Flash Chromatography Method Development

Transcription

1 Flash Chromatography Method Development Michael Ye, Boris Polanuyer, Lew Kurtzman, Daniel Vitkuske, and Tom Henderson Supelco, 595 North Harrison Road, Bellefonte, PA Shaoyin Wang, Brenda Nye, Becky Caproni, and Michael Singer Sigma RBI, 1 Strathmore Road, Natick, MS T405018H HST

2 Introduction What is Flash Chromatography? A type of preparative purification for rapid isolation of compounds including reaction mixtures, where the target (synthesized) molecule must be separated from excess reagents, by-products, and side-products; natural products compound of interest have be separated from matrix and impurities Sample range varies from several mg to over 150 g Linear flow rates up to 15 cm/min. Pressure psi

3 Early Flash Chromatography: Glass Flash Columns Michael Tswett reported separation of plant pigments using glass columns packed with calcium carbonate Clark Still reported fast flow glass column chromatography flash chromatography Do-by-yourselves style Significant labor involved in packing, unpacking and washing columns Safety risks in shattering glass columns Limited flow rate and pressure Reproducibility concerns

4 Modern Flash Chromatography (1994-Present) 1994 disposable cartridges for flash chromatography were introduced 1. Disposable plastic cartridges time and reproducibility 2. Cartridges of different size easy scale-up 3. Solid sample module and injection valve easy sample loading 4. Pressure up to 100 psi fast separation 5. Narrow particle size distribution - Low backpressure and higher efficiency

5 Modern Flash Chromatography Saves Time and Money Time Glass Cost Glass Flash Flash Purification Steps Areas

6 Experimental and Results Flash Chromatography System The Supelco VersaFlash System was used in the experiments. It fits a wide range of cartridge sizes and cartridge change-out is very convenient. Materials in contact with sample are stainless steel, Teflon, PEEK, and polypropylene, which are inert to most of samples.

7 Method Development Examples Purification of a synthetic compound Purification of plant pigments Purification of a synthetic peptide

8 Purification of a Synthetic Product 1. Product Properties This a derivative of 3-Indolylacetonitrile, which is a naturally occurring plant growth hormone. Total of 4 g of reaction mixture needs to be purified. The mixture was a semi-solid and soluble in dichloromethane and methanol. O CN H 2 N N H

9 Purification of a Synthetic Product 2. TLC Method Development H 2 N O N H CN Conditions: TLC plate: Silica Solvent: DCM:MeOH 5:95 Rf values: Product: 0.38 Impurity: 0.75 Impurity: 0.63 Impurity: 0.58 Impurity: 0.21 Impurity: 0.18 Impurity: 0.17 Origin P

10 Purification of a Synthetic Product 3. Transfer the Method from TLC to Flash Chromatography a. Under ideal conditions Vs = 1 Rf Vc Vs is the sample elute volume Rf is the TLC retention factor Vc is the cartridge bed volume b. In practice, we expect some deviations of the equation. Factors, such as sample quantity, particle size differences, and moisture content will affect the direct transfer.

11 Purification of a Synthetic Product 4. Flash Chromatography Conditions and Results Conditions: sample size: 4 grams cartridge size: 150 mm x 40 mm I.D. (96g silica) sample loading method: direct load with a syringe mobile phase: DCM:MeOH 5:95 flow rate: 50 ml/min. Results: Total solvent volume: 5 L Volume of fractions: 30 ml Yield: 56% Purity: 100% by TLC Total time (sample prep to purity confirmation): 3 hours

12 Flash Chromatography of Plant Pigments 1. Product Properties Chlorophylline (1), astaxanthene (2), and carotene (3) represents typical groups of plant pigments. There is 0.5 ml, 10 mg/ml of each compound in dichloromethane. All compounds are colored

13 Flash Chromatography of Plant Pigments 2. TLC Method Development A B C Development Solutions: A = dichloromethane:methanol, 4:1, v/v B = dichloromethane:methanol, 10:1, v/v C = dichloromethane Carotene (yellow) Astaxanthene (red) No single mobile phase can provide isocratic separation in a reasonable time Chlorophylline (green)

14 Flash Chromatography of Plant Pigments 3. Separation Strategy 2 1 1) Carotene - dichloromethane (100 ml) 2) Astaxanthene - dichloromethane:methanol 4:1, v/v, (100 ml) Carotene (yellow) Astaxanthene (red) 3) Chlorophyll - invert column to change direction of flow, methanol (60 ml) Chlorophylline (green)

15 Flash Chromatography of Plant Pigments 4. The Results Experimental Conditions: cartridge: sample: 75 x 40 mm I.D. silica 0.5 ml, 10 mg/ml of each compound in dichloromethane detection: by the compound color Samples from left to right Chlorophylline (green) Astaxanthene (red) Carotene (Orange)

16 Purification of a Synthetic Peptide 1. Product Properties Approximately 20 mg of synthetic peptide Val-Gln-Ala-Ala-Asp-Tyr-Ile-Asn-Gly In accordance with Expasy ProtParam analysis ( this peptide has the following properties Molecular weight: Theoretical pi: 3.80 Acidic Total number of negatively charged residues (Asp + Glu): 1 Total number of positively charged residues (Arg + Lys): 0 Aliphatic index: Hydrophobic, retention on C18

17 Purification of a Synthetic Peptide (contd.) 2. Selection of HPLC Conditions column: Discovery C18, 5 cm x 2.1 mm I.D., 5µm particles mobile phase: linear gradient of acetonitrile:water from 0 to 100% acetonitrile in 15 min. temp.: room temperature det.: UV at 254 nm Product mau Time (min)

18 Purification of a Synthetic Peptide (contd.) 3. Selection of Flash Chromatography Conditions Flash cartridge 75 x 40 mm I.D., packed with spherical silica (C18 bonded; carbon content 26%). Peptide samples (10 mg/ml in 0.02M KOH, 2mL) were injected using a stop-flow injector. Based on the HPLC results the following step gradient conditions were used: 1. 20% acetonitrile 200 ml 2. 60% acetonitrile 200 ml 3. 80% acetonitrile 200 ml % acetonitrile 200 ml

19 Purification of a Synthetic Peptide (contd.) 4. Results The compound of interest was found in the 80% acetonitrile fraction with 95%+ purity and 75% yield. Most of the impurities were concentrated in 60% acetonitrile, the 100% acetonitrile fractions contained no detectable components mau Time (min)

20 Summary of Method Development Understand the physical and chemical properties of the sample, such as solubility, hydrophobicity, sensitivity, and so on. Method development on TLC or HPLC. For isocratic conditions, TLC is more preferable. For gradient conditions, HPLC is more preferable. Select the appropriate flash chromatography cartridges, size and type of material. Transfer the method to the flash cartridge. Recover sample and characterize.

21 Conclusion Modern flash chromatography with disposable cartridges is applicable to a wide range of compound types. Saves time and solvents. HPLC linear gradients can be transformed into step gradients in flash chromatography. Flash chromatography can be a reliable and cost-effective alternative to preparative HPLC.