HPLC

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HPLC
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The best application fields of various
chromatographic modes
GC Volatile, thermostable compounds LC Polar,
non volatile. thermolabile EKC Ionic 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 (lt 100 000 tp). At least, a 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
Pump head
Motor Cam
Check valves
Plunger
Plunger seal
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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

Methanol / water 6 / 4
Long analysis time, low signal to noise ratio
Methanol / water 8 / 2
Poor separations
(Column ODS type)
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Aim of gradient - solution -

• Gradual change of the mixing ratio during analysis

Methanol concentration in mobile phase
Short analysis time Excellent
separation, 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.

Male nut
Dead volume
Tube
Poor connection
Excellent 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 gt spherical gt 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
• Diol
• Normal
• 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
OH
C18 (ODS)
weak
strong
CH3
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Mobile phase polarity - Retention time
Mobile phase Methanol /Water
Methanol / Water
60 / 40
Methanol / Water
70 / 30
Methanol / Water
80 / 20
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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
C Concentration
Cell
Eout
D2 / W Lamps
l
A eCl log (Eout / Ein)
(A Absorbance)
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External standard
Area
Concentration
A1
Calibration curve
C1
A4
A2
A3
C2
Peak area
A2
A3
C3
A1
A4
C1
C2
C3
C4
C4
Concentration
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Internal standard
Concentration
Area
Internal
Target
standard
A1
AIS
Calibration curve
C1
A4 /AIS
CIS
A2
AIS
A3 /AIS
C2
CIS
Area Target / Internal standard
A2 /AIS
A3
AIS
C3
CIS
A1/AIS
A4
AIS
C1/CIS
C2 /CIS
C3 /CIS
C4 /CIS
C4
CIS
Concentration Target / Internal 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)
Photodiode
Reference
W Lamp
Sample
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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 ionization Second
MS?fragmentation with EI ionization
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On line HPLC/MS coupling