Bioactive Natural Products

Zhao Qin , ... Xue-De Wang , in Studies in Natural Products Chemistry, 2021

Column chromatography

Column chromatography is the main method to obtain pure PAs. Stationary phases in column chromatography include dextran gel, macroporous resin, and polyamide [94,95]. Sephadex LH-20, a hydroxypropylated dextran gel, is universally used for the purification of PAs [96]. Unlike fractions are obtained via gradient elution using different mobile phases, east.chiliad., ethanol/water, methanol/water, or acetone/water. The purification of PAs using a macroporous adsorption resin is a suitable method for industrial production [97,98]. This method has some advantages, including depression operational expenses, easy regeneration of the adsorbent, and rubber usage.

Gel-permeation chromatography (GPC) was developed to judge the boilerplate molecular masses of PAs. However, adsorption tin occur betwixt the hydroxyl group of PAs and dextran via hydrogen bonds, and thus PAs are inappreciably separated on a typical GPC column. This problem needs to exist solved earlier conducting GPC. To this end, PA derivatives (methyl ether or acetylated derivatives) are unremarkably generated [99]. For example, the PAs are added to pyridine/acetic anhydride solution (1:i, v/v), and stirred at room temperature for ii   h. Then, water is added to the reaction solution, and the precipitates (acetylated PAs) are obtained past centrifugation. The PA derivatives tin can then be separated by GPC using tetrahydrofuran as a mobile phase. The GPC system is calibrated with polystyrene standards or procyanidin oligomer acetates, and the average values of the molecular mass of PAs are estimated from the constructed calibration curve.

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Bioassay-Guided Isolation and Evaluation of Herbal Drugs

Pulok Grand. Mukherjee , in Quality Control and Evaluation of Herbal Drugs, 2019

xiii.eight.1.one Column Chromatography

Column chromatography (CC) is one of the near useful methods for the separation and purification of both solids and liquids. This is a solid–liquid technique in which the stationary stage is a solid and the mobile phase is a liquid. The principle behind column chromatography is adsorption, in which a mixture of components dissolved in the mobile phase is introduced in to the column and the components motion depending on their relative affinities. The choice of the solvent depends on the solubility characteristics of the mixture. The solvents should too have sufficiently low boiling points to let ready recovery of eluted cloth. In CC, different mobile phases (in increasing order of polarity) can exist used, for example, petroleum ether, hexane, chloroform, and ethyl acetate. Even so, the polarity of both the stationary and mobile phases is the most important gene in adsorption chromatography. It is found to be very useful in the separation of mixtures of compounds, purification processes, the isolation of agile constituents, and the separation of diastereomers ( Gaudencio and Pereira, 2015).

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ION EXCHANGE | Theory Of Ion Exchange

R. Harjula , in Encyclopedia of Separation Science, 2000

Column chromatography

In column chromatography ions are separated from each other for assay or for chemical production purposes. Considering a simple example in which ions A and C are separated for analysis, a sample solution containing A and C is passed into the column containing an exchanger in the B form. The sample volume is so low that A and C have up only a very small fraction of the column chapters about the inlet. After sample injection, an eluent solution containing ion B is passed through the column. A and C in the exchanger are exchanged for B and brainstorm to move through the cavalcade at different velocities. At a given volume, the less preferred ion A first emerges in the eluent as a concentration peak followed by ion C. The eluent volumes at which A and C emerge, i.e. the volumes at the peak maxima, are called the retentiveness volumes (V R ) and they tin can be obtained from the relation:

[xx] V R = k d V S + V M

where V Southward is the book of ion exchanger bed and 5 M is the free solution volume in the bed. In analytical separations A and C are present at trace levels, so k d values are again easily calculated from eqn [nine]. In analytical work, efficient operation requires that the concentration peaks of A and B are well separated (the peaks are sharp). The retentiveness volumes V R should not be too large, because this leads to a long analysis time and to broadening of the peaks.

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Belittling Techniques for High Mass Materials: Method Development

R. Kandiyoti , ... G.D. Bartle , in Solid Fuels and Heavy Hydrocarbon Liquids, 2006

8.three.2 Column chromatography

A column chromatography method was developed, eluting fractions of the sample successively with acetonitrile and pyridine. NMP was used to sweep the remaining textile immobile in pyridine [ Islas, 2001; Islas et al., 2003b; Suelves et al., 2001a]. This method allows a quantitative interpretation of fractions and provides sufficient material for further analyses by a variety of methods. Some material could not be recovered and the silica remained colored; sample losses were most 10% overall as volatiles and insolubles.

The method was developed to produce upwardly to 1 gram of sample fractions; it would accept been difficult to produce comparable amounts of sample by planar chromatography. It was based initially on the use of silica gel with sequential elution using acetonitrile, pyridine and NMP. In subsequent work, pentane and toluene were added to the sequence. A total volume of 100 ml of each solvent was used, 50 ml with gravity elution and the 50 ml under vacuum so NMP (100 ml) with vacuum elution. H2o (100 ml) was added to launder out the NMP. Some loss of material in each fractionation was through a combination of high mass textile retained on the silica and low mass textile lost with evaporation of solvent during distillation and in the vacuum oven. Still, unlike during planar chromatography, an approximate mass residue could be achieved.

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Chromatographic analysis of phospholipids and glycosyldiacylglycerols

William W. Christie , Xianlin Han , in Lipid Analysis (Fourth Edition), 2012

1 Preparative-scale ion-exchange chromatography

While column chromatography on silica gel has been much used for preparative scale separations, information technology is of relatively little value every bit an belittling technique [ 728]. Column chromatography on DEAE-cellulose is preferable for isolation of circuitous lipids in comparatively large amounts [604]. The principle of the separation procedure is complex, involving partly ionic interactions between the packing fabric and the charged regions of complex lipids, and partly adsorption effects with the polar parts of the molecules. As a crude guide, about 300 mg of complex lipids can be applied to a 30 × two.5 cm column to yield fractions with distinctive compositions and little cross-contamination. Although it has been used mainly on this scale, information technology is possible to use the technique with much smaller columns, less solvent and a proportionately smaller amount of lipid.

It is offset necessary to catechumen the packing material to the acetate form past washing sequentially with 1M aqueous hydrochloric acrid, distilled water, 0.1M aqueous potassium hydroxide and water once more (each iii column volumes); the ion-commutation medium is left overnight in glacial acetic acrid, and then packed into a cavalcade in a slurry of this solvent. Finally, the acerb acid is washed out by elution with methanol, chloroform methanol (one:1 by volume), and chloroform alone. An elution scheme suitable for use in the separation of lipid extracts from animal tissues is shown in Table five.1 Chloroform elutes the simple lipids. All the choline-containing phospholipids are eluted with a chloroform-methanol mixture of relatively depression polarity, while a much higher proportion of methanol is required to recover the ethanolamine-containing phospholipids; phosphatidylserine is eluted with glacial acetic acrid, and a solvent of high ionic strength is required to recover the more acidic phospholipids (the salts tin can be removed later by a 'Folch' washing step; come across the adjacent Section and Chapter three).

With institute lipid extracts, monogalactosyldiacylglycerols tend to elute with the chloroform fraction, but digalactosyldiacylglycerols tin can be recovered on their ain if care is taken [550]. Further separation of the individual components of particular fractions tin be achieved later by ways of HPLC or TLC. At the end of the analysis, information technology is an piece of cake matter to regenerate the column for re-use, though it is important not to motion also abruptly from solvents of high to low polarity.

A number of modern polymer-based materials of the ion exchange type are now available that might exist usable in preparative applications, also as the bonded-NHtwo phases described in the adjacent Section. I such method appears promising [98].

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Sparse-LAYER CHROMATOGRAPHY | Overview☆

Eike Reich , Valeria Maire-Widmer , in Encyclopedia of Analytical Scientific discipline (Third Edition), 2019

Describing the Issue

In column chromatography, the chromatographic system has a fixed length. Each sample component can be characterized by the time it requires to pass through the column and reach the detector. This is the retention time t R and it is measured at the peak maximum. In TLC, the assay takes a fixed time (evolution fourth dimension) during which sample components tin can migrate. Depending on its retention each sample component will reach a specific migration distance (Physician) and remains there as a zone (spot) for detection when the TLC plate is dried (Fig. three). A compound with loftier Doc would in comparison have a short retention time in cavalcade chromatography, provided the separation mechanism is the same. The Physician of a zone is measured in millimeters from the awarding position to the bespeak of highest concentration (intensity). When the chromatogram is evaluated densitometrically this point represents the height maximum. As retention time is dependent on the mobile stage velocity, the migration distance is dependent on the position of the mobile phase forepart z f. Therefore, a relative measure has been introduced. The R F value (retardation factor) of a zone is the ratio of its migration distance to that of the mobile stage front. R F  =   Physician/z f.

Fig. 3.

Fig. iii. Schematic presentation of result in TLC. MD, migration distance; z f, distance between application and front; R F  =   MD/z f.

R F values are e'er <   one. For convenience it is common to multiply the R F value by 100 and report hr F with 2 digits. It is possible to compare the migration altitude of an unknown (A) to that of a reference compound (ref) to yield the R rel value. R rel  =   MDA/MDref.

Although the R F value is characteristic for a substance in a given TLC system, information technology must be treated with caution considering it is afflicted by several parameters. In practise, and without rigorous standardization, it is often difficult to reproduce R F values exactly. The R rel value has no physical pregnant.

The principle of qualitative assay is a comparison of R F values obtained from samples and standards on the same TLC plate. If ii substances are the aforementioned they volition have the same R F. Notwithstanding, different substances may likewise migrate to the same position. For further confirmation of identity a specific derivatization or recording of the ultraviolet (UV) spectrum of the substances on the plate can exist utilized. The size and intensity of the zone of the analyte is visually compared for interpretation of quantity to that of standards at several known levels on the same plate. A precise quantitation is possible past scanning densitometry or image analysis by which the assimilation or fluorescence of separated zones of each chromatogram rails is recorded. The resulting analog curves are integrated and evaluated based on elevation peak or expanse. The fundamental requirements for reliable quantitation are baseline resolved zones and symmetric peaks for the compounds in question. The resolution betwixt two zones or peaks can be calculated from the chromatogram (Fig. 4).

Fig. 4.

Fig. 4. The resolution (R s) between two separated peaks is calculated from the chromatogram: R s  =   2   ΔZ/W B1  + Westward B2.

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Fundamentals

D.A. Vicic , 1000.D. Jones , in Comprehensive Organometallic Chemical science III, 2007

1.07.3.v Chromatography

Under advisable weather, column chromatography tin be performed on air-sensitive organometallic compounds. Cannula transfer techniques allow chromatography to be washed on the more stable compounds without the need for custom-made glassware. For these cases, a standard column is packed dry in the fume hood and then a septum and vent needle are fastened. Degassed solvents are subsequently cannulated into the column and the adsorbent is packed tightly under a positive force per unit area of nitrogen. Eluted bands are so collected nether Schlenk-blazon conditions.

If access to a glovebox is available, chromatography on the more air-sensitive compounds tin can exist performed quite readily. Gravity elution is oft necessary, but if the glovebox is equipped with either a supplemental nitrogen or vacuum inlet, then flash chromatography would be possible. Figure 12 shows one possible adaptation of standard glassware to perform wink chromatography inside a glovebox using a vacuum source.

Figure 12. Exploiting a vacuum inlet for flash cavalcade chromatography within a glovebox. The tip of the column is fitted with neoprene filter adapters in order to accept a good seal with a standard filter flask. With plenty of solvent on the column and in reserve, a controlled vacuum is so applied to the filter flask in order to rapidly elute the various fractions.

Cavalcade chromatography of the highly delicate compounds requires additional care. Sometimes, the adsorbent needs to exist prepped before introducing a solution containing an organometallic compound. The writer's grouping has found that, in general, organometallic compounds are quite stable toward activated aluminum oxide (Brockman I, ∼150 mesh) that has been heated to 200   °C on a loftier vacuum line for 2 days before being used. The enhanced stability is perhaps due to the removal of trace amounts of oxygen, water, and/or acid. This rigorous preparation of the adsorbent does not always prevent decomposition, however, and for the extremely sensitive compounds depression temperature column chromatography can be performed using columns containing an boosted jacket for circulated coolants. 8

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Laboratory-Scale Preparative Chromatography

Colin F. Poole , in The Essence of Chromatography, 2003

11.3.two Dry-Cavalcade and Vacuum Chromatography

Dry-column chromatography is a variant of preparative-scale thin-layer chromatography with similar resolution merely a higher sample capacity. A drinking glass column or Nylon tube is packed with a thin-layer sorbent, unremarkably silica gel, to a meridian of 10 to 15 cm. Sample is added as a full-bodied solution or preadsorbed onto a small amount of sorbent. Separation is achieved by developing with a suitable volume of solvent to attain the lower end of the bed. Suction at the lesser of the cavalcade and/or slight overpressure at the tiptop may be required to supplement capillary forces in moving the mobile phase downwardly the column. Separated bands are removed by extrusion, slicing (if a Nylon column is used), or by digging out, and the products freed from the sorbent by solvent extraction. The separation is fast, requires footling solvent, and provides higher resolution than classical column techniques, owing to the use of sorbents with a smaller average particle size. Information technology is suitable for the recovery of modest quantities of material, since the loading capacity is only about 0.2 to 1.0% w/w of the sorbent used depending on the difficulty of separating the bands of interest. Thin-layer chromatography provides a suitable technique for method development in most cases, although significant differences in separations can arise for mixed solvents, especially when the solvent components differ in polarity and/or volatility. These differences result from the absenteeism of a vapor phase in the dry-column technique. Nylon columns can be more difficult to pack than glass columns, peculiarly when longer lengths are used, but Nylon columns are easier to department, and permit colorless bands to be observed with a UV lamp. Drinking glass columns, built upward of segments connected past footing glass joints, simplify the extrusion procedure.

Dry-cavalcade chromatography is not a widely used today. Preparative-scale thin-layer chromatography or flash chromatography is generally preferred. Although separations are fast, the recovery of separated zones is slow and labor intensive compared with elution methods.

Vacuum chromatography can be taken to mean the operation of a short column under suction to accelerate solvent migration. Either a short column, or a Buchner filter funnel fitted with a drinking glass frit, is dry out-packed with sorbent [26]. The sorbent bed is consolidated past borer the side of the cavalcade during filling, and pressing the acme layer of the sorbent bed with a flat object, such as a stopper, while suction is applied at the other stop. Consolidation is completed past releasing the vacuum and pouring a solvent of low polarity over the surface of the bed followed by restitution of the vacuum. If the column is packed correctly, the solvent front end volition descend the column in a horizontal line, otherwise the column should be sucked dry out, repacked, and tested again. When all the solvent has passed through the column, remainder solvent trapped betwixt particles is removed by suction. A solution of the sample in a suitable (weak) solvent or preadsorbed onto a small amount of sorbent or inert material, such equally Celite, is practical to the pinnacle of the column. The sample solvent, if used, is sucked gently into the column packing. A piece of filter paper with the same diameter as the within diameter of the cavalcade, or funnel, is placed on top of the packed bed to foreclose disruption of the bed during solvent addition. The column is eluted with appropriate solvent mixtures of gradually increasing solvent forcefulness. Between solvent applications, the column is sucked dry, and the eluent collected in examination tubes or round bottom flasks. A multiport manifold allows sequential fraction drove without having to disassemble the apparatus after each fraction is collected.

Vacuum chromatography is simple, rapid, and convenient. Optimum sample loads are similar to flash chromatography. Nonetheless, it is not unusual to utilise sample overload conditions to separate simple mixtures past stepwise gradient elution, or to simplify mixtures for further separation. Under these conditions the sample loads may achieve x % (w/west), or fifty-fifty higher, of the bed mass.

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Metal COMPLEXES | Ion Chromatography

D.J. Pietrzyk , in Encyclopedia of Separation Science, 2000

Ion Exchange

In ion exchange column chromatography the column is packed with either a cation exchanger, which will exchange cations, or an anion exchanger, which will exchange anions. The exchangers are like insoluble electrolytes and exchange ions rapidly, reversibly and stoichiometrically. Cation substitution is represented in eqn [4] while eqn [5] illustrates anion exchange.

[4] R − C + + M + = R − 1000 + + C +

[5] R + A − + X − = R + 10 − + A −

Here R is the ion exchanger matrix containing either an anionic or cationic ionogenic group, C+ and A− are co-cation and co-anion, respectively, and Chiliad+ and Ten− are the analyte cation and anion, respectively. The direction of the equilibrium in eqns [4] and [5] is determined by the selectivity coefficient for the exchanger towards the two competing ions. Thus, in the absence of mass action effects the exchanger will prefer the ion with the highest selectivity coefficient. For a mixture of analytes, for case a mixture of metallic ions and a cation exchanger, the metallic ion with the smallest selectivity coefficient would elute first and the metal ion with the largest selectivity coefficient would elute last when using a mobile phase containing an electrolyte that provides a cation of appropriate selectivity and concentration. To increment elution of the metallic ion the electrolyte concentration in the mobile phase is increased, or a different electrolyte that provides a cation of higher selectivity is used.

While low efficiency metallic ion separations are possible on cation exchangers, resolution is improved considerably when a ligand is included in the mobile stage and the separation occurs because of the properties of the metal complex. For example, elution of M+ from the cation exchanger is enhanced with a mobile phase containing the ligand, HL, because of the formation of metal ion–ligand complexes. As shown in eqn [4], M+ is retained on the cation exchanger through competition with the mobile stage cation C+. When the ligand is in the mobile phase, the ligand will form a circuitous with the metal ion, causing the equilibrium in eqn [4] to shift to the left with the germination of the metal ion–ligand complex. The overall effect of the ligand can exist represented past eqn [6]:

[6] R − M + + 2 HL + C + = R − C + + ML 2 − + two H +

From eqn [6] the best mobile phase ligand, assuming formation constants and solubility are favourable, will be ane that forms anionic complexes with the metallic ion.

On the other paw, if the metal ion–ligand complex that forms is anionic, the complex can exist retained by an anion exchanger, or

[7] M + + 2 HL = ML 2 − + two H +

[8] R + C − + ML 2 − = R + ML 2 − + C −

In this case the metallic ion is subsequently removed from the anion exchanger past reversing the equilibrium in eqn [7], which is washed by reducing the concentration of the ligand in the mobile phase. This causes the equilibrium in eqn [8] to shift to the left, thus removing the metal ion from the anion exchanger.

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Introduction to chemic analysis of forensic samples

Chaudhery Mustansar Hussain , ... Maithri Tharmavaram , in Handbook of Belittling Techniques for Forensic Samples, 2021

ii.2.ane High performance liquid chromatography

HPLC is a blazon of column chromatography technique in which the stationary stage is in a column and the mobile phase is passed through it nether high pressure. The unabridged process in HPLC is automated and currently several instruments come with autosamplers also. The stationary phase is of many types in HPLC, including normal stage, reverse phase, ion commutation, and size exclusion. In normal stage HPLC columns, the stationary stage, which is commonly silica gel, is more polar than the mobile phase. In opposite phase columns, the stationary phase is nonpolar, and the mobile phase used is polar. Ion substitution columns and size exclusion columns, use acidic/basic columns or porous stationary phases, respectively. Illicit drugs such equally opioids and cannabis as well every bit pesticides, poisons, plant toxins, and alkaloids are analyzed using HPLC. The analysis of each sample demands its ain protocol wherein factors such as the type of cavalcade to be used, the mobile phase, the pH of the mobile phase, flow rate, retentiveness time, and pressure are considered. The detectors used in HPLC too vary according to the blazon of sample to be analyzed. Unremarkably used detectors are UV/visible, fluorescence, refractive index, electrochemical, conductivity detector, evaporative low-cal scattering detector, and chiral detectors ( Ahuja, 2005; Daldrup et al., 1986; Ghosh, 1992).

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