Plug and Play
All FIAlab instruments can be ordered fully configured with a laptop PC loaded with software, and tested before shipping.
Sequential Injection Chromatography
In contrast to conventional chromatography, SI Chromatography (SIChrom) uses programmable flow, and sequential injection of sample and eluting solutions. The apparatus comprises a bidirectional syringe pump, multiposition valve, column and detector.
The principal advantages of SIChrom are:
1) versatility of operation as sample and eluant volumes are defined by software script and not by injector or gradient mixer volumes.
2) ability to accelerate auxiliary functions such as gradient elution column conditioning and washout.
3) ability to carry out additional functions such as derivatization prior to separations.
Flow scheme for Sequential
Injection Chromatography
SI Chromatography, as offered by FIAlab Instruments, is available in two variants:
1) low pressure system for SI microAffinity Chromatography.
2) medium pressure system for SI Liquid Chromatography.
microSI-LOV system
for micro SIchromatography
micro Sequential Injection Chromatography
The instrument for low pressure SI chromatography is identical to micro SIA–LOV or FIAlab 3200 LOV system. The only required modification is repositioning of one of the optical fibers deeper into the flow cell , in such a way, that it retains the stationary phase (Sephadex or Sepharose beads) forming the microcolumn. The unique property of this configuration, enabled by flow programming, is that the column can be packed, used and even discarded automatically by microfluidic manipulations. The volume of the column is 10 microliters and the dead
volume between the flow cell and the end of the column
is less than 0.1 microliter. The light path of the flow cell
can be selected between 8 to 1 mm. ( For details see
Renewable microcolumn and
flow cell within LOV module
Example: micro Affinity Chromatography
Micro Affinity Chromatography is a miniaturized version of conventional affinity chromatography, using traditional chromatographic supports of Sepharose and Sephadex, commercially available, derivatized with a wide variety of bioligands. These ligands can be specific so that the antibody binds to only one epitope, or maybe a group type ligands.
Separation and preconcentration of IgG are carried out on Protein A or Protein G Sepharose 6B microcolumns. Separation and assay are completed typically within 120 to 240 seconds, with detection limit of 6ng mouse IgG, monitored at 270nm. Target analytes are eluted from a microcolumn by pulses of eluant and monitored using UV-VIS spectrometry for quantification of native IgG or labeled biomolecules.. Required sample volume is typically in the range of 5 to 200 microliters.
Separation of mouse IgG from bovine serum albumin on a renewable microcolumn Protein A Sepharose 6B
Typical steps of an assay protocol:
1) Sepahrose beads are aspirated from a container and packed into the microcolumn.
2) Selected sample volume is aspirated into the holding coil and by flow reversal passed through the column. At this stage matrix components and impurities are washed out from the column.
3) Selected volume of eluant is aspirated into holding coil and perfused through the column by flow reversal. TARGET molecules are eluted and detected.
4) The column may be discarded by fast forward flow.
For further details consult page Bead Injection.
micro SI configured for
micro Affinity Chromatography
Recommended Instrument Configuration
micro SIA instrument or FIAlab 3200 equipped with Lab-on-Valve module two quartz optical fibers, OO spectrophotometer. Light source for visible spectrophotometry tungsten lamp, for UV either LED or Deuterium lamp.
OPTIONS
120 position conventional sample changer
8 position Microsip sample changer
stand for instrument and solution containers, external secondary pumps
are available in six or eight position versions, fabricated either from Plexiglass or ULTEM. In our experience the 8 position module is suitable for most applications such as microAffinity Chromatography.
Note: Plexiglas should not be used with organic solvents and is resistant only to max 10% ethanol solution. ULTEM is resistant to many organic solvents, but is less transparent and has significant fluorescence background.
Low pressure micro Sequential Chromatography can be performed with an external column using a dual pumping system for gradient elution.
For details on micro SI Chromatography with external colum consult Chapter SI chromatography in the complimentary CD Tutorial
Partial Compatibility list for ULTEM:
Acetic Acid, Acetone, Alconox, Carbon Tetrachloride, Cellosolve, Chromic Acid, Citric Acid, Clorox, Cyclohexane, Cyclohexylamine, Ethanol, Ethyl Acetate, Ethyl Ether, Formic Acid, Gasoline, Hexane, Hydrochloric Acid, Isopropanol, Kerosene, Lestoll 1, Methanol, Methylethyl Ketone (MEK), Naphtha, Nitric Acid, Phenol, Phosphoric Acid, Potassium Carbonate, Potassium Hydroxide, Propylene Glycol, Skydrol, Sodium Hydroxide, Sulfuric Acid, Tetrachloroethylene, Tin Chloride, Toluene, Trichloroethane (1,1,2-), Triethylphosphate, Xylene, Zinc Chloride
