1. GF v03 Aug 2000 1 Gel Filtration

2. GF v03 Aug 2000 2 Gel filtration What is gel filtration and why use it? Some typical results Why and when to choose gel filtration Model of the mechanism How to get the expected results Unexpected results and what to do about them

3. GF v03 Aug 2000 3 What is gel filtration? Gel filtration is a technique of liquid chromatography which separates molecules according to their sizes.

4. GF v03 Aug 2000 4 Simple to do Easy to understand Nothing to go wrong Gel filtration in the lab

5. GF v03 Aug 2000 5 Why use gel filtration? Some typical results Group separations: Desalting, Buffer exchange, Removing reagents Purification of proteins and peptides: complex samples, monomer/dimer Estimation size & size homogeneity

6. GF v03 Aug 2000 6 Desalting proteins Highly efficient desalting in less than 1 minute 10 20 30 40 50 sec

7. GF v03 Aug 2000 7 Desalting proteins Desalting in a simple column aa 2 Elution volume (ml) 0 4681012 AlbuminNaCl Column: Sample: Buffer: PD-10 HSA, 25 mg NaCl 0.5M

8. GF v03 Aug 2000 8 Desalting the sample 0 1 min Sample: His tagged protein eluted from HisTrap with 20 mM sodium phosphate, 0.5 M sodium chloride, 0.5 M imidazole, pH 7.4 Column: HiTrap Desalting 5 ml Buffer: 20 mM sodium phosphate, 0.15 M sodium chloride, pH 7.0 System: ÄKTAprime, 5 ml/min

9. GF v03 Aug 2000 9 Sample: BSA dissolved in 50 mM piperazine, 0.5 M sodium chloride, pH 6.2 Column: HiPrep 26/10 Desalting Buffer: 20 mM sodium phosphate, 0.15 M sodium chloride, pH 7.0 System:ÄKTAprime, 20 ml/min Buffer exchange using HiPrep 26/10 Desalting column

10. GF v03 Aug 2000 10 Purification of recombinant IGF-1 Column: HiLoad 16/60 Superdex 75 prep grade Sample: IGF-1, ZZ fusion protein and uncleaved material Buffer: 0.15 ammonium acetate, pH 6.0 Flow rate: 0.75 ml/min (22.5 cm/h) 1 2 Time (h) IGF-1 AU 280 0.1 0.05

11. GF v03 Aug 2000 11 Sample: 4 ml concentrated eluate from a HIC run containing rPhosphatase Column: HiLoad 16/60 Superdex 75 prep grade Buffer: 25 mM Tris-HCl, 0.3 M sodium chloride, 1 mM EDTA, 2 mM DTT, pH 7.4 System:ÄKTAprime, 0.5 ml/min (15 cm/h) Purification of recombinant phosphatase

12. 12 Enzyme purification - SDS-PAGE analysis M - LMW markers 1 - Start material 2 - Pool from ion exchange 3 - Pool from HIC 4 - Pool from Superdex 75 pg 4321M M 67k 43k 30k

13. GF v03 Aug 2000 13 Separating dimer and oligomers from monomer Column: Superdex 75 HR 10/30 Sample: A special preparation of rhGH in distilled water 0.025 0.05 Oligomer Monomer ime (min) Dimer T1020 V O V C 280nm A

14. GF v03 Aug 2000 14 Characterisation: Size homogeneity HiTrap Chelating 5 ml M r 160 000 Superdex 200 HR 10/30

15. GF v03 Aug 2000 15 Estimating molecular size Measure elution position Calculate molecular size

16. GF v03 Aug 2000 16 Why choose gel filtration? Pros l Fastest for buffer exchange l Very gentle, high yields l Works in any buffer solution l Removes dimers and aggregates l Separates by size Cons l Limited sample volume l Poor selectivity compared with SDS-PAGE

17. GF v03 Aug 2000 17 Use in group separations l Adjusting pH, buffer type, salt concentration during sample preparation, e.g. before an assay. l Removing interfering small molecules, e.g. EDTA, Gu.HCl l Removing small reagent molecules, e.g. fluorescent labels, radioactive markers. l But not when the protein will precipitate

18. GF v03 Aug 2000 18 Use in fractionation l Excellent during the polishing stage l Removes dimers and other aggregates l Transfers protein to buffer solution ready for the next set of experiments l Not so suitable if the sample volume is large

19. GF v03 Aug 2000 19 Use for size estimation l 'Free' information l Gives an estimate of molecular size in any practically any solution l Precision is not so good

20. GF v03 Aug 2000 20 Nomenclature Gel filtration (Porath, Flodin, 1959) Size exclusion chromatography (Pedersen, 1962) Molecular sieve chromatography (Hjertén, Mosbach, 1962) Gel permeation chromatography (Moore, 1964)

21. GF v03 Aug 2000 21 Separation mechanism Qualitative model Gel structure Steric exclusion

22. GF v03 Aug 2000 22 Gel structure AGAROSE A good gel for gel filtration contains about 95% water

23. GF v03 Aug 2000 23 Gel structure Agarose Dextran A hypothetical structure for Superdex

24. GF v03 Aug 2000 24 Steric exclusion Molecules are excluded from the gel bead to different extents according to their sizes. Gel bead Very large molecules are completely excluded Very small ones get everywhere

25. GF v03 Aug 2000 25 Steric exclusion leads to early elution Molecules elute in order of size. The largest molecules come first ; the smallest ones come last.

26. GF v03 Aug 2000 26 Terms used, defined Calibration curves Selectivity and efficiency Principles of gel filtration

27. GF v03 Aug 2000 27 Terms and explanations Void volume V o Volume of the gel matrix V s Pore volume V i V o = Void volume V r = Elution volume within the separation range of the gel V i = Inner pore volume = V c - V s - V o V c = Total (geometric) volume of the column 2 3 1 VoVo VrVr VtVt VcVc

28. GF v03 Aug 2000 28 The void volume V o VoVo VrVr Volume Concentration Elution volume for very large molecules, V o

29. GF v03 Aug 2000 29 V t and V c Volume Concentration VtVt VcVc VoVo VrVr Elution volume for very small molecules, V t Geometric volume of the gel bed, V c

30. GF v03 Aug 2000 30 The distribution coefficient, K d K d is difficult to get because V i is difficult to measure

31. GF v03 Aug 2000 31 The coefficient K av K av is easy to get and it is more useful in practice

32. GF v03 Aug 2000 32 K av for very large and very small molecules

33. GF v03 Aug 2000 33 K av should always be in the range 0 to 1 VtVt VrVr Volume Concentration Elution volume when K av = 1 Elution with K av > 1 Adsorption has occurred Elution with K av < 0 Serious problem!

34. GF v03 Aug 2000 34 The number of peaks is limited 200 400ml 0 Maximum number of peaks in RPC > 150 0 10 20 ml Maximum number of peaks in gel filtration ca 15 but we are not purifying peaks!

35. GF v03 Aug 2000 35 Resolution R s Rs = 4 Rs = 0.6 Rs = 1 Resolution = Peak separation Av. peak width

36. GF v03 Aug 2000 36 Resolution depends on efficiency and selectivity High efficiency Low efficiency High selectivity Low selectivity Efficiency is a measure of peak width Selectivity is a measure of peak separation

37. GF v03 Aug 2000 37 High efficiency can compensate for low selectivity. If selectivity is high, low efficiency can be tolerated (if large peak volume is acceptable). Resolution depends on efficiency and selectivity Low selectivity High selectivity high efficiency low efficiency high efficiency low efficiency

38. GF v03 Aug 2000 38 Calibration curves Plotting the elution volume versus the logarithm of molecular mass yields a calibration curve. VrVr log Mr

39. GF v03 Aug 2000 39 Selectivity curves Plotting K av versus the logarithm of molecular mass yields the selectivity curve of the matrix. The steeper this curve, the greater the difference in elution volume for two molecules of different sizes. K av log M r  K av  log M r  K av  log M r

40. GF v03 Aug 2000 40 Constructing a selectivity curve K av 1 0 log (M r ) 1. Run standards and determine the elution volume for each. 2. Calculate K av values. 3. Plot log (M r ) for each standard against the calculated K av.

41. GF v03 Aug 2000 41 Exclusion limit K av 1 0 log (M r ) Exclusion limit All molecules bigger than this elute in the void volume

42. GF v03 Aug 2000 42 Fractionation range K av 1 0 log (M r ) 0.7 0.1 Fractionation range A selectivity curve is usually fairly straight over the range Kav=0.1 to Kav=0.7

43. GF v03 Aug 2000 43 Separation ranges of modern supports 1: Superdex Peptide 2: Superdex 75 3: Superdex 200 4: Sephacryl S-100 HR 5: Sephacryl S-200 HR 6: Sephacryl S-300 HR 7: Sephacryl S-400 HR K av 1.0- 0.8- 0.6- 0.4- 0.2- ---------- 1000 10,000 100,000 1,000,000 10,000,000 1 2 3 4 5 6 7

44. GF v03 Aug 2000 44 Increasing exclusion limit Results depend on selectivity Sephacryl S-100 40 80 120 ml Sephacryl S-300 Sephacryl S-200 BSA Cyt C IgG  -L Cytidine AU 280 Better for larger proteins Better for smaller proteins Best for these proteins

45. GF v03 Aug 2000 45 Shape effects A gel has different fractionation ranges for native, globular proteins and denatured proteins in random coil Denatured proteins K av 1 0.7 0.1 log (M r ) Native proteins

46. GF v03 Aug 2000 46 Shape effects K av 1 log (M r ) Molecules with different shapes have different selectivity curves. Linear polysaccharides Globular proteins

47. GF v03 Aug 2000 47 V ) N = 5.54 ( WhWh r N = Number of theoretical plates Test: 1% solution of acetone (about 0.5% of column volume), 0.2 AUFS at 280 nm. Alternatively use 2 M NaCl and conductivity monitor. Efficiency W h = Peak width at half peak height VrVr AU 280 2

48. GF v03 Aug 2000 48 Efficiency Efficiency depends on: Particle size of matrix Particle size distribution of matrix Packing quality of the column Sample (volume and viscosity) Flow rate

49. GF v03 Aug 2000 49 Band broadening effects To increase efficiency: Lower the flow rate Flow rate Peak width Large particles/large sample molecules Small particles/small sample molecules

50. GF v03 Aug 2000 50 Band broadening effects Flow rate Peak width Large molecules Small molecules To increase efficiency: Increase the flow rate

51. GF v03 Aug 2000 51 To increase efficiency: Use uniform beads in well packed columns Band broadening effects Flow rate Peak width Uneven packing Irregular particles Good column packing Uniform spherical particles

52. GF v03 Aug 2000 52 peak width ml/min Diffusion and the impact of flow rate on peak width Increasing flow rates lead to broad peaks for large molecules. Very low flow rates are not suitable for very small molecules as column diffusion becomes too significant. Peak width Large molecules ml/min Mass transfer Diffusion along column Eddy Diffusion Peak width Small molecules Human serum albumin (Mr 68500) Myoglobin (Mr 16900) Tyrosine (Mr 181) ml/min Peak width ml/min

53. GF v03 Aug 2000 53 Peak width depends on particle size p Superdex Peptide 13-15 µm p Superdex 30 prep grade 24-44 µm

54. GF v03 Aug 2000 54 Superdex and Sephadex Bed length:60 cm Sample:Mouse monoclonal cell supernatant IgG 1 Sample size:1.2 ml (1 % V c ) Flow rate:0.2 ml/min (6 cm/h), Sephadex G-200 1.6 ml/min (50 cm/h), Superdex 200 prep grade Sephadex G-200 Superdex 200 prep grade 600 min 80 min Smaller beads can give both better resolution and shorter separation times

55. GF v03 Aug 2000 55 1 x Superdex ® Peptide HR 10/302 x Superdex ® Peptide HR 10/30 Resolution depends on column length Increasing column length increases resolution

56. GF v03 Aug 2000 56 Column: Superdex Peptide HR 10/30 Resolution depends on sample volume 25µl 200 µl 400 µl

57. GF v03 Aug 2000 57 Samples which are much more viscous than the eluent give very poor results even for a simple group separation. Resolution depends on sample viscosity Relative viscosity: 1.0 Relative viscosity: 4.2 Relative viscosity: 11.8 Haemoglobin and NaCl, viscosity changed by adding dextran

58. GF v03 Aug 2000 58 Recommended gels Desalting and other group separations l Sephadex G-25 Fine or Superfine grades Fractionation l Use a pre-packed column! l Superdex or Superdex prep grade l Native proteins: Superdex 200 l Recombinant proteins: Superdex 75 l Peptides: Superdex Peptide or Superdex 30

59. GF v03 Aug 2000 59 Column size Desalting and other group separations l Volume four times the expected sample volume l Length is not so important Fractionation l Volume ca 20-200 times the expected sample volume l Length 30-100 cm

60. GF v03 Aug 2000 60 Column packing Choose a properly designed column Prepare the gel slurry carefully Pack the column at the temperature at which it will be used Add all the slurry in one smooth movement Pack at constant pressure or constant flow rate Stabilise and equilibrate the packed bed Check the efficiency; re-pack the column if necessary A well-packed column is ESSENTIAL

61. GF v03 Aug 2000 61 Preparing and applying the sample Preparation Check the viscosity and remove nucleic acids and polysaccharides if necessay Filter or spin to remove particles Volume for desalting: up to 25% column volume Volume for fractionation: 0.5-5% column volume Application Through an adapter at the top of the column or Layering under the eluent

62. GF v03 Aug 2000 62 Increasing resolution Check the column efficiency Clean and/or re-pack Check that the separation is in the fractionation range Reduce the sample volume Reduce the flow rate Change to a gel with smaller beads Connect two columns in series

63. GF v03 Aug 2000 63 Trouble shooting Resolution and recovery See the Notes view for ideas which may be of use in answering questions on this topic

64. GF v03 Aug 2000 64 Trouble shooting Elution earlier than expected See the Notes view for ideas which may be of use in answering questions on this topic

65. GF v03 Aug 2000 65 Trouble shooting Elution later than expected See the Notes view for ideas which may be of use in answering questions on this topic