Overview of Analytical-to-Preparative Liquid Chromatography Method

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An overview of analytical-to-preparative liquid chromatography method development Jack Emanuel Silver ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00187 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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ACS Combinatorial Science

An overview of analytical-topreparative liquid chromatography method development Jack Silver Teledyne ISCO, 4700 Superior Street, Lincoln, NE 61504, USA Corresponding Author: [email protected] Abstract: Purification of compounds is a necessary aspect of chemical synthesis. Developing an efficient purification method is time consuming. A method that quickly calculates preparative gradients from analytical scouting runs is described. A solvent composition that provides a desired retention of a model compound is used to calibrate the analytical scouting run to determine an apparent gradient delay. This delay is applied to the retention times of compounds run with the same scouting gradient to yield a solvent composition for a preparative purification.

Preparative chromatography is an integral part of producing pure compounds in high-throughput synthesis labs. Efficient preparative chromatography is fast, allowing many purification of many compounds within a short time period. Short purification times reduce solvent usage and waste generation. It is also of importance to purify compounds as thoroughly as possible as impurities may lead to false positive or negative results in biological assays necessitating high resolution. Preparative chromatography only requires that the compound of interest be resolved, in contrast to analytical chromatography that requires resolution of all compounds for quantitation. A generic gradient is useful for purifying a mixture of compounds with a wide range of structures, but such gradients still need to be run sufficiently “flat” to allow resolution between compounds, requiring more time and solvent than an optimized method. The purification goals are similar for high-throughput purification labs and open-access purification instruments- rapid method development leading to rapid purification. Chemists work with a wide variety of compounds and use a variety of analytical columns, HPLC and UHPLC systems to evaluate the results of their synthesis, or natural product purification. A scouting gradient is used to quickly evaluate the complexity of the mixture. This scouting gradient is also useful to determine a solvent composition that elutes the desired compound(s). A scouting gradient, in reverse phase, is one from 5-10% organic up to 95-100% organic solvent over a short time period. The gradient length is typically 2-5 minutes for UHPLC columns, and 12 to 20 minutes for traditional HPLC columns.

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It is difficult to determine the solvent system that elutes a compound based solely on the peak retention time from a scouting run because the eluting solvent composition is delayed from the programmed gradient used for the scouting run. The gradient slope, mobile phase flow rate, internal column dimensions, and the analytical system dwell volume all play a role in the apparent gradient delay. Even when these delays are known, there is an additional delay requiring additional compensation. I will review the methods to determine this additional delay, and review various methods to calculate solvent compositions and gradients for preparative liquid chromatography systems.

It is assumed that, if a compound elutes during a scouting gradient, there is a shallow gradient or isocratic method which can elute the compound in a reasonable time. Such a purification method is desirable because it can be fast, while resolving the compound of interest from impurities. There are a number of ways to get to such a method. A common method is based on iterative runs using a small amount of sample until a reasonable purification is obtained.1 Briefly, the retention time of the compound is noted from a scouting run. Knowing the starting and ending solvent composition of the gradient and gradient length, it is possible to calculate the programmed solvent composition which eluted the compound. A gradient with a smaller solvent composition range is then run starting at 10% below the calculated solvent composition, to 20% above the determined composition. Depending on the elution time of the compound from this gradient, the gradient slope, start point, and end point are refined until the desired purification method is determined. Although this is a simple technique, it usually requires several runs to get a usable purification while using solvent and sample for each run. To save solvent and sample, the method development could be done on an analytical column and then scaled up using columns dimensions and solvent flow rate.

Computer methods are generally based on the Linear Solvent Strength Model2, which predicts a reliable calculation of separation in a gradient elution as a function of gradient conditions. These conditions include gradient slope (% B per minute), initial and final values of %B in the gradient, gradient shape, flow rate, and column dimensions. Calculations use two or more initial gradient separations, termed “calibration runs”, to predict isocratic or gradient separations as a function of different experimental conditions. The governing equation is: log 𝑘 = log 𝑘𝑤 ― 𝑆Φ Where k is the isocratic retention factor, kw is the value of k in water (or 0% B), S is a constant for a given set of experimental conditions equal to d(log k)/dΦ, and Φ is the fractional percent of strong solvent (%B/100). This model is incorporated into DryLab, and some vendor HPLC software. In general, two scouting gradients are needed with different gradient slopes to determine the purification method.

Blom3 et al described “compound-specific method optimization” which uses a set of focused preparative LC gradients, each covering a portion of the hydrophobic range. The crude mixture is run using a fast scouting gradient, and the retention time of the product within this gradient is used to determine the best of the focused gradient methods for purification.


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Time (minutes) Figure 1- Six compound mix divided into appropriate zones The preparative gradients were initially determined through the use of standard compounds. The gradients were modified by iteration until the purifications were satisfactory. The main disadvantage of this technique is that some compounds fall into a window where they elute early or late from the focused gradient. The zones aren’t optimized for any particular compound.

The “Accelerated Retention Window” (ARW) uses a scouting gradient to determine the retention of the desired compound. This retention time is used to calculate the starting and final solvent strength to elute a compound at a given time. The original algorithm4,5 uses a scouting gradient and the retention time of the desired compound is determined. The retention of the compound is used to determine the starting and ending solvent strength which elutes a compound at a fixed time using the same gradient steepness for both the analytical and preparative purifications. This algorithm allows a compound to elute at a particular time during the purification. A slight variation of this algorithm6 correlates the retention time of a compound during the scouting gradient linearly to the solvent composition that elutes that compound.



Figure 2- Retention time to % B correlation graph from Accelerated Retention Window After creating the graph correlating the analytical retention time to the solvent composition, focused gradients can be created according to the equation: 𝐶 = 𝑚𝑅𝑇 + 𝑏 + Δ Where C is the solvent composition used for the focused gradient, m and b are the slope and intercept for the relationship of the analytical retention time to the preparative eluting solvent composition, and Δ is an “instrument constant”. This “instrument constant” is partly due to dwell volume, but dwell and column volume corrections alone failed to create a focused gradient with a successful elution; the calculation yielded a solvent composition that is too high causing early elution of the desired compound, and reduced resolution from early eluting impurities. The value of Δ is determined iteratively by using the equation above to calculate the solvent composition for one of the standards, and then adjusting the value of Δ until the compound elutes at the desired time.

Another variation of the Accelerated Retention Window creates an isocratic method7. Gonnet et al also found that the solvent composition that eluted standard compounds correlated with the retention time according to the relationship:


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%𝑀𝑒𝐶𝑁 = Ω ∗ 𝑅𝑇 Where Ω is a conversion coefficient, and RT is the retention time for the desired compound in the scouting run. The coefficient Ω was found to vary linearly as a function of retention time: Ω = 𝐴 ∗ 𝑅𝑇 + 𝐾 Where A and K are constants determined by measurement using a series of standard compounds with different eluting times. The concentration of B solvent is: %𝑀𝑒𝐶𝑁 = Ω ∗ 𝑅𝑇 = (𝐴 ∗ 𝑅𝑇 ∗ 𝐾) ∗ 𝑅𝑇 A further correction was needed to move the retention time for the preparative run; this correction factor was less than 1. This correction was determined to be instrument dependent. The final equation for this method to calculate a preparative run is: %𝑀𝑒𝐶𝑁 = Ω ∗ 𝑅𝑇 = (𝐴 ∗ 𝑅𝑇 ∗ 𝐾) ∗ 𝑅𝑇 ∗ 𝐶𝐹

Where CF is the “correction factor”. Since the preparative method is an isocratic run, the correction factor cannot be a function of dwell volume in the preparative system since there is no gradient to be delayed. Although the Accelerated Retention Window algorithm shows a linear relationship between a compound’s retention time in a scouting gradient and the eluting solvent composition, the “First Time Right Time” shows a quadratic relationship; no explanation for the difference in relationship was suggested.

The “Time-on-Target”, or ToT, algorithm8 produces results similar to the Accelerated Retention Window, but the calibration procedure is different. Like the Accelerated Retention Window algorithm, a linear relationship between a compound’s scouting gradient retention time and preparative elution profile is assumed. The difference is that ToT assumes the slope of the relationship is the same as the scouting gradient, but the line defining the relationship is shifted to the right of the gradient. The calibration of the curve that defines the relationship of retention time in the scouting gradient and preparative solvent composition is defined by a single point. Time-on-Target also requires no “Correction Factors” or Δ values in order to calculate a solvent composition required to elute the compound. This technique is run with preparative HPLC or flash chromatography reverse phase, and preparative HPLC silica columns. Inputs needed are the dwell volume of the preparative system, and the preparative column volume. A model compound is run isocratically on the preparative system and the solvent composition is adjusted until the desired retention time is obtained. Isocratic runs eliminate the complication of system dwell volumes. The solvent composition determined by this isocratic run is used to “calibrate” the analytical system.


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Figure 3- A mixture of N-benzylbenzamide and phenacetin used to calibrate a 20x150 mm column in methanol (left) and acetonitrile (right). The desired retention time for purifications using this column is 6 minutes. Phenacetin is the first eluting peak.

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The next step is to calibrate the analytical system. Run a scouting gradient with the same compound used to determine the preparative isocratic retention, with the same solvent system using a column with chemistry matching the preparative run.

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Figure 4- Analytical calibration of a mixture N-benzylbenzamide and phenacetin in methanol. Phenacetin is the first eluting peak and was used for the analytical calibration. The retention time of the reference compound is compared to the time in the programmed gradient where the solvent composition matches the solvent composition determined in the isocratic run. The programmed gradient is apparently delayed by this time (Da). Compounds to be purified are then run with this same scouting gradient, although the solvents may be different from that used for the calibration. The preparative eluting solvent composition is determined by applying the offset of Da to the scouting gradient. A focused gradient is created from this solvent composition, with another correction applied for the preparative system dwell and column volumes that delay this gradient.

Compound

Scouting run Retention Time (Minutes)

Calculated Prep Gradient range Preparative (% mthanol) Retention Time


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Dimethyl yellow 5.800 87-97 5.49 butyl paraben 5.027 71-82 5.33 N-benzylbenzamide 4.349 58-69 6.34 Vanillin 3.087 34-45 6.18 Table 1- Scouting runs used a 2x50 mm C18 column at 0.5 mL/min with a water/methanol gradient. Preparative runs used a matching 20x150 mm column at 18 mL/min running water/methanol. Targeted retention time was 6.0 minutes.

Figure 5- Excel sheet to calculate focused gradients As an example of the ToT algorithm, refer to the Excel sheet in Figure 5. After the calibration of the analytical and preparative systems are complete, one needs only run scouting runs and enter the elution time in cell B2 (Compound elution time); values that are changed infrequently are on the right side of the spreadsheet. The spreadsheet, as an Excel file, is included with the supplemental materials. The preparative system Dwell Volume and Column Volume are in cells H2 and H3 respectively, and are used to adjust the final focused gradient. Cell H4 contains the isocratic solvent composition that provides the desired elution time for purified compounds. The parameters for the analytical scouting gradient are in column E; E2 and E3 are the starting and ending solvent composition for the scouting gradient while E4 is the gradient length. Cell E6 is the slope of the analytical scouting gradient and is used to determine the time the gradient was programmed to reach the solvent composition entered into H4; this time is stored in cell E7=(H4-E2)/E6. The time the model compound eluted from the scouting gradient is stored in cell E5; cell E7 is subtracted from this value and the results stored in cell E8 as the apparent gradient delay (Da). A compound to be purified is run on the analytical column with the scouting gradient used for the calibration. The elution time is entered in cell B2. The solvent composition which elutes the compound is calculated into cell B6=(B2-E8)*E6+E2; many compounds may be run with such a solvent composition isocratically. We find a focused gradient generally useful – the solvent composition for the start of the gradient, B10=B6+B8-B4/2 where the gradient is corrected for the prep system dwell and column volume. The solvent composition at the end of the focused gradient is calculated similarly into cell B11=B6+B8+B4/2.



Since the retention time of the peaks is determined by setting the solvent composition in an isocratic run, one can create a focused gradient with the desired retention time. Set Elution time (Phenacetin)

Isocratic solvent composition (% methanol)

Calculated focused Retention time gradient for dimethyl yellow dimethyl yellow (% (Minutes) methanol) 8.25 45% 79-89 8.75 4.75 55% 89-99 4.75 Table 2- Desired elution time of purified compounds can be changed by setting the retention time of a model compound. The same scouting run was used for the runs in Table 2; the only change was to change the iscocratic solvent composition entered into the spreadsheet (cell H4 in Figure 5). This suggests that Δ in the ARW algorithm, and “CF” in the “First Time-Right Time” calculation, is essentially an adjustment to the solvent composition to get the desired retention time, which isn’t needed for the ToT calculation. Compounds eluting from the analytical run within the period ~1.5x Da often elute later than the targeted time from the calculated gradient because they elute at very low solvent compositions. Except for the “zone” method, all of the methods described in this review assume the compound does not elute before a certain solvent composition in the scouting gradient. This is a reasonable assumption for most compounds, with the exception of weakly adsorbed samples. Early eluting compounds are handled by means of a defined gradient from the initial scouting gradient solvent composition, to that composition + 20% B. If the scouting method started at 5% B, the focused gradient is 5 to 25% B solvent.

The ToT calibration has had limited testing with normal phase silica gel with acceptable results. Compound

Calc. gradient range Elution time (Minutes) (% Ethyl Acetate) Methyl paraben 13-23 7.5 N-benzylbenzamide 18-28 7.6 Crude piperine (pepper extract) 44-54 7.1 Phenacetin 62-72 6.5 Table 3- silica gel runs (hexanes/ethyl acetate) calculated from Time-On-Target calibration. Isocratic elution solvent set to elute the model compound at 6 minutes. It may be desirable to run an initial purification on a smaller preparative column, with a scale-up to a larger column later. Scale-up is a matter of working in terms of column volumes rather than units such as minutes. Assuming the columns are packed with particles of the same size, the volume of the cylinder based in the column inside diameter and length provides the proportions needed. The flow rate (F) is scaled on column diameter (D): Fprep = Fanalytical * D2prep/D2analytical


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If the columns are the same length, the gradient profile remains the same. Scaling to a longer column requires the gradient segment volume to be the same to maintain the separation profile. In the Timeon-Target algorithm, entering the new column volume and flow rate adjusts the focused gradient to compensate for the change in column volume, although this adjustment is generally small.

Several methods were described to determine preparative gradients from scouting runs. As all, except for the iterative method, are based on relatively simple calculations, they are amenable to automation or use on shared instruments. They all need analytical and preparative columns with matching chemistry. Of the methods discussed, the ToT algorithm is the most versatile. A change in either the analytical or preparative LC system only needs a new calibration for that system, often just a single run. A change in column chemistry is also a simple calibration. A change in analytical scouting gradient requires a single calibration with a model compound. The main disadvantage is the poor calculation for early eluting compounds, but this is managed with a default gradient for these situations. The ARW technique requires a number of calibration runs to create the correlation between analytical retention time and preparative gradient method. It furthermore needs the value of Δ determined to adjust the final gradient method. Changes in the analytical or preparative chromatography systems require recalibration of the method. Likewise, the First Time Right Time algorithm has the same disadvantages. The Zone method requires determination of the zone boundaries with any change of chromatography system, requiring many injections. Its major advantage is that no assumptions are made about compound elution. The preparative methods aren’t optimized for any particular compound, and multiple methods need to be created for each column used on the preparative system, one method for each zone.

This work was supported by Teledyne ISCO, Lincoln, NE

Supplemental information contains chromatograms to support the Time-on-Target section and an MS Excel worksheet with the calculations.

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References 1

https://www.chromacademy.com/lms/sco8/Theory_Of_HPLC_Gradient_HPLC.pdf Retrieved 27 May 2019

Snyder, L. R.; Dolan, J. W. High-performance gradient elution: the practical application of the linear-solventstrength model; John Wiley: Hoboken, NJ, 2007. 3Blom, K. F.; Sparks, R.; Doughty, J.; Everlof, J. G.; Haque, T.; Combs, A. P. Optimizing Preparative LC/MS Configurations and Methods for Parallel Synthesis Purification. J. Comb. Chem. 2003, 5, 670-683 4 Irving, M.; Krueger, C.A.; Wade, J.V.; Hodges, J.C.; Leopold, K.; Collins, N.; Chan, C.; Shaqair, S.; Shornikov, A.; Yan, B. High-Throughput Purification of Combinatorial Libraries II: Automated Separation of Single Diastereomers from a 4-Amido-pyrrolidone Library Containing Intentional Diastereomer Pairs. J. Comb. Chem. 2004, 6(4), 478-486 5 Yan, B.; Collins, N.; Wheatley, J.; Irving, M.; Leopold, K.; Chan, C.; Shornikov, A.; Fang, L.; Lee, A.; Stock, M.; Zhao, J. High-Throughput Purification of Combinatorial Libraries I: A High-Throughput Purification System Using an Accelerated Retention Window Approach. J. Comb. Chem. 2004, 6(2), 255–261 6Blom, K. F.; Glass, B.; Sparks, R.; Combs, A. P. Preparative LC−MS Purification: Improved Compound-Specific Method Optimization. J. Comb. Chem. 2004, 6(6), 874–883 7Koza, P.; Gonnot, V.; Pelleter, J. Right-First-Time Isocratic Preparative Liquid Chromatography-Mass Spectrometry Purification. ACS Comb. Sci. 2012, 14, 4, 273−279 8 Silver, J.E. Calibration of analytical HPLC to generate preparative LC gradients for peptide purification. Proceedings of the 35th European Peptide Symposium. Patrick B. Timmons, Chandralal M. Hewage, Michal Lebl (Editors) European Peptide Society & PSP, 2018 2


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Overview of Analytical-to-Preparative Liquid Chromatography Method

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