MODH SUDIP C.

PA/2010/11

NIPER HYDERABAD

SOLID PHASE EXTRACTION AND APPLICATION

seminar on

Introduction

Principle

Types Solid phases

Experimental steps

Trace enrichment

Solid phase micro extraction (SPME)

Comparison with HPLC

Application

Contents

2

What is the solid phase extraction?

3

DEFINATION OF SOLID PHASE EXTRACTION (SPE):

“A solid phase extraction consists of bringing a liquid or gaseous test sample in contact with a solid phase, whereby the analyte is selectively adsorbed on the surface of the solid phase”

Other solvents (liquids or gases)added to remove possible adsorbed matrix components

Eluting solvent added to desorb analyte selectively

4

Liquid-solid extraction

Column extraction

Digital chromatography

Bonded phase extraction

Selective adsorption techniques

SYNONYMS

5

Active substance can be:

Unretained- while matrix interference are adsorbed

Retained-while matrix interference are washed through

Strategies for solid phase extraction

6

Retained-while matrix interference are washed through

7

Principle of Solid Phase Extraction:

Partitioning of compounds between two phases of solid and liquid

Must having greater affinity for the solid phase than for the sample matrix Compounds retained on the solid phase can be removed by eluting solvent with a greater affinity for the analytes

pH changes can be useful8

In modern SPE the adsorbent is packed between two flitted disks in polypropylene cartridge and liquid phases are passed through the cartridge either by suction or by positive pressure

9

Cartridge

10

11

OVERVIEW OF SPE

12

Activated charcoal

Alumina

Silica gel

Magnesium silicate (Florisil)

Chemically bonded silica phases and polymers

E.g. styrene divinylbenzene

Solid phases

13

According to chemical nature of The functional group bonded to the silica or the copolymer

The resulting phases are classified as Non-polar Polar Ion exchangers

It gives different mode of chromatography

Other solid supports Polymeric resins, cellulose and zirconia

14

Zirconia coated silica as a stationary phase

15

Reaction of phenobarbitone with pentafluorobenzyl bromide onto the adsorbent

Amphetamine by Chiral derivatization of solid Phases

Doxorubicin by the metal-loaded phases in which metal cation is loaded onto a reagent-labelled phase

Molecularly imprinted polymers synthetic polymeric materials with specific cavitiesdesigned for a template molecule

Examples of selective stationary phases

16

Technical Data - Solid Phase Extraction (SPE) Media ProductSorbent Abbreviations Description ODS Octadecyl silica 5% carbon loadODS-4 Octadecyl silica 14% carbon load, end capped*ODS-5 Octadecyl silica 18% carbon load, end cappedC-8 Octyl silica 8.5% carbon load, end cappedFLO Florisil™ Magnesium silicate NH2 Weak anion exchanger Primary amineSAX Strong anion exchanger Quaternary amine (-NR3

+)SCX Strong cation exchanger Aromatic benzene sulfonic acidSIL Normal phase silica

  * End capping masks residual silanol groups, reducing ionic affinity for amines. 17

1. Activation of sorbent by appropriate solvent that conditions the surface of the solid

2. Removal of solvent by liquid similar to the sample matrix

3. Application of sample, the analytes retained by the sorbent

4. Removal of interfering compounds retained in step 3 with a solvent, but shouldn’t remove the analytes (washing step)

5. Elution of the analytes with an appropriate solvent (desorption or elution step) and collecting for analysis 

Experimental procedure of five steps:

18

19

20

21

22

23

Other technique can be used

Supercritical fluid

Thermal desorption for analytes of high volatility and thermal stability

Thermal desorption with GC for occupational hygiene analysis

24

Analyte eluted with an organic, relatively volatile solvent is evaporated to dryness

Then residue dissolved in appropriate solvent

Due to evaporation step, speed with SPE is lost

25

 

IMMUNOAFFINITY PHASES:

Highly selective packings of Immunoaffinity phases of specific antibody immobilised on solid support such as agarose or silica

Useful for selective extraction of biological importanceSubstance

Diagnosis of cancer

ELISA TEST

 26

IMMUNOAFFINITY PHASES

27

TRACE ENRICHMENT WITH SPE

Sensitivity depends on

Physicochemical properties of the Analyte

Detection system

Selective clean-up

Isolation and concentration step

28

SPME is the technique in which by using special instrument, sampling is possible in a vapour state

Solid phase microextraction:

29

Design of SPME

Syringe like instrument

Fused silica fiber of a small size and cylindrical shape

connected to stainless-steel tube for additional mechanical strength and repeated sampling

30

31

Fused silica fibre coated with thin film of several polymeric stationary phases

Reusable and replaceable

Small size and cylindrical geometry of fiberPlacement into sample or headspace is easy

Loading in desorption chamber of GC orInterphase of the HPLC without any modification of Plunger

32

Working with SPME

Fiber is first drawn into the syringe needle

Lowered into the vial by pressing the plunger

Fiber cleaned before analysis to remove contaminants

Cleaning can be performed in the desorption chamber of HPLC by running solvent

Cleaned fiber coating is exposed to a sample matrix for a predetermined, fixed period

33

34

Limits of detection at the µg/L level with using a flame Ionization detector

Limits of detection as low as ng/ L can be reached with an ion-trap mass spectrometer

35

Extraction can be performed in two ways

1) Headspace SPME or HS-SPMEFiber is exposed in the vapour phase above a gaseous, liquid, or solid sample

2) Direct immersion or DI-SPMEFiber is directly immersed in liquid samples

36

Available SPME Fibres, by Film Type

•Absorption Fibres-Polydimethylsiloxane (PDMS) 7, 30, and 100μm Unpolar-Polyacrylate (PA) Polar-Polyethylene glycol (PEG) Polar •Adsorption fibres (with particles) -Carboxen-polydimethylsiloxane (CAR-PDMS) Adsorption

-Polydimethylsiloxane- divinylbenzene (PDMS-DVB) Adsorption-Divinylbenzene/ Carboxen-Polydimethylsiloxane (DVB-CAR-PDMS) Adsorption  

37

 

SPME applied to liquid, gaseous or heavily contaminated samples

chemicals like

Substituted benzene compounds

Polyaromatic hydrocarbons

Nitro- and chlorophenols

Naphthols

volatile organochlorine compounds

polychlorinated biphenyl congeners

caffeine

Metallic ions38

The SPE process can be performed in a two ways:

On-line Off-line

In offline SPE eluate from the cartridge is introduced into the chromatograph by means of an injection valve

In on-line SPE the extraction cartridge is inserted as part of chromatographic equipment, as loops or high pressure stream of the mobile phase

39

Types of online SPE:

SPE-GC(SPME-GC)

SPE-HPLC

40

ONLINE SPE-GC

41

ONLINE SPE-HPLC

42

10ppb Nitrosamines in Water: SPME-GC/MS

Chromatogram courtesy of J. Clark, Liggett Group, Inc.

43

ONLINE SPE-HPLC

THT= Tetra hydro thiophene 44

SPE-HPLC SPME-GC

Universality Compounds +++ + Detection Sensitivity ++ +++ Selectivity +++ ++ Identification + +++ Detection limit (µ/ L) 0.05-0.8 0.2-5   Reproducibility (%) 1-15 4-14Analysis time 90 20Sample volume (mL) 200 2Automation +++ +++Simplicity + +++   

Comparison of SPE-HPLC and SPME-GC

45

Theoretical basis as HPLC

Retention and selectivity remain unaffected by particle sizeEfficiency dependent on:

Particle sizeColumn geometry

Typical number of plates

HPLC ~ 10,000SPE < 50

Minimum Selectivity(alpha)for Rs=1.2

HPLC 1.06SPE 3.95

Comparison of SPE and HPLC

46

47

Advantages of SPE offers over LLE are

Higher selectivity

Cleaner extracts

More reproducibility

The avoidance of emulsion formation

48

Application of SPE in various fields

Impurity profiling of pharmaceuticals

Environmental applications

Applications in food chemistry

Analysis of wines and other alcoholic beverages

Application to biological fluids

Hair analysis49

Impurity profiling of pharmaceuticals

Residual solvent analysis by USP 467Involves head space solid phase micro extraction with GC and FID detector

50

Application to biological fluids:

Simultaneous qualitative and quantitative determination ofDrugs of abuse opiates, cocaine, or amphetamines Prescribed drugs tricycle antidepressants,phenotiazines, benzodiazepines in biological fluids was developed

Eg. A Weak Cation-Exchange Monolithic SPE Column for Extraction and Analysis ofCaffeine and Theophylline in Human Urine

51

Urinary Excretion Pattern of Benzophenone-3 and its Metabolite 2,4-Dihydroxybenzophenone in Human Urine

52

Hair analysis It is used for the long-term monitoring of drug and alcohol

53

References

Anal Bioanal Chem (2007) 388:1643–1651DOI 10.1007/s00216-007-1301-4K. Dettmer & D. Hanna

Chromatographia Vol. 41, No. 7/8, October 1995Comparison of On-Line SPE-HPLC and SPME-GC for theAnalysis of Microcontaminants in WaterC. Rivasseau / M. CaudeLaboratoire de Chimie Analytique (associ6 au CNRS, URA 437) de l'Ecole Sup6rieure de Physique et de ChimieIndustrielles, 10 rue Vauquelin, 75005 Paris, France

ChromatographiaTao Zhu, Kyung Ho Row&Department of Chemical Engineering, Inha University, 253 Yonghyun-Dong, Nam-Ku, Incheon 402-751, Korea; E-Mail: rowkho@inha.ac.krReceived: 10 October 2008 / Revised: 21 January 2009 / Accepted: 13 February 2009

54

INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRYANALYTICAL CHEMISTRY DIVISIONCOMMISSION ON GENERAL ASPECTS OF ANALYTICAL CHEMISTRYM. MOORS1, D. L. MASSART' and R. D. McDOWALL''Vrije Universiteit Brussel, Pharmaceutical Institute, Laarbeeklaan 103, B-1090 Brussels, Belgium'Department of Chemistry, University of Surrey, Guildford, Surrey, GU2 SHX, UK

SIGMA ALDRICH Chemie GmbH SIGMAEschenstraße 5, 82024 Taufkirchen GermanyAnalytical Chemistry Insights 2008:3 1–7

http://www.whatman.com/SPEColumnsandCartridges.aspx

55

THANK YOU

All of you

56