PEAK PURITY Determination by HPLC Diode Array Chromatography Software (UV/VIS): Limitations and Uses

"Peak Purity" software determination by HPLC UV/VIS detection 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 a fully resolved 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 very limited second dimension of data (retention time is the first dimension), but a scanning, multi-wavelength UV/VIS 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 recorded, allowing for a 3D plot of the spectra to be recorded much like a “fingerprint”. "Pure" compounds which do 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. Impure peaks may also NOT show any dissimilar spectra at all (because some compounds may not be detected). When a properly developed HPLC analysis method 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 detection of a sample's spectral profile to detect the presence of an "impurity" (that may have co-eluted with the sample). This additional dimension of analysis (full Spectra) is required to improve the confidence level that a peak may in fact be correctly identified (qualitatively) and does not contain any co-eluting compounds. IOW: "Peak Purity" does not actually test for purity.

Diode-Array 'Software' 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). In other words, it is impure by UV/VIS analysis. Note: It does not indicate that the compound is impure, but rather 'the peak' being measured is. As you can see, the concept makes sense, but the how it is used in many laboratories is flawed leading to invalid reports and data.

• “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 an estimated measure of PEAK 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 for this.

Points to consider when using "Peak Purity" software:

• The absence of any spectral differences across the sample peak are not an indication of actual purity;
• Compounds similar to your sample may have similar absorbance profiles (fooling the system);
• 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. Different people often get different results for the same sample. Inputting poor quality settings or using a poor quality method often leads to misleading purity results. This is an advanced software feature requiring many years of training to use. Again, it does NOT test for purity.
  
• 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(s) which elute at or near the column void volume (Low K prime values may demonstrate that good chromatography fundamentals were ignored. Poor quality methods fail validation). Poor quality HPLC method and poorly selected DAD "Purity" settings result in invalid results (audits, recalls etc may result from reliance on a subjective "software" feature).

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. You will not know what percentage of impurity level is (since you do not know what it is).

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. "Validation" does not in fact insure that the method follows good chromatography fundamantals). The correct detector sample rate, threshold, slope, signal wavelength and bandwidths need to have been properly selected and used (Reference Wavelength always OFF). The peaks shown in your chromatogram should have excellent symmetry with good on-column retention (K-prime, as applicable to mode), baseline separation (> 2.0 for non-SEC modes) and very low baseline noise levels. The two Peak Purity 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 (never use the instrument default settings for reference points!). 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 baseline or 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.

SUMMARY: 

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 regarding possible peak impurity levels. 

• Do NOT use the system default settings / values for 'Peak Purity' ! They are just place holders for actual values (which you must calculate and fill in the correct values for your method).
  

 

* Due to a general lack of formal training, I often see this software feature being used incorrectly by most chromatographers. This is 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 this advanced software 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. Years of 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. 

Due to very complex software setup needed for "Peak Purity" determination by UV/VIS spectra, the requirement for a high quality HPLC method and a high quality data-set,it is our opinion that few should ever use it. 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. Most of the methods that we professionally review where "Peak Purity" data have been used as part of the method have been found to be based on invalid methods, resulting in any "purity statements" issued as unscientific and invalid. Please proceed cautiously and request professional review of any methods which employ it BEFORE committing to relying on it.

©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.

Proper Wavelength Selection for HPLC Method Development (or Purity Determination)

Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires both knowledge and careful attention. Here is the basic procedure to use:

Step 1. Create the Method. To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains and resolves the compound(s) of interest on the column (goal is a K prime of >2.0, less than 10.0). Be sure and utilize a scanning diode array detector in full scan mode (often referred to as a photo-diode array detector, PDA or DAD) to scan all relevant wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.

Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the spectral data to determine which prominent peak wavelengths have the maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual wavelengths for integration and purity determination. By sure and double check that any detector options which use a “reference wavelength" are turned ‘OFF’ when running these methods (more info on “reference wavelengths” can be found on this blog in another post). With the wavelength selected, chose an appropriate bandwidth for use (narrow).

Step 3. Edit the HPLC method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the full scanning mode of the detector so you will know about any other components which absorb at wavelengths far away from and/or near the peak wavelengths. These compounds can add or subtract signal from the main peak making it appear to be more or less concentrated (or more or less pure) than it actually is. If you only monitored the sample with a single wavelength detector, then you would miss this vital information and make errors in your purity or concentration determinations.

Conclusion. (1) Using a multi-wavelength, scanning HPLC detector such as a DAD is one of the most important tools you can use to create accurate and reliable chromatography methods. Always use a scanning DAD for method development to prevent errors. (2) Learning how to correctly use and set up the detector's settings, parameters, special features and options may prevent false or misleading results. Only after you have developed a reliable and repeatable method with good sample retention and peak shape can you begin to report accurate integration and concentration values (and/or make UV/VIS "purity" determinations).