Chemistry II Chromatography Real samples are generally complex mixtures and substances interfere with each other in their analyses Chromatography ID: 1043301
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1. CHROMATOGRAPHYAnalytical Chemistry II
2. ChromatographyReal samples are generally complex mixtures and substances interfere with each other in their analyses. Chromatography is the set of techniques used for the analyses of mixtures.Stationary phase and mobile phaseBeginning of 20th century, TswettChroma:color graphein: writingColumn chromatography and planar chromatography
3. Intermolecular interactions+-+-Ionic interactionHydrogen bondingcationanionwaterδ+ δ- δ+ δ- δ+ δ- δ+ δ- Dipole-dipole interactionδ+ δ-δ+ δ-δ+ δ-Dispersion forces
4. POLARITYDipole moment, unit DebyeWater: 1.85 D Benzene: 0 DOctanol-water partition coefficient Kow = CA, Octanol / CA, waterLike dissolves likewateroctanolNonpolar solutePolar soluteSolute prefers octanolHigh Kow value Solute prefers waterLow Kow value
5. ClassificationSpecific methodStationary phaseType of interactionGas Chromatography (GC)Gas – liquid Gas – solid Liquid adsorbed or bonded to a solid surfaceSolidPartition between gas and liquidAdsorptionLiquid Chromatography (LC)Liquid – liquid Liquid – solid Ion exchangeSize exclusionAffinityLiquid adsorbed or bonded to a solid surfaceSolidIon-exchange resinLiquid in interstices of a polymeric solidGroup of specific liquid bonded to a solid surfacePartition between immiscible liquidsAdsorptionIon ExchangePartition / sievingPartition between surface liquid and mobile liquidSupercritical fluid chromatography (SFC)Organic species bonded to a solid surfacePartition between supercritical fluid and bonded surface
6. Elution is a process in which solutes are washed through a stationary phase by the movement of a mobile phase.The mobile phase that exits the column is termed the eluate.The eluent is the portion of the sample contained in the mobile phase moves down the columnDetectorcolumnsampleABMobile phaseABBSolvent carry solute molecules down the column in a continuous series of transfers between the two phases. Because solute movement can occur only in the mobile phase, the average rate at which a solute migrates depends on the fraction of time it spends in that phase. This fraction is small for solutes that are strongly retained by the stationary phase (component B) and large where retention in the mobile phase is more likely (component A). Ideally, the resulting differences in rates cause the components in a mixture to separate into bands, or zones, along the length of the column
7. Paper ChromatographyAbundant, cheap and porous material Inert support is cellulose which is polymer of glucosePolarity differencesApplication:Baseline must be drawn by pencil. Solution level must be under the level of base line.Never allow the running solvent to elute to the end of paper. This affects overall resolution by clustering components at the top of the paper.When the paper is dried, analytes are seen as colored plates. If the compunds do not have a color then they can be monitored under UV light or using sublimation of iodine. More intense color means higher concentration.
8. Thin Layer ChromatographyStationary phase: An adsorbent material is coated on the surface of glass or plastic. Adsorbent materials: alumina (Al2O3) and silica (SiO2).Alumina (more polar) for hydrocarbons, alkyl halogenides, ether, aldehydes and ketones. Silica (less polar) for alcohol, carboxylic acids and amines.TLC is used more than paper chromatography.Quantitatively, Spots can be scraped off and dissolved in a solvent for analysis.The size of spots can be usedOptical densitometryTwo way TLC
9. Column ChromatographyAdsorbents:Alumina, silica or calcinated diatomeSupport material glass columnBelow adsorbent porous glass or coton is placed.Separated compounds can be observed with a UV lamp or a derivatization with a coloring agent if they are colorless.Görsel: http://www.wikiwand.com/en/Column_chromatography Erişim Tarihi: 24.02.2019
10. High Performance Liquid Chromatography (HPLC)Automatized column chromatography Sensitive, suitable for both quantitative and qualitative analyses, applicable for non-volatile compoundsFor pharmaceuticals and food samples, the golden standard10-30 cm length 4-10 mm diameter steel columns filled with 2-25 µm diameter particles Stationary phase liquid or a thin film layer on solid particles (adsorbing or chemical binding)The liquid in the mobile phase and stationary phase must be immiscible.Liquid-liquid chromatography is based on partition.
11. High Performance Liquid Chromatography (HPLC)Mobile phaseStationary phaseNormal phase HPLCNon-polarPolarTers faz HPLCPolarNon-polarBenzene n-Heptane Toluene Chlorobenzene Metanol Water Acetonitrile 0 D 0 D 0.43 D 1.58 D 1.70 D 1.85 D 3.44 DH2OReversed phase liquid chromatographyNormal phase liquid chromatographyStationary phase polarityLowHighMobile phase polarityMedium to highLow to mediumRetention orderFirst polar compoundsFirst non-polar compoundsThe effect of increasing mobil phase polarityIncrease the elution timeDecrease the elution time
12. HPLCNormal phase liquid chromatographyReversed phase liquid chromatographySeparation of structural isomersUsed for compounds that are not separated in RP-HPLC.Equilibriation takes too much time.Fluctuation in retention times.Solvents are expensive.Wide application areas.Effective in separation of homologs.Long lifetime of stationary phasesSolvents are cheap.Quick equilibriation.
13. HPLCA chromatogram is a plot of some function of solute concentration versus elution time.Column efficiency is stated as theoretical plate height (H) and number (N). The lower the theoretical plate height and the greater the theoretical plate number, the higher the efficienct . H = L / N L is the length of column.ColumnABABBdetectorsamplemobile phase
14. Instrumentation in HPLCMain components of HPLC:Mobile phase bottlesPump: for mobile phase flowInjection unit: to inject samples to HPLC systemColumn: stationary phase Detector: to detect separated samplesAmplifier: to amplify the signals of detectorRecorder: to visualize and record the signals Görsel: https://laboratoryinfo.com/hplc/ adresinden alınarak düzenlenmiştir. Erişim tarihi: 24.02.2019
15. PumpPressure up to 400 atmPressure without pulse 0.1 – 10 mL/min flow rate≤0.5 % repeatibility in flowResistance to corrosionPump types:Reciprocating pumpthe screw-driven syringe typeNonlinear flow profile with pulseLinear flow profile without any pulse
16. Sample injection systemManual injectionAutomatic injection1-100 µL injection volume (different options are also available)
17. HPLC ColumnsGenerally,steel pipes 10 – 30 cm in length4 – 10 mm column inner diameter2 – 25 µm particle sizeDifferent columns can be produced depending on the needs.«250 x 4.6 x 5µm»Packing material:Pellicular: spherical, nonporous, glass or polymer beads with typical diameters of 30 to 40 mmPorous particles: porous microparticles having diameters ranging from 3 to 10 mm. The particles are composed of silica, alumina, the synthetic resin polystyrene-divinyl benzene, or an ion-exchange resin.
18. HPLC columns To produce a quantity of pure separated compound wide columns (preparative columns) are used and this technique is called as preparative HPLC.There are specific conditions (pH, solvent types, temperature etc) for each column to resist To increase the lifetime of column and to protect it from contamination, a guard column which is a short column packed with a similar stationary phase as the analytical column can be used.
19. HPLC Instrumentation - DetectorDetectorsStack (based on comparison) detectors; responds to the properties of mobile phase that are changed by analytes such as dielectric constant, refractive index and density.Analyte specific detectors, responds to the properties of analytes those mobile phases don’t have such as UV absorbance, fluorescence intensity or diffusion coefficient.
20. Absorbace detectors: Absorbance detectors with filtersProvides high sensitivity ↑Allows broad range of wavelength pass↓ Absorbance detectors with monochromatorDiode array detectors (DAD) are the most powerful detectors that collect full spectrum about one second.
21. Fluorescence detectorsLight sources: mercury lamp, xenon lamp or lazersWavelength selector: filters or monochromatorsCan be used for the substances at very low concentrationsOnly fluorescent substances can be analyzed (derivatization)Görsel: http://www.chromatographyonline.com/how-does-it-work-part-v-fluorescence-detectors Erişim tarihi:24.02.2019
22. Refractive index detector:In this detector, solvent (reference) pass through half of a cell while eluent pass through the other half of it. These two cells are divided by a glass plate which is placed at an angle for refraction of light if refractive index of two solution are different. When the light deviates from its ways through the surface of photosensitive detector, output signal changes. The changes in the output signal are amplified and recorded as a chromatogram.Photodiode detectorComputer –Monitoring detector responseLight sourceSample cellReference cell
23. Electrochemical detectorsAmperometry, polarography, coulometry and conductometryAdvantages: High sensitivity, simplicity and selectivity Disadvantages: only electroactive substances can be analyzedGörsel: https://community.asdlib.org/imageandvideoexchangeforum/2013/08/02/electrochemical-detector-for-hplc-separations/ Erişim tarihi: 24.02.2019
24. Mass spectrometric detectorsAnalysis using a mass analyzer after ionizationBroad ranges of applicationsLimit of detection upto 1 – 10 pgDue to high sensitivity, LC-MS (Liquid chromatography – mass spectrometry) is frequently used for investigation of the pharmacokinetic behaviours of pharmaceuticals and biological sample analyses in bioanalytical. For getting higher sensitivty and more precise results two mass spectrometers are used and this technique is called LC-MS/MS. LC-MS/MS is used in bioavailability and bioequivalance tests.
25. Other detectorsLight scattering detectors: Mobile phase passing through the columns is converted to small particles and solvent is evaporated and finally hey are detected in a light scattering cell.Conductometric detectors measures the conductivity of the mobile phase passing through two electrodes.
26. Mobile phase bottles and solvent treatment in LCGlass mobile phase bottlesTo remove dissolved gasses Degasser in the instrumentUltrasound application before analysesSolvent filtersIsocratic elution: mobile phase composition is held constant during separationGradient elution: mobile phase composition changes during separationdegasserGörsel: https://www.schambeck-sfd.com/en/hplc-gpc-products/hplc-gpc-online-degasser.php Erişim tarihi: 24.02.2019
27. System suitability parametersThe validity of chromatographic separationTheoretical plate number (N)Selectivity factor (α)Resolution(Rs)Capacity factor(k)Tailing factor (T)Assymetry factor (As)System is suitable: at least two of the parameters meet requirements.
28. Theoretical Plate Number (N)Theoretical Plate is the smalest plate at which separation takes place. Sharp and narrow peaks shows higher number of plates and highly efficient column. N = 16 []2tR retention time of the peak w: width of the peak at its baseİdeally N > 2000 Columnplate
29. Selectivity factor (α)Selectivity factor shows level of separation between two peaks. It is also called as relative retention.α = İdeally α > 1 .Although higher α values indicates better separation power, it is not the direct indicator of separation. Resolution is more useful for quality of separation. Görsel: Chromacademy, e-learning for the analytical chemistry community, The Theory of HPLC Chromatographic Parameters
30. Capacity factor (k)Capacity factor (k) is a measure of retention of an analyte in a column. It is the rate of retention of an analyte to the retention of and unretained species.k = Low k valueHigh k valueIdeally 2 < k < 8Capacity factor can be used for qualitative analysis of species in a sample.
31. Rezolution (Rs)Resolution is the measure of ability of a column to separate two analytes. The most important goal of a HPLC method is getting optimum resolution at shortest time.Rs = Rs=0.4Rs=1.6Ideally Rs > 1.5
32. Tailing factor(T) and Asymmetry factor (As)Both are indicators of the peak symmetry. In tailing factor peak width is measured at 5% of the peak height while it is measured at 10% of the peak height in asymmetry factor.T = A+B / 2A (at w0.05)As = B/A (at w0.10)Ideally 0.95 < As < 1.20 and T < 2
33. Instrument, peak area or peak height repeatibility Relative standard deviation of at least 6 times repeated experimentsIdeally,General separations < % 1.5 Bilogical samples < % 5.0Trace analyses %5 – 15 For instrument repeatibility, retention time %relative standard deviation < 5% or <1% for more experiments.
34. System suitability parameters in European PharmacopoeiaTheoretical plate number(N)Seçicilik faktörü (α)Resolution (Rs)Capacity factor(k)Tailing factor (T)Asymmetry factor(As)Relative standard deviation of peak height or areaInstrument repeatibility
35. Qualitative and quantitative analyses in HPLCQualitative analysisRetention time, tRQuantitative analysisPeak heightPeak areaInternal standardRegression analysisInstrument response factor
36. LC–MS combinationIn the combination of these two methods, first mixtures are separated in LC and more pure compounds are sent to MS for detection. LC-MS also can also analyze different molecules having the same retention times because the mass spectra of them are different and selective analysis can be provided. Therefore compounds that cannot be separated by chromatography can be determined. However for this something has to be taken into consideration: a. Interface between LC and MS should not reduce the performance because samples. b. Analyte should not undergo any uncontrolled modification while passing through the interface. c. The yield of sample transfer must be high. If transfer occurs in the interface then ionization efficienct must be high. d. The noise in the interface must be low. e. Interface must be reliable, repeatable and user friendly. f. Interface must be flexible to different chromatographic conditions. For example, flow rate of mobile phase can vary from 20 nL/min to 2 mL/min or composition of mobile phase composition can vary from 100% percent organic solvent to 100% aqueous solution. g. Interface should not change the vacuum of MS and it should not affect the performance of MS. h. Flow rate should be determined by LC. i. Mobile phase additives in LC must be volatile (such as acetic acid, formic acid, trichloroacetic acid (TCA), trifloroacetic acid (TFA), trimethylamin, triethylamin, ammonium acetate, heptaflorobutiric acid (HFBA)). Also concentraion of them must be low ( up to 10 mM). Nonvolatile compounds contaminate the interface and this can negatively affecy the analysis. Metal phosphates, carbonates, mineral acids and metal bases should never be used.
37. LC–MS combinationPhases in LC-MS combinationThe sample in LC is liquid (sometimes it contains high amount of water) and it cannot be transferred to MS directly. The role of interface is to remove the mobile phase.In MS analysis is performed in gas phase and samples must be gasified. Some samples may be nonvolatile and they can be decomposed when there are heated. For this reason different ionization techniques are developed rather than the ionization techniques in which decomposition can be observed such as chemical ionization and electron ionization.Detectors in HPLC are passive (sample does not change in the detector). On the other hand, MS is an active detector in which samples gasified and ionized.
38. REFERENCES: Skoog DA, Holler FJ, Nieman TA, Principles of Instrumental Analysis, Harcourt&Brace Company, USA, 1998.Khan JI, Kennedy TJ, Christian Jr DR, Basic Principles of Forensic Chemistry, Springer, New York, USA, 2012.Hage DS, Carr JD, Analytical Chemistry and quantitative analysis, Pearson Prentice Hall, New Jersey, USA,2011.Onur F, Analitik Kimya II, Ankara Üniversitesi Eczacılık Fakültesi Yayın No:101