Lc training basic_hplc_20_a modifi
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Lc training basic_hplc_20_a modifi
- 1. 1 Part I – Basic Principles of HPLCPart I – Basic Principles of HPLC Chromatography and HPLC Various HPLC modes and applications Normal phase Reversed phase Reversed phase ion pairing Ion exchange (IC) SEC (GPC/GFC) Chiral separation HPLC system modes Isocratic elution Gradient elution
- 2. 2 What is chromatography?What is chromatography? Chromatography is one of the separation technique. The main purpose of chromatography is to separateseparate and quantifyquantify the target sample in the matrix.
- 3. 3 Why mixed sampleWhy mixed sample can be separated?can be separated? river bed direction of flow
- 4. 4 What is the difference?What is the difference? Interaction is different by gravityStrong Weak
- 5. 5 How can the separationHow can the separation be carried out?be carried out? Separation can be carried out by the column. packing material, 3-5um column
- 6. 6 Interaction between packingInteraction between packing material and samplematerial and sample Due to difference interaction between packing material and sample, separation can be done. packing material sample A sample B
- 7. 7 column mixed sample Separation can be doneSeparation can be done by the columnby the column
- 8. 8 What kind of chromatography?What kind of chromatography? High Performance Liquid chromatography (HPLC) Gas Chromatography (GC) Thin-Layer Chromatography (TLC) Capillary Electrophoresis(CE)
- 9. 9 Advantage of ChromatographyAdvantage of Chromatography Simultaneous Analysis High Resolution High Sensitivity (ppm-ppb) Small Injection Volume (1-100uL)
- 10. 10 Advantage of HPLCAdvantage of HPLC Moderate analysis condition - no need to vaporize the sample like GC Easy to fractionate the sample and purify Good repeatability (C.V. < 1%)
- 11. 11 Flow Diagram of HPLCFlow Diagram of HPLC pump injector column oven detector
- 12. 12 Normal Phase ModeNormal Phase Mode Petroleum etherPetroleum ether CaCOCaCO33 Chlorophyll'sChlorophyll's ChromatoChromatograp h ColorsColors
- 13. 13 Effect of stationary phaseEffect of stationary phase C18 (ODS) Strong C8 sample sample sample C4 Medium Weak
- 15. 15 Relationship betweenRelationship between MW and RTMW and RT MolecularWeight( Exclusion limit Permeation limit Time
- 16. 16 Isocratic Elution SystemIsocratic Elution System pump injector column oven detector Single SolventSingle Solvent
- 17. 17 Gradient Elution SystemGradient Elution System pump injector column oven detector pump A B Time Bconcentr
- 18. 18 Isocratic Elution ModeIsocratic Elution Mode Long Time AnalysisLong Time Analysis Bad SeparationBad Separation MeOH / H2O = 6 / 4 MeOH / H2O = 8 / 2 ( column : ODS type )
- 19. 19 Gradient Elution ModeGradient Elution Mode 95% 30% MeOHcon
- 20. 20 Part II Instrumentation of HPLCPart II Instrumentation of HPLC Solvent delivery pump Sample injector Column oven Detector
- 21. 21 Flow Diagram of HPLCFlow Diagram of HPLC pump injector column oven detector
- 22. 22 Desirable Pump PerformanceDesirable Pump Performance High Pressure Resistance Precise Flow Rate Low Pressure Fluctuation
- 23. 23 Plunger reciprocating pumpPlunger reciprocating pump motor and cam plunger plunger seal check valve pump head 10 -100uL
- 24. 24 Advantage Low pressure fluctuation Very easy to replace the another solvent Disadvantage Change the plunger seal Plunger reciprocating pumpPlunger reciprocating pump
- 25. 25 Dual plungerDual plunger with tandem flow linewith tandem flow line check valve Main plunger Sub plunger Low pressure fluctuation UV / PDA detector Fluorescence detector The number of maintenance parts is less. So this design is suitable for routine analysis.
- 26. 26 Dual plungerDual plunger with parallel flow linewith parallel flow line check valve plunger head Very low pressure fluctuation Refractive index detector Conductivity detector Electrochemical detector MS detector The number of maintenance parts is more.
- 27. 27 Single plungerSingle plunger check valve High pressure-fluctuation Low sensitivity analysis using UV / PDA and Fluorescence detector The number of maintenance parts is minimized.
- 28. 28 HPLC InjectorsHPLC Injectors Manual injector Auto injector
- 29. 29 6 port valve system6 port valve system column load position inject position pump pump column sample loop
- 30. 30 Manual InjectorManual Injector Rheodyne Manual InjectorRheodyne Manual Injector
- 31. 31 Manual InjectorManual Injector [LOAD][LOAD] [INJECT][INJECT] Column Pump Column Pump
- 32. 32 Injection MethodInjection Method Partial Injection MethodPartial Injection Method better to inject less than half volume of sample loop Loop Injection MethodLoop Injection Method better to inject more than 3 times volume of sample loop
- 33. 33 Dispersion and Dilution EffectDispersion and Dilution Effect loop pum p colum n drain drain pum p colum n loop drain drain Sample Zone Dilution Zone Dispersion Zone Less than half volumeLess than half volume More than half volumeMore than half volume
- 34. 34 Dispersion and Dilution EffectDispersion and Dilution Effect drain drain pum p colum n loop Sample Zone Dilution Zone Less than 3 timesLess than 3 times More than 3 timesMore than 3 times drain drain pum p colum n loop
- 35. 35 INJECT / LOAD positionINJECT / LOAD position [INJECT ][INJECT ] positionposition [LOAD][LOAD] positionposition
- 36. 36 CautionCaution Do not use pointed or beveled needle tip. Must use square end type. Do not use more than pH 10 solution. Must change rotor seal.
- 37. 37 Column TemperatureColumn Temperature Control DevicesControl Devices Column Oven (heat /or cool) Heated Column Jacket Aluminum block Insulated Column Jacket Water bath Column temperature control devices are functioning to keep the column temperature constant. The temperature fluctuation of column will influence retention time reproducibility.
- 38. 38 Detectors for DetectorsDetectors for Detectors Ultraviolet / Visible detector (UV/VIS) Photodiode Array detector (PDA) Fluorescence detector (RF) Conductivity detector (CDD) Refractive Index detector (RID) Mass spectrometer detector (MS)
- 39. 39 Ultraviolet / Visible detectorUltraviolet / Visible detector Ein Eout A= εCl = - log (Eout / Ein) l C : concentration Lambert-Beer's law (A : absorbance) CellCell
- 40. 40 Ultraviolet / Visible detectorUltraviolet / Visible detector Sample Cell Reference Cell Photodiode Photodiode Ein EinEin Grating D2 / W lamp λ Eout
- 41. 41 Lambert-Beer's lawLambert-Beer's law A= εCl = - log (Eout / Ein) Absorbance Concentration linear range 2.5
- 42. 42 SpectrumSpectrum 275 nm275 nm 208 nm208 nm 275 nm275 nm 208 nm208 nm
- 43. 43 Additional FunctionsAdditional Functions Dual Wavelength mode Ratio Plot mode Wavelength Time Program mode Wavelength Scan mode
- 44. 44 Sample Cell 512 Elements Photodiode Array Grating D2 / W lamp Photodiode Array detectorPhotodiode Array detector One element can detect one absorbance at one wavelength.
- 45. 45 Photodiode Array detectorPhotodiode Array detector Time Wavelen Absorbanc Chromatogram Spectrum
- 46. 46 Photodiode Array detectorPhotodiode Array detector UV spectra of peaks are obtained, which are supportive for identification. By checking peak purity, one can know if any impurity is present.
- 47. Identification by SpectrumIdentification by Spectrum Elution sequence Peak 1 Peak 2 Peak 3 Acetonitrile 30% 15% Azelaic acidAzelaic acid Benzoic acidBenzoic acid Nitrobenzoic acidNitrobenzoic acid
- 48. 48 Comparison by 3 Point SpectrumComparison by 3 Point Spectrum Acetyl Salicylic Acid down slope up slope, peak top
- 49. 49 Cell designCell design of Fluorescence detectorof Fluorescence detector
- 50. 50 Conductivity detectorConductivity detector I V L A A K (conductivity) = I [A] / E [V] =A [cm2 ] / L [cm] * k (k : specific conductivity) k= (I/E)*(L/A)k= (I/E)*(L/A) electrode
- 51. 51 Conductivity detectorConductivity detector Conductivity is very affected to temperature. Must keep the cell in the temperature control devise.
- 52. 52 Type of Ion ChromatographyType of Ion Chromatography Non-Suppresser type simple system (easy maintenance) low conductivity mobile phase Suppresser Type low back grand current difficult maintenance
- 53. 53 Non-Suppresser TypeNon-Suppresser Type extremely low ion exchanger Low conductivity mobile phase
- 55. 55 Conductivity and Peak ResponseConductivity and Peak Response
- 56. 56 Chromatogram of CationsChromatogram of Cations in Tap waterin Tap water Analytical Conditions Column : Shim-pack IC-C3 Mobile phase : 2.0 mM Oxalic Acid Flow rate : 1.0 mL/min Temperature : 40 C Injection volume : 100 uL Peaks 1. Na (8.25 ppm) 2. NH4 (0.01 ppm) 3. K (1.66 ppm) 4. Mg (2.22 ppm) 5. Ca (11.85 ppm)
- 57. 57 LCMSLCMS Atmospheric Pressure IonizationAtmospheric Pressure Ionization Pneumatically Assisted ElectroSpray(ESI) Atmospheric Pressure Chemical Ionization(APCI) High Voltage 3)C oulon E xclusion Ion E vaporation 2)Evaporation ofSolvent Liquid Samples + - + - + - +- + -+ + + - - + -++ +-+- + -++ +-+- + + + + + + Liquid Samples Heater C oron aD ischarge Nee dle Ion molecular reaction Nebulizing gas Nebulizing gas
- 58. 58 Design of LCMSDesign of LCMS API Probe Atmosphere High Vacuum RP TMP1 TMP2 (Vacuum pump system) MS detector
- 59. 59 What kind of compoundsWhat kind of compounds can be analyzed ?can be analyzed ? ESI drugs and their metabolites peptides proteins many kinds of natural product (-OH, -NH2,-COOH, SO2, PO3 etc.) APCI pesticides steroids drugs
- 60. 60 What kind of benefitsWhat kind of benefits LC/MS users can get ?LC/MS users can get ? Powerful qualitative capabilityPowerful qualitative capability Selective quantitative capability M/Z M/Z M/Z
Notes de l'éditeur
- Reversed Phase Ion Pairing mode Ion Exchange mode SEC mode ( GPC / GFC ) Chiral separation mode
- Check the manual!!!
- Check the manual!!! Water bath?
- Check the manual!!! What is ratio plot mode?
- Although the elution sequence of peaks 1 and 2 is reversed by simply changing the solvent ratio in this example, the change in the sequence is made obvious by identification using PDA spectrums. Determination of analysis parameters is simplified by using the spectrums for identification in this way.
- The above figure is a 3 point spectrum for the target component (acetyl salicylic acid). For reference the spectrum of salicylic acid is shown below. The rise near 300nm in the down slope of the spectrum above is clearly from the affect of salicylic acid, an impurity.