Minimizing Solvent Impact on Purification of Nitrogencontaining

Transcription

1 Minimizing Solvent Impact on Purification of Nitrogencontaining Compounds J. Liu and P. C. Rahn Biotage Discovery Chemistry Group US 1725 Discovery Drive Charlottesville, VA USA 1

2 Abstract This paper evaluates solvent effect upon flash chromatography purification. The impact of sample mass overloading, volume overloading and sample dissolution solvents upon sample resolution are discussed. To maximize throughput, preparative columns are often overloaded. The purification efficiency depends on sample mass and loading volume. Sample dissolution is a major issue with preparative separations. To minimize the solvent s impact on the purification process, sample pre-treatment and loading techniques are investigated. Highest resolution and loading levels are achieved when the sample is dry loaded Methods developed in this study illustrate techniques that simplify post-reaction workup procedures for chemists 2

3 System, Material and Methods Separations were performed on the Biotage SP1 purification system using Touch Logic Control Variable UV detector utilized to detune the absorbance with high sample loads FLASH+ cartridges used in this study include: Biotage FLASH 12+ M Si Biotage FLASH 25+ M C18 Biotage Samplet Samples include: Crude 2-(4-chlorophenoxyentyl)-4,4- dimethyl-2-oxazoline Technical grade ethyl chrysanthemumate (mixture of trans- and cis-isomers) N-Phenylbenzylamine and 1- Nitronaphthalene Purity analysis performed by HPLC 3

4 Sample Loading Volume Impact on Band- Broadening AU A Desired product 150 mg in 1 ml Constant 150 mg load injected onto FLASH 25+M C18 cartridges using three different volumes Column void volume = 36 ml AU AU B 150 mg in 8 ml C 150 mg in 18 ml Minutes Chromatogram A: Loading volume equivalent to 3% of column void volume (1 ml) Chromatogram B: Loading volume equivalent to 22% column void volume (8 ml) Chromatogram C: Loading volume equivalent to 50% column void volume (18 ml) Sample: Dissolution solvent: Cartridge: Flow: Eluent: Detection: Crude 2-(4-chlorophenoxyethyl)-4,4- dimethyl-2-oxazoline 80/20 Methanol-water FLASH 25+ M C18 25 ml/min 80:20 (v/v) Methanol-water UV 254 nm 4

5 Volume Load Impact Upon Resolution Resolution (%) A Ην B Ηp Load Vol./Void Vol. (%) C The percentage of valley depth (Ην) over the peak height (Ηp) was used to define resolution (Ην / Ηp) x 100 Sample load volume, as a percentage of void volume (Sample Load Vol./Void Vol.), was plotted vs. resolution Graph illustrates the effect of sample volume on separation of component B from A Resolution significantly degrades when sample volume increases to 20% of the column void volume Sample: Dissolving solvent: Cartridge: Flow: Eluent: Detection: Crude 2-(4-chlorophenoxyethyl)-4,4- dimethyl-2-oxazoline 80/20 Methanol-water FLASH 25+ M C18 25 ml/min 80:20 (v/v) Methanol-water UV 254 nm 5

6 Load Volume Affects Peak Shape Width1/2 (min) Asymmetry (%) Width1/2 (min) Asymmetry (10%) Sample: Dissolving solvent: Cartridge: Flow: Eluent: Detection: Crude 2-(4-chlorophenoxyethyl)- 4,4-dimethyl-2-oxazoline 80/20 Methanol-water FLASH 25+ M C18 25 ml/min 80:20 (v/v) Methanol-water UV 254 nm Load Vol./Void Vol. (%) Peak volume vs. peak shape is plotted Sample load volume was illustrated as the percentage over the column void volume Peak parameters shown in the figure include: Peak width at half peak height (Width 1/2) Peak asymmetry factor at 10% peak height (Asymmetry) When loading volume is above 20% of column void volume Band-broadening increases significantly Peak symmetry degrades by 10% 6

7 Purification with Different Mass Load mv Desired component Mass loaded (mg) Minutes Sample: 95% Ethyl chrysanthemumate (mixture of trans- and cis- isomers) Cartridge: FLASH 25+ M C18 Flow: 30 ml/min Eluent: 80:20 (v/v) Acetonitrile-water Detection: UV 254 nm Sample volume kept constant at 2 ml volume 6% of column volume Dissolution solvent is 80:20 Acetonitrile:water As sample load increases from 80 mg to 1600 mg Peak fronts shift to earlier elution time Peak tails overlap as mass increases and purity of fractions decreases 7

8 Impact of Sample Mass on Separation R etention (m in.) Sample Mass/Packing (%) Ratio between loading mass and amount of packing material, (sample mass/packing (%)), is shown vs. retention behavior: When loading mass increases over 1.5% of the packing material, peak asymmetry significantly increases ( ) Retention shifts to front as loading mass increases ( ) As loading mass increases, peak tails overlap, but do not shift ( ) 8 Retention at Peak Max Retention at Peak Tail Asymmetry (10%) Sample: 95% Ethyl chrysanthemumate (mixture of trans and cis isomers) Cartridge: FLASH 25+ M C18 Flow: 30 ml/min Eluent: 80:20 (v/v) Acetonitrile-water Detection: UV 254 nm

9 Impact of Strong Solvent on a Preparative Separation Co-elution 0.8 ml injection A B UV Trace 0.4 ml injection Sample: N-Phenylbenzylamine (A) and 1-Nitronaphthalene (B) Cartridge: FLASH 12+ M Si Flow: 12 ml/min Eluent: 95:5 Hexane-Ethyl Acetate Total sample load 120 mg for components A and B Sample dissolved in either 0.4 (solid blue) or 0.8 ml DCM (semi-transparent blue) 95:5 Hexane-Ethyl Acetate as eluting solvent 1.3 ml/fraction collected As Sample Mass increased, the compounds resolution decreases and co-elution occurs due to the dissolution solvent Detailed fraction purity analysis for this purification was determined using HPLC, and the fraction purity was plotted vs. fraction number 9

10 Biotage Samplet Technology: Unique Sample Handling for Purification Samplet Loading Procedure Sample loaded onto new Samplet Loaded Samplet Syntage Heating Blocks New Samplet Evaporate Solvent Sample cavity Top frit Syntage media FLASH+ Cartridge Bottom frit Biotage-patented, Samplet cartridge technology improves the loading technique for liquid and solid samples for flash chromatography Crude reaction mixture is added directly to Samplet cartridges in any solvent Syntage Heating block uniformly distributes the heat to dry the Samplet cartridge after loading Each Samplet cartridge is isolated in a separate sleeve to reduce cross contamination Dried Samplet cartridge is placed directly into FLASH+ cartridge 10

11 Sample Loading Technique Comparison - Advantages of Samplet (Dry Sample Loading) Technology 120 mg with dried Samplet Purity of A = 98% Purity of B = 98% A B 120 mg in 0.8 ml DCM Purity of A = 37% Purity of B = 70% A B Sample: N-Phenylbenzylamine (A) and 1-Nitronaphthalene (B) Cartridge: FLASH 12+M Si Flow: 12 ml/min Eluent: 95:5 Hexane-Ethyl Acetate Advantage of Samplet (dry-loading) cartridge is obvious when comparing the UV traces Resolution between compounds is maintained No sample breakthrough at front of separation Higher purity and yield Blue trace Sample dissolved in DCM and loaded onto the Biotage FLASH 12 Samplet cartridge Dried under vacuum prior to purification Orange trace Sample dissolved in 0.8 ml DCM (120 mg) and directly loaded (injected) for purification 11

12 Differentiating Cartridge Overload vs. Detector Overload: Impact of De-tuning Detector Cartridge: FLASH 12+M Total load: 120 mg on dry Samplet cartridge Line: 0.1 mm path 254 nm Shade: 0.1 mm path 275 nm To maximize throughput, the chemist should de-tune the detector (monitor chromatogram not at the compounds maximum absorbance) to ensure the detector does not saturate and indicate merged peaks Detuning a detector helps the chemist to easily differentiate detector overload from column overload Easier to differentiate the valley between compounds and to use slope collection SP1 purification system allows the chemist to chose and display two wavelengths 12

13 Summary In flash chromatography, column overload is common technique to obtain maximum throughput and reduce overall purification costs Overloading the mass and volume injected in a cartridge maximizes throughput Maximum achievable overload depends on the chemist s purity criteria Sample loading factors including mass, volume and dissolution solvent affect sample resolution and purification efficiency Volume overload Causes significant band-broadening as sample loading volume increases Resolution degrades significantly when loading volume exceeds 20% of the column void volume Mass overload Increased mass load results in skewed peak shapes because of non-linear adsorption/partitioning phenomena Peaks shift towards the void volume with less retention Peak tails overlap at given point for different load mass Judicious choice of dissolution solvents is critical to resolution Stronger dissolution solvents can locally disrupt separation process by causing unnecessary band-broadening Use of the Biotage Samplet technology combined with the Biotage SP1 purification system allows the chemist to maximize resolution and loading capacity without sacrificing purity 13