Introduction:
The most powerful
technique to determine quantitatively and separate the mixture of composition
in today’s modern chemistry is Chromatography especially High-Performance Liquid
Chromatography or High-Pressure Liquid Chromatography. HPLC works on the
principle of Affinity chromatography. The solution of the sample is injected
into a column of a porous material (stationary phase) and a liquid (mobile
phase) is pumped at high pressure through the column. The mixture on travelling
through the stationary phase splits into its constituents and the component
with high affinity for stationary phase travels late whereas one with less
affinity elutes fast. This is also based partition coefficient of the material.
The actual separation of
each component in the sample is carried inside a column; however, this
separation needs to be “collected” for us to be able to see it. The detectors
are used for this purpose. The separated components are monitored and expressed
electronically. There is no universal detector that can monitor all compounds
and there are many detectors used for LC analysis.
What is Detector?
A detector is a major
component in HPLC, which is placed at the end of the system. Its work is to
analyze the solution which is eluting from the column. The concentration of
individual component of the analyte is proportional to the electronic signal
coming out of the component of the mixture.
There are many types of
HPLC detectors are used to quantify the analyte. Some commonly used detectors
are listed our below.
1. Refractive
Index Detector
2. Absorption
Detector (UV/Vis & PDA)
3. Fluorescence
Detector
4. Conductivity
Detector
5. Electrochemical
Detector
Refractive Index Detector:
The RI detector is one of
the few universal detectors available in LC.
They are also one of the
bulk property detectors and are based on the change of the refractive index of
the eluent from the column with respect to pure mobile phase.
Principle: The RI
detectors measure a bulk property of the mobile phase leaving the column: its
ability to refract to bend light (i.e., its refractive index). This property
changes as the composition of the mobile phase changes, such as when solutes
from the column. By detecting this change, the presence of solutes can be
detected.
One of simplest of RI
detectors is the deflection RI detector.
In this detector, light
is created by a source and passed through flow cells containing mobile phase
eluting from the column (sample stream) and a reference stream (usually mobile
phase with no solute in it). The light passing through these flow cells is
passed through a second time using a mirror and passed to a detector where its
intensity is measured.
When the refractive index
of liquid in the sample and reference flow cell are the same, little or no
bending of light occurs at the interface between the low-cells. This allows the
largest amount of light possible to reach the detector.
Application:
RI detector are universal applicable to the detection of any solute in LC. This makes them useful in preliminary work in LC where the nature or properties of a compound may not be known yet. They also the detector of choice for work with carbohydrates or in the separation of polymer by size-exclusion chromatography.
Some disadvantages:
(1) they do not have very good limits of detection,
(2) they cannot used with gradient elution, where the composition of the mobile phase is changing with time.
(3) The temperature of the system must also be controlled to avoid
baseline fluctuations with these detectors.
Sensitivity: The response
of a RI detector is approximately the same for all compounds.
Absorbance Detector (UV/Vis & PDA):
The UV, VIS, and PDA detectors are categorized as absorbance detectors. The
most common HPLC detectors used are UV detectors because of the fact that most
of the compounds absorb in UV or visible region. They give specific response to
the class of compounds or particular compounds depending upon the functional
group of eluting molecules.
Principle: Absorbance
detector measures the ability of solutes to absorb light at a particular
wavelength range. This absorbance is described by the Beer-Lambert Law.
A = e l
c
Where:
A = Absorbance of light
at a given wavelength
e = Molar
absorption coefficient of the solute
l = path length of the flow-cell
c = concentration of
solute
There are three types of
UV-Vis absorbance detector: fixed wavelength detectors, variable and diode
array detector. They are generally based on the following type of design:
In a variable wavelength
detector, a single wavelength is monitored at any given time, but any
wavelength in a wide spectral range can be selected. The wavelengths that can
be monitored can vary from 190 nm to 900 nm. The ability to use one instrument
for more than one wavelength is achieved by adding in more advanced optics to
the system.
Photo diode array
detectors operate by simultaneously monitoring absorbance of solutes at
different wavelength. The result is that an entire spectrum of a solute can be
taken in a minimum amount of time.
Absorbance
detector can be used to detect any compound absorbing at the wavelength
monitored. Absorbance detector can be sued with gradient elution. The response
of an absorbance detector depends on the molar absorption coefficient. The
larger this value is, the larger the response of the detector
Fluorescence Detector:
A fluorescence detector
is an example of a selective detector, with limits of detection smaller than
those by either RI or absorbance monitors.
Principle:
F = I F (1-e-
e l
c) = I F e l
c (at low concentration)
F = Fluorescence
intensity
I = intensity of the
excitation light
F= Fluorescence
quantum yield
e = Molar
absorption coefficient of the solute
l = path length of the
flow-cell
c = concentration of
solute
Applications:
It can be
used to detect any compound absorbing and emitting light At the given
excitation and emission wavelength.
Conductivity Detector:
Principle:
This detector measures the ability of a solution to conduct a current when placed in an electrical field. This ability depends on the number of ions or ionic compounds present in the solution. ii. The relationship between the current, electric field and conductivity of the solution is shown as follows:
I = C E
I = Current
C = conductivity
E = electric field strength
Applications: for any
compound that is ionic or weakly ionic. It is widely used in ion
chromatography.
Electrochemical detector:
Principle:
This
detector measure the ability of a solute to undergo either oxidation (i.e.,
loss of electrons) or reduction (i.e. gain of electrons)
Oxidation : A↔ A+
+ e-
Reduction : A + e-
↔A-
One way in which such a
reaction can be monitored is by measuring the change in current under a
constant electric field. Another way is to measure the change in the electric
field produced when a constant current is present.
Applications:
Electrochemical detectors can be used to detect any solute that can undergo oxidation or reduction.
Detection by reduction: aldehyes, kentones, nitriles, conjugated acid, etc..
Detection by oxidation: phenols, peroxides, purines, diols
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