CHAPTER 1 Gel-Filtration Chromatography Daniel M. Bollag 1. Introduction Gel filtration chromatography is a method for separating proteins and peptides based on their size (I). The chromatographic matrix consists of porous beads, and the size of the bead pores defines the size of macro- molecules that may be fractionated. Those proteins or peptides that are too large to enter the bead pores are “excluded,” and thus elute from the column first (Fig, 1). Since large molecules do not enter the beads, they have less volume to pass through, which is why they are the first to elute from the column. Smaller macromolecules that enter some, but not all of the pores are retained slightly longer in the matrix and emerge from the column next. Finally, small molecules filter through most of the pores, and they elute from the column with an even larger elution volume. This method is also called gel permeation, molecular sieve, gel-exclusion, and size-exclusion chromatography. Since no binding is required and harsh elution conditions can be avoided, gel-filtration chromatography rarely inactivates enzymes, and often is used as an important step in peptide or protein purification (see Note 1). The chief limitations of gel-filtration chromatography are that the separation may be slow and that the resolution of the emerging peaks is limited (see Note 2). The speed of sample elution is limited primarily by the requirement for a long, narrow column in order to permit suffi- cient component separation, although the procedure may be acceler- ated by the use of matrices permitting faster flow rates and by the use of pumps or high-pressure chromatography equipment if the matrix can tolerate the added pressure. The resolution is limited since the sample From: Methods in Molecular Biology, Vol. 36: Peptide Analysis Profoco/s Edited by: B. M Dunn and M. W. Pennmgton Copyright (81994 Humana Press Inc., Totowa, NJ 1 Bollag I moo.0 1 :.O I Fig. 1. Schematic representation of gel-filtration chromatography. Molecules of different size in the left frame are separated according to size during migra- tion through the gel-filtration matrix as shown in the middle and right frames. does not bind to the matrix. Therefore, careful selection of the matrix fractionation range is essential, and gel-filtration chromatography is fre- quently used as a separation step when only a small number of contami- nants remain. Gel-filtration chromatography separates proteins and peptides based on their diameter during chromatography. Thus, gel filtration allows an estimation of the molecular weight of a protein or multiprotein complex (2). However, a molecular-weight estimation is based on the assumption that the protein is generally globular in shape. Separation on the basis of size may also permit an approximation of a dissociation constant for a protein-protein or protein-l&and interaction (3). In addition, gel-filtra- tion chromatography may be used for sample desalting or for changing the buffer of the sample (see Note 1). The versatility of gel-filtration chromatography has made this separation technique an extremely useful and popular tool for protein or peptide purification and analysis. Gel-Filtration Chromatography 3 Fracticn Collector Fig. 2. SchematIc representation of chromatography equipment. 2. Materials The equipment required for gel-filtration chromatography is very simple, but a more sophisticated laboratory system may be preferable to save time and provide more reproducible results, The heart of a gel-filtra- tion chromatography setup (Fig. 2) is the column, which generally con- sists of a glass cylinder containing a column support. Columns for gel filtration are generally long and narrow, but the diameter should be at least 10 mm, so that anomalous effects from the protein and buffer inter- actions with the column wall can be avoided. Adaptors for the top and Bollag Table 1 Gel-Filtration Chromatographya Fractionation range, Linear flow rate, Name kDa Cm/h BioGel P-6 1-6 10 BioGel P-60 3-60 5 BioGel P- 100 5-100 5 Sephacryl S- 100 HR l-100 Sephacryl S-200 HR 5-250 15 Sephacryl S-300 HR 10-1500 15 Sephadex G-25 l-5 Sephadex G-50 1.5-30 5 Sephadex G- 100 4-150 5 Sephadex G-200 5-600 2 Sepharose CG6B 1 O-4000 18 The fractionation range defines the approximate protein and peptide molecular weights that can be separated with the matrix. The linear flow rate can be con- verted into a volumetric flow rate (n&/mm) by multiplying by the cross-sectional area (nr2) of the column. bottom of the column allow homogeneous and efficient delivery of sample or buffer to the column matrix. Tubing from the filtration column should be narrow bore to keep remixing of the separated components to a mini- mum. A reservoir for the buffer to be delivered to the cohunn can be con- nected via a pump that can control the column flow rate. A UV wavelength detector monitors the absorbance of the eluting sample, and the signal can be sent to a recorder or a personal computer for analysis. The eluting sample may be directed to a fraction collector that sequentially collects aliquots of the eluant either according to time or volume. All of this equip- ment can be purchased as individual components or as an integrated high- pressure chromatography system, depending on the needs of the user. The column matrix for gel filtration must be chosen carefully to allow the best resolved separation of the component of interest from the con- taminants. The matrix should be chosen so that the sample molecular weight falls in the middle of the matrix fractionation range or so that contaminating components are well resolved from the desired compo- nent. Table 1 provides information for selection of the proper matrix type; suppliers such as Bio-Rad and Pharmacia can be consulted for fur- ther information. Coarser matrices offer faster flow rates, which may, Gel-Filtration Chromatography 5 however, lead to reduced resolution of peaks. A coarse matrix will thus be better for such uses as desalting a protein or exchanging the buffer of the sample, whereas a fine matrix is preferred for separations. If the matrix is not supplied as a preswollen slurry, the dry powder needs to be swollen in buffer. Swelling is generally carried out by gently swirling the matrix in buffer. Using a magnetic stirrer may cause the matrix parti- cles to be broken into “fine” particles, which can cause irregularities in column packing and may also reduce the column flow rate. Thus, agitation by a rotary shaker or occasionally swirling the matrix by hand is recom- mended for swelling. Swelling can be carried out at room temperature or by boiling, which speeds the hydration process significantly; matrix manu- facturers should be consulted for swelling information. Fines are removed by swirling the slurry containing the gel-filtration matrix and, after most of the matrix particles have settled, pouring off the supernatant. This pro- cedure is repeated several times. A preswollen gel may only require reequilibration in the appropriate buffer. Degassing of the matrix is impor- tant to reduce the likelihood that air bubbles will form in the column, To degas the gel-filtration matrix, apply a vacuum to the matrix solution for up to an hour while agitating the matrix slurry. 3. Methods 3.1. Packing the Column 1. The chromatography matrix is first prepared and degassed as a thick slurry (the buffer supematant should comprise only 25% of the matrix volume). Space below the column support should be filled with buffer so no bubbles will form. 2. Add a small amount of buffer, and close the outlet after a small amount of buffer has been allowed to flow out. 3. Then, in a single step, the slurry is poured down a glass rod into the col- umn or along the side of a column that is temporarily tilted slightly, and the column outlet is opened. If necessary, a column extension or funnel is attached to the column in order to permit packing of the matrix in a single operation; otherwise, uneven beds can form. Care must be taken to be sure that air bubbles are not trapped as the matrix packs or the column will have to be repacked. If bubbles develop early during packing, they can be removed by gently stirring the matrix. 4. Once the matrix has been poured, it is possible to connect the pump and attach the reservoir (but do not exceed the maximum pressure recom- mended for the matrix). Two- or three-column bed volumes should pass through the packed matrix to stabilize and equilibrate the column. Bollag 3.2. Checking the Column Initially after packing the column, a visual inspection for air bubbles is necessary, since bubbles will cause mixing during chromatography that will reduce the resolution substantially. As a more rigorous test of column packing, 0.2% blue dextran ( 1% of the column bed volume) can be loaded on the column and should travel through the matrix as a well- defined, horizontal band. If the column is well packed, the blue dextran should elute in no more than twice the volume that was applied. 3.3. Sample Application 1, The sample should ideally be fairly concentrated (10-20 mg/mL), and the sample solution should be less than twice as viscous as the elution buffer or peaks may become too broad. Sample volume should be l-5% of the column bed volume; a larger volume may lead to poor resolution, whereas a sample volume smaller than 1% of the bed volume will not generally improve the separation. 2. The elution buffer should be chosen to preserve the protein’s activity and should contain a low ionic strength buffer (e.g., 20-100 mM) to mnnmize nonspecific ionic or hydrophobic mteractions. 3. The buffer in the column should be eluted until the buffer reaches the top of the matrix surface. Then the outlet should be closed. Remember that the chromatography matrix must never be allowed to run dry. 4. The sample 1s gently layered on top of the matrix, taking care not to disturb the packed matrrx. 5. Open the outlet, and allow the buffer to drain until the liquid level again reaches the matrix surface. Then close the outlet. 6. Add a small amount of buffer to the column, and run the buffer just into the column in order to wash the remaining sample into the matrix. 7. Finally, refill the column with buffer, and attach the pump and reservoir. At this point, elution of the sample may begin. 3.4. Column El&ion The buffer is simply run through the column until the peaks of interest have been eluted. Recoveries are typically over 85%. Slower flow rates generally yield better resolution, so some adjustments for optimal sepa- ration may be necessary. 3.5. Column Regeneration and Storage 1. Following elutlon of the sample, the gel-filtration matrix should be regen- erated to remove any of the remaining sample components. For most Gel-Filtration Chromatography 7 matrices, regeneration is carried out by washing 0.2M NaOH or noniomc detergent through the column, and then reequilibrating with the appropri- ate buffer for the next experiment. 2. If the column will be stored overnight or longer before the next use, it is advtsable to maintain the gel-filtration matrix in a solution contammg .an inhrbitor of microbial growth. For most applrcations, a buffer containing 0.02% sodium azide is effective for preventing the growth of microorgan- isms. Other inhibitors include O.Ol-0.02% trichlorobutanol or 0.002% hibitane (but do not use hibitane with Sepharose). 3. Finally, some matrices should not be stored in solutions of very high or low pH. 4. Notes 1. Gel-filtration chromatography, aside from its utility in protem and pep- tide purification, can also be employed for exchanging the buffer in which a macromolecule is found. Since the original sample buffer passes through a matrix, such as Sephadex G-25, much more slowly than a polypeptide, the protein or peptide can be eluted with a new buffer that has been used for column equilibration and elution. In this fashion, an ion-exchange chromatography fraction can be exchanged into a lower salt buffer (“desalting”) or a sample can be separated from low-molecular-weight contaminants, such as nucleotrdes or metals. This separation is a very distinct one, so the sample may be as large as 30% of the column bed volume without affecting the separation. Some matrix suppliers now offer spin columns, which allow desalting or nucleotide removal by passing the sample through the filtration matrix in a rapid centrrfugation step. 2. Poor peak resolution may be the result of: a. Improper selection of matrix: Use a matrix with a fractionation range that brackets the molecular weight of the desired protein (i.e., the molecular weight is in the middle of the separation range). Be aware that a nonglobular or denatured protein elutes differently from a globular protein. b. Wrong matrix grade: A finer matrix grade may be available that offers better resolution, although separation times will be longer. c. Column is too short: A longer column will allow better resolution: reso- lution increases as the square root of column length. d. Flow rate is too high: A faster flow rate reduces resolution. e. Large dead space before elution fractions are collected: Dead space is the region at the bottom of the chromatography column that allows the temporary accumulation of eluent before fraction collection occurs. If this space is large, protein peaks will remrx, reducing Bollag the resolution. A well-designed column will contain minimal dead space. f. The sample volume is too large: For a good separation, the sample should be between 1 and 5% of the column bed volume. g. The column is poorly packed: Uneven column packing or air bubbles trapped in the column matrix cause irregular flow patterns leading to poorer separation. 3. Skewed protein peaks may be the result of: a. Poor sample application: It is possible to practice sample application using blue dextran as described in Section 3.2. b. Protein adsorption to the matrix: Matrix adsorption can be suspected when peaks tail off slowly; adding a stronger ionic strength salt may reduce these undesired interactions. In addition, changing the buffer pH or composition may improve the situation. 4. A low flow rate can be traced to: a. Plugged filters or tubing: Such a situation can sometimes be remedied by adding some detergent or denaturant to the buffer or by reversing the buffer flow through the column. Otherwise, the column must be dis- mantled, cleaned, and repacked. b. A clogged matrix surface: If a residue has formed on top of the matrix, scrape off and remove the top layer of the matrix, then stir the top centimeter of the remaining matrix, and allow to settle slowly. c. A pump is poorly functioning. d. A matrix that is incompletely swollen, is compressed, or contains too many “fines”: If this is the case, the column must be repacked. e. Microbial growth in the matrix: A new chromatography column must be prepared. 5. Poor recovery of the sample might be caused by: a. Sample precipitation: Too little or too much salt can result in precipita- tion of the protein and poor entry into the column. b. Adsorption effects: See Note 3b. c. Elution conditions that are too harsh: This may release a necessary cofactor or damage the component of interest. d. Microbial growth: See Note 4e. e. Proteolysis: Include protease inhibitors in buffer. f. Slight adsorption of the sample to the matrix and very slow elution as a peak that cannot be distinguished from the background: A non- ionic detergent may disrupt this interaction without damaging the macromolecule. g. Dissociation from a complex or necessary cofactor during elution: Mix- ing fractionated aliquots may reactivate the sample. Gel-Filtration Chromatography 9 References 1. Stellwagen, E. (1990) Gel filtration. Methods Enzymol. 182,317-328. 2. Preneta, A. Z. (1989) Separation on the basis of size: gel permeation chromatogra- phy, in Protein Purtftcation Methods: A Practical Approach (Harris, E. L V. and Angal, S., eds.), IRL, Oxford, pp. 293-305. 3. Pharmacia Fine Chemicals (1991) Gel Filtration: Principles and Methods. Uppsala, Sweden. [...]... get broad peaks) (Fig 6) 3 Print the baseline and integration marks (Fig 6) 3.4 Determination of Peptide Content By comparing the integrals of the peptide concerned and a standard, it is possible to determine the content of the peptide in a sample Two requirements have to be met The standard must be the same peptide as the product concerned, and the content of the standard has to be exactly known 3.4.1... preparation 3.4.4 Analysis Before filling the sample loop, flush it with the solution you want to chromatograph (three- to fivefold loop volume) First inject the solvent (blank), then your standard, and subsequently chromatograph the two sample solutions Nirenberg 32 Fig 6 Expanded chromatogram of a peptide 3.4.5 Evaluation The content of the peptide in the sample is calculated as follows: Peptide content... the chiral center); cont.: 0.5 mg/mL in H,O; gradient: isocratic (93% A/7% B) 3.3 Purity To determine the purity of a peptide, two methods are described 1 The 100% method is a simple way to check the purity of a peptide m a smgle chromatographic run You get the amount of the interesting peptide and impurities as area% (integral) relative to the total integral area response With this method, you work over... dissolved in (blank) and then chromatograph the sample solution 3 Evaluation: In general, your data acquisition system (e.g., integrator) calculates the peptide purity and amount of impurities m area% automatically as follows: Peptide purity (area%) = [peak area (peptide) /peak area (total)] x 100 Impurity (area%) = [peak area (impurity)/peak area (total)] x 100 (1) See Fig 2 for an example of this procedure... (Hygroscopic peptides: it may be necessary to determine the content of H,O before standard preparation to correct the content of the standard.) 3.4.5.1 EXAMPLE (FIGS 7 AND 8) Determination of peptide content: Sample: weight = 12.32 mg Standard: weight = 12.72mg content = 78.5% Analytical HPLC 33 Fig 7 Chromatogram of the standard Integral: standard: I(std) = 348,312 product: I (p) = 325,864 Peptide content... American Peptide Symposium (Gross, E and Meienhofer, J , eds.), Pierce Chemical Company, Rockford, IL, p 121 5 Guo, D., Mant, C T., and Hodges, R S (1987) Effects of ion-pairing reagents on the prediction of peptide retention in reversed-phase high-performance liquid chromatography J Chromutogr 386,205-222 6 Guo, D., Mant, C T., Taneja, A K., Parker, J M R., and Hodges, R S (1986) Prediction of peptide. .. Analysm Protocols B M Dunn and M W Penmngton Copyright 01994 Humana Press Inc , Totowa, 23 NJ Nirenberg 24 or phosphate In some cases, triethylamine is added to suppress the interaction of residual free silanol groups of the matrix (stationary phase) and to reduce polar anionic groups Such an addition often results in a better peak shape The most favored solvent as organic modifier to elute peptides... organic modifier to elute peptides is acetonitrile All these eluent components meet one important requirement-they are transparent in the UV range down to 200-220 nm, where the peptides are detected because of the UV absorption of the peptide bond In some cases, it is possible to use longer wavelengths for detection, e.g., 280 nm where Trp and Tyr absorb because of their aromatic chromophores 2, Materials... seeSection 3.1.2 3.1.2 Preparation of the Eluents Three different eluent systems are described, the TFA and TEAP systems (1-8) as standard systems suitable for the most peptides, and the ion-pair (IP) system (9-11) for hydrophilic peptides that are not retained by the other two eluent systems Mix each eluent in the following way 3.1.2.1 TFA SYSTEM Eluent A: 2000 mL HZ0 + 20 mL ACN + 2 mL TFA; Eluent... Chromatography M BoZZag 1 Introduction Ion-exchange chromatography allows the separation of proteins and peptides by taking advantage of their net charge These macromolecules can also be concentrated by ion exchange either on a column or as a batch procedure (see Note 5) Although procedures for separating peptides or proteins vary according to each individual molecule, many basic rules apply to all ion-exchange . pressure. The resolution is limited since the sample From: Methods in Molecular Biology, Vol. 36: Peptide Analysis Profoco/s Edited by: B. M Dunn and M. W. Pennmgton Copyright (81994 Humana Press Inc.,. made this separation technique an extremely useful and popular tool for protein or peptide purification and analysis. Gel-Filtration Chromatography 3 Fracticn Collector Fig. 2. SchematIc. buffer passes through a matrix, such as Sephadex G-25, much more slowly than a polypeptide, the protein or peptide can be eluted with a new buffer that has been used for column equilibration