Uncovering Overlooked Factors Behind a Poor Baseline

Hello, everyone! In the last time, we learned that "Unraveling the Case: The Reason Behind Poor LC RSD!" I wonder if it was helpful for your work. Today, let's talk about the issue of baselines. As the saying goes, "Know yourself and know your enemy, and you will never be defeated." At the end of this article, there is a case study where we will continue to unravel the mystery. Let's go!

• Explanation of Relevant Concepts

Chromatogram : This refers to the graph formed by plotting the detector output signal against time, also known as a chromatographic elution curve. The raised portions of the curve are called chromatographic peaks.

Chromatographic Baseline : Under experimental conditions, the elution curve produced when only the mobile phase enters the detector after the chromatographic column is called the baseline. A stable baseline should be a horizontal straight line.

Baseline Noise : Baseline noise refers to the continuous or periodic electrical signals or peaks that appear in the chromatogram when no sample has been added or during baseline scanning.

Baseline Drift : Baseline drift refers to the slow directional change of the baseline over time.

Baseline noise

• Causes of Baseline Noise

1. Incomplete degassing of the mobile phase, presence of air in the pump, tubing, chromatographic column, or detector; poor miscibility of mobile phase components leading to incomplete mixing.

2. Contamination of the mobile phase, chromatographic column, or detector.

3. System leakage.

4. Insufficient energy from the detector light source.

5. When using a UV detector, the selected detection wavelength is near the solvent's UV absorption cutoff wavelength; excessively high data acquisition frequency.

Chromatographic Signal-to-Noise Ratio (S/N) : This refers to the ratio of the signal to the noise in chromatographic analysis. It is used to measure the strength of the signal relative to the level of noise interference. The signal-to-noise ratio is an important parameter for evaluating the sensitivity of a chromatographic method in system suitability testing, and it is commonly used to determine the method's limit of detection (LOD) and limit of quantification (LOQ).

•  Common Baseline Abnormalities and Solutions

When the baseline displays a regular sawtooth pattern, and the pressure trace and display show a thicker line with significant pressure fluctuations, this situation is generally caused by the following reasons:

1. Presence of Air in the Pump Head : Degas the solvent and refill the pump.

2. Issue with the Check Valve : Ultrasonically clean the check valve core or replace it.

3. Plunger Seal or Plunger Rod Issue : Clean or replace the plunger seal or plunger rod.

4. Insufficient Mixing : Replace with a larger volume mixer.

5. Insufficient Back Pressure on the Detector, Resulting in Air Bubbles : Ensure the outlet tubing of the flow cell is long enough to provide adequate back pressure.

6. Electrostatic Interference : Check grounding.

The issue of baseline with significant pulsation is generally caused by problems with the plunger rod seal. It requires corresponding maintenance such as cleaning and maintaining the plunger seal or plunger rod, and even replacing the corresponding components if necessary.

When the baseline is chaotic in the absence of sample injection, it is usually due to system contamination, requiring thorough cleaning of the system.

Baseline drift

1. Generally, it is influenced by column temperature. For some detectors, especially differential refractive index detectors, conductivity detectors, or high-sensitivity UV detectors, they are particularly sensitive to temperature. Control the column temperature, mobile phase temperature, flow cell temperature, and room temperature. Use a post-column heat exchanger if necessary.

2. Uneven or contaminated mobile phase leads to irregular baseline drift. Use solvents of HPLC grade or high-purity salts or additives during the experiment.

3. Contamination or air bubbles in the detector flow cell. Flush the detection cell with highly polar solvents (such as methanol).

4. The detector outlet tubing is too tightly twisted, resulting in excessive back pressure in the flow cell, which may cause cracks in the flow cell and thus elevate the baseline.

5. Insufficient equilibration of the chromatographic column. Equilibrate the chromatographic column fully according to the instructions provided with different chromatographic columns.

6. Strong retention properties of sample substances lead to severe peak broadening, resulting in an elevated baseline.

Case Analysis

A user encountered abnormal chromatographic peaks and baselines while using the chromatograph for analysis. They immediately consulted an engineer to investigate the cause.

In this case, the customer reported irregular and chaotic baseline. After communication with the customer, possible causes were investigated:

1. Air bubbles in the pump.
2. Issues with the mobile phase.
3. Problems with the detector.
4. Issues with the chromatographic column.

Since the user mentioned that the mobile phase was freshly prepared daily and the instrument was used normally, the investigation started with the pump:

Step 1:

Observed pressure fluctuations in the workstation software. After priming and purging operations, pressure fluctuations significantly reduced. However, after re-injection, peak shapes improved noticeably, but the baseline still fluctuated.

Step 2:

Replaced with a new chromatographic column, rebalanced the column, injected samples, and observed normal peak shapes. However, baseline fluctuations still persisted.

Step 3:

Potential contamination of the flow cell was considered. Initially, the flow cell was flushed with warm ultrapure water for 2 hours, followed by a 2-hour flush with methanol. After testing, it was found that the baseline returned to normal. Subsequent injections also showed normal baseline and peak shapes. The issue was resolved.