Sample preparation Samples must be clear and free from particulate matter,particularly when working with bead sizes of 34 um or less. Appendix 3 contains an overview of sample preparation techniques.For small sample volumes a syringe-tip filter of cellulose acetate or PVDF can be sufficient. Sample buffer composition The pH,ionic strength and composition of the sample buffer will not significantly affect resolution as long as these parameters do not alter the size or stability of the proteins to be re not outside the stability ra medium.The sample s not ha ange of the ave to be in exactly the same buffer as tha lth runing buffer during the seprion t u Sample concentration and viscosity Gel filtration is independent of sample mass,and hence sample concentration,as can be seen in Figure 12.Hence high resolution can be maintained despite high sample concentration and,with the appropriate medium,high flow rates. (30.cm/h) 24 48 72 96 120 155 mg/ml sample 0.6 08 10 2照 121n0. and G Superdex 200 prep grad
20 Sample preparation Correct sample preparation is extremely important for gel filtration. Simple steps to clarify a sample before applying it to a column will avoid the risk of blockage, reduce the need for stringent washing procedures and extend the life of the packed chromatography medium. Samples must be clear and free from particulate matter, particularly when working with bead sizes of 34 µm or less. Appendix 3 contains an overview of sample preparation techniques. For small sample volumes a syringe-tip filter of cellulose acetate or PVDF can be sufficient. Sample buffer composition The pH, ionic strength and composition of the sample buffer will not significantly affect resolution as long as these parameters do not alter the size or stability of the proteins to be separated and are not outside the stability range of the gel filtration medium. The sample does not have to be in exactly the same buffer as that used to equilibrate and run through the column. Sample is exchanged into the running buffer during the separation, an added benefit of gel filtration. Sample concentration and viscosity Gel filtration is independent of sample mass, and hence sample concentration, as can be seen in Figure 12. Hence high resolution can be maintained despite high sample concentration and, with the appropriate medium, high flow rates. Fig. 12. Influence of sample concentration on the resolution of transferrin and IgG on Superdex 200 prep grade. Medium: Superdex 200 prep grade Column: XK 16/70 Column volume: 140 ml Sample: Solution of transferrin (Mr 81 000) and IgG (Mr 160 000) by equal weight Sample volume: 0.8% × Vt Buffer: 0.05 sodium phosphate, 0.1 M sodium chloride, pH 7.2 Flow: 1 ml/min (30 cm/h) 0.0 0 mg sample/ml packed bed 0.0 Resolution, RS 0.5 1.0 1.5 0.2 0.4 0.6 0.8 1.0 1.2 24 48 72 96 120 155 mg/ml sample
be used running buffer,as shown by the change in elution profiles of haemoglobin and NaCl at different sample viscosities in Figure 13. Too high sample viscosity causes instability of the separation and an irregular flow pattern. This leads to very broad and skewed peaks and back pressure can increase. ution Volum Fig.13.Flution diagra obtained when hac lobin (blus the senaration Samples should generally not exceed 70 mg/ml protein.Dilute viscous samples,but note sample volume(refer to page 14 for more information on the importance of sample volume). Remember that viscosity varies with temperature. Sample volume Sample volume is one of the most important parameters in gel filtration.Refer to page 14 for more information Buffer composition and preparation Buffer composition does not directly influence the resolution obtained in gel filtration since the separation should depend only on the sizes of the different molecules.The most important consideration is the effect of buffer composition on the shape or biological activity of the molecules of interest.For example,the pH and ionic strength of the buffer and the presence of denaturing agents or detergents can cause conformational changes,dissociation of proteins into subunits,dissociation of enzymes and cofactors,or dissociation of hormones and carrier proteins. Select a buffer and pH that are cor apatible with protein stability and activity and in which the product of interest should be collected.Use a buffer concentration that is sufficient to 21
21 However, the solubility or the viscosity of the sample may limit the concentration that can be used. A critical variable is the viscosity of the sample relative to the running buffer, as shown by the change in elution profiles of haemoglobin and NaCl at different sample viscosities in Figure 13. Too high sample viscosity causes instability of the separation and an irregular flow pattern. This leads to very broad and skewed peaks and back pressure can increase. Elution Volume A B C Concentration Fig. 13. Elution diagrams obtained when haemoglobin (blue) and NaCl (red) were separated. Experimental conditions were identical except that the viscosities were altered by the addition of increasing amounts of dextran. A deterioration of the separation becomes apparent. (A lower flow rate will not improve the separation.) Samples should generally not exceed 70 mg/ml protein. Dilute viscous samples, but note sample volume (refer to page 14 for more information on the importance of sample volume). Remember that viscosity varies with temperature. Sample volume Sample volume is one of the most important parameters in gel filtration. Refer to page 14 for more information. Buffer composition and preparation Buffer composition does not directly influence the resolution obtained in gel filtration since the separation should depend only on the sizes of the different molecules. The most important consideration is the effect of buffer composition on the shape or biological activity of the molecules of interest. For example, the pH and ionic strength of the buffer and the presence of denaturing agents or detergents can cause conformational changes, dissociation of proteins into subunits, dissociation of enzymes and cofactors, or dissociation of hormones and carrier proteins. Select a buffer and pH that are compatible with protein stability and activity and in which the product of interest should be collected. Use a buffer concentration that is sufficient to
maintain buffering capacity and constant pH.Use up to 0.15 M NaCl to avoid non-specific ionic interactions with the matrix(shown by delays in peak elution).Note that some proteins may precipitate in low ionic strength solutions.Use volatile buffers such as ammonium nium bicarbonate or ethylenediamin acetate if the separated product is to be lyophilized. Use high quality water and chemicals.Solutions should be filtered through 0.45 um or 0.22 um filters.It is essential to degas buffers before any gel filtration separation as air bubbles can significantly affect performance.Buffers will be degassed if they are filtered under vacuum. When working with a new sample try these conditions first:0.05 M sodi m pho sphate 0.15 M NaCl,pH 7.0 or select the buffer into which the product should be eluted for the next step such as further purification,analysis or storage. Avoid extreme changes in pH or other conditions that may vation n If the a gel filt Denaturing(chaotropic)agents and detergents Denaturing agents such s guanidine hydrochloride or urea can beuscd for initial solubili el filt ion buffers in solubili y of the cause they will denature the protein,they should be avoided unle denaturation is required. Modern media such as Superdex,Sephacryl and Superose classical media such as Sepharose r dissociating or dena- turing conditions or at extreme pH values. Detergents are useful as solubilizing agents for proteins with low aqueous solubility such as membrane components and will not affect the separation. if denaturing agents or de maintain the solubility of the sa they should b ent in bot g buffe nd the s ple buffer.Note that high con trations of detergent will increase th vis. ityof the buffer that ower flow rate may be necessary to avoid over-pressuring the column packing. f proteins that have been solubilized in a den urant or detergent are seen to pre cipitate. elute later than expected or be poorly resolved during gel filtration,add a suitable concen tration of the denaturing agent or detergent to the running buffer. Urea o guanidine hydrochloride are v ery useful for molecular weight det mination.The presenc aturing age ts in th e runni g buffer maint ns proteins and r accu t determination the calibratio standa Note that selectivity curves are usually determined using globular proteins so they do not reflect the behavior of denatured samples. Gel filtration can be used to exchange a protein solubilized initially in,for example SDS, into a more gentle detergent such as TritonTM X-100 without losing solubility
22 maintain buffering capacity and constant pH. Use up to 0.15 M NaCl to avoid non-specific ionic interactions with the matrix (shown by delays in peak elution). Note that some proteins may precipitate in low ionic strength solutions. Use volatile buffers such as ammonium acetate, ammonium bicarbonate or ethylenediamine acetate if the separated product is to be lyophilized. Use high quality water and chemicals. Solutions should be filtered through 0.45 µm or 0.22 µm filters. It is essential to degas buffers before any gel filtration separation as air bubbles can significantly affect performance. Buffers will be degassed if they are filtered under vacuum. When working with a new sample try these conditions first: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 or select the buffer into which the product should be eluted for the next step such as further purification, analysis or storage. Avoid extreme changes in pH or other conditions that may cause inactivation or even precipitation. If the sample precipitates in a gel filtration column, the column will be blocked, possibly irreversibly, and the sample may be lost. Denaturing (chaotropic) agents and detergents Denaturing agents such as guanidine hydrochloride or urea can be used for initial solubilization of a sample and in gel filtration buffers in order to maintain solubility of the sample. However, because they will denature the protein, they should be avoided unless denaturation is required. Modern media such as Superdex, Sephacryl and Superose are in general more suitable than classical media such as Sepharose™ or Sephadex for working under dissociating or denaturing conditions or at extreme pH values. Detergents are useful as solubilizing agents for proteins with low aqueous solubility such as membrane components and will not affect the separation. If denaturing agents or detergents are necessary to maintain the solubility of the sample, they should be present in both the running buffer and the sample buffer. Note that high concentrations of detergent will increase the viscosity of the buffer so that lower flow rates may be necessary to avoid over-pressuring the column packing. If proteins that have been solubilized in a denaturant or detergent are seen to precipitate, elute later than expected or be poorly resolved during gel filtration, add a suitable concentration of the denaturing agent or detergent to the running buffer. Urea or guanidine hydrochloride are very useful for molecular weight determination. The presence of these denaturing agents in the running buffer maintains proteins and polypeptides in an extended configuration. For accurate molecular weight determination the calibration standards must also be run in the same buffer. Note that selectivity curves are usually determined using globular proteins so they do not reflect the behavior of denatured samples. Gel filtration can be used to exchange a protein solubilized initially in, for example SDS, into a more gentle detergent such as Triton™ X-100 without losing solubility
Column and media preparation To perform a separation,gel filtration medium is packed into a column 30-60 cm in height for high resolution fractionation and up to 10 cm in height for group separations.The volume of the packed bed is determined by the sample volumes that will be applied. Efficient column packing is essential,particularly for high resolution fractionation.The efficiency of a packed column defines its ability to produce narrow symmetrical peaks during ration takes cution Column efficincy is particulaly mpran in gelrationn which spara single colum s through the colun n.The niformi rticles influences th of the flow through the ual peak width.Gel filt (ower partice dis narrow peaks.Refe Chapeeyfo efficiency and column packing. Efficiency can be improved by decreasing the particle size of a medium.However,using a smaller particle size may increase back pressure so that flow rate needs to be decreased, lengthening the run time. Using prep packed columns is highly re ended to ensure the best perfo ormance and ible results.An eve nly packed colu mn ens cs th sample es down the peaks are not unn sarily Uneven packing causes Allow buffers,media or prepacked columns to reach the same temperature before beginning s in temperature,for example removing om tempe ature. an c d es should be washed away thoroughly befor starting a separation. Sample application The choice of sample application method depends largely on the volume to be applied and on the equipment available.Ensure that the sample is not diluted on the way to the column and that the top of the column bed is not disturbed during sample application.Samples can be applied automatically or manually. Apply samples directly to the column via a chromat nge.The choice of nds largel og em,a peristaltic pu amp or a y on and the mple vohe网 me.Fo the type o will be nereas a syringe car used wit rap D te that samples are applied by gravity feed to prepacked columns such as PD-10 Desalting. 23
23 Column and media preparation To perform a separation, gel filtration medium is packed into a column 30–60 cm in height for high resolution fractionation and up to 10 cm in height for group separations. The volume of the packed bed is determined by the sample volumes that will be applied. Efficient column packing is essential, particularly for high resolution fractionation. The efficiency of a packed column defines its ability to produce narrow symmetrical peaks during elution. Column efficiency is particularly important in gel filtration in which separation takes place as only a single column volume of buffer passes through the column. The uniformity of the packed bed and the particles influences the uniformity of the flow through the column and hence affects the shape and eventual peak width. Gel filtration media with high uniformity (lower particle size distribution) facilitate the elution of molecules in narrow peaks. Refer to Chapter 3 Gel filtration in theory and Appendix 1 for further information on column efficiency and column packing. Efficiency can be improved by decreasing the particle size of a medium. However, using a smaller particle size may increase back pressure so that flow rate needs to be decreased, lengthening the run time. Using prepacked columns is highly recommended to ensure the best performance and reproducible results. An evenly packed column ensures that, as the sample passes down the column, the component peaks are not unnecessarily broadened. Uneven packing causes peak broadening and high resolution results become impossible. Allow buffers, media or prepacked columns to reach the same temperature before beginning preparation. Rapid changes in temperature, for example removing packed columns from a cold room and applying buffer at room temperature, can cause air bubbles in the packing and affect the separation. Storage solutions and preservatives should be washed away thoroughly before using any gel filtration medium. Equilibrate the column with 1–2 column volumes of buffer before starting a separation. Sample application The choice of sample application method depends largely on the volume to be applied and on the equipment available. Ensure that the sample is not diluted on the way to the column and that the top of the column bed is not disturbed during sample application. Samples can be applied automatically or manually. Apply samples directly to the column via a chromatography system, a peristaltic pump or a syringe. The choice of equipment depends largely on the size of column, the type of gel filtration medium and the sample volume. For example, a chromatography system will be required for a Superdex column whereas a syringe can be used with small prepacked columns such as HiTrap Desalting. Note that samples are applied by gravity feed to prepacked columns such as PD-10 Desalting
Elution and flow rates Samples are eluted isocratically from a gel filtration colum,using a single buffer sy Afer sample applicaion the Use flow rates that allow time for molecules to diffuse in and out of the matrix (partitioning between the mobile phase and the stationary phase)in order to achieve a separation. The goal for any separation is to achieve the highest possible resolution in the shortest possible time.Figures 14a,14b and 14c show that resolution decreases as flow rate increases and each separation must be optimized to provide the best balance between these two parameters.Put simply,maximum resolution is obtained with a long column and a low flow rate whereas the fastest run is obtained with a short column and a high flow rate. Suitable flow rates for high resolution fractionation or group separation are usually supplied with each product. The advantage of a higher flow rate (and consequently a faster separation)may outweigh the loss of resolution in the separation 66900 1355 0.5 on15.2 m/ml I WN.O H70 0.05M5odiu 25 ul.0.25 ml/min (19 cm/h) 2)A3 25 ul 1.0 mlmin (76 cm/h) 02s 0.2s 0.2 0.1 0.1 0.0 00 25.0 50.0 75.0 100.0 00 5.0 10.015.0 20.025.0mi Fig.14a.Influence of flo
24 Elution and flow rates Samples are eluted isocratically from a gel filtration column, using a single buffer system. After sample application the entire separation takes place as one column volume of buffer (equivalent to the volume of the packed bed) passes through the column. Use flow rates that allow time for molecules to diffuse in and out of the matrix (partitioning between the mobile phase and the stationary phase) in order to achieve a separation. The goal for any separation is to achieve the highest possible resolution in the shortest possible time. Figures 14a, 14b and 14c show that resolution decreases as flow rate increases and each separation must be optimized to provide the best balance between these two parameters. Put simply, maximum resolution is obtained with a long column and a low flow rate whereas the fastest run is obtained with a short column and a high flow rate. Suitable flow rates for high resolution fractionation or group separation are usually supplied with each product. The advantage of a higher flow rate (and consequently a faster separation) may outweigh the loss of resolution in the separation. 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 Total sample concentration: 15.2 mg/ml Buffer: 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.0 Flow: 1) 0.25 ml/min (19.1 cm/h) 2) 1.0 ml/min (76.4 cm/h) 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.0 25.0 50.0 75.0 100.0 min 25 µl, 0.25 ml/min (19 cm/h) Vo Vt A280 nm 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 Fig. 14a. Influence of flow rate on resolution. 1) 2)