Nutraceutical Bioactives
Raj K. Keservani, Anil K. Sharma, Rajesh K. Kesharwani in Nutraceuticals and Dietary Supplements, 2020
Fractionation basically aims at further separating the compounds that are in the extracts in order to obtain pure compounds. Fractionation can be solvent–solvent, where the extract is put in a separating funnel, missed up, and shaken together with two immiscible solvents. After a short stay, the solvents will separate, each carrying with it some compounds that are as polar as the solvents. The other method is to use a fractionating column, where a polar stationary phase, for example, silica gel (chromatographic grade), is either first added to a column, followed by a nonpolar mobile phase, and then a sample, followed again by a lot of the mobile phase, or the stationary phase, first mixed up with a little mobile phase to form a slurry (Better way than the former) and then proceed as described above. The collected solutions at different time intervals contain various compounds. All principles of column packing need to be followed (not detailed in this chapter). Fractionation can also help in the isolation of pure compounds, but this can be confirmed using thin-layer chromatography or high-performance liquid chromatography.
Production of Essential Oils
K. Hüsnü Can Başer, Gerhard Buchbauer in Handbook of Essential Oils, 2020
Fractionation of essential oils on a commercial scale is carried out in order to isolate fractions containing a particular compound in very major proportions and occasionally even individual essential oil constituents in a pure state. In order to achieve the required separation, fractionations are conducted under reduced pressure (e.g., under vacuum) to prevent thermal decomposition of the oil constituents, using efficient fractionating columns. A number of different types of fractionating columns are known, but the one most commonly used in laboratory stills or small commercial stills is a glass or stainless steel column filled with Raschig rings. Raschig rings are short, narrow diameter, rings made of glass or any other chemically inert material. Examples of compounds produced on a commercial scale are citral (a mixture of geranial and neral) from L. cubeba, 1,8-cineole from eucalyptus oil (mainly E. polybractea and other cineole-rich species) as well as from Cinnamomum camphora oil, eugenol from clove leaf oil, α-pinene from turpentine, citronellal from citronella oil, linalool from Ho-oil, geraniol from palmarosa oil, and so on. A small-scale high-vacuum plant used for citral production is shown in Figure 5.25. The reflux ratio, for example, the amount of distillate separated and the amount of distillate returned to the still, determines the equilibrium conditions of the vapors near the top of the fractionation column, which are essential for good separation of the oil constituents.
The Noncollagenous Proteins of the Intervertebral Disc *
Peter Ghosh in The Biology of the Intervertebral Disc, 2019
Dissociative extraction using chaotropic salts or protein dénaturants, such as high molarity solutions of NaCl, LiCl, MgCl2, LaCl3, CaCl2, urea, GuHCl, or sodium thiocyanate has been commonly employed.17–20 Unfortunately, however, after removal of these denaturing salts, there is no guarantee that the native interactive properties of the extracted components will be restored. The relatively insoluble and self-aggregative nature of many of the structural glycoproteins may necessitate that a certain level of salt be present to maintain their solubility. This may impose limitations on the subsequent fractionation steps to be used.
Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells
Published in OncoImmunology, 2018
Francesca Tosetti, Roberta Venè, Caterina Camodeca, Elisa Nuti, Armando Rossello, Cristina D'Arrigo, Denise Galante, Nicoletta Ferrari, Alessandro Poggi, Maria Raffaella Zocchi
Subcellular fractionation. Lysosomal, endosomal and membrane fractions were isolated by differential centrifugation following published procedures42,43 with slight modifications (see Supplemental Data). Briefly, cell pellets obtained from 107 MSC16142 or MSC773 were washed in PBS, suspended in 1 ml homogenization buffer (250 mM sucrose, 0.5 mM EGTA), 20 mM Hepes-KOH (pH7) and passed sequentially through 21G1/2 and 25G needles. The homogenate was centrifuged 10 min at 1000 xg (nuclear fraction). Post-nuclear supernatants (cells) were centrifuged in an Eppendorf 5417 R refrigerated minicentrifuge at 8000 xg for 20 min (L: lysosome-enriched fraction); supernatants were further ultracentrifuged in TL-100 ultracentrifuge 10 min at 50000 xg (E: endosome-enriched fraction), 90 min at 100000 xg (M: membrane-enriched fraction) and the last recovered as supernatant (SN). All fractions were suspended in RIPA buffer with protease inhibitors (Sigma-Aldrich), stored at −20°C, or processed for western blot analysis. Protein content was assessed by the Lowry DC protein Assay (BioRad, Wadfirt, UK).
Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry
Published in mAbs, 2019
Rafael D. Melani, Kristina Srzentić, Vincent R. Gerbasi, John P. McGee, Romain Huguet, Luca Fornelli, Neil L. Kelleher
Even the simplest mixture of two unique polypeptide chains obtainable via disulfide bond reduction (without proteolysis), Lc and Hc, cannot be analyzed effectively by MS without some type of front-end fractionation that can isolate or partially separate the chains. Both the overlap of their charge state envelopes and ionization suppression effects can lower their spectral signal-to-noise ratio (S/N), particularly for the larger Hc, and could result in co-isolation during fragmentation experiments (tandem MS or MS/MS). Fractionation methods are typically based on liquid chromatography performed using reverse phase (RP),17 size exclusion,20,21 or ion exchange22,23 columns. Each LC-MS/MS run takes several minutes, only one fragmentation method is used per run generally, and multiple injections are needed to maximize sequence coverage.14 Furthermore, liquid chromatography instruments add expense, with elevated operational costs depending on the columns and extent of method development required. Front-end separation based on liquid chromatography also raises issues of sample carryover, contamination, and potential sample losses via irreversible adsorption.
In-line product quality monitoring during biopharmaceutical manufacturing using computational Raman spectroscopy
Published in mAbs, 2023
Jiarui Wang, Jingyi Chen, Joey Studts, Gang Wang
Protein A affinity capture fractionation experiments: Two affinity chromatography unit operations were conducted to generate calibration and validation datasets. These runs were used for both in-line and off-line analytics. A third run was carried out as the buffer blank and followed the same procedures as the previous two, except for the exemption of a load phase. Protein A affinity chromatography resin Mab Select PrismA was packed for operation on an Akta Avant 150 system (Cytiva, Uppsala, Sweden) in a 2.6 cm diameter column to a height of 19.4 cm (103 mL column volume). Equilibration was performed at 25 mM Tris, pH 7.5, 1.98 mS/cm, at a volumetric flow rate of 20.6 mL/min for five column volumes (CV). For the first calibration run, 1.274 L harvest material at 4.85 g/L (6.2 g) was loaded, while for the second validation run 1.483 L harvest material at 4.17 g/L (6.2 g) was loaded, for a column load density of 60 mg/mL. Following the loading phase, the column was washed with 3 CV of equilibration buffer, followed by 3 CV of 50 mM Tris, pH 7.5 wash buffer, followed by 3 CV of equilibration buffer. Following the washing phase, 3 CV of pH 3.5, 1.25 mS/cm buffer was used for elution. Fractionation was performed every 12 mL until the end of elution during the 26th fraction and a constant flow rate was maintained throughout fractionation as is typical of industrial processes. Following the elution phase, the column was regenerated and stored following conventional procedures.
Related Knowledge Centers
- Chromatography
- Column Chromatography
- Elution
- Enzyme
- Fractional Distillation
- Separation Process
- Mixture
- Suspension
- Chemical Purity
- Fractional Crystallization