The document provides an overview of various analytical instruments used in HPLC and GC, focusing on the principles and functionalities of detectors such as TCD, AED, ECD, FID, FPD, PID, and the general operation of HPLC systems. It explains how these instruments interact with mobile phases, sample injection, and the separation of analytes through columns. Lastly, it discusses challenges in liquid pumping and potential solutions for efficient operation.

1. PROJECT TRAINEE ONPROJECT TRAINEE ON
STUDY & MAINTENANCE OFSTUDY & MAINTENANCE OF
HPLC & GC ANALYTICALHPLC & GC ANALYTICAL
INSTRUMENTSINSTRUMENTS
&&
STUDY OF DIFFERENT HPLCSTUDY OF DIFFERENT HPLC
& GC DETECTORS& GC DETECTORS

2. Gas Chromatography (GC)Gas Chromatography (GC)
– InstrumentationInstrumentation
• Mobile phases are generally inert gasesMobile phases are generally inert gases
such as helium, argon, or nitrogen. Thesuch as helium, argon, or nitrogen. The
injection port consists of a rubber septuminjection port consists of a rubber septum
through which a syringe needle is insertedthrough which a syringe needle is inserted
to inject the sample. The injection port isto inject the sample. The injection port is
maintained at a higher temperture than themaintained at a higher temperture than the
boiling point of the least volatile componentboiling point of the least volatile component
in the sample mixture. Since the partitioningin the sample mixture. Since the partitioning
behavior is dependant on temperture, thebehavior is dependant on temperture, the
separation column is usually contained in aseparation column is usually contained in a
thermostat-controlled oven. Separatingthermostat-controlled oven. Separating
components with a wide range of boilingcomponents with a wide range of boiling
points is accomplished by starting at a lowpoints is accomplished by starting at a low
oven temperture and increasing theoven temperture and increasing the
temperture over time to elute the high-temperture over time to elute the high-
boiling point components. Most columnsboiling point components. Most columns
contain a liquid stationary phase on a solidcontain a liquid stationary phase on a solid
support. Separation of low-molecular weightsupport. Separation of low-molecular weight
gases is accomplished with solidgases is accomplished with solid
adsorbentsadsorbents

3. Thermal Conductivity DetectorThermal Conductivity Detector
(TCD)(TCD)
– PrinciplePrinciple
• A TCD detector consists of an electricallyA TCD detector consists of an electrically heated wireheated wire oror
thermistorthermistor . The temperature of the sensing element depends on. The temperature of the sensing element depends on
the thermal conductivity of the gas flowing around it. Changes inthe thermal conductivity of the gas flowing around it. Changes in
thermal conductivity, such as when organic molecules displacethermal conductivity, such as when organic molecules displace
some of the carrier gas, cause a temperature rise in the elementsome of the carrier gas, cause a temperature rise in the element
which is sensed as a change in resistance. The TCD is not aswhich is sensed as a change in resistance. The TCD is not as
sensitive as other dectectors but it is non-specific and non-sensitive as other dectectors but it is non-specific and non-
destructive.destructive.
• Two pairs of TCDs are used in gas chromatographs. One pair isTwo pairs of TCDs are used in gas chromatographs. One pair is
placed in theplaced in the column effluentcolumn effluent to detect the separated componentsto detect the separated components
as they leave the column, and another pair is placedas they leave the column, and another pair is placed before thebefore the
injectorinjector or in a separate reference column. The resistances of theor in a separate reference column. The resistances of the
two sets of pairs are then arranged in a bridge circuit.two sets of pairs are then arranged in a bridge circuit.
• The bridge circuit allows amplification of resistance changes due toThe bridge circuit allows amplification of resistance changes due to
analytes passing over the sample thermoconductors and does notanalytes passing over the sample thermoconductors and does not

4. Thermal ConductivityThermal Conductivity
Detector (TCD)Detector (TCD)

5. Atomic-Emission DetectorAtomic-Emission Detector
(AED)(AED) – principleprinciple
• The strength of the AED lies in the detector's ability to simultaneouslyThe strength of the AED lies in the detector's ability to simultaneously
determine the atomic emissions of many of the elements in analytes thatdetermine the atomic emissions of many of the elements in analytes that
elute from a GC capillary column called eluants. As eluants come off theelute from a GC capillary column called eluants. As eluants come off the
capillary column they are fed intocapillary column they are fed into a microwave powered plasmaa microwave powered plasma
cavity where the compounds are destroyed and their atomscavity where the compounds are destroyed and their atoms
are excited by the energy of the plasma.are excited by the energy of the plasma. The light that is emittedThe light that is emitted
by the excited particles is separated into individual lines via photo diodeby the excited particles is separated into individual lines via photo diode
array. The associated computer then sorts out the individual emission linesarray. The associated computer then sorts out the individual emission lines
and can produce chromatograms made up of peaks from eluants thatand can produce chromatograms made up of peaks from eluants that
contain only a specific elementcontain only a specific element..
• One of the newest additions to the gas chromatographer's arsenal is theOne of the newest additions to the gas chromatographer's arsenal is the
atomic emission detector (AED). This detector, while quite expensiveatomic emission detector (AED). This detector, while quite expensive
compared to other commercially available GC detectors, is an extremelycompared to other commercially available GC detectors, is an extremely
powerful alternative.powerful alternative.
• Instead of measuring simple gas phase ions created in a flame as with theInstead of measuring simple gas phase ions created in a flame as with the
flame ionization detector, or the change in background current because offlame ionization detector, or the change in background current because of
electronegative element capture of thermal electrons as with the electronelectronegative element capture of thermal electrons as with the electron
capture detector, the AED has a much wider applicability because it iscapture detector, the AED has a much wider applicability because it is
based on the detection of atomic emission.based on the detection of atomic emission.

6. InstrumentationInstrumentation
The components of the AED include
an interface for the incoming capillary GC column to the microwave induced plasma chamber
2) The microwave chamber
3) A cooling system for that chamber
4) A diffraction grating and associated optics to focus then disperse the spectral atomic lines
5) Position adjustable photo diode array interfaced to a computer.
The microwave cavity cooling is required because much of the energy focused into the cavity is
converted to heat

7. Atomic-Emission DetectorAtomic-Emission Detector
(AED)(AED)

8. Electron Capture DetectorElectron Capture Detector
(ECD)(ECD)
– IntroductionIntroduction
– The ECD is as sensitive as the FID but has a limited dynamicThe ECD is as sensitive as the FID but has a limited dynamic
range and finds its greatestrange and finds its greatest application in analysis organicapplication in analysis organic
molecules that contain electronegative functionalmolecules that contain electronegative functional
groups, such as halogens, phosphorous, and nitrogroups, such as halogens, phosphorous, and nitro
groups.groups.
• PrinciplePrinciple
• The ECD uses a radioactive Beta emitter (electrons) toThe ECD uses a radioactive Beta emitter (electrons) to
ionize some of the carrier gas and produce a currentionize some of the carrier gas and produce a current
between a biased pair of electrodes.between a biased pair of electrodes.

9. When organic molecules that contain electronegative functional groups, such as
halogens, phosphorous, and nitro groups pass by the detector, they capture some of the
electrons and reduce the current measured between the electrodes.
 The ECD is as sensitive as the FID but has a limited dynamic range and finds its
greatest application in analysis of halogenated compounds.
 Here tritium sources have a high specific activity but beta energy is so low. The max
work temp is 225*c.
 Nickel –63 is a high energy source and can be used up to 400*c.
 When an organic molecule that contains electro negative functional groups like
halogen, phosphorus they capture some of the electron and reduces current.

10. Electron Capture DetectorElectron Capture Detector
(ECD)(ECD)

11. Flame-Ionization DetectorFlame-Ionization Detector
(FID)(FID)
– PrinciplePrinciple FID consists of a hydrogen/air flame andFID consists of a hydrogen/air flame and
a collector plate. The effluent from the GC columna collector plate. The effluent from the GC column
passes through the flame, which breaks downpasses through the flame, which breaks down
organic molecules and produces ions. The ions areorganic molecules and produces ions. The ions are
collected on a biased electrode and produce ancollected on a biased electrode and produce an
electrical signal.electrical signal.
• The collector plate is kept 0.5-1cm above the flame tip.The collector plate is kept 0.5-1cm above the flame tip.
Across the two electrodes a potential is applied about 400V.ItAcross the two electrodes a potential is applied about 400V.It
reduces the resist and allow the current to flow freely.reduces the resist and allow the current to flow freely.
When –CH2- groups are introduced in to a flame a complexWhen –CH2- groups are introduced in to a flame a complex
process takes place in which positive charges carbon speciesprocess takes place in which positive charges carbon species
and electrons are formed since it the current is greatlyand electrons are formed since it the current is greatly
increased.increased.
It is insensitive to water and permanent gases like (CO, CO2,It is insensitive to water and permanent gases like (CO, CO2,
and SO2).and SO2).
The operating arrange is 100 ---420*c.The operating arrange is 100 ---420*c.

12. Flame-Ionization DetectorFlame-Ionization Detector
(FID)(FID)

13. Flame Photometric GCFlame Photometric GC
DetectorDetector
• PrinciplePrinciple
• The determination of sulfur or phosphorus containing compoundsThe determination of sulfur or phosphorus containing compounds
is the job of the flame photometric detector (FPD). This deviceis the job of the flame photometric detector (FPD). This device
uses the chemiluminescent reactions of these compounds in auses the chemiluminescent reactions of these compounds in a
hydrogen/air flame as a source of analytical information that ishydrogen/air flame as a source of analytical information that is
relatively specific for substances containing these two kinds ofrelatively specific for substances containing these two kinds of
atoms.atoms.
• The emitting species for sulfur compounds is excited SThe emitting species for sulfur compounds is excited S22 the lambdathe lambda
max for emission of excitedmax for emission of excited SS22 is approximately 394 nm.is approximately 394 nm.
• The emitter forThe emitter for phosphorusphosphorus compounds in the flame is excitedcompounds in the flame is excited
HPO (lambda isHPO (lambda is 510-526510-526 nm).nm).
• In order to selectively detect one or the other family of compoundsIn order to selectively detect one or the other family of compounds
as it elutes from the GC column, an interference filter is usedas it elutes from the GC column, an interference filter is used
between the flame and the photomultiplier tube (PMT) to isolatebetween the flame and the photomultiplier tube (PMT) to isolate
the appropriate emission band. The drawback here being that thethe appropriate emission band. The drawback here being that the
filter must be exchanged between chromatographic runs if thefilter must be exchanged between chromatographic runs if the

14. Flame Photometric GCFlame Photometric GC
DetectorDetector
Instrumentation
1) A combustion chamber to house the flame
2) Gas lines for hydrogen (fuel) and air (oxidant) and
3) An exhaust chimney to remove combustion products,
4) The final component necessary for this instrument is a thermal (bandpass) filter to isolate
only the visible and UV radiation emitted by the flame. Without this the large amounts of
infrared radiation emitted by the flame's combustion reaction would heat up the PMT and
increase its background signal.
It is mainly used in the determination of pesticides and to detect gaseous sulphur compound like
H2s, SO2.

15. Flame Photometric GCFlame Photometric GC
DetectorDetector

16. Photoionization DetectorPhotoionization Detector
• PrinciplePrinciple
• The selective determination ofThe selective determination of aromatic hydrocarbons oraromatic hydrocarbons or
organo-heteroatom species is the job of theorgano-heteroatom species is the job of the
photoionization detector (PIDphotoionization detector (PID ). This device uses ultraviolet). This device uses ultraviolet
light as a means of ionizing an analyte exiting from a GC column.light as a means of ionizing an analyte exiting from a GC column.
The ions produced by this process are collected by electrodes.The ions produced by this process are collected by electrodes.
The current generated is therefore a measure of the analyteThe current generated is therefore a measure of the analyte
concentrationconcentration..
– If the energy of an incoming photon is high enough, photo-If the energy of an incoming photon is high enough, photo-
excitation can occur to such an extent that an electron isexcitation can occur to such an extent that an electron is
completely removed from its molecular orbital.completely removed from its molecular orbital.
– R + hv ----> R + e-R + hv ----> R + e-

17. • Since only a small fraction of the analyte moleculesSince only a small fraction of the analyte molecules
are actually ionized in the PID chamber, this isare actually ionized in the PID chamber, this is
considered to be a nondestructive GC detector.considered to be a nondestructive GC detector.
• Therefore, the exhaust port of the PID can beTherefore, the exhaust port of the PID can be
connected to another detector in series with the PID.connected to another detector in series with the PID.
In this way data from two different detectors can beIn this way data from two different detectors can be
taken simultaneously, and selective detection of PIDtaken simultaneously, and selective detection of PID
responsive compounds augmented by responseresponsive compounds augmented by response
from, say, a FID or ECD.from, say, a FID or ECD.
• The major challenge here is to make the design ofThe major challenge here is to make the design of
the ionization chamber and the downstreamthe ionization chamber and the downstream
connections to the second detector as low volumeconnections to the second detector as low volume
as possible (read small diameter) so that peaks thatas possible (read small diameter) so that peaks that
have been separated by the GC column do nothave been separated by the GC column do not
broaden out before detection.broaden out before detection.

18. Photoionization DetectorPhotoionization Detector

19. HPLC INSTRUMENTATIONHPLC INSTRUMENTATION
BASIC OPERATION &ITSBASIC OPERATION &ITS
DETECTORSDETECTORS

20. HPLC INSTRUMENTATIONHPLC INSTRUMENTATION
 HPLC systems introductionHPLC systems introduction
• HPLC instrumentation includes a pump, injector, column, detector andHPLC instrumentation includes a pump, injector, column, detector and
recorder or data system. The heart of the system is the column whererecorder or data system. The heart of the system is the column where
separation occurs. Since the stationary phase is composed ofseparation occurs. Since the stationary phase is composed of
micrometer size porous particles, a high pressure pump is required tomicrometer size porous particles, a high pressure pump is required to
move the mobile phase through the column. The chromatographicmove the mobile phase through the column. The chromatographic
process begins by injecting the solute onto the top of the column.process begins by injecting the solute onto the top of the column.
Separation of components occurs as the analytes and mobile phasesSeparation of components occurs as the analytes and mobile phases
are pumped through the column. Eventually, each component elutesare pumped through the column. Eventually, each component elutes
from the column as a narrow band (or peak) on the recorder. Detectionfrom the column as a narrow band (or peak) on the recorder. Detection
of the eluting components is important, and this can be either selectiveof the eluting components is important, and this can be either selective
or universal, depending upon the detector used. The response of theor universal, depending upon the detector used. The response of the
detector to each component is displayed on a chart recorder ordetector to each component is displayed on a chart recorder or
computer screen and is known as a chromatogram.computer screen and is known as a chromatogram.
• To collect, store and analyze the chromatographic data,computers,To collect, store and analyze the chromatographic data,computers,
integrators, and other data processingintegrators, and other data processing equipment are frequently used.equipment are frequently used.

21. HPLC INSTRUMENTATIONHPLC INSTRUMENTATION

22. – Mobile phaseMobile phase ((Problem in pumping liquid)Problem in pumping liquid)
– The common problems encountered in pumpingThe common problems encountered in pumping
mobile phases aremobile phases are solvent degassing, corrosion,solvent degassing, corrosion,
and compressibility.and compressibility.
– REMEDIESREMEDIES::
– Subjecting the liquid to vacuumSubjecting the liquid to vacuum
– Heating the liquid until boiling occursHeating the liquid until boiling occurs

23. PUMPPUMP
• Reciprocating piston pumpReciprocating piston pump
– The piston expels liquid through a one-way valve (check valve). TheThe piston expels liquid through a one-way valve (check valve). The
pumping rate is usually adjusted by controlling the distance the pistonpumping rate is usually adjusted by controlling the distance the piston
retracts, thus limiting the amount of liquid pushed out by each stroke, orretracts, thus limiting the amount of liquid pushed out by each stroke, or
by the cam rotating speed.by the cam rotating speed.
– Dual Piston Pumps :Dual Piston Pumps :
• A more efficient way to provide a constant and almost pulse free flow is theA more efficient way to provide a constant and almost pulse free flow is the
use of dual-headed reciprocating pumps. Both pump chambers are drivenuse of dual-headed reciprocating pumps. Both pump chambers are driven
by the same motor through a common eccentric cam; this common driveby the same motor through a common eccentric cam; this common drive
allows one piston to pump while the other is refilling. As a result, the twoallows one piston to pump while the other is refilling. As a result, the two
flow-profiles overlap each other significantly reducing the pulsationflow-profiles overlap each other significantly reducing the pulsation
downstream of the pumpdownstream of the pump

24. INJECTORSINJECTORS
• Injectors for liquid chromatographic systems shouldInjectors for liquid chromatographic systems should
provide the possibility of injecting the liquid sampleprovide the possibility of injecting the liquid sample
within the range of 0.1 to 100 ml of volume with highwithin the range of 0.1 to 100 ml of volume with high
reproducibility and under high pressure (up to the 4000reproducibility and under high pressure (up to the 4000
psi). They should also produce minimum bandpsi). They should also produce minimum band
broadening and minimize possible flowbroadening and minimize possible flow
disturbances.Generally, the most useful and widelydisturbances.Generally, the most useful and widely
used sampling device for modern LC is theused sampling device for modern LC is the
microsampling injector valvemicrosampling injector valve

25. INJECTORSINJECTORS

26. ColumnColumn
• Today, column material is normallyToday, column material is normally
Type 316 stainless steel, once againType 316 stainless steel, once again
chosen because it offers the bestchosen because it offers the best
compromise of cost, workability, andcompromise of cost, workability, and
corrosion resistance. Mostcorrosion resistance. Most
commercial columns available todaycommercial columns available today
have internal diameters of eitherhave internal diameters of either
2.6-3 mm or 4.6-5 mm. Most leading2.6-3 mm or 4.6-5 mm. Most leading
manufacturers obtain a greatmanufacturers obtain a great
amount of interchangeabilityamount of interchangeability
between column sizes withoutbetween column sizes without
excessive fitting replacement byexcessive fitting replacement by
supplying columns with a variety ofsupplying columns with a variety of
internal diameters, but a uniform ¼internal diameters, but a uniform ¼
in. outside diameter. Naturally,in. outside diameter. Naturally,
preparative columns have largerpreparative columns have larger
diameters: usual O.D. values arediameters: usual O.D. values are
3/8 - 5/8 in., corresponding to3/8 - 5/8 in., corresponding to
internal diameters of 6.4-12.7 mm,internal diameters of 6.4-12.7 mm,

27. DETECTORSDETECTORS
BASIC DETECTORBASIC DETECTOR
REQUIREMENTSREQUIREMENTS
SensitivitySensitivity
SelectivitySelectivity
ResponseResponse
Linear dynamic rangeLinear dynamic range

28. REFRACTIVE INDEX DETECTORREFRACTIVE INDEX DETECTOR
• PrinciplesPrinciples
• The refractive index (RI) detector is the only universal detector inThe refractive index (RI) detector is the only universal detector in
HPLC.HPLC.
• The detection principle involves measuring of the change inThe detection principle involves measuring of the change in
refractive index of therefractive index of the column effluent passing through thecolumn effluent passing through the
flow-cellflow-cell. The greater the RI difference between sample and. The greater the RI difference between sample and
mobile phase, the larger the imbalance will become. Thus, themobile phase, the larger the imbalance will become. Thus, the
sensitivity will be higher for the higher difference in RI betweensensitivity will be higher for the higher difference in RI between
sample and mobile phase. On the other hand, in complexsample and mobile phase. On the other hand, in complex
mixtures, sample components may cover a wide range ofmixtures, sample components may cover a wide range of
refractive index values and some may closely match that of therefractive index values and some may closely match that of the
mobile phase, becoming invisible to the detector.mobile phase, becoming invisible to the detector.
• RI detector is a pureRI detector is a pure differential instrumentdifferential instrument , and any changes, and any changes
in the eluent composition require the rebalansing of the detector.in the eluent composition require the rebalansing of the detector.
This factor is severely limiting RI detector application in theThis factor is severely limiting RI detector application in the
analyses requiring the gradient elution, where mobile phaseanalyses requiring the gradient elution, where mobile phase
composition is changed during the analysis to effect thecomposition is changed during the analysis to effect the
separation.separation.

29. REFRACTIVE INDEXREFRACTIVE INDEX
DETECTORDETECTOR

30. REFLECTIVE DETECTORSREFLECTIVE DETECTORS
• The refractive index detectorThe refractive index detector
based on the Fresnel principle isbased on the Fresnel principle is
relatively rear .The light beam isrelatively rear .The light beam is
reflected from the liquid-glassreflected from the liquid-glass
interface in the detectinginterface in the detecting
photocell. As the introduction ofphotocell. As the introduction of
sample into onesample into one cell causes lightcell causes light
to be refracted at a differentto be refracted at a different
angle. The deflection of the lightangle. The deflection of the light
beam from thebeam from the photoresistorphotoresistor
cause the appearance of thecause the appearance of the
electrical signal. This diff betweenelectrical signal. This diff between
sample and reference signal issample and reference signal is
output to a recorder.output to a recorder.

31. ADV&DISADVANTAGEADV&DISADVANTAGE
ADVANTAGES
The major advantage of this type of detector is a very high sensitivity since the optics allow a higher
concentration of signal in a particular RI range than is possible in other wide-range detectors. Other
advantages include the ability to operate at extremely low flow rates with very low-volume cells, easy cell
accessibility, and low cost.
DISADVANTAGES
Its disadvantages are the incredible sensitivity to the flow and pressure fluctuations, and the need for
changing prisms to accommodate either high or low RI solvents and the need to manually adjust the optical
path when making solvent changes.

32. ULTRAVIOLET/VISIBLEULTRAVIOLET/VISIBLE
SPECTROSCOPIC DETECTORSSPECTROSCOPIC DETECTORS
Principles
Any chemical compound could interact with the electromagnetic field. Beam of the
electromagnetic radiation passed through the detector flow-cell will experience some change in its
intensity due to this interaction. Measurement of this changes is the basis of the most optical HPLC
detectors.
infrared (IR) 2,500 - 50,000 nm
near infrared 800 - 2,500 nm
Visible 400 - 800 nm
ultraviolet (UV) 190 - 400 nm

33. UV Absorbance
Absorbance is the logarithm of the ratio of the intensities of the incident light (Io) and the
transmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity
(molar extinction coefficient, ε), the
infrared (IR) 2,500 - 50,000 nm
near infrared 800 - 2,500 nm
Visible 400 - 800 nm
ultraviolet (UV) 190 - 400 nm
UV Absorbance
Absorbance is the logarithm of the ratio of the intensities of the incident light (Io) and the
transmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity
(molar extinction coefficient, ε), the

34. Name Chromophore Wavelength [nm] Molar extinction, e
Acetylide -C=C 175-180 6,000
Aldehyde -CHO 210 1,500
Amine -NH2 195 2,800
Azo -N=N- 285-400 3-25
Bromide -Br 208 300
Carboxyl -COOH 200-210 50 – 70
Disulphide -S-S- 194 5,500
Ester -COOR 205 50
Ether -O- 185 1,000
Ketone >C=O 195 1,000
Nitrate -ONO2 270 12
Nitrile -C=N 160 -
Nitrite -ONO 220 - 230 1000-2000
Nitro -NO2 210 Strong

35. Fixed wavelengthFixed wavelength
detectorsdetectors
• HPLC detectors which does notHPLC detectors which does not
allow to change the wavelength ofallow to change the wavelength of
the radiation called fixed-wavelengththe radiation called fixed-wavelength
detectors.detectors.
• low-pressure mercury vapor lamplow-pressure mercury vapor lamp
emit very intense light at 253.7 nm.emit very intense light at 253.7 nm.
By filtering out all other emittedBy filtering out all other emitted
wavelengths, manufacturers havewavelengths, manufacturers have
been able to utilize this 254 nm linebeen able to utilize this 254 nm line
to provide stable, highly sensitiveto provide stable, highly sensitive
detectors capable of measuringdetectors capable of measuring
subnanogram quantities of anysubnanogram quantities of any
components which containscomponents which contains
aromatic ring. The 254 nm wasaromatic ring. The 254 nm was
chosen since the most intense linechosen since the most intense line
of mercury lamp is 254 nm, andof mercury lamp is 254 nm, and
most of UV absorbing compoundsmost of UV absorbing compounds
have some absorbance at 254 nmhave some absorbance at 254 nm

36. Variable-wavelength detectorsVariable-wavelength detectors
• Detectors that allow theDetectors that allow the
selection of the operatingselection of the operating
wavelength called variablewavelength called variable
wavelength detectors andwavelength detectors and
they are particularly usefulthey are particularly useful
in three casesin three cases
• Depending on theDepending on the
sophistication of thesophistication of the
detector, wavelengthdetector, wavelength
change is done manually orchange is done manually or
programmed on a timeprogrammed on a time
basis into the memory ofbasis into the memory of
the systemthe system

37. Diode-array detectorsDiode-array detectors
• As already mentioned, a special feature ofAs already mentioned, a special feature of
some variable wavelength UV detectors issome variable wavelength UV detectors is
the ability to perform spectroscopicthe ability to perform spectroscopic
scanning and precise absorbancescanning and precise absorbance
readings at a variety of wavelengths whilereadings at a variety of wavelengths while
the peak is passing though the flowcell.the peak is passing though the flowcell.
Diode array adds a new dimension ofDiode array adds a new dimension of
analytical capability to liquidanalytical capability to liquid
chromatography because it permitschromatography because it permits
qualitative information to be obtainedqualitative information to be obtained
beyond simple identification by retentionbeyond simple identification by retention
time.time.
• There are two major advantages of diodeThere are two major advantages of diode
array detection. In the first, it allows for thearray detection. In the first, it allows for the
best wavelength(s) to be selected forbest wavelength(s) to be selected for
actual analysis. This is particularlyactual analysis. This is particularly
important when no information is availableimportant when no information is available
on molar absorptivities at differenton molar absorptivities at different

38. • The second major advantage is related to the problem ofThe second major advantage is related to the problem of
peak purity. Often, the peak shape in itself does not revealpeak purity. Often, the peak shape in itself does not reveal
that it actually corresponds to two (or even more)that it actually corresponds to two (or even more)
components. In such a case, absorbance rationing atcomponents. In such a case, absorbance rationing at
several wavelengths is particularly helpful in decidingseveral wavelengths is particularly helpful in deciding
whether the peak represents a single compound or, is inwhether the peak represents a single compound or, is in
fact, a composite peakfact, a composite peak
• In absorbance rationing, the absorbance is measured atIn absorbance rationing, the absorbance is measured at
two or more wavelengths and ratios are calculated for twotwo or more wavelengths and ratios are calculated for two
selected wavelengths. Simultaneous measurement atselected wavelengths. Simultaneous measurement at
several wavelengths allows one to calculate theseveral wavelengths allows one to calculate the
absorbance ratio. Evaluation can be carried out in twoabsorbance ratio. Evaluation can be carried out in two
ways:ways:
• In the first case, the ratios at chosen wavelength areIn the first case, the ratios at chosen wavelength are
continuously monitored during the analysis: if thecontinuously monitored during the analysis: if the
compound under the peak is pure, the response will be acompound under the peak is pure, the response will be a

39. FLUORESCENCE DETECTORSFLUORESCENCE DETECTORS
• principleprinciple
• Fluorescence detectors are probably the most sensitive amongFluorescence detectors are probably the most sensitive among
the existing modern HPLC detectors. It is possible to detect eventhe existing modern HPLC detectors. It is possible to detect even
a presence of a single analyte molecule in the flow cell. Typically,a presence of a single analyte molecule in the flow cell. Typically,
fluorescence sensitivity is 10 -1000 times higher than that of thefluorescence sensitivity is 10 -1000 times higher than that of the
UV detector for strong UV absorbing materials.UV detector for strong UV absorbing materials.
• Fluorescence detectors are very specific and selective among theFluorescence detectors are very specific and selective among the
others optical detectors. This is normally used as an advantage inothers optical detectors. This is normally used as an advantage in
the measurement of specific fluorescent species in samples.the measurement of specific fluorescent species in samples.
• When compounds having specific functionalWhen compounds having specific functional
groups are excited by shorter wavelength energygroups are excited by shorter wavelength energy
and emit higher wavelength radiation which calledand emit higher wavelength radiation which called
fluorescence. Usually, the emission is measuredfluorescence. Usually, the emission is measured
at right angles to the excitation.at right angles to the excitation.

40. Roughly about 15% of all compounds have a natural fluorescence. The presence of conjugated pi-electrons
especially in the aromatic components gives the most intense fluorescent activity. Also, aliphatic and
alicyclic compounds with carbonyl groups and compounds with highly conjugated double bonds fluoresce,
but usually to a lesser degree.
Most unsubstituted aromatic hydrocarbons fluoresce with quantum yeld increasing with the number of
rings, their degree of condensation and their structural rigidity.
Fluorescence intensity depends on both the excitation and emission wavelength, allowing selectively detect
some components while suppressing the emission of others.
The detection of any component significantly depends on the chosen wavelength and if one component
could be detected at 280 ex and 340 em, another 3+ be missed.
Most of the modern detectors allow fast switch of the excitation and emission wavelength, which offer the
possibility to detect all components in the mixture.
For example,
----->In the very important polynuclear aromatic chromatogram the excitation and emission wavelengths
were 280 and 340 nm, respectively, for the first 6 components, and then changed to the respective values of
305 and 430 nm; the latter values represent the best compromise to allow sensitive detection of compounds.

41. • Figure below shows theFigure below shows the
optical schematic of a typicaloptical schematic of a typical
fluorescence detector forfluorescence detector for
liquid chromatography. Theliquid chromatography. The
detectors available on thedetectors available on the
market differ in the method inmarket differ in the method in
which the wavelengths arewhich the wavelengths are
controlled. Less expensivecontrolled. Less expensive
instruments utilize filters;instruments utilize filters;
medium priced units offermedium priced units offer
monochromator control of atmonochromator control of at
least emission wavelength,least emission wavelength,
and full capability research-and full capability research-
grade instruments providegrade instruments provide
monochromator control ofmonochromator control of
both excitation and emissionboth excitation and emission
wavelengths.wavelengths.

42. ELECTROCHEMICALELECTROCHEMICAL
DETECTORSDETECTORS
• PrinciplePrinciple
• The chromatographer because ofThe chromatographer because of
the additional selectivity andthe additional selectivity and
sensitivity for some compoundssensitivity for some compounds
should consider it.should consider it.
• This detector is based onThis detector is based on
the measurements of thethe measurements of the
current resulting fromcurrent resulting from
oxidation/reduction reactionoxidation/reduction reaction
of the analyte at a suitableof the analyte at a suitable
electrode.electrode. Since the level of theSince the level of the
current is directly proportional tocurrent is directly proportional to
the analyte concentration, thisthe analyte concentration, this
detector coulddetector could used forused for
quantificationquantification..

43. • The eluent should contain electrolyte and be electricallyThe eluent should contain electrolyte and be electrically
conductive. Most of the analytes to be successfullyconductive. Most of the analytes to be successfully
detected require the pH adjustments.detected require the pH adjustments.
• The areas of application of electrochemical detection areThe areas of application of electrochemical detection are
not large, but the compounds for which it does apply,not large, but the compounds for which it does apply,
represent some of the most important drug, pollutant andrepresent some of the most important drug, pollutant and
natural product classes. For these, the specificity, andnatural product classes. For these, the specificity, and
sensitivity make it very useful for monitoring thesesensitivity make it very useful for monitoring these
compounds in complex matrices such as body fluids andcompounds in complex matrices such as body fluids and
natural products. Sensitivities for compounds such asnatural products. Sensitivities for compounds such as
phenol, catecholamines, nitrosamines, and organic acidsphenol, catecholamines, nitrosamines, and organic acids
are in the picomole (nanogram) range.are in the picomole (nanogram) range.
• The purity of the eluent is very important, because theThe purity of the eluent is very important, because the
presence of oxygen, metal contamination and halides maypresence of oxygen, metal contamination and halides may
cause significant background current and therefore, noisecause significant background current and therefore, noise

44. ELECTROLYTICELECTROLYTIC
CONDUCTIVITY DETECTORCONDUCTIVITY DETECTOR
• The conductivity of the columnThe conductivity of the column
effluent is continuosly measuredeffluent is continuosly measured
and the appearance of theand the appearance of the
analyte in the cell is indicated byanalyte in the cell is indicated by
a change in conductivity.a change in conductivity.
• Usually this is a very low volumeUsually this is a very low volume
flow-through capillary equippedflow-through capillary equipped
with two electrodes andwith two electrodes and
variations in conductivity of thevariations in conductivity of the
mobile phase due to the elutedmobile phase due to the eluted
sample components aresample components are
continuously recorded. Responsecontinuously recorded. Response
is linear with concentration over ais linear with concentration over a
wide range, quantitation of thewide range, quantitation of the
output signal is possible withoutput signal is possible with
suitable preliminary calibration.suitable preliminary calibration.

45. Best use is made of this detector in isocraticBest use is made of this detector in isocratic
analysis since solvent gradients will cause aanalysis since solvent gradients will cause a
proportional shift in the baseline. Such detectorsproportional shift in the baseline. Such detectors
have been used most successfully in ion-have been used most successfully in ion-
exchange chromatography of anions and cationexchange chromatography of anions and cation
but generally, they have found only limitedbut generally, they have found only limited
popular acceptance.popular acceptance.

46. EVAPORATIVE LIGHTEVAPORATIVE LIGHT
SCATTERINGSCATTERING
• LIGHT SCATTERING CELLLIGHT SCATTERING CELL
• The nebulized column effluentThe nebulized column effluent
enters the light scattering cell.enters the light scattering cell.
In the cell, the sample particlesIn the cell, the sample particles
scatter the laser light, but thescatter the laser light, but the
evaporated mobile phase doesevaporated mobile phase does
not. The scattered light isnot. The scattered light is
detected by a siliconedetected by a silicone
photodiode located at a 90ºphotodiode located at a 90º
angle from the laser. Theangle from the laser. The
photodiode produces a signalphotodiode produces a signal
which is sent to the analogwhich is sent to the analog
outputs for collection. A lightoutputs for collection. A light
trap is located 180º from thetrap is located 180º from the
laser to collect any light notlaser to collect any light not
scattered by particles in thescattered by particles in the
aerosol stream.aerosol stream.

47. • Analytical column outlet is connected directly to the nebulizer . TheAnalytical column outlet is connected directly to the nebulizer . The
column effluent is mixed with a stream of nebulizing gas to form ancolumn effluent is mixed with a stream of nebulizing gas to form an
aerosol. The aerosol consists of a uniform dispersion of droplets.aerosol. The aerosol consists of a uniform dispersion of droplets.
The lower the mobile phase flowrate, the less gas and heat areThe lower the mobile phase flowrate, the less gas and heat are
needed to nebulize and evaporate it. Reduction of flowrate byneeded to nebulize and evaporate it. Reduction of flowrate by
using 2.1mm I.D. column should be considered when sensitivity isusing 2.1mm I.D. column should be considered when sensitivity is
important. The gas flowrate will also regulate the size of theimportant. The gas flowrate will also regulate the size of the
droplets in the aerosol. Larger droplets will scatter more light anddroplets in the aerosol. Larger droplets will scatter more light and
increase the sensitivity of the analysis. The lower the gas flowincrease the sensitivity of the analysis. The lower the gas flow
used, the larger the droplets will be. It is also important toused, the larger the droplets will be. It is also important to
remember that the larger the droplet, the more difficult it will be toremember that the larger the droplet, the more difficult it will be to
vaporize in the drift tube.vaporize in the drift tube. Unvaporized mobile phase willUnvaporized mobile phase will
increase baseline noiseincrease baseline noise . There will be an optimum gas. There will be an optimum gas
flowrate for each method which will produce the highest signal-to-flowrate for each method which will produce the highest signal-to-
noise ratio. Volatile components of the aerosol are evaporated innoise ratio. Volatile components of the aerosol are evaporated in
the drift tube.the drift tube.

48. Principle of HPLC