Elution procedures

There are three principal elution procedures isocratic (from the Greek isochros, meaning equal strength) stepwise (or fractional) and gradient. 

1 Isocratic. By isocratic is meant the operation of the chromatographic column, by allowing a solvent mixture of unvarying composition to run through the column until separation is complete. The importance of mobile phase flow-rate has previously been discussed. 

There are available microprocessor controlled solvent delivery modules which can generate the required gradient profile, be it stepwise, linear, convex, concave or simply isocratic. Up to four solvents can be selected. A detailed discussion of the instrumentation is presented in Chapter 6. The pumping systems and accessories are equally adaptable to use with open tubular chromatography as with high pressure systems. 

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Catalyst Reactivation Using Propargyl Acetate. The Wiped-Film Evaporator/02 reactivation procedure and the Capture of Active Catalyst Using Solid Acidic Support with FI2 Elution procedure (see above) both involve the separation of uncomplexed phosphine from rhodium complex. Since the value of the uncomplexed phosphine is significant, technology that does not require separation of phosphine during catalyst reactivation is desirable. 

Most separations are performed using columns of resin and an elution procedure. The sample is introduced as a small band at the top of the column from where the various components are moved down the column at... 

To speed up the lipophilicity determination it was also proposed to use gradient elution procedures (for a review and guidelines, see references [5,101]). This generic approach is particularly useful when series of compounds with a broad lipophilicity range have to be tested since both polar and non-polar solutes can be retained with a reasonable elution time. 

HPLC-Like Methods Full Retention-Elution Procedures.486... 

HPLC-LIKE METHODS FULL RETENTION-ELUTION PROCEDURES... 

To elute the analytes from an lAC matrix, specific conditions within the column have to be applied. The elution procedure depends on the nature of the antibody-analyte bond, which is generally of weak physical nature. Since the types of physical bonds involved in immunochemical interactions vary considerably among the different anibody-analyte systems, each antibody-mediated cleanup procedure has its own optimal conditions for elution (168). 

Sega, G.A., Sluder, A.E., McCoy, L.S., Owens, J.G. Generoso, E.E. (1986) The use of alkaline elution procedures to measure DNA damage in spermiogenic stages of mice exposed to methyl methanesulfonate. Mutat. Res., 159, 55-63... 

Table I. Resin Cleanup and Elution Procedures for Resin Sampling...

Sorbent Elution. The loaded extraction columns were disconnected and eluted with solvents individually. The elution procedure was as follows First, 1 column bed volume of pesticide-grade pentane was added to the sorbent to displace any residual water. Then, 3 column bed volumes of ethyl ether were added. The eluting solvents were collected in a 2-mL volumetric flask. The eluates from the primary and secondary columns were combined in the volumetric flask. The internal standard solution was added, and the final extract volume was adjusted to 2 mL. Air pressure was again used to push the solvents through the sorbents to avoid volatility losses. 

The unacceptable taste and odor of drinking water from a tributary of the upper Ohio River led Frank Middleton and his collaborators in the U.S. Public Health Service to apply their granular carbon extraction-chloroform elution procedure to obtain residue samples of that water in 1962 (2). The first preliminary suggestions that such residues were carcinogenic were presented by Hueper and Payne (3) the following year. For whatever reason, those observations appeared to have had little impact. 

The membrane should not be allowed to dry before the completion of the elution procedure. 

Expose the column to several cycles of the low pH elution procedure (see step 10 below) to remove any noncovalently adsorbed antibody. 

Before using the column to purify antibody-toxin conjugate, apply a solution of the RIP at a concentration of 1 mg/mL to block any high affinity binding sites. Remove the RIP using the elution procedure (see Note 13)... 

The elution procedure using the low pH buffer removes 85-95% of the affinity-bound conjugate The remaining bound conjugate can be removed by a second pulse with the elution buffer (see Fig 1)... 

Collecting fractions during column chromatography. Column material and elution procedure are chosen to effect optimal separation of the desired protein. 

Thiocyanate can be recovered and recycled from this solution by the precipitation of iron as iron(III) hydroxide.366 Aurocyanide can also be eluted from a strong-base resin by chemical conversion of the gold to a cationic complex with the thiourea ligand, as shown above (equation 98). This method of gold elution is favoured in the Soviet Union, but suffers from the drawback that elution of the other metal cyanide complexes is generally poor, and multi-elution procedures are necessary. 

Two techniques were used to measure the apparent covalent grafting a Soxhlet extraction and an elution extraction. The Soxhlet procedure is well known. The elution procedure involved placing the graft... 

In 1973, Stewart and Doherty [9] resolved enantiomers of tryptophan on a column packed with BSA-succinoylaminoethyl-agarose in a discontinuous elution procedure. The mobile phase used was 0.1 M borate buffer (pH 9.2). The chromatograms of this classical research are shown in Figure 2. Several years later, this technique was applied for the chiral resolution of warfarin enantiomers [10]. In 1981, the enantiomers of tryptophan and warfarin racemates were resolved on various serum albumin CSPs [11,21,22]. The same method was used for the resolution of other drugs [12-14]. Allenmark et al. [23] studied the resolution of a series of active racemic sulfoxides on a BSA column using 0.08 M phosphate buffer (pH 5.8) as the eluting solvent. 

The IUPAC Compendium of Chemical Technology defines Normal Phase as an elution procedure in which the stationary phase is more polar than the mobile phase . In practice, the most widely used stationary phases for preparative HPLC are based on silica and the polarity of the underlying silyl ether and silanol provides the required hydrophilic surface. Amino and cyano bonded silica are also commonly used in normal phase mode though the latter also has some reversed phase properties. The predominant mechanism of interaction is hydrogen bonding. However, the silanol is mildly acidic so the silica surface will also have mild cation exchange properties. 

The IUPAC definition understandably states that reversed phase chromatography is an elution procedure in which the mobile phase is significantly more polar than the stationary phase . This is a somewhat simplistic statement that covers a wealth of... 

The protocol can be adapted to measure receptor recycling. If radiolabeled antibodies are internalized into cells by incubation at 37°C in the presence of chemokine or other agents, the acid-elution procedure can be used to remove radiolabeled antibodies remaining on the cell surface. If the acid-elution medium is not reduced below pH 3.0, and the washes kept brief and performed at 4°C, then the cells can be returned to 37°C culture (care should be taken to ensure that the endocytic-trafficking properties of cells are not perturbed by the low pH treatment). During a subsequent incubation at 37°C, receptors that recycle will return antibodies to the cell surface. These antibody molecules can be assessed by measuring the radioactivity that becomes accessible to a second round of acid elution. 

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