using HPLC process column (800 mm ((Lc Lc) x 200 mm ) x 200 mm ii.d. .d. (dc) (dc) (broken lines – fraction collection points)
NP
NP--w w chromatographic chromatographic system system
---- process process LC LC--chromatography chromatography --
---- process process LC LC--chromatography chromatography --
--Time Warunki NP-w
-- szczególnie korzystna produktywność rozdzielania w warunkach P-LC !
Fig 3. UV – 254 nm chromatogram of chloroform extract from Drosera aliciae obtained in preparative scale; A - NP-PLC conditions; B - RP-PLC conditions (see Tab. 1.);
Rośliny owadożerne -naftochinony
– plumbagina / ramantaceon
NP- EBF RP
in preparative scale; A - NP-PLC conditions; B - RP-PLC conditions (see Tab. 1.);
Symbols: R-ramentaceone, BF-backflush point of the eluent flow direction in the column.
Warunki NP-PLC RP-PLC
Column Lichrosorb Si 60 (silica gel 6 nm pore diameter), dp=10µm, 200 x 16.8 mm
Lichroprep RP-18 (10 nm), dp=12µm, 250 x 16.8 mm
Eluent n-hexane/methyl t-butyl ether 9/1 (v/v) methanol/water 8/2 (v/v)
Mobile phase flow rate 7mL/min. 5mL/min.
Sample concentration 100mg/ml 100mg/ml
Sample volume 500µl 150µl
Detector UV 254 UV 254
Recycling chromatography
... with „peak shaving“
Recycling of components until sufficient separation is
achieved
Simulation of a long column Advisable for a low separation factor
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 38
Recycle fraction
factor
Substances with a high
separation factor (to the target) are withdrawn in the first cycle Improved performance by withdrawal of the product fractions (peak shaving)
Recycling chromatography
Example (Paclitaxel purification)
Extraction
- Solvent extraction, Sohxlet - SFE (Supercritical CO2)
Extraction
- Solvent extraction, Sohxlet - SFE (Supercritical CO2)
Herbal or animal material
Herbal or animal material
crude extract crude extract Pre-Purification
Pre-Purification
8,48 10,19 11,31 14,11 15,25 16,6117,2318,03 18,9319,5719,92 20,7221,3922,24 23,04 24,2424,8825,12 26,00 27,2528,05 29,0429,4129,7330,37 31,31 32,80 34,08
0 5 10 15 20 25 30 35 40
Retention Time (min) 0,00
0,02 0,04 0,06 0,08
Intensity(AU) 8,48 10,19 11,31 14,11 15,25 16,6117,2318,03 18,9319,5719,92 20,7221,3922,24 23,04 24,2424,8825,12 26,00 27,2528,05 29,0429,4129,7330,37 31,31 32,80 34,08
0 5 10 15 20 25 30 35 40
Retention Time (min) 0,00
0,02 0,04 0,06 0,08
Intensity(AU) 28,00
1,2 1,4
28,00
1,2 1,4
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 39
Pre-Purification
- liquid/liquid-partitioning, solid phase extraction, silica
chromatography Pre-Purification
- liquid/liquid-partitioning, solid phase extraction, silica
chromatography
purified extract purified extract
Polishing
- chromatography in batch-, recycling- or SMB-mode
Polishing
- chromatography in batch-, recycling- or SMB-mode
pure product pure product
26,9927,2527,76 29,25
0 5 10 15 20 25 30 35 40
Retention Time (min) 0,0
0,1 0,2 0,3 0,4
Intensity(AU) 26,9927,2527,76 29,25
0 5 10 15 20 25 30 35 40
Retention Time (min) 0,0
0,1 0,2 0,3 0,4
Intensity(AU) 20,69 24,2124,5625,01 25,79 27,2027,47 28,7529,0129,5229,8130,29 32,88
0 5 10 15 20 25 30 35 40
Retention Time (min) 0,0
0,2 0,4 0,6 0,8 1,0 1,2
Intensity(AU) 20,69 24,2124,5625,01 25,79 27,2027,47 28,7529,0129,5229,8130,29 32,88
0 5 10 15 20 25 30 35 40
Retention Time (min) 0,0
0,2 0,4 0,6 0,8 1,0 1,2
Intensity(AU)
analytical chromatogram of a pre-purified Taxus extract
Chromatographic conditions:
Recycling chromatography
Example (Paclitaxel purification)
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 40
stationary phase: LiChrospher RP18, 5 µm column
dimension: 250 * 4 mm mobile phase: MeOH/water
60/40
Flow rate: 1.0 ml/min Detection: UV, 220 nm
Paclitaxel
Chromatographic conditions:
stationary phase: LiChroprep RP18, 15-25 µm column
dimension: 200 * 50 mm
Recycling chromatography
Example (Paclitaxel purification)
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 41
dimension: 200 * 50 mm mobile phase: MeOH/water
70/30
Flow rate: 150.0 ml/min Detection: UV, 210 nm
too short
retention
time for
sufficient
resolution
Chromatographic conditions:
stationary phase: LiChroprep RP18, 15-25 µm column
dimension: 200 * 50 mm mobile phase: MeOH/water
70/30
Recycling chromatography
Example (Paclitaxel purification)
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 42
70/30
Flow rate: 150.0 ml/min Detection: UV, 210 nm
Maximum fraction purity: 76%
Chromatographic conditions:
stationary phase: LiChrospher Si60 15 µm
column
dimension: 250 * 4 mm
mobile phase: Heptane/Dioxane 65/35
Recycling chromatography
Example (Paclitaxel purification)
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 43
65/35
Flow rate: 1.0 ml/min Detection: UV, 220 nm
recycling chromatogram of a pre-purified Taxus extract
Chromatographic conditions:
Stationary phase: LiChrospher Si 60, 10 µm
waste recycle w rec. w rec. w fraction w
Recycling chromatography
Example (Paclitaxel purification)
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 44
Stationary phase: LiChrospher Si 60, 10 µm Column type: Selfpacker NW 50
Column dimension: 220 * 50 mm
Mobile phase: EtAt/n-heptane 60/40 Flow rate: 80 ml/min
Detection: UV, 280 nm Feed concentration: 220 g/l (in Etat)
Injection: 20 ml
Cycle time: 75 min Run time with eluent
consumption: 42.4 min
In ideal conditions, transposition
from TLC to Flash Chromatography
Transposition, example 1 Flush Chromatography
Merck Chimie S.A.S.
25/11/2005 Page 45
Chromatography
should give such
results
Particle size comparisons
250000 300000
Test silice Si60 40-63 µm Test silice Si60 63-200 µm Test silice Si60 15-40 µm
Merck Chimie S.A.S.
25/11/2005 Page 46
0 50000 100000 150000 200000
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Przedmiot opracowania w Przedmiot opracowania w
PG w zakresie PG w zakresie aparatury
aparatury
„profesjonalnej” :
„profesjonalnej” :
-- Pompowy gradientowy Pompowy gradientowy moduł zasilania kolumny moduł zasilania kolumny
-- Komputerowy system Komputerowy system sterownia, rejestracji i sterownia, rejestracji i przetwarzania danych do przetwarzania danych do HPLC
HPLC
POMPOWY APARAT GRADIENTOWY POMPOWY APARAT GRADIENTOWY
moduł zasilania kolumny moduł zasilania kolumny HPLC z
HPLC z
oprogramowaniem oprogramowaniem sterownia programem sterownia programem elucji i przepływu eluentu elucji i przepływu eluentu
-- Projekt układu Projekt układu
wielowymiarowego wielowymiarowego modułu przełączania modułu przełączania kolumn i przepływu kolumn i przepływu zwrotnego eluentu w zwrotnego eluentu w kolumnie
kolumnie
Bez
Bez--pompowy moduł pompowy moduł
zasilania kolumny HPLC zasilania kolumny HPLC do pracy w warunkach do pracy w warunkach izokratycznych
izokratycznych
Bez
Bez--pompowy moduł pompowy moduł zasilania kolumny zasilania kolumny HPLC z
HPLC z
programowaniem programowaniem składu eluentu
składu eluentu -- do do pracy w warunkach pracy w warunkach elucji gradientowej elucji gradientowej
Aparaty
Aparaty bezpompowe bezpompowe do dydaktyki i nietrudnej analityki do dydaktyki i nietrudnej analityki -- do pracy w do pracy w warunkach izokratycznych
warunkach izokratycznych (po prawej) / (po prawej) / warunkach gradientowych warunkach gradientowych (po (po lewej)
lewej)
Pilotowa stacja rozdzielania
techniką
chromatografii cieczowej w
skali procesowej
Procesowa
instalacja PLC –
schemat ideowy
Chromatografia jako proces
ciągły ( MB / SMB)
Batch
Chromatography salad
SMB-Chromatography
basic principle
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 53
True Moving Bed
(TMB) salad
extract (A) feed raffinate (B)
SMB-Chromatography
basic principle
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 54
solid fluid
counter-current flow of solid and liquid feed is injected in the centre of the process
stronger retained component is carried with the solid stream less retained component is carried with the fluid stream
SMB
Simulated Moving Bed
SMB-Chromatography
running processes (enantioseparation)
Name: Levetiracetam Tetralon Escitalopram DOLE
Keppra® Zoloft® Cipralex®
Indication: antiepileptic antidepressant antidepressant CSE-inhibit.
CN
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 56
SMB-size: 100 + 45 60 2 * 80 30
Prod.amount: 145 to/a 45 to/a 160 to/a 10 to/a
Sales: 417 m€ (2004) 789 m$ (2003) 163 m€ (1-9/2004)
NH H
H Cl
Cl
O
N
F CN
N F
OH OH
CO2Et
Cyclosporines Cyclosporines
SP: Silica MP: ETAT New Process:
Batch separation + two SMB-separations
Isolation of Cyclosporine A
new process
Chromatography Workshop 03/06 (Poland)
Liquid
RAFFINATE ELUENT
FEED EXTRACT
Cyclosporines
A,U,L,B,C
Cyclosporines
D,G,A
Liquid
RAFFINATE ELUENT
FEED EXTRACT
Raffinate:
Cyclosporine
A
Raffinate:
Cyclosporine
A
RP Si
elution order
SMB-Chromatography
equipment (lab scale)
Chromatography Workshop
03/06 (Poland) © Merck KGaA Darmstadt/Germany Page 58