Unraveling the Case: The Reason Behind Poor LC RSD!

Hello everyone, liquid chromatography(LC) is one of the most commonly used large-scale analytical instruments for us, serving as a great companion in our work. However, due to improper usage or maintenance, it occasionally encounters some minor issues.

Recently, our after-sales engineer received another call for assistance from a customer. Today, I'll share with you an example of troubleshooting. Remember, having a clear train of thought and logical approach is crucial, so be sure to take notes.

1. Case Review:

One day, "ring ring ring" Welch's phone rang. A customer from a laboratory using Welch HPLC with a differential detector for sugar substances reported an issue: while retention time was stable, peak area repeatability was poor. The customer's method required an RSD of <0.3%, but the actual RSD was 0.8%. As a result, they sought assistance from Welch.

Following the customer's repair request, I promptly rushed to the customer's location within the response time specified by Welch to conduct an inspection.

1. Investigation of the case

Delving into the details of the case investigation,Upon arrival at the scene, after exchanging information, I gained a more specific understanding of the situation, and then we proceeded with the investigation.

First, we need to know what are the possible reasons for the poor repeatability of peak areas? In most cases, it is likely to be a problem with the autosampler, but of course, we cannot easily rule out the possibility of other modules, so we need to investigate them one by one.

Let's work together to unravel the details and understand the specific reasons.

Scrutinize every detail

Operate the instrument, activate the liquid phase pump, and observe whether there is any leakage at the connections of each module. After running for a period of time, inspect each module's connections to see if there is any liquid seepage, thereby eliminating the influence of peak area RSD caused by system leakage.

In this case, pure water is used as the mobile phase, and the retention time is stable, which can exclude the possibility of system leakage and ensure stable pump flow rate. This step is a routine check. Observe the automatic sampler injector for any bubbles. Upon confirming that there are no bubbles in the injector, the large RSD of the peak area is not caused by changes in injector sample volume. So, who is the real culprit?

Open the automatic sampler's needle washing operation and observe whether air enters the sample injector and whether liquid leaks out when sampling at the injection port. If liquid leakage is observed at the injection port, then the problem may lie here.

After rerunning the instrument and conducting sample replicability tests, the calculated result yielded an RSD of 0.7824%. 
Despite this, it still does not meet the customer's required RSD. Could there be another reason? Upon careful reinspection of the instrument, it was observed that there were debris from the sample vial spacer at the sealing point of the injection port. After dismantling and cleaning, following the recommendations in the chromatographic column manual, the chromatographic column and guard column were balanced for 12 hours before conducting the replicability test again.

After maintenance, the RSD is 0.2%.

After calculation, the RSD is 0.2047%. With this, our case has been clarified.

The real culprit turned out to be the deviation in peak area RSD caused by the HPLC column not being balanced according to the requirements! The case is finally solved.

1. Knowledge Sharing:

 Causes of large peak area RSD

Let's further discuss the issue of large peak area RSD. This part is crucial, so get ready to take notes.

01 Instrumental Issues

1. Autosampler

When encountering large peak area RSD, the first thing that comes to mind is the autosampler. The problems we encounter to some extent depend on the type of autosampler. Typically, air bubbles formed during the measurement of sample volume are the main source of the problem.

If we are using a manual injector, ensure that there are no bubbles in the syringe. When using a fixed-loop injector, ensure that no air enters the injector flow path.

There is another situation worth noting: if the sample vial spacer is sealed around the injection needle, negative pressure can be generated when drawing large sample volumes, causing bubbles to enter the subsequent flow path. The formation of any bubbles can result in differences in sample volume between each injection. In such cases, we can use pre-scored sample vial spacers.

We have summarized the situations related to autosamplers that cause abnormal RSD as follows:

 Repeatability issues caused by autosamplers 

1. Detector Issues

 Detector Issues Poor grounding of the detector can lead to fluctuations in peak area. The integration of peaks depends on their signal-to-noise ratio, and fluctuations in peak area caused by the mobile phase are relatively rare.
If there are any changes in the composition of the mobile phase, they will affect retention time. Ghost peaks or negative peaks caused by the mobile phase may also affect integration. This can typically be avoided by dissolving the sample in the mobile phase or using GB columns.

02 Sample Issues

1. If there is a significant difference in the concentration of the analyte and the peak area of a single sample remains constant only after two or three injections, sample residue may be the cause of the above problem. Thoroughly clean the autosampler after each injection to ensure that the solvent used for needle cleaning is well miscible with the sample.
2. A decrease in peak area may also be due to temperature changes, but this effect is relatively minor. When samples are taken out of the refrigerator and placed in the autosampler tray, they slowly warm up to the temperature of the autosampler, and the solvent expands.

Therefore, when the samples reach the temperature of the sample tray, the initial volume of the sample injected is larger than the subsequent ones. To eliminate this situation, samples should be allowed to stabilize at the autosampler temperature after being placed in the sample tray before injection.

3. Issues with the sample itself, such as certain proteins in reverse-phase chromatography not completely eluting during the initial gradient but eluting in subsequent blank gradients. If this occurs, a blank gradient should be run between two analyses.

Samples may also irreversibly adsorb onto certain chromatographic columns. The peak area obtained with a brand-new chromatographic column may slowly increase with repeated injections. This is because the sample binds to the active sites of the column tubing. Once these sites are saturated, reproducible peak areas are obtained. Other samples can be used to saturate the active sites, or diamond columns can be used to reduce specific adsorption.

03 Flow Rate Issues

• Random changes in flow rate can also cause fluctuations in peak area, usually caused by malfunctioning check valves or air in the pump head, often accompanied by fluctuations in retention time. This can be investigated by monitoring backpressure.

• Another potential cause of flow rate variation is leakage on the high-pressure side of the system. Most mainstream liquid chromatography pumps are equipped with leak sensors, making it relatively easy to troubleshoot this situation.

04 Integration Issues

Integration issues are challenging to resolve. While significant changes in the way peaks are integrated by visual inspection of the software can be detected, more subtle changes are harder to identify. Integration errors are often noticeable when tailing peaks occur, especially when the signal-to-noise ratio is 100 or lower. Baseline noise limits the precision of integration, but the problem becomes more severe with the appearance of fronting or tailing peaks.

Trying different integration parameters to reprocess the data and then checking if the issue can be resolved is a potential approach.