Purification Applications
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* Pharmaceutical - Drug Discovery, | * Pharmaceutical - Drug Discovery, | ||
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Revision as of 13:22, 21 April 2009
| Purification Applications |
| Application Type | |
| STANDARD AND SPECIAL | |
| Application ID | |
| Description | |
| Purification Applications |
General Description
The purpose of preparative-scale liquid chromatography is the isolation of materials conforming to a specified purity in the amounts that depend on the intended use of the product. Possible uses include the isolation of materials for structural elucidation, for biological or sensory evaluation (Eg:DMPKA), for organic synthesis or commercial applications. The scale of the operation includes laboratory, pilot plant and process-scale system. Process-scale separations will not be treated specifically in this section, since they represent a specialized area of chemical manufacturing and economic forecasting that the analytical chemist is infrequently exposed to.
From its inception liquid chromatography has been used as a preparative technique. Most chemist are familiar with the gravity feed glass column systems containing coarse adsorbent packings that are the main stay of laboratory practice. These low cost and easy to prepare and operate columns have many virtues. Not among them, however, are high resolution, short separation times and easy automation. When any of these factors are considered crucial then more sophisticated systems are required based on smaller particle sorbents with a narrow particle size distribution, that in turn are operated at above atmospheric pressure in the optimum mobile phase velocity range. These goals are best achieved using medium pressure and high pressure liquid chromatography.
Instead of gravity, a slight gas overpressure can be used to increase the sample throughput. This method is often called flash chromatography. Flash chromatography, also known as medium pressure chromatography, was popularized several years ago by Clark Still of Columbia University, as an alternative to slow and often inefficient gravity-fed chromatography. Flash chromatography provide a rapid (“over in a flash”) inexpensive general method for the preparative-scale separation of mixtures requiring only moderate resolution in amounts of 0.01 to 10 g depending on the difficulty of the separation and the column diameter (typically, 1-5cm).
When selecting the appropriate preparative-scale liquid chromatographic method for conditions requiring higher resolution or shorter processing times it is necessary to consider the intended use of the purified material. For example, 1-10 mg of a pure sample suffice for spectrocopic identification, while 1-10 g might be the minimum amount of an intermediate useful for an organic synthesis. The amount of sample required dictates the size of the column and the operating conditions necessary for the separation. The simplest approach to increasing the amount of sample recovered is to scale up an analytical separation using the same column packing, column length and linear mobile phase velocity while increasing the column diameter. The analytical separation should be optimized to maximize the separation factor between critical peak pairs at the expense of a longer separation time to permit the use of higher column loadings before the bands overlap. If the sample loading does not exceed the linear region of the sorption isotherm the results obtained are easily extrapolated from the analytical separation. Whith most analytical instruments the largest column size that can be used is about 25 cm long and 2.5cm internal diameter containing approximately 65 g of silica-based packing and requiring an optimun flow rate of 10-15 ml/min. For larger columns special purpose preparative scale instruments are usually neede to provide adequate flow and pressure capacity. Also, for the isolation of the maximum amount of sample a different approach to analytical separations is required. Success will be judged by the production rate and recovery yield obtained. The production rate is the amount of the purified fraction containing the corresponding component et the required degree of purity per unit of time. The recovery yield is the ratio between the amount of the component of interest that is collected in the product fraction and the amount injected in the column with the feed. the object is to maximize the production rate and the recovery yield, which invariably results in operating the column in a overloaded condition, such that the conditions predicted from an analytical separation have no direct meaning in establishing the optimum conditions for the separation. In fact, column operation under nonlinear or non-ideal conditions is very complex and it is not as easy to predict optimum operating conditions in this case as it is for the less demanding, although less powerful. scale-up approach to preparative liquid chromatography. several general reviews of high pressure preparative-scale liquid chromatography are available as well as application reviews for specific compound classes.
The scale-up approach to preparative HPLC is quite straightforward and is the approach likely to be taken in an analytical laboratory that requires sufficient material for identification purposes or to purify an analytical standard on an occasional basis. Separations are carried out in the linear region of the sorbent isotherm, which provide an upper sample mass limit of about 0.1-1.0 mg/g of sorbent. Increasing the column diameter at constant column length increases the weight of packing, and therefor the sample capacity of he column, without dramatically influencing the resolution obtained if the mobile phase linear velocity remains constant. The loading capacity of a column and the required mobile phase flow rate to maintain a constant linear velocity are directly proportional to the column cross-sectional area. Thus, to scale up an analytical separation a larger diameter column packed with the same material and operated at the same linear velocity as the analytical column used.
The pump requirements for preparative separations differ from those in analytical HPLC as the ability to generate high flow rates at moderate back pressures is crucial to the efficient operation of wide bore columns. A flow rate maximum of 100ml/min with a pressure limit of 3000 psi is considered adequate. Few analytical reciprocating piston-type pumps are capable of reaching this volume delivery rate; some are capable of operating at 30-60 ml/min, which suffices in many instances; stille others have a maximum delivery rate below 10 ml/min, which is barely adequate for all but semipreparative use.
The mode of injection differs appreciably from analytical methods where the object is to create discrete narrow bands by point or narrow zone injection of low sample amounts. Point injection of a large sample (mass or volume) in preparative liquid chromatography would result in a local overloading of the column packing with a deleterious effect on column performance. To minimize this effect the sample should be applied applied evenly over the entire column cross-sectional area of the column inlet. If the sample moving along the wall takes more time to enter the column than the sample moving in the center, because the path length is longer and/or the average velocity slower, the separated bands may be broadened considerably. To avoid this phenomenon, the column should be covered with a thin metal frit having a permeability lower than that, and the mobile phase evenly distributed over the frit using several methods, such as distributor plates with sector cut out, nozzles or a thin empty chamber above the frit.
Detection requirements in preparative-scale chromatography also differ from analytical operations where detectors are selected for their sensitivity. Sensitivity is not of overriding importance in preparative-scale chromatography; the ability to accommodate large column flow rates and a wide linear response range are more useful. The common analytical UV detectors can be detuned from the absorption maxima for the sample components to increase the dynamic range of the detector at the higher concentration end. some detectors have interchangeable flow cells which allow replacement of the analytical cell with one of shorter path length to reduce sensitivity. Finally, if only one or two fractions are to be collected and the number of run is low, then the sample can be collected manually in suitable-sized containers; a fraction collector is otherwise required.
Significant Markets
- Pharmaceutical - Drug Discovery,
