1. CHROMATOGRAPHY time or volume Detector Signal 12 Downloaded from www.pharmacy123.blogfa.com

2. Definition Chromatography is a separation process that is achieved by distributing the components of a mixture between two phases, a stationary phase and a mobile phase. Downloaded from www.pharmacy123.blogfa.com

3. Chromatography Historically, the word of chromatography was used by Tswett in 1906 He described the separation of plant pigments by percolating a petroleum-ether extract through a glass column packed with powdered calcium carbonate. Colored zones were produced by the various pigments migrating through the column at difference rates Downloaded from www.pharmacy123.blogfa.com

4. The technique, as described by Tswett was largely ignored for a along time and it was not until the late 1930s and early 1940s that Martin and Synge(2) introduced liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica in a packed bed and used it to separate some acetyl amino acids. In their paper, they recommended replacing the liquid mobile phase by a suitable gas, as the transfer of sample between the two phases would be faster, and thus provide more efficient separations. In this manner, the concept of gas chromatography was created Downloaded from www.pharmacy123.blogfa.com

5. Chromatography today : Today, chromatography is an extremely versatile technique; it can separate gases, and volatile substances by GC, involatile chemicals and materials of extremely high molecular weight (including biopolymers) by LC and if necessary very inexpensively by TLC. All three techniques, (GC), (LC) and TLC have common features that classify them as chromatography systems. Downloaded from www.pharmacy123.blogfa.com

6. Classification Chromatography GC GSCGLC LC Column C Adsorption chromatography Partition chromatography Ion exchange chromatography Pair ion chromatography Ligand exchange chromatography Size Exclusion chromatography Surface C TLC Paper chromatography Chromatography Column chromatography Paper chromatography Downloaded from www.pharmacy123.blogfa.com

7. The basic principle : Variation in the rate : - Which different components of a mixture migrate through a stationary phase under the influence of a mobile phase Rates of migration vary : - Because of differences in distribution ratios. Downloaded from www.pharmacy123.blogfa.com

8. Mechanism of chromatography During a chromatographic separation solute molecules are continually moving back and forth between the stationary and mobile phases. The rate of migration of each solute is therefore determined by the proportion of time in spends in the mobile phase, or in other words by its distribution ratio. Downloaded from www.pharmacy123.blogfa.com

9. Sorption : The process whereby a solute is transferred from a mobile phase to a stationary phase is called sorption. Downloaded from www.pharmacy123.blogfa.com

11. Mechanism of sorption Surface adsorption Partition Ion exchange Exclusion Downloaded from www.pharmacy123.blogfa.com

12. Surface adsorption The original method employed by Tswett involved surface adsorption where the relative polarities of solute and solid stationary phase determine the rate of movement of that solute through a column or across a surface. Downloaded from www.pharmacy123.blogfa.com

13. Adsorption systems : Separations in which surface adsorption is the predominant sorption process depend upon polarity differences between solute molecules. The approximate order of increasing strength of adsorption is : Paraffines<olefines<ethers<esters<ketones<al dehydes<amines<alcohol<acids Downloaded from www.pharmacy123.blogfa.com

14. Adsorption systems : During the separation process there is comptetion for adsorption sites between solute molecules and those of the mobile phase Solute and solvent molecules are continually being adsorbed and desorbed as the mobile phase travels through the system. Solute of low polarity spend more time in the mobile phase than those that are highly polarity. Consequently the components of a mixture are eluted in order of increasing polarity. Downloaded from www.pharmacy123.blogfa.com

15. Partition If a liquid is coated on the surface of an inert solid support the sorption process is one of partition and movement of the solute is determined solely by its relative solubility in the two phases or by its volatility if the mobile phase is a gas. Downloaded from www.pharmacy123.blogfa.com

16. Partition systems : In a partition system the stationary phase is a liquid coated on to a solid support. ( silicagel, cellulose ) Conditions closely resemble those of counter- current distribution so that in the absence of adsorption by the solid support. Solutes move through the system at rates determined by their relative solubilities in stationary and mobile phases. Downloaded from www.pharmacy123.blogfa.com

17. General aspect of chromatography Liquid chromatographyGas chromatography Downloaded from www.pharmacy123.blogfa.com

18. Stationary phase : The stationary phase is the part of the chromatographic system though which the mobile phase flows where distribution of the solutes between the phases occurs. The stationary phase may be a solid or a liquid that is immobilized or adsorbed on a solid. In general immobilization by reaction of a liquid with a solid is used in liquid chromatography and absorbtion of a liquid on a solid is used in gas chromatography but there are many exceptions to both of these generalizations. The stationary phase may consist of particles (porous or solid), the walls of a tube (eg. capillary) or a fibrous material (eg paper). Downloaded from www.pharmacy123.blogfa.com

19. Stationary phases : Almost and polar solid can be used - Silicagel - Alumina Downloaded from www.pharmacy123.blogfa.com

20. Activity of stationary phase : Activity is determined by the overall polarity and by the number of adsorption sites : In silicagel the adsorption sites are the oxygen atoms and silanol groups ( -S-OH ) which readily from hydrogen bonds with polar molecules. Removing water by oven drying Downloaded from www.pharmacy123.blogfa.com

21. mobile phase  In GC, the mobile is an inert Gas (argon, helium, nitrogen, hydrogen)  In LC, the mobile phase is a liquid (solvent or binary solvent mixture such as water, methanol, ethanol, acetonitryl etc).  In Chiral chromatography, the mobile phase is gas or liquid. Downloaded from www.pharmacy123.blogfa.com

22. Mobile phase : The eluting power of a solvent is determined by overall polarity, the polarity of the stationary phase and the nature of the sample components. Downloaded from www.pharmacy123.blogfa.com

23.  chromatography phase normal:  stationary phase is polar (silica grafting with NH 2, CN, diol……..)  mobile phase is a little polar or non polar (hexane, dichloromethane, )  solute : little polar or middle polar  reverse phase chromatography:  stationary phase is a polar (silica grafting of alkyl, C 8, C 18 ……)  mobile phase is polar (water, methanol, acetonitrile, ……….)  solute: a polar or middle polar Downloaded from www.pharmacy123.blogfa.com

24. Chromatographic development : A solute progresses through the chromatographic system, albeit through a column or along a plate, only while it is in the mobile phase. This process, whereby the substances are moved through the chromatographic system, is called chromatographic development. Downloaded from www.pharmacy123.blogfa.com

25. Chromatographic development : There are three types of chromatographic development : 1 - Elution development 2 - Displacement development 3 - Frontal analysis Downloaded from www.pharmacy123.blogfa.com

27. Evaluating column performance  Separation occurs mainly in column, so the accurate resolution is basis on column efficiency (the narrowness of peak), peak shape (whether it is tails or front) and the column ability to separate compounds.  factors involve in specimen separation:  Retention time  Death time or hold up time and average linear velocity  Retention factor  Separation factor  Number of theoretical plate  Separation (Trennzahl) number  Peak shape (symmetric or asymmetric) Downloaded from www.pharmacy123.blogfa.com

28. Retention time (t R )  Retention time (t R ): During sample molecule pass through the column, the solute spend a part of time in mobile phase and a part of time in stationary phase. This time is called retention time.  Adjusted time (t R ') : The time which the solute spend in stationary phase t’ r : adjusted time t r : retention time t m : hold up time or dead time t’ r = t r – t m Downloaded from www.pharmacy123.blogfa.com

29. Dead time or hold up time (t M / t 0 ) t M represent the time that the un retarded substance (mobile phase) spend in the column.  calculation equation t 0 = t R - t’ R شکل ’R’R t’ R Downloaded from www.pharmacy123.blogfa.com

30. Average linear velocity ( µ )  µ is the average speed of mobile phase (Gas or liquid), through the column. µ is expressed by cm/sec or mL/min.  Calculating equation: µ : is linear velocity of mobile phase L : is the column length t M : is the retention time of solute L tMtM µ = cm /s µ = L tMtM cm /s Downloaded from www.pharmacy123.blogfa.com

31. Retention factor (K)  K is the ratio of the amount of time that a solute spend in stationary and mobile phase.  K is calculating by equation below: t’ R K = t R - t M tMtM = tMtM Downloaded from www.pharmacy123.blogfa.com

32. Separation factor (α)  α is a measure of time interval between two peaks. Separation factor calculating by equation below α = K1K1 K2K2 Downloaded from www.pharmacy123.blogfa.com

33. Theoretical plate or column efficiency (N)  The plate theory needs to assume that the solute, during its passage through the column, is always in equilibrium with the mobile and stationary phases. But the equilibrium between the solute and phases never actually occurs. So to obtain this equilibrum, the column must divided in number of cell or plat. Every plat has a specific size and solute spend limite time in each plat. so in the existing of small plats, the solute will spend little time in each plat and it will elute fast. Downloaded from www.pharmacy123.blogfa.com

34. Equations of theoretical plat calculation N= 16 tRtR wbwb 2 N, is the theoretical plat t R, is retention time of solute w b, is the peak width at the Base, In unit of time. w h, is peak width at the half Of height in unit of time. N= 5.54 2 tRtR whwh Downloaded from www.pharmacy123.blogfa.com

35. Column Resolution : Downloaded from www.pharmacy123.blogfa.com

36. Mechanism of separation Downloaded from www.pharmacy123.blogfa.com

37. Dispersion interaction  It is the main interaction for all PSX and PEG stationary phase.  More volatile compound (low boiling point), elute first.  Effective for the solute with 30 difference in boiling point. Clark’s p 428 Downloaded from www.pharmacy123.blogfa.com

38. Dipole interaction  dipole interaction of PEG and cyanopropyl, trifluoropropyl substituted PSXs enable these phase to separate solute molecule, which has different dipole site.  this interaction is used for pesticide, halocarbons and drugs. Downloaded from www.pharmacy123.blogfa.com

39. Hydrogen bond  functional group that show strong hydrogen bond with stationary phase,such as alcohols, carboxylic acid, amines, aldehydes, esters and ketones, are less effective to separate.  hydrocarbons, halocarbons and ethers has produce weak hydrogen bonds. Downloaded from www.pharmacy123.blogfa.com

40. Mechanism of separation in liquid chromatography Downloaded from www.pharmacy123.blogfa.com

41. Mechanism of separation in adsorption chromatography  in sorption chromatography the stationary phase is solid and mobile phase is liquid and the analyte is adsorbed by stationary phase. Downloaded from www.pharmacy123.blogfa.com

42. Mechanism of separation in ion-exchange chromatography Sol +/- M + X +/- S Sol +/- S + X +/- M  in cation exchange chromatography  Sol + M + X - S Sol + S + X - M  SO - 3 for strong acids and CO - 2 for weak acid  in Ion exchange chromatography  Sol - M + X + S Sol - S + X + M  NR + 3 for strong bases and NHR + 2 for weak bases Downloaded from www.pharmacy123.blogfa.com

43. Mechanism of separation in affinity chromatography Downloaded from www.pharmacy123.blogfa.com

44. Mechanism of separation in size-exclusion chromatography resin are too severe, the incoming resin can be returned to the supplier as unacceptable. The Size Separation Mechanism Downloaded from www.pharmacy123.blogfa.com

46. Mechanism of separation in partition chromatography Downloaded from www.pharmacy123.blogfa.com

47. Mechanism of separation in Gas chromatography separation performing by adsorption and disorption mechanism.  separation according to difference between the boiling point of substance  separation according to dispersion of analyte in Liquid and Gas phase  separation of polar substance by using polar stationary phase and vice versa (packed column) Downloaded from www.pharmacy123.blogfa.com

48. Peak shape & asymmetry  Symmetric peak (needle shape peak), show the efficacy of column and enough theoretical plats.  tailing peak, show the affinity of solute to the stationary phase.  front peak (shark’s fin peak), show the column overload Downloaded from www.pharmacy123.blogfa.com

50.  polysiloxanes  polyethylene glycol  cyclodextrin Downloaded from www.pharmacy123.blogfa.com

53. Different form of columns Packed column Downloaded from www.pharmacy123.blogfa.com

57. Normal peakBoarding peakTailing peak Co-eluting peaks Downloaded from www.pharmacy123.blogfa.com

62. partition chromatography Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography) Downloaded from www.pharmacy123.blogfa.com

63. Detectors  Mass or MS-MS spectrometer  Flam ionization detector (FI D)  Electron capture detector (ECD)  Nitrogen Phosphor detector (NPD)  Atomic emission detector (AE D)  Infra-red detector (IR) Downloaded from www.pharmacy123.blogfa.com

64. Liquid chromatography column Monolithic columnSilica column Downloaded from www.pharmacy123.blogfa.com

65. Gas chromatography column Downloaded from www.pharmacy123.blogfa.com

66. Separation (Trennzahl) number (TZ)  TZ, is the measurement of how many resolved peaks can be accommodate between adjusted number of homologous series.  Calculating equation: TZ: separation number ∆ tR : the difference in retention time between A & B w h(A) and w h(B) are peak widths at the half height. TZ = ∆ t R w h(A) + w h(A) - 1 Downloaded from www.pharmacy123.blogfa.com