HPLC detectors Pharmawiki 122022 Desirable features of HPLC

HPLC detectors Pharmawiki 1/2/2022

Desirable features of HPLC Detectors – (1) it should response to all compounds in the mixture ( a general detector) or it should response with known sensitivity (a specific detector). – (2) It should not response to mobile phase. – (3) It should give linear response to solute concentration. – (4) It should be unaffected by variation in temperature and flow rate. – (5) It should not contribute to zone spreading. Pharmawiki 1/2/2022

Pharmawiki 1/2/2022

most common HPLC detectors: – – – Refractive index UV/Vis Fixed wavelength (no longer used) Variable wavelength (we have one) Diode array (we have one) Fluorescence (Weilin Huang has one) less common, but important – Conductivity (we used in IC) – Mass-spectrometric (LC/MS) – Evaporative light scattering Pharmawiki 1/2/2022

Good detectors… Regardless of the principle of operation, an ideal LC detector should have the following properties: – Low drift and noise level (particularly crucial in trace analysis). – High sensitivity. – Fast response. – Wide linear dynamic range (this simplifies quantitation). – Low dead volume (minimal peak broadening). – Cell design which eliminates remixing of the separated bands. – Insensitivity to changes in type of solvent, flow rate, and temperature. – Operational simplicity and reliability. – It should be tuneable so that detection can be optimized for different compounds. – It should be non-destructive. Pharmawiki 1/2/2022

Variable-wavelength UV detectors – Detectors which allow the selection of the operating wavelength called variable wavelength detectors and they are particularly useful in three cases: – offer best sensitivity for any absorptive component by selecting an appropriate wavelength; – individual sample components have high absorptivity at different wavelengths and thus, operation at a single wavelength would reduce the system's sensitivity; – Depending on the sophistication of the detector, wavelength change is done manually or programmed on a time basis into the memory of the system. Pharmawiki 1/2/2022

Pharmawiki 1/2/2022

Diode-array detectors – variable wavelength UV detectors can perform spectroscopic scanning. Helps in identification beyond simple identification by retention time. – can use best wavelength(s) for actual analysis. – The second major advantage is related to the problem of peak purity. Often, the peak shape in itself does not reveal that it actually corresponds to two (or even more) components. Absorbance rationing at several wavelengths can help decide whether the peak represents a single compound or, is in fact, a composite peak. Pharmawiki 1/2/2022

Pharmawiki 1/2/2022

Multi-Angle Light Scattering Detector – For the SEC analysis, MW of analyte is estimated from the calibration curve drown using a set of known standards. – However, by using a MALS, MW can be determined directly without the need of calibration curve. – Also MALS can provide an absolute MW of the analyte with very low detection limit. Pharmawiki 1/2/2022

Mass Spectrometer – The analytes are detected based on their MW. – The obtained information is especially useful for compound structure identification. – However, its use is not limited to structure identification and can be used to quantify very low detection limit of elemental and molecular components. Pharmawiki 1/2/2022