HPLC UV - VIS Wavelength Accuracy Check (" Calibration ") Notes

To verify correct detector wavelength accuracy of your HPLC UV / VIS module it is periodically necessary to measure the wavelength accuracy against know standards using an appropriate SOP ("fit for purpose"). This may be required as part of a Performance Verification (PV), Installation Qualification (IQ) or Operational Qualification (OQ). 

Wavelength accuracy may be adversely affected (or change) when an UV/VIS detector is serviced/repaired, moved, suffers a physical shock (bumped), large temperature changes occur, a lamp or other optical component is changed, a flow cell is changed, the optics become dirty or contaminated, or due to normal wear and age. The wavelength accuracy of any applicable detectors (e.g. UV, VIS, UV/VIS, DAD, PDA) should be measured on a regular basis as part of "Good Laboratory Practices" (GLP). Depending on the regulations or guidelines applied, most authorities require accuracy to be within 2 to 3 nm of a certified standard within the range used. In practice, we generally achieve accuracy of equal to or better than 0.5 nm across a range of UV / VIS wavelengths. Following good laboratory practice (GLP) requires that we establish the frequency and conditions which determine when they should be verified. Complete documentation of these wavelength checks which describe their purpose, specificity, application and detailed procedures (SOP) should be reviewed.

We present a few suggestions in how to measure the detector wavelength accuracy of your HPLC UV / VIS module. 

• Built-In Test Methods: Most instrument manufacturers incorporate one or more wavelength accuracy checks directly built into their detectors. This allows quick and accurate measurement of the detector's wavelength accuracy for one or more wavelengths in an automated fashion. Most instruments utilize built-in filters (e.g. holmium oxide) which have been treated with chemicals to provide repeatable wavelength spectra which can be used to determine the accuracy of the detector (and adjust it to within specification in most cases, too). If your instrument has one or more of these built-in test filters, then follow the manufacturer's instructions for using them to measure the wavelength accuracy of your detector. 

• Using a solution of high purity ANTHRACENE: Dissolved in an HPLC grade alcohol (i.e. Methanol ) or Acetonitrile (for low UV checks), anthracene has a lambda max of 251 nm. A solution concentration of ~ 1 ug / mL for HPLC use can be injected using a standardized method (SOP) and the area% evaluated, one-at-a-time, at several different wavelengths (for VWD or single wavelength detectors) as follows: 249, 250, 251, 252, 253 nm. Relative to the baseline, the areas should show a peak at 251 nm. If you have a scanning UV/VIS detector (aka: DAD or PDA), then you can scan all wavelengths around the 251 nm region and plot the results using just one run to obtain the same type of data.

• Using a solution of high purity CAFFEINE in HPLC grade water: Caffeine has two useful lambda maximums that we can use for wavelength accuracy checks in the ultraviolet region, 205 nm and 273 nm. We often prepare a range of solutions from 5 ug / mL to 500 ug / mL for linearity testing of UV/VIS detectors, but any of those same solutions could be used for wavelength accuracy checking (similar method as described above for anthracene).

• One of the most widely used methods requires a solution of HOLMIUM PERCHLORATE  solution (NIST). Available for purchase from many chemical suppliers, this acidic solution provides excellent signals for calibration at well documented transmittance bands (i.e. 241.1, 287.1, 361.5 nm and many others out to ~ 640 nm, depending on the solution it is dissolved in). The detector's flow cell can be filled with the solution and measurements made. The solution is also available coated onto quartz slides and is in fact what is found and used in many detectors today as part of their built-in verification. However, you can still prepare your own test solution.

Notes: A reminder that the solution used to prepare the wavelength check standard(s) in will directly affect the results obtained. If you prepare it in a solution which has strong absorbance at or near the region you test, the results obtained may be inaccurate (e.g. a test std dissolved in MeOH used to measure wavelength accuracy at 205 nm would not be an appropriate choice. A standard dissolved in ethyl acetate would obscure the UV wavelengths below its cutoff of ~ 256 nm). Make sure your SOPs state exactly which solutions are used, how they are prepared and which flow cell are used to make the measurements! Flow cells with different dimensions (i.e. path lengths, volumes) will result in different signal outputs. Different background solutions will also result in different results which can not be directly compared (invalid test). For each test, you must use scientifically appropriate methods and the same conditions to make all measurements.

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.

Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer

One of the many useful features of a UV/VIS scanning diode array detector is that it can be employed in flow injection mode to scan a sample and provide you with some useful data about the absorbance characteristics of the sample (which probably contains a mixture of components). Unlike a spectrophotometer, you only need about 1 ul of sample instead of a 1ml cuvette and only 15-20 seconds of time to gather the data.

Why do this? I use this feature often when I receive a new and unfamiliar sample for method development. I set up the detector to scan and store all wavelengths, in steps of 2nm, from 210nm to 450nm and inject the sample in flow injection mode (that means no-column is present and I easily do this using the By-Pass position on my column selector). In a very short amount of time I can view the resulting spectra of the sample which aids me in selecting the initial discreet wavelengths to monitor. For example: If I notice that the sample shows some absorbance at 410nm using the flow injection run, then notice while developing the analysis method that none of the peaks seen show absorbance near 410nm, then I can assume that I may still have some components retained on the column.

Setup Hints:
(1) For this to work well, you should have a high performance, low volume switching valve or automated column selection system (e.g. The LC Spiderling Column Selection System) installed so you can easily by-pass your column (otherwise, remove your column and place a high pressure, low volume union in its place).
(2) Set the diode array detector to a high sampling rate because the sample is going to fly through the flow cell quickly. Use a sampling rate that is faster than you would use if a column was there to disperse the sample and slow down the peaks.
(3) Choose a wide range of wavelengths to scan and store. If the sample appears colorless to the eye in solution and I am running in a UV transparent solvent such as acetonitrile, then I often use a range of 210 to 450nm.

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).

REFERENCE WAVELENGTHS (as used in HPLC UV/VIS):

One of the most common problems that I see as a consultant in laboratories which use chromatography for sample analysis relates to how to choose appropriate settings for the modern UV/VIS detectors. In addition to selecting scientifically appropriate UV/VIS wavelength(s) and Bandwidth signal values, selecting one optional feature may invalidate an entire HPLC method. This software feature, found in many Multi-Wavelength and Scanning Diode Array UV/VIS detectors (aka: "DAD" or a "PDA") is known as the “ReferenceWavelength” .

• Please do not confuse this specific software feature ("Reference Wavelength") with the initial reference scan ('zero') which the detector takes at the start of the analysis and is subtracted from your desired signal to show only one initial signal plot (and which is used as the initial signal value to compare to the measured signal during the rest of the analysis run. This is usually known as "zeroing" the detector and occurs just once, at the start of each run. When you manually press the 'Auto-zero', you are adjusting the displayed signal plot to a know reference point (often 0.0 volts). This is a one-time zero of the signal and has nothing to do with the special software feature we discuss in this article.

"Reference Wavelength" [Usually written as: Signal Wavelength/Bandwidth: Ref Wavelength/Bandwidth]. Most manufacturers of advanced HPLC UV/VIS (esp. DAD/PDA) detectors provide this extra software feature in their chromatography software, but its use and function are a mystery to most chromatographers. As with all advanced features, proper training is required to understand and use them successfully. Using advanced features without proper training can result in analysis errors, invalid methods and perhaps very expensive product recalls.

Allow me to provide a brief explanation of the “Reference Wavelength” software feature as seen and used with many DAD and/or PDA detectors (e.g. HP/Agilent and Waters brand HPLC systems).

If you are running a gradient analysis, then the change in solvent properties (RI and light absorption/transmission) and temperature over time can cause noticeable baseline drift during the run. This drift up or down relative to the starting baseline reference point is normal, but may cause a number of quantification problems with the analysis reporting software (as flat baselines are more easily and accurately integrated than sloped ones). 

Two scientifically correct methods were developed to deal with this slippery slope of a problem. Each proposed method has some limitations, but if optimized can improve the quality of the resulting baseline (flatter, allowing for better peak integration) and preserve the original acquired signal data for compliance.

(Method # 1) Run the same method again, but this time with no sample (a blank of mobile phase) and subtract the resulting signal to produce a "blank subtracted run". This preserves the original data and removes the observed drift from the resulting signal ('A' - 'B'  = 'C'), but due to the time difference between injections, you are unable to confirm if anything has changed between the time of the first and second injection. It is not perfect.

(Method # 2) Set up the detector to collect a second channel of data (2nd wavelength signal) that is close to the original wavelength selection, BUT far enough away from the original signal such that it will not overlap any of the peak spectra of interest or other compounds in the sample. This is tricky as you want it close enough to show the drift, but far enough away to not show any sample signal. If selected carefully, it can be used as a pseudo blank run for post-run baseline subtraction. You can then subtract the second acquired ‘blank’ signal run from your original signal run and the resulting chromatogram should have a flatter baseline (less drift) for quantification purposes. With this method, two separate signals, 'A' and 'B', are collected at the same time (this is the key). A third, baseline subtracted signal, 'C', can be generated from them. This method preserves the raw data obtained from all three signals (i.e. Original, Secondary, and Subtracted signals). The benefit of this method is that the signals are all acquired using the same time base (unlike Method #1).

Using the concept of Method # 2 described above, many HPLC manufactures added a software feature known as a the ‘Reference Wavelength’ to their systems. This feature allowed a chromatographer to include with each signal choice, 'A', a second wavelength value, 'B', (and bandwidth) as part of the method which would be used to subtract out raw data from the primary wavelength during the analysis. This subtraction occurs in real-time, on your raw data gathered from the detector and the resulting data reported to the user is in fact the result of the subtraction only. The original signal data is destroyed. You will never know what the original data looked like before the reference wavelength was subtracted from it (it has been destroyed). Only the newly manipulated (subtracted) result is provided, 'C'. If any sample peak(s) or impurities appeared in the region where you selected a reference wavelength/bandwidth, then the resulting data would have been subtracted from your actual sample and you would never know it happened or have any record of it! This brings up a serious validation issue as you are modifying the original data with no way of knowing (or documenting) how you have changed it. It is for this reason alone that we teach chromatographers to always turn this feature 'OFF' by default. If they want to make use of the feature, then we suggest that they simultaneously collect data from a second, separate wavelength channel such that the two raw data streams are preserved for validation purposes (Method # 2). IOW: To acquire scientifically useful data, turn 'OFF' the Reference Wavelength software feature and record all of the signal data. The separate signals can be compared, subtracted or manipulated as needed for integration and reporting purposes, but the original signal sample data, 'A', is left unchanged and secure. This allows you to monitor for contamination, impurities, problems or changes during the run. It also allows others to verify your method for accuracy.

Observational Notes:  I am often called in to diagnose what the client's refer to as 'a strange problem' where the area of a known sample peak changes in an unexpected way. That "way" often includes going NEGATIVE, below the baseline. Or even increasing in area, mass or decreasing in mass.The column is clean, pumps work fine, retention times are stable and everything appears to be working fine. *This anomaly is due to the reference wavelength software feature being turned 'ON' and another compound (peak) absorbing in the user selected Reference bandwidth region. Its absorption contributes to the final signal. If the data collected (area) for the 'reference peak' is larger than the sample peak the resulting chromatogram will show a negative peak (this tends to be noticed by most users as it is illogical and indicates a serious problem!), whereas if the reference peak is smaller than the sample peak, the resulting area signal decreases, which may or may not be noticed (incorrectly interpreted as a lower concentration sample). You can see the obvious danger posed by this situation. Companies can be put in a situation where all of their past data is found to be invalid and product recalls may result from this finding. The cause is directly related to a lack of understanding and proper training in the use of the software and/or HPLC system.

 
How to Solve The Problem: The reason we see this feature cause so many problems in laboratories appears to be due to the fact that the Reference Wavelength software feature is being turned 'ON' by default in the software for most DAD/ PDA modules (The real default value for "Reference Wavelength" should always be: 'OFF', not on).  To make matters worse, the default values for the wavelength and bandwidths often supplied by the manufacturers are actually used by most chromatographers (what are the odds that the random values placed in the system are even relevant to your analysis? Why would you use them?). We suggest using a ‘canned’ method template in most laboratories which includes a new default value for this feature... 'OFF' for all analysis methods. Most importantly of all, please obtain formal training in the use of a specialty detector such as a diode-array detector before using one for sample analysis.

Notes

1. The bandwidth chosen for each wavelength is also very important and if chosen poorly, can result in adding noise to your signal, reducing it or even enhancing it. Please refer to this article for more info: http://hplctips.blogspot.com/2011/09/uv-vis-hplc-detector-signal-bandwidth.html 
2. If you are still running HPLC methods with the “Reference Wavelength” turned 'ON' while awaiting approval to turn it 'OFF', then you can ADD additional signals to your method with the same primary settings as before, but with “Reference Wavelength” now set to 'OFF'. Adding the same signal w/o the “Reference Wavelength” will provide you with the original signal data for future comparison to the "collected/modified" signal (allowing you to see if the data was changed). Make sure you configure these extra channels to be saved with the analysis.