Part 1
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
The most common detection methods in HPLC are UV/VIS absorption, fluorescence, and refractive index detection. These optical detection methods are sometimes substance-specific for compounds with chromophores or fluorophores, but also non-selective when using the refractive index. The assignment of analytes is usually done via retention time. If the retention time fluctuates, it can lead to incorrect results. Therefore, the reproducibility of retention time is of great importance.
The following and the upcoming course unit will discuss the various influences.
1. Influence of Temperature
As with many problems in HPLC, temperature also affects retention time. The higher the temperature, the lower the viscosity of the mobile phase. Therefore, the back pressure in the system also decreases with increasing temperature. As a rule of thumb, a temperature change of 1°C results in a shift in retention time of 1 to 2%. Therefore, the temporal shift due to temperature fluctuations is greater for late-eluting analytes. The following shows four parabens measured at different temperatures.
Comparing the measurement at 20°C and 50°C, the total runtime is almost halved. This influence can, of course, also be used to optimize or shorten the method, provided it remains within the specifications of the column. However, at very high temperatures, the lifespan of most columns is expected to be reduced.
If the temperature is to be used for optimization, it should be noted that not all analytes react equally to a temperature change. As shown in the example on the right, they can behave very differently. While the analytes are separated at 20°C, sorbic acid and benzoic acid co-elute at 30°C. If the temperature is further increased, the elution order can even switch. If this is not noticed, it can also lead to incorrect results.
Ideally, you should use a column oven to keep the temperature of your column as constant as possible.
1: Sorbic acid, 2: Benzoic acid, 3: Methylparaben
2. Change in Flow Rate
Every pump has a certain number of consumable parts that should be replaced at regular intervals. For example, most piston seals are specified for delivering a certain volume. If wear is too advanced, they can become leaky and thus affect the flow rate. If retention times fluctuate, a defective pump could be the problem.
The simplest way to check the pumps is to calibrate them. Use a graduated cylinder, connect a backpressure capillary directly to the pump, and set a defined flow rate. Then compare the time it takes to deliver a certain volume with the set flow rate.
Regularly check the consumables of your HPLC. Also, keep spare parts on hand to quickly replace defective parts.
Non-robust methods and ineffective measurements lead to unstable chromatographic conditions. Under such conditions, even a small change, e.g., in temperature or the eluent, have a significant impact on retention time.
As a result, samples often need to be repeated, or the evaluation needs to be manually reworked. This is especially a big problem in regulated environments. Therefore, invest more time in developing robust methods. You will have less effort and more time in routine work later, and the time previously invested will quickly pay off.
Things to consider daily:
General:
- Conduct regular suitability tests
- Check the consumables at regular intervals
- Document retention times, resolution, etc., of the reference solutions
System:
- Use a column oven for temperature control
- Keep a column diary
- Document maintenance
The upcoming course unit will also address retention time fluctuations alongside the influences of eluent composition, the column, and equilibration time.
Your Shimadzu LC Team
