Chapter 10Introduction to Chromatography
SeparationImportant areas in this chapter:Basic concepts; partition coefficient; retention time;capacity factor; selectivity factor; band broadening;relationship between plate height and columnvariables; optimization of column performance;qualitative applications; quantitative applications:peak height; peak areas; internalcalibration.
Chromatographic separationsSample is dissolved in a mobile phase (a gas, aliquid or a supercritical fluid);The mobile phase is forced through an immisciblestationary phase which is fixed in place in acolumn or on a solid surface.The two phases are chosen so that the componentsof the sample distribute themselves between themobile and stationary phase to a varying degree.
Classification of chromatographic methods:1. physical meanscolumn chromatography: stationary phase is held ina narrow capillary through which the mobile phase isforced under pressure or by gravity;planar chromatography: stationary phase issupported on a flat plate or in the interstices of apaper. Themobile phase moves through the stationary phase bycapillary action or under the influence of gravity.
2. mobile and stationary phases Liquid chromatography Gas chromatography Supercritical-fluid chromatography
Separation of amixture ofcomponents A andB by column elutionchromatography. (b)The output of thesignal detector atthe various stages ofelution shown in (a).
A typical chromatogram for a two-component mixtureThe small peak on the left represents a species that is notretained on the column and so reaches the detector almostimmediately after elution is started. Thus its retention time tM isapproximately equal to the time required for a molecule of themobile phase to pass through the column.
Retention time, tR:tR is the time it takes after a sample injection for theanalyte peak to reach the detector.tM is the time for the unretained species to reach thedetector, dead time. The rate of migration of theunretained species is the same as the average rate ofmotion of the mobile phase molecules.
Retention time and retention volume
Concentration profiles of analyte bands A and B at two differenttimes in their migration down the column. The times t1 and t2are indicated in the figure above
Effects of migration rates and bandbroadening on resolutionlonger column distance enable peaks separatedbetter, but with more peak broadening, whichlowers the efficiency of the column as a separatingdevice.
Methods of improving separators(a) original chromatogram with overlapping peaks; improvements broughtabout by (b) an increase in band separation, and (c) a decrease in band spread.
Partition (distribution) CoefficientcoefficientPartition CoefficientIndependent of conc., butTemperature dependentMobile stationaryAs K increases the solute takes a longer time to elute
Partition Coefficient, K K should be constant over a large range ofconcentration range. Cs is directly proportional to CM Chromatography where this equation holds is linearchromatography
The relationship between retention time andpartition coefficients: relate tR with Kv u u x fraction of time an analyte spends inmobile phasex (moles of analyte in mobile phase/totalmole of analyte)uux (CM x VM / (CM x VM Cs x Vs ))x (1 / ( 1 Cs x Vs /CM x VM ))(1)tR vX L
The rate of solute migration: Capacity (Retention factor)(2)(2)(1)(3)
vSubstitutingandequation (3) yieldsufrom the above equations into
When capacity factor is less than 1,elution occurs so rapidly that accuratedetermination of tR is difficult as a resultof peak broadening When capacity factor is 20 to 30, elutiontimes become too long. Ideally, separation occurs underconditions of capacity factors in the rangeof 1 to 5
The selective factor aKB is the partition coefficient for the more strongly retainedspecies. Thus, a, is always 1
Since K’A can be expressed as:Then,sinceConsequently,
Retention Volume, Vr Vr : Volume of mobile phase required toelute sample component (move completelyfrom column) Vr tr X F Since
LXF LXF1 XVV1 k'rM1 1 XV VrM11 k'AAVr can be obtained from the chromatogramsince Vr tr X F
Mobile phase volume is proportional tocolumn length. So retention is alsoincreased for longer columns As peaks travel through the column, theybroaden. Peak width increases with the squareroot of column length Thus, longer columns wont give betterresolution
Methods for describing column efficiencyTheoretical plates In solvent extraction, a plate is represented by eachequilibrium (extraction) is conducted In chromatographic column, the plates aretheoretical The number of theoretical plates can be estimatedbased on peak retention times and widths
Column Efficiency - Theoretical Plates(Quantitative measures of separation efficiency)Distribution of molecules along the length ofThe column at the moment the analyte peak reachesthe end of the columnH plate heightN number of platesLN HH 2L is the standard deviation of measurementsEfficiency is defined in terms of variance per unit length
Relation between column length andretention timesL column length (distance) standard deviation in distancet R retention time standard deviation in time L tR L / tR
L tR LtRW 4 WL 4 tR 2W2 LH L16 t R2 LowH values achieve better separation In GC H 1 to 3 mm In HPLC, H is one to two orders of magnitude lower
Determining the Numberof Theoretical PlatesN number of pates tR N 16 W where2 L/Ht R retention timeW approximate base width of peakW is derived from the intersection of the baselinewith tangents drawn through inflection points on thesides of each peak
Experimental determination of N Measuring the peak width is not alwayssimple The peak may co-elute with another The low detector sensitivity may resultin a difficulty in finding the start andthe end of the peak Is there an alternative way?
The width at half height of the peak ismeasured Thus, the measurements are madewell above the background and awayfrom any detector sensitivity limitproblem
Since the peak is Gaussian, the number ofplates, N, can be determined by the followingmodified formulatRN 5.54()W1/ 2For a fixed length column the height equivalent to atheoretical plate , H, can be determined from theequation:H column height/N
Column Resolution Column resolution, Rs, provides aquantitative measure of the ability ofthe column to separate two analytes It is s measure of how completely twoneighboring peaks are separated fromone another
Column resolutionWA WB WRs (t R ) B (t R ) AW tR N 16 W 4tW RN2(t R ) B (t R ) ANRs (t R ) B4
Sample Capacity It is the amount of sample that can besorbed onto a particular stationaryphase before overloading occurs Exceeding the capacity results in :asymmetric peaks, change in retentiontime, loss of resolution. Sample capacity is proportional to Vs
Chromatographic Compromise Sample capacity, speed and resolution aredependent. Any one of the 3 can be improvedat the expense of the other 2. Always there is a compromise In LC, speed and resolution are desired.Sample capacity is not important providedthat detectable amount of sample isseparated In preparative LC, speed is usually sacrificed
Kinetic variables affecting band broadening Band broadening is a consequence of thefinite rate at which several mass-transferprocesses occur during migration of aspecies down a column. Some of theses rates are controllable byadjustment of experimental variables, thuspermitting improvement of separation
Effect of flow rate of mobile phaseLiquidchromatographyGaschromatography
Relationships between plate heights andcolumn variablesVan Deemter EquationModified Van Deemter Equation
(Longitudinal diffusion)to and from liquidStationary phase and in mobile phase
relationship between plate height and column variablesVan Deemter EquationModified Van Deemter EquationMultiple flow path(Eddy diffusion)Longitudinal diffusion Mass transfer between phases
Van Deemter eq.in the Table above
Multiple Pathways (Eddy diffusion)
Eddy DiffusionA 2ldRwhere l packing factordR average diameter ofparticle caused by many pathways minimized by careful packing
Longitudinal Diffusion: B/uColumnDiffusion
Longitudinal Diffusion B//uB 2yDM/uwhere y obstruction factorDM diffusion coefficient ofsolute in the mobile phase minimized by lowering temperatureof column oven and decreasing flowrate
(Cs CM)u Term: Resistance to mass transferIt takes time fora solute to reachequilibrium betweenthe two phasesThick or viscousstationary phaseshave larger termsThus, there is a lowerrate of mass transferand an increase inplate height
Csu Term Csu differs depending upon thestationary phase (l or s)– When stationary phase is liquid, Csu isdirectly proportional to d2f and inverselyproportional to diffusion coefficient ofspecies in the film– When stationary phase is solid, Csu isdirectlyproportional to the time required for thespecies to be adsoebed or desorbed
Cmu Term Cmu is inversely proportional to diffusion coefficient of theanalyte in the mobile phase and some function of the square ofthe particle diameter of the packing d2p
van Deemter plot for a packed liquidchromatographic columnH The points on the upper curve are experimental. The contributions of the various rate terms are shown by the lower curves: A,multiple path effect; B/u, longitudinal diffusion; Cu, mass transfer for bothphases.
Two important variables: diameter of the column andthe diameter of the particles packed in the columnEffect of particle size on plate height. The numbers to the rightare particle diameters.
Applications of Chromatography Qualitative Analysis Quantitative Analysis– Analyses Based on Peak Height– Analyses Based on Peak Areas– Calibration and Standards– The Internal Standard Method– The Area Normalization Method
GC & LC Qualitative andQuantitative Analysis
Qualitative analysis: Main approach GC is a blind method that indicates the presence of asubstance but not what it is. Qualitative analysis is based upon comparison ofretention data that are characteristics but not unique Retention data used :– Retention time,– retention distance,– retention volume These are dependent upon: Column dimensions, liquidphase (type and amount), column temperature, flowrate, type of carrier gas, packing density, pressure drop
Factors affect tR from two different columns ofthe same type Packing densityLiquid loadingActivity of the supportAge & previous use of the packingVariation in composition of the column wall Thus, when two separate columns must be used,Relative Retention date is preferred. Since tR values are characteristic of a particularsample in the column conditions but are notunique!!– run the unknown sample immediatelybefore or immediately after the standard;and all conditions are the same.
Component 1 is used as the reference; it should bepresent or added to the sample and compatible with thesample Peak of component 1 must be close (but resolved) to thesample peak
Relative retention eliminates variations in– Column diameter and length– liquid phase loading– Carrier flow rate– Others Referring the sample to 2 references givesdifferent results
When component 3 is suspected add more of this component to thesample and watch any change in its peak
Only 3 compounds are needed to establish the line The line can be used to identify other members of the Samehomologous series
Homologous and Pseudo-homologous iphatic aminesPyridine homologs.Aromatic hydrocarbons, dialkyl ethers,thiols, alkylnitrates, substitute tetrahydrofuran,tetrahydrofuran
Kovats Retention Index, R.I. R.I. indicates where a compound will appear withrespect to normal paraffins R. I (n-paraffins) (by definition) # C atoms x 100(regardless of column or conditions) R.I of any solute is derived from a chromatogram ofa mixture of the solute at least two normalparaffins having t'R close to that of the solute. R.I. values change with column They are used for qualitative analysis R.I values are meaningless without the conditionsbeing reported
Quantitative AnalysisSteps : Sampling Sample preparation Chromatography Integration Calculations Peak height or peak area are the basis forquantitative analysis
Basis for Quantitative Analysis The peaks in the chromatogram are the basis forquantitative analysisPeaks of interest should fulfill the following requirements: must be undistorted must be well separated Must have a large S/N ratio must have a flat aselinePeak shape: The ideal chromatographic peak issymmetric and narrowTo get symmetrical peaks following should emphasized:* Clean entry through the septum* Rapid depression of the syring plunger* Quick withdrawal of the syringe* Choosing proper column conditions* Temperature programming helps avoiding thebroadening of the later peaks in an isothermaltechniques.
Peak separation The resolution of at least 1 must be achieved for allpeaks of interest If the two peaks are fused together, dropline techniqueshould be considered first, where the fused peaks areseparated by dropping a perpendicular from the vallybetween them to the baseline.Peak size Most appropriate peak is a big one on a quite flatbaseline.Linear range It is desirable to operate in the linear range of thedetector system. When the linear range of the detector is narrow it isoften necessary to make several dilutions until a linearrange is found.
Response Measurements:The computer will take care of the chromatographicpeaks and give information like: retention times; peakheights; peak areas; calculations and comparison withmemory values
Calculation Methods126.96.36.199.5.Area normalizationNormalization with response factorsExternal standard methodInternal standard methodStandard addition method
1. Area normalization (Internal normalization)2. Normalization with response factors
column chromatography: stationary phase is held in a narrow capillary through which the mobile phase is forced under pressure or by gravity; planar chromatography: stationary phase is supported on a flat plate or in the interstices of a pape
2.3.1 Liquid chromatography (LC) 20 Size-exclusion chromatography (SEC) 20 Ion-exchange chromatography (IEC) 22 Reversed-phase chromatography (RP) 23 Reversed-phase ion pairing chromatography (RPIP) 24 2.3.2 Gas chromatography (GC) 25 2.3.3 Electrophoretic techniques 25 2.3.4 Isotope dilution analysis (IDA) 26 3 EXPERIMENTAL RESEARCH 27 Aims 27
Part One: Heir of Ash Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Chapter 11 Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17 Chapter 18 Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 Chapter 24 Chapter 25 Chapter 26 Chapter 27 Chapter 28 Chapter 29 Chapter 30 .
Chromatography 481 27.4.2 Partition (Liquid–Liquid) Chromatography 482 188.8.131.52 Introduction 482 184.108.40.206 Coated Supports 483 220.127.116.11 Bonded Supports 483 27.4.3 Ion-Exchange Chromatography 483 27.4.4 Size-Exclusion Chromatography 485 27.4.5 Afﬁnity Chromatography 488 27.5 Analys
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2. Gas Chromatography (Chapter 2 & 3) 3. Liquid Chromatography (Chapter 4 & 5) 4. Other Analytical Separations (Chapter 6-8) a. Planar chromatography b. Supercritical fluid chromatography c. Electrophoresis d. Centrifugation e. Field Flow Fractionation Separation Sciences
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4 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 11 4.1 Types of chromatography column 13 4.1.1 Reversed phase chromatography 13 5 LIQUID CHROMATOGRAPHY DETECTOR 14 5.1 Ultraviolet (UV) detector 14 6 INTRODUCTION TO SOLID PHASES EXTRACTION 16 6.1 Columns in solid phase extraction 17 6.1.1 Sorbent selectivity 17 6.1.2 Silica based sorbents 18
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