Prepline Sample Purification Systemsand 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

PumpDual-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)  

Column2 - 4.6 mm IDParticle size 3 - 7 µm 

Analytical HPLC

InjectorSample amounts 1-20 µl, automatic or manual

DetectorHighly sensitive, also for trace analysis  SoftwareSystem 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 µm), 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 options1. 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 Definitionanalytical semi-preparative prep.

Column ID [mm] 2 - 4.6 8 - 16 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] 0.01 - 2 0.1 - 50 1 - 700

UVD Flow Path [mm] 10 3 0.5 - 2

Column ID Cross section Linear velocity Flow rate(mm) (cm2) (cm/min) (cm3/min)

4 0.126 161

8 0.503 164

16 2.011 1616

32 8.042 1664

40 12.566 16100

62 30.191 16

240

Linear velocity (cm/min) =

Flow rate (cm3/min)

Cross section (cm2)

Linear velocityLinear velocity

OverloadingOverloading

AnalyticalAnalytical

Volume OverloadVolume Overload

Mass OverloadMass Overload

Separation EfficiencySeparation Efficiency

HETP as a function ofloading onColumns with differentparticle sizes

Mass OverloadNo smallParticles 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 of the column doubles the flow rate has to be quadrupled.

3. Final Step: Load as much as possible up to the highest productivity and purity level (non linear range)

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

PumpsInjectorsColumnsDetectorsFraction CollectorsSoftware

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 2, 3, 4, 4.6- Semi preparative 8, 16 - Preparative 20, 32, 40, 62

Columns available with lengths of 5 – 300 mm

Eurospher 100Eurospher 100

3, 5, 7, 10, 15 µm

C18, C8, C4, C1, CN, Amino, Diol, Si

For routine analysis

Eurosil Bioselect 300Eurosil Bioselect 300

5 µm - C4, C18

Separation of large molecules (proteins)

EurokatEurokat

pH stable styrene/divinyl zenzene copolymer

Modifications: H Pb Ca Ag

Particle size : 10 µm

Temperature for Pb, 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 µm)

1000 Angstroms pore sizeHigh loadability: up to 5 mg per g CSPUp to 200 bar stable

Amylose-based CSPs

O

OR

OR

RO

Kieselgel

nO

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

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

CH3

CH3

NH

O=R

=RN

H

O

CH3

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

Cellulose-based CSPsCellulose-based CSPs

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

OCH3R =

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

R =O

Cellulose-Tribenzoate)(Eurocel 03, „OB“)

R = N

H

O

Cellulose-Triphenylcarbamate(Eurocel 04 „OC“)

R = N

H

O

Cl

Cl

R = N

H

O

CH3

CH3

O

OOR

OR

RO

Kieselgel

n

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 Larger Systems in

CooperationCooperation

WithWith

TECHNITECHNIKROMKROM

Knauer - Technikrom

Knauer Instruments with Technikrom DAC Columns

Technikrom Systems with Knauer UV Detectors

Technikrom Systems with integrated Knauer Pumps andUV Detectors

Preparative HPLC ApplicationsPreparative 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 (+/-)

1996UOP, USA

Various manufacturers

Beta blocker, treatment and prevention of heart diseases, blood pressure regulation

Propranolol Purification

Column Eurocel 01 20 µm, 250 x 16 mmMobile phase n-Heptane 79.5, IPA 19.5, DEA 1 (v/v)Flow rate 8 ml/minDetector UV 254 nm

Column: 250x4.6 mm Eurocel 01, 5µm Eluent: MeOHFlow Rate: 1.0 ml/minTemp.: 25°CDetection:UV

Etozolin (ElkapinEtozolin (Elkapin®®))

0 3 6 9

Polar Organic

Mode

α: 1,54

Thiazolidone Derivative

Constipation Treatment

N

N

HN

NH

O

O

Br

Br

0 5 10 15 min

Dibromo Phakellstatine (R,S)

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

Flow Rate: 1.0 ml/minTemp.: 25°CDetection: UV

RP ModeRP Mode

α: 1,27

0 5 10 15 min

N-Methyl Ephedrine

Column: 250x4.6 mm Eurocel 01 Eluent: Hexane/IPA/DEA 80:20:0,1Flow Rate: 1.0 ml/minTemp.: 25°CDetection: UV

NP ModeNP Mode

α: 1,42

CH2

OH

CH

CH3

N

CH3

CH3

Peptides Purification

Reversed Phase C18 or C8, dependent on molecular sizeIon Exchange Chromatography

Peptide with 9 amino acids

Column: 120 x 16 mm Eurospher 100 C18 10 µm

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)

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mA

U

-250

0

250

500

750

1000

1250

1500

1750

2000

2250

mA

U

-250

0

250

500

750

1000

1250

1500

1750

2000

2250

UV Detectorpeptide

Peptides Purification

Reversed Phase C18 (Peptide overload)

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

mA

U

0

500

1000

1500

2000

2500

3000

3500

4000

mA

U

0

500

1000

1500

2000

2500

3000

3500

4000

UV Detectorpeptide

Separation of ProteinsSeparation of Proteins

Column: 120x4 mm Eurosil Bioselect 300-5 C4Eluent: A: H2O, 0.1 % TFA; B: MeCN, 0.1 % TFAGradient: 0% B to 20% B in 5 min

20% B to 100% B in 80 minFlow Rate:0.8 ml/minTemp.: ambientDetection: UV at 230 nmInj. Volume:20 µl

77 µg Ribonuclease94 µg Cytochrome C37 µg Lysozyme175 µg Katalase

Mixture in 20 µl water

0 10 20 30 40

Separation of MonosaccharidesSeparation of Monosaccharides

1 Sucrose2 Glucose3 Fructose4 Mannose5 Xylose6 Sorbitol

Column: 300x8 mm Eurokat PbEluent: H2O

Detection: Refractive Index

20 30 40 50

12

3

4

5

6

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

Summary

Isocratic and gradient modes (HPG, LPG) are possible.

Simple fraction collection can be achieved with the Knauer Valves K-6/12/16.

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