Part I Basic Principles of HPLCChromatography and HPLCVarious HPLC modes and applicationsNormal phaseReversed phaseReversed phase ion pairing Ion exchange (IC)SEC (GPC/GFC)Chiral separationHPLC system modes Isocratic elution Gradient elution
What is chromatography?Chromatography is one of the separation technique.
The main purpose of chromatography is to separate and quantify the target sample in the matrix.
Why mixed sample can be separated? river beddirection of flow
What is the difference?Interaction is different by gravityStrongWeak
Separation can be carried out by the column.How can the separation be carried out?packing material, 3-5umcolumn
Interaction between packing material and sampleDue to difference interaction between packing material and sample, separation can be done.packing materialsample Asample B
columnmixed sampleSeparation can be done by the column
What kind of chromatography?High Performance Liquid chromatography (HPLC)Gas Chromatography (GC)Thin-Layer Chromatography (TLC)Capillary Electrophoresis(CE)
Advantage of ChromatographySimultaneous AnalysisHigh Resolution High Sensitivity (ppm-ppb)Small Injection Volume (1-100uL)
Advantage of HPLCModerate analysis condition - no need to vaporize the sample like GCEasy to fractionate the sample and purifyGood repeatability (C.V. < 1%)
Flow Diagram of HPLC
Normal Phase ModePetroleum etherCaCO3Chlorophyll'sChromatograph Colors
Effect of stationary phaseC18 (ODS) StrongC8samplesamplesampleC4MediumWeak
Relationship between MW and RT
Isocratic Elution System
Gradient Elution System
Isocratic Elution ModeLong Time AnalysisBad Separation MeOH / H2O = 6 / 4MeOH / H2O = 8 / 2( column : ODS type )
Gradient Elution Mode95%30%MeOH concentration
Part II Instrumentation of HPLC Solvent delivery pump Sample injector Column oven Detector
Flow Diagram of HPLC
Desirable Pump PerformanceHigh Pressure ResistancePrecise Flow RateLow Pressure Fluctuation
Plunger reciprocating pump
AdvantageLow pressure fluctuationVery easy to replace the another solventDisadvantageChange the plunger sealPlunger reciprocating pump
Dual plunger with tandem flow lineLow pressure fluctuation UV / PDA detectorFluorescence detector
The number of maintenance parts is less. So this design is suitable for routine analysis.
Dual plunger with parallel flow line Very low pressure fluctuation Refractive index detector Conductivity detectorElectrochemical detectorMS detector
The number of maintenance parts is more.
Single plunger High pressure-fluctuation Low sensitivity analysis using UV / PDA and Fluorescence detector
The number of maintenance parts is minimized.
HPLC InjectorsManual injectorAuto injector
6 port valve systemcolumnload positioninject positionpumppumpcolumnsample loop
Manual InjectorRheodyne Manual Injector
Injection MethodPartial Injection Methodbetter to inject less than half volume of sample loop Loop Injection Methodbetter to inject more than 3 times volume of sample loop
Dispersion and Dilution EffectloopdraindraindrainLess than half volume More than half volume
Dispersion and Dilution EffectLess than 3 timesMore than 3 times
INJECT / LOAD position
Caution 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.
Column Temperature Control DevicesColumn Oven (heat /or cool)Heated Column JacketAluminum blockInsulated Column JacketWater bathColumn temperature control devices are functioning to keep the column temperature constant. The temperature fluctuation of column will influence retention time reproducibility.
Detectors for DetectorsUltraviolet / Visible detector (UV/VIS)Photodiode Array detector (PDA)Fluorescence detector (RF)Conductivity detector (CDD)Refractive Index detector (RID)Mass spectrometer detector (MS)
Ultraviolet / Visible detectorEinEoutA= eCl = - log (Eout / Ein) lC : concentration Lambert-Beer's law(A : absorbance)Cell
Ultraviolet / Visible detectorSample CellReference CellPhotodiodePhotodiodeEinEinEinGratingD2 / W lamplEout
Lambert-Beer's lawA= eCl = - log (Eout / Ein) AbsorbanceConcentrationlinear range2.5
Spectrum275 nm208 nm275 nm208 nm
Additional FunctionsDual Wavelength modeRatio Plot modeWavelength Time Program modeWavelength Scan mode
Sample Cell512 Elements Photodiode ArrayGratingD2 / W lampPhotodiode Array detectorOne element can detect one absorbance at one wavelength.
Photodiode Array detectorTimeWavelengthAbsorbanceChromatogramSpectrum
Photodiode Array detectorUV spectra of peaks are obtained, which are supportive for identification.By checking peak purity, one can know if any impurity is present.
Identification by SpectrumElution sequencePeak 1Peak 2Peak 3Acetonitrile30%15%Azelaic acidBenzoic acidNitrobenzoic acid
Comparison by 3 Point SpectrumAcetyl Salicylic Aciddown slopeup slope, peak top
Cell design of Fluorescence detector
Conductivity detector I V LAAK (conductivity) = I [A] / E [V] =A [cm2] / L [cm] * k (k : specific conductivity)
Conductivity detectorConductivity is very affected to temperature.Must keep the cell in the temperature control devise.
Type of Ion ChromatographyNon-Suppresser typesimple system (easy maintenance)low conductivity mobile phaseSuppresser Typelow back grand currentdifficult maintenance
Non-Suppresser Typeextremely low ion exchangerLow conductivity mobile phase
Conductivity and Peak Response
Chromatogram of Cations in Tap waterAnalytical ConditionsColumn : Shim-pack IC-C3Mobile phase : 2.0 mM Oxalic Acid Flow rate : 1.0 mL/minTemperature : 40 CInjection volume : 100 uLPeaks1. Na(8.25 ppm)2. NH4(0.01 ppm)3. K(1.66 ppm)4. Mg (2.22 ppm)5. Ca (11.85 ppm)
LCMSAtmospheric Pressure IonizationPneumatically Assisted ElectroSpray(ESI)
Atmospheric Pressure Chemical Ionization(APCI)
High Voltage3) Coulon Exclusion Ion Evaporation 2) Evaporation of Solvent Liquid SamplesLiquid SamplesHeaterCorona Discharge Needle
Ion molecular reaction Nebulizing gasNebulizing gas
Design of LCMS
What kind of compounds can be analyzed ?ESIdrugs and their metabolitespeptides proteinsmany kinds of natural product (-OH, -NH2,-COOH, SO2, PO3 etc.) APCIpesticidessteroidsdrugs
What kind of benefits LC/MS users can get ?Powerful qualitative capabilitySelective quantitative capability
Reversed Phase Ion Pairing modeIon Exchange modeSEC mode ( GPC / GFC ) Chiral separation modeCheck the manual!!!Check the manual!!! Water bath?Check the manual!!! What is ratio plot mode?1616Although 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.99The 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.Spectrum of Salicylic Acid