45
systempictured in Figure 2 is controlled by AutoMDS software (PDR-Chiral, Lake Park, FL USA) and includes a 20 bottle low pressure gradient mixer feeding an Agilent 1100, a 24 position heated/cooled column selector fitted with up to 22 columns and 2 bypass lines, and an Advanced Laser Polarimeter (ALP) for uniquely identifying enantiomers and tracking elution order.
In order to make method development fast and automated we build relatively large screening sequences that differ based on column type (coated vs bonded) and eluent phase (normal, polar, and reverse). The reason we separate based on column type is that coated phases can be easily ruined by strong solvents, as opposed to bonded phases which can handle almost any solvent. Depending on analyte characteristics, acid or base additives are included in eluents.
We suggest developing the master method libraries listed in Table 2 below. Sequences are written by selecting an appropriate master method, deciding which columns and solvents from the master set are to be screened, and then building a sequence of only those methods. We consider miscibility of solvents when arranging methods in a sequence and we include equilibration of about 1 column volume before each injection.
Column Eluent
Coated Normal Bonded Normal
Coated Bonded
Coated Bonded
Polar Polar
System HPLC HPLC
HPLC HPLC
Reverse HPLC Reverse HPLC
Coated Normal Bonded Normal
SFC SFC
Table 2: Suggested master method libraries for chiral screening
Gradient methods prevail in our screening method libraries because they save time and sample by exposing a single injection to varying eluent composition quickly. Multi solvent bottle gradients (more than binary) are often used to maintain constant eluent additive concentration without requiring additive to be in every solvent bottle. Figure 3 below shows an example where the eluent has a constant amount of TFA (5% of 2% = 0.1%) and a constant amount of Ethanol (5%), even though the percent contributions of Hexane and THF vary in a linear gradient from 90/5 to 40/55 between 1 and 6 minutes. Using this scheme, we can run acid, neutral, or base gradients against Hexane, for example, by adding only 1 bottle of Hexane with acid additive and one bottle of Hexane with base
Figure 3: Gradient Profile using Additive in only 1 Bottle
additive, rather than needing an acid, neutral, and base set of all solvents. These techniques allow us to screen a wide variety of normal, reverse, and polar organic phase eluents with acid, neutral, or base additives using only 20 bottles total. To avoid column + additive memory effects we load column selectors with 3 of each type of column, one for acid, neutral, and base eluents. Thus we do not need to spend time equilibrating our columns because of eluent additive changes.
Methods should be optimized based on application requirements and scale, e.g. analytical, semi-prep, prep, or process. Important considerations may include separation, elution order, solubility, stability, loading capacity, impurities, speed, and cost of eluents.
Preparative Purification After method optimization, scale-up to prep is usually straight forward if the method was developed and tested against appropriate requirements. Loading, cycle time, and other parameters can be determined using an analytical column and relatively simple mathematics can be used to scale performance up to prep. We developed a calculation worksheet called “Prep Predictor” (free on our web site) for pragmatically comparing methods for prep purification. Prediction accuracy is typically within 10% on a complete job. The Prep Predictor quickly summarizes important bounding parameters like total run time, total solvent consumed, and total solvent collected (for evaporation). This allows us to do prep chromatography in a very deterministic way with minimal method optimization at prep scale and minimal confusion about how much time and solvent we need, how big collection vessels should be, and how long rotovapping will take.
A modern HPLC/SFC prep purification system with 2 pumps configured for high pressure mixing, injector, column, detectors, and a collection valve is flow diagrammed below in Figure 4. The collection valve diverts eluent flow from waste to collection vessels and back
Figure 4: Prep Purification System
to waste under command of AutoPrep software (PDR-Chiral, Lake Park, FL USA). AutoPrep software controls peak collection based on a combination of parameters including: time, detector amplitude and sign, detector derivative and sign, and enantiomeric excess. Collection modes are very robust allowing us to routinely make multi-day continuous runs with no one on-site during evening and night hours. Most of our collection modes dynamically adapt to real time conditions and perform well even during changing conditions.
Chiral Detection We find chiral (optical activity) detectors necessary in chiral separations. Both polarimeter-based and circular dichroism- based chiral detectors can be useful to positively identify enantiomers in chromatograms with many peaks, monitor elution order during method development, and collect enantiomeric peaks during prep purification. Circular Dichroism detectors are absorbance-based, require a
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