Baran pipette column is a microscale variation of flash chromatography which is used for rapid purification of small reaction mixtures. It is mainly used when the amount of sample is low, generally 10 to 500 mg. This method was designed by Baran laboratory for making purification faster and more efficient.
It is based on the use of disposable column setup. The whole column is discarded after one use. So, washing of glass column is not needed and the chance of cross contamination is also reduced. This makes the method useful for high-throughput small scale reactions.
The column is prepared either from a Pasteur pipette or from a borosilicate test tube. Pasteur pipette is used for small sample amount about 10 to 50 mg. Test tube column is used for larger microscale amount about 50 to 500 mg.
For making test tube column, the bottom part of the glass test tube is heated with blowtorch and pulled into a narrow stem. The stem is then snapped to make a small spout. This gives a simple column shape for solvent flow.
The column is packed from bottom to top with a small plug, sand layer, silica gel bed, and sand cap. Kimwipe or similar material is used as plug. Flash chromatography grade silica gel is used as stationary phase.
The sample is loaded either by wet loading or dry loading method. After loading, the solvent is passed through the column by positive pressure. This pressure may be given manually by sealing the top with septum and pushing with syringe. It may also be given by using air or nitrogen line with proper adapter.
Baran pipette column is low-cost, fast and customizable method. It reduces dead volume and prevents loss of small amount of precious compound. It is useful alternative to large glass chromatography column for microscale purification.
Principle of Baran Pipette Column
Baran pipette column is based on the principle of flash chromatography in microscale form. In this method, the liquid solvent is passed through silica gel stationary phase packed inside a small disposable glass column.
It is based on applying positive pressure to move the solvent through the column. The pressure may be applied by pipette bulb, syringe, or regulated air line. This pressure helps the solvent to pass faster through the silica gel bed.
During this process, the compounds in the reaction mixture move at different rates through silica gel. The separation occurs due to different interaction of the compounds with stationary phase and mobile phase. The compound which interacts less with silica gel moves faster, while strongly interacting compound moves slowly.
The fast solvent flow reduces band broadening and helps in better separation. It also decreases the time for purification of small amount of sample. The column size is kept small according to the amount of sample, generally 10 to 500 mg.
This method also works by reducing dead volume in the column. Less dead volume prevents loss of precious microscale material. The column is used one time and then discarded, so washing is not required and contamination is avoided.
Components/Parts of Baran Pipette Column
The Baran pipette column is made up of glass column body, bottom plug, sand layer, silica gel bed, sand cap and pressure applying device. These parts together make a small disposable column for microscale purification.
Column hardware
The column hardware is the outer glass body of the column. It may be a disposable Pasteur pipette or a modified borosilicate glass test tube. Pasteur pipette is used for 10-50 mg sample and test tube column is used for about 50-500 mg sample.
In test tube column, the lower end of the glass tube is heated and pulled into narrow spout. This spout helps in controlled flow of solvent from the column.
Bottom plug
The bottom plug is placed at the lower narrow part of the column. It is generally made of small piece of Kimwipe, cotton or glass wool. It prevents silica gel and sand from coming out of the column.
The plug should not be very tight. It should hold the packing material but also allow the solvent to flow through it.
Base layer
The base layer is a thin layer of sand placed just above the plug. It is generally about 1-2 mm thick. This layer gives a level base for the silica gel bed.
It also prevents fine silica particles from clogging the plug. So, solvent flow remains smooth during elution.
Stationary phase
The stationary phase is the main adsorbent bed of the column. It is generally made up of flash chromatography grade silica gel. The silica gel used is commonly 230-400 mesh.
This layer helps in separation of compounds. The compounds interact differently with silica gel and solvent, so they move at different speed through the column.
Protective sand cap
The protective cap is the upper sand layer placed above the silica gel bed. It is generally about 5 mm thick. This layer protects the flat silica surface.
It prevents disturbance of silica gel when sample or solvent is added from the top of the column.
Pressure delivery tool
The pressure delivery tool is used to apply positive pressure from the top of the column. It may be pipette bulb, syringe with septum, or air or nitrogen line attached with glass adapter.
This pressure pushes the solvent through the silica gel bed. It helps in fast elution and gives rapid purification of small reaction mixture.
Construction of the Baran Pipette Column
- Selection of glassware
- Disposable Pasteur pipette is selected for 10-50 mg sample.
- Borosilicate test tube is selected for 50-500 mg sample.
- The test tube may be 16 x 150 mm or 25 x 150 mm.
- Fabrication of test tube column
- The bottom of borosilicate test tube is heated with butane or propane torch.
- Heating is continued until the glass becomes orange and soft.
- The softened bottom is pulled with tweezers to form a long narrow stem.
- The tube is cooled at room temperature.
- The extra stem is snapped off to make a narrow spout.
- Insertion of bottom plug
- A small plug is inserted into the narrow spout.
- Kimwipe, cotton or glass wool may be used as plug.
- The plug is pushed down with metal rod or wooden applicator stick.
- It holds the packing material inside the column.
- Addition of base sand layer
- A thin sand layer is added above the plug.
- It is generally 1-2 mm thick.
- It gives a level base for silica gel.
- It prevents fine silica from clogging the lower plug.
- Addition of silica gel bed
- Dry flash chromatography grade silica gel is added into the column.
- The silica gel is generally 230-400 mesh.
- It is filled until the bed height becomes just under 3 inches.
- This layer acts as the stationary phase.
- Tight packing of silica gel
- The sides of the column are tapped or slapped gently.
- This settles the silica gel properly.
- Air pockets are removed from the silica bed.
- The bed becomes compact and even.
- Equilibration of column
- The selected solvent system is passed through the column.
- One or two column volume of solvent is used.
- The solvent wets the whole silica gel bed.
- The column becomes ready for sample loading.
- Addition of protective sand cap
- A final sand layer is added on the top of wetted silica gel.
- It is generally about 5 mm thick.
- It protects the upper surface of silica bed.
- It prevents disturbance when sample or fresh eluent is added.
Types of Baran Pipette Column
- Pasteur pipette column
- Pasteur pipette column is made by using standard disposable Pasteur pipette.
- It may be 5.75 inch or 9.0 inch pipette.
- It is used for smallest sample amount.
- The sample range is generally 10-50 mg.
- Positive pressure is usually applied by pipette bulb.
- 16 x 150 mm custom modified tube
- This column is made from 16 x 150 mm borosilicate test tube.
- The bottom of the test tube is heated by blowtorch.
- The softened glass is pulled to make a narrow spout.
- It is used for intermediate microscale sample.
- The sample range is generally 50-200 mg.
- It can be attached with 14/20 inlet adapter for positive air pressure.
- 25 x 150 mm custom modified tube
- This column is made from 25 x 150 mm borosilicate test tube.
- A glass spout is formed at the bottom by heating and pulling.
- It is used for larger microscale sample purification.
- The sample range is generally 200-500 mg.
- It can be attached with 24/40 inlet adapter for air or nitrogen pressure.
Applications of Baran Pipette Column
The following are the applications of Baran pipette column-
- Rapid purification of small-scale crude reaction mixtures.
- Purification of sample amount ranging from 10-500 mg.
- Isolation of sensitive and fragile compounds.
- Isolation of highly reactive, oxidized and unstable intermediates.
- Use in total synthesis of complex natural products.
- Purification of Bipinnatin J and Vinigrol intermediates during synthetic work.
- Purification of heterocyclic products.
- Purification of alkaloid intermediates such as Stephacidin and Haouamine alkaloids.
- Use in high-throughput methodology development.
- Rapid monitoring of reaction mixtures.
- Screening and optimization of synthetic pathways.
- Use in drug discovery work where fast purification is required.
Benefits of Baran Pipette Column
The following are the benefits of Baran pipette column-
- Rapid construction and setup.
- Packing and running of column takes less time.
- Useful for high-throughput purification work.
- No washing of glass column is required.
- Disposable glassware is used only for single time.
- It saves time required for washing and drying of column.
- It prevents cross contamination between different samples.
- Fresh column is used for every purification.
- It reduces dead volume present in traditional glass columns.
- Loss of precious microscale sample is prevented.
- It is useful for highly reactive and sensitive compounds.
- Less amount of solvent is required.
- It is more solvent economical than large scale column.
- It is low-cost method.
- Pasteur pipette and standard test tubes are easily available.
- It reduces hidden cost of cleaning solvent and researcher time.
Limitations of Baran Pipette Column
The following are the limitations of Baran pipette column-
- It is useful only for limited sample scale.
- The sample amount is generally 10-500 mg.
- Very small sample needs preparative TLC.
- Larger sample needs standard glass column or automated flash system.
- The separation resolution is lower than traditional chromatography column.
- Short silica bed gives less separation power.
- It is mainly suitable for rapid elution.
- The column may dry out easily.
- If solvent level falls below the silica gel, cracking or channeling can occur.
- Channeling damages the separation.
- Manual pressure applying needs care.
- If pipette bulb seal is not broken before release, vacuum may be formed.
- This vacuum can pull the silica bed upward and destroy the column.
- Positive air pressure or syringe pressure must be controlled carefully.
- High pressure can force solvent too fast through the column.
- Construction of test tube column needs practice.
- Pulling narrow glass spout by blowtorch needs skill.
- Sample loading is sometimes difficult in microscale setup.
- Dry loading is not easily performed like large columns.
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