Purification of reaction mixtures using flash chromatography.

Purification of reaction mixtures using flash chromatography. This technical note details the use of ISOLUTE Flash chromatography columns for the purification of reaction mixtures. What is flash chromatography? Flash chromatography is a rapid purification technique used to purify compounds of wide ranging polarity; often reaction mixtures, where the target (synthesised) molecule must be separated from excess reagents and reaction by-products. As in standard liquid chromatography, the separation takes place in a column packed with stationary phase (sorbent). The sample is applied to the head of the column, and the components are separated as the mobile phase (solvent) flows through the column. ISOLUTE Flash columns are polypropylene columns, pre-packed with ISOLUTE Flash sorbents. Normal phase sorbents (e.g. Si, NH2), reversed phase sorbents (e.g. C18) and ion exchange sorbents (e.g. SCX-2) are available. This technical note describes the use of normal phase (silica) flash columns. There are various methods of processing samples using ISOLUTE Flash chromatography columns. Off-line flash chromatography In this mode, ISOLUTE Flash columns are processed using a vacuum manifold such as the FlashVac, in a similar way to solid phase extraction (SPE) columns. The columns should be attached to the manifold, and the sample applied. Elution solvent is then applied in successive aliquots to selectively elute the compounds of interest into suitable collection vessels. The elution solvent can be isocratic, where the successive aliquots applied consist of the same solvent mixture each time, or a step gradient where the successive aliquots applied consist of solvents of increasing polarity, and can elute compounds that are more strongly retained by the sorbent. On-line flash chromatography In this mode, the columns are mounted on a system that allows an external liquid pump to be connected to the column. A continuous flow of elution solvent is pumped through the columns, and depending on the capability of the pump, the solvent composition can be isocratic (a single solvent or solvent mixture) or a gradient with an increasing proportion of stronger solvent (either in a step gradient or a linear gradient). - 1 -

100 % Step gradient % organic solvent Linear gradient Fig. 1. Elution profiles of step and linear gradients 0 % Normal phase flash chromatography Traditionally, normal phase flash chromatography has been most popular. Sorbents: Elution solvents: polar (e.g. Silica, NH2) non-polar (e.g. hexane, heptane, dichloromethane), sometimes modified with small amounts of more polar solvents such as isopropanol) The sample is usually applied in a weak solvent (very non-polar), and separation occurs when elution solvent is applied. The elution order of compounds in normal phase flash chromatography is :- Non polar (early elution) polar (later elution) 1 2 3 4 1. Xylene (not used in TLC) 2. Dibutyl phthalate 3. Methyl benzoate 4. Dimethyl phthalate Fig. 2. Separation of standards using normal phase flash chromatography 0 3.75 7.50 11.25 15.00 Method development using ISOLUTE Flash columns Column equilibration Prior to sample loading, the column should be prepared for the separation by equilibration (pre-wetting) with a suitable solvent. Off-line Apply the equilibration solvent to the top of the column and allow to flow through the column under gravity. On-line We recommend that flash columns to be used on-line for the separation should be equilibrated in the off-line mode using a vacuum manifold such as the FlashVac. Alternatively, the column should be mounted on the instrument, and a suitable volume of equilibration solvent pumped through the column. - 2 -

Typical equilibration solvents For normal phase flash chromatography using Silica or NH2 columns, the optimum results are achieved when the column is pre-wetted. Suitable solvents are non-polar, for example, hexane or pentane. A suitable volume for column equilibration is approximately 2 bed volumes. Table 1 Column size Approximate column volume Typical solvent volume 2 g 2.5 ml 5 ml 5 g 6.5 ml 13 ml 10 g 12.5 ml 25 ml 20 g 25 ml 50 ml 25 g 31.3 ml 62.5 ml 50 g 62.5 ml 125 ml 70 g 88 ml 176 ml 100 g 125 ml 250 ml 150 g 188 ml 376 ml Normal phase flash columns can be used without pre-wetting, but some column to column variation may be experienced. Sample application There are two popular approaches to loading the sample in flash chromatography. a) Wet loading. Load the liquid sample directly onto the top frit of the column, and allow to percolate through to the top of the sorbent bed. For best results, load sample onto a pre-wetted column, in a non-polar solvent for e.g. Sorbent Si Typical solvent Hexane or other non-polar solvent or solvent mixture (as normal phase HPLC) Practical tips for wet-loading: Dissolve your sample in as non-polar a solvent as possible. If your compounds are not easily soluble in a non-polar solvent, either dissolve in a small volume of polar solvent, and dilute with a non-polar solvent to reduce the elution strength, or, consider dry loading of the sample see below Position your column on a suitable vacuum manifold (e.g. FlashVac or VacMaster, equipped with PTFE stopcock needles. Apply the sample evenly over the entire area of the top frit. To promote this, seal the column by closing the stopcock, then apply the sample so that it forms a pool on top of the frit. When the stopcock is opened, the sample will load evenly. Samples can alternatively be loaded onto the column in on-line mode when using the Flash module system, via a 3-way injection valve. - 3 -

b) Dry loading. Pre-absorb the reaction mixture onto a small amount of bulk material of the chosen sorbent. Evaporate off the majority of the solvent, leaving the compounds bound to the surface of the sorbent. Add this blend to the top of the pre-packed (and pre-wetted) flash column, settle, and add a further top frit to secure the blend in place. The top frit can be placed using a suitably sized frit inserter. This is the loading method of choice for reaction mixtures consisting of polar solvents when loading onto onto a silica or other normal phase column. N.B. A popular alternative sorbent for dry loading using the flash sorbent is to use a diatomaceous earth such as ISOLUTE HM-N. This can be used in the same way as the flash sorbent, but has several advantages including more efficient desorption of the compounds into the mobile phase. See technical note TN115 for full details of this approach. Practical tips for dry loading: Dissolve the sample initially in a suitable solvent, ensuring complete dissolution if possible. Use the smallest volume possible. Add the bulk material of choice. The ideal proportion of sample to bulk material is 1:1 to 1:3 by volume. Evaporate off the residual solvent using a rotary evaporator to ensure even adsorption of the sample on the bulk material. Pack the dry blend on top of the flash column (above the top frit) and add another frit. Push down to the new surface to prevent movement of the blend. When loading with ISOLUTE HM-N, ensure that the material is not crushed at this stage. When dry loading using bulk silica, ensure that it is identical to the material in the flash chromatography column. If this is not possible, use lower surface area material, ensuring that the surface ph and moisture content (activity) are as similar as possible to the column packing. All ISOLUTE Flash sorbents are available as bulk material for dry-loading. Capacity guidelines As a general guideline, the amount of sample loaded onto the flash column should be 5-10% of column bed mass, i.e. for purification of a reaction mixture, Reaction scale Column size 100 mg 1-2g 250 mg 2-5g 500 mg 5-10g 1g 10-20g 2.5g 25-50g Factors affecting the capacity of the column include: analytes, sample matrix, concentration of reaction products and elution solvent used. The elution solvent for the flash chromatography separation can now be applied. - 4 -

Optimisation of flash separation Prediction of elution profile using TLC In normal phase flash chromatography using silica columns, a popular method development approach is to use silica TLC plates to predict the best mobile phase composition. TLC data can be used to predict column elution behaviour using the relationship CV = 1 / R f Where R f is the retention factor for a particular component, and can be calculated as follows: R f = distance between origin and component distance between origin and solvent front CV is the number of column volumes required to elute the component from the column using that solvent system, regardless of column dimensions. [CV can be defined as the interstitial volume of the sorbent bed, i.e. (bed volume) (volume of packing material)] R f CV 0.90 1.10 0.70 1.40 0.50 2.00 0.30 3.33 0.10 10.00 i.e. For a particular set of separation conditions, a fast eluting component (weakly retained) with an R f value of 0.9 can be eluted in just over 1 column volume, whereas a more slowly eluting component (strongly retained) with an R f of 0.10 requires 10 column volumes to complete elution. For transfer of a separation from TLC to flash chromatography, the solvent system should be optimised in terms of solvent strength and selectivity so that R f values of between 0.15 and 0.35 are achieved for each component. This will allow isocratic elution within 7 column volumes. - 5 -

Relative solvent strengths for normal phase systems: Solvent Solvent strength Methanol 0.95 Ethanol 0.88 2-propanol 0.82 acetonitrile 0.65 Ethyl acetate 0.58 tetrahydrofuran 0.57 Acetone 0.56 Dichloromethane 0.42 Chloroform 0.40 Diethyl ether 0.38 Toluene 0.29 Hexane 0.01 Strong solvent Weak solvent Solvent front Hexane: EtOAc 1:1 Origin Hexane: DCM 1:2 Fig. 3. The selectivity of solvent system is as important as solvent strength For non-optimised TLC separations, a better separation can generally be achieved using a less polar solvent system (OR decreasing the proportion of polar modifier) TLC can also be used to predict the capacity of a column for separating a multi-component mixture using different solvent compositions. In general terms, the greater the difference in CV, the greater the capacity of the column for the separation, so the larger the amount of mixture that can be loaded. For example, for a 20 g / 70 ml ISOLUTE Flash Si column: CV Approx sample load 6 1g 2 0.5g 1 250mg R f A =.51 R f B =.39 R f =.12 CV = 0.6 B A R f A=.32 R f B =.21 R f =.11 CV = 1.6 R f A =.22 R f B =.14 R f =.08 CV = 2.6 Fig 4: CV vs. Rf to predict separation capacity. CV is an easier value to interpret - 6 -

Limitations of TLC in separation prediction TLC is a fast, effective and useful tool for prediction of the chromatographic behaviour of the sample on a normal phase flash column. However there are some limitations:- Accuracy of prediction can be reduced if the mobile phase contains very polar modifiers such as methanol or acetonitrile, or very volatile modifiers such as diethyl ether The activity (or moisture content) of the plate used can be different from the separation column The binding agent used in the TLC plate may affect the separation As well as being a useful tool for determining isocratic elution conditions, TLC can be used for prediction of separation conditions for mixtures that require gradient elution conditions for efficient separation. Step gradient Step gradients provide controlled elution with discrete changes in eluent strength. Each step is optimised to elute only those components that have solubility in that eluent. This technique can be applied to both off-line and on-line flash chromatography. 1. Use TLC to determine suitable solvent strengths to elute components at discrete intervals, choosing different solvent mixtures that elute each component separately with an R f = 0.2 to 0.5. 2. Calculate the volume of solvent required to elute each component using CV = 1 / R f. Column volumes for each column configuration are listed in table 1. 3. Apply between 2 and 5 column volumes of solvent for each step, starting with the solvent with weakest solvent strength. 4. Collect the eluent from each step in a separate vessel. Linear gradient Linear gradients are a quick way of separating complex mixtures, reducing the complexity of the subsequent purification of the fractions collected. This technique is suitable for online flash chromatography. 1. Use TLC to find both the weakest and strongest elution solvents. The weak solvent (solvent A) should give retention of the majority of components (R f of <0.1). The strong solvent (solvent B) should allow elution of all of the components of interest (R f >0.5). 2. Run a gradient starting with 100% solvent A and ending with 100% solvent B. 3. Collect the eluent at regular intervals. Method development can also be performed without TLC. This can be a useful approach to method development, particularly for non-silica based flash chromatography (e.g. reversed phase) where suitable TLC plates are not available. Generic approach to flash chromatography method development (off-line) 1. Load sample onto pre-wetted (equilibrated) flash column in as weak a solvent as possible (or use dry loading). For example, for normal phase work use hexane for loading. 2. Elute the column with aliquots (2 CV each) of successively increasing solvent strength. Example elution solvents are shown below: - 7 -

Solvent A = weak solvent (e.g. hexane) Aliquot # Solvent B = strong solvent (polar modifier) (e.g. isopropanol) %A %B 1 100 0 2 99 1 3 98 2 4 97 3 5 96 4 6 95 5 7 94 6 8 93 7 9 92 8 10 91 9 3. Collect each fraction, and analyse for the presence of the components of interest. 4. Using this data, identify the solvents that elute the components of interest separately, and set up a step or continuous gradient as described. Practical tips for gradient elution The use of a gradient will not improve the selectivity of a separation if the components are not separated isocratically using that solvent system. However, a gradient can be used to decrease the time taken for the separation. The gradient starting conditions must not cause chromatographic separation start the gradient with a weak solvent, to match the sample loading conditions. The flow rate at the beginning of a gradient can be high, but for best results, should be reduced to the optimum flow rate at the separation area. To speed up a gradient separation, either: Use a higher flow rate (most suited to sample with many components, high sample load) Or Use a steeper gradient (most suited to samples with few components, low sample load) Optimisation of flow rate The optimum flow rate for a flash separation is related to the particle size and dimensions of the column. Theoretical optimum flow rates for flash columns of different dimensions can be predicted. However, in practice, increasing the flow rate has not been found to significantly affect the separation, and has important productivity advantages. - 8 -

Alpha 1-4 7 6 5 4 3 2 1 0 Sample Load 20% w/w 0 10 20 30 40 50 60 70 80 Bed Mass (g) Flow rate 15 ml/min Flow rate 25 ml/min Flow rate 45 ml/min Fig. 5. α 1-4 values for 15 and 25 ml/min are comparable for all configurations, indicating little or no effect on the separation. In addition, the use of 45 ml/min for the larger 70 and 150ml columns again does not impact on the chromatography. Other factors also affect the flow rate that can be used, such as composition of mobile phase and back pressure. Recommended flow rates Column diameter (configuration) Recommended flow rate range 16 mm (D) 5-25 ml / min 20 mm (E) 5-25 ml / min 27 mm (F) 10-30 ml / min 37 mm (J) 20-50 ml / min 40 mm (V, W, X) 20-50 ml / min Fraction collection Once separated on the flash chromatography column, purified fractions / components must be collected for further processing and / or testing. Off-line flash chromatography When performing flash chromatography on a vacuum manifold such as the FlashVac, successive fractions can be collected as follows: Load collection rack with vials of a suitable volume in each position. Place a single flash column in position 1, and apply the first solvent aliquot. Collect the aliquot in the vial in position 1 of the collection rack. Move the column to position 2, and apply the second solvent aliquot. Collect the aliquot in the vial in position 2 of the collection rack. Continue until all the components of interest have been collected. Alternatively, multiple columns can be processed by replacing collection vials at each elution step. A typical volume for each fraction is 2 column volumes. On-line flash chromatography Using an automated system equipped with a fraction collector, fractions can be collected in a variety of ways, for example - - 9 -

Fixed volume fractions can be collected Individual peaks can be collected trigger collection via detector. ISOLUTE Flash chromatography products ordering information ISOLUTE Flash columns Column configuration Columns / box ISOLUTE Flash Silica ISOLUTE Flash C18 ISOLUTE Flash NH2 ISOLUTE Flash SCX-2 2 g / 15 ml 20 450-0200-D 451-0200-D 452-0200-D 456-0200-D 5 g / 25 ml 20 450-0500-E 451-0500-E 452-0500-E 456-0500-E 10 g / 70 ml 16 450-1000-F 451-1000-F 452-1000-F 456-1000-F 20 g / 70 ml 16 450-2000-F 451-2000-F 452-2000-F 456-2000-F 25 g / 150 ml 8 450-2500-J 451-2500-J 452-2500-J 456-2500-J 50 g / 150 ml 8 450-5000-J 451-5000-J 452-5000-J 456-5000-J 70 g / 150 ml 8 450-7000-J 451-7000-J 452-7000-J 456-7000-J ISOLUTE Flash-XL columns Nominal sorbent mass Units / pack ISOLUTE Flash Silica ISOLUTE Flash-XL Silica (4 x 10 cm) 40 g 12 450-040G-V ISOLUTE Flash-XL Silica (4 x 18 cm) 100 g 12 450-100G-W ISOLUTE Flash-XL Silica (4 x 22 cm) 100 g 12 450-100G-X ISOLUTE Flash C18 ISOLUTE Flash-XL C18 (4 x 10 cm) 70 g 1 451-070G-V ISOLUTE Flash-XL C18 (4 x 18 cm) 150 g 1 451-150G-W ISOLUTE Flash-XL C18 (4 x 22 cm) 150 g 1 451-150G-X ISOLUTE Flash bulk sorbents Pack size ISOLUTE Flash Silica ISOLUTE Flash C18 ISOLUTE Flash NH2 ISOLUTE FLASH SCX-2 25 g - 9451-0025 9454-0025 9456-0025 100 g 9450-0100 9451-0100 9454-0100 9456-0100 1 kg 9450-1000 9451-1000 9454-1000 9456-1000 ISOLUTE HM-N bulk Bulk ISOLUTE HM-N 1 Kg 9800-1000 Bulk ISOLUTE HM-N 5 Kg 9800-5000 Extra frits To fit column Units / pack 16 mm (5/8 ) D (15 ml) 100 120-1036-D 20 mm (13/16 ) E (25 ml) 100 120-1037-E 27 mm (1 1/16 ) F (70 ml) 100 120-1038-F 38 mm (1 1/2") J (150 ml) 50 120-1039-J 40 mm ( X (Flash-XL column) 50 120-1049-X - 10 -

FlashVac-10 FlashVac-10 complete with 19 mm, 10 position rack 122-1019 FlashVac-10 complete with 25 mm, 10 position rack 122-1025 FlashVac-20 FlashVac-20 complete with 19 mm, 20 position rack 122-2019 FlashVac-20 complete with 25 mm, 10 position rack* 122-2025 *The 25 mm positions in the FlashVac-10 are staggered in order to accommodate ten 150 ml columns Flash Modules Module with 16 mm (D column) plunger Module with 20 mm (E column) plunger Module with 27 mm (F column) plunger Module with 37 mm (J column) plunger 1 column module 5 column module 8 column module F-1CM-D F-5CP-D F-8CP-D F-1CM-E F-5CP-E F-8CP-E F-1CM-F F-5CP-F F-8CP-F F-1CM-J F-5CP-J F-8CP-J Contact your Argonaut distributor for spare part and accessories ordering information. TN 117 Local Distributor Dr. Wéber Consulting Kft. Kék Duna u. 34/a. 2132 Göd-Felsőgöd 2132 Göd, Pf. 7 27 / 330 149 Fax: 27 / 336 375-11 -