"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.
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.
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?
ReplyDeleteOne 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.
DeleteHere 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]