corrected reveleris app notebook 25-6-151).pdf · a full spectrum of purification solutions...
TRANSCRIPT
A Full Spectrum of Purification Solutions
Reveleris®
Technologies
�e puri�cation revolution continues...
Application Notebook
Table of Content
Sr. No. Title Pg. No.
Reveleris®
X2 Flash System in Natural Product Purification 1-11
1 Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves Extract 1
2 Separation and Isolation of α -Santalol and β -Snatalol from Sandalwood
Extract
2
3 Catechins from Green Tea Extract 3
4 Antioxidants in Caryophylli Flos (Dingxiang) Extract 4
5 Chromophoric and Nonchromophoric Compounds in Giant Knotweed
Rhizome
5
6 Ginsenosides from Red Panax Ginseng Extract 6
7 Puriication of Galla Chinensis (Wubeizi) 7
8 Isolation of Bioactive Diterpenoids from Tu-Jing-Pi Extract 8
9 Purification of Ferulic acid in Rhizoma Chuanxiong Extract with Greater
Recovery
9
10 Eugenol and Caryophyllene from Clove oil Extract with High Purity 10
Reveleris®
X2 Flash System in Non-chromophoric compound
Purification
12-18
11 Fast and Efficient Purification of a Mixture of Glycerides, Mono-, Di-, and
Tristearin
12
12 Isolation of Aminosugar, Acarbose along with its Sugar Components 13
13 Chromophoric and Non-chromophoric Component Isolation from Cat's
Claw Extract
14
14 Purification of Catechols in the Presence of Non-chromophoric Sugars 16
15 Fast and Efficient Purification of Sterols 17
16 Fast and Efficient Purification of Fatty Acid Methyl Esters (FAMEs) 18
Reveleris®
X2 Flash System in Drug Discovery 19-31
17 Purification of Conjugated Quercetin with Rutinose 19
18 Improved Isolation of Polar Compounds: Flavanone Glyciside Purification 20
19 Bile Acid Purification During Lead Generation in Drug Discovery 21
20 Anti-malarial Drug Purification in Drug Discovery 23
21 Isolation of Aminoglycoside Antibiotics in the Synthesis of Amikacin 24
22 Isolation of Valproic acid from Cyclodextrin during Encapsulation 25
23 Impurity Isolation During 16-ß Hydroxy boldenone Purification 26
24 Rapid Purification of a Diverse Range of Peptides 27
25 Mestranol Purification During Chemical Synthesis Using Reveleris
Navigator Software
28
26 Improved Purification Using Flash Chromatography During Drug
Purification
31
Reveleris®
Flash Cartridges for Purification 32-36
27 A New Wide Pore C18 Phase for Fast and Efficient Purification of Crude
Bacitracin
32
28 Loading Capacity of Reveleris®
C18 Flash Cartridges 33
29 A New Wide Pore C18 Phase for Fast and Efficient Purification of Peptides 34
Reveleris®
Prep System in Purification 37-50
30 Integrated Flash and Preparative LC Capabilities in a Single Instrument
Provide a Versatile Purification Platform
a. Vitamin E from Wheat Gram Oil
b. Citral from Lemongrass Oil
c. Eugenol from Clove Oil
37
31 Isolation of Capsaicin from Red Chili Using Reveleris®
Integrated Flash
and Preparative LC
42
32 Isolation of Stevioside & Rebaudioside-A Using Reveleris® Integrated
Flash and Preparative LC
44
33 Peptide Purification Using Reveleris®
Integrated Flash and Preparative LC 47
34 Complex Surfactant Purification Using Advanced Dual Detection
Preparative HPLC
49
Reveleris®
Solid Loader 51-54
35 One Step Extraction and Purification of Natural Products
a. Isolation of chlorogenic acid and caffeine from coffee bean
b. Isolation of Capsaicin from Red Chili
c. Isolation of Picroside I and Picroside II from Picrorhiza Kurroa
Rhizome
51
ELSD
UV 1
UV 2
6
16
9
0 6 123 9 15 18 21 Min.
0 5 7.5 10 152.5 12.5 17.5 20 Min.0 5 7.5 10 150 50 5 12.5 17.5 20 Min.0 5 7.5 10 152.5 12.5 17.5 20 Min.
Fraction 16
Fraction 9
Fraction 6
Isolation and Purifi cation of Flavonoids from Ginkgo Biloba Leaves Extract
®
Flow Rate:
Cartridge Equilibration:
Slope Detection:
ELSD Threshold: 3mV
UV Threshold:
UV1 Wavelength:
UV2 Wavelength: 280nm
Injection Type:
Solvent A:
Solvent B:
Gradient: Time:
%B: 0 0 50 100 100 0 0
®
Flash Chromatography Conditions:
Introduction
® ®
Conclusion
® ®
1
1
Separation and Isolation of -Santalol and -Snatalol from Sandalwood Extract
Flash Chromatography Conditions:
12 gram Reveleris cartridge
Flow Rate: 15mL/min
Cartridge equilibration: 4 min.
Slope detection: High
ELSD Threshold: 3mV
UV Threshold: 0.02AU
UV1 Wavelength: 254nm
UV2 Wavelength: 240nm
ELSD Carrier: Iso-propanol
Injection type: Liquid
Solvent A: Hexane
Solvent B: Ethyl AcetateSeparation of -Santalol and -Santalol in Sanadalwood by the Reveleris flash instrument.
Gradient: Time: 0 5 20 20 30 40 42
%B: 0 0 7 10 10 30 30
Introduction
Sandalwood oil is an essential oil obtained from the steam distillation and or alcohol extraction of chips and billets cut from the heartwood of
the Sandalwood (Santalum album) tree. Sandalwood oil is used in perfumes, cosmetics, and pharmaceuticals.
®
the two main non-chromophoric compounds, -Santalol and -Santalol, in Sandalwood.
Conclusion
® ®
-Santalol and
®
ELSD
UV 1
UV 2
0 14 21 35 49 Min.7 28 42
ELSD
UV 1
UV 2
0 14 21 35 49 Min.7 28 42
®
2
2
ELSD
UV 1
UV 2
0 4
7
6 10 142 8 12
12
16
16
18 Min.
0 4 6 10 142 8 12 16 18 Min.
Fraction 16
Fraction 12
Fraction 7
HPLC before flash
Catechins from Green Tea Extract
Flash Chromatography Conditions:
12 gram Reveleris RP C18 cartridge
Flow Rate: 30mL/min
Cartridge Equilibration: 3 min.
Slope Detection: Low
ELSD Threshold: 3mV
UV Threshold: 0.02AU
UV1 Wavelength: 254nm
UV2 Wavelength: 280nm
ELSD Carrier: Iso-propanol
Injection Type: Liquid
Solvent A: 0.5% Formic acid in Water
Solvent B: Methanol
Separation of catechins in green tea by flash chromatography.
HPLC Chromatograms of green tea fractions collected by Reveleris flash chromatography.
Gradient: Time: 0 2 15 16 17 18 19
%B: 0 0 50 100 100 0 0
Introduction
® ®
Conclusion
® ®
ELSD
UV 1
UV 2
0 4
7
6 10 142 8 12
12
16
16
18 Min.
®
0 4 6 10 142 8 12 16 18 Min.
Fraction 16
Fraction 12
Fraction 7
HPLC before flash
®
3
3
Extraction Conditions:
Extract Type: Pulverized
Weight: 2g
Extraction Solvent: Ethanol
Solvent Volume: 20mL
Ultra-sonication: 30 min
Flash Chromatography Conditions:
Cartridge: Reveleris® silica 12g
Solvent A: Hexane
Solvent B: Ethyl acetate
Flow Rate: 25mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 280nm
ELSD Carrier: Iso-propanol
Cartridge Equilibration: 5.0 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 0
2 5.0 5
3 8.7 56
4 1.7 83
5 0.6 100
Antioxidants in Caryophylli Flos (Dingxiang) Extract
Introduction
1
Conclusion
Natural product extracts have been shown to contain critical components that are both chromophoric and non-chromophoric. The Reveleris flash®
™
References
1467, (2000), pp. 1467-1469.
Figure: Separation of caryophylli flos extract shows the closely eluting non-chromophoric peak detected by the evaporative light scattering detector in the same run.
4
4
ELSD
UV 1
UV 2
0 4 6 10 142
3
5
8 12 16 Min.
Fraction 5
Fraction 3
HPLC before flash
0 4 6 10 142 8 12 16 Min.
Chromophoric and Nonchromophoric Compounds in Giant Knotweed Rhizome
Flash Chromatography Conditions:
12 gram Reveleris® RP C18 cartridge
Flow Rate: 30mL/min
Cartridge Equilibration: 3 min.
Slope Detection: Low
ELSD Threshold: 10mV
UV Threshold: 0.02AU
UV1 Wavelength: 254nm
UV2 Wavelength: 280nm
Injection Type: Solid
Solvent A: Water
Solvent B: Acetonitrile
chromatography.
HPLC Chromatograms of giant knotweed fractions collected by the Reveleris X2 flash system.®
Gradient: Time: 0 8.1 11.7 14.1 17 18 19
%B: 0 30 70 100 100 0 0
Introduction
Giant knotweed rhizome is a common medicinal plant in China, and is used for a range of purposes.
This application note shows the separation and isolation of the chromophoric and non-chromophoric compounds in giant knotweed
rhizome by using the Reveleris®
Conclusion
Using the proposed procedure with the Reveleris® ® cartridges, the separation and isolation of
chromophoric and non-chromophoric compounds in giant knotweed rhizome extract can be easily achieved.
5
5
Ginsenosides from Red Panax Ginseng Extract
ELSD
UV 1
UV 2
5
6 7
0 6 123 9 15 18 21 Min.
0 7.5 152.5 10 12.5 17.5 20 Min.0 5 7.5 152.5 10 12.5 17.5 20 Min.
Fraction 7
Fraction 6
Fraction 5
Flash Chromatography Conditions:
®
Flow Rate:
Cartridge Equilibration:
Slope Detection:
ELSD Threshold: 3mV
UV Threshold:
UV1 Wavelength:
UV2 Wavelength: 280nm
Injection Type:
Solvent A:
Solvent B:
®
Gradient: Time:
%B: 0 0 50 100 100 0 0
Introduction
® ®
Conclusion
® ®
Separation of ginsenosides in Red Panax ginseng extract by flash chromatography
HPLC Chromatograms of Red Panax ginseng fractions collected by Reveleris X2 flash chromatography
0 7.5 152.5 10 12.5 17.5 20 Min.000 55 7.7.57.57.7.57.557.7. 1515151522.52.52.2.52.55 10101010 12.512.512.12.512.55 17.517.5117.517.57.17.517.5517.57.7. 20 Min.20 Min.220 Min.20 Min.020 Min.20 Min.20 Min.20 Min.2220 Min.20 Min. Min20 Min.20 Min.0 . Min Min
Fraction 7Fraction Fraction
Fraction 6Fraction Fraction
Fraction 5Fraction Fraction
6
6
Extraction Conditions:
Extract Type: Pulverized
Weight: 6g
Extraction Solvent: Ethanol
Solvent Volume: 60mL
Ultra-sonication: 30 min
HPLC Chromatography Conditions:
HPLC System: Agilent 1100
Column: Alltima™ silica 5µm, 250 x 4.6mm,
Part No. 88171
Flow Rate: 1mL/min
Detector: UV@280nm
Mobile phase: A: 1% Formic acid in hexane
B: Ethyl acetate
Flash Chromatography Conditions:
Cartridge: Reveleris® silica 12g
Solvent A: Hexane
Solvent B: Ethyl acetate
Flow Rate: 25mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 280nm
Cartridge Equilibration: 4.0 min
Injection Type: Liquid
Figure : Fractions analyzed using HPLC show higher purity peaks as they are
well isolated during flash chromatography using the Reveleris® Silica cartridge.
Gradient Table
Step Time (min.) %B
1 0.0 0
2 15.0 100
3 5.0 100
Figure: The chromatogram shows the purification of Galla Chinensis
extract using the Reveleris® iES flash chromatography system.
Purification of Galla Chinensis (Wubeizi)
Introduction
Conclusion
Reveleris® cartridges provide greater loading capacity, allowing the use of smaller size cartridge beds and thus helping to reduce cost. As peaks are
™ detection technology using the Reveleris®
7
7
Figure : The chromatogram shows the purification of Tu-Jing-Pi extract using the Reveleris ® iES
flash chromatography system.
Extraction Conditions:
Extract type: Pulverized
Weight: 2g
Extraction Solvent: Ethanol
Solvent Volume: 20mL
Ultra-sonication: 30 min
Flash Chromatography Conditions:
Cartridge: Reveleris® silica 4g
Solvent A: Hexane
Solvent B: Ethyl acetate
Flow Rate: 18mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 280nm
Cartridge Equilibration: 5.0 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 0
2 0.6 0
3 2.6 25
4 3.2 40
5 3.8 100
6 1.8 100
Isolation of Bioactive Diterpenoids from Tu-Jing-Pi Extract
Introduction
1
Conclusion
The Reveleris® ™
References
and Drug Analysis, 14 (4), (2006), pp. 353-356.
8
8
Extraction Conditions:
Extract Type: Pulverized
Weight: 2g
Extraction Solvent: Ether
Solvent Volume: 40mL
Ultra-sonication: 20 min
Figure: Separation of Rhizoma chuanxiong extract shows the detection of peaks using the UV and the evaporative light scattering detectors.
Flash Chromatography Conditions:
Cartridge: Reveleris® silica 12g
Solvent A: Hexane
Solvent B: Ethyl acetate
Flow Rate: 25mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 280nm
Cartridge Equilibration: 3.0 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 0
2 3.0 0
3 8.8 5
4 0.5 6
5 2.6 33
6 5.2 98
7 2.6 98
Ferulic acid standard
Purified fraction
Ether extraction with Ferulic acid
Low levelcomponents
OH
O
OH
OCH3
Figure: Ferulic acid recovery = 97% (calculated by peak area)
HPLC Conditions:
Cartridge: Reveleris® silica 12g
Column: Alltima™ silica 5µm, 250 x 4.6mm
Mobile Phase A: 1% Formic acid in Hexane
Mobile Phase B: 1% Formic acid in
Ethyl acetate
Flow Rate: 1.0mL/min
UV Detector: 280nm
Gradient: Time: 0 10 20
%B: 0 50 100
Introduction
1
®
Conclusion
®
™
References
9
9 Purification of Ferulic acid in Rhizoma Chuanxiong Extract with Greater
Recovery
OCH3
OH
2 2CH CH=CH
CH3
CH3
H2C
H3C
Eugenol and Caryophyllene from Clove oil Extract with High Purity
Extraction Conditions:
Extract Type: Oil
™
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 media 12g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 30mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 3.0 min
Injection Type: Liquid
1. Eugenol 2. Caryophyllene
Gradient Table
Step Time (min.) %B
1 0.0 30
2 7.0 100
3 5.0 100
Eugenol
Caryophyllene
Introduction
1
various health disorders such as indigestion, cough, headaches, and stress.
is a rich source of eugenol and caryophyllene, both of which may contain medicinal properties. Besides its use in dentistry, it may be used for treating
Many plants contain “essential oils” extracted from them that have high boiling points and antibacterial properties. The clove oil extracted from cloves
Figure: Purification of clove oil extract shows peaks detected with the RevealX detection technology.
Figure: GC-MS analysis of the collected fractions of clove oil extract after flash chromatography identiies Eugenol (MW: 164) and Caryophyllene
(MW: 204) with greater than 99% purity.
10
10
Using the ELSD with UV allows for isolation of both chromophoric and non-chromophoric peaks in a
single run.
21
Natural product extracts have been shown to contain critical components that are both chromophoric and non-chromophoric. The Reveleris® ™ detection technology in having multiple peak detection capability and integrated
productive, even in the presence of low quantities of sample components.
1. Bioactive Natural Products; Detection, Isolation, and Structural Determination, 2nd edition; Colegate, S. M.; Molyneux, R. J.; CRC Press, Boca
Raton, Florida, 2008.
11
12
3
12
3
Tristearin
Introduction
three identical acyl chains. It is found in both plants and animals and may be used as a drug delivery vehicle for target drug compounds.
1
This application demonstrates purification of a mixture of glycerides (mono-, di-, and tristearin) using C18 and amino phase chemistries. The C18
fatty acyl chain of the analyte to determine separation. The amino phase cartridge can be used in a normal phase or a reversed phase mode,
depending on the solvents employed. When used in normal phase mode, the less polar compounds elute first, followed by the elution of the polar
- -
ones selectivity.
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 and Amino 12g
Solvent A: Acetonitrile
Solvent B: Methylene chloride
Flow Rate: 30mL/min
UV1 Wavelength: 210nm
UV2 Wavelength: 254nm
Cartridge Equilibration: 5.0 min
Injection Type: Solid
Gradient Table
Step Time (min.) %B
1 0.0 10
3 3.0 100
2 8.0 100
®
Gradient Table
Step Time (min.) %B
1 0.0 100
2 2.0 100
3 9.0 10
4 2.0 10
Compound ID:
1. Monostearin
2. Distearin
3. Tristearin
Figure : ®Separation of mono-, di-, and triglycerides using a Reveleris
amino cartridge. Note how altering polarity affects elution order.
Conclusion
Using the Reveleris X2 Flash Chromatography System with selective Reveleris cartridge phases, method development can be optimized for
compounds. The RevealX detection technology in the Reveleris X2 Flash Chromatography System helps chemists to isolate and purify lipid-
based compounds that are non-chromophoric with speed and high purity.
™
®
References
1. Medicinal natural products; a biosynthetic approach, 3rd edition; Dewick, P.; John Wiley & Sons, Inc., Hoboken, UK, 2009, pp 40-44.
®
Modifying®
Figure: Separation of mono-, di-, and triglycerides using a Reveleris C18 cartridge.
12
11 Fast and Effi cient Purifi cation of a Mixture of Glycerides, Mono-, Di-, and
Tristearin
OH
NH
OH
O
OH
OH
O
HO
O
OH
OH
OH
HO
HOHO
HO
HO
OO
HO
HO H
OHOH
OH
O
OH
OH
HO
HO
O
OH
OH
OH
HO
OO
Flash Chromatography Conditions:
Cartridge: Reveleris® amino 12g
Flow Rate: 36mL/min
Equilibration: 10.0 min
Run Length: 15.0 min
UV1 Wavelength: 220nm
UV2 Wavelength: 254nm
Solvent A: Water with 0.1% TFA
Solvent B: Acetonitrile
Gradient Table
Step Time (min.) %B
1 0.0 90
2 1.0 90
3 7.0 70
4 7.0 70
Acarbose
Maltose
Glucose Acarbose
™
during separation
Glucose Maltose
Isolation of Aminosugar, Acarbose along with its Sugar Components
Introduction
1
inhibitor than acarbose.
Conclusion
Using the Reveleris® amino phase cartridge and the RevealX™
®
References
1. Medicinal natural products; a biosynthetic approach, 3rd edition; Dewick, P.; John Wiley & Sons, Inc., Hoboken, New Jersey, 2009.
development of this drug with voglibose than valienamine leads to a more potent drug that has been found to be more effective as an enzyme
Figure: Using the ELSD of the RevealX detection technology, all three compounds can be detected
13
12
Extraction Conditions:
Extract type: Powder
Weight: 3g
Extraction Solvent: Methanol
Solvent Volume: 10mL
Ultra-sonication: 30 min at 60ºC
Figure: Using the Reveleris iES flash chromatography system and RevealX detection technology, ®
™ ®
shows the benefits of detecting peaks using the UV and the ELSD detectors.
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 media 12g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 30mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 5.0 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 30
2 3.0 30
3 3.0 100
4 4.0 100
Gradient Table
Step Time (min.) %B
1 0.0 30
2 3.0 30
3 9.0 100
4 1.0 100
Introduction
1
2
Cat’s Claw extract has been purified on a reversed phase Reveleris 12g cartridge. The chromatogram
Figure: Optimized gradient method improves resolution for higher purity fractions
14
13 Chromophoric and Non-chromophoric Component Isolation from Cat's
Claw Extract
Conclusion
by using the Reveleris ®
universal RevealX detection and the Reveleris Navigator software technology, chemists can isolate target compounds and low-level components
collected using ELSD isolate components with higher recovery and purity.Figure: Samples purified using UV signal only fails to detect non-chromophoric peaks leading to impure and diluted fractions. Fractions
15
™ ™®
Purification of Catechols in the Presence of Non-chromophoric Sugars
Introduction
1
Flash Chromatography Conditions:
Cartridge: Reveleris® amino 12g
Solvent A: Water
Solvent B: Acetonitrile
Flow Rate: 36mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
ELSD Carrier: Iso-propanol
Cartridge Equilibration: 3.0 min
Gradient Table
Step Time (min.) %B
1 0.0 99
2 1.0
3 3.0
4 4.0
99997070
Figure: RevealX technology is able to isolate the non-chromophoric sugars such as glucose in the
presence of dopamine and its precursor pyrocatechol.
™
Conclusion
™This works shows that by using high capacity amino and reversed phase flash cartridges along with universal RevealX detection technology,
chemists can separate sugar compounds that are polar from their reaction mixture saving time and labor when doing glycochemistry.
Using a non-chromophoric detector along with UV detection can help eliminate guesswork for the user during purification of carbohydrates, saving
time and sample. Isolating larger quantities of the target compound is fast with higher purity and recovery, when compared to traditional flash
chromatography systems.
16
14
DopaminePyrocatechol
Glucose
UV and ELSD collection
UV collection only
1
1
2
2
3
3
1) Ethyl Acetate
3) Cholesterol2) Cholesteryl Acetate
Fast and Effi cient Purifi cation of Sterols
Flash Chromatography Conditions:Cartridge: Reveleris® C18 media 12g
Solvent A: Acetonitrile
Solvent B: Methylene chloride
Flow Rate: 30mL/min
UV1 Wavelength: 210nm
UV2 Wavelength: 254nm
Cartridge Equilibration: 5.0 min
Injection Type: Solid
Gradient Table
Step Time (min.) %B
1 0.0 30
2 8.0 100
Figure :
Fractions are collected
Baseline drift masks peak 3.
Figure :
With RevealX™ detection
technology, the peaks can
be monitored by UV detector
ELSD, peak 3 is fully baseline
The integrated detection of RevealX detection technology gives confirmation of results by both UV and ELSD in a single run.
continuously when using
a UV detector only.
while fractions are collected based on the ELSD signal. With
Conclusion
The RevealX detection technology in the Reveleris X2 Flash Chromatography System allows chemists to isolate and purify sterol-based compounds®™
References
1. Davidson, M. H.; Toth, P. P.; Maki, K. C.; Therapeutic Lipidology: Phytosterolemia; Humana Press, Totowa, New Jersey, 2007, pp 291-319.
six
Introduction
1Sterols are waxy insoluble substances or lipids synthesized from acetyl coenzyme A (CoA). The most common sterol found among humans is
cholesterol; though there are other non-cholesterol sterols derived from plants (phytosterol). Cholesteryl ester is the major transport and storage
form of cholesterol in lipoprotein particles and most cells. Research work in sterols is of high interest to better understand human diseases such as
atherosclerosis and in development of novel drugs to treat coronary heart disease.
Separation and identification of cholesterol and cholesterol-based compounds is challenging. They lack chromophores and have poor response due
to higher background interference from certain organic solvents when detected at low wavelengths. Purification of such compounds may require
additional method development with other solvents or a ‘collect all’ approach, adding time to the purification process. This application shows the
benefit of RevealX detection technology for purifying a mixture of non-chromophoric sterols.
Flash Chromatography Conditions:
resolved.
™
™
17
15
Fast and Efficient Purification of Fatty Acid Methyl Esters (FAMEs)
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 media 12g
Solvent A: Methanol
Solvent B: Acetonitrile
Flow Rate: 30mL/min
UV1 Wavelength: 200nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 5.0 min
Injection Type: Solid
Gradient Table
Step Time (min.) %B
1 0.0 90
2 8.0 90
Figure : The methyl esters of the three fatty acids are separated using isocratic condition in
less than eight minutes. All three compounds lack a UV chromophore but are fully
detected by ELSD.
1
23
1. Methyl Myristate
2. Methyl Palmitate
3. Methyl Stearate
Introduction
Lipids play a major role in biological functions due to their presence in all cells. They are categorized into various classes and subclasses of molecules
based on their hydrophobic and hydrophilic elements. Fatty acids are commonly found in natural product extracts and have been shown to interfere with
noncellular assays. Fatty acid esters are fatty molecules containing nonpolar groups and may be used in drug development for lipid-based formulations.
Purifying complex mixtures containing non-chromophoric compounds such as lipids can be difficult. Traditional ultraviolet (UV) detection fails to detect
lipid targets and impurities that are present at low levels or lack chromophores, requiring a ‘collect all’ approach that can add significant time to the process.
With RevealX detection technology in the Reveleris X2 flash chromatography system, chemists can purify lipid-based compounds that are non-
chromophoric with speed and high purity.
1
®™
Conclusion
The RevealX detection technology in the Reveleris X2 Flash Chromatography System helps chemists to isolate and purify lipid-based compounds that®™
References
1. Eoin Fahy et al.; 2005. A comprehensive classification for lipids. J. Lipid Res. 46: 839-861.
to maximize purity. The separation is fast and efficient, resulting in three pure fractions within 8 minutes, meaning more time can be spent on discovery and less on purification.
®
18
16
OH
OH
O
O
OH
Glc-Rha
HO O
Flash Chromatography Conditions:
Cartridge: Reveleris® amino 12g
Flow Rate: 36mL/min
Equilibration:
Run Length:
UV1 Wavelength: 265nm
UV2 Wavelength: 220nm
Solvent A:
Solvent B:
Gradient Table
Step Time (min.) %B
1
2
3 70
4 70Rutin
Rhamnose
Quercetin
Glucose
Rutin
Purification of Conjugated Quercetin with Rutinose
Introduction
1
Conclusion
® ™
References
Using the Reveleris amino cartridge and the ELSD detector of the RevealX detection technology, sugar impurities from rutin can be isolated and
19
17
HO
HO H
OHOH
OH
OHO
OH
OMe
OH O
O
O
O
O
OH
OH
OH OH
HO
HO
HO
OMe
OH O
O
O
Improved Isolation of Polar Compounds: Flavanone Glyciside Purification
Flash Chromatography Conditions:
Cartridge: Reveleris® amino media 40g
Flow Rate: 40mL/min
Equilibration: 10.0 min
Run Length: 25.0 min
UV1 Wavelength: 220nm
UV2 Wavelength: 254nm
Solvent A: Water
Solvent B: Acetonitrile
Gradient Table
Step Time (min.) %B
1 0.0 99
2 2.0 99
3 10.0 70
4 13.0 70
Glucose Hesperitin Neohesperidin
Figure: Purification of neohesperidin isolates glucose in the mixture using the RevealX™
Hesperitin
Glucose Neohesperidin
Introduction
1
other sweeteners, such as aspartame, xylitol, and saccharin, for controlling the sugar uptake in human body.
® X2 flash chromatography system ™ ®
with UV detection can be isolated from the mixture.
Conclusion
Glycosidic compounds such as neohesperidin are not easily separated on a reversed phase column from other polar components while traditional
basic molecules and may be used in the separation of peptides, sugars, steroids, and triglycerides. Using the high capacity amino cartridge and the™ ®RevealX detection technology in the Reveleris X2 flash chromatography system, both polar and non-chromophoric compounds are separated and
References
1. Medicinal natural products; a biosynthetic approach, 3rd edition; Dewick, P.; John Wiley & Sons, Inc., Hoboken, New Jersey, 2009.
neohesperidin dihydrochalcones for diabetes related therapy. The compound is known to have a strong synergistic effect in combination with
with RevealX detection technology and the high capacity Reveleris amino cartridges, unreacted or even degraded reagents that may be undetected
20
18
detection technology
RevealX detection technology in the Reveleris X2 flash chromatography system, both polar and non-chromophoric compounds are
Bile Acid Purification During Lead Generation in Drug Discovery
Introduction
Bile acids, such as cholic acid, are steroidal acids secreted into the gastrointestinal tract to emulsify fats and encourage digestion. Hepatic conversion
of cholesterol into bile acids increases the breakdown of LDL cholesterol, adding value in treatment of coronary patients. Bile acids are starting materials
for the semi-synthesis of other medicinal steroids due to their availability as raw materials. Conjugation of cholic acid with glycine or taurine increases
Using the RevealX detection technology of the Reveleris X2 flash chromatography system and the Reveleris reversed-phase cartridge, chromophoric
and non-chromophoric target compounds can be separated and isolated in the presence of small impurities present in a chemical reaction by a single
chromatography run.
1
™
its water solubility for drug development.
® ®
(3) (1) (2)
NH2 CO2H
H
H
H
H
OH
OH
HOH
NH
O
CO2
-Na
+
H
H
H
H
OH
OH
H
OH
O
OH
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 12 g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 36mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 3.0 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 30
2 7.0 100
3 1.0 100
3
1
2
™ ®
1. Glycine 2mg2. Sodium Glycocholate 20mg3. Cholic acid 2mg
Figure: Using traditional flash chromatography system, non-chromophoric compounds have poor sensitivity or are undetected during purification.
21
19
RevealX detection technology of the Reveleris iES flash chromatography system can isolate such compounds using the ELSD in a reaction mixture during purification.
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 40 g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 40mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 3.0 min
Injection Type: Liquid
(3) (1) (2)
NH2
SO3H
H
H
H
H
OH
OH
H
OH
NH
O
SO3
-
H
H
H
H
OH
OH
H
OH
O
OH Na+
Gradient Table
Step Time (min.) %B
1 0.0 30
2 7.0 100
3 3.0 100
®
scattering detection of the RevealX detection technology.™
Figure: Non-chromophoric compounds on a larger scale are purified using the higher capacity Reveleris cartridge and the evaporative light
Conclusion
Using the high sensitivity Reveleris X2 flash chromatography system, all 3 components in a sample mixture have been separated and purified in a®
™
References
1. Medicinal natural products; a biosynthetic approach, 3rd edition; Dewick, P.; John Wiley & Sons, Inc., Hoboken, New Jersey, 2009.
with the UV detectors of the RevealX detection technology. Such isolation of compounds from their impurities provides accurate purity and quantity assessment, reducing post-run analysis time.
1. Taurine 1mg2. Sodium Taurocholate 400mg3. Cholic acid 30mg
22
1
2. Sodium Ta3. Cholic Acid 30 m
12
3
Figure 2: Non-chromophoric compounds on a larger scale are purified using the higher capacity Reveleris
SRC cartridge and the evaporative light scattering detection of the RevealX technology.
Conclusion:
Using the higher sensitivity Reveleris iES flash chromatography system, all 3 components in a sample
mixture have been separated and purified in a single run. Isolation of amounts as low as 1mg and as high as
400mg have been shown here using the ELSD along with the UV detectors of the RevealX technology.
Such isolation of compounds assists in further synthesis and development towards the target drug during
lead optimization in drug discovery.
mgurocholate 400 mg
Anti-malarial Drug Purification in Drug Discovery
Introduction
Cinchona and its alkaloids, such as quinine, have been used for the treatment of malaria, a disease caused by protozoa. A range of synthetic
anti-malarial drugs have been produced as alternatives to quinine. The emergence of Plasmodium falciparum strains resistant to the synthetic drugs
has resulted in reintroduction of quinine, such as the Primaquine with 8-aminoquinoline, in drug discovery.
1
(3) (2) (1)
Flash Chromatography Conditions:
N
NH2
+N
NH2
CH3O
N
NH N
CH3O
2 Amino-5-Diethylamino Pentane 20mg1.
2. 8-Amino-6-MethoxyQuinoline HBr 1mg
3. Primaquine 10mg
(1) (2) (3)
Gradient Table
Step Time (min.) %B
1 0.0 30
2 5.0 100
3 7.0 100
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 12g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 36mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 5.0 min.
Injection Type: Liquid
2
3
™ ®
Conclusion
Using the RevealX detection technology, separation of both chromophoric and non-chromophoric compounds can improve sample recovery and ™
References
1. Medicinal natural products; a biosynthetic approach, 3rd edition; Dewick, P.; John Wiley & Sons, Inc., Hoboken, New Jersey, 2009.
1
Figure: Using traditional flash chromatography system, non-chromophoric compounds have poor sensitivity or are undetected during purification.
23
20
RevealX detection technology of the Reveleris iES flash chromatography system can isolate such compounds using the ELSD in a reaction mixture during purification.
OH
OH
NH
HO
HO
H2N
O HO
HO
O
O
O
OH
HO
H2N
NH2
NH2
O
Isolation of Aminoglycoside Antibiotics in the Synthesis of Amikacin
Flash Chromatography Conditions:
Cartridge: Reveleris ® C18 12g
Flow Rate: 36mL/min
Equilibration: 5.0 min
Run Length: 10.0 min
UV1 Wavelength: 220nm
UV2 Wavelength:
Solvent A: Water with 0.1% TFA
Solvent B: Acetonitrile
Amikacin
Kanamycin
Glucose Amikacin
Gradient Table
Step Time (min.) %B
1 0.0 99
2 2.0 99
3 7.0 30
4 1.0 30
254nm
Introduction
Bacterial resistance to aminoglycoside antibiotics has been a problem leading to clinical failures. One such drug, kanamycin A, is a mixture of
aminoglycosides and has been used as a precursor to its semi-synthetic acyl derivative, amikacin. The later is a modification with 4-amino-2-
hydroxybutyryl group to protect against the enzymic deactivation while maintaining its activity.
1, 2
Conclusion
™
References
1. Medicinal natural products; a biosynthetic approach, 3rd edition; Dewick, P.; John Wiley & Sons, Inc., Hoboken, New Jersey, 2009.
ELSD of RevealX detection technology detects the non-chromophoric compounds and helps to isolate kanamycin A from amikacin and glucose present
in the mixture. The separation is done using a C18 reversed phase Reveleris cartridge with trifluroacetic acid as the modifier in the mobile phase.
2. Glycochemistry; principles, synthesis, and applications; Wang, P. G., Bertozzi, C. R.; Marcel Dekker, Inc., New York, 2001.
Figure: Purification of amikacin from its precursor, kanamycin A
24
21
®
Isolation of Valproic acid from Cyclodextrin during Encapsulation
Introduction
Valproic acid is one of the major antiepileptic drugs used in the treatment of different kinds of epilepsy.1 The drug has shown to be effective in treating
cancer due to its antitumor properties. Although water is usually used as the reaction medium for enzymes, organic solvents or cyclodextrins have
been added to improve the solubility of poorly soluble compounds. To increase its water solubility and bioavailability, the drug can be linked chemically
by physical entrapment with polymers such as cyclodextrin or to polymeric carriers such as dextran.
O
HO
Valproic acid -Cyclodextrin
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 12g
Solvent A: Water
Solvent B: Acetonitrile
Flow Rate: 36mL/min
UV1 Wavelength: 220nm
UV2 Wavelength: 254nm
Cartridge Equilibration: 3.0 min.
Gradient Table
Step Time (min.) %B
1 0.0 30
2 7.0 80Figure: In this application, the separation of valproic acid from its entrapment-carrier, -cyclodextrin,
®has been shown using the Reveleris flash cartridge with the RevealX detection technology.
reversed phase cartridge.Using a linear gradient with acetonitrile and water mobile phase, cyclodextrin is poorly retained on a
Flash Chromatography Conditions:
Cartridge: Reveleris® amino 12g
Solvent A: Water with 0.1 TFA
Solvent B: Acetonitrile
Flow Rate: 36mL/min
UV1 Wavelength: 212nm
UV2 Wavelength: 254nm
Cartridge Equilibration: 6.0 min.
%
Gradient Table
Step Time (min.) %B
1 0.0 90
2 1.0 90
3 4.0 70
4 7.0 70
Figure: Compared to a reversed phase cartridge separation as shown where the retention is based on the hydrophobic interaction, the water-soluble cyclodextrin has been retained and separated from
the free valproic acid using an amino phase media cartridge.
Conclusion
2
a key role in overcoming drug delivery challenges for those undeliverable molecules, nanospheres and nanocapsules designed as drug carriers can besynthesized with drugs linked through encapsulation or conjugation for better target delivery. There needs to be a better purification technique for isolation
water based encapsulated drug from its impurity either as free or degraded and unbound compounds from the reaction mixture.
References
1. Praveen, B., Shrivastava, P., Shrivastava, S. K.; In-Vitro release and pharmacological study of synthesized valproic acid-dextran conjugate;
Acta Pharmaceutica Sciencia, (2009), 51, pp. 169 – 176.
2. Vauthier, C. and Bouchemal, K.; Methods for the preparation and manufacture of polymeric nanoparticles; Pharmaceutical Research, (2008),
26, No. 5, pp. 1025 – 1058.
™
of these compounds when the drug is poorly soluble in water while its carrier is a polar molecule. Hence such a method helps medicinal chemists isolate
Polymer nanoparticles allow proper drug delivery and targeting with a wide range of properties under controlled conditions. As polymer technology plays
25
22
γ-Cyclodextrin
Valproic acid
γ-Cyclodextrin
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 12g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 36mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 3.0 min.
Injection Type: Liquid
®
™
16-
1. 16- Hydroxy boldenone
2. Impurity
Gradient Table
Step Time (min.) %B
1 0.0 20
2 7.0 100
3 5.0 100
Impurity Isolation During 16-ß Hydroxy boldenone Purification
Introduction
1Impurity isolation is critical during drug discovery. The purity of samples is compromised at times to meet the demands of increasing high throughputscreening. This can produce high false positive hit rates, resulting in waste of time and resources. Due to the stringent requirements from the ICH, all
impurities need to be identiied and reported, even at low levels.
Using the RevealX detection technology of the Reveleris X2 flash chromatography system, chromophoric and non-chromophoric target compounds
are isolated using multiple detection technology and advanced signal processing to recognize peaks and collect compounds during chromatography.
™ ®
Conclusion
™
References
1. Lead generation approaches in drug discovery; Rankovich, Z. and Morphy, R.; John Wiley & Sons, Inc. Hoboken, New Jersey, 2010.
Using the ELSD along with the dual UV detectors of the RevealX detection technology, isolation of impurities at low levels is possible during a
Figure: The Reveleris iES flash chromatography system shows the co-elution of an impurity peak as detected by the RevealX detection technology. Due to the high ELSD response for Hydroxy
chromatographic run.
26
23
boldenone, the non-chromophoric impuritycomponent is detected only by the secondary wavelength of the UV detector at 220nm.
1
2
d into the gut to
Rapid Purification of a Diverse Range of Peptides
Introduction
Newly discovered peptide sequences are providing novel drug development candidates for use in modern medicines. Purification of natural productsand synthetic peptides is an essential step in the drug discovery process, and is typically accomplished using preparative chromatography, which can
be expensive and time consuming. Flash chromatography is a fast and cost-efficient approach to purify synthetic peptides and other small molecules.Using flash chromatography can quickly increase overall purity of peptides prior to the next amino acid addition or final polishing on preparative HPLC.
This work demonstrates purification and recovery of a diverse range of peptides using a Reveleris X2 flash chromatography system with ELSD and
UV detection. The Reveleris C18 phase column shows high loading capacity and high resolution for peptide purification, allowing higher amounts of
peptides to be processed in a single injection compared to preparative HPLC. Automated flash chromatography reduces purification time and solvent
use compared to preparative HPLC.
®
®
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 12g
Solvent A: Water with 0.1 TFA
Solvent B: Acetonitrile with 0.05 TFA
Flow Rate: 25mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 210nm
Cartridge Equilibration: 3.0 min.
%
%
Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 2.5 30
4 2.0 30
5 3.0 100
6 2.0 100
0.0
0.0
0.5
1.0
1.5
2.0
3.0
2.5
0.5 1.0 1.5 2.0 2.5 3. 0 3 .5 4. 0 4 .5 5.0 Min.
0.0
0.0
0.2
0.4
0.8
1.0
1.4
1.2
0.5 1.0 1.5 2.0 2.5 3. 0 3 .5 4. 0 4 .5 5.0 Min.
Vancomycin Pre-purification HPLC Purity Check
Vancomycin Post-purification HPLC Purity Confirmation: >98%
HPLC Conditions:
Column: VisionHT HighLoad C18 1.5µm, 50 x 2.1mm
Mobile Phase A: Water with 0.1% TFA
Mobile Phase B: Acetonitrile with 0.1% TFA
Flow Rate: 0.2mL/min
UV Detector: 220nm
Gradient: Time: 0 5
%B: 5 100
AmbientColumn Temp:
Conclusion
A test mixture containing two large peptides (Vancomycin and Polymyxin B Sulfate) and a small amino acid (Taurine) demonstrated the ability of
flash purification to adequately resolve a mixture of peptides. The results show that flash purification using a 60Å, 40µm C18 media adequately
resolves the three diverse compounds with symmetrical peak shapes.
Another benefit of using a flash system with combined UV and evaporative light scattering detection (ELSD) is that unreacted amino acid
fragments and deletion products can be detected and collected. In this example, taurine is detected only by ELSD.
27
24
® HighLoad C18 1.5µm, 50 x 2.1mmVisionHT
Vancomycin
Taurine
Polymyxin B Sulfate
Introduction
Oestrogen compounds, along with progestogens, are the basis of combined oral contraceptives and hormone replacement therapy (HRT). Among
women, they are used to supplement natural oestrogen levels, suppress androgen formation in tumor growth of cancers, and are also used to help
provide protection against osteoporosis, heart attacks, and possibly Alzheimer’s disease.
Mestranol is one such compound which acts as a pro-drug that is formed from ethynylestradiol and methylsulfate. In this separation, mestranol can
be purified using a simple method optimized by the Reveleris Navigator software from the other two components that may be present in the
chemical reaction as impurities.
1
2
®
Step 1
Initially two separate isocratic runs at different methanol concentrations were carried out on an HPLC system using a Reveleris® C18 analytical
column. The retention times for the peaks to be resolved were obtained from the run. The chromatography conditions were as listed below.
1
1
2
2
3
3
1. Methyl Sulfate (RT: 1.46min, 1.26min)2. 17 - Ethynylestradiol (RT: 7.04min, 1.88min)3. Mestranol
HPLC Conditions:
Column: Reveleris® C18, 150 x 4.6mm
Mobile Phase A: Water 20% B: Methanol 80%
Flow Rate: 1.0mL/min
Detector 1: UV @ 254nm
Detector 2: ELSD (55º C, 1.5LPM, Gain 1)
HPLC Conditions:
Column: Reveleris® C18, 150 x 4.6mm
Mobile Phase A: Water 0% B: Methanol 100%
Flow Rate: 1.0mL/min
Detector 1: UV @ 254nm
Detector 2: ELSD (55º C, 1.5LPM, Gain 1)
Step 2
After selecting the appropriate HPLC conditions, the values for methanol solvent as percent B mobile phase concentration and the
retention times of the peak of interest were entered. The preferred flash column and optimize for purity were selected. Only the retention
times of the first two eluted peaks were used for flash chromatography optimization.
Mestranol Purification During Chemical Synthesis Using Reveleris Navigator
Software
®
28
25
™
Step 3
The Reveleris® Navigator software calculated the recommended gradient method to optimize for purity.
29
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 12g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 30mL/min
UV1 Wavelength: 220nm
UV2 Wavelength: 280nm
Cartridge Equilibration: 3.0 min
Injection Type: Liquid
®
appropriate gradient method for high purity isolation of all the three components that were present in the sample mixture.
Gradient Table
Step Time (min.) %B
1 0.0 64
2 0.9 64
3 6.0 88
4 3.5 88
17 – Ethynylestradiol(100%)
Mestranol (100%)
Figure : Analysis of mestranol and ethynylestradiol fractions show 100% purity using gas chromatography-mass spectrometry (GC-MS).
GC-MS Conditions:
AT-5ms (30m, 0.25mm, 0.25µm)
2µl injection at 250°C
30:1 split, 0.8mL/min He
Conclusion
Using the Reveleris Navigator software of the Reveleris flash chromatography system, mestranol in a sample mixture has been well resolved with
high purity from the other two components without any flash method development. Such an optimization tool minimized sample loss and eliminated
additional chromatographic runs using the flash system.
References
1. Medicinal natural products; a biosynthetic approach, 3rd edition; Dewick, P.; John Wiley & Sons, Inc., Hoboken, New Jersey, 2009.2. Lesniowski, A.; McCreary, D.; Anderson, S.; Lawrence, K.; Automating gradient method development in flash chromatography provides
productivity gains and minimizes solvent usage; ACS Fall 2010 poster, Boston, MA.
®
Figure: Using a traditional flash chromatography system, chromatography method optimization is dependent on the user. Reveleris Navigator software optimized the method purification by offering the
30
™
™®
1
23
1. Methyl Sulfate 2.5mg2. 17α - Ethynylestradiol 2.5mg3. Mestranol 2.5mg
Improved Purification Using Flash Chromatography During Drug Purification
Introduction
The Reveleris flash chromatography system detects and collects smaller quantities of compounds for scientists whose applications demand increased
lab productivity during purification in the drug discovery process.
Purification bottlenecks encountered when using flash chromatography can be eliminated by using the RevealX detection technology of the Reveleris
flash chromatography system, which independently triggers fraction collection from multiple detectors including UV and ELSD. Equipped with high
Using the Reveleris Navigator software optimizer tool helps minimize guesswork for the user during method development for flash chromatography,
helping to save both time and sample. Using a Reveleris flash chromatography system offers multiple benefits, such as having an integrated fraction
collector, user-friendly software, the Reveleris Navigator software optimizer tool, and higher capacity cartridges, making it simple and more productive
compared to a preparative chromatography system.
™ ®
™
®
®
®
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 media 40g
Solvent A: Water
Solvent B: Methanol
Flow Rate: 40mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
ELSD Carrier: Iso-propanol
Cartridge Equilibration: 5.6 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 80
2 6.0 80
Sample: 1mL injection of 10mg/mL
Estriol and Estradiol each
Preparative Chromatography:
Column: 250 x 22mm
Mobile phase: 20:80 Water:Methanol
Detector: ELSD (Gas: 1.5L/min,
Drift Tube: 40ºC, Gain: 1)
Sample: 1mL injection of 10mg/mL
Estriol and Estradiol each
Peaks lose baseline resolution on a preparative column compared to flash chromatography separation.
Recovery in mg
Flash Preparative
Estriol 7 5
Estradiol 5 2
Conclusion
Due to lower capacity of the preparative column, multiple injections are required to purify larger quantity of samples. Injected peaks begin to co-elute
on the standard reversed-phase preparative column, losing baseline resolution. With higher capacity flash cartridges, samples with solubility issues
can be purified in a single run using flash chromatography. Flash chromatography allows higher loading using a Reveleris cartridge over the
preparative column. At 100% purity, recovery of the two compounds was higher on the Reveleris flash chromatography system than on the preparative
system. Integrated RevealX detection technology with fraction collector made it less tedious and time consuming for collecting ELSD fractions.
®
®
™
®
31
26
performance flash cartridges and universal RevealX detection technology, chemists can isolate target compounds and low-level impurities from their
chemical mixture, saving time and labor.
™
™
Introduction
Bacitracin is a polypeptide antibiotic with a molecular weight of 1422. It is typically available as a mixture of several closely related polypeptides. Here
we show the purification of the main component of the peptide mixture, Bacitracin A, on the Reveleris C18-WP flash cartridge. Bacitracin A is present
in the mixed peptide sample in 71% amount.
®
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 WP 12g
Solvent A: Water with 0.1 TFA
Solvent B: Acetonitrile
Flow Rate: 20mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 220nm
Cartridge Equilibration: 3.0 min.
%
Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 8.0 40
4 3.0 40
5 2.0 100
6 2.0 100
HPLC Conditions:
Column: VisionHT HighLoad C18 3µm, 150 x 4.6mm
Mobile Phase A: Water with 0.1% TFA
Mobile Phase B: Acetonitrile with 0.05% TFA
Flow Rate: 1mL/min
UV Detector: 254nm
Gradient: Time: 0 10
%B: 5 100
AmbientColumn Temp:
Conclusion
The combination of automated flash chromatography and a wide pore C18 flash cartridge can help to make peptide purification a much easier step in
the peptide synthesis process.
The results show that the new wide pore media can be successfully used to purify Bacitracin. After purification, Bacitracin A was recovered in greater
than 99% purity with a yield of 86%.
A New Reveleris Wide Pore C18 Phase for Fast and Efficient Purification of
Crude Bacitracin
®
32
27
®VisionHTVisionHT HighLoad C18 3µm, 150 x 4.6mm HighLoad C18 3µm, 150 x 4.6mm HighLoad C18 3µm, 150 x 4.6mm Crude 71.7% purity
Fr. 4 79.9% purity
Fr. 5 99.5% purity
Loading Capacity of Reveleris C18 Flash Cartridges
Introduction
®
Flash chromatography is a fast and cost-efficient approach to purify synthetic peptides and other small molecules. To test loading capacity, variousamounts between 30mg - 1g of Polymyxin B Sulfate, a polypeptide antibiotic, were loaded on a 12g Reveleris C18, 60Å, 40µm flash cartridge.Typical prep HPLC loading is between 0.1- 1.0% of media bed mass. Loading capacity in flash purification is evaluated between 0.1 – 10% of flash
cartridge media bed mass.
Flash Chromatography Conditions:
Cartridge: Reveleris® C18 12g
Solvent A: Water with 0.1% TFA
Solvent B: Acetonitrile with 0.05% TFA
Flow Rate: 30mL/min
UV1 Wavelength: 254nm
UV2 Wavelength: 210nm
ELSD Carrier: Iso-propanol
Cartridge Equilibration: 3.0 min
Gradient Table
Step Time (min.) %B
1 0.0 5
2 7.0 100
3 3.0 100
30mg 27mg 90
52mg 87
120mg 119mg 99
250mg 242mg 97
500mg 95
1g 923mg 92
Conclusion
The data has shown that samples can be loaded in quantities up to 10% of the flash media bed weight and be successfully purified using flash
chromatography. This means that a higher quantity of crude peptide can be processed in a single injection using flash purification in less time than
using prep HPLC alone, which often requires multiple injections.
Another benefit of automated flash chromatography is the ability to use dry load sample injection techniques to enable higher loads for low solubility
samples.
®
33
28
Introduction
Peptides and proteins are becoming increasingly popular for their potential use as therapeutic drugs. To earn and maintain a share in the fast-growing
peptide market, peptide researchers and manufacturers are constantly trying to improve and optimize the various steps in peptide synthesis. A new
wide pore C18 phase expands flash purification capabilities to larger sized peptides, while providing better resolution. We present data to show the
benefits of higher loading and faster purifications in peptide purification. This rapid purification technique helps ensure less degradation of peptides
and provides better recovery, yields and purity.
Comparison of peptide retention and peak shapes
The below chromatograms show the comparison of peptide retention and peak shapes on Reveleris C18 flash cartridge and Reveleris C18-WP
cartridge. Representative chromatograms of small, medium and large sized peptide/protein are shown.
® ®
Lysozyme (MW 14700)
Bovine Serum Albumin (MW 66000)
Z-Gly-Phe (MW 357)
Reveleris® C18 WP cartridge
Reveleris® C18 WP cartridge
Reveleris® C18 WP cartridge
Reveleris® C18 cartridge
Reveleris® C18 cartridge
Reveleris® C18 cartridge
Loading Capacity Evaluation
Loading capacity is a very important consideration for a purification media. Here we evaluated the loading capacity of Reveleris C18 WP media
with a small peptide (Polymyxin B sulfate), as well as a larger protein (Lysozyme from egg). To test loading capacity, we loaded between 50 mg –
600 mg of Polymyxin B Sulfate, a polypeptide antibiotic, and 50 mg – 600 mg of Lysozyme, on a 12 g Reveleris C18 WP flash cartridge.
®
®
A New Reveleris Wide Pore C18 Phase Column for Fast and Efficient
Purification of Peptides
®
34
29
Polymyxin B Sulfate Loading Study (MW 1385)
Reveleris® C18 WP cartridge
50 mg Load
300 mg Load
Reveleris® C18 WP cartridge
600 mg Load
Reveleris® C18 WP cartridge
Cartridge: Reveleris ® C18 WP flash
cartridges, 12g
Flow Rate: 20 mL/min
Equilibration: 5.0 min
Run Length: 10.0 min
UV1 Wavelength: 254 nm
UV2 Wavelength: 220 nm
Solvent A: Water with 0.1% TFA
Solvent B: Acetonitrile
Flash Chromatography Conditions:
Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 3.0 50
4 3.0 100
5 2.0 100
50mg 38mg 76
89mg 89
150mg 141mg 94
300mg 276mg 92
600mg 77
Lysozyme Loading Study (MW 14700)
®
50 mg Load
300 mg Load
600 mg Load
Cartridge: Reveleris ® C18 WP flash
cartridges, 12g
Flow Rate: 20 mL/min
Equilibration: 3.0 min
Run Length: 12.0 min
UV1 Wavelength: 254 nm
UV2 Wavelength: 220 nm
Solvent A: Water with 0.1% TFA
Solvent B: Acetonitrile
Flash Chromatography Conditions:
Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 7.0 100
4 3.0 100
50mg 42mg 84
93mg 93
150mg 142mg 95
300mg 280mg 93
600mg 90
Reveleris C18 WP cartridge
® Reveleris C18 WP cartridge
® Reveleris C18 WP cartridge
35
Improvment in Resolution
Peptides are typically difficult to purify by liquid chromatography due to the presence of closely eluting impurities. High resolution is a highly valued
criterion for peptide media. The new Reveleris C18-WP flash cartridge is efficiently packed with smaller particle size media than regular flash cartridges.
As a result, the sample resolution is much better than typically observed in flash chromatography.
The below examples show the improved resolution of crude peptides on Reveleris C18-WP flash cartridge, in comparison with Reveleris C18 flash
®
cartridges.
® ®
Obestatin, mouse (crude peptide sample)
Reveleris C18 WP cartridge ® ® Reveleris C18 cartridge
Analytical Conditions Column: VisionHT High Load ®
C18 Column, 3 m, 4.6 x 150mm
Solvent A: Water + 0.1% TFA
Solvent B: Acetonitrile + 0.05% TFA Flow Rate: 1.0ml/min
UV: 220 nm
Cartridge:®
Reveleris C18 flash cartridges, 12g
Flow Rate: 20 mL/min
Equilibration: 3.0 min
Run Length: 12.0 min
UV1 Wavelength: 254 nm
UV2 Wavelength: 220 nm
Solvent A: Water with 0.1% TFA
Solvent B: Acetonitrile
Flash Chromatography Conditions:Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 7.0 100
4 3.0 100
Mixture of Vancomycin and Polymyxin B sulfate
®
Analytical Conditions Column: VisionHT® High Load
C18 Column, 3 m, 4.6 x 150mm
Solvent A: Water + 0.1% TFA
Solvent B: Acetonitrile + 0.05% TFA Flow Rate: 1.0ml/min
UV: 220 nm
Cartridge:®
Reveleris C18 flash cartridges, 12g
Flow Rate: 20 mL/min
Equilibration: 3.0 min
Run Length: 12.0 min
UV1 Wavelength: 254 nm
UV2 Wavelength: 220 nm
Solvent A: Water with 0.1% TFA
Solvent B: Acetonitrile
Flash Chromatography Conditions:Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 2.5 30
4 2.0 30
Reveleris® C18 WP cartridge
5 2.5 100
6 2.0 100
Reveleris C18 cartridge
Conclusion
The results show that the new wide pore media can be successfully used to purify both small and large peptides and proteins (approaching 70000
MW). The data shows that samples can be loaded in quantities up to 5% of the flash media bed weight and be successfully purified using flash
chromatography. The high loading capacity translates to better productivity as larger amounts of crude peptides can be processed using flash
purification, and in less time than preparative HPLC. Due to the smaller particle size media, the improved resolution makes separation of complex
peptide mixtures much more efficient. The combination of automated flash chromatography and a wide pore C18 flash cartridge can help to make
peptide purification a much easier step in the peptide synthesis process.
®
Reveleris C18 WP and
®
Reveleris C18 WP and
36
Integrated Flash and Preparative LC Capabilities in a Single Instrument
Provide a Versatile Purification Platform
Introduction
Delivering large quantities of high purity compounds in the shortest possible time is the goal of a purification chemist. Two of the most popular
purification techniques are Automated Flash Column Chromatography (AFCC) and Preparative LC. Traditionally, AFCC is characterized by the
ability to load large amounts of material and short purification times, while Preparative LC is valued for high resolution separations resulting in very
pure products. As a result, AFCC is typically used as a complementary technique whereby the crude sample is enhanced to a higher level of
purity before final purification using Preparative LC.
With recent advancements in instrumentation and cartridge technologies, AFCC has become a highly versatile and efficient purification technique
that can provide similar levels of purity and recovery to that of Preparative LC. Depending on the purification needs, AFCC can be used on its own
to provide high quality purifications at low cost, or used as a complementary technique together with Preparative LC to enhance purity.
In this study, we demonstrate the performance benefits of a new purification system that combines flash and preparative LC capabilities in a single
instrument. The flexibility of this new dual-mode purification system allows the user to perform either flash or preparative LC or to combine both
techniques to achieve the highest levels of purity and recovery.
Complex Essential Oil PurificationEssential oils are complex hydrophobic liquids formed by plants as secondary metabolites. They constitute heterogeneous mixtures that may contain
dozens of compounds at different concentrations, with each essential oil typically designated by its major component(s). Isolation and purification of
these molecules can be challenging and tedious, often requiring multiple steps and large quantities of organic solvents. These challenging compounds
make an ideal test system for evaluating the purifi cation and recovery performance of active components from essential oils using the Reveleris Prep
purification system.
®
Isolation of Vitamin E from Wheat Germ oil
Vitamin E is a powerful antioxidant that helps slow down processes that can cause cell damage from free radicals. It is also used for treating and
preventing heart disease and is sometimes used for improving physical endurance, improving muscle strength and increasing energy. Wheat germ
oil contains one of the most concentrated forms of natural Vitamin E available. Various approaches to the purification of Vitamin E using preparative
LC, often require multiple steps and large amounts of solvent. Using flash chromatography can help save time and be a cost effective approach
to purifying Vitamin E from Wheat germ oil. The purification of Vitamin E from wheat germ oil was performed on a conventional preparative system.
1-3
Cartridge: ®
Solvent A: Petroleum ether
Solvent B: 10% IPA in Petroleum ether
Flow Rate: 25mL/min
Detector:ELSD and UV @ 210,254,292nm
Cartridge Equilibration: 3 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 0
2 8.0 8
3 0.5 100
4 1.0 100
®Reveleris Prep System:
Flash Mode Conditions
Sample: Wheat Germ oil
Reveleris Silica cartridge 12g
HPLC Conditions:
Column: VisionHT C18 3µm, 150 x 4.6mm
Mobile Phase A: Water
Mobile Phase B: Methanol
Flow Rate: 0.9mL/min
UV Detector: 292nm
21 minRun Time:
Gradient Table
Step Time (min.) %B
1 0.0 90
2 10.0 100
3 20.0 100
4 21.0 90
Figure. HPLC analysis of Vitamin E standard and Wheat Germ oil sample before purification.
®
®
37
30
Figure. Purification of Wheat Germ oil using Reveleris flash chromatography system.
Fraction 16 and 17 corresponds to vitamin E.
The results show that acceptable purity and recovery of Vitamin E are attainable using flash purification alone. Using flash purification instead of
preparative LC purification can help save you time, use less solvent, resulting in lower costs and greater productivity.
Gradient Table
Step Time (min.) %B
1 0.0 0
2 3.0 20
3 27.0 55
4 5.0 100
5 5.0 100
Purification Technique Vitamin E (0.66mg/g) from Wheat Germ oil
Purity (%) Recovery (mg/g)
99.1 0.61
95.1 0.61
Conventional Prep System: 97.7 0.64
Table: Summary of purities and recoveries of Vitamin E for the three different purification techniques.
Evaluation of Purification Types
Reveleris Flash Mode:
Reveleris Prep Mode:
®
®
Figure. Preparative purification of Wheat Germ oil. Fraction 4 and 5 corresponds to vitamin E.
Gradient Table
Step Time (min.) %B
1 0.0 0
2 3.0 20
3 30.0 55
4 35.0 100
®
Isolation of Citral from Lemongrass oil
4
Citral is a component of lemongrass oil and is commonly used as a flavor enhancer in foods and beverages, and also used in cosmetics such as body
lotions and perfumes. Lemongrass oil contains several monoterpenes, with citral being the major component, present at levels between 65-85%. Citral
(3,7-dimethyl-2,6-octadienal) is the name given to a natural mixture of two isomeric acyclic monoterpene aldehydes: geranial (trans-citral, citral A)
and neral (cis-citral, citral B). In addition to citral, the lemongrass oil consists of small quantities of geraniol, geranylacetate, and monoterpene olefins,
such as myrcene.
Column: ®
Solvent A: n-Hexane
Solvent B: 20% Ethyl acetate in n-hexane
Flow Rate: 15mL/min
Detector:ELSD and UV @ 210,254,292nm
Prep Conditions
Sample: Wheat Germ oil
Davisil NP 710NW column, 2.5 x 28cm (10-14µ)
HPLC Conditions:
Column: Alltima C18 5µm, 250 x 4.6mm
Mobile Phase A: Water
Mobile Phase B: Acetonitrile
Flow Rate: 1mL/min
UV Detector: 210nm
21 minRun Time:
Gradient Table
Step Time (min.) %B
1 0.0 30
2 15.0 100
3 21.0 100
4 21.10 30
®
Figure. HPLC analysis of Citral standard and Lemongrass oil sample before purification.
38
Figure. Purification of Wheat Germ oil using Reveleris Preparative chromatography system. Fraction 5 and 6 corresponds to vitamin E.
min5 10 15 20 25 30
mAU
0
100
200
300
400
500
600
8.436
9.863
11.71
1
17.89
0 18
.452
20.86
5
25.16
2
26.65
8
27.70
0
fraction 5fraction 4
fraction 3
fraction 2fraction 1
purification instrument compared to conventional preparative LC purification. In this protocol, flash purification alone also gave satisfactory results.Result shows that high-quality purity and high-percentage recovery for citral from lemongrass oil is achieved using Reveleris preparative LC ®
Cartridge: ®
Solvent A: Petroleum ether
Solvent B: 20% EtOAc in Petroleum ether
Flow Rate: 15mL/min
Detector:ELSD and UV @ 210,240,254nm
Cartridge Equilibration: 3 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 1
2 8.0 18
3 0.5 100
4 3.0 100
®Reveleris Prep System:
Flash Mode Conditions
Sample: Lemongrass Oil
Reveleris Silica cartridge 4g
®
Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 26.0 30
4 30.0 30
Figure. Preparative purification of Lemongrass oil.
®Column: ®
Solvent A: n-Hexane
Solvent B: 20% Ethyl acetate in n-hexane
Flow Rate: 15mL/min
Detector:ELSD and UV @ 210,254,280nm
Prep Conditions
Sample: Lemongrass oil
Davisil NP 710NW column, 2.5 x 28cm (10-14µ)
Gradient Table
Step Time (min.) %B
1 0.0 0
2 2.0 0
3 28.0 30
4 47.0 30
5 10 15 20 25 30 35 40 45
0
500
1000
1500
2000
2500
3000
9.3
94 9
.916
10.
481
11.
744
13.
558
14.
756
24.
990
25.
435
25.
823
26.
088
26.
857
28.
103
29.
192
30.
131
30.
489
31.
469
32.
648
33.
074
33.
886
35.
412
36.
595
39.
339
Purification Technique Citral (391mg/g) from Lemongrass oil
Purity (%) Recovery (mg/g)
98.8 360
99.8 388
Conventional Prep System: 96.3 373
Table: Summary of purities and recoveries of Citral for the three different purification techniques.
Evaluation of Purification Types
Reveleris Flash Mode:
Reveleris Prep Mode:
®
®
39
Figure. Purification of Lemongrass oil using Reveleris flash chromatography system. Fraction 45 corresponds to Citral.
Figure. Purification of Lemongrass oil using Reveleris Preparative chromatography system. Fractions 21-27 corresponds to Citral.
Isolation of Eugenol from Clove Oil
Clove oil has been claimed to have antimicrobial, antioxidant, antifungal, and antiviral activity. It is also said to possess anti-inflammatory, cytotoxic,
insect repellent, and anesthetic properties. The main constituents of clove oil are phenylpropanoids, including eugenol, eugenol acetate, caryophyllene
and thymol.5HPLC Conditions:
Column: Alltima C18 5µm, 250 x 4.6mm
Mobile Phase A: Water
Mobile Phase B: Acetonitrile
Flow Rate: 1mL/min
UV Detector: 210nm
26 minRun Time:
Gradient Table
Step Time (min.) %B
1 0.0 30
2 15.0 100
3 25.0 100
4 26.0 30
®
Figure. HPLC analysis of Eugenol standard and Clove oil sample before purification.
Cartridge: ®
Solvent A: Petroleum ether
Solvent B: 20% EtOAc in Petroleum ether
Flow Rate: 25mL/min
Detector:ELSD and UV @ 210,254,282nm
Cartridge Equilibration: 3 min
Injection Type: Liquid
Gradient Table
Step Time (min.) %B
1 0.0 2
2 4.0 20
3 2.0 20
4 1.0 60
®Reveleris Prep System:
Flash Mode Conditions
Sample: Clove oil
Reveleris Silica cartridge 12g
Gradient Table
Step Time (min.) %B
1 0.0 10
2 10.0 60
3 10.0 78
4 2.0 100
5 2.0 60
6 1.0 100
7 5.0 100
Column: ®
Solvent A: n-Hexane
Solvent B: 20% Ethyl acetate in n-hexane
Flow Rate: 15mL/min
Detector:ELSD and UV @ 210,254,280nm
Prep Conditions
Sample: Clove oil
Davisil NP 710NW column, 2.5 x 28cm (10-14µ)
®
®
5 15.0 100
40
Figure. Purification of Clove oil using Reveleris flash chromatography system.
Fractions 21-23 corresponds to Eugenol.
Figure. Purification of Clove oil using Reveleris Preparative chromatography system.
Fraction 32 corresponds to Eugenol.
min5 10 15 20 25 30
mAU
0
200
400
600
800
1000
8.5
47
9.1
87
10
.09
2
12
.01
1
13
.41
8
14
.62
6
15
.67
3
18
.70
2
20
.64
6
25
.51
9 2
5.5
76
27
.69
2
29
.23
9
fraction 1
fraction 2
fraction 3
Gradient Table
Step Time (min.) %B
1 0.0 10
2 10.0 60
3 20.0 78
4 22.0 100
5 33.0 100
Figure. Preparative purification of Clove oil. Fraction 2 corresponds to Eugenol.
Purification Technique Eugenol (99.9mg/g) from Clove oil
Purity (%) Recovery (mg/g)
99.2 97.5
94.9 88.1
Conventional Prep System: 99.1 88.5
Table: Summary of purities and recoveries of Eugenol for the three different purification techniques.
Evaluation of Purification Types
Reveleris Flash Mode:
Reveleris Prep Mode:
®
®
Flash purification ensures the maximum recovery and purity of eugenol, favorably similar or better than the results obtained by using preparative
purification alone. In addition, using flash purification instead of preparative purification uses less solvent and reduces overall purification time.
References
1. Bruns, A.; Berg, D.; Werner-Busse A. Isolation of tocopherol homologues by preparative high-performance liquid chromatography. J. Chromatogr.
450, (1988), pp. 111–113.
2. Saito, M.; Yamauchi, Y. Isolation of tocopherols from wheat germ oil by recycle semipreparative supercritical fluid chromatography. J. Chromatogr.
505, (1990), pp. 257–271.
3. Shin, T.S.; Godber, J.S. Isolation of four tocopherols and four tocotrienols from a variety of natural sources by semipreparative high performance
liquid chromatography. J. Chromatogr A, 678, (1994), pp. 49-58.
4. Ferreira, M. S. C.; Fonteles, M. C. Aspectos etnobotânicos e farmacológicos do Cymbopogon citratus Stapf (capim limão). Revista Brasileira de
Farmácia, 70, (1989), pp. 94-97.
5. Chaieb, K.; Hajlaoui, H.; Zmantar, T.; Kahla-Nakbi, A. B.; Rouabhia, M.; Mahdouani, K.; Bakhrouf, A. The chemical composition and biological
activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res., 21, (2007), pp. 501–506.
41
Isolation of Capsaicin from Red Chili Using Reveleris Integrated Flash and
Preparative LC
®
Introduction
Capsaicins, an active compound of chili peppers, are N-vanillylamides of fatty acids. It is commonly used as an essential component of certain
cuisines, and can also used as an analgesic in topical ointments and dermal patches to help relieve pain. Due to similarity in structures, isolation of
capsaicin and dihydrocapsaicin with traditional methods such as silica gel column chromatography and normal-phase thin-layer chromatography (TLC)
becomes difficult. Isolating pure capsaiscins from chili peppers tends to be a lengthy process. Traditional flash chromatography alone is not able to
adequately separate the capsaicin in the extract, therefore a two step purification using flash chromatography and preparative chromatography is
required.
1
2
HPLC Conditions:
Column: VisionHT C18 3µm, 150 x 4.6mm
Mobile Phase: Water : Acetonitrile (50:50)
Flow Rate: 1.5mL/min
UV Detector: 227nm
6 minRun Time:
®
Figure: HPLC profile for a) Capsaicin standard, b) Red chili skin extract before purification
Cartridge: ®
Mobile Phase: DCM: ACN (90:10)
Flow Rate: 10mL/min
Detector: ELSD and UV @ 227,254,280nm
Cartridge Equilibration: 6 min
Injection Type: Dry sample
®Reveleris Prep Purification System:
Flash Mode Conditions
Sample: Red chilli skin extract
Reveleris Silica cartridge 12g
Figure. Reveleris flash purification of Red chilii skin extract. Fractions 9-11 corresponds to capsaicinoids.®
Figure: HPLC confirmation of the Capsaicinoid fractions after flash purification.
To further separate the capsaicinoids, we did a second purification using the Reveleris Prep purification system in preparative mode.®
42
31
Column: ®
Flow Rate: 15mL/minDetector:ELSD and UV @ 227,254,280nm
Prep Conditions
Sample: Flash Purified Capsaicinoid Fractions
Davisil 710 C18E column, 2.5 x 36cm (10µ)
®
Mobile Phase: Water : Acetonitrile (50:50)
Figure. Flash fractions of red chili extract purified using the preparative mode of the Reveleris
Prep purification system.
Figure: HPLC profiles for preparative purified fractions.
Conclusion
This application demonstrates a two step purification of capsaicin from red chili extract. Flash purification alone was not able to adequately separate
the individual capsaicinoids in the extract. By cleaning up the sample with flash purification first and following it with preparative purification, capsaicin
was well resolved and purified.
References
1. Anand, P.; Bley, K. Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin
8% patch; Br. J. Anaesth., 107, (2011), pp. 490–502.
2. Sass, N.L.; Rounsavill, M. Combs, H.; A high-yield method for the extraction and purification of capsaicin; J. Agric. Food Chem., 25, (1977),
pp 1419–1420.
43
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Capsicum F-Prep fr 21; Purity: 78.57%
Capsicum F-Prep fr 22; Purity: 84.22%
Capsicum F-Prep fr 23; Purity: 92.06%
Capsicum F-Prep fr 24; Purity: 95.31%
Capsicum F-Prep fr 25; Purity: 95.18%
Capsicum F-Prep fr 26; Purity: 89.54%
Isolation of Stevioside & Rebaudioside-A Using Reveleris Integrated Flash
and Preparative LC
®
Introduction
Stevioside and Rebaudioside-A are major low-calorie diterpene steviol glycosides found in the leaves of S. rebaudiana and used as a sweetener in
food and beverages. These compounds range in sweetness level from 250 to 450 times sweeter than sugar. They are widely used as natural
This application shows the benefit of new Reveleris Prep purification system and Davisil NNH purification media for the separation and isolation
of the two main compounds, Stevioside and Rebaudioside-A, in S. rebaudiana. The flexibility of this new dual-mode Reveleris Prep purification
system allows the user to perform either flash or preparative LC or to combine both techniques to help achieve the highest levels of purity and recovery.
sweeteners for diabetic patients.1
2® ®
®
Extraction Conditions:
Extract Type: Hot Percolation
Weight: 50g
Extraction Solvent:
Solvent Volume: 500mL
30 min
Ethanol 70%
Extraction Time:
HPLC Conditions:
Column: ™
Mobile Phase A:
Mobile Phase B: Acetonitrile
Flow Rate: 1.5mL/min
Detector: UV @ 210nm and ELSD
ELSD Conditions:
Prevail NH 4.6 x 250mm, 5µm2
Water, pH 5 Adjusted with AA Temperature:
Gain:
Gas Flow: 1.6
1
60° C
Figure: HPLC profile of Stevioside standard.
to desired compounds which are not detected with UV.
Column: ™
Mobile Phase A:
Mobile Phase B: Acetonitrile
Flow Rate: 1.5mL/min
Detector: UV @ 210nm and ELSD
ELSD Conditions:Prevail NH 4.6 x 250mm, 5µm™
2vail NH 4.6 x 250mm, 5µmvail NH 4.6 x 250mm, 5µm
Water, pH 5 Adjusted with AA Temperature:
Gain:
Gas Flow: 1.6
1
60° C
Figure: HPLC profile of Rebaudioside-A standard.
Figure: HPLC analysis of crude stevia extract with ELS and UV detection. ELS detection shows additional impurity peaks close
Flash Chromatography Conditions:
Cartridge: Reveleris® amino 40g
Solvent A:Acetonitrile
Solvent B: Water
Flow Rate: 30mL/min
UV@ 210,254,280nm and ELSD
Sample:
Injection Type:Dry sample (3g loader)
Cartridge Equilibration: 2.0 min
Gradient Table
Step Time (min.) %B
1 0.0 99
2 10.0
3030
Detection:
Figure: Flash chromatogram for stevia extract. Fractions 7-11 correspond to the mixture
of Stevioside and Rebaudioside-A.
Stevia extract (500mg adsorbed on celite)
44
32
ELSD
UV
ELSD
UV
ELSD
UV
Column: ®
Solvent A:Acetonitrile
Solvent B: Water
Flow Rate: 25mL/min
UV@ 210,254,280nm and ELSD
Sample:
Injection Type:Liquid (10mL loop)
5.0 min
Gradient Table
Step Time (min.) %B
1 0.0 99
2 43.0
2020
Detection:
Flash purified fractions 7-11(dissolved in ACN: water, 1:1)
Reveleris Prep Conditions
Column Equilibration:
Davisil 710N NH2 Column, 250x25mm, 10-14µ
Figure: Prep chromatogram for combined fractions 7-11. Fractions 17-18 and 28-33 correspond to
Stevioside and Rebaudioside-A.
Flash Chromatography Conditions:
Cartridge: Reveleris® amino 40g
Solvent A:Acetonitrile
Solvent B: Water
Flow Rate: 30mL/min
UV@ 210,254,280nm and ELSD
Sample:
Injection Type:Dry sample (3g loader)
Cartridge Equilibration: 2.0 min
Gradient Table
Step Time (min.) %B
1 0.0 99
2 9.0
3030
Detection:
Figure: Flash chromatogram for stevia extract. Fractions 6-11 correspond to the mixture of
of Stevioside and Rebaudioside-A.
Stevia extract (500mg adsorbed on celite)
Column: ®
Solvent A:Acetonitrile
Solvent B: Water
Flow Rate: 25mL/min
UV@ 210
Sample:
Injection Type:Liquid (2mL loop)
Gradient Table
Step Time (min.) %B
1 0.0 99
2 50.0
2020
Detection:
Flash purified fractions 6-11(dissolved in ACN: water, 1:1)
Agilent Prep Conditions
Davisil 710N NH2 Column, 250x25mm, 10-14µ
Figure: Prep chromatogram for combined fractions 6-11. Fractions 1 and 2 correspond to
Stevioside and Rebaudioside-A.
Purification System Stevioside
98.65 24.42
99.65 28.54
# Average of UV and ELSD Signals * Davisil 710 NNH2 Media
Table: Purity and recovery results of Stevioside and Rebaudioside-A.
Purity and Recovery Results
Reveleris Prep System*:
Agilent Prep System*:
®
®
Rebaudioside-A
98.50 10.47
98.46 8.32
#Purity (%)
Recovery (mg/0.5g extract)
# #Purity (%)
Recovery (mg/0.5g extract)
#
®
45
Conclusion
For complex mixtures, the Reveleris Prep purification system allows both flash and prep LC to be used for purification. This means that the relatively
expensive prep LC column is protected from contamination and degradation by other components in the sample. These elements are retained or
separated on the low cost flash cartridge.
References
1. Genus, J.M.C.; Augustijns, P.; Mols, R.; Buyse, J.G.; Driessen B.; Metabolism of Stevioside in pigs and intestinal absorption characteristics of
Stevioside, Rebaudioside-A and steviol. Food and Chemical Toxicology, 41 (2003), pp. 1599-1607.
®
In this application, the initial sample cleanup was done with flash using a Reveleris amino cartridge, followed by a final preparative purification using
Davisil 710 NNH2 media. Comparison of the results also showed that the performance of the Reveleris Prep purification system in preparative LC
mode, performs as well as traditional preparative LC systems.
The flexibility of the Reveleris Prep purification system allows the user to perform either flash or preparative LC or the ability to combine both
techniques on the same system to achieve the highest levels of purity and recovery.
®
® ®
®
46
Peptide Purification Using Reveleris Integrated Flash and Preparative LC®
Introduction
47
Solid phase peptide synthesis (SPPS) is one of the most commonly used approaches for synthesizing peptides used in research, clinical, and
commercial applications. Purification of these reaction mixtures can be challenging, as reactions typically yield various peptide chain lengths.
Preparative HPLC using wide porosity reversed phase media has been the purification method of choice as it generally yields high purity results.
However, peptide reactions can often be complex and contain unwanted impurities. Introducing these samples onto expensive preparative HPLC
columns and systems can require additional cost and cleaning over time. The Reveleris Prep purification system is capable of running in both
“Prep-HPLC” mode as well as in “Flash Chromatography” mode, giving the user a choice to use prep HPLC columns or less expensive, disposable
flash chromatography columns. Reveleris wide pore flash cartridges are packed with high efficiency 20µm particles that yield high resolution
separations, which are especially needed for closely related molecules such as peptides. Here we demonstrate how both Reveleris flash cartridges
and HPLC columns can be used to purify complex peptide reaction mixtures to greater than 95% purity on the same system.
®
®
®
Flash Chromatography Conditions:
Cartridge: Reveleris® WP C18 12g
Solvent A:Water
Solvent B: Acetonitrile + 0.1% TFA
Flow Rate: 20mL/min
UV@ 214 and ELSD
Sample:
Injection Type:Liquid
Cartridge Equilibration: 6.0 min
Detection:
25mg reaction mixture
CQQYNRPYxxxGGTKLEIKR-NH2
(Target peptide length-20 amino acids)
Gradient Table
Step Time (min.) %B
1 0.0 0
2 3.0 25
3 9.9 38
4 0.0 100
5 8.1 100
HPLC Conditions:
Column: Kromasil C18 3µm, 100 x 4.6mm
Mobile Phase:
10-50% B in 20min
UV Detector: 214nm
®
A = Water + 0.1% TFAB = 20% Water, 80% ACN + 0.1% TFA
Gradient:
Figure. Crude peptide prior to purification
Cartridge: ®
Solvent A:Water
Solvent B: Acetonitrile + 0.1% TFA
Flow Rate: 15mL/min
UV@ 214, 256 and ELSD
Sample:
Injection Type:Liquid
Cartridge Equilibration: 5.0 min
Detection:
15mg reaction mixture
CAKHYYYGGxxxMDY-NH2
(Target peptide length-15 amino acids)
Reveleris Prep Conditions®
Vydac 150HC, 22x150mm, 10um
Figure. After flash purification
fraction 13
Figure: Flash chromatogram for peptide mixture.
Figure: Prep chromatogram for peptide mixture.
33
48
Gradient Table
Step Time (min.) %B
1 0.0 0
2 4.0 15
3 15.0 45
4 3.0 100
HPLC Conditions:
Column: Kromasil C18 3µm, 100 x 4.6mm
Mobile Phase:
15-55% B in 20min
UV Detector: 214nm
®
A = Water + 0.1% TFAB = 20% Water, 80% ACN + 0.1% TFA
Gradient:
Figure. Crude peptide prior to purification Figure. After Prep purification
fraction 15
Purity before Purification
58.5%
55.9%
Table: Purity results
Purity Results
Flash Chromatography
Prep Chromatography
Purity after Purification
95%
98.9%
Conclusion
As demonstrated above, successful peptide purification using reversed phase flash cartridges and HPLC columns was carried out on the same
system. Purity of greater than 95% was obtained with simple fraction collection. High performance wide pore flash chromatography media provides
comparable levels of purity to that of prep HPLC columns. The Reveleris Prep system is capable of running in both “Prep-HPLC” mode as well as®
in “Flash Chromatography” mode giving the user a choice to use prep HPLC columns or less expensive, disposable flash chromatography columns.
49
Introduction
Polysorbate 80 or Tween 80 (TW80) is a nonionic surfactant and emulsifier often used in foods and cosmetics. Tween is a registered trademark
of ICI Americas,Inc. for Polysorbate 80. The sample contains several compounds that are non-chromophoric, closely related, and can be challenging
to purify. The Reveleris Prep purification system integrates flash and prep chromatography with multiple UV and ELSD signals that can detect
UV active or non-active compounds. High resolution separations of complex sample mixtures can be achieved with Grace Alltima semi-preparative
HPLC columns packed with high efficiency 10µm particles. Here we demonstrate how a Reveleris Prep purification system and Alltima HPLC prep
columns can be used to purify complex TW 80 mixture to greater than 94.5% purity in a single run.
®
® ®
Complex Surfactant Purification Using Advanced Dual Detection Preparative
HPLC
®
®®
®
HPLC Conditions:
Column: Alltima C18 5µm, 250 x 4.6mm
Mobile Phase A: Methanol
Mobile Phase B: Tetrahydrofuran
Detector: ELSD
®
Gradient Table
Step Time (min.) %B
1 0.0 0
2 5.0 10
3 19.0 80
4 19.1 0
Figure. Crude TW 80 prior to purification
Cartridge: ®
Methanol
Tetrahydrofuran
Flow Rate: 8mL/min
UV@ 220, 254, 280 and ELSD
Sample:
Injection Type:Liquid
Cartridge Equilibration: 6.0 min
Detection:
TW80 mixture including several
compounds
Reveleris Prep Conditions®
Alltima C18, 10x150mm, 10um
Solvent A:Water
Solvent B:
Solvent C:
Figure: Prep chromatogram for TW80 mixture.
Gradient Table
Solvents
Time (min.)
%2nd
AB AB AB AB AB AB AB BC BC BC BC BC
0.0 9.1 2.9 0.6 0.1 0.3 5.0 0.0 0.3 15.0 30.0 10.0
70 97 97 99 99 100 100 100 0 10 55 80
Figure. HPLC analysis of fraction 44 Figure. HPLC analysis of fraction 46
34
Figure. HPLC analysis of fraction 52 Figure. HPLC analysis of fraction 77
Purity before Purification
32.1%
Table: Purity results
Purity Results
Prep Chromatography
Purity after Purification
94.5%
Conclusion
As demonstrated above, successful TW80 purification using HPLC columns was carried out on the Reveleris Prep purification system. Purity of
greater than 94.5% was obtained using simple fraction collection. The Reveleris Prep purification system is capable of detecting and separating
both UV-active and non-active compounds in complex mixtures leading to very high purity of individual components.
®
®
50
One Step Extraction and Purification of Natural Products Using a Reveleris
Solid Loader
®
Introduction
The extraction and recovery of a solute from a soild matrix can be rather complex. In order to obtain reproducible quantitative recoveries, careful
control and optimization of the extraction process is required. Conventional solvent extraction methods can be tedious, slow and use large amounts of
thermal, oxidative and photo decomposition of some constituents. New technologies have emerged that will supersede traditional extraction methods.
Choosing the right technique for the application requires a consideration of the matrix and of the analytes. Using Reveleris screw cap solid loaders,
solvent, and typically require subsequent clean-up and further purification to obtain the final pure compound. In addition, these procceses may lead to
2
®
®together with a Reveleris flash chromatography system, will perform the extraction and purification in a single step, when compared to traditional
extraction methods.
Isolation of chlorogenic acid and caffeine from coffee bean
Coffee is one of the most popular and widely consumed beverages throughout the world due to it's pleasant taste and stimulant effect. Coffee beans
are rich in several bioactive compounds such as phenolic acids, which are absorbed by the body and may act as antioxidants or as free radical
scavengers. Caffeine and chlorogenic acids are the main components present in green coffee beans. Caffeine is an attractive compound because of
its extensive applications in pharmacological preparations, including analgesics, diet aids and cold and flu remedies. In addition, it can be applied as
an additive in many popular carbonated drinks. About 120,000 tons of caffeine is consumed worldwide every year. Green coffee extract has been sold
1
as a nutritional supplement, and has been clinically studies for its chlorogenic acid content and it's lipolytic and weight-loss properties.
2
3
HPLC Conditions:
Column: VisionHT C18 HL 3µm, 150 x 4.6mm
Mobile Phase:
Flow Rate: 1 mL/min
UV Detector: 275 nm
10 minRun Time:
®
MeOH : 0.5% Acetic acid in water (25:75)
Figure. HPLC chromatogram for mixed chlorogenic acid and caffeine standard
Flash Chromatography Conditions:
Cartridge: Reveleris®C18 12g
Solvent A:Water
Solvent B: MeOH
Flow Rate: 15mL/min
UV@ 210,275,330nm and ELSD
Sample:
Injection Type:
Cartridge Equilibration: 4.0 min
Detection:
Ground coffee bean (1g mixed with 8g celite)
Gradient Table
Step Time (min.) %B
1 0.0 3
2 12.0 3
3 1.0 25
4 20.0 25
Dry sample (12g solid load cartridge)
Figure. Chromatogram shows the purification of ground coffee beans using a Reveleris screw cap solid loader
during flash chromatography. Reveleris empty screw cap solid loader filled with coffee bean powder.
®
®
Figure. Flash fractions analyzed using HPLC.
Using the single step isolation and purification procedure with a Reveleris screw cap solid loader and a Reveleris Prep purification instrument,
for the separation and isolation of chlorogenic acid and caffeine from coffee beans, high purity and recovery can be easily achieved.
® ®
51
35
4.02
2
5.50
4
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Chlorogenic Acid
Caffeine
2.60
7
4.05
14.
338
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
4.06
3
5.74
4
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Flash Fraction 4-5
Chlorogenic Acid
Purity 88.36%
Recovery: 18.22 mg/g
Flash Fraction 11-14
Caffeine
Purity 99.52%
Recovery: 14.98 mg/g
Isolation of Capsaicin from Red Chili
Capsaicinoids are all N-vanillylamides of fatty acids. When evaluating the pungency of a pepper, capsaicin and dihydrocapsaicin constitute
approximately 80-90% of the total capsaicinoids present and account for the majority of the hotness. The Capsaicinoids group produce a large
number of physiological and pharmacological effects on the gastrointestinal tract, the cardiovascular and respiratory system as well as the sensory and
thermoregulation systems. Capsaicin is the main active component that accounts for the pharmaceutical properties of peppers.
Capsaicin is reported to possess an analgesic action in arthritis pain and inflammation. It has also been reported to show anticancer effect and to be
4
active against neurogenic inflammation (burning and stinging of hands, mouth and eyes). It also shows protective effects against high cholesterol levels5and obesity.
This application shows the benefit of new Reveleris screw cap solid loader in single step extraction free separation and isolation of the capsaicin from
red chili.
HPLC Conditions:
Column: VisionHT C18 HL 3µm, 150 x 4.6mm
Mobile Phase:
Flow Rate: 1.5 mL/min
UV Detector: 227 nm
8 minRun Time:
®
Water : ACN (50:50)
Flash Chromatography Conditions:
Cartridge: Reveleris®C18-WP 12g
Solvent A:ACN : MeOH (60:40)
Solvent B: Water
Flow Rate: 15mL/min
UV@ 227,254,280nm and ELSD
Sample:
Injection Type:
Cartridge Equilibration: 2.0 min
Detection:
Red chili skin powder (3g mixed with 6g celite)
Gradient Table
Step Time (min.) %B
1 0.0 40
2 15.0 40
Dry sample (12g solid load cartridge)
Figure. HPLC chromatogram for standard capsaicin
Figure. Chromatogram shows the purification of red chili skin powder using a Reveleris screw cap solid
loader during flash chromatography. Fractions 23-24 corresponds to the capsaicin.
®
®
Reveleris empty screw cap solid loader filled with red chili.®
3.24
3
3.64
73.
884
4.67
7
5.40
5
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Flash Fraction 23-24
Capsaicin
Purity 89.79%
Recovery: 4.48 mg/g
Figure. Flash fractions analyzed using HPLC.
This application demonstrates a single step purification of capsaicin from red chili skin powder. The capsaicin was well resolved and purified
using a Reveleris screw cap solid loader on a Reveleris Prep purification system.® ®
52
3.63
23.
861
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Isolation of Picroside I and Picroside II from Picrorhiza Kurroa Rhizome
Picrorhiza kurroa is an important herb in the traditional Chinese and Ayurvedic systems of medicine, which is used to treat liver and upper respiratory
conditions. It’s traditional uses include treatment of a wide range of conditions, including fevers, chronic diarrhea, constipation, dyspepsia and jaundice.
Picroside I and picroside II are the active components of P. kurroa along with other identified active constituents such as apocynin, drosin, iridoid and
cucurbitacin glycosides.
6
7
HPLC Conditions:
Column:
Mobile Phase:
Flow Rate: 1 mL/min
UV Detector: 270 nm
15 minRun Time:
®
Flash Chromatography Conditions:
Cartridge: Reveleris®C18 40g
Solvent A:Water
Solvent B: ACN
Flow Rate: 20mL/min
UV@ 220,254,270nm and ELSD
Sample:
Injection Type:
Cartridge Equilibration: 4.0 min
Detection:
P. kuroa Powder (3g mixed with 6g celite)
Dry sample (12g solid load cartridge)
Figure. HPLC chromatogram for standard Picroside I and Picroside II
Figure. Chromatogram shows the purification of P. kurroa powder using a Reveleris screw cap solid loader during flash chromatography.
Fraction 17-23 and 27-32 corresponds to Picroside II and Picroside I respectively.
®
Reveleris empty screw cap solid loader filled with P. kurroa powder.®
Figure. Flash fractions analyzed using HPLC.
Alltima C18 HL 5µm, 250 x 4.6mm
ACN : Water pH 5, adjusted
with acetic acid (25:75)
3.33
33.
414
5.84
1
8.05
3
8.60
38.
993
9.61
5
10.6
28
11.9
71
13.6
07
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
5.38
45.
847
6.15
9
8.04
7
8.61
4
9.61
7
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Picroside I Picroside II
Gradient Table
Step
Time (min.)
%B
1 2 3 4 5 6 7 8 9 10 11 12 13 14
0.0 6.0 4.0 3.0 2.0 8.0 1.5 7.0 2.5 28.0 1.0 2.0 5.0 16.0
5 5 8 8 10 10 12 12 14 14 15 15 18 18
3.15
33.
634
4.10
7
4.83
4
5.35
95.
725
6.07
16.
334
6.59
2
7.36
77.
651
7.94
18.
437
8.86
5
9.42
6
10.4
38
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Flash Fraction 17-23
Picroside II
Purity 47.67%
Recovery: 18.82 mg/g
5.76
26.
178
6.65
4
7.70
2
8.46
9
9.06
69.
427
10.4
64
13.3
23
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Flash Fraction 27-32
Picroside I
Purity 97.53%
Recovery: 36.78 mg/g
53
Flash Chromatography Conditions:
Cartridge: Reveleris® Silica 12g
Solvent A:DCM
Solvent B: MeOH
Flow Rate: 15mL/min
UV@ 220,254,270nm and ELSD
Sample:
Injection Type:
Cartridge Equilibration: 4.0 min
Detection:
Flash fraction 17-23 (fromstep 1) on celite
Dry sample (12g solid load cartridge)
Figure. Chromatogram shows the purification of flash fractions 17-23 from step 1. Fractions 8-10 are Picroside II.
Gradient Table
Step
Time (min.)
%B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0.0 2.0 9.0 1.0 2.0 1.0 2.5 2.5 1.0 4.5 1.0 8.0 1.0 13.0 1.0 6.0
1 3 3 4 4 5 5 6 8 8 10 10 12 12 15 15
4.57
9
5.31
05.
698
6.05
8
7.27
8
8.25
6
9.19
7
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Figure. Flash fractions analyzed using HPLC.
Using the Reveleris screw cap solid loader with the Reveleris Prep purification system, bioactive components Picroside I and Picroside II, present
in P. kurroa Rhizome have been separated and collected with high purity and recovery.
® ®
Conclusion
Using the Reveleris screw cap solid loader with the Reveleris Prep purification system can isolates and collects the critical components of the plant
products. Using this methodology, chemists can purify and isolate compounds present in natural products that are used for medicinal purposes.
It reduces extraction and post extraction clean-up procedures. The short operation time without any heating procedure makes the purification process
References
®
more productive and effective.
®
1. Bastos, D.H.M.; Fornari, A.C.; de Queiroz, Y.S.; Soares R.A.M.; Torres, E.A.F.S.; The chlorogenic acid and caffeine content of Yerba maté
(Ilex paraguariensis) beverages. Acta Farm. Bonaerense., 24 (1), (2005), pp. 91-95.
2. Mohammed, M.J.; Al-Bayati, F.A.; Isolation, identification and purification of caffeine from Coffea arabica L. and Camellia sinensis L.:
A combination antibacterial study. Int J Green Pharm., 3, (2009), pp. 52-57.
3. Hausenblas, H.; Huynh, B.; Effects of green coffee bean extract on weight loss: An updated meta-analysis of randomized clinical trials.
Natural Medicine Journal, 6 (3), (2014).
4. Govindarajan, V.S.; Sathyanarayana, M.N.; Capsicum. Production, technology, chemistry, and quality. Part V. Impact on physiology,
pharmacology, nutrition, and metabolism; structure, pungency, pain, and desensitization sequences. Crit. Rev. Food Sci. Nutr., 29,
5. Othman, Z.A.A.; Ahmed, Y.B.H.; Habila M.A.; Ghafar, A.A.; Determination of capsaicin and dihydrocapsaicin in Capsicum fruit samples
using high performance liquid chromatography. Molecules, 16, (2011), pp. 8919-8929.
6. Luper S.; A review of plants used in the treatment of liver disease: Part 1. Alternative Medicine Review, 3, (1998), pp. 410-421.
7. Rathee, D.; Rathee, P.; Rathee, S.; Rathee, D.; Phytochemical screening and antimicrobial activity of Picrorrhiza kurroa, an Indian
traditional plant used to treat chronic diarrhea. Arabian J Chemistry, In Press. DOI: 10.1016/j.arabjc.2012.02.009
(1991), pp. 435-475.
54
MODcolSpringColumnInnovation
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Tel: +61 3.9237.6100
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Tel: +55 15.3235.4705
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Tel: +86 21.5467.4678
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Tel: +91 20.6644.9900
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Tel: +1 847.948.8600
Contact us today for more information, or to request a demo:
The information presented herein is derived from our testing and experience. It is offered for your consideration and verification. Since operating conditions vary significantly, and are not under our control, we disclaim all warranties on the results that may be obtained from the use of our products. W. R. Grace & Co.-Conn. and its subsidiaries can not be held responsible for any damage or injury occurring as a result of improper installation or use of its products. Grace reserves the right to change prices and/or specifications without prior notification. GRACE® and DAVISIL® are trademarks, registered in the United States and/or other countries, of W. R. Grace & Co.-Conn. MODCOL®, REVELERIS®, ALLTIMA®, VISIONHT® and VYDAC® are trademarks, registered in the United States and/or other countries of Alltech Associates, Inc. REVEALX™ and PREVAIL™ are trademarks of Alltech Associates, Inc. TALENT|TECHNOLOGY|TRUST is a trademark of W. R. Grace & Co.-Conn. KROMASIL® is a trademark, registered in the United States and/or other countries, of EKA Chemicals AB. This brochure is an independent publication and is not affiliated with, nor has it been authorized, sponsored, or otherwise approved by EKA Chemicals AB. This trademark list hasbeen compiled using available published information as of the publication date of this brochure and may not accurately reflect current trademark ownership or status. Alltech Associates, Inc. is a wholly owned subsidiary of W. R. Grace & Co.-Conn. Grace Discovery Sciences is a product group of W. R. Grace & Co.-Conn., which includes all product lines formerly sold under the Alltech brand. Grace Materials Technologies is a business segment of W. R. Grace & Co.-Conn., which includes products formerly sold under the Grace Davison brand. © Copyright 2015 W. R. Grace & Co.-Conn. Inc. All rights reserved.
AP0008 06/2015
grace.com
Global HeadquartersW. R. Grace & Co.-Conn
7500 Grace Drive
Columbia, Maryland 21044 USA
countries, of W. R. Grace & Co.-Conn. MODCOL®, REVELERIS®, ALLTIMA®, VISIONHT® and VYDAC® are trademarks, registered in the Unicountries, of W. R. Grace & Co.-Conn. MODCOL®, REVELERIS®, ALLTIMA®, VISIONHT® and VYDAC® are trademarks, registered in the Unicountries, of W. R. Grace & Co.-Conn. MODCOL®, REVELERIS®, ALLTIMA®, VISIONHT® and VYDAC® are trademarks, registered in the Uniand PREVAIL™ are trademarks of Alltech Associates, Inc. TALENT|TECHNOLOGY|TRUST is a trademark of W. R. Grace & Co.-Conn. KROMAand PREVAIL™ are trademarks of Alltech Associates, Inc. TALENT|TECHNOLOGY|TRUST is a trademark of W. R. Grace & Co.-Conn. KROMASIL® is a trademark, registered in the United States and/or other and PREVAIL™ are trademarks of Alltech Associates, Inc. TALENT|TECHNOLOGY|TRUST is a trademark of W. R. Grace & Co.-Conn. KROMASIL® is a trademark, registered in the United States and/or other SIL® is a trademark, registered in the United States and/or othcountries, of EKA Chemicals AB. This brochure is an independent publication and is not affiliated with, nor has it been authorized, sponsored, or otherwise approved by EKA Chemicals AB. This trademark list hasbeen compiled using available published information as of the publication date of this brochure and may not accurately reflect current trademark ownership or status. Alltech Associates, Inc. is a wholly owned