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.

HPLC Detector Optical SLIT WIDTH Selection

A few notes on HPLC Optical Slit Width selection:

   Notes: 

1. The chosen slit width setting determines the amount of light which is directed to the detector.
2. For most HPLC methods, a slit width value of 4 nm is suggested. 
3. Bandwidth should be set at least as wide as the optical slit width.

Characteristics of Narrow Optical Slit Widths:

• Less light falls on the detector
• Less signal intensity
• Increased baseline noise
• S/N ratio decreases
• Spectral resolution improves which allows for more accurate spectral identification.

Characteristics of Wide Optical Slit Widths:

• More light falls on the detector
• Greater signal intensity
• Decreased baseline noise 
• S/N ratio improves
• Spectral resolution decreases and detail is lost. Less accurate spectral identification and an increase in errors for spectral library matching.

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.

UV / VIS, VWD, DAD, PDA HPLC DETECTOR SIGNAL BANDWIDTH (bw) SELECTION

Modern chromatography UV/VIS detectors offer the operator a choice of one to several hundred different signal wavelength choices (as is the case for Diode Array Detectors). Besides being able to specify a single wavelength, you can often choose a signal BANDWIDTH (bw) to associate with each wavelength [e.g. for a 280 nm signal with 10 nm bandwidth. This is often written as: 280 (10) or [280:10]. In many detectors, Signal Bandwidth is a variable, not fixed and represents the total number of nanometers across the specified signal value chosen. For example: If you select a signal wavelength of 280 nm and choose a bandwidth value of 10 nm, then you are actually gathering all signal data between 275 nm and 285 nm (5 nm to the left of the apex and 5 nm to the right for a total of 10 nm). Using a narrow bandwidth has the advantage of increasing the signal selectivity of the detector as you are only collecting data within a tight window. If you were to increase the bandwidth to 60 nm in the same example you would now be collecting data between 250 nm and 310 nm. The additional data collected over this wider range may reduce the total noise (by averaging it over a wide range), improve the S/N ratio (which may increase sensitivity), but it also reduces the selectivity. Large bandwidths also increase the chance you may include peak signal data from other co-eluting components into your signal data. You must select a bandwidth range for each signal wavelength which is located 'safely' away from any other potentially interfering peak. As with many things in life, balance is important. In this case, bandwidth choice is the balance between selectivity and sensitivity.

• When developing new methods we recommend that you choose an initial bandwidth value of 10 nm for each signal. This provides a nice balance between selectivity and sensitivity. It is also a common bandwidth value used on many older UV/VIS detectors which have a fixed signal bandwidth (such as many single or variable wavelength detectors).

• If you have determined the exact signal maximum for your sample and you would like to gain additional sensitivity for your sample (and thus decrease selectivity), re-run the analysis using several different, but increasing signal bandwidth values (e.g. 10, 20, 30, 50 and 100 nm). Choose bw values that are safely within the range of the detector, within the limits of the mobile phase's absorption region and also away from any potential co-eluting peaks. *To confirm which value is best, be sure and calculate the actual measured signal to noise ratio of the peak of interest after each analysis. This is a critical step! Do not be fooled by increases in the peak height or area alone as these changes are not always synonymous with better signal to noise ratios. Only by measuring the actual baseline noise level for each run and comparing it with the actual peak signal obtained will you be able to determine if increasing the bandwidth has provided you with better noise reduction and signal strength.

• To increase spectral signal selectivity choose a bw value that is very narrow. A value such as 2 or 4 nm would allow the detector to collect only signal data that is at or near the apex of your selected wavelength. This can be very useful when trying to discriminate your signal from nearby signal peaks, especially at low wavelengths such as 210 nm.

• When reporting your method conditions always include the wavelength AND bandwidth used for each signal. In order to accurately reproduce your method, this information is needed. *The flow cell dimensions, wavelength and bandwidth should always be included in your method.