gelchromatography presentation ..........

chromatography

1. GEL CHROMATOGRAPHY Presented by, Shruthy. P 1year , M Pharm Pharmacology 1

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3. Differences with other techniques GPC must operate free of interaction and separation based on size only. Only the entropy effects should influence the separation. HPLC methods rely on interactions between sample and stationary phase; those interactions result from ion exchange, bio-affinity or chirality 3

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5. PRINCIPLE 5 The separation of molecules on the basis of their molecular size and shape is achieved by gel filtration chromatography. It uses the molecular sieve properties of various porous resins . Large molecules that are completely excluded from the pores pass through the void space, interstitial spaces between the resin particles and thus, they elute first. While smaller molecules get distributed between the mobile phase inside and outside the beads and therefore pass through the column at a slower rate. Thus, they elute in the last .

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7. Trapping of the mobile phase inside the beads depends upon the porosity of the resin beads and the size of the molecules to be separated. Thus, the distribution of a molecule in a gel filtration column of cross-linked beads is determined by the total volume of mobile phase , both inside and outside the beads. In case of gel filtration chromatography, the distribution coefficient of a molecule between the inner and outer mobile phase is a function of its molecular size . 7

8. Theory of SEC The total volume V t of a column packed with a porous polymer or silica gel is given by V t = total volume of packed column V = void volume V i = internal volume of beads V g = volume of gel (solid material) 8 V t =V + V i +V g

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10. Volume of mobile phase required to elute a component which is too small than pores. (V e )-Elution volume , Elution volume of component given by V e = V + K d (Kd) Distribution Coefficient which describes how much of the internal volume is available for the compound. For molecule too larger to enter the pores K d =0 . For molecule that can enter the pores unhindered, K d = 1 10

11. 11 The useful molecular mass range for a size-exclusion packing is conveniently illustrated by means of a calibration curve . Here, molecular mass , which is directly related to the size of solute molecules, is plotted against retention volume VR. where, VR= retention time × volumetric flow rate.

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13. Note that the ordinate scale is logarithmic. The exclusion limit is the maximum molecular mass of a species that will penetrate the pores. All species having greater molecular mass than the exclusion limit are so large that they are not retained and elute together to give peak A in the chromatogram shown. 13

14. Below the permeation limit, the solute molecules can penetrate into the pores completely. All molecules below this molecular mass are so small that they elute as the single band labelled D. As molecular mass decrease from the exclusion limit, solute molecules spend more time in the particle pores and thus move progressively more slowly. It is in the selective permeation region that fractionation occurs, yielding individual solute peaks such as B and C in the chromatogram. 14

15. INSTRUMENTATION The instrumentation of the SEC contains the following components. They are as follows : Mobile phase reservoir Pump Injector Column Detector Recorder 15

16. Mobile phase reservoir Glass or stainless steel reservoirs are commonly employed as reservoirs. In GPC, the commonly employed mobile phases are of non-polar organic mobile phases. Example : Tetra hydro furan, toluene, dimethylformamide, etc. To improve the GPC separations, additives are added to improve the efficiency of separation. Example : An antioxidant is added to the trichlorobenzene to the increase the stability in high temperatures . 16

17. In GFC, the commonly employed mobile phases are aqueous mobile phases . In general, buffer solutions are used. Example : Sodium acetate buffer, sodium phosphate buffer, Sodium borate buffer, etc. Based on the separation of the compounds, the following mobile phases are used Compound to be separated Mobile phase Synthetic elastomer Toluene Polyvinyl carbons Epoxy resin tetra hydro furan Poly olefins Trichlorobenzene Proteins&Polysaccharides Water & buffer solutions 17

18. Pumps : This is mainly used for the continuous flow of mobile phase through the injector with constant pressure of 6,000 psi. The flow rate is ranging from 1 to 10 ml per minute. There are two types of pumps commonly employed in the SEC. 18

19. They are as follows: 1)Syringe pumps: works as a normal syringe and delivers the mobile phase by the pulse less flow. 19

20. 2)Reciprocating pumps: There are two types of reciprocating pumps. single piston reciprocating pumps dual piston reciprocating pumps. Single piston pumps are cheap but these are not suited for the SEC. The dual piston pumps are easy to maintain and frequently used in the SEC. 20

21. In dual piston reciprocating pump suction and delivery strokes occur together. When the plunger moves forward, it makes the water to move out from the cylinder through the discharge valve at right side and the adjacent suction valve is closed in position. At this moment, the suction will be created inside the cylinder which draws the water into the cylinder through left side suction valve whereas the adjacent delivery valve is closed. During the reverse stroke, the water moves out from the cylinder through the delivery valve at left side, and the adjacent suction valve is closed in position. 21

22. Injector : This is manly used for the injection of the solvent through the column. The sample is dissolved in the solvent. Volumes of the mobile must be small that is 1–500 μl . 22

23. Column Smooth bore steel or heavy walled glass tubing is used as column. Column temperature is maintained by the thermostats (105 °C) and is cooled by fitting it in the cooling jacket containing water. The column is packed with the porous silica or organic gels such as styrene and divinyl benzene. Based on the internal diameter and length of the column, they are divided as the following: 23

24. 1)Analytical columns : 8 mm in diameter and 300 mm in length . 2)Microcolumns : 4.6 mm in diameter and 300 mm in length. 3)Preparative columns : 20 mm in diameter and 300 or 600 mm in length . 24

25. The materials used in the column packing: Gels : Gels which are commonly used packing materials There are in essence two approaches to the preparation of these materials A macromolecular substance is brought into solution and allowed to react with a bifunctional reagent in order to render it insoluble, or the solution of a mixture of suitable monofunctional and bifunctional monomers is allowed to copolymerize . 25

26. In either case a gel is obtained the porosity which is a function of the concentration of the bifunctional reagent. This concentration effect on the degree of cross-linking is easily explained by the assumption that more cross-linkages between the polymer chains are formed in concentrated solutions. fewer cross-linkages are formed in dilute solutions because the bifunctional reagent reacts with one and the same molecule. 26

27. With the two techniques described, bead-shaped gel particles of different porosities may be produced. Gels of high porosity which are applicable to the separation of very large molecules and it is relatively soft and difficult to handle them, particularly in organic solvents . The cross-linked polymers are used as stationary phases for the packing of the columns . 27

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29. Types of stationary phases used in SEC. They are as follows: Soft gels such as poly acrylamide gels or dextran gels are used. These gels are mainly used for the separation of the proteins. These are prepared by imbibing the gel into the liquid for the complete swelling. Rigid gels such as polystyrene gels which are mainly used for the separation of the non-polar polymers. 29

30. Column packing The vertically mounted tube is close at its lower end. Glass wool and layer of sand or glass beads are placed into the bottom of the tube, before it is filled to one third of its height with solvent observing that no air bubbles are trapped. 30

31. The gel to be packed has been allowed to swell properly before it is applied in the form of suspension that allows the easy escape of air bubbles The entire tube is filled in one pouring. As soon as a layer of several centimeters height of the gel particles has settled the valve of the column is gradually opened. As the solvent flows out of the tube, new gel suspension can be added to the top. 31

32. It must be carefully observed during the filling operation that three zones have at all times to be in the column: The growing layer of gel particles, the gel suspension above it, and the clear solvent at the top. If the sedimentation is relatively fast with regard to flow rate, the clear supernatant of solvent may be removed from time to time from the column in order to be replaced by new gel suspension. This prevents the formation of density layers along the gel packing solvent The gel and the solvent allowed to acquire the environmental temperature before the operation is started. Frequently it is advisable to counteract the subsequent formation of air bubbles by evacuation or boiling of solvent. 32

33. Detectors : The detectors are mainly used to detect the sample constituents and concentration of the sample. The detectors should posses the following ideal characters: Adequate sensitivity Stability and reproducibility High reliability Low zone broadening Short time process Non-destructive . . 33

34. The detectors are of following types: Concentration sensitive detectors Example: Refractive index detectors UV detectors Molecular weight sensitive detectors Example: Low-angle light scattering detectors. Multi-angle light scattering detectors 34

35. UV-Visible detector Ultraviolet visible detector Most of the organic compounds absorb light in the region of UV (190-400nm) and in the visible region(400-750nm). It is based on Beer-Lamberts law Deuterium and high pressure xenon lamp are the sources of the UV. 35

36. 36 Advantages: High sensitivity Relative robust to temperature Compatible with gradient elution Disadvantages: Only compounds with UV or Visible absorption could be detected.

37. R efractive index (RI) detector A refractive index (RI) detector in GPC (Gel Permeation Chromatography) is  a commonly used detector that measures the concentration of a sample by detecting the difference in refractive index between the sample solution and the pure solvent flowing through the column . 37

38. Advantages: Responds to nearly all solutes Unaffected by the flow rate Disadvantages: Not as sensitive as most other types of detectors Could not be used with a gradient elution 38

39. Low-angle light scattering (LALS) Measure at a very low angle, close enough to zero that the angular effects are negligible and no correction is needed. 39

40. Multi-angle light scattering ( MALS ) Measure the scattered light at two or more angles and extrapolate the data back to estimate the scattered light intensity at zero degrees. 40

41. Recorders : Recorders are commonly employed for the interpretation and recording the data obtained from the detector . 41

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43. P rocess of gel filtration Buffer selection – Any buffer in which the analyte to be separated is stable can be used as buffer in GC. pH, ionic strength and composition do not significantly affect resolution, though the buffer should maintain the buffering capacity and constant pH . Low strength salt can be added to the buffer to avoid any interaction with gel particle. Extreme pH conditions and ionic strengths should be avoided because it may affect the gel as well as separation. The sample buffer does not have to be the same buffer as the column, but prior to running the column should be equilibrated with sample buffer. 43

44. 2 . Sample preparation & loading – The sample should not be diluted as it may cause band broadening. Ideally, 1% of bed volume of sample should be loaded onto the column. The sample should be clear, free of any debris therefore it should be filtered prior to loading. To load the sample manually, remove the upper adapter and remove the excess of the buffer on the top of the gel without disturbing the gel surface. Then, gently pipette in the sufficient amount of sample and allow the sample to pass through the surface . 44

45. 3.Flow rate – Ideally the flow rate should be slow in the range 6-12mL/hr. The slower flow rate may cause diffusion and high flow rate may cause poor resolution. 4.Elution – A fraction collector is attached to the system to collect the fractions of the elution. The sample is eluted isocratically using a single buffer system. The small fractions of size 1mL or less should be collected to avoid mixing of peaks. 45

46. 46 The detector connected to the fraction collector analyses the separation. The total elution should be equal to or more than one bed volume. The volume of the buffer that elutes from the column before a particular peak in the elution profile appears is called elution volume ( Ve ). The distribution coefficient ( Kd ) of an analyte in gel filtration chromatography can be mathematically defined as, Kd =Ve –V0/ Vt – V After completion of each run, the column should be washed thoroughly to remove any analytes left in the gel. Ideally, it should be done by 1-2 bed volumes of buffer. For long term storage, antimicrobial agents should be added to the buffer.

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48. Resolution is a function of selectivity of the resin and the efficiency of that resin to produce sharp, narrow peaks with minimal peak broadening . Resolution (Rs) can be expressed as follows: Rs=Ve2 – Ve1/(W1+ W2 ) 2 Resolution Parameters used to define resolution (Rs ) 48

49. where: Ve1 and Ve2 are the elution volumes for two adjacent peaks measured at the center of the peak. W1 and W2 are the respective peak widths. (Ve2 – Ve1) represents the distance between the peaks and (W1 + W2)/2 the mean peak width of the two peaks as shown in Figure. 49

50. The final resolution, that is the degree of separation between peak depends both on the selectivity on the resin and the degree of peak broadening (Fig ). The resin efficiency and other experimental factors contribute to peak broadening . Resolution depends on both selectivity & efficiency of the resin 50

51. 51 Short analysis time Well defined separation Small amount of mobile phase required The flow rate can be set Narrow bands and good sensitivity There is no sample loss ADVANTAGES

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53. Applications Molecular weight determination Gel filtration chromatography is widely applied for determination of the molecular weight of proteins. The standard curve is constructed by plotting logarithm of the molecular weights of standard proteins against their respective ratios of elution volume to column void volume (Ve/V0). This standard curve is then used to determine the molecular weight of unknown proteins. 53

54. 54 (ii) Desalting Salt molecules and buffer components are several times smaller in comparison to macromolecules. For this reason, gel filtration chromatography using a resin with smaller size exclusion limits can be used for desalting and buffer exchange applications. Desalting is the process of salt removal from a protein sample, while in buffer exchange a protein solution is replaced by more appropriate buffer. This is one of the most applied applications of gel filtration chromatography. The separation limits for resins are generally in range of up to 10 kDa . Resins with large exclusion limits are not suitable for buffer exchange and desalting application.

55. (iii) Separation of macromolecules – Gel filtration chromatography is commonly applied in research laboratories for separation of proteins and peptides. It is applied for detection and separation of oligomers. (iv) Group separation - Gel filtration chromatography is also applied for fractionation of crude samples into low and high molecular weight protein groups. 55

56. 56 Reference : Gel filtration, Principles&methods . http://www.waters.com/waters/en_US/GPC---Gel-Permeation- Chromatography/nav.htm? I. Willard H H , Merritt l l , Dean J A, Settle F A. Instrumental Methods of Analysis. Size exclusion chromatography, cytivia

57. Thank you 57