Introduction to Over Pressured Layer Chromatography
By William Amoyal, CEO and Founder Disruptive Technologies
OPLC combines benefits of different separation techniques Automated analytical
Rapid preparative Parallel screening
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Slide 2
Principle of Over Pressured Layer Chromatography (OPLC) 22 tons
720 psi
50 bars Eluent in
Eluent out Teflon layer
HTSorb column
Metal base
20 cm
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Slide 3
Efficiency improves with higher external pressure
Plate height in µm
60 50
10 bar
40
25 bar
30
50 bar Nopt~15,000 for 17 cm column, 5µm particle size
20 10 0 0
0,2
0,4
0,6
0,8
1
1,2
1,4
Linear flow rate in mm/s
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Slide 4
Forced-flow pushes solvent linearly with time limiting diffusion and increasing resolution OPLC 16
OPLC
14
Distance, cm
HPTLC
12 10 8
TLC
6 4 2
0 0
20
Time, min
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40
60
Diffusion
Slide 5
The simple components of an OPLC system make the technique easy to use Cassette
OPLC layer
Pump Pressurisation chamber
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Off-line mode allows for the separation of multiple samples in a single run
Spotting samples
Inserting OPLC layer in cassette
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Detecting compounds by densitometry on cassette
Inserting cassette in pressurized chamber
Slide 8
On-line mode allows for the separation and collection of a single sample at a time pump solvent
Sample is injected or spotted
OSU 50 Detection and fractionation
sample injector
Eluent in
Separation on the layer
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Fractions elute out of the OPLC layer Slide 9
Scale-up of a separation from analytical to semi prep Once a separation has been developed at the analytical scale …
Sample : Dyes mixture
50 µL
250 µL
Sorbent : SiO2, 5 µm Flow rate : 1 mL/min Eluent: Toluene
… scaling up the sample size by keeping the same mobile phase and the same method is simple 20/02/2010
Slide 10
Over-running technique improves separation and simplifies fractionation
Separation is not limited by column length Over-running is used to « push » products further along the column and eventually elute and collect them
Initial 20/02/2010
1 CV
3 CV
5 CV Slide 11
Different cassette designs allow for different elution modes Standard 1D
Bi-directional
2D
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circular
Slide 12
Capacity of OPLC layers in function of size 1-10
1-20
Max. loading
50mg
100mg
5 cm
10 cm
1-50 (or 100 bidirect.)
200mg total
20 cm
# samples
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20 cm
Slide 13
OPLC is a reproducible technique Methyl paraben
Retention time (min.)
Peak area
Ethyl paraben
Propyl paraben
Butyl paraben
Mean
11.2
14.40
18.95
26.46
Standard deviation
0.094
0.118
0.158
0.197
CV%
0.81%
0.82%
0.84%
0.75%
Mean
1918
1574
1046
1192
22
16
13
17
1.16%
1.0%
1.27%
1.46%
Standard deviation CV%
Courtesy of Institut de Chimie Organique et Analytique, Prof. Dreux, Orleans, France
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Injection: 20 µL of a mixture of 4 parabens at a concentration of 100 µg/mL Column: OPLC layer, RP C18, 5x20 cm Mobile phase: ACN/Water 50/50 Detection: on-line UV at 254 nm (Shimadzu UV detector) Slide 18
Disruptive Technologies offers 2 solutions for high throughput screening and purification Personal OPLC50 OPLC stand alone instrument comprising a separation chamber and a solvent delivery pump for off-line preparative and semi-quantitative applications
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Personal OSU50
OPLC separation accessory that integrates in any HPLC system in place of the column for on-line preparative and quantitative applications
Slide 19
OPLC features and benefits
Speed and productivity
Up to 100 samples separated in 5-20 min off-line simultaneously 4 or 8 samples separated in 5-20 min on-line simultaneously No or little sample prep required
Easy scale-up
Visualization of all compounds
High resolution and reproducibility
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All retained compounds can be seen on the column All eluted samples are detected by any HPLC detector (e.g. UV/Vis, MS, NMR, Fluo, ELSD)
Column is under 50 bar pressure and eluent is forced through it Separations are automated
As simple as injecting more sample using same method developed at analytical scale Quantity of sample applied not limited by injection loops contrary to HPLC High capacity sorbent beds available to scale up to 10-200 mg level
Ease of use
OSU 50 integrates in any HPLC system OPLC 50 chromatograph menus to program methods are very simple
Low cost
Reasonable investment cost Inexpensive OPLC layers 10 to 1000x less solvent consumption than HPLC and less solvent disposal cost
Slide 20
Applications
Our focus is on a limited number of applications despite the broad range of possibilities Determination of impurities in pharmaceutical products and reaction mixtures Extraction of compounds of pharmacological interest from natural products Purification of reaction mixtures to extract the compound of interest for additional studies such as isomers (e.g. NMR, MS etc) Determination of breakdown components from stability testing in drugs Screening of multiple samples and standards in a single run in biological samples, natural products or reaction mixtures Determination of impurities in manufacturing vessels for cleaning validation procedures
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Slide 22
Screening for drugs of abuse substances in urine
TLC
OPLC
Pelander et Al, dept. of forensic medicine, Helsinki, Finland J. Liq. Chrom. & Rel. Techol., 24 (10), 1425-1434 (2001) 20/02/2010
Slide 23
Profiling of Da-Huang (Rhubarb) by OPLC vs TLC OPLC (2 cv solvent) Pet Ether/AcOEt/HCO2H (15:5:1) Densitometry @ 360 nm
High efficiency is maintained over a longer development distance … … even beyond 1 plate length of solvent
TLC Courtesy of Prof. Li Man Ling, Institute of Chinese Materia Medica, China Academy of Traditional Chinese Medicine, 2002.
50 20/02/2010
100
150 mm Slide 29
Separation of Scaligeria oils by OPLC
SSL 2 mL
SSL 4 mL
SF 2 mL
SF SR 4 mL 2 mL
SR NE231C NE231C 4 mL 2 mL 4 mL
(a) 1st run with hexane-Et2O, 95:5, v/v
(b) 2nd run with hexane
(C) 2nd run with hexane
UV 254 nm
UV 254 nm
vanillin-sulfuric acid, visible
Chromatogram of off-line OPLC separation of Scaligeria oils under UV and visible with (a) hexane-Et2O, (b) hexane, and (c) visualized by vanillin-sulfuric acid reagent. 2 and 4 µL of 16 and 18 mg/mL with 10mm band size of SSL, SF and SR oils were applied at 27mm measured from the bottom edge of the adsorbent layer. LA001 SiO2 layer 20x20 cm, 5 µm particle size, 500 µl/min, 4.4 mL of solvent used SSL: Scaligeria tripartita stems and leaves oil; SF: S. tripartita fruits oil; SR: S. tripartita roots
oil
Separation of Scaligeria oils by TLC Chromatogram of classical TLC separation of Scaligeria oils using hexane and Et2O(95:5, v/v). Oils were applied as a 20 mg/ml in 4 µL of hexane onto a silica TLC plate. The plate was visualized by vanillin-sulfuric acid reagent. SSL: Scaligeria tripartita stems and
leaves oil; SF: S. tripartita fruit oil; SR: S. tripartita root oil
Nurhayat Tabanca, Betul Demirci, K. Husnu Can Baser, Emil Mincsovics, Shabana I. Khand, Melissa R. Jacob, David E. Wedge Journal of Chromatography B, 850 (2007) 221–229 20/02/2010
Slide 31
Comparison of TLC and OPLC for the separation of Scaligeria oils
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Nonpolar compounds co-migrated together in the top of the classical TLC chromatogram while those compounds were well separated by OPLC OPLC gave better overall separation of Scaligeria oils than did classical TLC Longer separation distances increases zone capacity compared to classical TLC OPLC faster elution speeds limits the diffusion effects, making the separation of closely related compounds cleaner than classical TLC, thereby concentrating bioactive compounds into a smaller more compact zone or band, increasing compound/mm² and enhancing direct bioautography detection.
Slide 32
Chromatograms of OPLC and classical TLC separations CHCl3:MeOH (4:1)
Courtesy of Dr. Tabanca & Dr. Wedge, USDA 20/02/2010
Slide 33
Semi-preparative isolation of Chamomile oil by OPLC a-Bisabolol chamazulene Bisabolol oxydes
50 bar Hexane:ethyl acetate (90:10, v/v) Flow rate: 1000 mL/min Sample: 225 mg/250 mL Reagent: Vanillin-sulfuric acid
No need to scrape sample from the plate ! Courtesy of Dr. Tabanca & Dr. Wedge, USDA 20/02/2010
Slide 34
Separation of Erythroxylum catuaba by OPLC 7:3:0.5 CHCl3:MeOH:FA
OH HO
OH OH
OH O
OH
O O
O
OH
O O
O OH
OH
OH OH
OH
catuabin A (new) OH
cinchonain Ia OH
7:3:0.5 CHCl3:MeoH:FA
OH
OH
O
OH
O
OH
O
O
O
O
OH
OH OH
OH
OH
OH
HO
HO
OH
OH O OH
O OH OH
OH OH
cinchonain IIa Courtesy of Dr. Tabanca & Dr. Wedge, USDA
OH
kandelin A1 Submitted to Planta Medica
Slide 35
Off-line modeling of loading capacity of RP 18 layer using different amount of Pinot noir red wine extracts 366 nm
254 nm
trans-resveratrol
1
2
3
4
cis-resveratrol
1
2
3
4
Loaded volume /10 mm; 1, 4 µl; 2, 8 µl; 3, 12 µl; 4, 16 µl 20/02/2010
Slide 36
Visualization of synthetic intermediates to antibiotics in process chemistry Diastereo-isomers produced by catalytic hydrogenation of ketones Need rapid screening tool to aid process chemists define optimal conditions
Diastereo-mixture
Pure active isomer
OPLC for analysis and fractionation 20/02/2010
Slide 37
Over-running : Elution and fraction collection
A µ-preparative separation with 10mg was performed in about 60 minutes including fraction collection and control.
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Slide 38
OPLC is more selective than HPTLC for inprocess purity testing of Nandrolone
Eluent :
HPTLC
OPLC NP 5µm silica
50% Cyclohexane 25% EtOAc 25% CHCl3
Bagocsi, B; Fabian, D; Lauko, A; Mezei, M; Maho, S; Végh, Z; Ferenczi-Fodor, K J Planar Chromatog. 2002, 15, 252.
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Slide 43
OPLC selectivity is close to HPLC for inprocess purity testing of Nandrolone Access to column after separation allows derivatization with sulfuric acid at 120°C to detect compounds which lack chromophores or poorly absorb in the UV HPLC
OPLC
NP 5µm silica
Bagocsi, B; Fabian, D; Lauko, A; Mezei, M; Maho, S; Végh, Z; Ferenczi-Fodor, K J Planar Chromatog. 2002, 15, 252. 20/02/2010
Slide 44
Quantitative and high resolution analysis of cannabinoids
Separation of Cannabinoids by OPLC Mobile phase: Isooctane-Diethyl Ether
Densitogram of Cannabinoids test mixture comprising neutral cannabinoids, extract of hashish cannabis and the calibration curve Mobile phase: Toluene:Dioxane (90:10)
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Slide 47
High throughput analysis of sugars by OPLC
Separation of glucose in urine Stationary phase: NP, 5-10 µm Mobile phase: CH3CN:Water (85:15) Flow rate: 300 µL/min Total vol. 2.4 mL Sample: 1 µL
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Separation of carbohydrate standards Stationary phase: NP, 5-10 µm, Mobile phase: CH3CN:Water (85:15), Flow rate: 75 µL/min, Total vol. 2.35 mL, Sample: 1 µL (0.2 mg) of each sugar
Slide 53
2D separation of a complex natural product extract 2
After a 1D analysis, the column is taken out of the cassette, dried at 90°C and inserted again to do a second elution
>16 red spots, 3 green and 4 blue can be identified
One absorbent-non fluorescent black compound can be seen
This 2D indicates that one method is not enough to fractionate and collect the green compounds
366nm
1
254nm
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Slide 55
Fractionation of a crude plant extract by OPLC Fraction of interest is green fluorescent compound, typically recovered by Prep-TLC (elute, scratch, extract, filter, …..) 40mg deposited with a Desaga AS30 spotter Elution with 1 column volume of solvent Observe Continue elution to recover green compound in 96% Preparative run: 200 µl of chemical reaction mixture injected on OPLC layer RP18 5x20cm Flow rate: 270 µl/mn Detection: UV/Vis 307 nm Main component was collected
Analytical run: 20 µl of collected fraction was analyzed on HTSorb RP18 The main peak represents 98.5%
D Papillard, et al. LCGC, Application Book April 2003, 1. 20/02/2010
Slide 58
Detection of minor metabolites by OPLC-NMR by injecting up to 10x more pure urine than in HPLC 10x larger volume of crude urine can be injected without any prior prep vs HPLC In this case 500 µL in OPLC vs 70 µL max in HPLC was injected A maximum of 800 µL was successfully injected in OPLC
Thanks to its high sample loading capacity, OPLC can compensate NMR low sensitivity Possible to detect metabolites impossible to “see” with HPLC-NMR Sample: 500 µL of urine collected 4 h after drug intake Column: OPLC layer RP18, 6 µm 20x20 cm Eluent H2O/ACN 0,1%TFA (1% to 99%) Flow rate: 800 µl/min Gradient volume: 40 ml
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Slide 59
Universal separation and detection of sugars and fatty acids by OPLC-ELSD
Separation of carbohydrates by OPLC
Separation of fatty acids by OPLC
Sample 20 µL: 1-Xylose 0.3 mg/mL, 2-Arabinose 0.3 mg/mL, 3-Fructose 0.3 mg/mL, 4-Galactose 0.3 mg/mL, 5Sucrose 0.15 mg/mL, 6-Maltose 0.3 mg/mL, 7-Lactose 0.3 mg/mL
Sample 5 µL: 1-Linoleic acid 1.1 mg/mL, 2-Palmitic acid 2.5 mg/mL, 3-Stearic acid 2.5 mg/mL, 4-Arachnidic acid 1.9 mg/mL
Detection conditions: N2 flow = 1.5 L/min, T nebulizer = 50°C, T evaporator = 75°C
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Detection conditions: N2 flow = 1.6 L/min, T nebulizer = 30°C, T evaporator = 35°C
Slide 61
OPLC customers are : Synthesis chemists Natural products chemists
Medicinal chemists Chemical process development engineers Process R&D chemists Catalysis chemists Forensic scientists 20/02/2010
Slide 67
Some OPLC customer references
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Research Institute for Medicinal Plant, Hungary Nihon University, Japan St Stephen University, Hungary Plant Protection Institute, Hungary University of Tennessee, US Kossuth Lajos University, Hungary Fruit Growing Research Development, Hungary Meat Research Institute, Hungary Sklodovska University, Poland University of West Hungary Scynexis, US University of Ferrara, Italy Katholic Univeristy of Brussels, Belgium School of Pharmacy, Louvain la Neuve, Belgium Certech, Belgium Carlsberg, Denmark LVMH/Dior, France L’Oreal, France Charabot, France
Pharmaceutical Sciences Univ Toulouse, France Pharmaceutical Sciences Univ Rennes, France Pharmaceutical Sciences Univ Montpellier, France Pharmaceutical Sciences Univ Tours, France Pharmaceutical Sciences Univ Paris, France Pharmaceutical Sciences Univ Chatenay, France Pharmaceutical Sciences Univ Limoges, France Faculté des Sciences Lens, France University of Toulouse, France Faculté des Sciences Orléans, France Faculté des Sciences Rangueil, France
Slide 68
Some OPLC customer references
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Cirad Montpellier, France IRBD Nancy, France Pharmacie Centrale des Hôpitaux (Paris), France Serobiologiques/Cognis Labs, France CIRT Rennes, France Avanti Polar Lipids, US Archer Daniels Midland Company, US University of Helsinki, Finland Queens College of CUNY, US Bruker Analytik, Germany SKC, South Korea Bioventure 21, South Korea USDA Mississippi, US USDA Gainsville, Florida, US McGill University, Canada Richter Gedeon, Hungary GSK, France Pierre Fabre, France Ipsen-Beaufour, France
Sanofi Aventis, Lyon, France Sanofi Aventis, Vitry, France UCB Pharma, Belgium Lilly Development, Belgium Sanofi Synthelabo, US Barr Laboratories, US Wyeth Ayerst, US Pfizer, US Bristol-Myers Squibb, US Daewoog Pharma, South Korea
Slide 69
Conclusion
OPLC is a modern, fast, easy to use and cheap analytical and semipreparative technique that is complementary to TLC, HPTLC and HPLC 20/02/2010
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THANK YOU FOR YOUR ATTENTION
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Details of Disruptive Technologies
Contact: William Amoyal Disruptive Technologies 3 allée des camélias 94440 Villecresnes France Mobile: +33 6 98 64 98 81 Fax: +33 1 72 70 38 10 Email:
[email protected] Web: www.disruptechno.com
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