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.

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 and shape of the stationary phase (irregular or spherical), other parameters must be considered including: Porosity (fully porous or superficially porous) the coating chemistry and degree of 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 or non-covalently coated onto the support, plus 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 exact 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 specific column in your method if the results are not reproducible ?

Pressure Drop Across an HPLC / UHPLC Column

Many of you prefer tables of data over equations that you must work out. So, instead of providing you with another equation, I have done some basic measurements for you to provide a general overview of how particle size (porous) effects System backpressure.

For simplicity, let us start with a few parameters. Pore Volume = 0.70; Linear Velocity = 1.44 mm/sec; Solvent Viscosity = 0.89 cP at 25C (Water). 

Pore Volume and Flow Resistivity will vary by column type. Obviously the back pressure will be higher with more viscous solvents (e.g. EtOH is 1.20 cP) and lower with less viscous solvents (e.g. ACN is 0.34cP). A Table of HPLC Solvent Viscosity values can be found here [ http://www.hplctools.com/lcsolvent.htm ]. Linear flow rates have been used for all column I.D.'s to better illustrate the relationship between column dimensions and flow rate. If you double the flow rate, then the pressure will approximately double as well. 

Note that when run at traditional linear velocities, most 2.5u particles are within the maximum pressure limits of most HPLC systems (under 400 bars). Only the newer sub 2.0 micron particles used in long columns exceed the 400 bar limit. The higher maximum pressure limits of many UHPLC systems allow the use of higher flow rates with these particles. Naturally, you should optimize both column efficiency and system dwell volume when developing any UHPLC method. Failure to optimize the dwell volume (and minimize all volumes) may result in very poor chromatography separations. Meeting any/all backpressure requirements to run a method does not translate to success in sample analysis. Successful ultra-fast separations require ultra-low system dwell volumes, higher sampling rates and usually smaller flow cell volumes.

HPLC Column I.D. (mm)	Particle Size (u)	Column Length (mm)	Flow Rate (mL/min)	Observed System Back Pressure (Bars)
4.6	5	250	1.000	89
4.6	5	150	1.000	54
4.6	5	100	1.000	36
4.6	5	50	1.000	18
4.6	3.5	250	1.000	182
4.6	3.5	150	1.000	109
4.6	3.5	100	1.000	73
4.6	3.5	50	1.000	36
4.6	2.5	250	1.000	357
4.6	2.5	150	1.000	214
4.6	2.5	100	1.000	143
4.6	2.5	50	1.000	71
4.6	1.9	250	1.000	618
4.6	1.9	150	1.000	371
4.6	1.9	100	1.000	247
4.6	1.9	50	1.000	124
3.0	5	250	0.430	90
3.0	5	150	0.430	54
3.0	5	100	0.430	36
3.0	5	50	0.430	18
3.0	3.5	250	0.430	184
3.0	3.5	150	0.430	110
3.0	3.5	100	0.430	74
3.0	3.5	50	0.430	37
3.0	2.5	250	0.430	361
3.0	2.5	150	0.430	217
3.0	2.5	100	0.430	144
3.0	2.5	50	0.430	72
3.0	1.9	250	0.430	625
3.0	1.9	150	0.430	375
3.0	1.9	100	0.430	250
3.0	1.9	50	0.430	125
2.1	5	250	0.210	90
2.1	5	150	0.210	54
2.1	5	100	0.210	36
2.1	5	50	0.210	18
2.1	3.5	250	0.210	184
2.1	3.5	150	0.210	110
2.1	3.5	100	0.210	73
2.1	3.5	50	0.210	37
2.1	2.5	250	0.210	360
2.1	2.5	150	0.210	216
2.1	2.5	100	0.210	144
2.1	2.5	50	0.210	72
2.1	1.9	250	0.210	623
2.1	1.9	150	0.210	374
2.1	1.9	100	0.210	249
2.1	1.9	50	0.210	125

* The results obtained in this table from are from one of our HPLC systems and reflects the total system backpressure (what the pressure gauge reads), with the column inline. Your results may vary due to differences in HPLC system used, flow path, tubing ID, column choice and mobile phase selected.