Sample Prep note

8/10/2019 Sample Prep note

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Course Learning Outcomes

Students should be able to:

1. Discuss the importance of sample preparation prior

to sample analysis.

2. Explain the principles & procedures of separation

involve in the extraction method.

3. Describe the advantages & limitations of each

extraction method as compared to other extractionmethod.

4. Choose the right & suitable extraction method forsample analysis.


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STEPS IN MEASUREMENT

1. SAMPLING

Process of collecting a representativesample for analysis.

2. SAMPLE PREPARATION Extraction

Clean-up & preconcentration

3. SAMPLE ANALYSIS

Chromatographic / spectroscopic methods

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Despite the great technological advances in

the analytical field, most sophisticated

instruments cannot handle complex samplematrices directly &, as a result, a sample

preparation step is commonly involved in

an analytical procedure.

SAMPLE PREPARATION

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Also known as sample treatment/samplepretreatment.

Mean : Is a series of step required to bring thesample into the correct size & form that issuitable for analysis.

Often the bottleneck of analysis.

Time consuming (~ 61 %).

Laborious step.

Error prone.

SAMPLE PREPARATION


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Efficient sample preparation is therefore

important for a successful analysis.

Generally,

a clean sample helps to improve theseparation & detection,

while poorly treated sample may invalidatethe whole assay.

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To isolate & concentrate the analytes of interest

from interfering sample components.

To convert the analytes to a form that is

compatible with the instrument for the final

analysis.

To provide the analytes of interest at a

concentration appropriate for detection or

measurement.

Objective

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Extraction methods

Liquid samples1. Liquid-liquid extraction (LLE)

Liquid, gaseous samples

1. Solid phase extraction (SPE)

2. Solid phase microextraction (SPME)

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Solid, semi solid samples1. Soxhlet

2. Sonication extraction

3. Microwave assisted extraction (MAE)

4. Pressurized liquid extraction (PLE)

5. Supercritical fluid extraction (SFE)

Extraction methods

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Liquid-Liquid Extraction (LLE)

A method to separatecompounds based on the their

relative solubility in 2 different

immiscible liquids, usuallywater & organic solvent.

An extraction of a substance

from one liquid phase into

another liquid phase.


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Commonly performed after a chemical

reaction as part of the work-up.

A distribution ratio (D) is often quoted as a

measure of how well-extracted a species is.

Concentration of a solute in the organic phase

Concentration in the aqueous phase

Distribution ratio (D) =

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Disadvantages :

Operate manually.

Large volume of organic solvents.

Formation of emulsion.

Time consuming.

Different polarity of samples result in lowrecoveries.

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Solid Phase Extraction (SPE) An extraction method that uses a solid phase & a

liquid phase to isolate analyte from a solution.

Commercially available sorbents with differentphases, including non polar (C8 & C18), polar

(cyano & amino) & ion exchange sorbents.

Different interaction (Van der Waals, H bonding,

cation or anion exchange).


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A selection of SPE cartridges, available in

many sizes, shapes & types of stationaryphase.

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http://en.wikipedia.org/wiki/File:SPE_Cartridges.jpg


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Column

Cartridge

Disk
http://en.wikipedia.org/wiki/File:SPE_Manifold.jpg


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How to use SPE ???

5 step processes to follow

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An SPE disk is recommended

for large volume samples,

samples containing high

amounts of particulates, orwhen a high flow rate is

required during sampling.

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Conditioned with a water-miscible organic solvent

such as methanol, followed

by water or an aqueousbuffer.

Methanol wets the surface

of the sorbent & penetrates

bonded alkyl phases,allowing water to wet the

silica surface efficiently.

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Accurately transfer the

sample to the tube or

reservoir, using avolumetric pipette or

micropipette.

The sample must be in a

form that is compatible withSPE.

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If compounds of interest are

retained on the packing, wash offunwanted, unretained materials

using the same solution in which

the sample was dissolved, oranother solution that will not

remove the desired compounds.

To remove unwanted, weakly

retained materials, wash the

packing with solutions that arestronger than the sample matrix,

but weaker than needed to remove

compounds of interest.

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Rinse the packing with a smallvolume (typically 200L to 2mL

depending on the tube size, or

5- 10mL depending on the disksize) of a solution that

removes compounds of

interest, but leaves behind anyimpurities not removed in the

wash step.

Collect the eluate.

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Relying on mainly non polar, hydrophobicinteractions, only non polar or very weakly polar

compounds will absorb to the surface.

When silica particles are bonded with a

hydrophobic phase, they become waterproof &

must be conditioned in order to interact with the

aqueous samples.

This is accomplished by passing methanol or a

similar solvent through the sorbent bed.


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This penetrates into the bonded layer & permitswater molecules & analytes to diffuse into the

bonded phase.

After conditioning, water is passed to remove the

excess solvent prior to adding the sample.

Following conditioning, the analyte & other

sample constituents are adsorbed on the sorbent

bed.


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A rinsing step removes some of the undesiredconstituents, while elution removes the desired

analytes, perhaps leaving other cosituent

behind, depending on the relative strength ofinteraction with the solid phase or the solubility

in the eluting solvent.


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Applications :

SPE is used most often to prepare liquid

samples & extract semivolatile or nonvolatile

analytes.

Also can be used with solids that are pre-

extracted into solvents.

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Advantages :

Simplicity.

High analyte recovery.

Extraction reproducibility. Lower solvent consumption.

Ability to automate the extraction process.

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Soxhlet Extraction

Typically, a Soxhlet

extraction is only required

where the desired

compound has a limitedsolubility in a solvent, &

the impurity is insoluble in

that solvent.

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http://en.wikipedia.org/wiki/File:Soxhlet_Extractor.jpg


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1: Stirrer bar2: Round bottom flask (the flask

should not be overfilled & the

volume of solvent should be 3 to 4times the volume of the soxhlet

chamber)

3: Distillation path4: Thimble 5: Solid

6: Siphon top 7: Siphon exit

8: Expansion adapter

9: Condenser

10: Cooling water in 11: Coolingwater out

A schematic representation of a Soxhlet extractor

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http://en.wikipedia.org/wiki/File:Soxhlet_extractor.svg


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The solid sample to be extracted is placed in athimble.

The solvent is added to the round bottom flask.

The solvent is heated to reflux.

Step in soxhlet extraction

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The solvent vapour travels up a distillation arm &floods into the chamber where it comes into

contact with the sample.

The condenser ensures that any solvent vapour

cools & drips back down into the chamber housingthe solid material.

Step in soxhlet extraction

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The chamber containing the solid material slowly

fills with warm solvent.

Some of the desired compound will then dissolve

in the warm solvent.

When the Soxhlet chamber is almost full, the

chamber is automatically emptied by a siphon

side arm, with the solvent running back down to

the distillation flask.

This cycle may be allowed to repeat many times,

over hours (12-18 hrs).31


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During each cycle, a portion of the non-volatile

compound dissolves in the solvent.

After many cycles the desired compound is

concentrated in the distillation flask.

The advantage of this system is that instead of

many portions of warm solvent being passed

through the sample, just one batch of solvent is

recycled.

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After extraction the solvent is removed, typically

by means of a rotary evaporator, yielding theextracted compound.

The non-soluble portion of the extracted solid

remains in the thimble & is usually discarded.

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Type of solvent used : less polar solvent(e.g. hexane, acetone).

Advantage of Soxhlet extraction :

Cheap apparatus.

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Disadvantages of Soxhlet extraction :

1. Large solvent consumption (50-200 mL for a

10g sample).

2. Long extraction time (16-24 hours).

3. Dirty extracts require tedious clean up

steps.

4. Not suitable for thermally labile compounds.

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Vibrational disruption of samples.

Sonication Extraction

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Advantages : Cold extraction - good for thermally labile

compounds.

Quick (a few hours).

A sequence of solvents may be used.

Disadvantages :

Physically violent process -may cause

breakdown of macromolecules & clays.

Labor intensive.

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Microwave radiation is utilized to heat up theextraction solvent & therefore also the sample

through ionic conduction & dipole rotation.

The solvent must be able to absorb the

microwave radiation & pass it to the samplemolecules in a form of heat.

Microwave Assisted Extraction (MAE)

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Suitable solvents : polar solvent with high dipole

moment (water, MeOH, acetone).

Step in MAE :

The solid samples & solvent is placed in a

vessel & heated using microwave radiation in

a closed system.

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Extraction perform in a closed vessel :

1. Temp. can be higher than the atmospheric b.p

of the solvent, thus extraction is faster due tothe increased diffusion.

2. Vessels are irradiated simultaneously (henceextraction) up to 12 vessels.

3. Volume of solvent : 20 50 mL.

4. Extraction time : 20 40 min.

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Microwave reaction system

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Advantages :

Wide applicability for fast extractions ofanalytes including some thermal instable

substances.

Enhance solvent extraction method.

Simultaneous & automatic extraction (up to

12 vessels).

Disadvantage :X Sample vessel has to been cooled before

the extract can be obtained.

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Pressurized Liquid Extraction (PLE)

PLE also known as pressurized solvent extraction

(PSE), accelerated solvent extraction (ASE),pressurized fluid extraction (PFE).

Utilized solvent at high temperature & pressure.

Higher pressure causes the solvent to remain liquefiedabove boiling point & allows solvent to penetrate the

sample matrix.

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High temperatures can disrupt the strong solute-

matrix interactions & decrease the viscosity of

organic solvents, allowing improved penetration ofthe matrix & thus improved extraction.

The solubility & diffusity of the analytes are

increased by the increased temperature, thus

making the extraction faster & more efficient.

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5 steps in PLE :

1. Load sample into the extraction cell.

2. Fill the cell with solvent.

3. Increase temperature & pressure (for static

extraction, maintain temperature &pressure for a specific time).

4. Pump solvent into the cell.

5. Purge solvent from cell using N2.

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PLE can be performed :

1. Dynamic mode

The solvent continuously flows throughthe sample in the extraction vessel.

2. Static mode

The supercritical fluid circulates in the

extraction vessel for some period of timebefore being released to the trapping

vessel.

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Advantages :

Fast analysis (approximately 15 min/sample).

The high temperature & pressure increase the

capability of solvent to penetrate the sample

matrix.

The solubility & diffusity of the analytes are

increased by the increased temperature, thusmaking the extraction faster & less solvent is

required.

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Low solvent consumption (30-50 mL).

Use of less hazardous/environmental friendly

solvent such as acetone & hexane in the placeof methylene chloride.

Solvent systems similar to Soxhlet extraction,

so easy to optimize.

Automated (>20 samples can be processed as

a batch).

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Disadvantages :

May not be appropriate for thermally labilecompounds.

Expensive.

Produce dirty extracts which may need

clean-up.

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Introduced by Pawliszyns group (1990).

Was developed to address the need for a fast,

solvent-free, cost effective & field compatiblesample preparation method.

Is a microextraction technique, which means thatthe amount of extraction solvent is very small

compared to the sample volume.

Solid phase microextraction (SPME)

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All steps of the LLE such as extraction,concentration, (derivatization) & introduction to

the chromatograph are integrated into single

step & 1 device, considerably simplifying the

sample preparation procedure.

SPME reduces the time necessary for sample

preparation, decreases purchase & disposalcosts of solvents & can improve detection limits.

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The SPME technique enables the simultaneous

extraction & pre-concentration of volatile & non

volatile analytes from gaseous, liquid & solidsamples & even inorganic species.

SPME not suitable for sample with organic

solvent because organic solvent adsorb onto

the fiber as well & might damage it.

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The SPME apparatus is a very simple device.

The device consisting of a fiber holder & a fiber

assembly, with built-in fiber (12 cm long) inside

the needle which looks like a modified syringe.

The SPME fiber itself is a thin fused-silica optical

fiber, coated with a thin polymer film (such as

PDMS).

This film acts like a sponge, concentrating the

analytes on its surface during adsorption from

the sample matrix.54


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Design of the commercial SPME device

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When the coated fiber is placed into an aqueousmatrix for a pre-determined time, the analyte is

transferred from the matrix into the coating.

The extraction is considered to be complete when

the analyte has reached an equilibrium distributionbetween the matrix & fiber coating.

SPME Principle

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SPME relies upon the extraction of solutes from asample into the SPME absorptive layer.

After a sampling period - during which extractionhas ideally reached equilibrium - the absorbed

solutes are transferred into an inlet system that

desorbs the solutes into a gas (for GC) or liquid(for LC) mobile phase.

During desorption of the analyte, the polymericphase is cleaned & therefore ready for reuse.

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1. Direct Immersion (DI-SPME)

In DI-SPME, the fiber is directlyimmersed/inserted in liquid samples.

DI-SPME works best for low concentration

water based sample matrices.

Position the fiber just below the sample

surface & maintain this position consistently

for all extractions.

Two Basic Types of Extractions

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2. Head Space (HS-SPME)

In HS-SPME, the fiber is exposed in the vapor

phase above a gaseous, liquid or solid sample.

HS-SPME is suitable for the analysis of VOCs.

It is extremely important to keep the headspace

volume constant & keep the fiber position at thesame depth every time.

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SPME procedure (extraction)

1. Pierce septum on samplecontainer;

2. Expose SPMEfiber/extract analytes;

3. Retract fiber/withdrawneedle.

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SPME procedure (desorption)

4. Pierce septum in GC inlet

(or introduce needle intoSPME/HPLC interface);

5. Expose fiber/desorbanalytes;

6. Retract fiber/withdraw

needle.

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Parameters affect the extraction efficiency :

Types of fiber coating

Thickness of the fiber coating

Extraction time

Extraction temperature

Sample agitation

Sample pH

Salt addition

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1. Type of fiber coating

The fiber is coated with a thin polymeric film,

which concentrates the analytes.

The type of fiber used affects the selectivity of

extraction (in general, polar fibers are used forpolar analytes & non-polar fibers for non-polar

analytes).

Optimizing extraction condition

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2. Thickness of the fiber coating

The thickness affects both the equilibrium

time & sensitivity of the method.

The use of a thicker fiber requires a longer

extraction time but the recoveries are

generally higher.

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The thinnest film is employed to reduce

extraction times (faster the partition equilibriumcan be reached).

Volatile compounds require a thick film & a thin

film is effective for semivolatile compounds.

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3. Extraction time

The longer the extraction time, the more

solutes adsorbed onto the fiber until it reach

optimum extraction time whereby the amountof the adsorbed solutes is max.

Increasing the extraction time then will not

increase the amount anymore.

If the extraction is lower than the optimum

time, possibility only a fraction of solutes will

be adsorbed.70


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The extraction time depends on the size of the

compounds, fiber coating, type of extraction used &sample concentration.

Extraction times can be shorter when :

Analyzing small compounds (< 150 MW)

Using thinner fiber coatings. Using the headspace technique.

Working with high concentration samples (high

ppb or ppm range).

Extractions typically take 15-20 minutes.

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4. Extraction temperature

Optimization of extraction temperature is

generally more important in the headspace mode.

The use of heat during headspace SPME will help

release the analyte from the sample, improve

sensitivity & shorten the extraction time.

A constant temperature is advisable for all

extractions to obtain good precision.

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5. Sample agitation

Agitation accelerates the transfer of analytes

from the sample matrix to the fiber & reduce

the equilibrium time.

Agitation methods include magnetic stirring,sonication & vibration.

Vigorous or harsh agitation modes such as

sonication may affect the coating, thus they

should be used with caution.73

6 Sample pH


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6. Sample pH

Adjustment of sample pH may improve the

extraction yield for compound that can be

protonated.

pH is adjusted in order to obtain the analyte in

its neutral form to enhance the extraction

yield.

E.g., a sample may be acidified for extracting

acidic compounds or made alkaline for

extracting basic compounds.74


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f S


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Advantages of SPME

It is a rapid (analysis time very fast).

The configurations & operation of the SPMEdevices are very simple.

Reducing cost.

Solvent free & sensitive method for the

extraction of analytes.

Clean extracts & do not need any clean-up

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It gives highly consistent, quantifiable results

from very low concentration of analytes.

SPME is becoming widely used as anextraction & concentration step prior to MS

analysis.

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It has small size, which is convenient for

designing portable devices for field or on-site

sampling.

On-site sampling can be done even by

nonscientists without the need to have

equipment at each location.

When properly stored, samples can beanalyzed days later in the laboratory without

significant loss of volatiles.

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Limitations of SPME

The volume of the polymer extraction phase isvery small & requires extreme precision during

manufacturing of the coating.

The quality of the fibers depends on the

manufacturer & sometimes the performance is

different from batch to batch.

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Some level of degradation of the fiber occurs

during repeated usage.

The carry-over of the fiber is also a problem

that in some cases is difficult to eliminate.

Fibers are fragile & can easily be broken.

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Application of SPME in various fields

of analytical chemistry

Environmental (water analysis).

Food samples (analysis of wines & other

alcoholic beverages).

Biological fluids (blood, urine).

Forensic, clinical & pharmaceutical (analysis

of drugs).

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Sample problem

Briefly discuss how the used of fiber give advantages to

SPME method when compared to SPE method.

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How if we use LLE ???(in term of extract quality, may or may

not require clean-up & concentration

step)

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Separation methods

Method Basic of method

Extraction Different in solubility in two immiscible

liquids

Ion exchange Difference in interaction of reactantswith ion exchange resin

Chromatography Difference in rate of movement of a

solute through a stationary phaseElectrophoresis Difference in migration rate of

charged species in an electric field


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Derivatization A process of chemically modifying a compound

to produce a new compound which has

properties that are suitable for analysis.

Organic compounds with readily exchangeable

H (acids, alcohols) generally exhibit poor

chromatographic behavior on a polar GCstationary phases.

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Advantages of derivatization :

Improved chromatography (better sensitivity

& quantification).

Prevents GC column degradation.

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Common derivatization methods:1. Fatty Acids

Methylation (BF3/MeOH or MeOH/HCl) to yield

Fatty Acid Methyl Esters (FAMES). Silylation (BSTFA) to yield trimethylsilyl (TMS)

esters.

2. Alcohols (n-alkanols, sterols)

Acylation (pyridine, acetic anhydride) to yield

acetates. Silylation (BSTFA) to yield trimethylsilyl (TMS)

ethers.

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Pyrolysis

Pyrolysis is a form of incineration that

chemically decomposes organic materials byheat in the absence of oxygen.

Pyrolysis typically occurs under pressure & atoperating temperatures above 430 C (800 F).

Organic materials are transformed into gases,small quantities of liquid & a solid residue

containing carbon & ash.

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Application

Pyrolysis treats & destroys :

Semi-volatile organic compounds (SVOCs),

fuels & pesticides in soil.

The process is applicable for the treatment of

organics from refinery wastes, coal tar wastes,

contaminated soils, hydrocarbons & volatileorganic compounds (VOCs).

Documents

Sample Prep note