Preparative chromatography recovery

For preparative chromatography, the main goal is the recovery of pure mixture components. Therefore, the localization of separated bands is an important issue. The localization of bands directly on plates in daylight (for colored substances) or mostly in UV light is more convenient. The majority of adsorbents and commercially available precoated plates have a flnorescent indicator, e.g., silica gel 60 F254 -t 366. In several cases, separated bands may be localized in iodine vapors if snbstances form only unstable complexes. Brown or yellow zones produced in this way shonld be immediately outlined. 

Multiobjective optimization is an optimization strategy that overcomes the limits of a singleobjective function to optimize preparative chromatography [45]. In the physical programming method of multiobjective optimization, one can specify desirable, tolerable, or undesirable ranges for each design parameter. Optimum experimental conditions are obtained, for instance, using bi-objective (production rate and recovery yield) and tri-objective (production rate, recovery yield. 

In addition to requiring significant bulk material, the timeframe to complete the isolation is considerable. If the maximum analytical load for a 4.6 mm x 150 mm column has been determined to be 5 mg, assuming the isolation will be performed using semi-preparative chromatography (20 mm x 300 mm column), approximately 190 mg of sample can be loaded onto the preparative column. For a 0.1% level unknown, this translates to 190 pg of unknown injected onto the preparative column. Therefore, a total of 27 injections are required. If the assay time were estimated to be 1 hr, it would take at least 27 hr to perform the injections needed to obtain 5 mg (once again assuming 100% recovery). This timeframe does not include the time needed for method scale-up development, concentration and... 

Sample Recovery from Preparative Chromatography COSHHA009 ... 

The main fraction collected from a sample injection of 100 mg was returned to the synthesising chemist for recovery from the solvent. Only 40 mg of pure compound was recovered and this was considered unacceptable by the chemist (especially since the analytical trace shown in Fig. 8..3 suggested a purity, from a normalised UV chromatogram, somewhere in the region of 80 — see discussion later). Seemingly poor practical sample recoveries (compared to expected recoveries) are not uncommon in preparative chromatography and systematic examination of the process is required to determine the reason for this as and when it occurs. 

Whatever the approach selected, in order to optimize a nonlinear, preparative chromatographic system, one needs to consider four different factors, the objective function selected, the experimental parameters that can be optimized, the decision variable, and the constraints that must be satisfied [1]. The objective functions are discussed later, in Section 18.2.2). They include the production rate, its cost, the recovery )deld, the specific solvent consumption, or some combination of the above. Many experimental parameters of a preparative chromatography separation cannot be changed during the optimization process. Typically, such parameters are the nature of the feed components, their relative compositions, and the nature or even the brand of packing material. Sometimes, the coliunn diameter is also fixed by prior investments. The decisions variables are those parameters that can be changed during the optimization process, in order to maximize the ob-... 

Once the analytical scale method conditions are optimized, the next step is to choose a column and scale up the analytical HPLC parameters so that preparative chromatography can be performed and the unknown compound(s) can be isolated for identification by MS and NMR. For ease of transition, a preparative column consisting of the same packing material and particle size should be chosen. The column is the most important component of the process because it determines the amount of material that can be loaded for the desired purity and recovery. An important step in the scale-up procedure is determining the maximum load on the analytical column. The maximum analytical load is essential in determining the loading capacity of the preparative column. When an appropriate column is chosen, the analytical isolation can be scaled up using Eq. (5) 2 ... 

Hostettmann K, Marston A, Hostettmann M (1998) Preparative chromatography techniques applications in natural product isolation. Springer, Berlin, Heidelberg, New York, 244 pp Hua ZC (1997) Renaturation and purification of recombinant tissue-type plasminogen activator expressed in E. coli. lUBMB Life 41(4) 815-820 Huang RB, Andrews BA, Asenjo JA (1991) Diflerential product release (DPR) of proteins from yeast a new technique for selective product recovery from microbial cells. Biotechnol Bioeng 38 977-985... 

The conventional preparative chromatograph, although certainly more massive, is generally less complex than the analytical chromatograph. Although gradient elution has been used in preparative chromatography, it is avoided, if possible, due to the cost of solvents and solvent recovery. 

While an examination of the chromatogram, shows that the 10-mm diameter column was overloaded at the 50-mg sample the data in Table 10.1 indicate excellent recovery independent of sample or column size. In the preparative chromatography nonlinear effects caused by column overload are often observed, " and this affects the separation resolution as sample... 

Preparative chromatography can be practised on two different scales laboratory (recovery of grams of sample) and production (recovery of kilograms of sample). Preparative Scale Supercritical Fluid Chromatography (PS-SFC) has been practised on the laboratory scale from the very beginning of SFC in 1962 [1], More information on these historical experiments has been gathered by Berger et al. [2] and Bevan [3]. 

---