ENrich™ SEC 70 ENrich™ SEC 650 High-Resolution Size Exclusion Columns Instruction Manual Catalog numbers 780-1070 780-1650 Please read these instructions before you use ENrich SEC high-resolution size exclusion media. If you have any questions or comments regarding these instructions, please contact your Bio-Rad Laboratories representative.
Table of Contents Section 1: Characteristics of the ENrich Size Exclusion Columns .......................................... 1 1.1 Introduction .............................................................1 1.2 The ENrich SEC Separation Media ........................... 1 1.3 Connection to the NGC™ and Other Chromatography Systems ....................................... 2 Section 2 : Use of the ENrich SEC Columns ............................ 3 2.1 Preparation for Initial Use .............................
Section 1: Characteristics of the ENrich™ Size Exclusion Columns 1.1 Introduction ENrich prepacked columns for size exclusion chromatography are designed for rapid and reproducible high-resolution separation of biomolecules. Each column contains a unique, high-performance size exclusion media based on spherical polymer beads modified to provide quick, differential size–based diffusion. 1.2 The ENrich SEC Separation Media ENrich SEC media is available with different exclusion limits and separation ranges.
The ENrich support is compatible with aqueous solutions of 6 M guanidine-HCl and 8 M urea. Detergents and organic solvents such as methanol, ethanol, and isopropanol may also be used. In some cases, alcohols and stability agents such as glycerol may increase the column backpressure. Care should be taken to maintain a flow rate at which the backpressure is below the maximum operating pressure. Table 1. ENrich SEC media and column characteristics.
Section 2: Use of the ENrich Columns 2.1 Preparation for Initial Use The column is supplied in 20% ethanol. To remove the storage buffer, pump deionized, filtered water at a flow rate of 0.5 ml/min or less for 30 ml, or just over one column volume (CV). The backpressure will decrease as the ethanol is washed from the column. After this water step you may equilibrate your column as described below. Warning: Do not exceed a maximum pressure of 600 psi (4.1 MPa, 41 bar). 2.
2.3 Sample considerations 1. Always filter (0.22–0.45 μm filter) and/or centrifuge your sample to remove any particulates. 2. In size exclusion chromatography, mass loading is less critical than the volume of the sample. As a general rule, to obtain maximum performance, the sample volume should not exceed 1% of the bed volume (that is, ~0.25 ml for a 24 ml CV) and the sample concentration should not exceed 20 mg protein/ml. Larger volumes can be applied, but resolution may decrease. 2.
ENrich SEC 70 media ENrich SEC 650 media Figure 1. Typical chromatograms for ENrich SEC media. Detection at 280 nm. Absorbance at 280 nm may be greater depending on sample concentration. Time may change depending on flow rate. A. thyroglobin; B. IgG; C. ovalbumin; D. myoglobin; E. vitamin B12.
Section 3: Care of the ENrich SEC Column 3.1 Regular Maintenance To maintain performance, the column should be stored in 20% ethanol, as described below, for storage periods longer than 2 days. Always run at least one CV of water between 20% ethanol and running buffers. If the column is exposed to air or if air bubbles are trapped in the column. 1. Wash the column in the forward direction with 30 ml of water at 0.25–0.5 ml/min. 2. Wash the column with 30 ml of 20% ethanol at 0.25–0.5 ml/min.
3.3 Column Cleaning Careful preparation (especially filtration) of the sample and the buffers will maintain the column’s performance and extend its lifetime. Normally, washing with a few CV of running buffer with an ionic strength of at least 100 mM NaCl will remove most contaminants. However, if there is a decrease in column performance (that is, increasing backpressures or a drop in resolution), then steps 1 and 2 of the cleaning protocol described below should be used.
Adjusting nut Lock nut Upper retainer Lower retainer Adaptor Fig. 2. Column diagram. 1. Start a slow flow rate of buffer (0.5 ml/min or less) through the column. 2. Remove the lower end tubing and fitting from the column. Firmly hold the bottom of the column over a sink or container. 3. Loosen the lock nut by turning it clockwise. 4. Raise the adaptor a few millimeters by slowly by turning the adjusting nut counterclockwise. 5. Unscrew the upper retainer. Let it rest on the bottom retainer. 6.
3.5 Storage Conditions To prevent bacterial growth in the column, it must be stored correctly. For short-term storage (up to 2 days), store it in freshly prepared buffer. For long-term storage, first wash the column with 30 ml of deionized water and then with a storage solution of 20% ethanol. Use a flow rate of 0.5 ml/min or less. Ensure that the backpressure stays below the maximum operating pressure. Always store the column with plugs at each end.
Section 4: Ordering Information Catalog # Description 780-1070 ENrich SEC 70 10 x 300 Column, 10 x 300 mm 780-1650 ENrich SEC 650 10 x 300 Column, 10 x 300 mm 780-0093 ENrich 10 Frit Kit, includes 2 frits, 1 frit remover, 2 O-rings 151-1901 Gel Filtration Standard, 6 vials 10 ENrich SEC Instruction Manual
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