YAZAWA HPLC COLUMN SCHOOL

Even when HPLC analysis is performed, failure to detect a peak corresponding to the target compound is a problem that can occur not only for beginners but also for experienced analysts.
This phenomenon can be broadly classified into two categories:
issues that arise during the early stage of method development, and issues that occur during the routine operation stage after a method has been established.

1) Causes of “No Peak Detected” During Early Method Development

At the early stage of method development, the absence of a peak should not necessarily be regarded as an abnormal result.
Rather, failure to elute a peak often provides important information for identifying conditions under which the compound can be eluted.

One of the most fundamental causes is that the mobile phase composition is not suitable for the analyte.
If analytical conditions are selected without sufficient consideration of the compound’s polarity (hydrophobicity), ionic properties (pKa, pI), and detection method, the following situations may occur:

• The analyte interacts too strongly with the stationary phase and does not elute from the column.

• The analyte is not retained at all and is buried in the unretained (solvent front) peak.

• The analyst does not notice that the analyte is not dissolved in the injection solvent.

• The analyte elutes, but the detection method or sensitivity is inappropriate, and the peak is overlooked.

An important diagnostic step is flow injection analysis (FIA), in which a standard solution is injected without a column installed.
By removing the column and delivering the sample directly to the detector, the following points can be evaluated:

• Whether the compound can be recognized as a peak by the detector at all

• Whether the detection wavelength or detection mode (UV, MS, ELS, etc.) is appropriate

If no peak is observed at this stage, the problem lies upstream of the separation conditions.

When a column is installed, it is advisable not to judge the result based solely on isocratic conditions from the beginning.
During early method development, the basic approach is to apply gradient elution and check whether a peak appears at any retention time.

If no peak is detected even under gradient conditions, the following possibilities should be considered:

• The elution strength of the mobile phase is insufficient

• The selected separation mode itself is not suitable for the compound

Possible countermeasures for insufficient elution strength include:

• Switching to a stronger organic solvent, such as THF or acetone

• Changing the stationary phase, for example:

  • C18 → C8 (e.g., Imtakt Unison UK-C8)

  • C18 → C1 (e.g., Imtakt Unison UK-C1)

With regard to changing the separation mode, the following options may be considered:

• Reversed-phase → normal-phase

• Reversed-phase → ion-exchange mode

• Combination of multiple separation modes (e.g., Imtakt Scherzo C18)

Once the elution position of the analyte has been confirmed, it is desirable to optimize various conditions while considering resolution and analysis time, such as:

• Column length and internal diameter

• Mobile phase composition, pH, and ionic strength modifiers

• Flow rate and column temperature

• Peak shape and linearity

2) Causes of “No Peak Detected” After Method Establishment

When a peak suddenly becomes undetectable using an already established analytical method, the cause is often related to operational errors.

The most frequent causes are mistakes in the preparation or selection of the mobile phase, such as:

• Confusion between mobile phases A and B

• Incorrect mixing ratio of organic solvent and water

• Errors in pH or ionic strength adjustment

These mistakes can significantly alter retention behavior and ultimately lead to peak disappearance.

If no error is found in mobile phase preparation, instrumental problems should be considered, including:

• Insufficient solvent delivery by the pump

• Malfunction of check valves

• Clogging or injection failure at the injector

• Deterioration of the detector lamp or incorrect detector settings

• Failure in signal transmission to the data processing system

Although all of these issues manifest as “no peak detected,” their causes are unrelated to chromatographic separation conditions.

3) Method Robustness

(From the Viewpoint of Method Validation and Reproducibility)

When an analytical method is used for routine analysis, method validation is essential.
Methods that lack robustness—where peaks are not detected or reproducibility cannot be ensured—often reveal problems under the following circumstances:

• When the analyst changes

• When the instrument or laboratory changes

In particular, for inter-laboratory reproducibility, it is crucial to verify whether the same results can be obtained regardless of who performs the analysis, where it is conducted, and which instrument is used.

To achieve this, attention should be paid to the following points:

• Clear definition of mobile phase preparation procedures

• Quantification of analytical conditions (instrument settings, gradient initialization time, column washing time, etc.)

• Elimination of ambiguous or operator-dependent procedures

If these aspects are insufficient, problems such as peak disappearance and poor retention reproducibility are likely to occur.

Rather than immediately suspecting the column when “no peak is detected,” it is essential first to review one’s own operation and analytical environment.

(Reference)
Two types of HPLC gradient systems