Hplc detectors

1. Presented by: IRFAN AHMED
PHARM.D
3rd proff.
Akhtar saeed college of pharmaceutical sciences,lahore

2.  INTRODCTION
 Detectors with high sensitivity are required
in HPLC, usually with sensitivities in
microgrames to nanogrames.
 There are following types of detectors which
are commonly used
 1)differential refractometer detector
 2)ultraviolet detector
 3)Diode array detector

3.  4)Fluorescence detector
 5)amperometric detector

4.  It is often called as universal detector
Principle
 This measures changes in refractive index of the
eluent that result from the presence of solutes
as they emerges from the column
Draw back
 It can not be used effectively gradient elution
due to a change in baseline(a change in the
solvent index of refraction as the gradient is
change) nor when the solvent has an index of
refraction close to that of solute
 This detector is very sensitive to temperature
 This can detect concentrations of about 10-5
to10-6gml.

6.  They have much better sensitivity ,about 10-
8gml
 It is not temperature sensitive
 It is relatively inexpensive and can be used with
gradient elution
 It is sensitive to a large number of organic
compounds
 It can not be used with solvents that have
significant absorption with ultraviolet or with
sample components that do not absorb in u.v
 many UV detectors are simple interference
devices that can measure only a few selected
wavelengths

7.  The more expensive detector have a
monochromator that allows the selection of
particular wavelength.
 Scanning of spectrum can even be achieved
for qualitative identification by momenterily
stopping the flow of the mobile phase
 The most popular HPLC detector is variable
wavelength UV-vis detector
 It can measure nanogram amounts of UV
absorbing analytes or those with suitable
chromophores that absorb in the visible
region

10.  It is a common feature of modern HPLC
instruments
 The instantaneous recording of absorption
spectra provide a powerful qualitative tool
 Principle
 The focused radiation source passess through
the detector flow cell and is dispersed by a
grating to a photo diode array for detection
 The ability to mathematically resolve
overlapping spectra can provide additional
separating ability when a chromatographic
peak may consist of two or more analyte

12.  It can give improved selectivity over UV
absorption detector bz fewer compounds
fluoresce then absorb
 Sensitivities are at least good as perhaps
better then UV detector achieved
 Depending upon the geometry of excitation
of source-detector arrangement , the
intensity of the source .


13.  It is useful for detecting electroactive
substances and has found considerable use in
biological applications
 Example
 In the HPLC separation and detection of
trace quantities of catecholamines from
brain
 In arranging the apparatus ,there must be
minimum of dead volume bw injection port
and column and bw column and detector to
minimize spreading of peaks and to obtain
maximum efficiency

14.  A 20cm length of stainless steel capillary
tubing can generally be used to connect the
column to the detectors without significantly
affecting column performance
 The detector volume must be small and
typical volumes are on the order of 1uL or
so,with high performance detector used with
capillary LC systems having submicroliter
volumes