principles and application of chromatography by asheesh pandey
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PRINCIPLES AND APPLICATION OF CHROMATOGRAPHY
PRINCIPLES AND APPLICATION OF CHROMATOGRAPHYAsheesh Pandey1
CHROMATOGRAPHY
Laboratory technique for the Separation of mixturesChroma -"color" and graphein - "to write.Colour bands - separation of individual compounds Measured or analysed.
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PURPOSE OF CHROMATOGRAPHYAnalytical Determine Chemical composition of a sample
Preparative Used to purify sufficient quantities of a substanceAsheesh Pandey3
TSWETT EXPERIMENT Asheesh Pandey4
Sample clean up is usually much less of a problem with HPLC than GLC and biological fluids can often be directly onto an HPLC column. Much sample pretreatment can also be avoided because aqueous solvents can be used in HPLC.because of all these advantages, HPLC has already made a significant impact in pharmaceutical, clinical, forensic and environmental analysis and it is now an ideal complementary technique to GLC.2. TYPES OF HPLC TECHNIQUES:A. Based on modes of chromatography1. Normal phase mode2.Reverse phase modeB. Based on principle of separation1. Adsorption chromatography2. Ion exchange chromatography3. Ion pair chromatography4.Size exclusion(or)Gel permeation chromatography5. Affinity chromatography6. Chiral phase chromatography
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C. Based on elution technique1. Isocratic separation2. Gradient separationD. Based on the scale of operation1. Analytical HPLC2. Preparative HPLCE. Based on the type of analysis1. Qualitative analysis2. Quantitative analysis3. PRINCIPLE: The principle of separation in normal phase mode and reverse phase mode is adsorption. When a mixture of components are introduced into a HPLC column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster. Since no 2 components have the same affinity towards the stationary phase, the components are separated.Asheesh Pandey6
High-Performance Liquid Chromatography (HPLC)
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Chromatograph - equipment that enables a sophisticated separation EX. Gas chromatography or Liquid chromatographyEluent - Fluid entering column/ solvent that carries the analyte.Eluate - Mobile phase leaving the column.Stationary phase - Immobilized phase Immobilized on the support particles or on the inner wall of the column tubing.Examples : Silica layer - Thin Layer Chromatography
Asheesh Pandey8CHROMATOGRAPHY TERMS
Mobile phase Moves in a definite direction. Liquid (LC), Gas (GC).The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.Retention time : Time takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. Sample (Anylate) :Substance analyzed in chromatography. Solvent : Any substance capable of solubilizing another substance.
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Chromatogram Visual output of the chromatograph. Separation - Different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
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X- axis - Retention time
Y-axis - Signal
Signal is proportional to the concentration of the specific analyte separated.
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HOW TO DESCRIBE A CHROMATOGRAMAsheesh Pandey12
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Separation of PeaksAsheesh Pandey14
Retention Asheesh Pandey15k = (tr to)/ to
Where tr = the retention time of the compound, and to = the dead time
Higher values of k mean the analyte will stay in the column longer. The longer it stays, the more time there is for the peak will widen.
SelectivityAsheesh Pandey16a = kB/kA
the selectivity factor and is an indication of how well the compounds will separate. Higher means larger difference in retention time and more separation
EfficiencyAsheesh Pandey17The term that is generally used to describe column efficiency is number of theoretical plates or NN = L/H
Where: L =column lengthH = plate height (both in the same units)
N in Practical Terms...Asheesh Pandey18Units for tr and to.?Units for W1/2 ..?N can be measured from the peaks on a chromatogram..N = 5.54 trw1/2()2
ResolutionAsheesh Pandey19The purpose of chromatography is to separate or resolve compounds. The separation or distance between two peaks is known as their resolution and is a function of the 3 factors discussed previously: retention (the time it takes for the analytes to elute, related to k), selectivity (how different the analytes are from each other and related to ), and efficiency (how good the column is, related to N)
ResolutionAsheesh Pandey20Rs = (a-1/a) (k/k+1) NThe effect on Rs of:increasing a?increasing k?increasing N?EfficiencySelectivityRetention
ResolutionAsheesh Pandey21Rs = 2 (tR-B tR-A)/(wb-A + wb-B)
Where: A and B are the two peakstR = retention time andwb = the peak width at the base of each peak
Rs can also be calculated from actual measurements of peak retention times and measured peak widths
Retention Time: The time from the start of signal detection by the detector to the peak height of the elution concentration profile of each different sample.Curve Width: The width of the concentration profile curve of the different samples in the chromatogram in units of time.RESOLUTION (RS) :Rs = 2(tRB tRA)/(wB + wA)Where:tRB = Retention time of solute BtRA = Retention time of solute A wB = Gaussian curve width of solute B wA = Gaussian curve width of solute APlate Number (N):N = (tR)2/(w/4)2Plate Height (H):H = L/NWhere L is the length of the column.Asheesh Pandey22
ResolutionAsheesh Pandey23With a resolution value of 1.0, two peaks that overlap by about 4%. Values less than 1.0 indicate peaks that overlap, while at a resolution of 1.5, the peaks are considered fully separated.
Retention factor : R = Distance travelled by a Solute Distance travelled by a Solvent R = zero, - Solute remains in the stationary phase and thus it is immobile. R = 1 - Solute has no affinity for the stationary phase and travels with the solvent front. Asheesh Pandey24
Going back to N.Asheesh Pandey25N = L/HThe value of N is greatly dependent on the value of H.The value of H depends primarily on four factors:1) the velocity of the mobile phase,2) eddy diffusion or multipath diffusion, 3) the diffusion of the compound in the mobile phase4) the transfer of the compound between the stationary phase and the mobile phase.
H - Theoretical Plate HeightH = A + B/u + (Cs + Cm) uu = the average linear mobile phase velocity A is a term expressing multipath diffusionB/u is the term for longitudinal diffusionCs is the mass transfer term in the stationary phase Cm is the mass transfer term in the mobile phaseH = A + B/u + (Cs + Cm) uAsheesh Pandey26
A Multipath
12FlowDirection
Pathways of two molecules during elution. Distance traveled by molecule 1 is longer than that traveled by molecule 2, thus molecule 1 will take longer toelute.The amount of spreading is affected by the nature of the column material and how well the column is packed. This factor is generally proportional to the particle size of the packing material. This factor must be taken into account for packed columns, but for capillary columns, this term is not needed since there are no particles.Asheesh Pandey27
B Longitudinal Diffusion
Flow
Flow
Molecules diffuse from areas of highconcentration to areas of low concentration.
Over time.
At low velocities longitudinal diffusion has a negative effect on resolution, but this effect is negligible at higher velocities. This term is very important in gas chromatography as diffusion coefficients in gasses are orders of magnitude higher than in liquids. In liquid chromatography, this term is typically close to zero relative to the other terms.Asheesh Pandey28
Equilibrium between the mobile and stationary phases is never realizedMass Transfer Terms Cs & CmIt takes time for analytes to move from the mobile phase into the stationary phase. Because no equilibrium is reached, some of the analytes are swept ahead of the of the main band. It also takes time for molecules to move back out of the stationary phase, and some of the analyte molecules will be left behind by the rapidly moving mobile phase.Asheesh Pandey29
Mass Transfer Terms Cs & CmThe faster the mobile phase moves, the less time there is for equilibrium between the phases and the mass transfer effect on peak broadening is directly related to mobile phase velocity.Asheesh Pandey30
van Deemter PlotAsheesh Pandey31
Linear Velocity, uPlate Height, H
Multipath Term, A
Mass Transfer (both), CuLongitudinal diffusion, B/u
A + B/u + Cu
H = A + B/u + (Cs + Cm) u
PRICNIPLES OF CHROMATOGRAPGHY
Physical method of separation that distributes components to separate between two phases moves in a definite direction.
Substances are separated based on their differential distribution between two phases
Substances will move with the mobile phase at different rate depending upon their Partition or Distribution co-efficients.
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PRINCIPLESThe samples are subjected to flow by mobile liquid phase onto or through the stable stationary phase. Separation of fractions of mixture based on their relative affinity towards the two phases during their travel.The fraction with greater affinity to stationary phase travels slower and shorter while that with less affinity travels faster and longer.The separation is based on Differential partitioning between the mobile and stationary phases.
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FACTORES AFFECTING THE SEPARATIONIntermolecular interaction between the two phases
Extent of dispersion of solute molecules over the stationary phase
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CLASSIFICATION OF CHROMATOGRAPHYTechniques by Chromatographic bed shape Column chromatography Planar chromatography Paper chromatography Thin layer chromatographyTechniques by Physical state of mobile phase Gas chromatography Liquid chromatographyAffinity chromatography Supercritical fluid chromatography Asheesh Pandey37
TECHNIQUES BY CHROMATOGRAPHIC BED SHAPE
A.COLUMN CHROMATOGRAPHYPRINCIPLESSolid materials (Adsorbants) Ability to hold the molecules at their surfaceAttractive forces (Vanderwalls & Hydrogen )Functional groups (Hydroxyl/ Aromatic)Silica
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Stationary bed is within a tube. Solvent is driven through the column by applying Positive pressure.Separations - 20 minutes
Modern flash chromatography :Pre-packed plastic cartridges, Solvent is pumped through the cartridge. Quicker separations Less solvent usage.
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Column : Diameter - 5mm to 50mm Height - 5cm to 1 m with a tap Filter (a glass frit or glass wool plug)The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. During the entire chromatography process the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g., UV absorption, or fluorescence.
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STATIONARY PHASE
Asheesh Pandey41 Silica gel, Alumina. Cellulose
SOLVENTSHydroxyl groups - AlcoholCarboxyl group - AcetoneNon polar Compounds Hexane Heptane TouleneAsheesh Pandey42
Flow rate - Separation. Pump or compressed gas (e.g. Air, Nitrogen, Argon)A faster flow rate of the eluent: Minimizes the time required to run a column Minimizes diffusion Better separation.
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B. PLANAR CHROMATOGRAPHYSeparation technique - Stationary phase is present as or on a plane.Paper Paper ChromatographyLayer of solid particles spread on a support such as a glass plate - Thin layer Chromatography.Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. Retention factor (Rf)Asheesh Pandey47
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PAPER CHROMATOGRAPHYAsheesh Pandey49
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This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.
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b) THIN LAYER CHROMATOGRAPHY
Widely employed laboratory technique Stationary phase - Adsorbent - Silica gel Alumina CelluloseWidely used in pharmaceutical & food stuff industry Advantages :Simple, Rapid and CheapFaster runs Better separationsChoice between different adsorbents. Better resolution Allow for quantification
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Used to identify the unknown compounds and to determine the purity of mixture.
TLC Plate - Aluminium or glass - coated by stationary phase.Coated material : 0.1-0.3mm in thickness Fluorescent indicator that will make it florescence during the UV light exposure.Asheesh Pandey55
MOBILE PHASE
Volatile Organic solventsAsheesh Pandey56STATIONARY PHASESilica gel, Alumina, or Cellulose on a flat, inert substrate.
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SPRAYSAsheesh Pandey59
2.TECHNIQUES BY PHYSICAL STATE OF MOBILE PHASEA. GAS CHROMATOGRAPHYGas-Liquid chromatography, (GLC)Mobile phase Gas (Helium) Carrier Gas Pressure = 4 kg/cm2 Stationary phase - Column, which is typically "packed" or "capillary".The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column).Partition Coefficient of Volatile analyte between a solid stationary phase (Silicone) and a mobile gas (Helium).
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AdvantagesHigh sensitivity, High Resolution, High speedHigh Accurasy, Highly Quantitative APPARATUS Gas Chromatograph, GC analyzer, Normal syringes and one micro syringe, Beakers, Sample bottles and Electronic weight.
CHEMICALS Methanol, Isopropyl Alcohol and water
SAMPLE: Gases, Liquid, SolidsM.Wt: 2-800Volatile
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APPLICATIONQuantitative & Qualitative analysis of low polarity compoundsAnalytical chemistry, Biochemistry, Petrochemical, Environmental monitoringMeasure picomoles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samplesMeasuring toxic substances in soil, air or water. Asheesh Pandey63
APPLICATION OF GC- MSEnvironmental monitoring : Oraganic PollutantsCriminal forensics : Analyze the particles (Fibre) from a human body in order to help link a criminal to a crime.Law enforcement : Detection of illegal narcotics, Forensic toxicology : Find drugs and/or poisons in biological specimens of suspects, victims, or the deceased.Sports anti-doping analysis : Test athletes' urine samplesSecurity : Explosive detection (September 11 development) systems have become a part of all US airports.Food, beverage and perfume : from spoilage or Adultration - aromatic compounds, esters, fatty acids, alcohols, aldehydes, terpenesMedicine : Congenital metabolic diseases In Born error of metabolism
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B. LIQUID CHROMATOGRAPHYMobile phase - Liquid. Column or a plane. Very small packing particles and a relatively high pressure -High Performance Liquid Chromatography (HPLC).
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LC- MSMass spectra is obtained rapidlySmall amount of material is required to form the spectra.Data collected is highly informative with respect to molecular structure.
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APPLICATION Pharmacokinetics : How quickly a drug will be cleared from the hepatic blood flow and organs of the body. Proteomics : Peptide mass fingerprintingDrug development: Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite Identification, Impurity Identification, Degradant Identification, Quantitative Bioanalysis, and Quality Control.Fungal toxinsPesticides, HerbicidesAsheesh Pandey72
HIGH PERFORMANCE LIQUID CHROMATOGRAPHYHPLC V/S LC TECHNIQUEColumns : Small diameter (4.6 mm), stainless steel, glass or titanium. Column packing with very small (3, 5 and 10 m) particles Relatively high inlet pressures and controlled flow of the mobile phase.Detecting very small amountsHigh resolution Rapid analysisSpeed, efficiency, sensitivity and ease of operationHigh degree of versatilityEasily separate a wide variety of chemical mixtures400atmospheres. PUMP PRESSURE"Ultra High Performance Liquid Chromatography" systems 1000atmospheres.
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ELUTION : Isocratic and Gradient.
ISOCRATIC :ISO ==> SAME- Solvent Composition Stays the Same for the Entire Run EX: 60:40 Alcohol:Water
GRADIENT : Solvent Composition Changes Throughout the Run
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TYPES OF HPLCNature of the stationary phase Separation process
Adsorption chromatographyIon-exchange chromatographySize exclusion chromatographyAsheesh Pandey79
APPLICATIONProtein separationInsulin purificationPlasma fractionationEnzyme purificationAsheesh Pandey80
SIZE EXCLUSION CHROMATOGRAPHYGel filtration or gel permeation chromatography Separation - Molecular size of its components.Larger molecules are rapidly washed through the column, smaller molecules penetrate inside the porous of the packing particles and elute later.
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APPLICATIONS
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AFFINITY CHROMATOGRAPHY
Based on specific & non-covalent binding of the proteins to other molecules Ligands ( His-tags, biotin or antigens)Physical properties of the analyte.
Biochemistry in the purification of proteins (Enzymes) bound to tags.
After purification, some of these tags are usually removed and the pure protein is obtained.
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SUPERCRITICAL FLUID CHROMATOGRAPHYUsed for the analysis and purification of low to moderatemolecular weight , thermally labile molecules. Principles are similar to those of(HPLC)Mobile phase - High pressure liquid orSuper critical Carbon Dioxide.Modifiers Methanol, Ehanol, isopropyl alcohol, acetonitrile and Chloroform.APPLICATIONUse in industry primarily for separation ofChiral (Asymmetric Carbon atoms) molecules.Serine Soman Glyceraldehyde Phosphours (Phosphine) Sulfar metal Cobalt Enkephalins
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DETECTORGas Chromatography or liquid Chromatography To visualize components of the mixture being eluted off the chromatography column.
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DETECTORSFlame ionization detectorAerosol-based detector Flame photometric detector (FPD). Atomic-emission detector (AED).Mass spectrometer ( MS) detectorNitrogen Phosphorus Detector, Evaporative Light Scattering Detector (ELD) : LC.
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DETECTORSUV detectorsThermal conductivity Detector, (TCD)Fluorescence detectorElectron Capture Detector, (ECD)Photoionization Detector, (PID)Refractive indexDetector (RI or RID)Radio flow DetectorChiral Detector
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HPLC InstrumentationAsheesh Pandey90
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In This Section, We Will Discuss:General components of a high performance liquid chromatograph.HPLC solvent delivery systems.How automatic injectors work.Common HPLC detectors.Asheesh Pandey91
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HPLC Instrumentation OverviewAsheesh Pandey92
Principle PatternAn Example
DetectorThermostatted Column CompartmentAutosamplerBinary PumpVacuum DegasserSolvent CabinetSolvent ReservoirsController
Solvent FiltersSolvent Inlet FilerStainless Steel or glass with 10 micron porosity.Removes particulatesfrom solvent.Precolumn FilterUsed between the injector andguard column.2 to 0.5 micronRemoves particulates from sampleand autosampler wear debris.Must be well designed to preventdispersion.Asheesh Pandey93Guard column
Injector
Analytical ColumnPrecolumn Filter
Solvent Inlet Filter
Vacuum DegassingAsheesh Pandey94
Functions of the Solvent Delivery SystemThe solvent delivery system has three basic functions:
Provide accurate and constant flow.Provide accurate mobile phase compositions.Provide the force necessary to push the mobile phase through the tightly packed column.Asheesh Pandey95
Multichannel Gradient ValveDetermines mobile phase composition.
Largest solvent plug fills first.
Agilent 1100 and 1200 quaternary pump.96
Dual Piston Parallel PumpAsheesh Pandey97
SinglePistonDelivery
CombinedDelivery
Piston 'A' AdvancingPiston B Retracting
CheckValves
AB
PumpheadPistonRotarySwitchingValve
Dual Piston in Series PumpAsheesh Pandey98
First piston displaces solvent at twice the speed and stroke volume of the second piston.
Provides constant flow and the pressure necessary to get through column.
Ballvalves for Reciprocating Piston PumpsAsheesh Pandey99
Pump Seals and PistonsAsheesh Pandey100
PistonSupport RingsSeal KeepersSealsWear Retainers
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Frits and FiltersAsheesh Pandey101Frits, Filters, and Sieves are used to protect other parts of the LC from pump and seal material.
Purge valvePTFE Frit
Damping UnitsFilled with compressible liquid separated from the mobile phase by a membrane.Pressure ripples reduced to < 2% original value.102
Damping Unit
Pressure2% P/PPump Ripple
Gradient FormationAsheesh Pandey103
Low Pressure GradientHigh Pressure Gradient
SummaryThe pump is the most critical piece of equipment for a successfully operating HPLC.
Performance parameters for HPLC pumps:
Flow PrecisionFlow RangeDelay VolumePressure PulseComposition PrecisionAsheesh Pandey104
Sample InjectorsRequirements:
Reproducible introduction of the sample volume into the mobile phase flow.
Two major designs:Automatic Injectors or Manual InjectorsAsheesh Pandey105
Manual InjectorsAsheesh Pandey106
Front ViewInjectRear ViewLoad - Inject
Sample Loop
Manual InjectorsAsheesh Pandey107
Sample inSolvent inSolvent outSample LoadSample Inject
From PumpTo columnSample inSolvent inSolvent out
From PumpTo column
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Step 1Step 2Step 3
Rotor SealsAsheesh Pandey109
Rotor Seal found withinvalve
Column OvenConstant temperature for solvent and column is required to perform reproducible results.110
Common HPLC DetectorsAsheesh Pandey111
UV-VISDiode ArrayMultiple WavelengthVariable Wavelength
Mass Spectrometers
Refractive Index
Fluorescence
Light Scattering
Electrochemical
Radioactivity
Conductivity
Necessity for More Than One Detector - SensitivityAsheesh Pandey112
Necessity for More Than One Detector - SelectivityAsheesh Pandey113
Necessity for More Than One Detector - Qualitative InformationAsheesh Pandey114Qualitative Information
Take peak spectrum (UV)
Chlortoluron ?
44685896132138158172215200Take peak spectrum (MS)104
Mass/ChargeAtrazine ?
Wavelength (nm)6080100120140160180200220
HPLC Detector CharacteristicsDetector performance characteristics: Sensitivity (LoD, LoQ) Selectivity Linearity Qualitative information Reliability Ease of use UniversalityAsheesh Pandey115
LODThe limit of detection for a detector can be characterized by its signal to noise ratio (S/N) for an analyte under a given set of conditions.Asheesh Pandey116
NoisePeak
Limit of Detection - Limit of QuantitationLimit of detection (LOD) is a result of the whole chromatography system, not only the detector performanceLimit of quantification (LOQ) is a defined limit for a method used for a specific purpose.117
Linear rangeSlope = sensitivityMQLMDLResponseAmountIntercepte.g.,RSD 20e.g., S/N > 3
UV-Vis DetectorsAsheesh Pandey118
bc
Detector Flow CellI0ILog I0 = A = abcI
Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beers Law.Characteristics: Specific, Concentration Sensitive, good stability, gradient capability.Special: UV-Vis Spectral capability (Diode Array Technology ).
UV-Vis Detectors - Design PrinciplesSingle wavelength detection of multi wavelength detection possible. Wavelength calibration is done automatically using a holmium filter.119
UV LampGratingFlow cellReference diodeSample diodeCut-off filterHolmium oxide filterSlitMirror 2Mirror 1
Variable WavelengthDetector
UV-Vis Detector with Spectral CapabilityDiode Array UV-Vis Detector allows online measurement of spectra.Wavelength range 190 - 950 nm. Wavelength Resolution: Up to 1 nm.Wavelength calibration with Holmium oxide filter.120
Diode ArrayGratingOpticalSlitDetectorFlow CellHomiumFilterAchromaticLensUVLampVisLamp
Fluorescence DetectionAsheesh Pandey121
Electrochemical DetectorsGold for carbohydrates.Platinum for chlorite, sulfate, hydrazine, etc.Carbon for phenols, amines.Silver for chloride, bromide, cyanide.122
Thin-layer design
Porous flow-through design
Wall-jet design
HPLC-MSD API- ElectrosprayAsheesh Pandey123
Refractive Index Detector DesignThe Refractive Index Detection is strongly influenced by:Pressure changesTemperature changes Flow pulseGradient elution is not possible!124
Conductivity DetectorsAsheesh Pandey125
ref.capacitorcellvariable resistancesfixed resistorCrBalancecontrol AEFDB
~Schematics
Applications
water
soap products
detergents
soft drinks
blood
plating baths
nuclear fuel reprocessing
streamsIonsAcidsBasesSaltsin}