Separation coupled column

Coupled-column separations or multidimensional chromatography can be considered as a sample preparation form, as one column is used to derive fractions for the second column. It provides a two dimensional separation in which sample substances are distributed over a retention plane formed by the operation of two independent columns. This type of two dimensional based separation method is more powerful than a single dimensional based one. A retention plane has more peak capacity than a retention line and so can accommodate much more complex mixtures. Component identification is more reliable because each substance has two identifying retention measures rather than one. These type of combinations offer high selectivity and high sensitivity, and could be used with less expensive and more robust detectors (e.g., flame ionization). ... 

The specific reasons for coupling column separations with thin-layer chromatography are to increase peak capacity and to take advantage of layer attributes summarized... 

Zorbax PSM Bimodal and Trimodal columns are packed with mixed pore-size packing to achieve linear size separations over a broad molecular weight range (Table 3.3). Zorbax PSM Bimodal columns are packed with PSM 60 and PSM 1000 particles, and Trimodal columns contain PSM 60, PSM 300, and PSM 3000 particles (Fig. 3.4). Carefully selecting and mixing different pore-size particles in columns provide much better linearity than coupling columns that are each packed with single pore-size particles. 

As with other size-exclusion techniques, the pore size of the selected Zorbax GF column should provide resolution over the molecular size range of the proteins that are to be separated. The Zorbax GF-250 column separates proteins in the range of 4000 to 400,000 Da. The Zorbax GF-450 provides separation over the range of 10,000 to 1,000,000 Da. When these two columns are coupled, they can be used to separate proteins with molecular weights of 4000 to 1,000,000. 

Figure 4.1 Schematic diagram of a coupled column system. The first column (ID) is connected to the second column (2D) tlirough the interface or valve system. The interface can be a diiect coupling, a live T-union, a complex multiport valve, or a thermal or cryogenic modulation system. The stimulus can be the switching of the valve, abalancing pressure to divert flow towards 2D, an added flow that is used in pressure tuning, or the drive mechanism for the modulator. The line to detector 1 will normally be a non-retaining section of column. In a two-oven system, ID and 2D will be in different ovens the dotted line indicates separately heated zones.

LC-LC coupling systems are also employed to perform separations requiring very large plate numbers. However, it has been demonstrated (see equation (5.20) that for coupled columns peak capacity increases linearly with the square root of n... 

A. Walhagen and L.-E. Edholm, Chiral separation on acliiral stationary phases with different functionalities using /3-cyclodextiin in the mobile phase and application to bioanalysis and coupled columns , Chromatographia 32 215-223 (1991). 

A. Walhagen and F.-E. Edholm, Coupled-column cliromatography of immobilized protein phases for direct separation and determination of dmg enantiomers in plasma , 7. Chromatogr. 473 371-379 (1989). 

Figure 12.11 Coupled SEC-RPLC separation of compound Chemigum mbber stock (a) SEC ti ace (b) RPLC trace of fraction 1, dibutylphthalate (c) RPLC trace of fraction 2, elemental sulfur. Coupled SEC conditions MicroPak TSK 3000H (50 cm) X 2000H (50 cm) X 1000 H (80 cm) columns (8 mm i.d.) eluent, THE at a flow rate of 1 mL/min UV detection at 215 nm (1.0 a.u.f.s.) injection volume, 200 p-L. RPLC conditions MicroPak MCH (25 cm X 2.2 mm i.d.) column flow rate, 0.5 mL/min injection volume, lOpL gradient, acetonitrile-water (20 80 v/v) to 100% acetonitrile at 3% acetonitrile/min UV detection at 254 nm (0.05 a.u.f.s.). Reprinted from Journal of Chromatography, 149, E. L. Jolmson et al., Coupled column cliromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

Figure 13.5 Schematic presentation of the procedure involved in coupled-column RPLC AS, autosampler C-1 and C-2, first and second separation columns, respectively M-1 and M-2, mobile phases S-1 and S2, interferences A, target analytes HV, high-pressure valve D, detector. Reprinted from Journal of Chromatography, A 703, E. A. Hogendoom and R van Zoonen, Coupled-column reversed-phase liquid cliromatography in environmental analysis , pp. 149-166, copyright 1995, with permission from Elsevier Science.

A rule of thumb has been developed after a large number of analytes were tested. Once the selectivity was observed on the coupled column, a baseline separation can always be achieved on a 25 cm column under optimized conditions. Since the screening procedure already indicates the separation conditions, optimization is straightforward and requires a minimum amount of time. 

Rizzi, A. M. and Plank, C., Coupled column chromatography in chiral separations systems employing P-cyclodextrin phases for chiral separation, /. Chromatogr., 557, 199, 1991. 

Detector selectivity is much more important in LC than in GC since, in general, separations must be performed with a much smaller number of theoretical plates, and for complex mixtures both column separation and detector discrimination may be equally significant in obtaining an acceptable result. Sensitivity is important for trace analysis and for compatibility with the small sizes and miniaturised detector volumes associated with microcolumns in LC. The introduction of small bore packed columns in HPLC with reduced peak volume places an even greater strain on LC detector design. It is generally desirable to have a nondestructive detector this allows coupling several detectors in series (dual... 

Consider the use of a side-rectifier or side-stripper for the separation of a three-product mixture. Assume that thermally coupled columns operate at the same pressure. Also, assume the feed to be saturated liquid. Data for the operation of the two arrangements are given in Tables 21.9 and 21.10. 

Gray, M.J., Dennis, G.R., Slonecker, P.J., Shalhker, R.A. (2005). Utilising retention correlation for the separation of oligostyrenes by coupled-column liquid chromatography. J. Chromatogr. A 1073, 3. 

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