Solid phase micro extraction.pptx

Solid phase micro extraction

1. Solid Phase Micro extraction (SPME)

2. CONTENTS SPME INTRODUCTION DISCOVERY PRINCIPLE PROCEDURE TYPES OF HOLDERS SPME FIBERS ADVNTAGES AND DISADVANTAGES APPLICATIONS HEADSPACE TECHNIQUE TYPES APPLICATIONS

3. Discovery of SPME Solid Phase Micro extraction was invented in 1990 by Dr. Janusz Pawliszyn . He invented this technique to “address the need for a fast, solvent-free, and field compatible sample preparation method”, which is faster and more efficient. Its also a solvent less technique for isolation of analyte from a sample matrix. SPME was developed from the technique of solid phase extraction.

4. What is SPME? It consists of coated fibers that are used to isolate and concentrate analytes into a range of coating materials. After extraction, the fibers are transferred to an analytical instrument for separation and quantification of the target analytes. This is accomplished with the help of a syringe-like handling device that protects the sample while transferring from sample to the instrument. This syringe-like device also protects the fiber during storage. SPME, also known as a solvent-free adsorption/desorption technique.

5. PRINCIPLE SPME is also a micro extraction technique that, when compared to the sample volume, contains a very small amount of extraction solvent. SPME allows for an equilibrium to be reached between the sample matrix and the extracting phase rather than an exhaustive removal of the analytes to the extracting phase occurring. The extracting phase is permanently attached to a rod that is made out of different material. The amount of analyte adsorbed by the fiber depends on the thickness of the coating and on the distribution constant of the analyte . Extraction time depends on the length of time required to obtain precise extractions for the analytes with the highest distribution constants. Selectivity can be changed by altering the type of fiber - Volatile compounds require - thick coating - semi volatile analytes - thin coating.

6. How does SPME work? First, we draw the fiber into the needle. The needle is then passed through the septum that seals the vial. Then depress the plunger to expose the fiber to your sample or headspace above the sample. Organic analytes are then adsorbed to the coating on the fiber. After adsorption equilibrium is attained, which can be anywhere from 2 minutes to 1.5 hours, the fiber is drawn back into the needle and is withdrawn from the sample vial. Finally, the needle is introduced into the GC injector or SPME/HPLC interface, where adsorbed analytes are thermally desorbed and delivered to the instruments column.

7. Components of SPME Holder Plain Hub The O ring Adjustable needle guide/depth gauge Plunger Plunger retaining Screw SPME manual holder Septum piercing needle Where fiber is exposed in headspace/liquid sample

8. Other SPME holders available SPME Portable Field Sampler Contains an internal septum that stores your fiber after sampling by sealing it. Comes with a PDMS/ Carbowax fiber for volatile analysis Or a PDMS fiber for -concentrating polar analytes

9. Automated or HPLC holder - This holder is used with an autosampler or an SPME/HPLC interface. Contains a needle that moves freely for control by an automated system, and for depth regulation in the interface desorption chamber.

10. Conditions affecting SPME fiber Performance a) Choice of stationary phase For non-polar analytes – poly( dimethyl siloxane ) PDMS For more polar compounds-poly- acrylate phase. Carbowax-divinylbenzene for alcohols Divinyl benzene poly- dimethylsiloxane for volatile amines. The poly- dimethylsiloxane phase is available in various thicknesses. A thick stationary phase -volatile compounds. while a thinner phase -larger molecules. b) Extraction conditions Exposing the SPME fiber to2-10 ml of aqueous sample or headspace for a set time. Sample is mixed to achieve faster equilibration, generally by using a magnetic stirrer. The rate of stirring should be constant for all samples.

11. The sample can also be mixed by using ultrasound. Raising and lowering of the needle in the sample, to minimize the static layer around the fiber. The extraction time is dependent on the partition coefficient of the analyte and on agitation of the sample, and is generally shorter for extractions from the headspace. Sample temperature has a double impact: at higher temperatures, diffusion coefficients in water are higher -the extraction time is shorter, but the partition coefficients are also lower. Heating of the sample is therefore not recommended, except to speed up the release of analytes into the gaseous phase especially, for extraction from the headspace above solid samples.

12. pH of the sample is important for slightly acidic or basic compounds. Extraction is more effective if these compounds are kept undissociated. For example, the polydimethylsiloxane phase is not resistant to media with pH below 4 or above 10 . Addition of a soluble salt into the sample - increases the ionic strength of the solution, - makes organic compounds less soluble, and -increases the partition coefficients . Salts commonly added are NaCl or Na2SO4. presence of organic solvents in the sample suppresses the analyte's adsorption onto the fiber. Humidity of the air has small impact, at a relative humidity above 90%, adsorption of analytes onto the fiber can be reduced by as much as 10% .

13. c)Extraction from solid samples Solid samples cannot be extracted directly using SPME, and extraction from the headspace is necessary. The partition of analytes into the gaseous phase is often reduced, owing to interactions of the analytes and the matrix. These are dependent mainly on the type of solid sample. Improved transport to gaseous phase can be achieved by heating the sample. Another approach is to prepare a suspension of the sample in water . If the suspension is the analytes are not too strongly bonded to the matrix too thick, the fiber coating can be damaged during agitation.

14. d) Influences on desorption from the SPME fiber The temperature, time of desorption, and the position of the needle in the GC also affect the thermal desorption of analytes from the SPME fiber. The desorption temperature is usually between 150 and 250³C. Generally, the optimal desorption temperature is approximately equal to the boiling point of the least volatile analytes . Position of the needle in the injector is important as the injector is not heated uniformly.

15. SPME fibers available Fiber coating available: PDMS PDMS/DVB Polyacrylate CAR/PDMS CW/DVB CW/TPR Stable Flex DVB/CAR/PDMS

16. Fibers preparation The methods of depositing coatings onto the fibers- The dipping technique typically consists of placing a fiber in a concentrated organic solvent solution of the material to be deposited for a short time. After removal of the fiber from the solution, the solvent is evaporated by drying and the deposited material can be cross linked . An extension of this method is electro deposition , which can be used to deposit selective coatings on the surface of metallic rods . The limitation of this approach is coating thickness variance from fiber to fiber.

17. Stable Flex Fibers These type of fibers are coated on a flexible fused silica core instead of the standard fused silica core used on the other fibers. This coating partially bonds to the flexible core which results in: a more stable coating a more durable and longer lasting fiber. These special coated fibers are for GC use only . They also have the same temperature, conditioning, and cleaning requirements as the other fiber of its same coating and thickness. These are available in every coating EXCEPT for PDMS, Polyacrylate, and CW/TPR.

18. Polydimethylsiloxane (PDMS) Film Thickness Description Hub Description Recommened use 100 μm Non-bonded Red/plain Volatiles on GC/HPLC 30 μm Non-bonded Yellow/plain Nonpolar semivolatiles on GC/HPLC 7 μm Bonded Green/plain Moderately polar to non polar semi volatiles on GC/HPLC

19. Polydimethylsiloxane/Divinylbenzene (PDMS/DVB) * This fiber is more durable due to it not containing any epoxy Film Thickness Description Hub Description Recommended use 65 μm Partially crosslinked Blue/plain Polar volatiles on GC 60 μm * Partially crosslinked Brown/notched General purpose on HPLC StableFlex Fiber 65 μm Highly crosslinked Pink/plain GC

20. Polyacrylate Film Thickness Description Hub Description Recommened use 85 μm Partially crosslinked white/plain Polar semivolatiles on GC/HPLC

21. Carboxen/Polydimethylsiloxane (CAR/PDMS) Film Thickness Description Hub Description Recommened use 75 μm Partially Crosslinked Black/plain Trace-level volatiles on GC StableFlex Fiber 85 μm Highly crosslinked Lt.Blue/plain GC

22. Carbowax/Templated Resin (CW/TPR) Film Thickness Description Hub Description Recommened use 50 μm * Partially crosslinked Purple/plain Surfactants on HPLC * This fiber is more durable due to it not containing any epoxy

23. StableFlex Divinylbenzene / Carboxen /PDMS (DVB/CAR/PDMS) Film Thickness Description Hub Description Recommened use 50/30 μm Highly crosslinked Gray/plain GC 50/30 μm Highly crosslinked Gray/notched GC

24. Carbowax / Divinylbenzene (CW/DVB) Film Thickness Description Hub Description Recommened use 65 μm Partially crosslinked Orange/plain Polar analytes on GC

25. SPME sampling performed in three ways Direct extraction Headspace trapping Extraction with membrane protection

26. DIRECT EXTRACTION In this the coated fiber is inserted into the sample and the analyte are transported directly from the sample matrix to the extracting phase . To facilitate rapid extraction, some agitation is required to transport the analytes from the bulk of the sample to the vicinity of the fiber. HEADSPACE MODE In this the analytes are extracted from the gas phase equilibrated with the sample . Reason of this modification is to protect the fiber from the adverse effect caused by non volatile, high molecular weight substances present in the sample matrix.

27. EXTRACTION WITH MEMBRANE PROTECTION The fiber is separated from the sample with a selective membrane ,which lets the analytes through while blocking the interferences. The main purpose for the use of the membrane barrier is to protect the fiber against adverse effects caused by high molecular weight compounds when dirty samples are analysed . It also enables analysis of less volatile compounds.

28. Reaching Equilibrium The extraction is considered to be complete when it reaches equilibrium and the conditions can be described by the following equation: This equation shows the relationship between the analyte concentration in the sample and the amount extracted by the coated fiber. If the amount of analyte extracted on to the fiber is an insignificant portion of that present in the sample, this equation simplifies to n=K fs V f C , where the amount of extracted analyte is independent of the volume of the sample. This means that: there is no need to collect a defined amount of sample prior to analysis the fiber can be exposed directly to whatever is being analyzed and the amount of extracted analyte will correspond directly to its concentration in the matrix.

29. Recommended Temperature and Conditioning for GC Use Maximum Operating Conditioning Time P hase Thickness Temperature Temperature Temperature (Hrs.) PDMS 100 μm 280 °C 200 °C -270 °C 250 °C 1 30 μm 280 °C 200 °C -270 °C 250 °C 1 7μm 340 °C 220 °C -320 °C 3 20 °C 2-4 PDMS/DVB 65 μm 270 °C 200 °C -270 °C 260 °C 0.5 Polyacrylate 85 μm 320 °C 220 °C -310 °C 300 °C 2 CAR/PDMS 75 μm 320 °C 240 °C -300 °C 280 °C 0.5 CW/DVB 65 μm 265 °C 200 °C -260 °C 250 °C 0.5 DVB/CAR/PDMS 50/30 μm 270 °C 230 °C -270 °C 270 °C 4 The Polyacrylate, or white fiber, will turn brown as a result of condition and will not hurt the performance of the fiber.

30. Injecting and Running a Sample on GC This is where you inject your SPME needle on the GC-MS

31. Maintenance on SPME DO NOT USE CHLORINATED SOLVENTS EVER. Use caution when handling PDMS/DVB and CW/DVB fibers because the coating can be inadvertently stripped off. Cleaning your fiber depends on the fiber phase coating: Bonded can be taken to maximum temperature and thermally cleaned for 1 hour to overnight, or can be rinsed in an organic solvent and then thermally cleaned. Non-bonded can only be thermally cleaned and can be taken to the maximum temperature for 1 to 2 hours or baked overnight at 10-20 degrees under the maximum temperature. Partially bonded fibers can be rinsed in water-miscible organic solvents.

32. Advantages of SPME During desorption of the analytes, the polymeric phase is cleaned and ready for reuse. Absence of solvent makes SPME environmentally friendly separation is faster. Small in size Amount of extracting phase is small and equilibrium of system is not disturbed . Very small objects can be studied. Range of analytes that can be analyzed include volatile, semi volatile, nonvolatile, and inorganic species. coupled with other instruments besides GC and MS. When compared to similar extraction methods, SPME has a better detection limit, precision, cost, time, solvent use, and simplicity.

33. Disadvantages of SPME Can get relatively expensive if one is not careful with fibers due to the cost being roughly $108 per fiber. Polymer coating is fragile, easily broken, and have limited lifetime. Its main limitation is its reduced concentration capability due to the small volume of polymer coating on the fiber.

34. APPLICATIONS It can be used to analyze various types of analytes from gaseous, liquid, and solid samples . Very cheap compared to other extraction methods. Reduces sample preparation times and disposal costs due to being solvent-free, also a bonus for the environment. Improves detection limits. A very simple methods that almost anyone could perform .

35. Different fields using SPME Applications SPME is applied to include: Food and drug Environmental Clinical/Forensics

36. Clinical/Forensic Application Used for the detection and quantitative determination of illicit and therapeutic drugs, pesticides, solvents, and other poisons from blood, urine, hair, and human tissue. SPME conditions were determined by structure and properties of the analyte .

37. Environmental Application Air sample Analytes are extracted by the fiber wither by direct exposure or by use of the headspace method. On-site air sampling can be performed by the equilibrium methods or by the non-equilibrium method. Rapid air sampling can be performed with controlled air-flow rate .

38. Water samples The SPME has been used for analysis of pesticides, and herbicides in aqueous samples. The fibers have also been used to analyze environmental pollutants in aqueous samples. Soil and sediment sample SPME has also been used for determination of herbicides in sewage-sludge samples.

39. Conclusion SPME is a solvent-free microextraction technique that is: Cost efficient Simple to understand and use High sensitivity Low detection limits Can be used to sample analytes of many types Used in many areas of industry.

40. HEADSPACE TRAPPING Headspace sampling is a means of introducing volatile compounds associated with a sample material without the use of solvent extraction. Types of headspace trapping 1) STATIC HEADSPACE TRAPPING 2)DYNAMIC HEADSPACE TRAPPING 1)In static headspace , the sample is sealed into a vessel, warmed, and then a sample of the atmosphere surrounding the sample is withdrawn and injected into the injection port of the GC.

41. 2)Dynamic headspace sampling is a technique which uses a flow of carrier gas through the sample vessel to increase the headspace sample size, and thus the sensitivity of the technique. Instead of allowing the sample to come to equilibrium in a sealed container, the sample is warmed and the headspace atmosphere is constantly purged out of the sample vessel and through a trap. After the collection step, the trap is heated and back flushed to transfer the adsorbed compounds to the GC for analysis.

42. Static Headspace  - In static headspace analysis, a liquid or solid sample is placed into a vial, sealed, and heated to a specific temperature.  All of the components that are volatile at or below the pre-set temperature escape from the sample to form a gaseous "headspace" above the sample.  The term "static headspace" refers to the sealed environment in which the out gassed products are collected. After a certain period of time, the headspace gas is extracted from the vial and injected into a gas chromatograph which separates the various components of the sample based on size and/or polarity.  STATIC HEADSPACE TECHNIQUE

43. The separated components then go into a mass selective detector.  The resulting mass spectrum allows for the identification of the components using standard reference libraries. Static headspace analysis is an ideal choice for volatile compounds, such as residual solvents or low molecular weight additives. The sensitivity for static headspace is typically in the sub microgram range; however this is dependent on the volatility of the compounds.

44. Dynamic Headspace  - DHS is a form of headspace analysis that utilizes a "purge and trap" method to collect and concentrate out gassed materials for analysis by GC/MS.   In this method, the sample is purged with ultra-pure nitrogen while being heated in a Teflon vessel.  As the nitrogen stream exits the vessel it passes through a thermal desorption tube filled with an adsorbent material.  The out gassed products are collected onto the adsorbent material.  Following the predetermined collection time, the tubes are transferred to a thermal desorption unit which is in line with the gas chromatograph and mass selective detector (GC/MS). DYNAMIC HEADSPACE TECHNIQUE

45. The thermal desorption unit heats the individual tubes while a flow of gas is applied through the tube.  The collected materials are flushed from the sorbent material and collected onto a cold trap within the thermal desorption unit.  After the entire sample has been purged from the sample tube and collected on the cold trap, the cold trap is heated rapidly.  The collected materials are then swept from the cold trap into the GC/MS for analysis as a volatile material. 

46. APPLICATIONS Medicinal materials are tested by headspace trapping. Volatile constituents of white Hyacinths are isolated by dynamic headspace trapping. For the analysis of flavor and fragrance compounds. Also used for air and field analysis. Volatiles components are easily extracted by this method. These technique are also useful for the semi vola tile compounds.