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 COLUMN TEST MIXTURE SOLUTIONS; NP & RP EXAMPLES

When selecting standards for use in testing an HPLC column OR for evaluation of an HPLC system’s performance (e.g. System Suitability, Performance Verification, Retention), in addition to selecting special high purity chemical compounds, consider using the actual sample which is specific to the method or application. Characterize the sample’s retention time (esp. K prime), peak shape and spectra (if applicable) and use the data to measure, compare and detect changes in  performance over time. This can be combined with a suitable test mixture to provide more comprehensive results.

Common HPLC Column Test Mixtures:

Running Sugars on an Amino Column? Use simple and complex sugars as standards. Example: D-Fructose; D-Glucose; Lactose; Maltose; Sucrose.

NP (prepare the test solution in a mixture of Hexane/Ethanol, as appropriate)

Diethyl phthalate; Dimethyl phthalate; Toluene; Benzene.

RP (prepare a fresh test solution in ACN/Water or Methanol/Water, as applicable)

Select 4 or 5 of these compounds for use in a mix. Uracil; Benzene, Acetophenone; Toluene; Naphthalene; N,N-Diethyl-m-toluamide; Phenol;  diethyl phthalate; diamyl phthalate; di-n-hexyl phthalate; dioctyl phthalate.

Include a Void Marker in your Test Solution:

Always measure the actual void volume of your specific HPLC column with a compound which is un-retained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well. Monitor by UV detection. 

 

• You must know what the Method's void time is for every HPLC method that you run BEFORE starting to analyze any samples. This is one of the most fundamental aspects of using HPLC so make sure you understand and can show what this value is.

Reference: “Determination of HPLC Column Dead Volume/ Dead Time (T zero)”

Gradient Mixing Test For Your HPLC Pump (Step Gradient)

The most popular type of gradient pumping module used to perform HPLC analysis utilizes a low pressure mixing valve in their design. These valves are electronically controlled and proportion the amount of mobile phase from one of several solvent channels into a mixer for introduction to the pump head (*the solenoid valves used for this are sometimes called gradient proportioning valves). They provide random access to multiple solvents (e.g. 4) for method development and column flushing. The mobile phase solutions are mixed at low pressure before entering the high pressure side of the pump head (where they undergo compression). This design requires only one high pressure pumping head and can allow for very high mixing accuracy (often 0.1% per channel) of the mobile phase. This allows for the formation of mobile phase gradients over time which greatly aid in resolving samples apart on the column.

The gradient proportioning valves need to be tested along with the other parts of your HPLC system on a regular basis to insure they are operating within the manufacturer's specifications. They should also be tested anytime you suspect a problem may be present. One quick way to check the operation of two of the valves is to use a tracer compound and STEP gradient to monitor their operation. You can set up a method to perform this test as suggested below.

QUICK GRADIENT COMPOSITION TEST:

Bottle A = 100% DH20;
Bottle B = 0.1 % Acetone in DH20 (*Acetone is the tracer compound);

Flow Rate = 1.000 ml/min;
Column = No column. Install a restriction capillary in place of the column to obtain a backpressure of > 60 Bars;

Detection = 265nm (10 nm bandwidth) UV;

STEP Gradient Program:
    0 to 2.00 min, 0 % B
    2.01 min, 20% B
    4.01 min, 40% B
    6.01 min, 60% B
    8.01 min, 80% B
  10.01 min 100 % B
  12.01 min 20% B
  14.00 min 20% B

Note: If the delay volume (dwell volume) of your system is large, then you may want to adjust the time values shown to LARGER values (i.e. 2 minutes delays are used in this example, but 5 or even 10 minute delays between steps may be more appropriate if your system has > 1 ml dwell volume.

Running the above method should result in a signal trace which shows a step-wise rise to 12.00 minutes (as the acetone concentration increases). The edges of the "steps" should be sharp and the risers should also be close to vertical. The final step change which starts at 10.01 minutes shows a linear gradient change back down to the 20% B level. This line should not have any bumps or dips in it and should transition smoothly back down. The height of the baseline at this point should match the height seen between 2.01 and 4.00 minutes (same 20% B). The height of the proportional steps (e.g. 20, 40, 60, 80) should also be the same. You can use your CDS to measure these height values.

Another useful aspect to view is the S/N ratio at each step. Use your CDS to establish noise windows within each range (e.g. 2.50 to 3.5 minutes). This data is useful when comparing the performance of the pump at different intervals.

If you observe deviations in the height of the proportional steps or dips in the lines, these can be caused by leaking or sticking check valves as well as leaking or sticking gradient proportioning valves. *If you have a quaternary pump, be sure and test all four of the valves used (2x per test).

Lastly, the above example is a generalized method and may or may not be applicable to your specific HPLC pump. Be sure and customize a test method which takes into account the pressure ranges, flow rates, delay volume, mixing volume, and number of low pressure channels used in your pump.