Peak Area Fluctuations

HPLC Troubleshooting Course

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1 - Basics of Systematic Troubleshooting

2 - Preventive Maintenance Measures

3 - Baseline Disturbances

4 - Pressure Fluctuations

5 - Changes in Peak Shape - Part 1

6 - Changes in Peak Shape - Part 2

7 - Ghost Peaks

8 - Peak Area Fluctuations

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

In most cases, quantification is done using the area of a peak. If this fluctuates due to external influences, it leads to over- or underestimation. The result does not match reality. Since this must be prevented, pretreatment or the temperature of the detector cell are important points.

1. Variability of Injection Volume

When peak areas fluctuate, the cause is often found in the injector. To confirm this, repeated injections of a reference with a known concentration are recommended (gravimetrically assessed water can be used). Constant conditions should always be maintained, as even the rinsing of the needle can have a significant impact on the deviation.
For highly viscous samples, the draw speed should be reduced, as air bubbles could otherwise be sucked in.

Air bubbles in the rinse solution or an empty rinse solution can also lead to fluctuations in peak areas. Therefore, it should also be purged regularly.

2. Adsorption in the Injector

Depending on the substance, different adsorption effects can occur on the injection needle or the seal. If five repetitions of a caffeine and thiamine standard are injected and measured using the same method, the thiamine shows twice the standard deviation. In this case, the rinse solution of the injector and the pretreatment program should be adjusted to the analytes.

To check repeatability, an adsorption-resistant internal standard should always be used.

3. Detector - Temperature - Fluctuations

The detection temperature in the measuring cell has a significant impact on the signal. If this fluctuates, the signal also fluctuates in its height. The refractive index is particularly sensitive to temperature. However, UV/VIS detectors or fluorescence detectors also react to temperature changes. For example, if the temperature of a fluorescence detector cell increases, the signal decreases. Therefore, tempering the detector cells is essential.
Compare the signal with the temperature of the detector cell and the room temperature to identify effects disturbing the signal. Additionally, the room temperature should be kept as constant as possible. The system should not be placed in a draft, directly under an air conditioner, or in direct sunlight.

 

First, you should check the autosampler. If this is not the cause of the peak area fluctuation, the detector should be checked. In rare cases, the adsorption of analytes on the column packing material can be the cause. This is especially the case with ODS columns and basic, chelating substances. Check the suitability of the column used for your analysis. If sufficient information is not found in the column's datasheet, contacting the column manufacturer is recommended.

Things to consider daily:

General:

  • Conduct regular suitability tests
  • Check the system with multiple repetitions of a reference
  • Document retention times, resolution, etc. of the reference solutions

System:

  • Keep a column diary
  • Document maintenance
  • Adjust pretreatment of the sample matrix
  • Select a suitable rinse solution
  • Use heated detector cells

In addition to peak area fluctuations, retention time fluctuations are a common problem. Therefore, the next two parts of the troubleshooting course will address the possible reasons for retention time fluctuations and how they can be resolved.

Your Shimadzu LC Team

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