Elution profile separation

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Sometimes elution profiles may also depend on the number of cycles. This can cause problems in the reproducibility of SEC separations. It should be mentioned that no regeneration procedure will give a completely new SEC support. 

Preswelled Sephacryl S-1000 was prepared in a K26/100 column (88 X 2.6 cm). Equilibration with 0.005 M NaOH containing 0.002% NaN3 at a flow rate of 0.67 ml/min was achieved after 20 hr. Sample solutions were applied with a 5-ml injection loop. The mass and iodine-complexing potential of separated glucan components was determined off-line for each of the subsequently eluted 5-ml fractions. Based on the determined mass of carbohydrate for each of the fractions, elution profiles such as Fig. 16.1 were constructed. 

Reproducibility of separation for a Dextran T-500 sample was tested on a semipreparative Sephacryl system S-500/S-1000 (65 + 95x1.6 cm) over a period of 6 months. The elution profiles of Dextran T-500 could be superimposed with deviations in the elution axis of 3 ml ( 1 fraction), and deviations in carbohydrate content within 5% referring to the maximum value at V,e, = 213 ml (Fig. 16.8). 

Figure 16.15 shows the resulting chromatograms for the three glucan fractions obtained by previous preparative separation on Sephacryl S-200/S-1000 (Fig. 16.14). From the normalized fraction chromatograms, the elution profile of the initial mixture has been reconstructed by mixing 50% fraction 1, 40% fraction 2, and 10% fraction 3. Compared to the chromatogram of the preparative Sephacryl S-200/S-1000 system, separation with the TSK/ Superose system yields improved resolution in the low dp (high V, ) domain.

These combined HDF and GPC separations require the use of detectors such as static light scattering or viscometers to help sort out the convoluted elution profiles seen in those type of experiments. It should also be remembered in these situations that the typical refractive index or ultraviolet detector responses may not be representative of the actual mass fraction of insolubles eluting from the column because of the significant light scattering that can occur with those large particles in the detector cell. 

Figure 4. Elution profile on HPAEC of pea shoot pectin fractions obtained after Biogel P4 separation.

SEC-ESI-FTMS combines the size separation based technique of SEC with one of the most powerful mass spectrometric techniques of FTMS offering high mass accuracy (ppm), ultrahigh resolving power (>10(i) 6) and the capability to perform tandem mass spectrometry. The technique enables generation of oligomer elution profiles, which can be used for accurate calibration of standard SEC data. Coupling of SEC to ESI-MS is further described in ref. [710],... 

Gradient elution is a procedure whereby the conditions under which the sample is eluted are progressively varied throughout the separation so as to speed up the process. This can be achieved by altering the composition of the mobile phase or increasing the temperature or flow rate. The effect is to elute components more rapidly in the latter stages and sharpen their elution profiles. Stepwise elution is a similar procedure in which elution conditions are changed at predetermined times rather than continuously. 

A typical elution profile the separation of saturated esters by gas-liquid chromatography 1. methyl formate 2. methyl acetate 3. ethyl formate 4. ethyl acetate 5. -propyl formate 6. iso-propyl acetate 7. w-butyl formate 8. sec-butyl acetate 9. iso-butyl acetate 10. n-butyl acetate... 

Fig. 2.145. Semipreparative HPLC separation of the subunits from P. cruentum B-PE. The elution profile monitored at 226 nm is shown. The identity of each peak was detemined from the visible absorption spectra, the known distribution and content of PEB (phycoeryhtrobihn) and PUB (phu-courobihn) and SDA-PAGe. Dotted line symbolizes the gradient in percentage of acetonitrile (for quantitative details see text). The order of subunit elution is y. a, p. The elution profile shows a partial resolution of at least three j- species. Reprinted with permission from R. Bermejo et al. [317].

FIGURE 1.36 General scheme for the process of CPC and mobile phase flow regime for the descending mode of CPC (insert bottom left) used for the CPC separation of dichlorprop with [mono-ll-octadecylthio-h ls-10,ll-dihydroquinidinyl)]-l,4-phthalazine as chiral selector. Elution profiles for dichlorprop after injection of 366 mg racemate, amolar ratio r = 1 of loaded dichlorprop to total selector present in the rotor, and arotor speed of 1100 rpm. Stationary phase, 10 mM selector in methyl tert-butylether mobile phase, 100 mM sodium phosphate buffer (pH 8) flow rate 3 mLmin temperature, 25°C. (Reproduced from E. Gavioh et ah. Anal. Chem., 76 5837 (2004). With permission.)... 

Fig. 4 Elution profiles for (A) propranolol (a), promethazine (b), and chlorprom-azine (c) applied separately on a 5-mm ILC column containing cytoskeleton-depleted red blood cell membrane vesicles entrapped in dextran-grafted agarose gel beads (1.4 /amol phospholipid, 0.5 mL/min) and (B), from left to right, acetylsalicylic acid, salicylic acid, warfarin, and pindolol on a capillary continuous bed containing liposomes immobilized by use of C4 ligands (1.0 /xmol phospholipid, 10 /xl./min). The elution volumes in the absence of lipid are shown (a0, b0, and c0, and the arrow, respectively). (Part A is reprinted with permission, with slight modification, from Ref. 26. Copyright 1999 Elsevier Science. Part B is reprinted with permission from Ref. 23. Copyright 1996 Elsevier Science.)...

Reversed-phase chromatography is often used to separate both neutral and ionic organic compounds. In this section, some important aspects for the understanding of the behavior of ionic compounds in reversed-phase chromatography are discussed. The important concepts introduced here are the electrical double layer and the electrostatic surface potential. It will be shown that they are essential for the understanding of the elution profile of ionic compounds. These concepts are further explored in the next section where theoretical models for ion-pair chromatography are discussed. 

Figure 11 illustrates an SEC separation of a sample of 3-conponent polystyrene mixture with the dual concentration and viscosity detectors of Figure 10. The top trace shows the concentration elution profile of the SEC separation as detected by a uv-photometer. The bottom trace records the same SEC separation, except with the viscometer signal from the log-amplifier output.

Figure 25 Elution Profiles for Oxytocin (O), Carbetocin (C ), and Desmopressin (D) Separated by Capillary...

Examples for and have been observed under certain experimental conditions for reactive and/or strained chiral oxiranes which were separated by complexation gas chromatography (Figure 21)133. The first eluted peak was diminished in the separation of racemic 2-methyl-3-phenylo.xirane. In this case two enantioselective processes are mediated by the chiral metal chelate, i.e., chromatographic resolution and kinetic resolution (in favor of the first eluted enantiomer). Since two enantioselective processes are involved, the elution profile will be the same svhen the chirality of the metal chelate is inverted. 

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