Low Sensitivity in HPLC and UHPLC Systems

High-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) are widely used analytical techniques for trace-level qualitative and quantitative analysis. One of the most frequently reported performance issues in both systems is low sensitivity, characterized by reduced peak height, poor signal-to-noise ratio, and elevated detection limits.

Low sensitivity directly impacts method robustness, quantitative accuracy, and regulatory compliance. Identifying the underlying cause requires a systematic evaluation of sample introduction, detector configuration, extracolumn effects, system integrity, and mobile-phase conditions.

Symptom and Observable Problem

Low sensitivity in HPLC or UHPLC may present as one or more of the following:

• Reduced peak height or peak area compared to historical performance

• Broad, distorted, or flattened chromatographic peaks

• Early analyte elution with poor resolution

• Increased baseline noise relative to analyte response

• Poor reproducibility of peak response between injections

These symptoms often occur even when system pressure and flow appear nominal, making low sensitivity a diagnostic challenge without a structured approach.

Root Cause Analysis

Low sensitivity is typically caused by analyte dilution, peak broadening, or signal loss occurring either at injection, during separation, or at detection. The most common contributors are outlined below.

1. Sample Overload Leading to Early Analyte Elution

Excessive sample concentration or injection volume can exceed the stationary phase loading capacity. When this occurs, analytes elute prematurely with broadened and distorted peaks due to incomplete retention. The result is a lower peak height and reduced detector response despite higher analyte mass.

2. Detector Cell Volume Too Large

Large detector flow cells increase band broadening within the detection zone. This internal dispersion reduces peak height and dilutes the analyte signal, directly decreasing sensitivity. This effect is especially pronounced in UHPLC systems where peak volumes are inherently small.

3. Injection Volume Too Large or Incompatible Injection Solvent

Large injection volumes or the use of a strong injection solvent relative to the mobile phase prevents proper analyte focusing at the column inlet. Poor focusing results in broader peaks and reduced signal intensity, even when analyte mass is sufficient.

4. Excessive Extracolumn Volume (Dead Volume)

Extracolumn volume from tubing, fittings, connectors, and detector cells causes additional band broadening outside the column. In UHPLC systems, where column volumes are small, even minor dead volume can significantly degrade sensitivity.

5. System Leaks

Leaks at fittings, injector seals, or detector connections can reduce effective analyte mass reaching the detector. Leaks may also introduce baseline noise, pressure instability, and flow inconsistencies that further reduce sensitivity.

6. Mobile Phase Viscosity and Composition

Highly viscous mobile phases increase mass-transfer resistance and can broaden peaks. Inappropriate solvent strength may lead to insufficient retention, early elution, and poor peak shape, all of which reduce sensitivity.

7. Pressure Instability During Separation

Fluctuations or unexpected drops in column pressure may indicate partial blockages, leaks, or pump irregularities. These conditions disrupt flow stability and compromise peak integrity and detector response.

Diagnostics

A disciplined diagnostic workflow improves root-cause identification and minimizes unnecessary component replacement.

• Evaluate chromatograms for peak broadening, early elution, and signal-to-noise degradation

• Review baseline stability for noise or drift indicative of leaks or flow irregularities

• Trend system pressure for sudden drops, instability, or abnormal behavior

• Confirm injection parameters, including volume and injection solvent strength

• Inspect detector flow cell specifications and cleanliness

• Estimate extracolumn volume by reviewing tubing length, internal diameter, and fitting geometry

• Perform a dilution test by diluting the sample (e.g., 1:10) and reinjecting to assess overload effects

• Review mobile phase composition and temperature settings

Corrective Actions

Corrective actions should be implemented incrementally and verified by reinjection after each change.

Sample Overload Mitigation

• Dilute the sample (commonly 1:10) and reinject

• Reduce injection volume

• Use a column with higher loading capacity if required

Detector Optimization

• Select the smallest practical detector cell volume compatible with the method

• Verify flow cell cleanliness and proper installation

Injection Optimization

• Reduce injection volume to less than one-sixth of the volume of the first-eluting peak

• Match or weaken injection solvent strength relative to the mobile phase to improve focusing

Extracolumn Volume Reduction

• Replace fittings with low- or zero-dead-volume connectors

• Use the shortest tubing lengths possible

• Select appropriate internal diameters (<0.010 inch for HPLC; <0.005 inch for UHPLC)

• Ensure proper fitting seating and alignment

Leak Elimination and Pressure Stabilization

• Reseat or replace leaking fittings

• Replace worn injector seals or pump components as needed

• Remove blockages and confirm unrestricted flow through the system

Mobile Phase and Temperature Optimization

• Increase column temperature to reduce solvent viscosity and improve mass transfer

• Adjust mobile phase composition to improve retention without excessive viscosity

Related Issues

Low sensitivity is often associated with or may contribute to additional chromatographic problems, including:

• Poor quantitative accuracy and precision

• Method robustness failures during validation

• Increased variability between injections

• Shortened column lifetime due to overload conditions

• Misinterpretation of detection limits and reporting thresholds

Summary

Low sensitivity in HPLC and UHPLC systems is a multifactorial problem most commonly caused by sample overload, detector cell volume mismatch, excessive extracolumn volume, leaks, and suboptimal mobile phase conditions. A structured diagnostic approach focusing on injection behavior, system integrity, and dispersion sources allows accurate identification of the root cause. Targeted corrective actions restore signal intensity, improve peak shape, and enhance overall method performance.