HPLC
description
Transcript of HPLC
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HPLC
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The best application fields of various chromatographic modes
GCVolatile, thermostable compounds
LCPolar, non volatile. thermolabile
EKCIonic compounds
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The role of interaction types in various chromatographic modes
Types GC SFC HPLC EKC
Dispersion ++++ +++ ++ +
- ++ ++ ++++ +
Dipole-dipole ++ ++ ++++ ++
Hydrogen bridge + ++ +++ +++
Ionic / / ++ ++++
Repulsion ++ ++ +++ ++
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Advantages of various chromatographic modes
Tulajdonság GC SFC HPLC EKC
Efficiency ++++ +++ ++ ++++
Analyses temperature
+ +++ ++++ ++++
Variability of mobile phase
/ + ++++ +++
Speed of analyses ++++ ++ + +++
Sensitivity ++++ ++ +++ +
Established instrumentation
+++ + ++++ ++
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Resolution as function of other chromatographic parameters
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Resolution-efficiency- selectivity
HPLC can produce high selectivity, but moderate efficieny (< 100 000 tp).
At least, α = 1.3 is required for baseline separation.
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Band broadening in HPLC
The HPLC uses packed columns.The diffusion processes are much slower in HPLC than GC.
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Van Deemter curve in HPLC
The slow diffusion causes increasing HETP values as function of linear flow of mobile phase.
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Schematic view of high performance liquid chromatography (HPLC) instrument
Degassing is important to gain smooth baseline.
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An pp to date HPLC instrument
• Pumps upto 300 bar
• The degassing is important
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Pump
Motor & Cam
Plunger
Plunger seal
Check valves
Pump head
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Pump of HPLC instrument
Pulsation of system is decreased with two pumps, working in opposite periods.
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Gradient system
• Isocratic system– Fixed (un-changeable) mixing ratio during
analysis
• Gradient system– Changeable mixing ratio during analysis
• HPGE (High Pressure Gradient, mixing after pumps)
• LPGE (Low Pressure Gradient, mixing before pumps)
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Mobile phase pump with 4 eluents
Low Pressure Gradient
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Aim of gradient - problems in isocratic mode -
• in isocratic mode
Long analysis timeLong analysis time, low signal to noise ratio, low signal to noise ratio
Poor separationsPoor separations
Methanol / water = 6 / 4
Methanol / water = 8 / 2
(Column : ODS type)
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Aim of gradient - solution -
• Gradual change of the mixing ratio during analysis
95%
30%
Methanol concentrationin mobile phase
Short analysis timeShort analysis time &&Excellent separationExcellent separation, good signal to noise ratio, good signal to noise ratio
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Polarity of eluents
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Rotary valve injection in HPLCben
The loop injector introduces exact volume of sample.
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On-line SPE-HPLC arrangement
Precolumn is in the loop. Precolumn is good for sample concentration.
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HPLC analyses of polar pesticides with precolumn concentration
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Integrated precoumn HPLC
The precolumn protect the main column, against the deposition of matrix components, and dissolution of stationary phase.Main columns have 15-25 cm length and 2- 4,6mm I.D.
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Dead volume
• Dead volume may cause problems such as poor peak separations and poor reproducibility.
Tube
Male nut Dead volumeDead volume
Excellent connection Poor connection
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Sample vs. HPLC mode
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The diameters and porosity of sample influence of efficieny
The efficiency increase with the decrease of packing diameter. However the mobile phase pressure has limits (~ 250 att), wich allows 3-5 µm size of packing material. The increased porosity increased the loadability. However the deep holes are badly washed. Spherical particles are the best.
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Various HPLC packings
Goodnes: monolith > spherical > irregular
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New type of packings
The limited depths of holes improves the efficiency.
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New trend the use of 1.8 µm diameter packings
Very high pressure, short columns and fast analyses
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Different molecular weight molecules requires different poremsizes
Bigger molecules need bigger pore size..
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Most frequently used HPLC
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Normal phase / Reversed phase
Stationary phase Mobile phase
Normal phase
High polarity(hydrophilic)
Low polarity(hydrophobic)
Reversed phase
Low polarity(hydrophobic)
High polarity(hydrophilic)
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Retention order on reverse vs. normal phase packings
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Polarity of solvent
The strongest mobile phase is hexane in reversed phase mode. The strongest mobile phase is acetic acid in normal phase mode.
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Bonded silica (Reversed phase HPLC packing)
Revers phase s are used in 80 % of HPLC analyses.
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Stationary phase
Reversed phase packings: • C18
• C8
• C4
• Cinao• DiolNormal
Specials: chiral, ion exchange, gel
Increasing polarity→
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Most frequently used HPLC stationary phase C18
Apolar compounds have big retention Mobile phases are mixture of water, methanol acetonitrile.
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Condition process of C18 stationary phase
A methanol wash reqires for the activation of C18 stationary phase.
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Column polarity - Retention time
C18 (ODS)
CH3
strongstrongweakweak
OH
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Mobile phase polarity - Retention time
60 / 40
Mobile phase: Methanol /Water
80 / 20
70 / 30
Methanol / Water
Methanol / Water
Methanol / Water
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Influence of strength of mobile phes on C18
stationary phase
A decrease of mobile phase strength results in increases of resolution values and retention times.
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HPLC analysis of basic herbicides
Amines need specially deactivated packings
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Ionic compounds analysed as ion pairs on C18.
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Cianopropyl Stationary phases
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Stationary phase vs. sample
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Normal phase,Adsorption chromatography
The molecules of sample is solved in mobile phase, but they touch only in the surface of stationary phase.
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Ion excange chromatography
The ions of stationary phase interact with the oppositely charged molecules of sample.
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Ion chromatogram of anaions
The stationary phase is anionic ionexchange resin.
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Analysis of anions in ppb level using supressor
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Size excusion (gel) chromatography
The voluminous molecules elute fast because they are excluded from the small diameter pores, therefore they interact in less extent.
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Size excusion (gel) chromatography
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Specially designes stationary phase for carbamate pesticides
Carbamate can not be analysed with GC, because they are thermolabiles.
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Molecular imprintesd (MIP) stationary phases
They are very selective, but low efficiency packings
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Various HPLC detectors
Electrochemical S
Mass spectrometric U
Fluorescent S
Ultraviolett S
Refractive U
Light scaterring U
S, selective; U, univeral
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UV/UV-VIS detector
A ·C·l= –log (Eout / Ein)
(A : Absorbance)
l
C : ConcentrationCell
Ein Eout
A
C
D2 / W Lamps
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External standard
C1
C4
C3
C2
Concentration Area
A1
A2
A3
A4C1 C2 C3 C4
A1
A2
A3
A4
Concentration
Pea
k ar
ea
Calibration curveCalibration curve
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Internal standard
C1
C4
C3
C2
ConcentrationArea
A1
A2
A3
A4C1/CIS C2 /CIS C3 /CIS C4 /CIS
A1/AIS
A2 /AIS
A3 /AIS
A4 /AIS
Concentration: Target / Internal standard
Are
a: T
arge
t /
Inte
rnal
sta
ndar
d Calibration curveCalibration curveTarget
Internal
CIS
CIS
CIS
CIS
AIS
AIS
AIS
AIS
standard
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Diodarray (DAD) UV-VIS detector
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HPLC-UV detection of pesticides
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Recommended detection wave length for various functional groups
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Light scattering HPLC detector
Universal, sensitive
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Refractive index detector(RID-10A)
Sample
Reference
Photodiode
W Lamp
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Ionization in HPLC/MS
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LC/MS-MS is appropriate for compound identification
First MS→Ionic adduct with soft ionizationSecond MS→fragmentation with EI ionization
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On line HPLC/MS coupling