Determination of a Selectivity Curve for Ultrogel® AcA 54 Resin Using a Mixture of Known Molecular Weight Proteins
Column: 1.6 x 40 cm; Buffer: 0.05 M Tris HCl, pH 7.4 containing 0.17 M sodium chloride; sample constituted of Blue-Dextran 2000 (a) for the determination of the void volume (Vo); bovine serum albumin (b) (MW 68,000); ß-lactoglobulin (c) (MW 35,000); myoglobin (d) (MW 17,800); and cytochrome c (e) (MW 12,400); Sample volume: 0.6 mL; Flow rate: 4.8 cm/h. The insert represents the elution profile of the above protein standards from the above column.
Ultrogel AcA resins are based on semi-rigid particles composed of a polyacrylamide and agarose composite gel matrix, which possess good mechanical properties. They can be used at high flow rate while maintaining a good resolution. These resins offer a choice between maximum resolution at recommended flow rates or rapid separations at higher flow rates with minimal loss of resolution. Resolution by molecular size-by-size exclusion chromatography
is influenced by a number of factors:
- Exclusion limit of the media, defining the molecular weight of the smallest molecule that cannot penetrate the pores of the gel matrix.
- Fractionation range corresponding to the linear region of the selectivity curve.
- Particle shape, ideally being spherical and able to pack in a column to form a highly uniform bed volume.
- Particle size and particle size distribution, which can have a significant impact on volumetric flow and resolution in the packed column bed.
Presence of very small particles or “fines” can lead to reduced eluent flow rates and should be removed during manufacture. The Ultrogel AcA family of pre-swollen gel media have been developed to meet the above critical success factors and five different resin types are available for fractionation of molecules with molecular weights ranging from 1,000 to 1,200,000. A summary of the properties of the Ultrogel AcA size exclusion resins are summarized in Table 4.26.
Properties of Ultrogel AcA Size Exclusion Resin
|Exclusion Limit (kda)
||45% gel. vol.|
||SDS-ND; Triton♦ X-100-ND; Deoxycholate-ND|
||Guanidine HCl < 2.0 M; Urea < 2.0 M|
*Height Equivalent to a Theoretical Plate (HETP).
ND – detergent limits have not been determined. Preliminary experiments should be conducted at the concentration required to ensure that the Ultrogel AcA beads are compatible. Beads should be examined for swelling or fragmentation which will impact the flow characteristics of this medium in a packed bed configuration
In this section, the application focus will be on larger-scale protocols (> 10 mL) for size exclusion and desalting applications employing Ultrogel resins on a liquid chromatography
workstation in glass columns. Smaller-volume size-based pre-fractionation (0.5-10 mL volume) of complex samples in packed glass columns gravity fed or pumped with a liquid chromatography workstation or peristaltic pump are described in Section 2.2.5
Protocol for Ultrogel® AcA Size Exclusion Chromatography Resin
A. Materials Required
- Glass column where the column diameter/length ratio is > 20 to provide the optimal geometry for peak resolution in size exclusion chromatography. Two examples are described in this protocol section to cover the 10-60 mL sample volume range. Other column configurations are possible as long as the above diameter/length ratio is met when size based fractionation is the intended application. In the case of desalting with Ultrogel AcA 202 resin, it is possible to use lower column diameter/length ratio due to the unique nature of this media and its application. In this process, the sample of interest is excluded from entering the gel-based media and rapidly passes through the packed media bed (see Figure 4.25).
- Glass column 1.5 cm ID x 50 cm length, max 83 mL bed volume (e.g., Omnifit PN 006CC-15-50-AF) for a 2-10 mL sample volume load.
- Glass column 3.5 cm ID x 100 cm length, max 693 mL bed volume (e.g., Omnifit PN 006CC-35-75-AF) for a 10-60 mL sample volume load.
Large-Scale Desalting on Ultrogel AcA 202 Resin
Column: 5 x 37 cm; volume: 730 mL; Sample: bovine serum albumin (5 mg/mL) containing NaCl (6.5 mg/mL); Flow rate: 7 cm/h (i.e. 140 mL/h). Sample volumes: A = 36 mL (5 % of sorbent volume); B = 220 mL (30 % of sorbent volume); C = 327 mL (45 % of sorbent volume); 280 nm UV detection.
- Column packing reservoir for 1.5 cm x 50 cm column (e.g. Omnifit PN 006-PS-15 and empty glass column PN 006-RG-15-50).
- Column packing reservoir for 3.5 cm x 75 cm column (e.g. Omnifit PN 006-PS-35 and empty glass column PN 006-RG-35-75).
- Suitable gravity flow system of adjustable height to achieve flow in the above columns.
- Peristaltic pump or liquid chromatography workstation able to provide buffer linear velocities in the above columns at up to 4.8 cm/h.
- Degassed* 50% (v/v) slurry of the Ultrogel® AcA resin.
- Degassed* suitable buffer, such as 0.05 M Tris HCl, pH 7.4 containing 0.17 M NaCl.
Tips on Handling Ultrogel AcA Resin:
Some BioSepra media are supplied as concentrated slurries and may be difficult to resuspend. DO NOT use magnetic stir bars with BioSepra media as they can damage the beads. When adding slurry to any device, mix well between additions.
If it is necessary to prepare 50% (v/v) slurry, use a clean spatula to remove some of the packed media and transfer to a graduated glass cylinder containing buffer. DO NOT add any media back to the storage bottle to avoid contamination of the bulk media. Thoroughly mix and allow settling. Note the volume of settled resin. Decant the supernatant and add back an equal volume of buffer to make 50% (v/v) slurry.
For packed columns, removal of fines may be necessary. Prepare the slurry in desired buffer, mix, and allow settling for approximately 5 minutes or enough time that the beads have settled but that small particles are still in solution. Decant off the suspension of fine particles and add fresh buffer and re-mix. Repeat the process until particles settle within approximately 5 minutes and leave a clear supernatant.
B. Packing Ultrogel AcA Resin
For successful size exclusion chromatography, it is very important to prepare a properly packed Ultrogel AcA media bed. The final column should present a low and uniform (with respect to cross sectional area) resistance to eluant flow and needs to be free of air bubbles and channels that may lead to uneven flow.
- Packed glass column format
- Equilibrate column, packing accessories, degassed 50% (v/v) gel slurry, and degassed buffer solution to room temperature. This is very important to prevent a volume change as the media or column adjusts to temperature.
- Install the fixed bottom fitting onto the glass column and attach the column packing adapter to the top of the column followed by the second length of glass column. Clamp the column and packing adapter upright in a stable laboratory stand. It may be necessary to use more than one clamp to hold the column. Check that the column is vertical in two dimensions with a suitable leveling device, such as a carpenter’s level.
- Add a sufficient volume of degassed buffer to the column to fill it up to the reservoir (wide-mouth) portion, and then gently tap the end and side of the column to dislodge any air bubbles.
- Open up the bottom fitting and allow flow to displace air form underneath the bottom support frit. Drain down to about 20% of the expected bed height. Stop flow.
- Add sufficient volume of 50% (v/v) degassed gel slurry to obtain the desired settled gel volume of 69-83 mL (15 x 50 cm column) or 616-693 mL (35 x 75 cm column). The slurry should fill the combined column and packing adapter. Initial brief gentle stirring with a long plastic rod will ensure uniform settling of the media.
- Allow gel to settle in the column for at least 15-30 minutes with the outlet closed.
- Open the column outlet and allow the supernatant eluant to drain to waste, until approximately 1 cm of buffer remains above the gel bed. If the bed height is low, add more of the 50% gel slurry after resuspending the top 2 cm of the packed bed with the plastic rod. If the bed height is too high, excess media can be removed by suction. At this stage it is important to allow the media to settle to a constant height and measure the final gel bed volume accurately.
- Drain and leave 1 cm of buffer on top of the packed bed.
- Remove the column adapter and set aside.
- Attach the top adjustable length fitting onto the column and screw down the top fitting until the top of the packed gel bed is reached. The top fitting should not be screwed down too far, compressing the packed bed that can lead to reduced flow rates.
- The bottom outlet should then be connected to pump and buffer flowed up through the column at 0.5 mL/min to displace air from the top fittings.
- Packed column is now ready for storage at 4 ºC for no more than one week or for immediate use.
- Refer to Section C for use instructions.
Tip: Size exclusion chromatography can be carried out at a range of temperatures, including 4 ºC. The column must reach the desired temperature and equilibrate for 8-16 hours to ensure the column packing is uniform. Some slight bed height adjustment may be required, adjusting to the different temperatures. When a column is moved to a new location, for example, a cold room, it should be supported with a stable clamp stand and maintained in an upright orientation (check with a level) for storage.
Store the packed column upright at 4 °C with the gel bed submerged under 1-2 mL of buffer and a top column cap securely in place. Sodium azide added to the storage buffer to a concentration of 0.02% (w/v) will help prevent microbial growth. Always remove the top cap before the bottom cap to avoid drawing air bubbles down into the gel bed. Prevent air bubbles from forming in the gel bed by using only degassed buffer and sample solutions. Degassing involves subjecting a solution to vacuum to remove excess dissolved air. Use of too high a vacuum can lead to evaporation of solvent from the solution. Check the final volume after degassing and, if necessary, add more solvent to return to original volume.
C. Size-based Purification
One of the following approaches can be used:
- Varying the binding conditions, such as pH and salt concentration of the sample to influence the size or shape of the proteins in the complex sample.
- Size exclusion can be carried out in the presence of limited amounts of detergents, such as sodium dodecyl sulfate (SDS), Triton♦ X-100 and sodium deoxycholate and denaturants such as urea or guanidine hydrochloride.
These conditions may be used where sample solubility is an issue, such as in the case of bacterial exclusion bodies; varying the size exclusion limit and fractionation range of the Ultrogel® AcA media; or temperature can have a significant impact on eluant flow, peak shape and molecular shape. Combinations of the above approaches are useful in developing an optimal size exclusion purification polishing step and are summarized in Table 4.27. Either strategy or a combination of both can be applied to purify a component isolated from a complex sample. During size exclusion chromatography
there will be some dilution of the samples fractionated on the media due to peak diffusion. If necessary, the sample (10-60 mL) can be concentrated with a centrifugal UF devices, such as a Pall Microsep™, Macrosep®, or Jumbosep™ with 10 K MWCO UF membrane (see Section 4.4
). Or for larger volumes (25 mL-1 L) Minimate™ Tangential Flow Filtration Systems with a 10K MWCO UF membrane (contact Pall for more information). In the case of desalting with Ultrogel AcA 202, the excluded volume fraction will only experience slight dilution (peak volume may increase by 100-150% of the sample volume loaded) and may not required re-concentration.
Summary of Pre-Fractionation Options for Ultrogel® AcA Size Exclusion Resin
*Initial conditions recommended: 0.05 M Tris HCl, pH 7.4 containing 0.17 M sodium chloride. The presence of salt helps to minimize non-specific adsorption to the size exclusion resin. Increasing salt concentration during loading can be employed to alter the shape or size of molecules in the complex sample that may impact the size fractionation.
**Presence of detergents may be necessary for sample solubilization. Proteins in the presence of detergents such as SDS exist in micellar structures when used at concentrations above the critical micellar concentration (CMC) of the detergent. These structures in solution are much larger than the true size of the molecule of interest and will require a higher size fractionation range of Ultrogel AcA resin. It may be necessary to use two Ultrogel AcA size fractionation range beads for native and denatured forms of the same protein sample. For removal of detergents from the final samples, SDR HyperD resin is recommended (see Section 2.3, page 141).
- Packed glass column format
Sample introduction onto the packed bed of the size exclusion column can have a significant impact on the resulting peak shape and resolution. The volume of sample loaded for most analytical separations should be 5-10% of the media volume to achieve optimal resolution and acceptable dilution. Too high a volume sample leads to reduced resolution and too small a volume leads to high sample dilution and poor recovery. In the case of desalting with Ultrogel® AcA 202 resin, this volume can be up to 45% due to the unique uniformity of the bead shape and pore size distribution. See Figure 4.25 for an example of this application.
- Prepare a size exclusion column as described above and equilibrate with a suitable buffer of the same composition as the sample. Exception is in the case of a buffer exchange application (see Section 2.4) with Ultrogel AcA 202 resin, where the column equilibration buffer will be different.
- Prepare a sample in the appropriate buffer (see Table 4.27 for options) and prefilter with a suitable volume 0.45 μm pore size microfiltration device (see Section 2.4). The sample should not contain any particulate material that could get trapped on the head of the packed bed column. If a large amount of material is lost during the microfiltration, it may be necessary to evaluate different sample buffer conditions or homogenization conditions to render the sample more soluble.
- Load the sample onto the column using the adjustable plunger method which requires the use of a three-way valve attached as close to the top fitting as possible. The column is set up and the top fitting left in place at all times. To prevent sample dilution and “tailing” (peak shape asymmetrical), the tubing length between the valve and the adjustable top fitting should be as short as possible. Sample is introduced with a syringe or a peristaltic pump via the three-way valve without disturbing the packed column bed.
- For fractionation applications, load a sample volume 5-10% of the column volume; then elute with column buffer and collect the effluent in 1 mL fractions. Measure absorbance at 280 nm to locate eluting peaks. If a void volume marker, such as Dextran blue, has been included in the sample, only start fraction collection when this colored marker reaches the bottom of the column. Fractions eluting after this point are being fractionated by the size exclusion resin with the largest size fractions eluting first. As the volume approaches Vt or the media volume, fraction collection should be stopped as these fractions should be only very low molecular weight. Peaks can either be pooled or processed singly and some dilution of the sample will occur during elution. If necessary, the sample can be concentrated.
- For desalting or buffer exchange applications, load up to 45% of the column volume then elute with column buffer (in the case of buffer exchange this will be a different buffer to the sample) and collect fractions. Monitor the A280 to locate eluting peaks.
- The peak eluting with the void volume Vo will be the desalted sample.
- If a high loading is used (up to 45% of the column volume), the desalted sample will continue eluting and should return to baseline absorbance before the low molecular weight fraction elutes (see Figure 4.25, Panel C).
- If the desalted sample elutes after the low molecular weight fraction, the column was overloaded. Fractions containing low molecular weight material should be pooled separately and re-run on the column to achieve complete desalting.
- After protein has eluted, the column can be washed with 2 CV (5 CV in the case of desalting to remove the low molecular weight fraction from the column bed) of loading buffer before re-use is attempted.
- Packed column is now ready for storage at 4 ºC for no more than one week or for immediate use.
Tip: Sodium azide can be added to the buffer to a concentration of 0.02% to prevent microbial growth if storage > 1 week is required. Before re-use at a different temperature after storage at 4 ºC, the packed bed column should be re-equilibrated overnight. Before re-use, any sodium azide in the column that may be present should be removed by washing with 2 CV of buffer.
Application Data for Ultrogel® AcA Size Exclusion Chromatography Resin
Selectivity Curve for Size-based Pre-fractionation Using Ultrogel AcA 54 Resin
Molecular weights of proteins may be reliably determined by choosing an Ultrogel AcA media which has the required fractionation range. The first step in the process consists in the creation of a selectivity curve from the elution curves of known standard proteins for the media and column configuration (see Figure 4.24
). If the media or column configuration is changed, a new selectivity curve will need to be established. The second step consists of the size exclusion chromatography
of the protein of interest and the comparison of its elution volume or calculated Kav with the selectivity curve to obtain an estimate of the molecular weight.
Ultrogel AcA resin provides excellent separation efficiency, as demonstrated by the low HETP (Height Equivalent to a Theoretical Plate) values in Table 4.26
. The HETP of approximately 0.15 mm corresponds to over 1,500 theoretical plates per meter; except for Ultrogel AcA 202 resin where the HETP is 0.3 mm (eq. 3,000 plates per meter). There are only moderate HETP variations as a function of flow rate.
Desalting with Ultrogel AcA 202 Resin
In desalting applications, the separation of proteins from salts can be achieved with Ultrogel AcA 202 resin. When optimal column geometry is used, very large volumes may be processed. The sample volume must theoretically correspond to the difference between the total media volume and the void volume (generally 60% of the media volume). For traditional media, the recommended sample volume loaded is only 30% of the column volume. Because peak broadening is reduced with Ultrogel AcA 202 resin, as a result of the uniformity of the particles and the pores, the sample volume with this media may be as high as 40% of the media volume, or even 45% under certain circumstances. An example of a loading study on Ultrogel AcA 202 resin is shown in Figure 4.25
. The data clearly show that loading up to 327 mL of 5 mg/mL BSA in Panel C (45% of the column volume) still achieved elution of the desalted protein before the conductivity due to salt increased.
The concentration of sample may be as high as 40 g/L without affecting the desalting quality. Peak fractions eluting after the void volume Vo, showed minimal dilution and may not require further re-concentration processing. In addition to the removal of salts, desalting can be extended to:
- Removal of low molecular weight sugars, such as lactose from whey.
- Removal of small aromatic compounds, such as phenol during the purification of nucleic acids.
- Removal of certain agents used for the solubilizing proteins, such as urea and guanidine salts.
Detergents, such as SDS, CHAPS and ASB-14, used above their critical micellar concentration form large micelles in aqueous systems. These micelles co-elute with the protein in the void volume Vo and cannot be desalted on an Ultrogel® AcA 202 column. At lower than their critical micellar concentration, where equilibrium favors the free-detergent state, it may be possible to use this bead-based desalting approach. At these concentrations, proteins may be less soluble, which can lead to aggregation or precipitation in the column. Trial experiments are recommended to screen for sample stability at these reduced detergent levels. As an alternative, removal of these detergents up to and above their critical micellar concentration limit can be achieved with SDR HyperD® resin (see Section 2.3
Ordering Information for Ultrogel AcA Size Exclusion Chromatography Resin
||Ultrogel AcA 22
||Ultrogel AcA 34
||Ultrogel AcA 44
||Ultrogel AcA 54
||Ultrogel AcA 202
References for Ultrogel AcA Size Exclusion Chromatography Resin
- 1. Gomi, K., & Jajiyama, N. (2001). J. Biol. Chem., 276(39), 36508-13.
- Sehgal, N., Goswami, S., et al. (2001). J. Biochem. Biophys., (38)4, 263.
- Knutson, V.P., et al. (1991). J. Immunol. Meth., (145), 263.
- Diesterhaft, M. (1984). Biochem. Biophys. Res. Commun., (125)3, 888.
- Boschetti, E., et al. (1972). Biochimie, (54), 439.