Sample Preparation 123

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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.

Documents

Sample Preparation 123