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
Linear range
Detector response
Slope = sensitivity
LOQ = 10 * noise
LOD = 3 * noise
Cmax
Limits of
Detection & Quantification
Concentration of the solute
Detection issues in chromatography
Derivatisation
For
Solute
- not detectable solutes
- increased sensitivity
- selectivity
+
Reactant
Detectable 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-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
O
H
OH
+
OH
R
N
COOH
NH2
O
OH
O
lmax=570 nm
e=20000
O
O
OH
+
OH
O
HN
HOOC
+
N
O
UV-visible detection
COO-
lmax = 440 nm
Detection issues in chromatography
Derivatisation
Fluorescamine for amino acid and peptide derivatisation
Fluorescent detection
Detection issues in chromatography
Quantification
1. External standard
Sample
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
Standard
All analysis conditions must be
identical for sample and standard
Csample  Cst
Asample
Ast
Detection issues in chromatography
Quantification
2. Calibration curve
Sample
Area
Asample
Csample
Standard
Cstandard
Requires at least 3 standard points
Csample must be between smallest and
largest Cstandard
Calibration curve is not necessarily a line
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
Standard
+
Internal standard
Internal standard accounts for variations
due to injection volume (GC), detector
variability, slow column change
Detection issues in chromatography
Quantification
4. Titrated additions
Sample
Area
Asample
Csample
Sample
+
Increasing
quantities
of standard
Cadded standard
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=εlC
Detector response
Linear range
Absorbance of MP
Cmax
Concentration of the solute
Diode-array detector (DAD)
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)
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
Fluorescence detector
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
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.
number of regents have been
developed specifically for this purpose
relatively inexpensive
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