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Prepline Sample Purification Systems and Selected Applications


Overview Introduction Analytical vs Preparative Chromatography Components of Preparative HPLC Systems Proposed System Configurations Applications Summary


Introduction In the chemical production, separation methods, especially for the purification of value products, are well established and inevitable. The liquid batch chromatography is necessary when the classic methods as crystallization and distillation fail. Goal of this talk is to introduce Preparative Chromatography Equipment for LC purification processes. Features, Benefits and Applications of the versatile Preparative HPLC systems „made by Knauer“ are presented.


Purification Methods (Chemical Industry) Crystallization Distillation Chromatography - Highest Purity - Highest Productivity - Smallest Process Costs (Eluent Consumption, Eluent Recycling)


Analytical Chromatography Qualitative and quantitative determination of components in mixtures


Analytical HPLC Pump Dual-piston pump with constant flow. The flow accuracy deviation shall be less than 1% and the variation of the flow precision less than 0.5%. Typical flow rates are in the range of 0.5 – 2 ml/min. Isocratic or Gradient (HPG, LPG)

Column 2 - 4.6 mm ID Particle size 3 - 7 Âľm


Analytical HPLC Injector Sample amounts 1-20 Âľl, automatic or manual Detector Highly sensitive, also for trace analysis Software System control and complex data management


Preparative Chromatography Purification and isolation of value products: fine chemicals, pharmaceuticals, ‌


Preparative Chromatpgraphy Demand for high productivity and high purity, Systems have to be much larger Mass overload, concentration overload Peaks are no longer resolved Highest solubility for the target compound Largest possible sample size


Preparative vs Analytical Chromatography


Preparative vs Analytical Chromatography Pump with a high accuracy and precision with flow rates from 5-1000 ml/min, preferably isocratic due to the need for mobile phase recycling Column with a large diameter (8-100 mm), larger particles (10-50 Âľm), high efficiency/resolution is not important, preferably normal phase, less expensive, easy transfer of TLC conditions Injection with sample loop (up to 45 ml) or Feed pump (up to 50 ml/min), autosampler possible as well


Preparative vs Analytical Chromatography Fraction collector with 4 options 1. Collect, 2. Waste, 3. Mobile Phase Recycling (back into the mobile phase reservoir), 4. Peak Recycling (re-introduction of contaminated fractions onto the column) Due to high sample concentration the detector must not be sensitive. Software for instrument control and data management and sophisticated fractionation management (time, slope, peak purity)


Scale Definition Column ID [mm]

analytical 2 - 4.6

semi-preparative 8 - 16

prep. 20 - 62

Particle Size [µm]

≤5

5 - 10

10- 30

Stationary Phase [g] ≤ 5

5 - 30

50 - 900

Flow Rates [ml/min] 0.1 – 2

5 - 50

100 - 1000

Sample Size [mg]

0.1 - 50

1 - 700

3

0.5 - 2

0.01 - 2

UVD Flow Path [mm] 10


Linear velocity Flow rate (cm3/min) Linear velocity (cm/min) = Column ID (mm) 4 8 16 32 40 62

Cross section (cm2) 0.126 0.503 2.011 8.042 12.566 30.191

Cross section (cm2) Linear velocity (cm/min)

Flow rate (cm3/min)

16 16 16 16 16 16

1 4 16 64 100 240


Overloading Analytical

Volume Overload

Mass Overload


Separation Efficiency HETP as a function of loading on Columns with different particle sizes Mass Overload No small Particles required!!!

Dr. Birger Holz General Product Manager holz@knauer.net


Successful Preparative Scale 1. Optimize the separation under analytical conditions:preferably isocratic, high solubility / loadability 2. Upscale chromatographic parameters (consistent retention times) while maintaining the linear velocity of the mobile phase. If the diameter 3. Final Step: Load as much as possible up to the highest productivity and purity level (non linear range)

of the column doubles the flow rate has to be quadrupled.


Features of KNAUER Prepline Systems Systems can be tailored to the requested application Flexible configuration, modular structure Everything for the Preparative HPLC available Easy servicing and maintenance Excellent instrument specifications Compact design Reasonably priced Long lifetime


Equipment for Preparative HPLC


Selected Components

Pumps Injectors Columns Detectors Fraction Collectors Software


Knauer Preparative Assistant 6000


Assistant 6000 Footprint of Smartline analytical HPLC and Prepline Modules Combination of pumps, valves and detector for different applications Pump 100, Detector 200, Valves K-6, K-12, K-16 Prepline: feed pump, injection valve and rraction collector in one housing!


Features of Knauer Software Chrom Gate Easy to use Control of all instruments Data acquisition and saving of all raw data Easy data management Sophisticated fractionation management (FRC Option) - time, slope, level peak purity

Fully capable with FDA 21 CFR Part 11 guidelines


ChromGate FRC Option – FRC Setup


ChromGate FRC Option – FRC Setup


Columns for Preparative HPLC


Knauer Column Hardware Columns available with ID 2 – 62 mm - Analytical - Semi preparative - Preparative

2, 3, 4, 4.6 8, 16 20, 32, 40, 62

Columns available with lengths of 5 – 300 mm


Eurospher 100 3, 5, 7, 10, 15 Âľm C18, C8, C4, C1, CN, Amino, Diol, Si For routine analysis


Eurosil Bioselect 300 5 Âľm - C4, C18 Separation of large molecules (proteins)


Eurokat pH stable styrene/divinyl zenzene copolymer Modifications:

H

Particle size :

10 Âľm

Pb

Temperature for Pb, Ca, Ag:

Ca

Ag

60 - 90°C

For organic acids, carbohydrates, sugar alcohols


Eurocel Polysaccharide-based, on silica coated CSP For NP, RP and Polar organic mode Columns and bulk material (3, 5, 10, 20 Âľm) 1000 Angstroms pore size High loadability: up to 5 mg per g CSP Up to 200 bar stable


Amylose-based CSPs CH3 H

R =

N O

CH3

Amylose-Tri(3,5 Dimethylphenyl carbamate)* (Europak 01 „AD“)

OR O RO OR O

n

Kieselgel CH3

H

R =

N O

Amylose-Tri(α-Methylbenzylcarbamate) (Chiralpak AS)


Cellulose-based CSPs Cellulose-Tri(3,5 Dimethylphenyl carbamate)* (Eurocel 01 „OD“)

CH3 H = R N O

CH3

Cellulose-Tri(3,5 Dichlorphenylcarbamate) (not yet commercially available)

Cl H R = N

OR O

O RO OR

Cl

O

n

R = O

CH3

Cellulose-Tri(4-Methylbenzoate)* (Eurocel 02 „OJ“)

Kieselgel R = O

H R = N O

Cellulose-Tribenzoate) (Eurocel 03, „OB“) Cellulose-Triphenylcarbamate (Eurocel 04 „OC“)


Preparative System Proposals


Semi Preparative Chromatography


Isocratic System Feed Pump Fraction Collector Eluent Recycling Peak Recycling


Low Pressure Gradient System Feed Pump Fraction Collector


High Pressure Gradient System Feed Pump Fraction Collector


ScaleUp System no modification required when switching from analytical to preparative scale optimized flow paths for analytical and preparative scales two separate detectors for analytical and preparative LC collection of up to 16 large volume fractions with the built-in fractionation valve easily upgradeable to multi-fraction Fraction Collector


Prepline Equipment from Knauer Pumps up to 1 l/min (1/16“, 1/8“) UV Detectors up to 10 l/min´(1/16“, 1/8“, 1/4“‘) Flanged Columns up to 62 mm ID


Larger Systems in Cooperation With TECHNIKROM


Knauer - Technikrom Knauer Instruments with Technikrom DAC Columns Technikrom Systems with Knauer UV Detectors Technikrom Systems with integrated Knauer Pumps andUV Detectors


Preparative HPLC Applications


Applications Chiral Separations (Pharmaceutical Industry) - Special Chiral Stationary Phases required - Eurocel and Europak

Peptides / Protein Purification - Reversed Phase C18 or C8 - Pore size accorging to molecular size - Ion Exchange Chromatography


Applications Fine Chemicals Purification - Normal Phase Si - Reversed Phase (C8/C18)

Sugar Separation - Polystyrene-based ion-exchanger - Pb or Ca form


Semi Preparative LC Application Research and Development for Characterization of newly synthesized compounds Pure product required for HNMR, MS, IR, Elemental Analysis or even X-Ray structural analysis)


Chiral Separation Propranolol (+/-) 1996 UOP, USA Various manufacturers Beta blocker, treatment and prevention of heart diseases, blood pressure regulation


Propranolol Purification

Column Mobile phase Flow rate Detector

Eurocel 01 20 Âľm, 250 x 16 mm n-Heptane 79.5, IPA 19.5, DEA 1 (v/v) 8 ml/min UV 254 nm


Etozolin (Elkapin®) Polar Organic Mode α: 1,54

Thiazolidone Derivative Constipation Treatment

0

3

6

9

Column: 250x4.6 mm Eurocel 01, 5µm Eluent: MeOH Flow Rate: 1.0 ml/min Temp.: 25°C Detection:UV


Dibromo Phakellstatine (R,S) α: 1,27 O

RP Mode HN

Br

NH N Br N O 0

5

Column: Eluent:

10

250x4.6 mm Eurocel 01 MeOH/H2O(75:25)

Flow Rate: 1.0 ml/min Temp.: 25°C Detection: UV

15 min


N-Methyl Ephedrine α: 1,42

NP Mode CH3

0

Column: Eluent: Flow Rate: Temp.: Detection:

5

10

250x4.6 mm Eurocel 01 Hexane/IPA/DEA 80:20:0,1 1.0 ml/min 25°C UV

15 min

CH2

CH

OH

CH3

N CH3


Peptides Purification Reversed Phase C18 or C8, dependent on molecular size Ion Exchange Chromatography Peptide with 9 amino acids Column: 120 x 16 mm Eurospher 100 C18 10 Âľm Eluents: A: phosphate buffer with pH 3 and B:mixture of acetonitrile and water (70/30 v/v) Flow rate: 20 ml/min Gradient: 70/30 (A/B) which changes to 10/90(A/B) in 12 minutes (3 min sample loading)


Peptides Purification Reversed Phase C18 (Peptide small concentration) UV Detector peptide

2000

2000

1750

1750

1500

1500

1250

1250

1000

1000

750

750

500

500

250

250

mAU

2250

mAU

2250

0

0

-250

-250 0

2

4

6

8

10

12

14

16 Minutes

18

20

22

24

26

28

30


Peptides Purification Reversed Phase C18 (Peptide overload)

4000

4000

3500

3500

3000

3000

2500

2500

2000

2000

1500

1500

1000

1000

500

500

0

0

0

2

4

6

8

10

12

14

16 Minutes

18

20

22

24

26

28

30

mAU

mAU

UV Detector peptide


Separation of Proteins

77 µg Ribonuclease 94 µg Cytochrome C 37 µg Lysozyme 175 µg Katalase

Column: 120x4 mm Eurosil Bioselect 300-5 C4 Eluent: A: H2O, 0.1 % TFA; B: MeCN, 0.1 % TFA Gradient: 0% B to 20% B in 5 min 20% B to 100% B in 80 min Flow Rate:0.8 ml/min Temp.: ambient Detection: UV at 230 nm Inj. Volume:20 µl

0

10

Mixture in 20 µl water

20

30

40


Separation of Monosaccharides 1

2 3 4 5 6

20

30

Column: Eluent:

40

50

300x8 mm Eurokat Pb H2O

Detection: Refractive Index

1 2 3 4 5 6

Sucrose Glucose Fructose Mannose Xylose Sorbitol


Summary Preparative Chromatography is a highly applicable method to generate highly pure value products in the food and pharmaceutical industries (chiral compounds) as well as for the production of fine chemicals. Knauer‘s Preparative Systems can be tailored to the requested application and cover flow rates up to 1000 ml/min with Knauer columns up 62 mm ID. For larger equipment, Knauer and/or Technikrom can provide the most suitable solution to the customers.


Summary Isocratic and gradient modes (HPG, LPG) are possible. Simple fraction collection can be achieved with the Knauer Valves K6/12/16. Collection can be managed by time, slope or spectra comparison (PDA Detector 2800 or Multi-wavelength Detector 2600). Solvent recycling and peak recycling can be applied ChromGate software with FRC module is a strong partner for an efficient sample purification and fractionation


Prepline Sample Purification Systems