1. Theory, Application of TLC, HPLC, CE Separation Methods: Part 1 September 2014 Pharmaceutical Analysis Course (#8002)

2. Why Separate?  Complex samples cause interference problems with direct spectroscopic measurements  Chromatography can help improve quantitative and qualitative measurements by separating these components prior to making the spectroscopic measurements  Increased time for analysis and subsequent costs hv Direct spectroscopic measurements Complicated via sample matrix

3. History of Chromatography 3  1906: Mikhail Tswett separated leaf pigments in a glass column packed with calcium carbonate  1940s: paper chromatography  1952: Nobel Prize to Martin and Synge  1942 work on partition chromatography  1969: J.Gas Chrom.  J. Chrom. Sciences.  1970s: HPLC  non-volatile compounds  thermally labile compounds  1990s: Capillary Electrophoresis (Human Genome project)  Today/2014: high resolution, pressure, precision, excellent HPLC columns (pure silica gel, bonding techniques), capillary GC columns, sensitive detectors, mass spectrum and UV spectrum detectors

4. Importance of Chromatography 4  Why was the development of chromatography so important to science in the 20th century?  “Chemistry today is to a large extent concentrated upon the study of natural products, which are obtained from animals, plants, or even bacteria and other microorganisms. A starting material of this type contains a great number of widely varied substances, some simple, others more complicated. The first thing the chemist must do is to isolate the substances he is interested in from the material and prepare them in a pure state. The next step is, if possible, to identify these substances and find out what they consist of and how they are built up from simple constituents.”  (1952 Nobel Prize Presentation Speech)  Some other separation techniques (can’t separate very similar cpds)  Distillation  Centrifugation  Extractions

5. Chromatography (USP’s definition) 5  “…dynamic differential migration process…  …in a system consisting of two or more phases…  …one of which moves continuously…  …substances exhibit different mobilities  …by reason of differences in  Adsorption  Partition  Solubility  Vapor pressure  Molecular size  or Ionic charge density. ”

6. Separation Methods 6 ModeStationary Phase Mobile Phase Separation Mechanism Applications HPLCSolidLiquid Adsorption Absorption/Partition Molecular size Ionic charge density Potency Impurities TLCSolidLiquidAdsorption Impurities Monitor Rxns GCLiquid adsorbed on solid (or just solid) GasAdsorption Partition Boiling pt. (vap pressure) (Potency) Residual Solvents SFCSolidSuper- critical CO 2 Adsorption PartitionChiral CESolidAqueous buffers Adsorption, flow in an electric field Proteins Chiral

7. Basics 7  Data collected in a chromatography experiment = chromatogram  t r = retention time  t m = dead time (time spent in MP)  t s = corrected ret time (time spent in SP)  Qualitative info = retention time  Quantitative info = peak size  Migration Rates – depend on equilibrium constant for distributing between MP & SP.  Partition coefficient : K = C S /C M (aka: distribution constant)  Retention factor: k= K x Vs/Vm  k= 0 unretained peak  k= 1 retained 2 times the unretained

8. Concept: Partitioning  General principle in chromatography is likes dissolve likes  Separation of analytes can be accomplished based on relative solubility and associative partitioning between two phases A+B Dissolved in Hexane (50:50 ratio) Assume 20% of A and 40% of B will partition out of hexane and into the water Add Water Extract water and repeat

9. Concept: Partitioning  General principle in chromatography is likes dissolve likes  Separation of analytes can be accomplished based on relative solubility and associative partitioning between two phases A+B Dissolved in Hexane (50:50 ratio) Assume 20% of A and 40% of B will partition out of hexane and into the water Add Water Extract water and repeat

10. Column Chromatography  Mechanism to facilitate sample component separation  Mobile phase: Gas or Liquid  Stationary phase: Solid  Process of separation of mixtures into individual components via:  Passing said mixture dissolved in a mobile phase through a stationary phase which separates the analytes to be measured into individual bands based on the partitioning between the mobile and stationary phases  Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. T=0 min Mobile Phase Flow hv

11. Column Chromatography  Mechanism to facilitate sample component separation  Mobile phase: Gas or Liquid  Stationary phase: Solid  Process of separation of mixtures into individual components via:  Passing said mixture dissolved in a mobile phase through a stationary phase which separates the analytes to be measured into individual bands based on the partitioning between the mobile and stationary phases  Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. hv T=5 min

12. Column Chromatography  Mechanism to facilitate sample component separation  Mobile phase: Gas or Liquid  Stationary phase: Solid  Process of separation of mixtures into individual components via:  Passing said mixture dissolved in a mobile phase through a stationary phase which separates the analytes to be measured into individual bands based on the partitioning between the mobile and stationary phases  Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. hv T=15 min

13. Column Chromatography  Mechanism to facilitate sample component separation  Mobile phase: Gas or Liquid  Stationary phase: Solid  Process of separation of mixtures into individual components via:  Passing said mixture dissolved in a mobile phase through a stationary phase which separates the analytes to be measured into individual bands based on the partitioning between the mobile and stationary phases  Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. hv T=20 min

14. Separation Performance Mechanics behind a separation and how to measure Rate Theory (General) Van Deemter Equation (Specific) First principals Kinetic Performance How temperature, pressure, volume and heat flow effects chemical and physical processes Retention mechanisms Thermodynamics

15. Separation Performance Mechanics behind a separation and how to measure Rate Theory (General) Van Deemter Equation (Specific) First principals Kinetic Performance How temperature, pressure, volume and heat flow effects chemical and physical processes Retention mechanisms Thermodynamics

16. Rate Theory 16  It is clear that symmetrical peaks are preferred.  What causes peak shape?  The “perfect chromatographic peak” resembles a normal statistical distribution. Why?  The “perfect chromatographic peak” is also narrow, analogous to a small standard deviation.  What causes peaks to widen?  What causes peaks to deviate from symmetry?

17. Rate Theory 17  Helps define basis for optimization of a particular chromatographic process based on first principles  Uses a random walk mechanism to model the migration of molecules through a column  Takes into account:  band broadening  effect of rate of elution on band shape  availability of different paths for different solute molecules to follow  diffusion of solute along length  Several “Derivations” of the Rate theory have been developed:  van Deemter Equation, the Giddings Equation, the Huber Equation, the Horvath Equation and the Knox Equation  We’ll focus on the van Deemter  most commonly used

18. Rate Theory 18 Rate Theory Eddy Diffusion Longitudinal Diffusion Resistance to Mass Transfer van Deemter Equation Huber Equation Horvath Equation Knox Equation

19. Van Deemter Equation Time for unretained peak to elute Column Length  Band broadening within the column can be modeled using the van Deemter Equation  A: Describes the broadening attributed to eddy diffusion  B: is the contribution due to longitudinal diffusion  C: contribution due to mass transfer   : mean linear velocity

20. A Term Eddy Flow Contributes to Band Broadening 20 Some molecules take longer more erratic paths while others take more direct paths, for instance those traveling close to the walls may exit first. In the example above X elutes before Y (band broadening). Not affected by flow rate. Can be affected by particle shape. Paths around uniform spherical particles are more uniform than irregular larger particles. Capillary columns improve A term.

21. B Term: longitudinal diffusion of the peak (band broadening) 21 Inversely related to flow rate (1 / ц ) : the longer the solute stays in the system, the more diffusion can occur.  Think of the extreme case: stopping flow  Occurs while solute resides in mobile phase  Insignificant when solute is in stationary phase  Keeping solute in stationary phase improves H  Gradient HPLC  GC with temperature programming time Actually It looks like this:

22. C Term: Mass Transport (resistance to mass transport, rmt) Most important term, most significant improvements in chromatography 22  As the solute travels, it interacts with the stationary phase. The resistance to mass transfer term relates to the rate at which the solute can exchange between the 2 phases.  The solute comes to equilibrium (according to its partition coefficient) but the equilibrium is disrupted as the solute is swept downstream.  The solutes left behind must desorb, returning to the mobile phase to “rejoin the peak”. The solutes that are swept ahead must return to the stationary phase to allow the others to “catch up”.  The rate of these exchanges determines the C term.  The C term is directly proportional to linear flow rate (as the flow is increased, it becomes more challenging to keep the solute molecules together, resulting in band broadening.)  C term is proportional to diameter of particle, film thickness in capillary columns.

23. Plate Theory  The concept of theoretical plates, or the height equivalent to a theoretical plate, was introduced by chemical engineers interested in comparing the efficiency of distillation columns  To calculate height equivalent to a theoretical plate (sometimes called HETP or simply H)  Smaller the H the more efficient your chromatographic/separation process Efficiency Column Length

24. Plate Theory 24  Used to quantitatively measure columns ability  View column as divided into a number (N) of adjacent imaginary segments called theoretical plates  Within each theoretical plate complete equilibration of analytes between stationary and mobile phases occurs  Significance? Greater separation occurs with:  In the most efficient modern systems, a theoretical plate count of several hundred thousand can be achieved in minutes or less.  greater number of theoretical plates (N)  as plate height (H or HETP) becomes smaller

25. Plate Theory  Direct measure of band broadening  Efficiency  Measure of the band broadening during its retention in the column  Assuming that the band spreading follows a Gaussian distribution efficiency (N) can be defined by:  For a Gaussian band (normal distribution) N= t R 2 σ t 2 Standard deviation of the distribution at time t Time band is retained in the column Width at half height of the band Time band is retained in the column 2 Peak Area Peak Height 2

26. or N = 5.54 (t/w 1/2 ) 2 What happens when t changes? What happens when w changes? 26 2 Calculating Theoretical Plates

27. A, eddy diffusion C, mass transport B, long. diffusion Plate height Linear flow sum Related to flow rate: A is constant, B can be minimized C slope influences limits of system Bad Good 27

28. How to Increase Plate Count 28  Well-packed columns (A term)  Lower flow rates (C term)  Higher flow rates (B term)  Smaller particles or capillary stationary phase (C term)  Higher temperature (C term)  Lower mobile phase viscosity (C term)  No extra-column effects (A term)  Column length (plate count)  Gradient HPLC, Temperature program GC (B term)

29. Relationship to particle size

30. Consequences of particle size

31. Consequences of particle size  Efficiency starts to grow exponentially as particle size goes lower  Graph shows:  Flow= 1 mm/sec  Flow Resistance factor = 1000  Viscosity = 1 centipoise  Length = 15 cm 31 Gruska, E., Snyder, L. R., and Knox, J. H. J. Chromatogr. Sci., 1975, 13, 25.

32. But…Consequences of particle size  Pressure drop increases exponentially at particle sizes < 3um  Graph shows:  Flow= 1 mm/sec  Flow Resistance factor = 1000  Viscosity = 1 centipoise  Length = 15 cm 32

33. Separation Efficiency 33  A compromise must be reached between analysis time and flow rate.  In practice the contributions of the A term are a function of the column.  At low flow the B term makes more of a contribution but can be easily minimized.  The C term can be optimized with flow rate.  The C term gets the most attention by column and instrument designers.

34. Separation Performance Mechanics behind a separation and how to measure Plate Theory Van Deemter Equation First principals Kinetic Performance How temperature, pressure, volume and heat flow effects chemical and physical processes Retention mechanisms Thermodynamics

35. Variables to effect separations 35  How to effect a separation (achieve R) by considering:  N, k, α, and T  Theoretical plates, N  Retention Factor (k)  Retardation factor, R f (TLC)  Resolution, R (or R s )  Selectivity, α  tailing factor, T N= t R 2 σ t 2

36. Retention Factor (k’) k’ = 0 means no retention k’ = 1 means double the time of an unretained compound

37. Separations occur because of differences in solute behavior: 37  Partition/Absorption: Reversed-phase HPLC; solute dissolves in the bulk of stationary phase  Adsorption: Normal phase HPLC or TLC; solute stays on surface  Vapor pressure: GC  Molecular size: size exclusion (gel filtration)  Ionic charge density: ion exchange retention

38. The capacity factor of B > A If k’ = 4, the V o of a column =1 ml and the flow is 1 ml/min, the total time taken for the compound to pass through the column would be 5 min, i.e. for the 1 min required to pass through the void space in the column, 4 min would be spent in the stationary phase. (note: the time axis increases from right to left) 38

39. Void Volume and Retention Factor  A compound may not appreciably partition into the stationary phase, if so it will travel through the column at the same rate as the mobile phase.  The time taken for an unretained molecule to flow through the column is determined by the void volume of the column V o 39 A compound may not appreciably partition into the stationary phase: so it will travel through the column at the same rate as the mobile phase. Solvent front t 0 The time (t 0 ) taken for an unretained molecule to flow through the column is determined by the void volume of the column V o

40. Selectivity factor ( α ) 40  Also known as the separation factor  describes the separation of two species (A and B) on the column  Selectivity will always be >1 by definition  Higher the selectivity the more robust of a separation  Critical pair: two species with the minimal resolution between the peaks  hence  lowest selectivity

41. Strategies to influence thermodynamics 41 Changing Stationary phase Change the surface chemistry Changing mobile phase Solvent strength Temperatures

42. Fundamental Physical-Chemical Interactions 42 TypeExampleChrom. example In waterIn Hexane (organic) ElectrostaticRC00 - --- + H 3 NR’Ion exchangeWeakerStronger Hydrogen Bond SiO--H----OH 2 R 3 N:----H--OR’ Normal phase, Silanol/tailing WeakerStronger Hydrophobic Forces (van der Waals) aromatic, aliphaticReversed phase chromatography StrongerWeaker

43. Most Common HPLC and TLC Stationary Phases (Bonded Silica) Electrostatic? Hydrogen bonding? Hydrophobic?

44. Hydrogen bonding in Normal phase chromatography Hydrophobic interactions in Reversed-phase chromatography Effect of Stationary phase 44

45. Polarity 45 What makes a molecule polar?  Linear molecule (CN)  Single H (HCl, HF)  Alcohol group (CH 3 OH)  Oxygen at one end (H 2 O)  Nitrogen at one end (NH 3 )

46. Electrostatic Interactions in Ion Exchange Chromatography 46

47. Solvent Strength 47 Strong Solvent: K is small [X] s < [X] m Weak Solvent: K is large [X] s > [X] m

48. Summary 48  Multiple ways to effect separation performance  Leverage to help optimize separation  Many combinations of:  Mobile phases  Stationary phases  Instrumentation factors (Temperature, flow rates)  Leverage literature to gain starting point

49. Separation Performance Mechanics behind a separation and how to measure Plate Theory Van Deemter Equation First principals Kinetic Performance How temperature, pressure, volume and heat flow effects chemical and physical processes Retention mechanisms Thermodynamics

50. Resolution  Mathematically describes the separation between two peaks taking into account:  Selectivity factor ( α )  Retention factor (k’)  Efficiency (N)

51. Resolution  Mathematically describes the separation between two peaks taking into account:  Selectivity factor ( α )  Retention factor (k’)  Efficiency (N)

52. Other methods to calculate R 52 R = 2 (t 2 – t 1 ) (w 1 + w 2 )

53.  Without calculation A and B are obviously well resolved.  Incomplete separations make resolution determinations more difficult since overlap obscures the start and finish.  If peak shape is good it is possible to assume starts and finish as in B and C above. 53

54. Resolution, R 54 Relative Peak Areas of the two components Larger peak can swamp out smaller peaks

55. Resolution  As column efficiency increases the resolution between two closely eluting peaks will increase.  A R s of 1 indicates a separation of 4  between the apex of 2 adjacent peaks.  A R s >1.5 indicates complete baseline resolution for peaks of similar size. 55

56.  (selectivity or separation factor) = k 2 /k 1 k 1 and k 2 are the k for adjacent peaks (k=capacity or retention factor) k = average k for two peaks N = column plate count Consider : If α = 1, then R=0 If N doubles, R increases by 1.4 If retention (k) doubles, then R increase depends on k. Therefore, deciding what to change to get a separation, depends on the situation. To resolve two peaks: Resolution can be expressed in terms of selectivity, plate count, and retention: 56

57. As retention time (k) is increased, resolution increases. If k is small then there can be big gains in R. (assuming some resolution already exists, i.e. α > 1) If k is already large, increasing k will have minimal effects. Improve R by changing k (assuming same column) 57

58. If α = 1, then R=0 If increasing k doesn’t work, then try to change (increase) selectivity. With same stationary phase: 1.Change B solvent (methanol, acetonitrile) 2.Change pH 3.Change buffer, ionic strength 4.Additive or complexing agent (TEA, EDTA) Change stationary phase: 1.Reversed-phase to normal phase 2.Column brand or end-capping 3.GC column packing/coating Improve R by changing selectivity, alpha 58

59. Example of Selectivity Change by changing pH (same column, same gradient, different pH) 59

60. If N doubles, R increases by 1.4 N is a function of the column, therefore an increase in N, is not the most convenient way to increase R. If there are >>2 peaks to resolve, then increase in N may be best choice. Increase N: Smaller particles Change from packed to capillary GC column Improve R by increasing N 60

61. Next 4 slides show 4 examples of changing HPLC conditions, k, selectivity, N: and affect on Resolution 61 Change: C18 to C8 k: decrease selectivity: same N: same Run time:decrease Change: C8 to Phenyl k: change selectivity:different N: same Run time:improved Change: C8 to Phenyl to CN k: change selectivity:same (except for 5,6,7) N: same Run time:improved Change: non endcap to endcap k: different selectivity: different N: increase Peak shape:improved

62. Change: C18 to C8 k: decrease selectivity: same N: same Run time:decrease Columns: 4.6 x 150 mm, 5µm Mobile Phase: 80% 25 mM NaH 2 PO 4, pH 3.0 20% MeOH Flow Rate: 1.0 mL/min Temperature: 35ºC UV Detection: 254 nm Sample: 1. Theobromine 2. Theophylline 3. 1,7- dimethylxanthine 4. Caffeine 62

63. Change: C8 to Phenyl k: change selectivity:different N: same Run time:improved Columns: Rapid Resolution 4.6 x 150 mm, 3.5 µm Mobile Phase: 40% H 2 O 60% MeOH Flow Rate: 1.0 mL/min Temperature: 35º Sample: 1. Acetophenone 2. Cinnamaldehyde 3. Eugenol 4. Cinnamaldehyde impurity 5. Ethyl cinnamate 6. p-cymene 63

64. Change: C8 to Phenyl to CN k: change selectivity:same (except for 5,6,7) N: same Run time:improved 64

65. Change: non endcap to endcap k: different selectivity: different N: increase Peak shape:improved 65 Columns: Rapid Resolution 4.6 x 75 mm, 3.5 µm Mobile Phase: 70% 25 mM NaH 2 PO 4, pH 3.0 30% Methanol Flow Rate: 1.0 mL/min Temperature: 35ºC Sample: 1. Barbital 2. Sulfamethoxazole 3. Caffeine

66. Tailing factor, T (symmetry) 66 T = 1peak is symmetrical T < 1 peak is fronting T > 1 peak is tailing

67. Effect of Tailing on Resolution and Quantitation (10:1) 67 Tailing Factor of First Peak: (a) 1.0 (b) 1.2 (c) 1.5 (d) 2.0

68. Peak Symmetry Tailing, fronting, splitting, atypical band broadening  Common causes of asymmetry  Column overload: sample mass is greater than column’s capacity  Sample decomposition  Sample matrix is incompatible with mobile phase (sample solvent should be mobile phase or weaker in solvent strength)  Dirty system, where analyte adsorbs onto active sites other than the column  Secondary retention mechanism  Inefficient trapping of analyte when it is loaded onto the column, too much dead volume  System leaks (GC, HPLC)  Non-homogeneity of stationary phase (e.g. impurities)  Extracolumn effects 68

69. Gas Chromatography

70. Chromatography Instrumentation“Generic” Chromatograph Gas Chromatography (GC) - In GC (generally) compounds are separated by boiling points, and interactions with the stationary phase. MPdetector signal processor readout injector column oven injector signal processo r readout detector pressure regulator MP = carrier gas flow controller split vent

72. 2.Injector: microflash vaporizer injector split/splitless Injection port held ≥50 o C higher than highest bp Sample size: 0.1-20  L (packed) 0.001-1  L (capillary) 3. Column (heart of the chromatograph, where separation occurs) **SP later!! A. packed -made of glass, Al, stainless steel or Cu - L = 2-3 m, id = 2-4 mm -SP = thin liquid film (.05-1  m) coated on solid support particles -particles must be chemically inert, thermally stable 149-170  m (80-100 mesh) 170-250  m (60-80 mesh) -surface areas ≥ 1m 2 /g (smaller more uniform particles = ↑ N)

74. 4.Oven Controls temperature of the separation (thermostated to ±0.1 o C) Optimum column/oven temp. depends on BP of cmpds in sample a.Isothermal = constant temperature throughout separation -use temp at or slightly above the average bp in sample -in general, higher temp = lower resolution -General Elution Problem (*show PP) b.Temperature programming = ramp temp during separation -used for samples with broad range of bp’s. -to optimize resolution & efficiency Rate o C/min Temp o C Time min 802 101004 Time (min) Temp ( o C) Isothermal 80 o C Signal 2468

75. 1.) -effluent from column ignited in H2/air flame -hydrocarbons produce ions & e-:CH (radical) + O → CHO + + e- (except carbonyl, C=O and carboxyl, O=C-OH) conducts electricity! -potential (~300V) applied bet. burner tip & collector electrode -current (~10 -12 A) prop. to #reduced carbons amplified and read (mass sensitive, not concentration sensitive) 2.)most widely used GC detector -sensitive to hydrocarbons -not sens. to functional groups (halogens, amines, carbonyls, alcohols) -not sens to non-combustible gases (H 2 O, SO 2, CO 2, NO, NO 2 ) 3.) + high sensitivity + large LDR (10 7 )- destructive + low noise+/- versatile but + low LOD (pg) not selective GC Detectors: Flame Ionization Detector (FID)

76. B.Thermal Conductivity Detector (TCD) 1.) -electrically heated element (Pt, Au, or W wire) -temp of wire depends on thermal conductivity of gas surrounding it -when solute present, TC of gas ↓, temp of wire ↑, resistance ↑ 2.) -first GC detector -”universal” detector (responds to everything) -low sensitivity (best with packed columns) -use with H 2 or He carrier gas only (they have high TC’s) 3.) + simple operation + non-destructive + responds to both organic & inorganic + large LDR (~10 5 ) - low sensitivity - highLOD - non-selective (?)

77. C.Electron Capture Detector (ECD) 1.) -effluent passes over  -emitter (Ni-63) [  particle = e - ] -ionization of carrier gas (N 2 ) causes constant current -when solute present, captures e - and current ↓ 2.) -sensitive toward electronegative functional groups (halogens, peroxides, nitrogroups) -insensitive toward hydrocarbons, amines, alcohols -main application = environmental (Chlorinated pesticides!!) 3.) + very selective + high sensitivity + non-destructive (but alters sample) + lower LOD than FID (for Cl – cmpds) - not versatile (?) - low LDR (~10 2 )

78. D.Thermionic Detector (TID or NPD = Nitrogen-phosphorus detector) 1.) - operation similar to FID except: - gas flows around alkali metal salt (rubidium silicate bead) plasma forms (600-800 o C) [plasma = gas w/ ions & e - ] - potential applied between bead and collector electrode - current increases in presence of N & P atoms 2.) - 500 x more sensitive than FID for P-cmpds - 50 x more sensitive than FID for N-cmpds - application = environmental (P-containing pesticides & soil) 3.) + very selective- destructive + very sensitive (N&P)- not versatile (?) Other GC detectors: 1.Sulfur Chemiluminescence Detector (SCD) 2.Electrolytic Conductivity Detector (ELCD) 3.Flame Photometric Detector (FPD) 4.Atomic Emission Detector (AED) 5.Photoionization Detector (PID) 6.Infrared Detector (IR) 7.Mass Spectrometer (MS)

79. Ideally characteristics of SP: Low volatility (bp 100 o C higher than highest oven temp) Thermally stable Chemically inert Solutes should “match” SP in polarity…so elution order based on bp Types of SP: 1.Polyethylene glycol (e.g., Carbowax 20M) General purpose for polar compounds Acids, alcohols, ethers, oils, glycols 250 o C max temp 2.Polydimethyl siloxane (PDMS; ov-?) Can be substituted (e.g., 5% phenyl) to change polarity General purpose nonpolar Hydrocarbons, aromatics, PCB’s, drugs, steroids, pesticides 350 o C max temp (lower for highly substituted) GC Stationary Phases:

80. 3.Chiral SP’s – need to separate enantiomers ($expensive!) General purpose for polar compounds Acids, alcohols, ethers, oils, glycols 250 o C max temp 4.PLOT (porous-layer open tubular; gas – solid chromatography) For small gases, get trapped in pores

81. Thin Layer Chromatography (Planar Chromatography)

82. Thin Layer Chromatography (TLC) 82  Planar system: earliest technique along with paper chromatography.  Good for trace impurities.  Flexible: can detect almost any compound including some inorganic materials.  Flexible detection: the spots can be viewed by an unlimited variety of general or specific reagents, UV fluorescence, UV quenching. Detection reagents can be sequential.  Pure compounds can be easily retrieved for subsequent structure confirmation or elucidation.  Upon completion of a run all the material is evident, thus there is no doubt that a compound did not elute as in GC or HPLC.  Because of its simplicity it is often used prior to other techniques.

83. “Instrumentation” 83  Glass or plastic plate coated with silica gel of particles in the 2-25  m range.  Typical Method: A few  l of sample solution are slowly spotted onto the plate at the origin. Loading must be slow and a period between each application should exist for drying. Typical sample load is 20  g.  The bottom 0.5 cm of the plate is immersed in the MP contained in a tank and allowed to travel up the plate by capillary action.  The more polar a compound the more it adsorbs (partitions into) the silica SP, the less time it spends in the MP as it travels up the plate and thus the shorter the distance it travels up the plate in a given time.

84. 84

85. 85  A is less polar than B since it travels further.  The distance traveled by the compound from the origin divided by the distance traveled by the solvent from the origin is called the R f value of the compound.  For A, R f = a/S and for B, R f = b/S  R f is usually quoted as R f X 100.  The area/intensity of a spot on a plate is logarithmically related to the concentration of the analyte producing it.

86. Stationary Phases 86  Silica gel is most common.  The rate of compound migration up the plate depends on polarity.  In a given time the most polar compound moves the least while the least polar moves the most.

87. Elutropic Series and Mobile Phases  The “strength “ of a MP depends on the particular solvent mixture used.  The more polar the solvent or solvent mixture, the further it will move a polar compound.  With non-polar compounds there will not be a marked increase in the distance migrated with increasing MP polarity because they migrate towards the solvent front.  Although water is polar it is not used alone since many organics are miscible. 87

88. Detection following development 88  UV light: silica gel impregnated with fluorescent material is often used to prepare TLC plates in order to observe the UV absorption by an analyte.  Developed plate is irradiated with light at 254nm  UV absorbing species appear as a black spot on a yellow-green background where the UV absorbance is quenched.  Natural fluorescence: samples are irradiated with light at 365nm.  Chemical Methods: Many reagents exist that react with various types of solutes on a TLC plate to yield colored or fluorescent species for visualization.  Iodine: most common, produces a light brown spot with most organic compounds.  Appropriate reagent can be chosen based on known properties of the analyte.

89. Applications of TLC Analysis 89  Qualitative identity tests.  Limit tests: A TLC limit test is based on comparison between a concentrated solution of an analyte and a dilute solution of an impurity. Below is a test where the structure of the impurity is known. In this case the limit test failed because the impurity is more intense (bigger) than the standard.

90. Limit tests where impurity is not known 90  Often used with natural products with a wide variety of impurities that are similar to the analyte.  Assumption is made that the related unknowns will produce a spot of similar intensity to the test substance when at equal concentrations.  To pass this limit test: There should be no spots in solution 1 more intense than 2. There should be no more than one secondary spot with a R f higher than that of codeine and more intense than 3

91. Summary Wrap Up! 91

92. Separation Goals  Develop to answer a specific question?  New lot of material has “new peak” then it may simply be an investigation into development  New product and need to develop a method for clinical release  Do you have to resolve all components in the sample?  What sort of sample throughput will ultimately be required? Good practice develop a method that would be mass spec compatible volatile buffers versus inorganic buffers Good practice develop a method that would be mass spec compatible volatile buffers versus inorganic buffers Routine practice Two orthogonal methods developed to ensure the analytical method is not fooling you Routine practice Two orthogonal methods developed to ensure the analytical method is not fooling you

93. Define Separation Goals  What is the question you are trying to answer?  Why are you developing the method in the first place? 1. Define/Identify the Problem 2. Form a Hypothesis 3. Make Observations or Test Hypothesis and Perform Experiments 4. Organize and Analyze Data 5. Do Experiments and Observations Support Hypothesis?  If No, Perform New Experiments and Repeat Step 4 6. Draw Conclusions 7. Communicate Results

94. Basic Questions for Sample  Number of compounds present in the sample matrix  2 or 200?  Chemical structures known of compounds?  Molecular weight range  pKa of the compounds  Spectroscopic response (UV/VIS)  Concentration range expected?  Sample Solubility?  Quantity of sample available?  1 mg or 1 kilogram  proportional to the number of experiments you can run  Representative diversity of samples available for the methods development work  Stability of samples?  Light, oxygen or temperature sensitivity  ICH Q1: Stability … but need a method to measure stability Chicken or egg?  Must develop methods in parallel to help answer questions on stability, but also challenge analytical rigor of method as a function of development!

95. Steps to develop method 1 Information on Sample, define separation goals 2 Define separation mode, procedure, pretreatment 3 Choose Detector and settings 4 Choose LC Method, preliminary run 5 Optimize separation conditions 6 Check for problems, challenge method 7 Validate method for release

96. Theory, Application of TLC, HPLC, CE Separation Methods: Part 2 September 2014 Pharmaceutical Analysis Course (#8002)