SAMPLE

PREPARATION
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Why ?

Concentrating analytes to improve detection More accurate results Remove interferences from sample Protecting equipment to reduced costs

Sample preparation steps

1. Isolation or Extraction. 2. Dehydration.

3. Sample cleanup.
4. Sample concentration. 5. Derivatization (optional). 6. Qualitative & Quantitative analysis.
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Sample preparation techniques

Method

Basis for Selectivity

Precipitation Liquid-Liquid Extraction Solid-Liquid Extraction (SPE) Dialysis / Ultra filtration Electrophoresis Distillation/Evaporation Supercritical Fluid Extraction

Solubility Partitioning in one of two liquid phases Adsorption/partitioning onto solid sorbent Molecular weight/size Charge

Boiling point/ vapour pressure

Partitioning into supercritical fluid6

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 separate components (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 chromatography. the principle of liquid

Involves strong but reversible interactions between the analyte & the surface of the stationary phase.

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TYPES OF SORBENT- ANALYTE INTERACTIONS

Non

polar:

van der Waals Dipole / Dipole Hydrogen bond Ionic

~20 KJ/mole ~ 40 KJ/mole ~40 KJ/mole ~600 KJ/mole

Polar:

Electrostatic:

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TYPES
OF

BASE MATERIALS
FOR

SPE PACKING

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Silica Na and K Silicates Fluorosil Mg Silicates Alumina Carbon Polystyrene Polystyrene Di vinyl benzene Polystyrene N-Vinyl pyrrolidone Cellulose Hydroxy apatite Fullerenes Cyclo dextrin Agarose

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Silica-based SPE cartridges

Tails solvate during step 1 (conditioning), will collapse if the cartridge goes dry. Extraction is ruined

C18 tails

Si

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Polymeric SPE cartridges

No tails to solvate, and so extraction can continue even if cartridge goes dry

F F F

F FF FF F F F F F F FF F

O P O O

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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

HPLC

SPE 40-80 m

Particle size

~5 m

Packed bed efficiency

Extra-column volume Column length Number of plates (N)

High
Low 5-30 cm ~10,000

Low
High ~1 cm < 50
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Semi volatile compound extraction

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Soxhlet

extraction Ultrasonic extraction

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SOXHLET
EXTRACTION

Not suitable for thermally unstable compounds.

How does it works ?

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SOXHLET
EXTRACTION
THE SOXHLET EXTRACTOR CONTINUOUS
EXTRACTION OF A COMPONENT FROM A SOLID MIXTURE. BOILING SOLVENT VAPORS RISE UP THROUGH THE LARGER SIDE-ARM

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SOXHLET
EXTRACTION
CONDENSED DROPS OF
SOLVENT FALL INTO THE POROUS CUP, DISSOLVING OUT THE DESIRED COMPONENT FROM A SOLID MIXTURE
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SOXHLET
EXTRACTION
WHEN THE
SMALLER SIDE-ARM

FILLS TO
OVERFLOWING, IT INITIATES A

SIPHONING ACTION.
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SOXHLET
EXTRACTION
THE SOLVENT,
CONTAINING THE DISSOLVED

COMPONENT, IS
SIPHONED INTO THE BOILER

BELOW
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SOXHLET
EXTRACTION
RESIDUAL SOLVENT
THEN DRAINS OUT OF THE POROUS

CUP, AS FRESH
SOLVENT DROPS CONTINUE TO FALL

INTO THE POROUS

CUP.
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SOXHLET
EXTRACTION

. . . AND THE CYCLE REPEATS . . .

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ULTRASONIC EXTRACTION

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. most commonly achieved by using anhydrous sodiumsulfate (Na2SO4).
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Dehydration

is

CONCENTRATION

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Kuderna-Danish(KD)
or rotary evaporator to concentrate the chromatography eluant.

concentrator
or column

extract

KD method or rotary evaporation

Depends 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 loss of 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 GC and 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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VOLATILE ORGANIC COMPOUNDS

Headspace method. Purge and Trap method.

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HEADSPACE METHOD (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 analyzing
volatiles present in non-volatile solids and liquids Needle

Sample is equilibrated in a sealed

container at elevated temperature

Headspace

The headspace in the container is

sampled and introduced into a GC

Liquid/solid

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Qualitative & Quantitative analysis:

Measurement by highly selective and Sensitive analytical Equipments such as GC or GC-MS, HPLC or LC-MS.

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