P es that are ut of t le to diffu s part am nd the Diffusio out of the entire separa valent to P9
10 1. Spherical particles of gel filtration medium are packed into a column. Vo V – V t o Vt Low molecular weight Interacting with medium AbsorbanceSample injection High molecular weight Intermediate molecular weight Ve 2. Sample is applied to the column. 3. Buffer (mobile phase) and sample move through the column. Molecules diffuse in and out of the pores of the matrix (also described as partitioning of the sample between the mobile phase and the stationary phase). Smaller molecules move further into the matrix and so stay longer on the column. 4. As buffer passes continuously through the column, molecules that are larger than the pores of the matrix are unable to diffuse into the pores and pass through the column. Smaller molecules diffuse into the pores and are delayed in their passage down the column. 5. Large molecules leave the column first followed by smaller molecules in order of their size. The entire separation process takes place as one total column volume (equivalent to the volume of the packed bed) of buffer passes through the gel filtration medium. Diffusion Diffusion out of the pores Buffer Fig. 3. Process of gel filtration. Diffusion into the pores Buffer
Group separation Gel filtration is used in group separation mode to remove small molecules from a group of larger molecules and as a fast,simple solution for buffer exchange.Small molecules such as excess salt(desalting)or free labels are easily separated.Samples can be prepared for storage or for other chromatography techniques and assays.Gel filtration in group separation mode is often used in protein purification schemes for desalting and buffer exchange.For further details refer to Chapter 2,page 57 and the Protein Purification Handbook from Amersham Biosciences. Sephadex G-10,G-25 and G-50 are used for group separations.Large sample volumes up total o me (packed bed)can 4 sh the ge mo ules are eluted in or ust after the the now molecules such as salts that have full access to the pores move down the column,but do not separate from each other.These molecules usually elute just before one total column volume,Ve of buffer has passed through the column.In this case the proteins are detected by monitoring their UV absorbance,usually at Aso and the salts are detected by monitoring the conductivity of the buffer. 20 0.1 Sample: Refer to Chapter 2,page 57 for detailed info molecula rmation on group separation of high and low ar weight substances,i.e.des lting,buffer exchange and sample clean up using Sephadex. Refer to Chapter 3 for detailed information on the theory of gel filtration
11 Group separation Gel filtration is used in group separation mode to remove small molecules from a group of larger molecules and as a fast, simple solution for buffer exchange. Small molecules such as excess salt (desalting) or free labels are easily separated. Samples can be prepared for storage or for other chromatography techniques and assays. Gel filtration in group separation mode is often used in protein purification schemes for desalting and buffer exchange. For further details refer to Chapter 2, page 57 and the Protein Purification Handbook from Amersham Biosciences. Sephadex G-10, G-25 and G-50 are used for group separations. Large sample volumes up to 30% of the total column volume (packed bed) can be applied at high flow rates using broad, short columns. Figure 4 shows the elution profile (chromatogram) of a typical group separation. Large molecules are eluted in or just after the void volume, Vo as they pass through the column at the same speed as the flow of buffer. For a well packed column the void volume is equivalent to approximately 30% of the total column volume. Small molecules such as salts that have full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt , of buffer has passed through the column. In this case the proteins are detected by monitoring their UV absorbance, usually at A280nm, and the salts are detected by monitoring the conductivity of the buffer. Sample: (His)6 protein eluted from HiTrap™ Chelating HP with sodium phosphate 20 mM, sodium chloride 0.5 M, imidazole 0.5 M, pH 7.4 Column: HiTrap Desalting 5 ml Buffer: Sodium phosphate 20 mM, sodium chloride 0.15 M, pH 7.0 Fig. 4. Typical chromatogram of a group separation. The UV (protein) and conductivity (salt) traces enable pooling of the desalted fractions and facilitate optimization of the separation. Refer to Chapter 2, page 57 for detailed information on group separation of high and low molecular weight substances, i.e. desalting, buffer exchange and sample clean up using Sephadex. Refer to Chapter 3 for detailed information on the theory of gel filtration. 0 0.05 0.10 0.15 UV 280 nm Conductivity (His) protein Salt 6 Inject 0 1 2 min Vo Vt A280 nm void volume Vo, total column volume Vt
High resolution fractionation oraeile componmple size.The goal ma be t erto Chapterfor ht derermnatnd ditribt The best results for high resolution fractionation will be achieved with samples that originally contain few components or with samples that have been partially purified by other chromato- graphy techniques(in order to eliminate proteins of similar size that are not of interest). High resolution fractionation by gel filtration is well suited for the final polishing step in a purification scheme.Monomers can be separated from aggregates(difficult to achieve by any other technique)and samples can be transferred to a suitable buffer for assay or storage. Gel filtration can be used directly after any of the chromatography techniques such as ion exchange,chromatofocusing,hydrophobic interaction or affinity since the components from any elution buffer will not affect the final separation.For further details on using gel ostrategy,reertoChapter6and the Pron riftion H Figure5shows the theoretical ution profile (chromatogram)of a high resolution ctionat me,V irectly through flow of packed column the void I volume is equivalent to approximately 30%of the total column volume (packed bed).Molecules with partial access to the pores of the matrix elute from the column in order of decreasing size.Small molecules such as salts that have full access to the pores move down the column,but do not separate from each other.These molecules usually elute just before one total column volume,V of buffer has passed through the column. Column Volumes (cv) Fig.5.Thec ctical chromatogram on fractionation (UV absorbance) 12
12 High resolution fractionation Gel filtration is used in fractionation mode to separate multiple components in a sample on the basis of differences in their size. The goal may be to isolate one or more of the components, to determine molecular weight, or to analyze the molecular weight distribution in the sample (refer to Chapter 4 for details of molecular weight determination and distribution analysis). The best results for high resolution fractionation will be achieved with samples that originally contain few components or with samples that have been partially purified by other chromatography techniques (in order to eliminate proteins of similar size that are not of interest). High resolution fractionation by gel filtration is well suited for the final polishing step in a purification scheme. Monomers can be separated from aggregates (difficult to achieve by any other technique) and samples can be transferred to a suitable buffer for assay or storage. Gel filtration can be used directly after any of the chromatography techniques such as ion exchange, chromatofocusing, hydrophobic interaction or affinity since the components from any elution buffer will not affect the final separation. For further details on using gel filtration in a purification strategy, refer to Chapter 6 and the Protein Purification Handbook from Amersham Biosciences. Figure 5 shows the theoretical elution profile (chromatogram) of a high resolution fractionation. Molecules that do not enter the matrix are eluted in the void volume, Vo as they pass directly through the column at the same speed as the flow of buffer. For a well packed column the void volume is equivalent to approximately 30% of the total column volume (packed bed). Molecules with partial access to the pores of the matrix elute from the column in order of decreasing size. Small molecules such as salts that have full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt , of buffer has passed through the column. Fig. 5. Theoretical chromatogram of a high resolution fractionation (UV absorbance). high molecular weight intermediate molecular weight low molecular weight Column Volumes (cv) sample injection volume equilibration Absorbance 1 cv V Vt o void volume Vo total column volume Vt
Resolution in gel filtration Many factors influence the final resolution (the degree of separation between peaks of a gel filtration separation):sample volume,the ratio of sample volume to column volume,column dimensions,particle size,particle size distribution,packing density,pore size of the particles, flow rate,and viscosity of the sample and buffer.The molecular weight range over which a gel filtration medium can separate molecules is referred to as the selectivity of the medium (see selection guide for gel giltration media on page 18).Resolution is a function of the selectivity of the medium dthe efficienev fha mdmtoproduceow peak aden ng),as illus that give suffic t depends low efficiency Fig6.Dependence of resolutionon selectivity and the of peak broadening. After selecting a gel filtration medium with the correct selectivity,sample volume and columr two of th most critical parameters that will affect the resolution of the separation
13 Resolution in gel filtration Many factors influence the final resolution (the degree of separation between peaks of a gel filtration separation): sample volume, the ratio of sample volume to column volume, column dimensions, particle size, particle size distribution, packing density, pore size of the particles, flow rate, and viscosity of the sample and buffer. The molecular weight range over which a gel filtration medium can separate molecules is referred to as the selectivity of the medium (see selection guide for gel giltration media on page 18). Resolution is a function of the selectivity of the medium and the efficiency of that medium to produce narrow peaks (minimal peak broadening), as illustrated in Figure 6. The success of gel filtration depends primarily on choosing conditions that give sufficient selectivity and counteract peak broadening effects during the separation. high efficiency low efficiency Fig. 6. Dependence of resolution on selectivity and the counteraction of peak broadening. After selecting a gel filtration medium with the correct selectivity, sample volume and column dimensions become two of the most critical parameters that will affect the resolution of the separation
Sample volume and column dimensions percentage of the ota ovome (packed bed) umes he verlap i te how sample volume can influence a high resolution fractionation 25 ul1.0 mVmin (76 cm/h) 200HR100V24m 02 Conc.(mg/ml 0.20 0.10 n B12 1355 05 ation:15.2 mg/ml 00050100150200250mm 05M te,0.15 M NaCl,pH7.0 250 ul.1.0 ml/min (76 cm/h) 10 ml/min(76.4cnh】 0.15 0.0 0.00050100150200250m 3引 1 000 ul.1.0 ml/min (76 cm/h) 0.10 00005010015020.025.0mm For group separations sample volumes up to30%of the total column volume can be applied For high resolution fractionation a sample volume from 0.5-4%of the total column volume is recommended,depending on the type of medium used.For most applications the sample volume should not exceed 2%to achieve maximum resolution.Depending on the nature of the specific sample,it may be possible to load larger sample volumes,particularly if the eaks of inte res are well reso olved.This only be der ined by expe rimentation
14 0.05 0.10 0.15 0.20 0.25 0.00 0.0 5.0 10.0 15.0 20.0 25.0 min 25 µl,1.0 ml/min (76 cm/h) Vo Vt A280 nm 0.05 0.10 0.15 0.00 0.0 5.0 10.0 15.0 20.0 25.0 min 250 µl,1.0 ml/min (76 cm/h) Vo Vt A280 nm 0.05 0.10 0.00 0.0 5.0 10.0 15.0 20.0 25.0 min 1 000 µl,1.0 ml/min (76 cm/h) Vo Vt A280 nm Column: Superdex™ 200 HR 10/30 (Vt : 24 ml) Sample: Mr Conc. (mg/ml) Thyroglobulin 669 000 3 Ferritin 440 000 0.7 IgG 150 000 3 Transferrin 81 000 3 Ovalbumin 43 000 3 Myoglobin 17 600 2 Vitamin B12 1 355 0.5 Sample concentration: 15.2 mg/ml Sample volumes: 1) 25 µl (0.1% × Vt ) 2) 250 µl (1% × Vt ) 3) 1000 µl (4.2% × Vt ) Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 1.0 ml/min (76.4 cm/h) Sample volume and column dimensions Sample volumes are expressed as a percentage of the total column volume (packed bed). Using smaller sample volumes helps to avoid overlap if closely spaced peaks are eluted. Figure 7 illustrates how sample volume can influence a high resolution fractionation. Fig. 7. Influence of sample volume on resolution. For group separations sample volumes up to 30% of the total column volume can be applied. For high resolution fractionation a sample volume from 0.5–4% of the total column volume is recommended, depending on the type of medium used. For most applications the sample volume should not exceed 2% to achieve maximum resolution. Depending on the nature of the specific sample, it may be possible to load larger sample volumes, particularly if the peaks of interest are well resolved. This can only be determined by experimentation. 1) 2) 3)