Detectors Used in HPLC, desirable features, detector performance criteria, uv-visible detector, photo diode array detector, fluorescence detector, refractive index detector, elsd detector, ms detector

1. DETECTORS OF HPLC
Presented by:
Aditya Sharma
M.S. (Pharm)
Pharmaceutical Analysis
NIPER Guwahati

2. DETECTORS
• Purpose: The purpose of the detector in an HPLC system is to identify the
presence of certain compounds of interest in the eluent from the HPLC column
• The analyte undergoes physico-chemical interaction. The detector provides the
electrical output signal which is proportional to the quantity of analyte within
detector at a given moment. Integrator quantifies the amount of analyte present.
• Two broad classes of Detectors:-
A. Bulk property detectors : Respond to any change in certain physical property
of the HPLC eluent which is common to both analyte and mobile phase and its
magnitude is altered by the presence of analyte. Example: RI, ELSD
B. Solute property detector : They respond to certain unique properties of the
analyte such as fluorescence or electrochemical property

3. Desirable features
• Should be equally sensitive to all eluted peaks
• Should not be affected by changes in temperature
• Should not be affected by changes in mobile phase composition
• Should be able to monitor small amounts of analytes i.e. highly sensitive
• Should not contribute to band broadening, hence the flow cell should be small
• Easy to operate and cheap

4. Detector Performance Criteria
• Detector performance characterized by sensitivity, noise, limit of detection, linear
and dynamic range and detection volume.
• Noise : is a random short-term variation in the output signal. Limits the size of
signal and thus amount of analyte.
• Short term noise: occurs at sub second level, much more rapidly than variations
in response to elution of an analyte peak. Fuzzy baseline.
• Long term noise: comprises random variations in the output signal with a time
scale similar to peaks due to elution of compounds from HPLC column and leads
to confusion as to the identity of the peak.

5. Sources of Noise:
Shot noise: electrical optical devices.
Flicker noise: lamps and lasers
• Pulsation of the pump pistons
• Dirty flow cell
• Detector noise increases with the age of the instrument.
Drift: Drift is very slow variation in the detector output signal, occurring on a
longer time scale of minutes or hours. Makes integration difficult.

6. • Sensitivity: LOD: The signal should be 3 to 5 times of the noise
• Sensitivity: LOQ: The signal should be 10 to 12 times of the noise
• Linearity and dynamic range: Linear range is the range of analyte concentrations
over which the detector output signal varies in linear proportions to the analyte
concentration. The region where a change in analyte concentration produces a
change in output signal is the dynamic range, although without this necessarily
being a linear relationship.
• Band broadening: It is due to two main detector parameters
1. Cell volume: Too small a cell volume impairs the sensitivity (as a certain amount
of product is needed to produce any signal). A standard cell volume is 8 – 10µl.
2. Detection time constant: Time constant can be defined as the minimum time
required by a system to reach 98% of its full scale value.

7. Types of Detectors:
UV / Visible
detector
Photodiode
array detector
(PDA)
Fluorescence
detector
Electro-chemical
detector (ECD)
Refractive index
detector (RID)
Evaporative
light scattering
detector (ELSD)
Mass
spectrometry
(MS)

8. UV / VISIBLE DETECTOR
• They are the work horses of HPLC and constitute
about 70% of all detection system
• Sensitive, wide linear range, relatively unaffected
by temperature fluctuations and is also suitable for
gradient elution and relatively inexpensive.
• It records compounds that absorb UV or visible
light. Absorption occurs above 200 nm if the
molecule has at least:.
1. A double bond adjacent to an atom with lone
pair of electron (X=Y-Z:)
2. Bromine, Iodine or sulphur
3. Ketone group or a nitro group
4. Two conjugated double bonds (X=Y-Z=A)
5. An aromatic ring

9.  Lamps or Light source : Fixed wavelength or variable wavelength
Fixed wavelength :
i. Mercury Lamps : emit at 253.7 nm
ii. Cadmium Lamps: emit at 229 nm
iii. Zinc Lamps: emit at 214 nm
Variable wavelength :
i. Deuterium Lamp: emit continuous UV spectrum up to 340 nm
ii. Tungsten Lamp: emit in the near UV & visible ranges (340 to 800nm)

10. PHOTODIODE ARRAY DETECTOR
• It is UV technique but produces a 3D output i.e. On X axis time, Y-axis absorbance
and Z axis wavelength
Chromatogram: On X axis time and Y-axis absorbance
Spectrum: On X axis wavelength and Y-axis absorbance
• Allows simultaneous collection of chromatograms at different wavelengths during
a single run. Main application is in the field of method development
• Used for peak purity testing.

11. FLUORESCENCE DETECTORS
• When light is absorbed by a molecule and an electron
is promoted to a higher energy state and while coming
back to its ground state it loses energy by emission of
a photon, this process being called fluorescence.
• Design: Light from the lamp (D2 or a xenon) passes
through an excitation filter, which provides essentially
monochromatic light of the desired wavelength for
excitation of sample molecules.
• This light passes through the flow cell causing sample
molecules to fluoresce at a higher wavelength than
that used for excitation.
• Resultant light passes onto the detector for
quantitation of the emission signal.
• As the fluorescent emission is very low,
photomultiplier rather than photodiode is used.

12. ELECTROCHEMICAL DETECTORS
Electrochemical detectors are based on
amperometric measurements. Also called
amperometric detectors.
Principle of operation is the oxidation or reduction of
analyte in a flow -through electrolysis cell with a
constant applied electrical potential. e.g. oxidation of
hydroquinone
• Detectors where only a low percentage of the analyte
is reacted- amperometric.
• All the analyte reacts-coulometric.
• Catecholamines, phenols,aromatic amines are easily
oxidized.
• Quinones and some nitro-aromatics are easily
reduced.

13. • The detector cell in which the electrochemical reaction takes place has three
electrodes namely working, auxiliary and reference.
• Electrolysis of analyte occurs at working electrode and auxiliary electrode
supplies the current.
• Reference electrode measures the solution potential.
• Potentiostat is used to control the voltage at auxiliary electrode in order to
maintain the constant potential difference between the solution and the
working electrode.
• Sensitivity is high. Low detection limits can be achieved.

14. REFRACTIVE INDEX DETECTOR
• The velocity of electromagnetic wave varies as it
passes from one medium to another.
• The ratio of its velocity in vacuum to that in given
medium is known as RI of the medium.
• The RI detector measures the change in RI of the
mobile phase due to the presence of dissolved
analyte.
• RI detector is very useful for analysis of sugars
which have poor UV absorbance or fluorescence
measurements without chemical derivatization.
• RI detector can not be used in case the solute and
mobile phase has the same RI.

15. Disadvantages
Low sensitivity
Factors like
temperature,
pressure, minor pump
pulsations etc. cause
major noise in RI
detectors
Gradient elution
practically impossible
Use premixed mobile
phase in isocratic
mode

16. EVAPORATIVE LIGHT SCATTERING DETECTOR
(ELSD)
• A universal detector. The effluent
from column is nebulized and
evaporated as it passes through
the drift tube. Analyte particles are
detected as they pass through light
scattering cell.
• Non-volatile analytes and volatile
mobile phase.
• Compatibility with gradient elution
possible. Useful for impurity
analyses.

17. Advantages
No ‘solvent front’
peaks to interfere
with early eluting
sample
components
Higher sensitivity
Stable baselines that
are not affected by
changes in column or
laboratory
temperature
Compatibility with
gradient elution for
improved resolution
and
faster separations
Uses a simple 3 step
process that produces
a signal for any non-
volatile sample
component

18. MASS SPECTROMETER (MS) DETECTOR
• LC-MS is a hyphenated technique, combining separation power of HPLC with the
detection power of MS.
• For using MS there is a need for an interface that will eliminate the solvent and
generate gas phase ions, for MS.
• MS detector has three distinct parts:
i. Ion source
ii. Analyzer
iii. Detector
• For all MS techniques, an analyte is first ionized in the ion source since the MS
can only detect charged species. Based on their mass to charge (m/z) ratio the
ions are separated and focused in the mass-analyzer.
• Mass Analyzers: There are many types of mass analyzers in MS. E.g. Triple
Quadrupole