Automated Liquid Chromatography Method Development Design and Practice

Method development bottlenecks can be minimized with good planning, setup, procedures and techniques. For each method development station to be automated, users should start by building and optimizing a table of useful columns and solvents appropriate for the column selector and eluent mixer. Table 1 provides an example of useful columns and solvents for automated chiral method development using a 12-position column selector and a 10-bottle eluent mixer. Once the table of columns and solvents is optimized (see guidelines below), a useful list of eluents must be built and optimized. Table 2 (coated columns) and Table 3 (bonded columns) show examples of useful eluents for chiral method screening, based on the example of columns and solvents shown in Table 1. Eluents are arranged by decreasing solvent strength; miscibility is considered. Notice the ethanol wash in line 8 of Table 2; this is included because acetonitrile and heptane are not miscible.

Table 1 – Useful columns and solvents
Table 2 – Useful coated column eluents
Table 3 – Useful bonded column eluents

An automated ultrahigh-performance liquid chromatography/high-performance liquid chromatography/ supercritical fluid chromatography (UHPLC/HPLC/SFC) method development system flow is illustrated in Figure 1 and an HPLC example is pictured in Figure 2. The HPLC system includes a 10-bottle eluent mixer feeding an Agilent 1100 (Agilent Technologies, Santa Clara, Calif.) and a 12-position column selector fitted with 11 columns and bypass tubing.

Figure 1 – Automated method development flow diagram for UHPLC/HPLC/SFC.
Figure 2 – Automated method development for HPLC (11 columns, 10 solvents).

When optimizing tables of useful columns and solvents for specific final applications (i.e., UHPLC, HPLC, SFC, simulated moving bed [SMB], process, countercurrent chromatography [CCC] and centrifugal partition chromatography [CPC]), it is important to consider the requirements of the final application. Possible considerations include separation, scale, impurities, throughput, solubility, stability, loading capacity, elution order, cost of eluents and cost of columns. Method development requirements can be quite different—the final application may be UHPLC analysis or large-scale purification. During the early planning stages, it is best to consider long-term needs and where flexibility can be important, for example, as new columns enter the market.

After final review of column, solvent and eluent lists for the intended application, master sequences are built and optimized. In addition to separation methods, sequences should include bypass methods so that lines can be quickly flushed when changing eluents. The bypass column selector position (usually #1) is occupied only by tubing. Bypass methods are followed by separation methods for each appropriate column using the same eluent. If sequences hold the column constant and eluents are varied, more time will be wasted flushing lines. Thus, it is recommended that eluent be held constant and columns varied. Before each injection, an equilibration period must be included to wash and condition columns with new eluent. Methods should track pumped volume to correlate flushing, equilibrating and eluting volumes with system and column volumes and characteristics. Nonseparating steps (bypass, equilibrate and wash) are very important to autonomous operations and column lifetimes. It is usually sufficient to screen with fast gradients, since chromatograms need not be beautiful, just elucidate adequate separations.

Sequences should wash all columns at the end to prepare for the next sample. For example, in normal phase, all columns should be washed with methanol, a strong solvent, to remove retained samples, followed by isopropanol, which is miscible with all solvents. Water is typically used to wash off salts or other water-based additives. Well-written sequences typically serve as master libraries for many years, mitigating the amount of required labor and errors from method and sequence writing. Flush sequences should be built to flush air from solvent lines going into the eluent mixer from solvent bottles; this is particularly important if the system is not used continuously. Column wash sequences wash columns more rigorously than what is included at the end of every sequence (important if samples are dirty) by pumping more solvents for longer periods of time.

Near the end of initial setup, it is recommended that isopropyl alcohol (IPA), water or other high-viscosity solvent is used to record pressure and flow for each column position from the solvent list. Subsequently, column pressure and flow can be recorded and compared at regular intervals to anticipate system and column degradations. Increasing pressure is an early indicator of system and column problems. Autonomous systems perform with greater accuracy and consistency when preventative maintenance is practiced. Test samples can be run to evaluate overall performance and identify degrading columns before they fail.

Gradient methods should prevail in screening sequences because they save time and sample by exposing a single injection to varying eluent composition. Gradients of three or more solvents can be used to maintain constant eluent additive concentration without requiring additive in every solvent bottle (Figure 3). The example in the figure shows a gradient of methyl tert-butyl ether and tetrahydrofuran combined with a constant proportion of solvent from a different bottle of methyl tert-butyl ether with 1% trifluoroacetic acid (TFA). The methyl tert-butyl ether with 1% TFA is proportioned at 10% by the gradient mixer to achieve a target eluent TFA concentration of 0.1%. This mixing technique allows a wider variety of eluents with additives to be created with fewer bottles.

Figure 3 – Gradients using additive in only one bottle.
Figure 5 – Automated method development and semipreparative purification combination CCC (10 solvents).

As a dramatically different example, consider CCC using long spirals of tubing wound on bobbins similar to fishing reels. These bobbins rotate and revolve to create forces that retain a stationary phase eluent while allowing an immiscible mobile phase eluent to flow past, even while being mixed. Compounds at the immiscible boundaries selectively exhibit either more or less preference (retention) for the stationary phase, thus creating separation. Think of CCC as a liquid–liquid column. In a typical CCC method, first stationary, then mobile, phase is pumped into the tubing, sample is injected and eluted, and column contents are extruded. A new “column” is created for each method. A flow diagram of a combined CCC method development and semipreparative purification system is illustrated in Figure 4 and an example is shown in Figure 5. CCC can have advantages in purification, when compared to packed-column chromatography, including high loading capacity, recovery and resolution. The technique is especially suited for dirty samples because there is no packing material to foul and the extruded phase cleans the tubing for the next method.

Figure 4 – Automated method development and semipreparative purification flow diagram for combination CCC.

Equipment/software

Systems shown and described include software, eluent mixers, column selectors, flow selectors and injector/collectors from PDR-Separations (PDR-Separations LLC, Palm Beach Gardens, Fla.). Standard HPLC components include 1100/1200 LC systems (Agilent Technologies) and the Spectrum CCC (Dynamic Extractions Ltd., Tredegar, Gwent, U.K.).

Summary

Method development has been automated successfully for all types of liquid chromatography. After automation, method development becomes largely a background task, freeing staff for other duties. PDR-Separations software includes offers hardware-independent methods, a convenient sequence editor designed for large sequences and a data processing tool (DPT) with adjustable filters. DPT can display thumbnail chromatograms of an entire sequence or show only chromatograms that pass filter tests like minimum peak area, minimum number of peaks and peak area ratios.

Gary W. Yanik is president, PDR-Separations, 3 Old Meadow Way, Palm Beach Gardens, Fla. 33418, U.S.A.; tel.: 561-818-8445; fax: 561-429-4541; e-mail: [email protected]www.pdr-separations.com

Related Products

Comments