2. CHROMATOGRAPHY  A laboratory technique in which the components of a sample are separated based on how they distribute between two chemical or physical phases, one of which is stationary and other of which is allowed to travel through the separation system.

3. CHROMATOGRAPHY  Introduced first by the Russian botanist Mikhail Semenovich Tswett.  Mixtures of solutes dissolved in a common solvent are separated from one another by a differential distribution of the solutes between two phases.

4. PRINCIPLE  Fractionalism of mixtures of substances  In the operation of the chromatogram, a mobile gaseous or liquid phase is use to wash the substances to be separated through a column of a porous material.

5. PRINCIPLE  The rate of migration of the solute depends upon the rate of interaction of the solute with the two phases, one being the mobile phases and the other stationary phase as the compounds travel through the supporting medium.

6. DEFINITION OF TERMS:  Capillary Action – the movement of liquid within the spaces of a material due to the forces of adhesion, cohesion, and surface tension.  Retention time  Peak  Viscosity

7. DEFINITION OF TERMS:  VOCs  Stationary phase  Mobile phase

8. COMPONENTS OF A CHROMATOGRAPH  MOBILE PHASE – A phase that is flowing through the column and causes sample components to move toward the column’s end.  STATIONARY PHASE- A fixed phase that is coated or bonded within the column; it always remain in the system.  It is responsible for delaying the movement of compounds as they travel through the column.  SUPPORT- onto which the SP is coated or attached.

9. COMPONENTS OF A CHROMATOGRAPH Receiving vessel COLUMN ORIGINAL SAMPLE AND MOBILE PHASE SUPPORT AND STATIONARY PHASE

10. TYPES OF CHROMATOGRAPHY CAN BE CLASSIFIED ACCORDING TO: ∞MOBILE PHASE ∞STATIONARY PHASE ∞SUPPORT

11. GAS CHROMATOGRAPHY  A type of Chromatography in which the mobile phase is a GAS.  First GC system was developed by Erika Cremer  The presence of a gas mobile phase makes GC valuable for separating substances like VOCs that occur naturally as gases that can easily be placed into gaseous phase.

12. GAS CHROMATOGRAPHY It can separate nanograms or pictograms of volatile substances. It is principally a method for the separation and quantitative determination of gases and volatile liquids and substances.

13. GAS CHROMATOGRAPHY HOW IS IT PERFORMED? A System called Gas Chromatograph is used to perform GC. COMPONENTS: GAS SOURCE INJECTION SYSTEM COLUMN DETECTOR

15. GAS CHROMATOGRAPHY ∞ GAS SOURCE- supplies the mobile phase. It is typically a gas cylinder equipped with pressure regulators to deliver the mobile phase at a controlled state. ∞ INJECTION SYSTEM- consists of a heated loop or port into which the sample is placed and converted into a gaseous form.

16. GAS CHROMATOGRAPHY ∞ COLUMN- contains the stationary phase and support material for the separation of components in the sample. This column is held in an enclosed area known as the column oven. ∞ COLUMN OVEN- maintains the temperature at a well-defined value. ∞ DETECTOR- monitors sample components as they leave the column.

17. GAS CHROMATOGRAPHY FACTORS THAT AFFECT GC:  Requirements for the Analyte Volatility and Thermal Stability Chemical Derivatization

18. GAS CHROMATOGRAPHY Common Mobile Phases in GC:  Hydrogen  Helium  Nitrogen  Argon

19. GAS CHROMATOGRAPHY GC SUPPORT MATERIALS  Packed Column filled with small support particles that acts as an adsorbent or that are coated with the desired stationary phase.  Open- Tubular Columns stationary phase coated on or attached to its interior surface.

20. GAS CHROMATOGRAPHY GC STATIONARY PHASES  Gas- Solid Chromatography ( solid adsorbents)  Gas-Liquid Chromatography (liquids coated on solids)  Bonded phases

21. GAS CHROMATOGRAPHY ◊ Gas-Solid Chromatography o Solid adsorbent is used as a stationary phase. o Uses the same material as both the support and stationary phase, with retention occurring through the adsorption of analytes to the support’s surface. o Example of support is a MOLECULAR SIEVE. o Other supports include: o ORGANIC POLYMERS - porous polystyrene o INORGANIC SUBSTANCES – Silica or Alumina

22. GAS CHROMATOGRAPHY o Increasing the surface area of the GSC support will increase the phase ratio and result in higher retention for analytes o Pore size is important because only compounds smaller than the pores are able to contact the surface are within the space. o Polarity of Support and its functional groups will also affect how analytes will bind to them.

23. GAS CHROMATOGRAPHY ◊ Gas-Liquid Chromatography o A chemical coating or layer is placed onto the support and used as the stationary phase. o Most Common types of GC. o 100% dimethylpolysiloxane, 5%phenyl-95% methyl polysiloxane – Examples of liquids that are used as Stationary phase.

24. GAS CHROMATOGRAPHY ◊ Gas-Liquid Chromatography o All of these liquids have High boiling points and low volatilities, which allows them to stay within the column at relatively high temperatures that are often used in GC for sample injection and elution. o Liquids are also wettable- easy to place onto a support in a thin, uniform layer.

25. GAS CHROMATOGRAPHY ◊ Gas-Liquid Chromatography o All of these liquids have High boiling points and low volatilities, which allows them to stay within the column at relatively high temperatures that are often used in GC for sample injection and elution. o Liquids are also wettable- easy to place onto a support in a thin, uniform layer.

26. GAS CHROMATOGRAPHY ◊ Bonded Phases o Column Bleed- most nonvolatile liquid will slowly vaporize or break apart and leave the column over time. o Column bleed changes the retention characteristics of the column. o It can also cause some GC detectors to have a high background and noisy signal as the stationary phase leaves the column and enters the detector. o Use of bonded phase minimize column bleed.

27. GAS CHROMATOGRAPHY ◊ Bonded Phases o Produced by reacting groups on a polysiloxane stationary phase with silanol groups that are located on the surface of a silica support.

28. GAS CHROMATOGRAPHY Types of Gas Chromatography Detectors General Detectors x Thermal Conductivity Detector -used for both organic and inorganic compounds -measures the ability of the eluting carrier gas and analyte mixture to conduct heat away from hot-wire filament-”thermal conductivity”. -example: Wheatstone bridge

29. GAS CHROMATOGRAPHY Flame Ionization Detector - detects organic compounds by measuring their ability to produce ions when they are burned in flame.

30. GAS CHROMATOGRAPHY Selective Detectors x Nitrogen-Phosphorous Detector - selective for the determination of nitrogen- or phosphorous containing compounds. - Similar to FID, but does not use a flame for ion production.

31. GAS CHROMATOGRAPHY Electron capture detector - detects compounds that have electronegative atoms or groups in their structure, such as halogen atoms ( I,Br,Cl and F) and Nitro Groups. -can also be used to detect polynuclear aromatic compounds, anhydrides and conjugated carbonyl compounds.

32. GAS CHROMATOGRAPHY Applications:  Most effectively used for analyses of organic compounds, space related, complex mixtures of volatile substances at column temperature of less than -40 °C to greater than 550° C.  Geochemical research projects such as determination of various environmental pollutants at extremely low concentrations.

33. GAS CHROMATOGRAPHY ADVANTAGES: o Ability to provide qualitative information and quantitative information o FAST ANALYSIS o Efficient, providing high resolution o Sensitive o Nondestructive o Requires small samples o Inexpensive DISADVANTAGES: o LIMITED to volatile samples o Not suitable for thermally labile samples o Fairly difficult for large preparative samples o Requires spectroscopy usually mass spectroscopy for confirmation of peak identity

34. LIQUID CHROMATOGRAPHY √ A Chromatographic technique in which the mobile phase is a liquid. √ Originally developed by Russian botanist Mikhail Tswett in 1903. √ Its popularity is due to the ability of this method to work directly with liquid samples, which makes it valuable in such areas as food testing, environmental testing and biotechnology.

35. LIQUID CHROMATOGRAPHY HOW IS LIQUID CHROMATOGRAPHY PERFORMED?  A System known as a Liquid Chromatograph is used to perform LC.

36. LIQUID CHROMATOGRAPHY √ Components of the LC System:  Support – enclosed in a Column  Stationary phase- enclosed in a Column  Liquid mobile phase-delivered to Column by means of Pump  Injection device- on analytical applications it is being used,to apply samples to the column.  Collection Device (optional)- placed after the column to capture analytes as they elute.

38. LIQUID CHROMATOGRAPHY FACTORS THAT AFFECT LIQUID CHROMATOGRAPHY: *requires both a difference in retention and good efficiency for it to separate two given chemicals *Sample *Analyte Requirements *Formats *Role played by the Mobile phase

39. LIQUID CHROMATOGRAPHY Requirements for the Analyte:  Must be possible to place this chemical into a liquid that can be injected onto the column.  There must be a difference in retention between the analytes to be prepared.

40. LIQUID CHROMATOGRAPHY Types of Liquid Chromatography:  Adsorption Chromatography  Partition Chromatography  Ion-Exchange Chromatography  Size-Exclusion Chromatography  Affinity Chromatography

41. LIQUID CHROMATOGRAPHY 1. ADSORPTION CHROMATOGRAPHY A chromatographic technique that separates solutes based on their adsorption to the surface of a support.  Also known as Liquid-Solid Chromatography  Equivalent method in GC is Gas-Solid Chromatography  Uses the same material as both stationary phase and support.  Retention process can be explained on the ff below:  A+ n M-Surface A-Surface + n M

42. LIQUID CHROMATOGRAPHY  Elutropic strength- strength of a mobile phase in adsorption chromatography  It is a measure of how strongly a particular solvent or liquid mixture will absorb to the surface of a given support.  Examples: silica and Alumina supports  A liquid with large elutropic strength will strongly adsorb to the given support, which will prevent the analyte from binding to the support.

43. LIQUID CHROMATOGRAPHY

45. 2. PARTITION CHROMATOGRAPHY o It is a Liquid Chromatographic technique in which solutes are separated based on their partitioning between a liquid mobile phase and a stationary phase coated on a solid support. o Support used is usually Silica o Originally, it involves coating of support with a liquid stationary phase that was immiscible with the mobile phase

46. LIQUID CHROMATOGRAPHY Two Main Categories of Partition Chromatography: Normal-phase- uses polar stationary phase Reversed phase-uses nonpolar stationary phase

47. LIQUID CHROMATOGRAPHY Applications:  Used in analytical laboratories  Use of NPLC for separating analytes in organic solvents and chemicals that contain polar functional groups.

48. LIQUID CHROMATOGRAPHY 3. ION- EXCHANGE CHROMATOGRAPHY Solutes are separated by their adsorption onto a support containing fixed charges on its surface.  Routinely used in Industry for the removal or replacement of Ions in products.  Used for the separation of charged compounds ( inorg. Ions, org. ions, AA, Proteins and Nucleic Acids)