Chromatographic detectors for Liquid Chromatography

Detection issues in chromatography Universality ≠ Specificity (discrimination) Sensitivity (baseline noise, LOD, LOQ) Linear range Dispersion (cell volume, response time) Solvent compatibility, possibility of using elution gradients Destructive or not (if recovery or second detection is required)

Detection issues in chromatography Baseline noise Limit of Detection (LOD) = 3 x noise Limit of Quantification (LOQ) = 10 x noise

Detection issues in chromatography Detector response Concentration of the solute Slope = sensitivity Limits of Detection & Quantification LOD = 3 * noise LOQ = 10 * noise C max Linear range

Detection issues in chromatography Derivatisation For - not detectable solutes - increased sensitivity - selectivity Solute + ReactantDetectable reaction product UV-visible Fluorescence Electrochemical detector

Detection issues in chromatography Derivatisation 2 possibilities : before or after the separation + + Different solutes = different separations!

Detection issues in chromatography Derivatisation Pre-columnPost-column Slow reactions possibleRapid reactions necessary Stable derivatives necessaryNon-stable derivatives possible Detectable reactant possibleReactant must not be detectable No additional dispersionDispersion in the reaction chamber Other solutes = other separationsSeparation of the original solutes No constraints on the mobile phaseMobile phase = reaction bath Reaction must be quantitative

Detection issues in chromatography Derivatisation Ninhydrin for amino acid and peptide derivatisation UV-visible detection

Detection issues in chromatography Derivatisation Fluorescamine for amino acid and peptide derivatisation Fluorescent detection

Detection issues in chromatography Quantification 1. External standard Standard solution contains the analyte to be quantified Standard analyte should be at similar concentration as unknown The standard and sample matrix should be as similar as possible All analysis conditions must be identical for sample and standard Sample Standard

Detection issues in chromatography Quantification 2. Calibration curve Sample C standard Area A sample C sample Requires at least 3 standard points C sample must be between smallest and largest C standard Calibration curve is not necessarily a line Standard

Detection issues in chromatography Quantification 3. Internal standard Sample + Internal standard Internal standard is a known substance added to sample and standard solutions at constant concentration Internal standard should elute close to the target species but be well resolved Internal standard must be stable under analysis conditions Internal standard accounts for variations due to injection volume (GC), detector variability, slow column change Standard + Internal standard

Detection issues in chromatography Quantification 4. Titrated additions Sample + Increasing quantities of standard C added standard Area A sample C sample

Fixed wavelength UV-detector wavelength depends on the type of lamp wavelength ranges from 210 nm to 280 nm least expensive detector high intensity = high intrinsic sensitivity Pre-requisite Sample must exhibit absorption in UV-visible range Solvent must not absorb significantly at the measured wavelength

Fixed wavelength UV-detector A = A MP + A solute Problem if A MP is elevated = out of linear range of Beer-Lambert law A = ε l C Detector response Concentration of the solute C max Linear range Absorbance of MP

Sample is subjected to light of all wavelengths generated by a broad emission source (D 2 + W) Dispersed light from the grating is allowed to fall on to a diode array Array may contain many hundreds of diodes For any time of the analysis, a total UV-visible spectrum can be obtained For any wavelength recorded, a total chromatogram can be visualised Diode-array detector (DAD)

Not very useful for qualitative analysis as spectra tend to be broad bands with little structure Quantitative analysis is the major use Always attempt to work at the wavelength of the maximum absorbance = point of maximum response = better sensitivity and lower detection limits

one of the most sensitive LC detectors often used for trace analysis response is only linear over a relatively limited concentration range (three orders of magnitude) the majority of substances do not naturally fluoresce fluorescent derivatization number of regents have been developed specifically for this purpose relatively inexpensive Fluorescence detector Light from a fixed wavelength UV lamp passes through a cell, through which the column eluent flows and acts as the excitation source. Any fluorescent light emitted is sensed by a photo electric cell positioned normal to the direction of exciting UV light.

Evaporative Light Scattering Detector A spray atomizes the column eluent into small droplets The droplets are allowed to evaporate, leaving the solutes as fine particulate matter suspended in the atomizing gas The suspended particles pass through a light beam and the scattered light is measured at 45° to the incident light beam Responds to all solutes that are not volatile = Nearly universal Response is proportional to the mass of solute present Magnitude of response does vary widely between different substances

Electrochemical Detector Based on a redox reaction (oxidation or reduction) A known potential is applied accross a set of electrodes Typically limited to working with a specific class of materials per analysis Very specific