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 noiseLimit of Quantification (LOQ) = 10 x noise

Detection issues in chromatography

Detector response

Detector response

Concentration of the solute

Slope = sensitivity

Limits of Detection & Quantification

LOD = 3 * noise

LOQ = 10 * noise

Cmax

Linear range

Detection issues in chromatography

Derivatisation

For - not detectable solutes- increased sensitivity- selectivity

Solute + Reactant Detectable reaction product

UV-visibleFluorescenceElectrochemical detector

Detection issues in chromatography

Derivatisation

2 possibilities : before or after the separation

+

+

Different solutes = different separations!

Detection issues in chromatography

Derivatisation

Pre-column Post-column

Slow reactions possible Rapid reactions necessary

Stable derivatives necessary Non-stable derivatives possible

Detectable reactant possible Reactant must not be detectable

No additional dispersion Dispersion in the reaction chamber

Other solutes = other separations Separation of the original solutes

No constraints on the mobile phase Mobile phase = reaction bath

Reaction must be quantitative Reaction must be quantitative

Detection issues in chromatography

Derivatisation

Ninhydrin for amino acid and peptide derivatisation

O

O

OH

OH

H

R

NH2

COOH

OH

N

O O

O

+

lmax=570 nme=20000

O

O

OH

OH

NH

HOOC

N+

O

COO-O

+

lmax = 440 nm

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

st

samplestsample A

ACC

Detection issues in chromatography

Quantification

2. Calibration curve

Sample

Cstandard

Area

Asample

Csample

Requires at least 3 standard points

Csample must be between smallest and largest Cstandard

Calibration curve is not necessarily a line

Standard

Detection issues in chromatography

Quantification

3. Internal standard

Sample+

Internal standardInternal 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 additionsSample

Sample+

Increasing quantities

of standard

Cadded standard

Area

Asample

Csample

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 soluteCmax

Linear range

Absorbance of MP

Sample is subjected to light of all wavelengths generated by a broad emission source (D2 + 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)

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