PROJECT TRAINEE ONPROJECT TRAINEE ONSTUDY & MAINTENANCE OF STUDY & MAINTENANCE OF HPLC & GC ANALYTICAL HPLC & GC ANALYTICAL INSTRUMENTSINSTRUMENTS&&STUDY OF DIFFERENT HPLC STUDY OF DIFFERENT HPLC & GC DETECTORS& GC DETECTORS

Gas Chromatography Gas Chromatography (GC)(GC)

– InstrumentationInstrumentation• Mobile phases are generally inert Mobile phases are generally inert

gases such as helium, argon, or gases such as helium, argon, or nitrogen. The injection port consists of nitrogen. The injection port consists of a rubber septum through which a a rubber septum through which a syringe needle is inserted to inject the syringe needle is inserted to inject the sample. The injection port is sample. The injection port is maintained at a higher temperture maintained at a higher temperture than the boiling point of the least than the boiling point of the least volatile component in the sample volatile component in the sample mixture. Since the partitioning mixture. Since the partitioning behavior is dependant on temperture, behavior is dependant on temperture, the separation column is usually the separation column is usually contained in a thermostat-controlled contained in a thermostat-controlled oven. Separating components with a oven. Separating components with a wide range of boiling points is wide range of boiling points is accomplished by starting at a low oven accomplished by starting at a low oven temperture and increasing the temperture and increasing the temperture over time to elute the temperture over time to elute the high-boiling point components. Most high-boiling point components. Most columns contain a liquid stationary columns contain a liquid stationary phase on a solid support. Separation of phase on a solid support. Separation of low-molecular weight gases is low-molecular weight gases is accomplished with solid adsorbentsaccomplished with solid adsorbents

Thermal Conductivity Detector Thermal Conductivity Detector (TCD)(TCD)

– PrinciplePrinciple• A TCD detector consists of an electrically A TCD detector consists of an electrically heated wireheated wire or or

thermistorthermistor. The temperature of the sensing element depends on the . The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal thermal conductivity of the gas flowing around it. Changes in thermal conductivity, such as when organic molecules displace some of the conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other as a change in resistance. The TCD is not as sensitive as other dectectors but it is non-specific and non-destructive. dectectors but it is non-specific and non-destructive.

• Two pairs of TCDs are used in gas chromatographs. One pair is placed Two pairs of TCDs are used in gas chromatographs. One pair is placed in the in the column effluentcolumn effluent to detect the separated components as they to detect the separated components as they leave the column, and another pair is placed leave the column, and another pair is placed before the injectorbefore the injector or or in a separate reference column. The resistances of the two sets of in a separate reference column. The resistances of the two sets of pairs are then arranged in a bridge circuit.pairs are then arranged in a bridge circuit.

• The bridge circuit allows amplification of resistance changes due to The bridge circuit allows amplification of resistance changes due to analytes passing over the sample thermoconductors and does not analytes passing over the sample thermoconductors and does not amplify changes in resistance that both sets of detectors produce due amplify changes in resistance that both sets of detectors produce due to flow rate fluctuations, etcto flow rate fluctuations, etc. .

Thermal Conductivity Thermal Conductivity Detector (TCD)Detector (TCD)

Atomic-Emission Detector Atomic-Emission Detector (AED)(AED)

– principleprinciple• The strength of the AED lies in the detector's ability to simultaneously The strength of the AED lies in the detector's ability to simultaneously

determine the atomic emissions of many of the elements in analytes determine the atomic emissions of many of the elements in analytes that elute from a GC capillary column called eluants. As eluants come that elute from a GC capillary column called eluants. As eluants come off the capillary column they are fed into off the capillary column they are fed into a microwave powered a microwave powered plasma cavity where the compounds are destroyed and their plasma cavity where the compounds are destroyed and their atoms are excited by the energy of the plasma.atoms are excited by the energy of the plasma. The light that is The light that is emitted by the excited particles is separated into individual lines via emitted by the excited particles is separated into individual lines via photo diode array. The associated computer then sorts out the photo diode array. The associated computer then sorts out the individual emission lines and can produce chromatograms made up of individual emission lines and can produce chromatograms made up of peaks from eluants that contain only a specific elementpeaks from eluants that contain only a specific element. .

• One of the newest additions to the gas chromatographer's arsenal is the One of the newest additions to the gas chromatographer's arsenal is the atomic emission detector (AED). This detector, while quite expensive atomic emission detector (AED). This detector, while quite expensive compared to other commercially available GC detectors, is an extremely compared 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 Instead of measuring simple gas phase ions created in a flame as with the flame ionization detector, or the change in background current the flame ionization detector, or the change in background current because of electronegative element capture of thermal electrons as with because of electronegative element capture of thermal electrons as with the electron capture detector, the AED has a much wider applicability the electron capture detector, the AED has a much wider applicability because it is based on the detection of atomic emission.because it is based on the detection of atomic emission.

InstrumentationInstrumentation

The components of the AED include an interface for the incoming capillary GC column to the microwave induced plasma chamber

2) The microwave chamber3) A cooling system for that chamber4) A diffraction grating and associated optics to focus then disperse the spectral atomic lines5) Position adjustable photo diode array interfaced to a computer.The microwave cavity cooling is required because much of the energy focused into the cavity isconverted to heat

Atomic-Emission Atomic-Emission Detector (AED)Detector (AED)

Electron Capture Detector Electron Capture Detector (ECD)(ECD)

– IntroductionIntroduction– The ECD is as sensitive as the FID but has a limited The ECD is as sensitive as the FID but has a limited

dynamic range and finds its greatest dynamic range and finds its greatest application in application in analysis organic molecules that contain analysis organic molecules that contain electronegative functional groups, such as electronegative functional groups, such as halogens, phosphorous, and nitro groups. halogens, phosphorous, and nitro groups.

• PrinciplePrinciple• The ECD uses a radioactive Beta emitter (electrons) The ECD uses a radioactive Beta emitter (electrons)

to ionize some of the carrier gas and produce a to ionize some of the carrier gas and produce a current between a biased pair of electrodes.current between a biased pair of electrodes.

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.

Electron Capture Electron Capture Detector (ECD)Detector (ECD)

Flame-Ionization Flame-Ionization Detector (FID)Detector (FID)

– PrinciplePrinciple FID consists of a hydrogen/air flame and FID consists of a hydrogen/air flame and a collector plate. The effluent from the GC column a collector plate. The effluent from the GC column passes through the flame, which breaks down passes through the flame, which breaks down organic molecules and produces ions. The ions are organic molecules and produces ions. The ions are collected on a biased electrode and produce an collected 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.It Across 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 complex When –CH2- groups are introduced in to a flame a complex

process takes place in which positive charges carbon species process takes place in which positive charges carbon species and electrons are formed since it the current is greatly and 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.

Flame-Ionization Flame-Ionization Detector (FID)Detector (FID)

Flame Photometric GC Flame Photometric GC DetectorDetector• PrinciplePrinciple• The determination of sulfur or phosphorus containing compounds The determination of sulfur or phosphorus containing compounds

is the job of the flame photometric detector (FPD). This device is the job of the flame photometric detector (FPD). This device uses the chemiluminescent reactions of these compounds in a uses the chemiluminescent reactions of these compounds in a hydrogen/air flame as a source of analytical information that is hydrogen/air flame as a source of analytical information that is relatively specific for substances containing these two kinds of relatively 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 S2 2 the lambda the lambda max for emission of excited max for emission of excited SS22 is approximately 394 nm. is approximately 394 nm.

• The emitter for The emitter for phosphorusphosphorus compounds in the flame is excited compounds in the flame is excited HPO (lambda is HPO (lambda is 510-526510-526 nm). nm).

• In order to selectively detect one or the other family of compounds In order to selectively detect one or the other family of compounds as it elutes from the GC column, an interference filter is used as it elutes from the GC column, an interference filter is used between the flame and the photomultiplier tube (PMT) to isolate between the flame and the photomultiplier tube (PMT) to isolate the appropriate emission band. The drawback here being that the the appropriate emission band. The drawback here being that the filter must be exchanged between chromatographic runs if the filter must be exchanged between chromatographic runs if the other family of compounds is to be detected. other family of compounds is to be detected.

Flame Photometric GC Flame 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.

Flame Photometric GC Flame Photometric GC DetectorDetector

Photoionization DetectorPhotoionization Detector• PrinciplePrinciple• The selective determination of The selective determination of aromatic hydrocarbons aromatic hydrocarbons

or organo-heteroatom species is the job of the or organo-heteroatom species is the job of the photoionization detector (PIDphotoionization detector (PID). This device uses ). This device uses ultraviolet light as a means of ionizing an analyte exiting ultraviolet light as a means of ionizing an analyte exiting from a GC column. The ions produced by this process are from a GC column. The ions produced by this process are collected by electrodes. The current generated is therefore collected by electrodes. The current generated is therefore a measure of the analyte concentrationa measure of the analyte concentration. .

– If the energy of an incoming photon is high enough, If the energy of an incoming photon is high enough, photo-excitation can occur to such an extent that an photo-excitation can occur to such an extent that an electron is completely removed from its molecular electron is completely removed from its molecular orbital.orbital.

– R + hv ----> R + e-R + hv ----> R + e-

• Since only a small fraction of the analyte Since only a small fraction of the analyte molecules are actually ionized in the PID molecules are actually ionized in the PID chamber, this is considered to be a chamber, this is considered to be a nondestructive GC detector. nondestructive GC detector.

• Therefore, the exhaust port of the PID can be Therefore, the exhaust port of the PID can be connected to another detector in series with the connected to another detector in series with the PID. In this way data from two different detectors PID. In this way data from two different detectors can be taken simultaneously, and selective can be taken simultaneously, and selective detection of PID responsive compounds detection of PID responsive compounds augmented by response from, say, a FID or ECD. augmented by response from, say, a FID or ECD.

• The major challenge here is to make the design The major challenge here is to make the design of the ionization chamber and the downstream of the ionization chamber and the downstream connections to the second detector as low connections to the second detector as low volume as possible (read small diameter) so that volume as possible (read small diameter) so that peaks that have been separated by the GC peaks that have been separated by the GC column do not broaden out before detection.column do not broaden out before detection.

Photoionization DetectorPhotoionization Detector

HPLC INSTRUMENTATIONHPLC INSTRUMENTATIONBASIC OPERATION &ITS BASIC OPERATION &ITS DETECTORSDETECTORS

HPLC INSTRUMENTATIONHPLC INSTRUMENTATION HPLC systems introductionHPLC systems introduction

• HPLC instrumentation includes a pump, injector, column, detector HPLC instrumentation includes a pump, injector, column, detector and recorder or data system. The heart of the system is the column and recorder or data system. The heart of the system is the column where separation occurs. Since the stationary phase is composed of where separation occurs. Since the stationary phase is composed of micrometer size porous particles, a high pressure pump is required micrometer size porous particles, a high pressure pump is required to move the mobile phase through the column. The chromatographic to move 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 phases Separation of components occurs as the analytes and mobile phases are pumped through the column. Eventually, each component elutes are pumped through the column. Eventually, each component elutes from the column as a narrow band (or peak) on the recorder. from the column as a narrow band (or peak) on the recorder. Detection of the eluting components is important, and this can be Detection of the eluting components is important, and this can be either selective or universal, depending upon the detector used. The either selective or universal, depending upon the detector used. The response of the detector to each component is displayed on a chart response of the detector to each component is displayed on a chart recorder or computer screen and is known as a chromatogram. recorder or 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 equipment are frequently used. used.

HPLC INSTRUMENTATIONHPLC INSTRUMENTATION

– Mobile phaseMobile phase ( (Problem in pumping liquid)Problem in pumping liquid)

– The common problems encountered in The common problems encountered in pumping mobile phases are pumping mobile phases are solvent solvent degassing, corrosion, and compressibility.degassing, corrosion, and compressibility.

– REMEDIESREMEDIES: : – Subjecting the liquid to vacuum Subjecting the liquid to vacuum – Heating the liquid until boiling occursHeating the liquid until boiling occurs

PUMP PUMP • Reciprocating piston pumpReciprocating piston pump

– The piston expels liquid through a one-way valve (check valve). The piston expels liquid through a one-way valve (check valve). The pumping rate is usually adjusted by controlling the The pumping rate is usually adjusted by controlling the distance the piston retracts, thus limiting the amount of liquid distance the piston retracts, thus limiting the amount of liquid pushed out by each stroke, or by the cam rotating speed. pushed out by each stroke, or by the cam rotating speed.

– Dual Piston Pumps :Dual Piston Pumps :

• A more efficient way to provide a constant and almost pulse free A more efficient way to provide a constant and almost pulse free flow is the use of dual-headed reciprocating pumps. Both pump flow is the use of dual-headed reciprocating pumps. Both pump chambers are driven by the same motor through a common chambers are driven by the same motor through a common eccentric cam; this common drive allows one piston to pump while eccentric cam; this common drive allows one piston to pump while the other is refilling. As a result, the two flow-profiles overlap each the other is refilling. As a result, the two flow-profiles overlap each other significantly reducing the pulsation downstream of the pumpother significantly reducing the pulsation downstream of the pump

INJECTORS INJECTORS

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

INJECTORS INJECTORS

ColumnColumn• Today, column material is normally Today, column material is normally

Type 316 stainless steel, once again Type 316 stainless steel, once again chosen because it offers the best chosen because it offers the best compromise of cost, workability, compromise of cost, workability, and corrosion resistance. Most and corrosion resistance. Most commercial columns available commercial columns available today have internal diameters of today have internal diameters of either 2.6-3 mm or 4.6-5 mm. Most either 2.6-3 mm or 4.6-5 mm. Most leading manufacturers obtain a leading manufacturers obtain a great amount of interchangeability great amount of interchangeability between column sizes without between column sizes without excessive fitting replacement by excessive fitting replacement by supplying columns with a variety of supplying columns with a variety of internal diameters, but a uniform ¼ internal diameters, but a uniform ¼ in. outside diameter. Naturally, in. outside diameter. Naturally, preparative columns have larger preparative columns have larger diameters: usual O.D. values are diameters: 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, respectivelyrespectively

DETECTORSDETECTORSBASIC DETECTOR BASIC DETECTOR REQUIREMENTS REQUIREMENTS

Sensitivity Sensitivity Selectivity Selectivity Response Response

Linear dynamic range Linear dynamic range

REFRACTIVE INDEX DETECTORREFRACTIVE INDEX DETECTOR• Principles Principles • The refractive index (RI) detector is the only universal The refractive index (RI) detector is the only universal

detector in HPLC. detector in HPLC. • The detection principle involves measuring of the change The detection principle involves measuring of the change

in refractive index of the in refractive index of the column effluent passing column effluent passing through the flow-cellthrough the flow-cell. The greater the RI difference . The greater the RI difference between sample and mobile phase, the larger the between sample and mobile phase, the larger the imbalance will become. Thus, the sensitivity will be higher imbalance will become. Thus, the sensitivity will be higher for the higher difference in RI between sample and mobile for the higher difference in RI between sample and mobile phase. On the other hand, in complex mixtures, sample phase. On the other hand, in complex mixtures, sample components may cover a wide range of refractive index components may cover a wide range of refractive index values and some may closely match that of the mobile values and some may closely match that of the mobile phase, becoming invisible to the detector.phase, becoming invisible to the detector.

• RI detector is a pure RI detector is a pure differential instrumentdifferential instrument, and any , and any changes in the eluent composition require the rebalansing changes in the eluent composition require the rebalansing of the detector. This factor is severely limiting RI detector of the detector. This factor is severely limiting RI detector application in the analyses requiring the gradient elution, application in the analyses requiring the gradient elution, where mobile phase composition is changed during the where mobile phase composition is changed during the analysis to effect the separation. analysis to effect the separation.

REFRACTIVE INDEX REFRACTIVE INDEX DETECTORDETECTOR

REFLECTIVE DETECTORS REFLECTIVE DETECTORS • The refractive index detector The refractive index detector

based on the Fresnel principle is based on the Fresnel principle is relatively rear .The light beam is relatively rear .The light beam is reflected from the liquid-glass reflected from the liquid-glass interface in the detecting interface in the detecting photocell. As the introduction of photocell. As the introduction of sample into onesample into one cell causes cell causes light to be refracted at a light to be refracted at a different angle. The deflection different angle. The deflection of the light beam from the of the light beam from the photoresistor photoresistor cause the cause the appearance of the electrical appearance of the electrical signal. This diff between sample signal. This diff between sample and reference signal is output to and reference signal is output to a recorder.a recorder.

ADV&DISADVANTAGEADV&DISADVANTAGE

ADVANTAGESThe major advantage of this type of detector is a very high sensitivity since the optics allow a higherconcentration of signal in a particular RI range than is possible in other wide-range detectors. Otheradvantages include the ability to operate at extremely low flow rates with very low-volume cells, easy cellaccessibility, and low cost.DISADVANTAGESIts disadvantages are the incredible sensitivity to the flow and pressure fluctuations, and the need forchanging prisms to accommodate either high or low RI solvents and the need to manually adjust the opticalpath when making solvent changes.

ULTRAVIOLET/VISIBLE ULTRAVIOLET/VISIBLE SPECTROSCOPIC DETECTORSSPECTROSCOPIC DETECTORS

PrinciplesAny chemical compound could interact with the electromagnetic field. Beam of the

electromagnetic radiation passed through the detector flow-cell will experience some change in itsintensity due to this interaction. Measurement of this changes is the basis of the most optical HPLCdetectors.

infrared (IR) 2,500 - 50,000 nmnear infrared 800 - 2,500 nmVisible 400 - 800 nmultraviolet (UV) 190 - 400 nm

UV AbsorbanceAbsorbance is the logarithm of the ratio of the intensities of the incident light (Io) and thetransmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity(molar extinction coefficient, ), the

infrared (IR) 2,500 - 50,000 nmnear infrared 800 - 2,500 nmVisible 400 - 800 nmultraviolet (UV) 190 - 400 nm

UV AbsorbanceAbsorbance is the logarithm of the ratio of the intensities of the incident light (Io) and thetransmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity(molar extinction coefficient, ), the

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

Fixed wavelength Fixed wavelength detectors detectors

• HPLC detectors which does not HPLC detectors which does not allow to change the wavelength allow to change the wavelength of the radiation called fixed-of the radiation called fixed-wavelength detectors. wavelength detectors.

• low-pressure mercury vapor low-pressure mercury vapor lamp emit very intense light at lamp emit very intense light at 253.7 nm. By filtering out all 253.7 nm. By filtering out all other emitted wavelengths, other emitted wavelengths, manufacturers have been able manufacturers have been able to utilize this 254 nm line to to utilize this 254 nm line to provide stable, highly sensitive provide stable, highly sensitive detectors capable of measuring detectors capable of measuring subnanogram quantities of any subnanogram quantities of any components which contains components which contains aromatic ring. The 254 nm was aromatic ring. The 254 nm was chosen since the most intense chosen since the most intense line of mercury lamp is 254 nm, line of mercury lamp is 254 nm, and most of UV absorbing and most of UV absorbing compounds have some compounds have some absorbance at 254 nmabsorbance at 254 nm

Variable-wavelength Variable-wavelength detectorsdetectors

• Detectors that allow the Detectors that allow the selection of the operating selection of the operating wavelength called variable wavelength called variable wavelength detectors and wavelength detectors and they are particularly useful they are particularly useful in three casesin three cases

• Depending on the Depending on the sophistication of the sophistication of the detector, wavelength detector, wavelength change is done manually change is done manually or programmed on a or programmed on a time basis into the time basis into the memory of the systemmemory of the system

Diode-array detectorsDiode-array detectors• As already mentioned, a special As already mentioned, a special

feature of some variable wavelength feature of some variable wavelength UV detectors is the ability to perform UV detectors is the ability to perform spectroscopic scanning and precise spectroscopic scanning and precise absorbance readings at a variety of absorbance readings at a variety of wavelengths while the peak is passing wavelengths while the peak is passing though the flowcell. Diode array adds a though the flowcell. Diode array adds a new dimension of analytical capability new dimension of analytical capability to liquid chromatography because it to liquid chromatography because it permits qualitative information to be permits qualitative information to be obtained beyond simple identification obtained beyond simple identification by retention time. by retention time.

• There are two major advantages of There are two major advantages of diode array detection. In the first, it diode array detection. In the first, it allows for the best wavelength(s) to allows for the best wavelength(s) to be selected for actual analysis. This is be selected for actual analysis. This is particularly important when no particularly important when no information is available on molar information is available on molar absorptivities at different wavelengths.absorptivities at different wavelengths.

• The second major advantage is related to the problem of The second major advantage is related to the problem of peak purity. Often, the peak shape in itself does not reveal peak 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 at several components. In such a case, absorbance rationing at several wavelengths is particularly helpful in deciding whether the wavelengths is particularly helpful in deciding whether the peak represents a single compound or, is in fact, a peak represents a single compound or, is in fact, a composite peakcomposite peak

• In absorbance rationing, the absorbance is measured at two In absorbance rationing, the absorbance is measured at two or more wavelengths and ratios are calculated for two or more wavelengths and ratios are calculated for two selected wavelengths. Simultaneous measurement at selected wavelengths. Simultaneous measurement at several wavelengths allows one to calculate the absorbance several wavelengths allows one to calculate the absorbance ratio. Evaluation can be carried out in two ways: ratio. Evaluation can be carried out in two ways:

• In the first case, the ratios at chosen wavelength are In the first case, the ratios at chosen wavelength are continuously monitored during the analysis: if the compound continuously monitored during the analysis: if the compound under the peak is pure, the response will be a square wave under the peak is pure, the response will be a square wave function (rectangle),. If the response is not rectangle, the function (rectangle),. If the response is not rectangle, the peak is not pure. peak is not pure.

FLUORESCENCE DETECTORSFLUORESCENCE DETECTORS • principleprinciple

• Fluorescence detectors are probably the most sensitive among Fluorescence detectors are probably the most sensitive among the existing modern HPLC detectors. It is possible to detect the existing modern HPLC detectors. It is possible to detect even a presence of a single analyte molecule in the flow cell. even a presence of a single analyte molecule in the flow cell. Typically, fluorescence sensitivity is 10 -1000 times higher Typically, fluorescence sensitivity is 10 -1000 times higher than that of the UV detector for strong UV absorbing materials. than that of the UV detector for strong UV absorbing materials.

• Fluorescence detectors are very specific and selective among Fluorescence detectors are very specific and selective among the others optical detectors. This is normally used as an the others optical detectors. This is normally used as an advantage in the measurement of specific fluorescent species advantage in the measurement of specific fluorescent species in samples. in samples.

• When compounds having specific functional When compounds having specific functional groups are excited by shorter wavelength energy groups are excited by shorter wavelength energy and emit higher wavelength radiation which and emit higher wavelength radiation which called fluorescence. Usually, the emission is called fluorescence. Usually, the emission is measured at right angles to the excitation. measured at right angles to the excitation.

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.

• Figure below shows the Figure below shows the optical schematic of a optical schematic of a typical fluorescence typical fluorescence detector for liquid detector for liquid chromatography. The chromatography. The detectors available on the detectors available on the market differ in the market differ in the method in which the method in which the wavelengths are wavelengths are controlled. Less expensive controlled. Less expensive instruments utilize filters; instruments utilize filters; medium priced units offer medium priced units offer monochromator control of monochromator control of at least emission at least emission wavelength, and full wavelength, and full capability research-grade capability research-grade instruments provide instruments provide monochromator control of monochromator control of both excitation and both excitation and emission wavelengths.emission wavelengths.

ELECTROCHEMICAL ELECTROCHEMICAL DETECTORSDETECTORS• PrinciplePrinciple

• The chromatographer because The chromatographer because of the additional selectivity of the additional selectivity and sensitivity for some and sensitivity for some compounds should consider it.compounds should consider it.

• This detector is based on This detector is based on the measurements of the the measurements of the current resulting from current resulting from oxidation/reduction oxidation/reduction reaction of the analyte at a reaction of the analyte at a suitable electrode.suitable electrode. Since the Since the level of the current is directly level of the current is directly proportional to the analyte proportional to the analyte concentration, this detector concentration, this detector couldcould used for quantificationused for quantification..

• The eluent should contain electrolyte and be electrically The eluent should contain electrolyte and be electrically conductive. Most of the analytes to be successfully conductive. Most of the analytes to be successfully detected require the pH adjustments.detected require the pH adjustments.

• The areas of application of electrochemical detection are The 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 and represent some of the most important drug, pollutant and natural product classes. For these, the specificity, and natural product classes. For these, the specificity, and sensitivity make it very useful for monitoring these sensitivity make it very useful for monitoring these compounds in complex matrices such as body fluids and compounds in complex matrices such as body fluids and natural products. Sensitivities for compounds such as natural products. Sensitivities for compounds such as phenol, catecholamines, nitrosamines, and organic acids phenol, 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 the The purity of the eluent is very important, because the presence of oxygen, metal contamination and halides presence of oxygen, metal contamination and halides may cause significant background current and therefore, may cause significant background current and therefore, noise and drift in the base line. noise and drift in the base line.

ELECTROLYTIC ELECTROLYTIC CONDUCTIVITY DETECTOR CONDUCTIVITY DETECTOR

• The conductivity of the column The conductivity of the column effluent is continuosly measured effluent is continuosly measured and the appearance of the and the appearance of the analyte in the cell is indicated analyte in the cell is indicated by a change in conductivity. by a change in conductivity.

• Usually this is a very low Usually this is a very low volume flow-through capillary volume flow-through capillary equipped with two electrodes equipped with two electrodes and variations in conductivity of and variations in conductivity of the mobile phase due to the the mobile phase due to the eluted sample components are eluted sample components are continuously recorded. continuously recorded. Response is linear with Response is linear with concentration over a wide concentration over a wide range, quantitation of the range, quantitation of the output signal is possible with output signal is possible with suitable preliminary calibration. suitable preliminary calibration.

Best use is made of this detector in Best use is made of this detector in isocratic analysis since solvent gradients isocratic analysis since solvent gradients

will cause a proportional shift in the will cause a proportional shift in the baseline. Such detectors have been used baseline. Such detectors have been used

most successfully in ion-exchange most successfully in ion-exchange chromatography of anions and cation but chromatography of anions and cation but generally, they have found only limited generally, they have found only limited

popular acceptance. popular acceptance.

EVAPORATIVE LIGHT EVAPORATIVE LIGHT SCATTERINGSCATTERING

• LIGHT SCATTERING CELL LIGHT SCATTERING CELL • The nebulized column effluent The nebulized column effluent

enters the light scattering cell. enters the light scattering cell. In the cell, the sample particles In the cell, the sample particles scatter the laser light, but the scatter the laser light, but the evaporated mobile phase does evaporated mobile phase does not. The scattered light is not. The scattered light is detected by a silicone detected by a silicone photodiode located at a 90º photodiode located at a 90º angle from the laser. The angle from the laser. The photodiode produces a signal photodiode produces a signal which is sent to the analog which is sent to the analog outputs for collection. A light outputs for collection. A light trap is located 180º from the trap is located 180º from the laser to collect any light not laser to collect any light not scattered by particles in the scattered by particles in the aerosol stream. aerosol stream.

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

Principle of HPLC