Solid - Phase Extraction By GUNTI SHASHIKANTH

Solid - Phase Extraction
PRINCIPLE
INSTRUMENTATION
APPLICATIONS

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 1 ST YEAR II -SEM ASSISTANT PROFESSOR PHARMACEUTICAL ANALYSIS DEPARTMENT OF PHARMACY Roll no:1008-22-885-007 UNIVERSITY COLLEGE OF TECHNOL OGY,OU UCT,OU 1

2. CONTENTS: INTRODUCTION PHYSICOCHEMICAL PROPERTIES OF DRUG AND THEIR BIOLOGICAL MATERIAL METHODS OF EXTRACTION SOLID – PHASE EXTRACTION PRINCIPLE PROCEDURE MECHANISM OF SOLID PHASE EXTRACTION APPLICATIONS CONCLUSION REFERENCES UCT,OU 2

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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). UCT,OU 7

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. UCT,OU 8

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. UCT,OU 9

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. UCT,OU 10

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 i n 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. UCT,OU 11

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. UCT,OU 12

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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 UCT,OU 14

15. Normal Phase SPE : Involves a Mild polar - Non Polar Solvent matrix ( Mobile Phase) , Polar Stationary Phase & The Polar Analyte . Intially Stationary Phase or Catridge is Equilibrated with Solvent matrix which penetrate into the Stationary phase. The stationary phase is then washed with water or Buffer of same composition as that of sample. The Analyte is then loaded onto the Stationary Phase. As the Analyte passes through SP, Hydrophilic interaction b/w Polar Functional Groups of Analyte or Polar groups of stationary Phase takes place. The interaction could be dipole - dipole interaction , dipole - induced dipole interactions or Hydrogen Bonding. UCT,OU 15

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. UCT,OU 16

17. 2. Reverse Phase SPE : In which Non Polar SP, a polar or Moderately Polar Solvent matri x & 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 . UCT,OU 17

18. Carbon-Based media: It Consist of Graphitic, Non- porous carbon with a high attraction for organic polar & non - polar Compounds f rom 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. UCT,OU 18

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. UCT,OU 19

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. UCT,OU 20

21. Based on Charge of Compounds to be separated ion exchange SPE is 2 types. I. Anionic Exchange SPE II. Cation Exchange SPE. 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. UCT,OU 21

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. UCT,OU 22

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. UCT,OU 23

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.... UCT,OU 24

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 . UCT,OU 25

26. REFERENCE 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 . 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. Principles of instrumental Analysis – Doglas A Skoog ,F.James Holler,Timothy A.Nieman , 5 th edition.Eastern press , Bangalore,1998 Analysis of drugs in biological fluids- Joseph Chamberlain ,2 nd Edition .CRC press,Newyork.1995. Pharmaceutical analysis –Higuchi ,Brochmman and Hanseen , 2 nd edition ,Wiley – intersciences publication,1961. Pharmaceutical analysis –modern methods-Part B –J W Munson,Volume 11,Marcel Dekkel Series . UCT,OU 26

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