Technical data

Agilent 1290 Infinity LC System Manual and Quick Reference 61

Optimization of the Agilent 1290 Infinity LC System

How to Achieve Higher Sensitivity

For example, a signal at wavelength 250 nm with a bandwidth of 16 nm will be

an average of the absorbance data from 242 nm to 258 nm. Additionally, a

reference wavelength and reference bandwidth can be defined for each signal.

The average absorbance from the reference bandwidth centered on the

reference wavelength will be subtracted from its equivalent value at the signal

wavelength to produce the output chromatogram.

The signal wavelength and bandwidth can be chosen so that they are

optimized for:

• Broad band universal detection

• Narrow band selective detection

• Sensitivity for a specific analyte.

Broad band or universal detection works by having a wide bandwidth to

detect any species with absorbance in that range. For example, to detect all

absorbing molecules between 200 nm and 300 nm set a signal at 250 nm with a

bandwidth of 100 nm. The disadvantage is that sensitivity will not be optimal

for any one of those molecules. Narrow band or selective detection is used

most often. The UV spectrum for a particular molecule is examined and an

appropriate absorbance maximum is selected. If possible, the range where

solvents absorb strongly should be avoided (below 220 nm for methanol, below

210 nm for acetonitrile). For example, in Figure 23 on page 62, anisic acid has

a suitable absorbance maximum at 252 nm. A narrow bandwidth of 4 nm to

12 nm generally gives good sensitivity and is specific for absorbance in a

narrow range.

The narrow band can be optimized for sensitivity for a specific molecule. As

the bandwidth is increased the signal is reduced but so is the noise and there

will be an optimum for best S/N. As an approximate guide, this optimum is

often close to the natural bandwidth at half-height of the absorption band in

the UV spectrum. In the anisic acid example this is 30 nm.

The analytical wavelength is usually set at a wavelength maximum to increase

sensitivity to that molecule. The detector is linear up to 2 AU and beyond for

many applications. This offers a wide linear range for concentration. For high

concentration analysis the concentration linear range can be extended by

setting the wavelength to one with a lower absorbance such as a wavelength

minimum or by taking a wider bandwidth which usually includes lower

absorbance values. The use of wavelength maxima and minima for

quantitation dates back to conventional UV detectors which because of

mechanical tolerances in moving gratings needed to avoid steeply sloping

parts of the spectrum. Diode-array based detectors do not have this limitation