Volume solvent elution

C. Isolation and purification of XK-62-2 100 g of the white powder obtained in the above step B are placed to form a thin, uniform layer on the upper part of a 5 cm0X 150 cm column packed with about 3 kg of silica gel advancely suspended in a solvent of chloroform, isopropanol and 17% aqueous ammonia (2 1 1 by volume). Thereafter, elution is carried out with the same solvent at a flow rate of about 250 ml/hour. The eluate is separated in 100 ml portions. The active fraction is subjected to paper chromatography to examine the components eluted. XK-62-2 is eluted in fraction Nos. 53-75 and gentamicin Cja is eluted in fraction Nos. 85-120. The fraction Nos. 53-75 are combined and concentrated under reduced pressure to sufficiently remove the solvent. The concentrate Is then dissolved in a small amount of water. After freeze-drying the solution, about 38 g of a purified preparate of XK-62-2 (free base) is obtained. The preparate has an activity of 950 units/mg. Likewise, fraction Nos. 85-120 are combined and concentrated under reduced pressure to sufficiently remove the solvent. The concentrate is then dissolved in a small amount of water. After freeze-drying the solution, about 50 g of a purified preparate of gentamicin Cja (free base) is obtained. 

Extraction of sample using LSE solvent elution volume reduction... 

By contrast, it is often not possible to standardize cleanup steps based on adsorption chromatography. Altered volumes of elution solvent, small deviations in the water content of the adsorbent and minor changes in the composition of binary eluents are often necessary and should be regarded as minor changes. 

The choice of solvent directly influences the retention of the analyte on the sorbent and its subsequent elution, whereas the solvent polarity determines the solvent strength (or ability to elute the analyte from the sorbent in a smaller volume than a weaker solvent). Dean [272] gave solvent strengths for normal- and reversed-phase sorbents. The elution solvent should be one in which the analytes are soluble and should ideally be compatible with the final analysis technique. For example, for HPLC analysis, a solvent similar to the mobile phase is a good choice of elution solvent. For the elution step it is also important to consider the volume of the solvent. A minimum volume of elution solvent (typically 250 xL per 100 mg of sorbent) allows maximum concentration of the analytes. 

Figure 26-11 (a) Large molecules cannot penetrate the pores of the stationary phase. They are eluted by a volume of solvent equal to the volume of mobile phase. (fc>) Small molecules that can be found inside or outside the gel require a larger volume for elution. V, is the total column volume occupied by gel plus solvent. V0 is the volume of the mobile phase outside the gel particles. 

The use of commercially available SPE cartridges is an attractive technique, because the volume of elution solvent is reduced and the cartridges can be used in different cleanup methods. C 8 retains OPPs because of its apolar characteristics, and it allows polar analytes to pass. This method has been used to clean up extracts from beef tissues (60,61,63). The opposite is true of polar stationary phases such as Florisil, which has been used to clean up extracts from marine mammal tissues (62). 

It is also necessary to monitor the volume of solvent which has passed through the GPC column set from the time of injection of the sample (this is called the elution volume or the retention volume). Solvent flow is conveniently measured by means of elapsed time since sample injection, relying implicitly on a con.stant solvent pumping rate. As an added check on this assumption, flow times may be ratioed to those of a low-molecular-weight marker that provides a sharp elution peak at long flow times. 

Second, the elution step introduces a dilemma Removal of all the trace metal on the column theoretically requires an infinite volume of eluting solvent, yet the concept of preconcentration demands that eluent volume be kept to a minimum. In practice, a highly reproducible fraction of a trace metal is removed from a chelating column by the first several milliliters (21) thus, elution is carried out with this volume, the elution efficiency is factored into the final results, and a second portion of eluting solvent is passed to complete elution of the remaining tail into the waste container. 

Column load rate Milli-Q wash volume Eluting solvent Elution volume Solvent wash volume Milli-Q rinse volume... 

The actual volume of eluting solvent required is dependent on the void volume of the SPE sorbent and on the retention of the solute in the eluting solvent. A typical SPE cartridge that contains 500 mg of sorbent that is 60 pm in diameter will have a void volume of approximately 120 pL per 100 mg of sorbent (Hennion and Pichon, 1994), which is approximately 600 pLor 0.6 mL. Given that a k (the column capacity factor) of many of the solutes could be 2 to 3 in the elution solvent, the prediction of the elution volume is given by the equation below ... 

Thus, the maximum concentration of an analyte that has a k of 3 on the C-18 sorbent would elute from 500 mg of sorbent in approximately 2.4 mL. A k of 3 is a high value for most solutes so that this case should represent an extreme case of retention. Because the compound is at its maximum concentration at 2.4 mL, if this concentration were doubled, then recovery of the analyte should be nearly 100%. Thus, 5 mL would be a good first estimate for the volume of elution solvent that is required for elution. If the k were less than 1, then an elution volume of only 2.5 mL would be required. Typically, experience shows that syringe barrels and cartridges containing 500 mg of C-18 sorbent require approximately 3 to 5 mL of elution solvent. From Eq. (3.5), one recognizes that the amount of void volume per 100 mg of sorbent is an important number and has a direct influence on the amount of solvent required to elute the SPE sorbent. Thus, it is important to pack SPE columns as efficiently as possible and to minimize the void volume in order that the least amount of solvent may be used to elute the sorbent. 

Increase the volume of eluting solvent or use back elution of the sorbent. 

BaA and BaP is added and the entire sample is extracted with methylene chloride (250-, 100-, and 100-mL portions). The extracts are combined, 10 mL of isooctane added, and the solution evaporated to about 10 mL. When the solution approaches 10 mL, a second 10-mL volume of isooctane is added. This is repeated three times. The isooctane solution is made up to 75 mL and extracted with three 100-mL portions of dimethyl sulfoxide (DMSO) that contains 20% phosphoric acid. The DMSO extracts are each washed with three 30-mL portions of fresh isooctane, combined and diluted with 500 mL of water. This solution is extracted with three 80-mL portions of isooctane. The isooctane extracts are combined, rinsed once with water, and evaporated to 25 mL. At this point, a 1-mL portion of the solution is removed for intermediate radioassay and a UV scan. Usually the concentrate is sufficiently free from interferences and is evaporated down to about 0.05 mL and an aliquot injected into the GC. If further purification is indicated, the solution is chromatographed on a 1.1-cm X 90-cm column containing 75 g of Woelm Neutral Alumina deactivated with 2% water. Before deactivation, the alumina is dried in an oven for 1 hr at 150°C. The solvent elution schedule is as follows 25 mL cyclohexane prewash, 25-mL sample in cyclohexane, 100 mL cyclohexane, 100 mL cyclohexane-methylene chloride (9 1), 100 mL cyclohexane-methylene chloride (1 1), 100 mL methylene chloride. After the first 125 mL has eluted, 10 cuts are then radioassayed and combined into a single PNA fraction. This fraction is evaporated to about 0.05 mL in preparation for the GC-UV finishing step. Figure 2 is a diagram of the wastewater procedure. 

Concentration to a small volume, solvent exchange, clean-up on a sihca-alumina column 2 1, elution with 100 mL hexane/... 

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