In both industrial and academic labs, complex mixtures, either biochemically generated or naturally occurring, are routinely separated and analysed during discovery, development and manufacturing. Obtaining a pure and high-quality end product is the ultimate goal with chromatography, the method of choice for these complex separations.
Chromatography refers to the scientific laboratory techniques used to separate components from a mixture. It involves passing a compound mixture dissolved in a “mobile phase” through a “stationary phase,” separating and collecting the analyte(s) to be purified from other molecules in the mixture. This technology spans a breadth of diverse applications, including basic research, drug discovery, forensics, environmental testing, athlete drug testing and food/beverage quality control.
Two-step preparative purification involves using the faster yet often crude purification method of flash liquid chromatography (flash-LC) followed by high-performance liquid chromatography (HPLC) to achieve larger quantities and higher-quality purified material with potential time savings and reduced costs.
Although there are many ways to perform chromatography, the focus of this article is flash-LC followed by HPLC. There isn’t one perfect protocol suited for every separation purpose; therefore, optimizing a method for your particular application is recommended. With the suggestions listed in this article and a little upfront reading and testing, you will easily purify the component(s) you’re looking for.
First step: Flash-LC purification
Flash-LC is routinely used at a research-lab scale (using milligram to gram amounts), as well as at a larger, industrial scale (using grams to kilograms amounts). Advances made by instrument manufacturers have provided scientists with reliable, automated flash-LC purification hardware for the laboratory, easy-to-use software and dependable pre-packed columns. The drawback to using flash chromatography for purification is the lower resolution inherent with this method. Flash-LC columns use larger particle sizes and column capacity, allowing for a larger amount of starting material, but at the cost of resolution and purity. If the compound does not require higher purity, or if HPLC will be performed in the next step, this might be the choice for you. Here are some considerations and tips for performing flash-LC.
Determining the solvent system
The purity of a sample (i.e., how many compounds are in the mixture) can be qualitatively determined using thin-layer chromatography (TLC) and the relative polarities of the components. Screening for the optimal mobile and stationary phases upfront with TLC will save time and money. Depending on the solvent being used, you might need to run the column in the hood because of potentially hazardous fumes. Instruments on the market today offer containment and exhaust systems for the fraction collector, so the instrument can be run outside of a hood without jeopardizing safety.
The separation achieved with a TLC plate can be used to determine the best method for the first purification step using flash chromatography. Some manufactures have software that lets the user enter the values generated from the TLC plates and then use this information to calculate the best flash-LC method for the compound.
Determining the column to use
Consider the sample size when choosing the separation column. Column-loading capacities vary and are determined by particle size and volume. In general, the maximum loading capacity of flash-LC columns is approximately 10% of the silica weight. The larger columns have the capacity to handle kilogram quantities, but if you don’t have that much starting material, you are only wasting money on the silica and extra solvents to run these large columns. After deciding on a column, matrix and solvent, you are ready to perform a flash separation.
Packing or purchasing a column
Whether you pack your own column or purchase one pre-packed, determining the ratio of silica gel to compound (i.e., silica: compound) by weight will help you achieve a more effective separation. Separation of less-complex solutions requires ratios of 30:1 to 50:1, but more complex separations may call for ratios of up to 120:1. When running a column manually, it is critical to never allow the solvent line to drop below the silica, or air holes will form as long lines in the silica that look like cracks. This greatly reduces the efficiency of the separation.
If you are manually packing the column, be aware that silica gel is an inhalation hazard. Dispense dry silica gel in the hood. Alternatively, consider the option of pre-packed columns to avoid this hazard. Pre-packed columns are convenient and cost-effective, particularly if you are not experienced in packing columns or don’t have the time to do it. Using pre-packed columns with an automated system may give better results and lessen the chance of the column matrix cracking.
Running the column and collecting fractions
A rapid flow rate helps provide a good separation. But this rate needs to be optimized for each sample, because it will affect separation results as well as system pressure. Adjust the flow rate until separation time and resolution have been optimized and ensure that system pressure remains within specifications of the hardware and column. Watch the progress carefully and change the collection tubes after they become filled. Remember to replenish the solvent in the column frequently.
Recent flash-LC systems are sold as pre-configured systems, and the solvent is pumped through the cartridge via an integrated, binary gradient pump, resulting in quicker separations and less solvent use. Systems typically include UV-VIS detectors, built-in fraction collectors and manual injectors. These systems also have intuitive, touch-screen software, enabling even a novice user to perform a separation with ease.
If flash-LC purification is sufficient for your needs, the fractions containing the purified compound can be used for further downstream research, discovery or quality control. Flash fractions dry down easily for compound storage, as they are made of 100% organic solvent. If the sample needs further purification, advance to the next steps of running it on an HPLC.
Second step: HPLC purification
Running samples on an HPLC is fairly straightforward, because many parameters identified during the pre-purification on a flash-LC column can be applied to this process. If you are running HPLC separation without a pre-purification step, consider using the guidance listed above to develop an appropriate method for HPLC. An additional consideration when using HPLC is the calculated back pressure. Because HPLC columns have smaller particle sizes for higher-quality separation, these systems tend to run under high-pressure conditions, in contrast to the relatively low pressure seen with flash separations. Calculate the estimated back pressure using information about the particle size and shape, the column’s internal diameter and the specifications of your solvent. Several companies offer systems that calculate this information using intuitive software.
The framework provided here will help you get started developing a purification method. Consider using the websites of chromatography vendors to discover up-to-date equipment, columns and software that can make preparative liquid chromatography easy, even for novices.
Because of the increased demand for ease of use, increased purification efficiency and higher-quality separated product, some manufacturers have combined the two-step method (flash-LC followed by HPLC) into one instrument with intuitive software, such as Gilson’s PLC Purification Systems. These innovations provide outstanding performance and efficiency for process development, as well as homogenous molecule purification, all in a small-footprint layout that saves space, time and money.
Finally, look for a company with strong customer-service programs. Sometimes a brief discussion with technical-support experts can save you valuable time and enable you to separate your compound in a flash!
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