Post on 04-Apr-2018
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SAMPLE
PREPARATION
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Why ?
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Concentrating analytes to improve
detection
More accurate results
Remove interferences from sample Protecting equipment to reduced
costs
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1. Isolation or Extraction.
2.Dehydration.
3.Sample cleanup.
4.Sample concentration.
5.Derivatization (optional).
6.Qualitative & Quantitative analysis.
Sample preparation steps
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Sample preparation techniques
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Precipitation
Liquid-Liquid Extraction
Solid-Liquid Extraction (SPE)
Dialysis / Ultra filtration
Electrophoresis
Distillation/Evaporation
Supercritical Fluid Extraction
Solubility
Partitioning in one of two liquidphases
Adsorption/partitioning onto solid
sorbent
Molecular weight/size
Charge
Boiling point/ vapour pressure
Partitioning into supercritical fluid
Method Basis for Selectivity
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Precipitation
Can you change the solubility property of the
compound?
How?
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Recovery
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Acidic compound recovery:
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Base compound recovery:
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Liquid- Liquid Extraction
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Liquid- liquid Extraction is useful method to separatecomponents (compounds) of Mixture
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Polar Solvents:
Water> Acetonitrile>Methanol>Ethanol
Non-polar Solvents:
n-Decane > n-Hexane>n-Pentane>Cyclohexane
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Solid Phase Extraction
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How to define SPE? Separation or removal of an analyte or
analytes from a mixture of compounds by
selective partitioning of the compounds between a
solid phase(Sorbent) and a Liquid phase
(Solvent).
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SPE COLUMN
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SPE works.well .
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Whats the principle behind it?
SPE works on the principle of liquid
chromatography.
Involves strong but reversible interactions
between the analyte & the surface of the stationary
phase.
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Non polar: van der Waals ~20 KJ/mole
Polar: Dipole / Dipole ~ 40 KJ/moleHydrogen bond ~40 KJ/mole
Electrostatic: Ionic ~600 KJ/mole
TYPESOF SORBENT- ANALYTE INTERACTIONS
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TYPES
OF
BASE MATERIALSFOR
SPE PACKING
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SilicaNa and K Silicates
Fluorosil Mg Silicates
Alumina Carbon
Polystyrene
PolystyreneDi vinyl benzene PolystyreneN-Vinyl pyrrolidone
Cellulose
Hydroxy apatite
Fullerenes
Cyclo dextrin
Agarose
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Si
C18 tails
Silica-based SPE cartridges
Tails solvate during step 1
(conditioning), will collapse if the
cartridge goes dry. Extraction is
ruined
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Polymeric SPE cartridges
No tails to solvate, and so
extraction can continue even if
cartridge goes dry
P
O
O
O
F F
F F
F F
F F
F F
F F
F
FF
F F
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Experimental procedure
Conditioning the SPE tube.
Loading the sample to conditioned SPE tube.
Washing of the packing to remove impurities.
Elution of compounds of interest.
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Conditioning of silica-based sorbents
Add an organic solvent to rinse and activate the
alkyl-chains (C18, C8 etc.)!
Dont let the column run dry during conditioning!
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DIFFERENCES BETWEEN HPLC AND SPE
Particle size
Packed bed efficiency
Extra-column volume
Column length
Number of plates (N)
HPLC SPE
~5 m 40-80 m
High Low
Low High
5-30 cm ~1 cm
~10,000 < 50
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Semi volatile
compound extraction
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Soxhlet extraction
Ultrasonic extraction
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SOXHLETEXTRACTION
Not suitable for thermallyunstable compounds.
How does it works ?
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THE SOXHLETEXTRACTORCONTINUOUS
EXTRACTIONOFACOMPONENTFROMASOLIDMIXTURE.BOILINGSOLVENT
VAPORSRISEUPTHROUGHTHELARGERSIDE-ARM
SOXHLETEXTRACTION
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CONDENSEDDROPSOFSOLVENTFALLINTOTHEPOROUSCUP,DISSOLVINGOUTTHEDESIREDCOMPONENT
FROMASOLIDMIXTURE
SOXHLETEXTRACTION
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WHENTHE
SMALLERSIDE-ARMFILLSTOOVERFLOWING, IT
INITIATESASIPHONINGACTION.
SOXHLETEXTRACTION
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THESOLVENT,CONTAININGTHE
DISSOLVEDCOMPONENT, ISSIPHONEDINTO
THEBOILERBELOW
SOXHLETEXTRACTION
S
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RESIDUALSOLVENTTHENDRAINSOUT
OFTHEPOROUSCUP, ASFRESHSOLVENTDROPS
CONTINUETOFALLINTOTHEPOROUSCUP.
SOXHLETEXTRACTION
S
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. . . ANDTHECYCLEREPEATS . . .
SOXHLETEXTRACTION
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ULTRASONICEXTRACTION
It uses ultrasonic vibrations
to extract samples with polar
solvents in an ultrasonic bath.
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DEHYDRATION METHODS
From liquid-liquid extractions often
contain water originating from the
sample.
Dehydration is most commonly
achieved by using anhydrous
sodiumsulfate (Na2SO4).
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CONCENTRATION
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Kuderna-Danish(KD) concentratororrotary evaporator
to concentrate the extract or column
chromatography eluant.
KD methodorrotary evaporationDepends on the boiling point of the target
compounds, their sublimation character, timeframe
for analysis etc.
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KD CONCENTRATION& ROTARY EVAPORATION
KD concentration
takes longer time than
rotary evaporation.
But there is less lossof target chemical
Applicable to low
boiling & high boiling
compounds.
Rotary evaporation can
concentrate large
volumes of samples in a
relatively short period of
time. It has big evaporative
losses and is not suitable
for low boiling point
compounds
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CLEAN-UP METHOD
Components of the mixtures can interfere with GCand HPLC analysis by giving poor separation,
because
over separation capacity of a column or compounds that elute at the same time as, and
interfere with, the target compounds peaks.
therefore, necessary to clean up, or remove, non
target compounds as much as possible.
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PURITY
of isolated active principle via specific tests:
melting point
boiling point
optical rotation
chemical tests*
chromatographic data (Rf, Rt values)
spectral data (UV, IR, MS)biological evaluation
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VOLATILEORGANICCOMPOUNDS
Headspace method.
Purge and Trap method.
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HEADSPACEMETHOD (HS)
Also known as the static headspace method.
Less sensitive (ppb level) compared to the purge &
trap method.
But operation is simple, easily automated.
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HEADSPACE GC
A very useful method for analyzingvolatiles present in non-volatile solids
and liquids
Sample is equilibrated in a sealed
container at elevated temperature
The headspace in the container is
sampled and introduced into a GC
Needle
Liquid/solid
Headspace
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Qualitative & Quantitative analysis:
Measurement by highly selective and
Sensitive analytical Equipments such as GCor GC-MS, HPLC or LC-MS.