Translator for HPLC HINTS and TIPS for Chromatographers

Friday, September 30, 2011


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 often can 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]. Signal Bandwidth is the total number of nanometers across the specific signal value chosen. For example: If you selected a signal wavelength at 280 nm and chose a bandwidth value of 10 nm, then you are actually gathering all signal data from 275 nm to 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 starting from 250 nm through 310 nm. The additional data collected over this wide range can reduce the total noise (by averaging it over a wide range), improve the S/N ratio (increases 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 which is always 'safe' from any other potential peak. As with many things in life balance is important. In this case, 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 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. *It should always be included in your method.

Wednesday, September 14, 2011

How Do C18 HPLC Phases Differ ?

Reversed phase HPLC columns which utilize the octadecyl functional group often differ in many ways. Besides the particle size, shape of the stationary phase (irregular or spherical), the coating chemistry and end-capping used. Two other  very important ways that columns can differ from one another are in their available surface area and the extent to which those surfaces are covered with the phase coating (i.e. covalently bonded, non-covalently coated and the total carbon %). When comparing columns for use in validated methods, be sure and consider these factors to minimize the number of changes to your method. Always test several columns of the same type to determine the batch to batch reproducibility and variation. Some manufacturers have mastered the art of preparing and packing columns which achieve high batch to batch reproducibility. After all, what good is a column in your method if the results are not reproducible when you purchase another column some time later?