Preparative chromatography is the process of isolating material from a sample using HPLC, usually in large amounts, by collecting separated peak fractions as they emerge from the detector. The basic objective of an analytical-scale separation is to provide a chromatogram with crisp, well-resolved, symmetrical peaks that provide the necessary analytical information. The purpose of preparative-scale high performance liquid chromatography (HPLC) is to create a quantity of pure substance as easily and economically as feasible, eventually depositing the sample into a sample collecting vessel before recovery from the eluent. Preparative HPLC is more expensive than classic purification processes like distillation, crystallization, or extraction, making it only suitable for rare or expensive items. Preparative HPLC is evolving to meet the growing demand for high-purity chemicals for activity, toxicity, and pharmaceutical screening.
In the chemical and pharmaceutical industries, as well as in biotechnology and biochemistry, preparative high-performance liquid chromatography (HPLC) is employed in the isolation and purification of important products. The amount of substance to isolate or purify varies greatly depending on the working area. When it comes to enzyme isolation in biotechnology, it begins in the µg range. It is at this scale that we discuss micro purification. In order to identify and clarify the structure of unknown chemicals in synthesis or natural product chemistry, one must get pure molecules in milligram-to-milligram-scale concentrations. Standards, reference compounds, and chemicals for toxicological and pharmacological testing require larger quantities, measured in grams. The main objective of preparative is preparative runs are evaluated based on three key parameters: product purity, yield, and throughput. Preparative HPLC methods cannot be optimized for all three parameters due to their interdependence. The column is critical for establishing a solid, reproducible preparative HPLC technique. In analytical separations, Langmuir-like isotherms and rigorous adherence to the resolution equation are common assumptions. In preparative HPLC, where columns are regularly and substantially loaded, isotherms and commonly recognized relationships are no longer applicable quantitatively.
In analytical HPLC, column capacity is typically defined as a 10% decrease in efficiency due to injected sample mass. In preparative chromatography, the amount of injected sample might surpass the column capacity by an order of magnitude, hence it is rarely given an absolute value. In preparative HPLC, overload occurs when the product cannot be isolated at the necessary purity or recovery levels due to excessive loading. Column capacity must account for other molecules in the sample, including the matrix. When discussing capacity, we refer to sample capacity rather than the capacity factor, which measures analyte retention. Preparative purification aims to produce as much purified product per injection as possible.
To develop a method, optimize the initial separation on an analytical size column, overload it while maintaining adequate separation of components of interest, and scale-up to a preparative column of appropriate dimensions based on the amount of compound to purify, following previously outlined guidelines. Analytical columns are commonly chosen based on the availability of preparative columns with the same packing material, either prepackaged or in bulk media for self-packing. Before developing and optimizing a preparative method, it's important to have both analytical and preparative columns from the same packing material accessible.
Fraction collectors are commercially available in various sizes and types. Agilent Technologies provides specific fraction collectors for three flow rate ranges, while others can be utilized at low to high flow rates. The micro fraction collector is suitable for flow rates below 100µL/min, the analytical scale fraction collector for flow rates under 10 mL/min, and the preparative scale fraction collector for flow rates up to 100 mL/min. Some instruments combine the auto-sampler and fraction collector on a single platform, using a single needle and valve for both injection and fraction collection. Others use two devices, one for injection and one for fraction collection.