Noise, HPLC detector response

Direct analysis with the on-line ORD detector linked to the HPLC was hampered by peak broadening, which led to incomplete resolution of the two peptides. Diffusion in the large flow cell may be a major factor in this difficulty. Because detector response can be either positive or negative, it is important to have an excellent signal-to-noise ratio and baseline HPLC resolution of the peptide isomers to be able to quantitate the degree of racemization by this technique. While the on-line ORD detector is promising, further improvements in resolution as well as sensitivity are required for it to be useful for routine peptide analysis. 

An ideal on-line detector for HPLC or CE has versatility, high sensitivity, continuous monitoring of the column effluents, low noise level, wide linearity of response, stable baseline, insensitivity to flow rate and temperature changes, and response to all types of compounds. It should be rugged and not too expensive. The detector should be able to measure accurately a small peak volume without increasing its volume appreciably. Of these parameters, the terms noise, sensitivity, and linearity are typically used in describing detector performance. 

One of the major advantages of SFC over HPLC (high-performance liquid chromatography) is its compatibility with the flame ionization detector (FID), a universal and sensitive detector for carbon compounds. Unfortunately, most modifiers used in SFC are incompatible with FID. Therefore, a search for polar modifiers that have less response to FID led to the use of water, formic acid, and formamide. These modifiers produce acceptably low background noise and enable the use of FID. Because both water and formic acid have poor solubilities in carbon dioxide, they have been used as modifiers at very low concentrations. However, the modifier effect is significant even at this low level. For example, when water or formic acid was used as modifiers, the resolution of free fatty acids was significantly improved. 

Increasing the electrode length may, however, result in a much poorer response, as the signal to noise ratio (S N) is reduced. Recalling that the flow-through detector is on line to a HPLC column, the concentration time profile may be considered as a Gaussian distribution. The chromatographic plate number may be used to characterize the column ... 

---