lc training basic_hplc_20_a modifi
TRANSCRIPT
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Part I – Basic Principles of HPLCPart I – Basic Principles of HPLC
Chromatography and HPLCVarious HPLC modes and applications
Normal phaseReversed phaseReversed phase ion pairing Ion exchange (IC)SEC (GPC/GFC)Chiral separation
HPLC system modes Isocratic elution Gradient elution
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.
3Why mixed sample Why mixed sample can be separated? can be separated?
river bed
direction of flow
4
What is the difference?What is the difference?
Interaction is different by gravityStrong
Weak
5How can the separation How can the separation be carried out?be carried out?
Separation can be carried out by the column.
packing material, 3-5um
column
6Interaction between packing Interaction 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
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colum
n
mixed sample
Separation can be done Separation can be done by the columnby the column
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What kind of chromatography?What kind of chromatography?
High Performance Liquid chromatography (HPLC)
Gas Chromatography (GC) Thin-Layer Chromatography (TLC) Capillary Electrophoresis(CE)
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Advantage of ChromatographyAdvantage of Chromatography
Simultaneous Analysis
High Resolution
High Sensitivity (ppm-ppb)
Small Injection Volume (1-100uL)
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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%)
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Flow Diagram of HPLCFlow Diagram of HPLC
pumpinjector
column
ovendetector
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Normal Phase ModeNormal Phase Mode
Petroleum etherPetroleum ether
CaCOCaCO33
Chlorophyll'sChlorophyll's
ChromatoChromatograph
ColorsColors
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Effect of stationary phaseEffect of stationary phase
C18 (ODS)
Strong
C8
sample
sample
sample
C4
Medium
Weak
14
Elution orderElution order
SECColumn
15Relationship between Relationship between
MW and RTMW and RT
Molecu
lar Weigh
t (LogM
W)
Exclusion limit
Permeation limit
Time
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Isocratic Elution SystemIsocratic Elution System
pump injector
column
ovendetector
Single SolventSingle Solvent
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Gradient Elution SystemGradient Elution System
pumpinjector
column
ovendetector
pump
A
B
Time
B con
centration
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Isocratic Elution ModeIsocratic Elution Mode
Long Time AnalysisLong Time Analysis
Bad SeparationBad Separation
MeOH / H2O = 6 / 4
MeOH / H2O = 8 / 2
( column : ODS type )
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Gradient Elution ModeGradient Elution Mode
95%
30% MeO
H con
centration
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Part II Instrumentation of HPLCPart II Instrumentation of HPLC
Solvent delivery pump
Sample injector
Column oven
Detector
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Flow Diagram of HPLCFlow Diagram of HPLC
pumpinjector
column
ovendetector
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Desirable Pump PerformanceDesirable Pump Performance
High Pressure Resistance
Precise Flow Rate
Low Pressure Fluctuation
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Plunger reciprocating pumpPlunger reciprocating pump
motor and cam
plungerplunger seal
check valve
pump head
10 -100uL
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AdvantageLow pressure fluctuationVery easy to replace the another
solvent
DisadvantageChange the plunger seal
Plunger reciprocating pumpPlunger reciprocating pump
25Dual plunger Dual plunger
with tandem flow linewith tandem flow line
check valve
Main plunger Sub plunger
Low pressure fluctuation UV / PDA detectorFluorescence detector
The number of maintenance parts is less. So this design is suitable for routine analysis.
26Dual plunger Dual plunger
with parallel flow line with parallel flow line
check valve
plunger head
Very low pressure fluctuation Refractive index detector Conductivity detectorElectrochemical detectorMS detector
The number of maintenance parts is more.
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Single plunger Single plunger
check valve
High pressure-fluctuation Low sensitivity analysis using UV / PDA and Fluorescence detector
The number of maintenance parts is minimized.
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HPLC InjectorsHPLC Injectors
Manual injector
Auto injector
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6 port valve system6 port valve system
column
load position inject position
pump pump column
sample loop
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Manual InjectorManual Injector
Rheodyne Manual InjectorRheodyne Manual Injector
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Manual InjectorManual Injector
[LOAD][LOAD] [INJECT][INJECT]
Column
Pump
Column
Pump
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Injection MethodInjection Method
Partial Injection MethodPartial Injection Methodbetter to inject less than half volume of
sample loop
Loop Injection MethodLoop Injection Methodbetter to inject more than 3 times
volume of sample loop
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Dispersion and Dilution EffectDispersion and Dilution Effect
loop
pump
column
drain
drainpump
column
loop
drain
drain
Sample ZoneDilution ZoneDispersion Zone
Less than half volume Less than half volume More than half volume More than half volume
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Dispersion and Dilution EffectDispersion and Dilution Effect
drain
drain
pump
column
loop
Sample ZoneDilution Zone
Less than 3 timesLess than 3 times More than 3 timesMore than 3 times
drain
drain
pump
column
loop
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INJECT / LOAD positionINJECT / LOAD position
[INJECT ][INJECT ] position position
[LOAD][LOAD] position position
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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.
37Column Temperature Column 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.
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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)
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Ultraviolet / Visible detectorUltraviolet / Visible detector
Ein Eout
A= εCl = - log (Eout / Ein)
l
C : concentration
Lambert-Beer's law
(A : absorbance)
CellCell
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Ultraviolet / Visible detectorUltraviolet / Visible detector
Sample Cell
Reference Cell
Photodiode
Photodiode
Ein
EinEin
Grating
D2 / W lamp
λ Eout
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Lambert-Beer's lawLambert-Beer's law
A= εCl = - log (Eout / Ein)
Absorb
ance
Concentration
linear range2.5
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SpectrumSpectrum
275 nm275 nm
208 nm208 nm275 nm275 nm
208 nm208 nm
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Additional FunctionsAdditional Functions
Dual Wavelength modeRatio Plot modeWavelength Time Program modeWavelength Scan mode
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Sample Cell
512 Elements Photodiode Array
Grating
D2 / W lamp
Photodiode Array detectorPhotodiode Array detector
One element can detect one absorbance at one wavelength.
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Photodiode Array detectorPhotodiode Array detector
TimeW
avelength
Ab
sorban
ce
Chromatogram
Spectrum
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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.
Identification by SpectrumIdentification by SpectrumElution sequence
Peak 1
Peak 2
Peak 3
Acetonitrile30% 15%
Azelaic acidAzelaic acid
Benzoic acidBenzoic acid
Nitrobenzoic acidNitrobenzoic acid
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Comparison by 3 Point SpectrumComparison by 3 Point Spectrum
Acetyl Salicylic Acid
down slope
up slope, peak top
49Cell design Cell design
of Fluorescence detectorof Fluorescence detector
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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
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Conductivity detectorConductivity detector
Conductivity is very affected to temperature. Must keep the cell in the temperature control devise.
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Type of Ion ChromatographyType of Ion Chromatography
Non-Suppresser typesimple system (easy maintenance)low conductivity mobile phase
Suppresser Typelow back grand currentdifficult maintenance
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Non-Suppresser TypeNon-Suppresser Type
extremely low ion exchanger
Low conductivity mobile phase
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Suppresser TypeSuppresser Type
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Conductivity and Peak ResponseConductivity and Peak Response
56Chromatogram of Cations Chromatogram 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)
57LCMSLCMS
Atmospheric Pressure IonizationAtmospheric Pressure Ionization
Pneumatically Assisted ElectroSpray(ESI)
Atmospheric Pressure Chemical Ionization(APCI)
High Voltage
3) Coulon Exclusion
Ion Evaporation
2) Evaporation
of Solvent
Liquid Samples
+ - + -+-
+-
+ -+
+
+
-
-+-++
+-+-+-+++-+-
++
++
+
+
Liquid Samples HeaterC oron a D ischarge
Nee dle
Ion molecular reaction
Nebulizing gas
Nebulizing gas
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Design of LCMSDesign of LCMS
API Probe
Atmosphere High Vacuum
RP TMP1 TMP2(Vacuum pump system)
MSdetector
59What kind of compounds What 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
60What kind of benefits What 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