Flash chromatography
Objectives
At the end of the lecture, student will be able to
Discuss the principle of flash chromatography
Explain the instrumentation of flash chromatography
Flash chromatography
Most of the sophisticated analytical techniques like NMR, MS and FT-IR need sample in the extreme pure form which is usually achieved by Preparative column chromatography but it is time consuming
From past few decades, technique called as Flash chromatography is developed which is a modification of Preparative column chromatography
Flash chromatography is basically an air pressure driven
hybrid of medium pressure and shorter column chromatography which has been optimized for particularly rapid separation
Flash chromatography is a technique used to separate mixtures of molecules into their individual constituents, frequently used in the drug discovery process
Column chromatography is separated into two categories,
depending on how the solvent flows down the column
If the solvent is allowed to flow down the column by
gravity, or percolation, it is called gravity column chromatography.
If the solvent is forced down the column by positive air
pressure, it is called flash chromatography
Flash chromatography is basically an air pressure driven
hybrid of medium pressure and shorter column chromatography which has been optimized for particularly rapid separation
Flash chromatography is a technique used to separate
mixtures of molecules into their individual constituents, frequently used in the drug discovery process
Flash chromatography differs from the conventional technique in two ways: first, slightly smaller silica gel particles (250-400 mesh) are used
Second, due to restricted flow of solvent caused by the small gel particles, pressurized gas (ca. 10-15 psi) is used to drive the solvent through the column of stationary phase.
The net result is a rapid (“over in a flash”) and high resolution chromatography
The absorbent has a much smaller particle size
Different kinds of chromatographic columns- If the column contains a porous plate to support packing, no additional support such as cotton, glasswool and sand is necessary
Components:
1. Sorbent Selection:
The basic prerequisite for successful separations is the
choice of the proper adsorbent
Silica gel and alumina
These adsorbents are sold in different mesh sizes, “silica
gel 60” or “silica gel 230-400” etc
This number refers to the mesh of the sieve used to size the
silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed
Adsorbent particle size affects how the solvent flows through the column
Smaller particles (higher mesh values) are used for flash chromatography; larger particles (lower mesh values) are used for gravity chromatography
For example, 70-230 silica gels are used for gravity columns
and 230-400 mesh for flash columns
Adsorbents used in flash chromatography:
Silica: Slightly acidic medium. Best for ordinary compounds,
good separation is achieved
Florisil: Mild, neutral medium. 200 mesh can be effective
for easy separations. Less than 200 mesh best for purification by filtration
Alumina: Basic or neutral medium, effective for easy separations, and purification of amines
2. Solvent Systems:
Flash column chromatography is usually carried out with a
mixture of two solvents, with a polar and a nonpolar component. Occasionally, just one solvent can be used
One component solvent system:
Hydrocarbons: pentane, petroleum ether, hexane
Ether , dichloromethane (very similar polarity)
Ethyl acetate
Two-component solvent systems
Ether/Petroleum Ether, Ether/Hexane, and Ether/Pentane
Ethyl Acetate/Hexane: The standard, good for ordinary
compounds and best for difficult separations
Methanol/Dichloromethane: For polar compounds
For basic compounds- Add a small amount of triethylamine or pyridine to the solvent mixture (about 0.1%).
For acidic compounds, a small amount of acetic acid is
sometimes useful
In this case, be very careful in concentrating the solvent
as trace amounts of acids can be very dangerous when they are concentrated with a product
Acetic acid can often be safely removed away by adding
portions of toluene
Column Selection
Select a column that is 10, 20, 40 mm ID based upon
preparative requirements
SINGLE Step Flash Columns – purification of organic
compounds
Thomson flash columns – wide variety of sizes ranging from 4g to 300g silica-based for easy scalability of synthetic reactions
C18 flash columns which enable the end-user to utilize these
flash columns for a broad range of reactions
TYPICAL VOLUME OF ELUANT REQUIRED FOR PACKING AND ELUTION
|
Column Diameter |
Volume of |
Sample Load |
|
|
Rf > 0.2 |
Rf >0.1 |
||
|
10 |
100 |
100 |
40 |
|
20 |
200 |
400 |
160 |
|
30 |
400 |
900 |
360 |
|
40 |
600 |
1600 |
600 |
|
50 |
1000 |
2500 |
1000 |
Packing of Column:
Obtain a glass column and make sure that it has either a
glass frit or a plug of cotton wool directly above the stopcock to prevent the silica gel from escaping from the column through the stopcock
Next, put a 1/2 inch layer of clean sand above the plug of
glass wool to obtain a flat surface, with the same diameter as that of the body of the column.
Add dry silica gel adsorbent, 230-400 mesh One
way to fill the column is to invert it into the jar of silica gel and scoop it out & then tamps it down before scooping more out
Another way to fill the column is to pour the gel into the
column using a 10 mL beaker and tamp it down on the bench top to pack the silica gel
Tap gently and evenly the sides of the column with a piece
of rubber tubing to settle the silica gel
Pour a good amount of elution solvent onto the silica gel.
Use pressurized gas to force the solvent through the silica
Continue to flush solvent through the silica gel until the
entire silica plug becomes homogeneous in appearance
Recycle the solvent coming through the column onto the top
of the column several times before all the silica gel is solvated
Loading of sample onto the silica gel column: Two different methods are used to load the column: the wet method and the dry method
Wet Loading Method: In the wet method, the sample to be purified is dissolved in a small amount of solvent, such as hexanes, acetone, or other solvent and loaded onto the column.
Amount of solvent used is critical, too much solvent, the
loading solvent will interfere with the elution and hence the purification orbseparation of the mixture
Dry Loading Method:
First dissolve the sample to be analyzed in the minimum
amount of solvent and add about 100 mg of silica gel
Swirl the mixture until the solvent evaporates and only a
dry powder remains
Place the dry powder on a folded piece of weighing paper and transfer it to the top of the prepared column
Add fresh eluting solvent to the top and now it is ready to
begin the elution process
Elution of the column
Add a good part of elution solvent to the column
Apply pressure to force the solvent through the column by
pressing on the top of the Pasteur pipette with a pipette bulb
Only force the solvent to the very top of the silica: do not
let the silica go dry and add fresh solvent as necessary
Pressure should be the minimum necessary to keep a steady stream coming out of the column
Analysis of fractions: If the fractions are colored, simply combine like-colored fractions, although TLC before combination is usually advisable.
If the fractions are not colored, they are analyzed by TLC
(usually)
Once the composition of each fraction is known, the fractions containing the desired compound(s) are combined.
Cleaning the Column:
Flush all the remaining solvent out of the column using
pressurized gas
Flowing air through the column for 2 hours will give dry,
free flowing silica gel.
In most cases, washing the column with water and acetone is sufficient. If necessary, a small amount of liquid soap can be used
When all liquid solvent has been removed from the reservoir,
remove the last remnants of solvent by applying a vacuum (from aspirator) to the bottom of the column
Try to avoid scratching the columns with abrasive brushes or soaps
When all liquid solvent has been removed from the reservoir,
remove the last remnants of solvent by applying a vacuum (from aspirator) to the bottom of the column
Try to avoid scratching the columns with abrasive brushes or soaps
Advantages:
Fast and economic methods for the synthesis laboratory
Ideal for the separation of compounds up to gram quantities
No expensive equipment required
Automated changes between normal phase and reversed phase chromatography
Applications:
It is used for Purification of Peptides
It is used for Separation of Closely Related Organic
Compounds
Flash systems are powerful tools for purification of trace
compounds from organic mixtures.
It is used as a tool to monitor the reaction progress and to
isolate and identify a mixture’s compounds.
It is used for High Speed Flash Fractionation of Natural
Products – Tocopherols
To purify, identify and collect the isomers of an aqueous
modified antibiotic precursor
Continuous Gradient Purification of Closely Related Drug
Intermediates Using Flash Chromatography
It has received increased attention as a lead investigation
and optimization tool in drug discovery
INSTRUMENTATION OF FLASH CHROMATOGRAPHY
1. Pump Systems
2. Sample Injection Systems
3. Glass Columns, Filling Sets
4. Pre columns, Column Valves
5. Fraction Collector
6. Detectors and Chart Recorders
7. Computerized LCD Display
Pump system – Pump Controller C610, Control Unit C620, Pump Manager C615.
The pump modules can be controlled by three different units. A pressure range up to either 10 bar or 50 bar gives optimum separation results for a broad range of applications
Pump Controller – Delivered with a overpressure sensor for
maximum safety and the flow rate can be easily adjusted by turning a knob and is indicated by a large illuminated LCD-display
The Pump Controller C-610 is designed for isocratic
separations
The unit has Input/Outputs for 2 solvent valves and level
sensors and includes a pressure sensor and mixing chamber
The Pump Manager C-615 is designed for both isocratic and gradient separations
SAMPLE INJECTION SYSTEMS
Injection systems are designed to facilitate column loading
with liquids and low solubility oils and solids
COLUMNS:
Glass Columns: Depending on the nature and the quantity of
the sample offers a series of column types which vary in form, size and performance
A wide range of columns offer maximum flexibility for every
situation
Plastic+Glass-coated Glass Columns are available for larger
amounts of samples and higher pressure applications on a high safety level
PRECOLUMNS:
The large Pre column, fits to Glass Columns of ID 70 and 100 mm inner diameter. The small Pre column, fits to Glass Columns of inner diameter of ID 15, 26, 36 and 49 mm
Pre column are minimizing dead volumes and enhance the life time of the main column by trapping contaminants
FILLING SETS:
Dry Filling Set : Silica gel in the size range of 25 –200micro meters can be packed with this method
The Dry Filling Set is employed for filling glass columns with silica gel using compressed gas.
Slurry Filling Set is used for wet filling and conditioning of glass columns with silica gel particles smaller than25 micro meter
Modern cartridges:
DETECTORS AND RECORDERS/SOFTWARE:
UV Photometer C-635 and UV Monitor C-630
Both detectors are delivered in combination with a preparative flow cell
For most applications one of the robust UV/Vis detectors
would be sufficient for the systems detection needs
RI detector
FRACTION COLLECTOR:
Fraction of known quantities are collected in a fraction
collector
Computerized LCD Display
Summary
Solvent is forced down the column by positive air pressure –
flash chromatography
Flash chromatography – An air pressure driven hybrid of
medium pressure and shorter column chromatography
Adsorbents – Silica, florisil, alumina
Solvent – single component and two component
Column – SINGLE Step Flash Columns and Thomson flash column
Solvent is forced down the column by positive air pressure –
flash chromatography
Packing of column
Loading of column – wet packing and dry packing
Elution of the column
Cleaning of column
Advantages and applications
Pump Systems
Sample Injection Systems
Glass Columns, Filling Sets
Pre columns, Column Valves
Fraction Collector
Detectors and Chart Recorders
Computerized LCD Display
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