Baseline separation

Proteins are separated on Zorbax GF columns based on their hydrodynamic size, which may be related to the proteins molecular weights (Fig. 3.10). Under ideal conditions, two proteins whose molecular sizes differ by a factor of 2 can be baseline separated. 

The chromatogram of the protein mixture should show the partial separation of serum albumin and ovalbumin with a trough of at least 30% of height between their peak signals and baseline separation between ovalbumin and cytochrome c. If present in the sample, the dimeric form of serum albumin should also appear as an individual peak signal before elution of the monomeric form. 

Another application shows the preparative purification and polishing of a therapeutic fusion protein with a humanized recombinant IgG protein. The fusion protein was expressed by the fermentation of baby hamster kidney cells. The filtered culture supernatant (155 liters) contained 2.2 g of IgG and 75.5 g of total protein. After the immunoglobulins were isolated by expanded bed adsorption and rebuffering, the IgG fraction was bound to Fractogel EMD SOj (M). This column achieved baseline separation of complete antibodies (fusion protein) from small amounts of antibodies lacking the fusion part. The resulting highly purified IgG fraction (110 ml) was diluted to 150 ml and... 

In planar ehromatography, the fraetions are not always transferred to another separation system, but rather a seeondary separation is developed, orthogonally on the same ehromatographie plate. Therefore, for all substanees not eompletely separated it is possible that baseline separation ean be aehieved by means of a seeond separation proeess with an appropriate mobile (stationary) phase. Figure 8.2 shows that in the seeond dimension a theoretieally unlimited number of seeondary eolumns ean be applied. Beeause of this, the terminology two-dimensional PC is not suffieiently... 

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. 

An example of the effect of acid and base concentration on the separation of propranolol is shown in Fig. 2-13. In this case, the baseline separation is achieved by adjusting the concentration without changing the acid/base ratio. 

In a flame-dried Schlenk tube 0.37 g(1.88 mmol) of (-)-3-exo-(dimethylamino)isoborneol (C) and 200 mL of dry toluene are placed under an atmosphere of argon. 27 mL of 4.2 M diethylzinc (113 mmol) in toluene are added and the resulting solution is stirred at 15°C for 15 min. After cooling to — 78°C, lOg (94.2 mmol) of benzaldehyde are added and the mixture is wanned to O C. After stirring for 6 h, the reaction is quenched by the addition of sat. NH4C1 soln. Extractive workup is followed by distillation yield 12.4 g (97%) 98% ee [determined by HPLC analysis. Baseline separation of rac-1 -phenyl-1 -propanol was achieved on a Bakerbond dinitrobenzoyl phenylglycine column (eluent 2-propanol/hexanc 1 3 flow rate l.OmL/ min detection UV 254 nm)] [a] 0 —47 (c = 6.11, CHC13). 

The dominant interactions that take place with this novel phase are not clear. There will obviously be dispersive interactions with the propyl chain and some polar interactions with the amino group. Whether there are ionic interactions taking place as well is uncertain but, nevertheless, the material affects an excellent separation of glucose and fructose and this remains a baseline separation even when the two sugars are both present at about the 5% level. 

Although some normal phase methods have been used, the majority of carotenoid separations reported in the literature were carried out by reversed phase HPLC. Among the Cjg columns employed for determination of complete carotenoid compositions in foods, the polymeric Vydac brand is preferably used for separation of cis isomers. Several examples of different C,g columns and mobile phases are cited in the literature, but not aU carotenoids are baseline separated in most systems. Table 6.2.1 shows some examples employing different brands of Cjg columns." Acetonitrile did not improve selectivity toward separation of carotene isomers in a Vydac 201TP column and resolution was strongly dependent on the Vydac column lot. ... 

In the crystalline state of a substance, the molecules are arranged in a defined unit cell that is repeated in a three-dimensional lattice [1], Since the crystal lattice can act as a diffraction grating for X-rays, the X-ray diffraction pattern of a crystal consists of a number of sharp lines or peaks, often with baseline separation. Figure 1 shows the X-ray powder diffraction pattern of the crystalline and amorphous forms of nedocromil sodium trihydrate. 

Ferretti et al. (1988) used an amino column coupled to a derivatized amylose column (Chiralpak AS) operated in the reverse-phase mode to separate the enantiomers of the antifungal agent voriconazole from several chiral impurities and one achiral impurity. Three of the chiral impurities are the other enantiomer and corresponding diastereomers of voriconazole. More chiral impurities result from a chlorinated voriconazole. Additionally, this multidimensional method could baseline separate all but two of the chiral impurities into their respective enantiomers. These separations are shown in Figure 14.5. 

For a reference experiment the mixture was separated by LCCC using conventional column technology resulting in a baseline separation of all components, see Fig. 17.8. The time requirement for this separation, however, was about 140 min. 

Chromatographic conditions should be optimized wherever possible to achieve baseline separation of the analyte peak from other peaks produced by co-extracted compounds. The retention time of the analyte should be at least twice the column... 

Calculate the number of theoretical plates required to achieve baseline separation (Rs = 1.5) for an (= kjk2) value of 1.10 in... 

The chiral recognition ability of a CSP is quantitatively evaluated from the results of chromatographic separation of enantiomers. Figure 3.4 shows a chromatogram of the resolution of benzoin (19) on cellulose tris(3,5-dimethylphenylcarbamate). The (+)-isomer elutes first followed by the (—)-isomer complete baseline separation is achieved. The results of the separation can be expressed by three parameters—capacity factors (k1), separation factor (a), and resolution factor (Rs)—defined as follows ... 

The analysis time for protein hydrolysates is 85 min using standard columns. For extra high resolution a high-resolution lithium cation exchange column is recommended which achieves baseline separation of virtually all 40 amino acids (Fig. 1.3). 

Zhou and Pietrzyk [41] found that increasing the mobile-phase ionic strength not only increases the retention of AS and AES on a reversed stationary phase, but also improves the resolution since the peak widths are significantly reduced. The authors achieved baseline separation of a multicomponent alkane sulfonate and alkyl sulfate mixture from C2 to Cis using a mobile-phase gradient whereby acetonitrile concentration increases and LiOH concentration decreases. 

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