PDA (Photo Diode Array Detector)/DAD: How to Perform HPLC Peak Purity Analysis

PDA (Photo Diode Array) Detector/DAD is a widely used technique to check the HPLC peak purity in the pharmaceutical industry due to its simplicity and fast result. In this article, you will learn about the peak purity concept, different methods for calculating peak purity, advantages, limitations, and applications of peak purity. You will also learn the concept of the PDA detector. After reading the article, your doubts will be cleared and you will be able to answer several questions related to peak purity and the PDA detector.

What is the peak purity?

The process of assessing the spectral uniformity of a chromatographic peak is called peak purity.

Why is peak purity needed?

HPLC is used at almost all stages during pharmaceutical development to control the quality of APIs. It may be possible that other impurity peaks may co-elute with the peak of interest. Peak purity is performed to check the covariation/interference of other peaks with the peak of interest. In the following HPLC chromatogram (Figure1) impurity A and impurity B are coeluting with the main peak

PDA or DAD Detector

PDA detector or photodiode array detector acquires absorbance data over the entire uv-visible range (190 nm to 900 nm). Therefore an analytical scientist can extract chromatograms at multiple wavelengths within the selected wavelength range as per requirement during method development. Peak spectra can also be generated as per the required wavelength range. It is very helpful during HPLC method development. It is also called DAD or Diode array detector.

PDA wavelength range

Most PDA instruments are available with wide UV-visible ranges such as 190 to 900nm. But some of the instruments may have narrow UV ranges like 190 nm to 650 nm or 200 nm to 650 nm

Advantages and Applications of PDA Detectors

The following are the advantages of the PDA detector:

• To know peak maxima in HPLC chromatogram
• To know the peak homogeneity or peak purity
• It tells about the coelution of the peak
• To decide operational wavelength during HPLC method development
• It is cost effective
• It gives fast result
• Very helpful during HPLC method development

Limitations of the PDA detector

The following are the limitations of the PDA detector:

• Not suitable for molecules having no UV absorption
• Less suitable for related substances or impurity profile testing due to less sensitivity
• Not suitable for inorganic compounds
• A PDA detector is low sensitive detector

Peak Purity concept

The process of assessing the spectral uniformity of a chromatographic peak is called the peak purity. This is the analysis of absorption spectra across peaks to determine whether they are all similar or have differences. It tells about the coelution of the peak in the main peak. This concept is based on the UV principle.

Peak Purity by PDA Detector

Peak purity by PDA detector is based on the comparison of spectra recorded during the elution of the peak (see Figure-1). Typically, the following three spectra are taken and compared to assess the peak purity by the PDA detector:

1. Two spectra at the inflexion points (the upslope and downslope spectrum) and
2. One at the apex (see Figure 2).

All the above three spectra satisfy the Peak purity suitability criteria.

Figure-2

One can select more than three points for spectra generation and can use all spectra for peak purity calculation.

Peak purity suitability criteria

1. If all the above three spectra are similar or match each other, then the peak theoretically has no spectral impurities and the peak is said to be pure.
2. If the above three spectra are not similar or do not match each other then theoretically the peak contains spectral impurity, and then the peak is said to be impure

In the figure-3:

• All the three-point spectra (upward slope, peak apex and downward slope) of peak A are matching and hence peak A is pure
• All the three-point spectra (upward slope, peak apex and downward slope) of peak B are not matching and hence peak B is impure

Factors affecting peak purity

Peak purity test may be affected:

• If close eluting peaks are not separated base to base
• Noise or background absorption
• If spectral absorption co-eluting peaks are low of
• If the impurity and main compound have the same or almost the same spectra with similar elution time

Sample Concentration optimization for Peak Purity

• Sample concentration should be adjusted in such a way that absorbance should be below 1000 mAu
• Peak must-have area response value ≥ QL (Quantification limit)

Peak Purity Test Wavelength-Range Setting

Wavelength selection plays an important role in peak purity determination. If the wavelength has not been chosen scientifically then the peak purity result may be incorrect. The following factors must be considered while selecting the wavelength range:

• Buffer concentration: Higher buffer concentration in the mobile phase may affect peak purity. Always put a lower wavelength above the buffer UV cut-off, If possible.
• Operating wavelength: If the operating wavelength is 254 nm and all impurities and the main analyte have maxima ≥ 240 nm then select a wavelength range like 220 nm to 350 nm and not like 190 nm to 650 nm
• Buffer quality: Poor buffer quality may lead to failure of peak purity due to formation of noise. Always use HPLC grade buffer
• Type of buffer: Peak purity also depends upon the type of buffer. If buffer-like ion pairs (such as Hexane sulfonic acid sodium salts, tetrabutylammonium hydroxide etc) are used in that case keep the lower wavelength range limit more than the UV cut-off of the buffer
• Solvent quality: Poor quality of solvent may lead to failure of peak purity. Always use gradient grade HPLC solvent while determining peak purity

Difference between PDA and UV detector

Difference between Peak purity and Chemical purity

3 Widely used peak purity calculation techniques

The following techniques are widely used for peak purity:

1. Spectral normalisation method
2. Spectral similarity method and
3. Ratio gram generation method

Among the above methods, the spectral normalisation method and Spectral similarity method are widely used in industries for peak purity calculation

Spectral normalisation method

This technique involves normalising and comparing spectra taken across different points of the peak. This technique involves the following steps;

1. First spectra are acquired at the upslope, the apex and the downslope and
2. The spectra are normalised and overlaid for graphical presentation

Peak purity suitability criteria

1. If all the above three spectra are similar or match each other, then the peak theoretically has no spectral impurities and the peak is said to be pure.
2. If the above three spectra are not similar or do not match each other, then theoretically the peak contains spectral impurity and the peak is not said to be pure

This technique very good for interactive data evaluation and for automated routine analysis

Figure-3

• In the above HPLC chromatogram, the spectra of all the points on the peak match across all points for peak A and hence peak A is spectrally pure.
• The spectra of all the points on the peak are matching across all points for peak B, and hence, peak B is spectrally impure.

Spectral Similarity

In this technique, all spectra from the concerned peak are compared with one or more selected spectra (reference spectra). Generally, the apex spectrum is taken as the reference spectrum. The degree of match or spectral similarity is plotted over time during elution.

The ideal profile for a pure peak is a flat line, and it is denoted as 1000. It is called a similarity curve.

At the start point and end point of each peak, spectra may vary due to the contribution of background noise take place. Due to this noise interference flat line is not always possible. That is the reason the threshold is kept. Generally, 980 is set as the threshold value for peak purity calculation.

Spectral Similarity peak purity acceptance criteria

The following criteria must be satisfied for peak purity:

1. If the peak purity factor or purity angle is within the threshold value or purity threshold, the peak is classified as pure
2. If the peak purity factor exceeds the threshold value, the peak is classified as impure.

Peak purity terminology

Match factor

The spectral comparison of the two spectra is called the match factor. The following are the relationships between the match factor and the spectra similarity:

• Match factor = 0, indicates no matching between the two spectra
• Match factor = 1000, indicates 100% matching between two spectra (identical spectra)
• Match factor = 990, which indicates two spectra are similar
• Match factor = 900 to 990, which indicates some similarity between the two spectra
• Match factor = < 900, which indicates two spectra are different

Purity factor or Purity level

The purity factor is the mean value of the match factor. It is the measure of the similarity in the shape of the spectra of a given peak.

HPLC Peak Purity by PDA Detector: How Authentic?

The peak purity by the PDA detector is not a foolproof technique for peak purity checking of any peak. It has the following limitations and advantages:

Limitations of Peak Purity

The following are the limitations of the peak purity test:

• The coeluting impurity must have UV absorbance
• The coeluting impurity must have a value more than the quantification level (QL)
• The coeluting impurity must have spectra different from the main analyte
• The coeluting impurity must not be the inorganic compounds
• The coeluting impurity must not be the optical isomer

Advantages/Applications of Peak Purity Test

The following are the advantages/applications of the peak purity test:

• Covers both UV and visible range (200 nm to 900 nm)
• Very helpful during method development and forced degradation studies
• Cost effective technique
• Fast analysis
• Tells about stability indicating nature of the HPLC method

PDA is a basic technique to check HPLC peak homogeneity. It is also recommended that LC-MS be used while performing the peak purity.

Conclusion

HPLC peak purity assessment using a PDA/DAD detector is a fast and effective way to confirm chromatographic separation in pharmaceutical analysis. By understanding spectral comparison principles and the available purity calculation methods, analysts can quickly determine whether a peak represents a single compound or contains co-eluting impurities.

The PDA detector’s ability to capture full UV spectra across the peak makes it a practical and powerful tool during method development, validation, and routine QC. While not as selective as MS, its simplicity, speed, and accessibility make it ideal for everyday purity checks.

With the concepts explained in this article, you can now confidently evaluate peak purity using a PDA detector and apply this knowledge to strengthen the reliability of your HPLC methods.

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FAQs

What is PDA scan?

PDA scan is 190 nm to 900 nm

References

• PDA detector: Waters
• Chromatography online

Abbreviations

• UV: Ultra-violet
• PDA: Photodiode array
• DAD: Diode array detector
• QL: Quantitation limit