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.

Internal Standard (ISTD) HPLC Calculation Notes:

Why Use an ISTD?

Two main reasons: (1) The addition of an internal standard to all vials containing standards and any unknown samples takes into account any changes caused by the sample preparation process. This is useful when the samples are run through various media or filters as part of a pre-treatment or clean-up initial phase (e.g. clinical or biological samples). (2) Retention time drift over the day can be compensated for through the use of ISTDs.

Key Points To Keep In Mind: 

• The ISTD must be of known purity (by certified method), have a similar response as the sample and not interfere with the analysis.

•  The calculated amount of your unknown samples is directly related to the amount of the ISTD used. The results are calculated based on the ratio of the responses for both peaks (i.e. std and unknown). The amount of ISTD used in all vials must be kept constant.

• Relating peak retention times based on the ISTD (as a ratio) instead of establishing retention time windows makes it much easier to transfer methods to other systems and also account for variation seen. This is because with ISTD and Relative Response Ratios you can define peak retention based on the elution time of the ISTD and not the actual retention times. 

• You must use identical integration parameters to calculate the areas of the standards, samples and/or unknowns. Failure to do this may invalidate the process.

• For Multi-level Calibrations you will calculate an amount and response ratio. You will do this for each calibration level (and each std type if multiple standards are used). Note: Most professional chromatography data systems are designed with special fields for the internal standard data and will perform these calculations for you once you load the data for each level into the calibration table. Response ratios are then used for measurements (Response Ratio = Sample/Std Area / ISTD Area). The accuracy of your calibration curve fit and the overall reproducibility of the entire method used will impact your final results. Poor quality curve fit and/or RSD equals poor accuracy.

• Highest accuracy is achieved using a professionally developed method which first retains (with proper K prime values), then elutes all samples off the column during the run. Column wash and equilibration steps should be separate runs, not part of the analysis method. Poor quality method development is the most common reason why calibration results are poor or RSD is high. 

• Use the same injection volume for all samples (unknowns) and standards (knowns).

• Base the amount of ISTD concentration such that it is between 1/3 and 1/2 of the expected concentration of the sample(s). *The sample's target range is best.

Calculations:

Response Ratio = Sample Response (Area) / ISTD Response (Area)

Response Factor = Amount Ratio / Response Ratio

Summary:

The best way to guarantee success and generate high quality data is to first develop a stable and chromatographically correct HPLC method which resolves apart all of the compounds AND elutes everything off the column during the run. Do not start the calibration or quantification process until the method has been demonstrated to be stable, reliable and working perfectly. Problems seen with calibration methods are often caused by poor quality methods. Invest the time needed to produce an excellent quality method first and then you should have few to no problems later on.

*Related Reading: "External vs. Internal Standard Calibration in HPLC";