Gel filtration columns and media

Transcription

1 GE Healthcare Life Sciences Gel filtration columns and media Selection guide

2 General information Principles of gel filtration Gel filtration (GF) separates molecules on the basis of differences in size as they pass through a gel filtration medium packed in a column. GF media consists of spherical particles with pores of different sizes where molecules small enough to enter the pores are retarded as compared to larger molecules (Fig ). Samples are eluted isocratically (single buffer, no gradient). uffer conditions can be varied to suit the sample type or the requirements for the next purification, analysis, or storage step. A variety of media with different selectivities are available and cover a molecular weight range from M r 00 to , from peptides to very large proteins, protein complexes, and virus. GF can be applied in two ways:. Group separations where the components of a sample are separated into two major groups according to size range (Fig ). A group separation can be used to remove high or low molecular weight contaminants, such as phenol red from culture fluids, or for desalting and buffer exchange. A80 nm Inject V o Protein Salt V t UV 80 nm Conductivity 0 0 Min. Large molecule cannot enter the pores of chromatography beads Target protein can use a fraction of the pore volume of the beads Salt or other low molecular weight substances can use the entire pore volume of the beads Fig. Typical group separation.. High-resolution fractionation of biomolecules where the components of a sample are separated according to differences in their molecular size (Fig ). High-resolution fractionation can be used to isolate one or more components, to separate monomers from aggregates, or to perform a molecular weight distribution analysis. Highresolution gel filtration is most suitable for samples that originally contain few components or for samples that have been partially purified by other chromatography techniques so that most of the unwanted proteins of similar size are eliminated. Absorbance V o Absorbance Sample injection High molecular weight Intermediate molecular weight V R Vt Low molecular weigh High molecular weight Sample injection Intermediate molecular weight Low molecular weigh V C Equilibration CV Columns volumes (CV) Equilibration CV Columns volumes (CV) Fig. Schematic process of GF. Fig. Typical high-resolution GF separation.

3 Media selection Group separation dex* is excellent for rapid group separations such as desalting and buffer exchange, before, between, or after other chromatography purification. This medium can be used at both laboratory and production scale. High-resolution fractionation rdex* is the first choice for high-resolution fractionation, short run times, and high recovery. cryl* is suitable for fast, high recovery separations at laboratory and industrial scale. rose* offers a broad fractionation range, but is not suitable for large scale or industrialscale separations. Rapid purity check and screening rdex 75 5/50 GL and rdex 00 5/50 GL are short columns with small bed volumes that are suitable for rapid protein homogeneity analyses or purity checks. They save time when screening many samples, and require less buffer and sample than longer columns. Practical considerations Selection of medium Resolution is a function of the selectivity of the medium, that is the distanced between two peaks, and the efficiency of the medium, that is the ability to produce narrow peaks. The fractionation range defines the range of molecular weights that have access to the pores of the matrix; molecules within this range can be separated by high-resolution fractionation. The exclusion limit for a GF medium indicates the size of the molecules that are excluded from the pores of the matrix and therefore elute in the void volume. The selectivity of a GF medium depends solely on its pore size distribution and is described by a selectivity curve (Fig 4). The steeper the selectivity curve, the higher the resolution that can be achieved. In cases where two media have a similar fractionation range, select the medium with the steepest selectivity curve for best resolution of all components in the sample. When you are interested in a specific component, select the medium where the target protein falls in the middle of the selectivity curve. The success of GF depends primarily on choosing conditions that give sufficient selectivity and counteract peak broadening effects during the separation. After the selection of a GF medium, sample volume and column dimensions are the two most critical parameters that affect the resolution of the separation. ead size For a given column dimension, the resolution can be improved by using smaller bead size. However, using a smaller particle size can increase in back pressure so that flow rate must be decreased and run time extended. Column dimensions The height of the packed bed affects both resolution and the time taken for elution. The resolution in GF increases with the square root of bed height. Doubling the bed height gives an increase in resolution equivalent to =.4 (40%). For high resolution and fractionation, long columns will give the best results. The effective bed height can be increased by coupling columns containing the same media in series. For maximum resolution, the dead volume should be kept at a minimum; short, narrow capillaries should be used and unnecessary system components should be bypassed. This is especially important for micro preparative and analytical applications. Sample and buffer preparation Removal of particles in the sample is extremely important for GF. Clarifying a sample by centrifugation and/or filtration before application onto a column will avoid the risk of blockage, reduce the need for stringent washing procedures, and extend the life of the medium. uffer composition will generally not directly influence the resolution unless the buffer affects the shape or biological activity of the molecules. Select buffer conditions that are compatible with protein stability and activity and include between 5 and 50 mm NaCl to avoid nonspecific ionic interactions with the matrix which can result in delays in peak elution and poor reproducibility. Always use high quality water and chemicals and filter all solutions through 0.45µm or 0. µm filters before use. K av Mr logarithmic scale Fig 4. Selectivity curves for rdex. rdex peptide rdex 75 rdex 00 rdex 0 prep grade rdex 75 prep grade rdex 00 prep grade

4 Sample volume Smaller sample volumes help to avoid overlap between closely spaced peaks. For high-resolution fractionation, a sample volume from 0.5% to 4% of the total column volume (CV) is recommended, depending on the type of medium used. For most applications the sample volume should not exceed % to achieve maximum resolution. For group separations, use sample volumes up to 0% of the total CV. Flow rate The resolution depends on the flow rate for mainly two reasons: A flow rate that is too high gives insufficient time for the molecules to equilibrate between the gel matrix and the elution buffer, while a flow rate that is too low gives broadening of the peaks as a result of diffusion. The practical optimum for proteins is often in the range of to 0 cm/h. Viscosity High sample viscosity causes instability of the separation and an irregular flow pattern, leading to very broad and skewed peaks. To increase the capacity of a GF separation, the sample may need to be concentrated. Note that the solubility or the viscosity of the sample can limit the concentration that can be used. Transport device Prepacked gel filtration columns are delivered with a storage/shipping device that keeps the pressure in the column and thereby prevents it from drying out. We recommend that you connect the storage/ shipping device according to instructions supplied with the column for long-term storage. Column efficiency test GE Healthcare packs columns to the highest standards, and each column is thoroughly tested with regard to the number of theoretical plates (N, Fig 5). Column performance should be checked at regular intervals to determine column efficiency and peak symmetry, either by injecting acetone as above or by running a set of standard proteins relevant for the application used. Optimization Perform a first run as described in the enclosed Instructions for the column. If the results obtained are unsatisfactory, consider the following: Action Decrease flow rate Decrease sample volume Effect Improved resolution for high molecular weight biomolecules. The resolution for small biomolecules may be decreased. Improved resolution Sample: Sample volume: Eluent: Flow rate: Temperature: Absorbance V R Fig 5. Column efficiency test. Acetone 0 mg/ml 5 µl (0/00 GL), 00 μl (HiLoad*/HiPrep* 6/60) or 500 μl (HiLoad/HiPrep 6/60) Distilled water 5 cm/h 0. ml/min (0/00 GL) 0.8 ml/min (6/60) or. ml/min (6/60) Room temperature (5ºC) Column efficiency is calculated using the equation: N = 5.54 (V R /w h ) where, V R = Peak retention (elution) volume, w h = Peak width at half peak height, V R and w h given in same units Maintenance Note: The description of regular and more rigorous cleaning below refers to a rdex column; for other media please read the respective instruction. Regular cleaning Perform the following regular cleaning cycle after every 0 to 0 separation cycles. Wash the column with 0.5 to CV of 0.5 M NaOH at a flow rate of 5 cm/h to remove most nonspecifically adsorbed proteins. Wash with CV of distilled water. Re-equilibrate the column with at least CV of buffer. Further equilibration is necessary if your buffer contains detergent. Wait until the UV base line stabilizes before applying next sample. More rigorous cleaning Reverse the flow. Wash the column at a flow rate of 5 cm/h at room temperature with the following solutions:. 4 CV of M NaOH for removal of hydrophobic proteins or lipoproteins followed by 4 CVs of distilled water CV of 0% isopropanol (removal of lipids and very hydrophobic proteins), followed by CV of distilled water. efore a new chromatography experiment, equilibrate the column after cleaning with at least 5 CVs of buffer. W h

5 As an alternative to more rigorous cleaning or if the column performance is still not restored, replace the filter at the top of the column, contaminants introduced with the liquid flow can be caught by the filter. After replacement of the filter, clean the column according to Regular cleaning. Storage If the column is to be stored more than two days after use, wash the column with 4 CV of distilled water, and then equilibrate with 4 CV of 0% ethanol. Flow rate conversion Flow rate is measured in volume terms, for example ml/min, but when comparing results between columns of different sizes it is useful to use the linear flow, cm/hour. To convert between linear flow and volumetric flow rate use the formulas below: From linear flow (cm/h) to volumetric flow rate (ml/min) Volumetric flow rate (ml/min) From volumetric flow rate (ml/min) to linear flow (cm/h) Linear flow = (cm/h) Linear flow (cm/h) = 60 Volumetric flow rate (ml/min) 60 column cross-sectional area (cm ) column crosssectional area (cm ) column crosssectional area (cm ) For more information, please refer to the handbook Gel Filtration, Principles and Methods, which can be ordered from GE Healthcare or downloaded at Figure 6 summarizes which column to choose in terms of scale of purification, sample volume, and desired resolution. Troubleshooting Symptom Increased back pressure Loss of resolution and/or decreased sample recovery Air in the column Space between adapter and medium Low resolution Remedy Clean the column according to the section Maintenance Clean the column according to the section Maintenance Reverse flow direction and pump 5 CV of well degassed water through the column at a flow rate of 50 cm/h (5 ml/min for XK 6/60 or ml/min for XK 6/60). Stop the flow. Close the outlet tubing with the domed nut and then disconnect the inlet tubing. Adjust the adaptor to the medium surface according to instructions for the specific column. Reconnect the inlet tubing immediately avoiding to get air into the column. Note that some prepacked columns cannot be opened. Minimize dead volumes in the chromatographiy system by decreasing the capillary length between the injector and the detector. You can also change to capillaries with smaller diameter given even less dead volume. For best performance and convenience use pre packed gel filtration columns Analytical and micropreparative scale Preparative scale rdex PC./0 rose PC./0 rdex 0/00 GL rose 0/00 GL Resolution rdex 5/50 GL HiLoad 6/600 rdex pg HiPrep 6/60 cryl HiLoad 6/600 rdex pg HiPrep 6/60 cryl Note: Dead volume in the system must be minimized! µl ml Sample volume Fig 6. Schematic overview of resolution and sample volume for prepacked, high-resolution gel filtration columns.

6 Ordering information Columns Code no. rdex Peptide PC./ rdex Peptide 0/00 GL rdex 75 PC./ rdex 75 0/00 GL rdex 75 5/50 GL rdex 00 PC./ rdex 00 0/00 GL rdex 00 5/50 GL HiLoad 6/600 rdex 0 pg HiLoad 6/60 rdex 0 pg HiLoad 6/60 rdex 75 pg HiLoad 6/60 rdex 75 pg HiLoad 6/60 rdex 00 pg HiLoad 6/60 rdex 00 pg rose PC./ rose 0/00 GL rose 6 PC./ rose 6 0/00 GL HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-400 HR HiPrep 6/60 cryl S-400 HR HiPrep 6/60 cryl S-500 HR HiPrep 6/60 cryl S-500 HR HiTrap Desalting (5 5 ml) HiTrap Desalting (00 5 ml) HiPrep 6/0 Desalting ( 5 ml) HiPrep 6/0 Desalting (4 5 ml) Media Pack size Code no. PD-0 Desalting Columns (0 pcs) rdex 0 prep grade 50 ml rdex 75 prep grade 50 ml rdex 00 prep grade 50 ml rose prep grade 5 ml rose 6 prep grade 5 ml cryl S-00 HR 50 ml ml cryl S-00 HR 50 ml ml cryl S-00 HR 50 ml ml cryl S-400 HR 50 ml ml cryl S-500 HR 50 m ml cryl S-000 SF 750 ml dex G-0 00 g g dex G-5 rfine 00 g dex G-5 Fine 00 g g dex G-5 Medium 00 g g dex G-50 Fine 00 g g dex LH-0 5 g g g Related products Gel Filtration Calibration Kit, LMW Gel Filtration Calibration Kit, HMW Handbook: Gel Filtration, Principles and Methods For contact information for your local office, please visit, GE Healthcare io-sciences A jörkgatan Uppsala Sweden imagination at work GE, imagination at work and GE monogram are trademarks of General Electric Company. * ÄKTA, ioprocess, HiLoad, HiPrep, HiTrap, Labmate, cryl, dex, rdex, and rose are trademarks of GE Healthcare companies General Electric Company All rights reserved. First published 000. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place Little Chalfont, uckinghamshire, HP7 9NA UK GE Healthcare Europe, GmbH Munzinger Strasse 5, D-79 Freiburg Germany GE Healthcare io-sciences Corp. 800 Centennial Avenue, P.O. ox 7, Piscataway, NJ USA GE Healthcare Japan Corporation Sanken ldg., -5-, Hyakunincho, Shinjuku-ku, Tokyo Japan AF 06/0

7 Product Ordering information Fractionation range globular proteins M r Prepacked column/ulk media Code no. Column dim. Pack size (relative molecular id bed height weight) (mm) Group separation HiTrap* Desalting HiTrap Desalting HiPrep 6/0 Desalting HiPrep 6/0 Desalting PD-0 Desalting Columns dex G g < g dex G-5 rfine g dex G-5 Fine g g dex G-5 Medium g g dex G-50 Fine g g dex LH g 00 g 500 g < 5000 Fra dex mo High resolution fractionation rdex Peptide PC./0 rdex Peptide 0/00 GL rdex 75 PC./0 rdex 75 0/00 GL rdex 75 5/50 GL rdex 00 PC./0 rdex 00 0/00 GL rdex 00 5/50 GL HiLoad 6/600 rdex 0 pg HiLoad 6/600 rdex 0 pg HiLoad 6/600 rdex 75 pg HiLoad 6/600 rdex 75 pg HiLoad 6/600 rdex 00 pg HiLoad 6/600 rdex 00 pg < < rdex 0 prep grade ml < rdex 75 prep grade ml rdex 00 prep grade ml rose PC./0 rose 0/00 GL rose 6 PC./0 rose 6 0/00 GL rose prep grade ml rose 6 prep grade ml HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR HiPrep 6/60 cryl S-00 HR cryl S-00 HR ml ml cryl S-00 HR ml ml cryl S-00 HR ml ml HiPrep 6/60 cryl S-400 HR HiPrep 6/60 cryl S-400 HR HiPrep 6/60 cryl S-500 HR HiPrep 6/60 cryl S-500 HR cryl S-400 HR cryl S-500 HR ml 750 ml 50 ml 750 ml cryl S-000 SF ml 500 ioprocess* Media Made for bioprocessing. Process scale quantities are avaible. Please contact GE Healthcare for further information. Pack size available by special order. PC./0 is designed for analytical and micropreparative use. Precision Column Holder (Code No ) allows the columns to be used with ÄKTA* design systems and other high-performance chromatography systems.

8 ctionation range trans M r (relative lecular weight, Daltons) Exclusion limit DNA (base pairs) Particle size range (μm) Number of theoretical plates ph stability (working and long term) Maximum operating back pressure (MPa/psi) Approx. maximum operating flow rate Recommended sample volume Not specified 0./44 5 ml/min ml (dry) Not specified 0.5/ 40 ml/min.55 ml Not specified.5.5 ml < (dry) 40 cm/h (dry) Can be 0 cm/h calculated (dry) using 60 cm/h Darcy s Law (dry) 50 cm/h (dry) 0 60 cm/h 76 (dry) 0.5/ 0 cm/h > > > 000 > 000 > 000 > 000 > 000 > /90.8/60.4/50.8/60.8/60.5/0.5/0.5/0 0./44 0./44 0./44 0./44 0./44 0./ ml/min. ml/min 0. ml/min.5 ml/min 0.7 ml/min 0. ml/min.0 ml/min 0.8 ml/min.7 ml/min 4.4 ml/min.7 ml/min 4.4 ml/min.7 ml/min 4.4 ml/min 55 μl 550 μl 5 μl 550 μl 450 μl 5 μl 550 μl 450 μl ml ml ml 44 0./44 90 cm/h /44 90 cm/h /44 90 cm/h > > /50 /45 0. ml/min.5 ml/min 5 μl 550 μl /75.5/0 0. ml/min.0 ml/min 5 μl 550 μl /00 40 cm/h /58 40 cm/h > 5000 > / 0.5/.0 ml/min.7 ml/min ml > 5000 > / 0.5/.0 ml/min.7 ml/min ml > 5000 > / 0.5/.0 ml/min.7 ml/min ml /9 60 cm/h /9 60 cm/h /9 60 cm/h / 0.5/.0 ml/min.7 ml/min ml / 0.5/.0 ml/min.7 ml/min ml /9 60 cm/h /9 50 cm/h Not determined 40 cm/h Flow rate is calculated from measurement in packed columns with an i.d. of.6 cm. A column heigth of 60 cm is used for rose, rdex and cryl. For dex the column i.d. is.6 cm and the height 0 cm. Labmate* buffer reservoir (Code No ) can be used with PD-0 Desalting Columns for easier and more convenient equilibration. ph stability long term refers to the ph interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. All ranges given are estimates based on our knowledge and experience. At room temperature in aqueous buffer. The flow rate giving optimal resolution depends on the sample. Refer to instructions for each column and media.

9 Approx. bed volume (ml) Applications 5 Fast and convenient group separation between high and low molecular weight substances 5 Fast and convenient group separation between high and low molecular weight substances 8.5 Disposable column for group separation and buffer exchange Fast and convenient group separation between peptides and low molecular weight substances Fast and convenient group separation between high and low molecular weight substances Separation of natural products, such as steroids, terpenoids and lipids, in organic solvents Micropreparative and analytical high-resolving separation of peptides and other small biomolecules Micropreparative and analytical high-resolving separation of proteins, peptides, polynucleotides, and other biomolecules. Rapid size analysis of protein homogeneity in screening experiments Micropreparative and analytical high-resolving separation of proteins, especially monoclonal antibodies, DNA fragments- and other biomolecules. Rapid size analysis of protein homogeneity in screening experiments Preparative separation of peptides and other small biomolecules Rapid, preparative separation of proteins, peptides, polynucleotides, and other biomolecules Rapid, preparative separation of proteins, especially monoclonal antibodies, DNA fragments, and other biomolecules Preparative separation of peptides and other small biomolecules Rapid, preparative separation of proteins, peptides, polynucleotides, and other biomolecules Rapid, preparative separation of proteins, especially monoclonal antibodies, DNA fragments, and other biomolecules.4 Micropreparative and analytical high-resolving separation of proteins, peptides, oligonucleotides, and polysaccharides 4.4 Micropreparative and analytical high-resolving separation of proteins, peptides, oligonucleotides, polysaccharides and 4 nucleic acids Preparative high-performance separation of proteins, peptides, oligonucleotides, and polysaccharides Preparative high-performance separation of proteins, peptides, oligonucleotides, polysaccharides, and nucleic acids 0 Preparative separation of proteins and peptides 0 0 Preparative separation of proteins e.g., small serum proteins such as albumin 0 0 Preparative separation of proteins e.g., membrane proteins and serum protein such as antibodies 0 Preparative separation of proteins and peptides Preparative separation of proteins e.g., small serum proteins such as albumin Preparative separation of proteins e.g., membrane proteins and serum protein such as antibodies Preparative separation of polysaccharides and other macromolecules with extended structures e.g. proteoglycans and liposomes Preparative separation of large macromolecules e.g., group separation of DNA restriction fragments Preparative separation of polysaccharides and other macromolecules with extended structures e.g. proteoglycans and liposomes Preparative separation of large macromolecules e.g., group separation of DNA restriction fragments Preparation of DNA and separation of very large polysaccharides, proteoglycans, and small particles e.g. membrane-bound vesicles and viruses

10 6 Selection guide - Gel filtration media Start here High resolution (M r ) Wide range (M r ) rdex High/medium pressure system High recovery High stability High selectivity 0 High resolution (M r ) rose Medium pressure system High recovery Wide M r fractionation range Wide range (M r ) Analytical (M r ) Preparative (M r ) cryl Low/medium pressure system Macromolecule separation Product line covering wide fractionation range 0 Preparative and analytical (M r ) dex Desalting uffer exchange Preparative/larger biomolecules (M r ) 0 0 High resolution fractionation dex LH Separation in (nonpolar) organic solvent Group separation

11 Peptide M r (approx). Thyroglobulin (M r ). Ferritin (M r ). Aldolase (M r ) 4. Albumin (M r ) 5. Ovalbumin (M r 4 000) 6. Chymotrypsinogen A (M r 5 000) 7. Ribonuclease A (M r 700) 8. Cytochrome C (M r 500) 9. Aprotinin (M r 6500) 0. Gastrin I (M r 6). Substance P (M r 48). (Gly) 6 (M r 60). (Gly) (M r 89) 4. Gly (M r 75) Micro preparative Analytical separation Preparative separation Peptides Small proteins Polynucleotides Proteins DNA-fragment Peptides Small proteins Polynucleotides rose 6. Thyroglobulin (M r ). Ferritin (Mr r ). Aldolase (M r ) 4. Albumin (M r ) 5. Ovalbumin (M r 4 000) 6. Chymotrypsinogen A (M r 5 000) 7. Ribonuclease A (M r 700) Micro preparative Analytical separation Proteins DNA fragment Small proteins up to DNA fragments Small/medium proteins M r (approx) rose Preparative separation Small proteins up to DNA fragment Small/medium proteins S - 00 S Thyroglobulin (M r ). Ferritin (M r ). Aldolase (M r ) 4. Albumin (M r ) 5. Ovalbumin (M r 4 000) 6. Chymotrypsinogen A (M r 5 000) 7. Ribonuclease A (M r 700) Proteins Small proteins Medium proteins Large proteins M r (approx) S - 00 Macro molecules Small macro molecules Large macro molecules Small particles viruses NaCl SA Small peptides Peptides/small proteins Proteins Time (seconds). C O OH NH C CH O 50 mg 54 mg. CH C HN C OH O O Low molecular steroids Terpenoids, lipids, and peptides 8 Time (h)

12 Fraction range (globular proteins) rdex Peptide rdex 75 rdex 00 rdex 0 prep grade rdex 75 prep grade rdex 00 prep grade rose 6 rose rose 6 prep grade rose prep grade Resolution cryl S-00 HR cryl S-00 HR cryl S-00 HR cryl S-400 HR cryl S-500 HR cryl S-000 SF dex G-0 dex G-5 SF dex G-5 F dex G-5 M dex G-50 F Exclusion limit Exclusion limit Exclusion limit Exclusion limit Exclusion limit dex LH-0