This document discusses solid-phase extraction, an improved form of liquid-liquid extraction used to separate analytes from biological matrices. Solid-phase extraction uses a solid stationary phase and liquid mobile phase to separate analytes based on physical and chemical properties. The procedure involves loading a sample onto a cartridge containing a solid stationary phase, washing away contaminants, and then eluting the analyte for analysis. Common mechanisms for retention include normal phase, reverse phase, and ion exchange interactions. Solid-phase extraction has advantages over liquid-liquid extraction like lower solvent use and easier analyte collection.

1. EXTRACTION OF DRUGS AND METABOLITES
FROM BIOLOGICAL MATRICES : SOLID – PHASE
EXTRACTION
UNDER THE GUIDENCE OF PRESENTED BY
Mrs.Dr.V.NARMADA GUNTI.SHASHIKANTH
M.Pharm,( Ph.D) M.PHARMACY 1ST YEAR II -SEM
ASSISTANT PROFESSOR PHARMACEUTICAL ANALYSIS
DEPARTMENT OF PHARMACY Roll no:1008-22-885-007
UNIVERSITY COLLEGE OF TECHNOL OGY,OU
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2. CONTENTS:
1. INTRODUCTION
2. PHYSICOCHEMICAL PROPERTIES OF DRUG AND THEIR
BIOLOGICAL MATERIAL
3. METHODS OF EXTRACTION
4. SOLID – PHASE EXTRACTION
5. PRINCIPLE
6. PROCEDURE
7. MECHANISM OF SOLID PHASE EXTRACTION
8. APPLICATIONS
9. CONCLUSION
10. REFERENCES
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7. Solid - Phase Extraction
 This technique is an improved form of Liquid - Liquid
Extraction.
• It Employs Solid Phase & Liquid Phase to Separate the
Analytes from the Solution.
• Solid Phase Extraction is a separation technique in which
dissolved or Suspended compounds in Solution are
Separated from other Component in the mixture based
on their physical & chemical properties.
• A Solid Phase Extraction consist of bringing a liquid or
gaseous test sample in contact with solid phase ( packed
in Catridge)where by the Analyte is selectively adsorbed
on the surface of the solid phase.
• Eluting Solvent is added to desorb the analyte selectively
from stationary phase (adsorbent).
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8.  Solid Phase Extraction Techniques also known as
1. Liquid - Solid Extraction. 2. Column Extraction
3. Bonded Phase Extraction
4.Selective Adsorption Technique
5. Digital Chromatography
 Principle: The principle involved in the Solid Phase Extraction
is Partitioning of Solute (analyte) b/w Solid & Liquid Phase.
• The Liquid in which Solutes are dissolved or Suspended acts as
the Mobile Phase.
• Whereas the Solid Through which the sample is passed acts as
a stationary phase.
• The sample Solution is loaded on to the Stationary phase
( packed in Catridge) where the mixture gets separated into
desired & undesired Components.
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9. • The desired Analytes may be adsorbed on the stationary
Phase or may directly flow through it.
• If it gets retained then packing material ( adsorbent in
Cartridge) has to be washed with a suitable solvent .
• If it passes through the stationary phase then it is
collected Directly.
• In Both cases the desired Analyte is collected in collecting
tube & Concentrated, Then Purify.
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10. Procedure
 In modern SPE the adsorbent is packed b/w 2 fitted Disks
in Polypropylene cartridge .
 Liquid phase are passed through the cartridge either by
suction or by Positive pressure.
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11. Steps involved in SPE
 Generally following steps are followed for developing
the method for extracting the Analyte from plasma.
1. Pretreatment of Sample: Which includes dilution of
sample or PH adjustment, filteration to avoid the clogging
cartridge for better adsorption.
2. Conditioning of the Catridge: It is main step in case of
reverse phase SPE Catridge.
 Precondition is mainly done by solvent such as
methanol, acetonitrile, Isopropyl alcohol, tetra hydro
furan which is necessary to obtain reproducible results.
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12.  Without this step, a highly aqueous solvent can't
penetrate the pores & wet the surface. Hence only Small
fraction of Analyte is available for interaction with
stationary phase.
3. Loading of Sample:
• Sample size must be scaled to suit the size of Catridge
bed.
• A typical RP Catridge may have capacity for up to 100 mg
of Very strongly retained substances.
4. Wash: In this Step suitable solvent or Water mixture is
passed through the SPE bed to remove the Contaminants.
5. Elution of Fraction: In this a Suitable Solvent or Buffer is
used to elute the Analyte from SPE bed for Analysis.
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14. Mechanism of Solid Phase Extraction
 The Separation mechanism is the function due to the
intermolecular interaction b/w the Analyte & the
functional groups of the Sorbent.
 The most common retention mechanism in SPE are
based in Vanderwaal Forces(Non - Polar Interactions),
Hydrogen Bonding, dipole -dipole forces ( Polar
Interactions) , Cation - anion Interactions.
 Based on these interactions 3 types of mode of
separation possible.
1. Normal Phase SPE.
2. Reverse Phase SPE
3. Ion Exchange SPE
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15. 1. Normal Phase SPE:
I. Involves a Mild polar - Non Polar Solvent matrix
( Mobile Phase), Polar Stationary Phase & The Polar
Analyte.
II. Intially Stationary Phase or Catridge is Equilibrated with
Solvent matrix which penetrate into the Stationary
phase.
III. The stationary phase is then washed with water or
Buffer of same composition as that of sample.
IV. The Analyte is then loaded onto the Stationary Phase.
V. As the Analyte passes through SP, Hydrophilic
interaction b/w Polar Functional Groups of Analyte or
Polar groups of stationary Phase takes place.
VI. The interaction could be dipole - dipole interaction,
dipole - induced dipole interactions or Hydrogen
Bonding.
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16. VI. These Reactions causes retention of an Analyte while
the Solvent, Salts & other impurities passed out the catridge.
VII. Finally Polar Analyte molecules adsorbed onto SP &
Eluted when Binding Mechanism is disrupted by Passing
Non - Polar Solvent or Buffer of Suitable PH.
VIII. In which Polar - Functionalized Bonded Silicas ( Ex: LC-
CN, LC-NH2, LC - Diol)& Polar Adsorption media ( LC-SI, LC-
Alumina, LC- Florisil, ENV1- Flourosil) are usuay Employed as
Stationary Phase in Normal Phase SPE.
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17. 2. Reverse Phase SPE:
 In which Non Polar SP, a polar or Moderately Polar Solvent
matrix & Mild - Non polar Analyte is used.
• These forces b/w Analyte & Sorbents are distrupted by Eluting
Solvents for the Elution of sample from SP Catridge.
• Several SPE materials such as the Alkyl or Aryl Bonded Silicas
( LC-18, ENV1-,LC-18,ENV1-8,LC-4) are used in RP category.
• Retention occurs Via Non polar interaction b/w Carbon -
Hydrogen (C-H) bonds of both the Analyte & Sorbent
functional groups due to Vanderwaals or Dispersion forces.
 Non polar solvents disrupts these forces hence these are
used as Eluting Solvent for the Elution of adsorbed Compound
from the RP SPE Catridge.
 Materials that can be used as RP sorbents are
1. Carbon-Based media 2. Polymer - Based Media
3. Polymer - coated & Bonded Silica media.
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18. 1. Carbon-Based media:
• It Consist of Graphitic, Non- porous carbon with a high attraction
for organic polar & non - polar Compounds from both polar &
non -polar matrices.
• Retention of Analyte is primarily is based on the Analytes
structure, rather than on interaction of functional groups in the
Analyte with the sorbent surface.
• This type media mainly used when bonded silicas don't work
efficiently.
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19. • II .Polymer - Based Media: These are Styrene or divinyl
Benzene material.
• It is used for retaining hydrophobic compounds containing
hydrophilic functionality especially aromatics.
• This type of media efficiently retains phenols.
• III. Polymer coated & Bonded Silica media : It is
hydrophobic - Bonded Silica that is coated with Hydrophilic
Polymer.
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20. • The pores in the polymer allow small Hydrophobic organic
compounds of interest ( EX: Drugs) to reach the bonded Silica
surface while large interfering compounds ( Ex: Protiens) are
Shielded from the Bonded Silica by the Polymer which are
flushed through the SPE tube.
3. Ion Exchange SPE: In Which Separation of Analyte is based
mainly on the Electrostatic interaction b/w charged groups
bonded to SP & Charged Groups of Analyte.
• The PH of Sample Matrix plays an important role in this
technique.
• It Should be such that both SP & the Analyte are Charged.
• For Elution of Analyte, a Solution is used that neutralizes
either the functional group in the SP or The Analyte.
• Neutralizes of any of these functional groups results
disruption of binding forces b/w the Analyte & stationary
phase with the Elution of Analyte.
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21. Based on Charge of Compounds to be separated ion exchange SPE
is 2 types.
I. Anionic Exchange SPE
II. Cation Exchange SPE.
I. Anion Exchange SPE:
• In which anionic ( negatively charged) species are isolated by
using suitable sorbents like KC-SAX, LC-NH2.
• The mechanism involved is Derivatization of anion Exchange
sorbents with positively charged functional groups.
• These +vely charged Groups interact with anions & retain on
them.
• Ex: Strong A.E.Sorbents comprise of quaternary ammonium
groups, that possess permanent positive charge in Aqueous
Solution.
• These Sorbents binds to the Strong acidic impurities ( if
Present), hence prevent their Elution along with Analyte.
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22. • II. Cation Exchange Resins:
• In which Cations ( +vely charged) are isolated by using like
LC- SCX & LC- WCX.
• The mechanism involved is Derivatization of cationic
Exchange Sorbents with negatively charged functional
groups, that interact with Cations &retain on them.
• Ex: Strong Cationic Exchange Sorbent Possess Aliphatic
Sulfonic acid groups that consist permanent negative
charge in Aqueous Solution.
• These Sorbents binds to Strong Basic impurities , hence
prevent their. Elution along with Analyte.
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23. Advantages of SPE over LLE:
1. Preparation of Samle is Rapid.
2. Consumption of Solvent is Low.
3.Recovery rate is High & Separation rate is More Efficient.
4. Collection of Analyte is Easy.
5. By Using Catridge Interferences can easily removed.
6 Higher Selectivity & increases Sensitivity.
7.SPE is lesss time consuming & not tedious as compared to LLE.
Disadvantages:
1. It is an Expensive Process
2. It is difficult to control the process due to its greater
complexity.
3. It is not Suitable for concentrating metabolites from large
samples due to Catridge capacity.
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24. Applications:
1. It can be used to isolate the Analyte of interest from
blood, urine, water, animal tissues etc..
2. Used for Impurity Profiling for Pharmaceuticals
3. Applications in Food Chemistry
4. Analysis of Wines & Other Alcoholic Beverages
5. Used in Environmental Studies
6. Qualitative & Quantitative Analysis of Cocaine, opiates,
Amphetamines etc....
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25. 9.CONCLUSION
Automated SPE is widely used to increase the SPE
throughput for qualitative or quantitative analysis in
the food industry, environmental and biomedical
research, as well as the pharmaceutical industry.
The advantages of automated SPE include the
standardization of the sample extraction procedure,
increased assay throughput, improved assay
reproducibility, and reduction of the overall cost.
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26. REFERENCE
1. Prabhu S .L , Suriyaprakash, T.N.K extraction of drug from the
biological matrix : A Review applied biological engineering –
principles and practice.2012 , 479 – 502 .
2. Mali ,N Karpe , M , Kadam , V.A review on biological matrices and
analytical matrices and analytical methods used for determination
of drug abuse ….journals of applied pharmaceutical
sciences.2011,01 (06).58.65.
3. Principles of instrumental Analysis – Doglas A Skoog ,F.James
Holler,Timothy A.Nieman , 5th edition.Eastern press ,
Bangalore,1998
4. Analysis of drugs in biological fluids- Joseph Chamberlain ,2nd
Edition .CRC press,Newyork.1995.
5. Pharmaceutical analysis –Higuchi ,Brochmman and Hanseen , 2nd
edition ,Wiley – intersciences publication,1961.
6. Pharmaceutical analysis –modern methods-Part B –J W
Munson,Volume 11,Marcel Dekkel Series .
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