Resolution series connected columns

The resolution is theoretically proportional to the square root of the bed length. This is also sometimes encountered in practice (Hagel et ai, 1989). Therefore, adding column in series may yield the resolution that is needed. However, some resolution is generally lost in the tubings, fittings, and distribution systems when connecting columns and therefore it may not be reasonable to expect to achieve more than 80% of the theoretically calculated improvement in resolution. 

If adequate resolution is still not obtained it means that the molecular size differences are too great to be dealt with on a single column, and two or more columns of varying pore sizes should be connected in series, the columns being placed in order of increasing pore size so that the sample first enters the column of smallest pore size. 

Phenylthiocarbamoyl derivatives of 18 chiral amino acids were separated on a C8 column connected in series to a phenylcarbamoylated (3-cyclodextrin column (Iida et al., 1997). The Cg column separated the derivatized amino acids from one another entering the chiral column. Under this configuration several enantiomers of adjacent amino acids coeluted resulting in poor resolution. However, this configuration was successful in determining the amino acid sequence and chirality of the amino acids in a D-amino acid containing peptide. 

Fig. 27 High-resolution separation of TGs in olive oil. Column and mobile phase were as described in the text, except three columns were connected in series. Mobile-phase flow rate and inlet pressure, 1.8 ml/min, 28 MPa (4100 psi). Refractive index detector.

MA systems can easily add a module to increase membrane capacity or resolution. Alternatively, the series addition of a traditional chromatography column for improvement of capacity or resolution is seldom practiced. Thus, membrane chromatography offers the chromatographer a new flexibility of easy expansion of inadequate systems. Alternatively, the easy connectibility also enables a simple way to couple multiple units of different chemistries for a mixed-mode separation.43 The next section describes these configurations in greater details. 

Once retention optimization has been achieved as in Fig. 26, further increase in / s can usually be achieved by increase in column length (see Ref. 44). This is shown for the separation of Fig. 26 in Fig. 27, where column length is doubled by connecting two 25-cm columns in series. The time required for separation is now doubled, but the separation of band pair 7-11 is improved, as is the resolution of an impurity on the side of band 9. 

To further improve resolution increase column length by connecting two columns in series... 

One system we used for this work was comprised of four 30-cm 100-A /x-Styragel columns (from Waters Associates) connected in series with a Rheodyne Model 7105 injector valve and a Varian 4200 syringe pump that maintained a constant flow of either 1 or 2 mL/min. At 1 mL/min the overall resolution efficiency was about 15,000 theoretical plates and the run time was about 50 min. By doubling the flow rate, the run time was cut in half with only a slight loss of column efficiency. The pressure required for a flow rate of 2 mL/min was usually about 1200 psi. Tetrahydrofuran (UV grade from Burdick and Jackson Laboratories) was used as the eluting solvent. The amount of sample solution injected into the system varied from 10 /xL to 500 /xL. 

Figure 3 Chromatograms of optical resolution of poly(D2PyMA) (DP = 30, [a] 365+389°) on a (+)-poiy(TrMA) coiumn. UV (1.0-mm cell) and polari-metric (5 x 0.2 (i.d.) cm cell) detectors were connected in series (column, 50 X 0.72 (i.d.) cm eiuent, CHCI3 fiow rate, 1.0 mi min" temperature, 15°C amount of sampie, 4 mg). Reproduced with permission from Okamoto, Y. Mohri, H. Nakano, T. Hatada, K. J. Am. Chem. Soc. 1989, 111, 5952. ° Copyright 1989 American Chemical Society.

Figure 1 shows the test configuration having the form of two back-to-back cantilever beams with a central column. Load was applied at a distance of 1 m from the connection and measured by a 9 kN load cell connected in series with a manual hydraulic jack. Rotations of the column and connections were recorded using three electronic clinometers with a resolution of 0.02 mrad (linear to 1% over a i0° range). One clinometer was located along the centre-line of each beam and close to the end with the connection, while the third clinometer was located on the column above the joint. The two connection rotations were determined from the clinometer readings. 

More recently, the effect of Coriolis force was demonstrated in the separation of organic acids with organic/aqueous two-phase solvent systems in a centrifugal partition chromatograph equipped with a separation column consisting of rectangular partition compartments connected in series.As shown in Fig. 4, clockwise column rotation (CW) shows substantially better peak resolution than... 

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