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Saturday, December 31, 2016

Peak Purity Determination by HPLC Diode Array Chromatography Software: Limitations and Uses



"Peak Purity" Determination by HPLC is one of the most abused and easily misunderstood features found in advanced liquid chromatography systems (e.g. HPLC, UHPLC and CE).

For HPLC, one or more inline detectors can be used which provide additional data about the peak’s physical or chemical properties. The data obtained can be compared to that of a pure standard, or known impurity. For compounds which absorb light in the region of most UV/VIS detectors (~ 200 to 900 nm), a single wavelength detector (e.g. UV/VIS) provides a second dimension of data, but a scanning, multi-wavelength detector can add a second and third dimension of data to the retention time. Scanning detectors, commonly known as Diode-Array Detectors (aka: DAD or PDA) are commonly used in HPLC and CE analysis (they are required for routine method development). A scanning DAD can provide detailed sample UV/VIS spectra across a range of wavelengths for each peak, at any retention time seen, allowing for a 3D plot of the spectra to be recorded much like a “fingerprint”. Pure compounds which absorb light across a pre-defined wavelength range should show identical spectral profiles (“slices”) across the upslope, apex and down slope of the resolved peak. "Impure" peaks may show dissimilar spectra across the width of the peak revealing the presence of a co-eluting peak (or impurity).  When a properly developed HPLC separation method or analysis is used to evaluate the purity of a sample, the single dimension of “retention time” is evaluated with additional dimensions of analysis such as the UV/VIS peak spectra. Peak Purity relies on the sample's spectral profile to detect the presence of an impurity that has co-eluted with the sample. This additional dimension of analysis is required to improve the confidence level that a peak is in fact correctly identified and does not contain any co-eluting compounds.



Diode-Array based Peak purity determination by HPLC is a qualitative assessment of the impurity profile of the sample. It is designed to reveal impurities, not prove peak purity. BTW: We really should rename it “Peak Spectral Impurity Assessment" because that is in fact what we are measuring. The algorithm used for Peak Purity determination is designed to confirm the presence of one or more impurities by comparing spectral data slices (multiple slices taken at the apex and both the upslope and down slope sections of the peak).  A mismatch would indicate the peak has not been fully resolved (one or more co-eluting peaks are present). It is impure by UV/VIS analysis. Note: It does not indicate that the compound is impure, but rather 'the peak' being measured is.

“Peak Purity” does not in fact indicate the actual purity of the compound, but instead indicates when a peak may be found to contain impurities. It is a measure of Impurity.



In simple terms, if the spectral slices obtained from one peak are not identical, than the peak may contain one or more impurities. Co-elution is the most likely reason.

Points to consider when using "Peak Purity" software:


  • The absence of any spectral differences across the peak is not an indication of actual purity;
  • Compounds similar to your sample may have similar absorbance profiles;
  • The relative concentration of actual impurities may not be high enough to detect;
  • The compounds / impurities may not absorb light at the wavelengths scanned;
  • The HPLC method used, the software settings and the parameters that you chose in the ‘Peak Purity’ software menu have a huge effect on the results obtained. Inputting poor quality settings or using a poor quality method often leads to misleading purity results.
  • The peak of interest must be retained on the column (K prime > 2) and resolved apart from any observed peaks. Don't use peak purity to analyze peak which elute at or near the column void volume (demonstrates lack of a method and specificity, fails validation).


We prefer to think of HPLC Peak Purity Assessment as a null test. If the recorded peak spectral data slices are different, than you probably have co-elution and/or impurities present (so try and develop a better method to resolve the peaks apart). If no differences in the spectra are seen (they are similar), then the peak may be pure or may contain compounds with similar spectra as are commonly seen with related reaction synthesis products or compounds. So only when you detect differences in the acquired spectra can you be confident that there is a qualitative difference or impurity present.

When configuring the Peak Purity parameters for your sample, you must start with a very high quality HPLC method (A "validated method" is not necessarily a high quality method). The correct detector sample rate, signal wavelength and bandwidths need to have been selected and used (Reference Wavelength OFF). The peaks shown in your chromatogram should have excellent symmetry with good on-column retention (K-prime), baseline separation (> 2.0) and very low baseline noise levels. The two spectral reference points should be manually selected and placed at times before and after the peak of interest in clear baseline areas where no other peaks or spectra are seen. Select at least 7 spectra from the sample peak for comparison (more detail can be provided with more spectra, but be careful not to select spectra near the noise limits). If your method and chromatogram are not of the highest quality, then please do not use the automated peak purity analysis feature, instead spend time improving your method.



The HPLC UV/VIS Peak Purity Analysis (“Peak Spectral Purity”) feature is very complex and has many software settings which must be set up correctly to obtain any scientifically useful data. * In many major laboratories, due to a lack of training, I see it being used incorrectly by most chromatographers on a regular basis. Worth repeating... the HPLC method used to obtain the original data must be of the highest quality and the training of the operator must also be at the highest level. To use the feature successfully, an advanced understanding of the fundamentals of chromatography are required as are a detailed understanding of all of the peak purity software features (how to set the correct threshold, obtain reference baselines, Set sampling rate, noise levels, signal extraction, normalization settings…). Routine HPLC training classes do not cover these types of tasks. Specialized training and practical experience are required to use these tools. Never use the “automated” versions or the manufacturer’s default values to find “Peak Purity”. The only correct way to use these features is to manually tune the method and settings to your specific sample. Failure to customize the method and settings used may result in invalid data and incorrect purity determinations. 

In general, the recommendation for most chromatographers is to not use this feature unless first having demonstrated the required skills and advanced understanding of the fundamentals of chromatography.

©Copyright, March 1, 1996 by William Letter of Chiralizer Services (Plainsboro, NJ) from a portion of material presented in an HPLC Diode Array Method Development Class.

2 comments:

  1. We are getting different purity values for the same sample using the same method. Our retention times also change for each run. 12.1 minutes, then 12.9 minutes and later each time. The earlest one had the highest purity. What are possible reasons for this?

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    1. One of the most fundamental guidelines of chromatography is to have reproducible retention times. You indicate that your sample elutes at different retention times during the method. This indicates that your method is not reproducible. You should not use a special feature or tool such as 'Peak Purity' unless you have excellent, reproducible results. This should be demonstrated statistically by making several runs and calculating the results. Your results are only as good as the method you use. You must first improve the method.

      Here is a link to an article, "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it" which may help you troubleshoot the reason for the variation in retention time [http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html]

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