Chromatography Focus
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.
Figure 3: Gradient Profile using Additive in only 1 Bottle
arranging methods in a sequence and we include equilibration of about 1 column volume before each injection.
Figure 2: AutoMDS on Agilent 1100 with 22 Columns & 20 Bottles
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
Table 2: Suggested master method libraries for chiral screening
Column Eluent System
Coated Normal HPLC Bonded Normal HPLC
Coated Polar Bonded Polar
HPLC HPLC
Coated Reverse HPLC Bonded Reverse HPLC
Coated Normal SFC Bonded Normal SFC
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 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 optimiSed 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 optimisation, scale-up to prep is usually straight forward if the
Figure 4: Prep Purification System
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 summarises 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 optimisation 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
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