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Green BioPak® MicroColumns Filled with C-18 Resin
BioPak C-18 filled MicroColumns utilize reversed phase chromatography principles to isolate and purify biomolecules based on their hydrophobicity. Applications include purifying proteins, peptides, ssDNA, and biological or synthetic oligonucleotides. These purification applications are typically performed prior to protein digestion, gel electrophoresis, HPLC, mass spectrometry and other techniques.
General Principles
The target molecule binds to the C-18 resin based primarily on its hydrophobic nature, and secondarily on characteristics such as size and charge. In a polar solution, the molecule of interest binds to the C-18 resin, and elutes in an organic solvent. The ideal solution for binding and elution is determined empirically for a given molecule in a given environment. Solutions for binding and elution typically contain a buffer component, an organic modifier, and often, an ion-pairing agent. Altering the types and concentrations of these components optimizes binding and elution efficiency. The organic solvent is then easily removed from the recovered molecule in a centrifugal evaporator. After elution and drying of the target molecule, subsequent steps are not necessary to prepare the DNA for downstream reactions such as as restriction enzyme digests, ligations, transformations, sequencing, and amplifications.
Advantages
BioPak C-18 filled MicroColumns are a rapid and inexpensive technique for isolating and purifying a wide range of biomolecules from various sources. The capture of solutes, that vary in their hydrophobicity, is readily achieved. Using appropriate organic solvents, this process ensures excellent recovery.
The high speed of the C-18 filled MicroColumn purification process increases the potential to recover biologically active molecules. BioPak MicroColumns are easily adapted for various applications using a 200ul hand-held pipettor. And, because they are mounted from a rack onto a pipettor, MicroColumns never need to be handled directly, eliminating enzyme contamination.
Also, MicroColumns with radiolabeled probes may be purified with less exposure to radiation. Not only may radiolabeled probes be purified very quickly using MicroColumns, there is less need to handle and manipulate microcentrifuge tubes, as well.
Technical Information
Matrix: Silica
Particle Size: 55-105um
Pore Size: 125A
Binding Efficiency: 95-100%
Elution Efficiency: 85-90%
Ion-Pairing Agents
Typical concentration ranges for ion-pairing agents are 0.01% to 0.1%. Ion-pairing agents are thought to bind to the solute molecules to increase their hydrophobicity, by neutralizing their charge. This alters selectivity and allows the capture of charged molecules. Ion-pairing agents vary according to charge, volatility, and UV transparency.
Commonly used volatile ion-pairing agents include: trifluoroacetic acid (TFA) (anionic), pentafluoroproprionic acid (PFPA) (anionic), and heptafluorobutyric acid (HFBA) (anionic). Commonly used non-volatile ion-pairing agents include: ammonium acetate (anionic), phosphoric acid (anionic), tetramethylammonium chloride (cationic), tetrabutylammonium chloride (cationic), and triethylamine (cationic).
Phosphoric acid and trifluoroacetic acid (TFA) (anionic) may act as buffers and ion-apring agents. Trifluoroacetic acid (TFA) is more widely used because it is volatile, and essentially UV trasparent at low wavelengths.
The concentrations for the buffer, and ion-paring agents, if used, are generally the same in both binding and elution solutions. Volatile buffers and ion-pairing agents ensure that sample recovery will not require a seperate desalting step.
Buffers
Buffer concentrations ranging from 0.05% to 0.1% are commonly used in the purification process of biomolecules such as proteins and peptides. Buffers vary according to pH, volatility, and UV transparency. Low pH facilitates ion suppression of acidic groups and may improve solubility of the sample components. Although it may be advantageous to isolate certain peptides in conditions above pH 8.0, a basic solution is not compatible with the C-18 silica matrix in these MicroColumns.
Some commonly used volatile buffers are hydrochloric acid (pH 2-3) and trifluoracetic acid (TFA) (pH 2-3). Commonly used non-volatile buffers include phosphoric acid (pH 2-3), triethylammonium phosphate (TEAP) (pH 6) and sodium hydroxide (pH 12).
Solvents
The lower the solvent's polarity, the higher its elution strength. The polarity of the solvent used is increased, by adding ddH20, to achieve solvent concentration for binding and elution. Solvents vary according to elution strength, viscosity, volitility, and the tendency to cause loss of biological activity.
Acetonitrile is a widely used solvent because it is volatile and therefore easily removed from the recovered molecule. It also has low visocity, and is esentially UV transparent at low wavelenghts. Disadvantages are that it is a strong denaturant, and is toxic.
Isopropanol has strong eluting properties, but is viscous. A mixture of 50:50 isopropanol to acetonitrile will reduce the viscosity of isoproponal, while retaining its hydrophobic character. Isoproponal is also the best solvent for retaining biological activity.
Methanol and ethanol are nearly as effective, as isoproponal, in retaining biological activity. Methanol has advantages over ethanol in that it has low viscosity, and is essentially UV transparent at low wavelenghts. Methanol or ethanol may be prepared in a 50:50 mixture with acetonitrile.
Protocol Guidelines
In summary, preparation for cloning quality templates of ssDNA and oligonucletides simply requires the following steps. First, to enhance the binding capability of the C-18 resin, prepare the MicroColumn by pipetting up and down with the several different solutions. Next, increase the hydrophobicity of the target molecule, in order to bind it to the C-18 resin, by pipetting the appropriate ion-pairing agent up and down in the MicroColumn. Then, wash with ddH20, and elute with the solvent of choice, simply by pipetting up and down in the same MicroColumn.
Molecules of greater hydrophobicity bind more favorably to C-18 resin. It is important that the BioPak MicroColumn does not become saturated to capacity, with undesired molecules present in the seperation mixture, before the molecule of interest has the opportunity to bind. If this does occur, consider the following options:
Perform a prior purification step to separate the target molecule from more hydrophobic molecules. This can be accomplished with a BioPak ion-exchange MicroColumn filled with DEAE resin, or a BioPak MicroColumn packed with less hydrophobic resin than C-18. Such a resin will remove the more hydrophobic molecule, and leave the target molecule behind in solution.
Conditions for loading, washing, and elution are not critical. For example, some researchers use a 25mM buffer as a binding agent, and some use ethanol for washing and elution. Alternative buffers may also be used, like TEA or TEAC. The buffer's pH range can be varied, as well. But, the key issue is the percentage of organic solvents used for washing and elution. For example, washing with 5-10% acetonitrile will wash away some oligonucleotides. Therefore, it just depends on the particular requirements of the researcher using BioPak C-18 filled MicroColumns.
Green C-18 Resin Filled MicroColumn Protocol
Green C-18 Resin Filled MicroColumn Protocol for Radiolabeled Oligonucleotides
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