prepline sample purification systems
DESCRIPTION
Prepline Sample Purification Systems and Selected Applications Introduction Analytical vs Preparative Chromatography Components of Preparative HPLC Systems Proposed System Configurations Applications Summary OverviewTRANSCRIPT
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
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