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Reveleris® Amino and Diol Flash Cartridges0 pages
Discovery Sciences
®
Reveleris Amino and Diol
Flash Cartridges
Silica flash cartridges, while effective for many applications, have their limitations for some
compound classes like carbohydrates and lipids. For these applications, Grace Discovery
Sciences has introduced the Reveleris® Amino and Diol Flash Cartridges.
Reveleris® Amino Flash Cartridges
•t Ideal for polar and basic molecules
•t Compounds such as carbohydrates, nitrogen containing heterocycles, amines, etc. which
t exhibit peak streaking and tailing with unbonded silica cartridges.
Reveleris® Diol Flash Cartridges
•t Alternative selectivity to unbonded and reversed phase media
•t Broad application to separate a variety of molecule polarity types including lipids
Reveleris® Bonded Flash Cartridges (up to 200 psig*)
Size
Qty
Amino Part No.
Diol Part No.
C18 Part No.*
4g
1
5157330
5157335
5152990
12g
1
5157331
5157336
5152103
40g
1
5157332
5157337
5152104
80g
1
5157333
5157338
5152105
120g
1
5157334
5157339
5152991
5164430
330g*
1
Reveleris® cartridges also available in Reverse Phase C18 Bonding
*330g cartridges have a pressure rating of 100 psig
Better Cartridges
Using Grace Silica
Better Together - Further Streamline Your Purifications by Combining Reveleris® Cartridges with
the Most Advanced Flash System
Reveleris® Cartridge
+
Reveleris® X2 System
=
Enhanced Purification Benefits
Plug & Play
Automatic cartridge identification populates the
system for size and phase of cartridge in use
Confidence in Results
Serial tracking of cartridges allows for tracking of
cartridge use
Simplifying Purification
Specifically for compounds like carbohydrates and
lipids, the RevealX™ technology allows maximum
purity with minimal effort in a single system
Faster Runs
Optimize your purification with the Navigator
software
Reveleris iES Application Note:
Bile acid purification during lead generation in drug discovery
Grace Davison Discovery Sciences
Introduction:
Grace Davison Discovery Sciences
Bile acids such as cholic acid are steroidal acids, which occur, in salt form in bile, secreted into the gut 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 its water solubility for drug development.
Reveleris™ Flash System
Reveleris® iES Flash System
Isolation and Purification of Catechins from Green Tea Extract
Using the RevealX platform of the Reveleris iES flash chromatography system and the Reveleris reversed
target
Bile Acid Purification During phase cartridge,small impuritiesand non-chromophoric reactionDavisonbe separated and isolated in
Lead of chromophoric present DrugGrace compounds can Discovery Sciences
Discovery chromatography run.
the presenceGeneration in in a chemical
by a single
Grace Davison Discovery Sciences
Over the last few decades, green tea has been subjected to many scientific and medical studies to determine the extent of its longpurported health benefits, with research centering on polyphenols, particularly the catechins, in green tea.
HPLC and UHPLC Methods for Melamine
Reveleris iES Flash System
Isolation and purification of natural products is a challenging, tedious, and lengthy process requiring multiple steps and a large amount of
organic solvent. The use of the Reveleris™ automated flash chromatography instrument and Reveleris™ reversed-phase cartridges can
ease the process.
RevealX Technology Allows Both Chromophoric and Non-chromophoric
Component Isolation from Cat’s Claw Extract
N
Cartridge: Reveleris® C18
Solvent A: Water
Extraction Conditions:
O
OH
Extract type: Powder OH
OH
H
NH2
H
UV1 Wavelength: 254nm
H
UV2 Wavelength: 220nm
HO
ELSD Carrier: Iso-propanol
CO2H
Weight: 3g
4
6
8
10
12
14
16
After 2007 and more recent melamine contamination outbreaks, there is an urgent need for analytical methods that can identify
and quantify melamine in food. Current melamine detection methods involve LC-MS and GC-MS. GC-MS methods require
derivatization, and LC-MS methods generally use gradient conditions that require column clean up and re-equilibration.
Grace has developed MS-compatible LC methods for Melamine using an HILIC media platform that can be applied to both
traditional HPLC as well as UHPLC systems. Melamine was analyzed with a standard HPLC system using a 5µm particle HILIC
phase packed into a 250 x 4.6mm column. The 1.5µm version of this phase was then packed into a high throughput format
conducive to UHPLC and fast LC systems. Both methods deliver excellent linearity and use isocratic elution for fast analysis
without the need for re-equilibration.
N
N
OH
Cyanuric Acid
HPLC Method for Melamine
ELSD Carrier: Iso-propanol
16
This HPLC analytical method for melamine fulfills the FDA requirements using a HILIC column
and an ionizable mobile phase compatible with mass spec. Low UV detections offers excellent
linearity between 40ng and 100µg.
Injection Type: Liquid
Solvent A: 0.5% Formic acid in Water
Solvent B: Methanol
Gradient: Time: 0 2 15 16
Cartridge: Reveleris C18 media 12g
Step Time (min.)
%B
®
1
Solvent A: Water0.0
2
7.0
Solvent B: Methanol
3
1.0
Flow Rate: 30mL/min
UV1 Wavelength: 254nm
30
100
100
%B:t0t 0t 50t 100t100t 0t 0
Glycine 2 mg
Sodium Glycocholate 20 mg
Gradient
Cholic Acid 2 mg
Injection Type: Liquid
0
Fraction 12
Table
Fraction 16
Step
Time (min.)
%B
1
2
3
4
0.0
3.0
3.0
4.0
30
30
100
100
1
0.5
Figure 1: Using the Reveleris® iES flash system and RevealX™ technology, Cat’s Claw extract has been
purified on a reversed phase Reveleris® 12g cartridge. The chromatogram shows the benefits of detecting
peaks using the UV and the ELSD detectors.
1.
2.
3.
UV2 Wavelength: 220nm
Figure 1: Using traditional flash chromatography system, non-chromophoric compounds have poor
ELSD Carrier: Iso-propanol
sensitivity or are undetected during purification. RevealX technology of the Reveleris iES system can
Cartridge Equilibration: 5.0 min
isolate such compounds using the ELSD in a reaction mixture during purification.
2
17 18 19
0
2
4
6
8
10
12
14
16
18 Min.
0
2.5
5
7.5
10 12.5 15 17.5 20 Min.
HPLC Column: VisionHT™ HILIC, 5µm, 4.6 x 250mm (Part No. 86466)
Mobile Phase: Acetonitrile:10mM Ammonium Acetate in Water (95:5)
Flow Rate: 1mL/min
Detection: UV@240nm
Column Temperature: 30ºC
Injection: 40µg/mL x 20µL
10
8
6
4
2
0
HPLC Chromatograms of green tea fractions collected by Reveleris™ flash chromatography.
0 20 40 60 80 100 120
Sample Weight (µg)
Conclusion
Using the proposed procedure with the Reveleris™ instrument and Reveleris™ RP cartridges, the separation and isolation of the various
chatechins in green tea extract can be easily achieved.
UHPLC Method
1
Peak Area (x 106 mAU x sec.)
Gradient Table
Flash Chromatography Conditions:
1. Glycine 2mg
2. Sodium Glycocholate 20mg
3. Cholic Acid 2mg
y = 146800x + 87120
R2 = 0.998
14
12
mV(x100)
2.0
1.5
1.0
Fraction 7
HPLC before flash
Injection Type: Liquid
for Melamine
Compared to the conventional HPLC method, the UHPLC method is 4 times faster. With the use of 1.5μm particles, optimal linear velocities extend over a
wider range. Therefore, it is possible to maintain efficiency and resolution while running samples at faster flow rates.
3
www.discoverysciences.com
mAU
1
UHPLC Column: VisionHT™ HILIC, 1.5µm, 2 x 50mm (Part No. 5141919)
Mobile Phase: Acetonitrile:10mM Ammonia Acetate in Water (95:5)
Flow Rate: 0.2mL/min
Detection: UV@240nm
Column Temperature: 30ºC
Injection: 50µg/mL x 0.5µL
5
ALLTECH® is a trademark, registered in the United States and/or other countries, of Alltech Associates, Inc. REVELERIS™ is a trademark of Alltech Associates, Inc. GRACE®, and GRACE DAVISON® are trademarks, registered in the United States and/or other countries, of W. R. Grace & Co.-Conn.
GRACE DAVISON DISCOVERY SCIENCES™ is a trademark of W. R. Grace & Co.-Conn. This trademark list has been compiled using available published information as of the publication date of this brochure and may not accurately reflect current trademark ownership. Alltech Associates, Inc. is a wholly
4
owned subsidiary of W. R. Grace & Co.-Conn. Grace Davison Discovery Sciences is a product group of W. R. Grace & Co.-Conn., which now includes all product lines formerly sold under the Alltech brand. © Copyright 2010 Alltech Associates, Inc. All rights reserved. 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 3
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.
2
01/2010 M328
1
0
-1
W. R. Grace & Co.-Conn.
7500 Grace Drive, Columbia, mD 21044
Grace Davison Discovery Sciences Regional Headquarters:
Figure 1: Using traditional flash chromatography system, non-chromophoric compounds have poor sensitivity or are undetected during purification.
RevealX™ technology of the Reveleris® iES system can isolate such compounds using the ELSD in a reaction mixture during purification.
Figure 2: Optimized gradient method improves resolution for higher purity fractions.
In THE AmERICAS:
2051 Waukegan Road
Deerfield, IL 60015-1899
Tel: 1 847 948 8600
Fax: 1 847.948.1078
Email: discoverysciences@grace.com
In EURoPE:
Brandstraatt12
B-9160 Lokeren, Belgium
Tel: +32 (0)9-340-65 65
Fax: +32 (0)9 340 65 60
Email: discoverysciences.EU@grace.com
In ASIA:
19th Floor, K.Wah Center
1010 Huai Hai Zhong Road
Shanghai, 200031 PRC
Tel: 86 21 5467 4678
Fax: 86 21 5405 1500
Email: dsbiz.asia@grace.com
In InDIA:
17 Commerce Center
Opposite Krishna Hospital, Paud Road
Pune, 411038 India
Tel: +91 20 6644 9900
Fax: +91 20 2544 1740
Email: pune@grace.com
1400
0
1
2
3
In AUSTRALIA/nEW ZEALAnD:
2 Kerr Court
Rowville, 3178
4
5 Min.
9 injections in parallel shows good reproducibility
Victoria, Australia
Tel: +61 3 9237 6100
Fax: +61 3 9237 6101
Email: discoverysciences.AU@grace.com
40
30
20
Gradient Table
10
Step
Time (min.)
%B
1
2
3
4
www.discoverysciences.com
Toxicology studies show ingestion of melamine in large quantities may lead to reproductive damage or bladder cancer due to the
formation of bladder/kidney stones. At lower levels, melamine and cyanuric acid are absorbed into the bloodstream. Together,
they concentrate and form melamine cyanurate in the urine-filled renal microtubules. Cystallization blocks and damages the
renal cells that line the tubes, causing the kidneys to malfunction.
NH2
N
18 Min.
Separation of catechins in green tea by flash chromatography.
UV2 Wavelength: 280nm
(1)
(2)
Ultra-sonication: 30 min at 60ºC
Cartridge Equilibration: 3.0 min.
OH
UV 2
UV 1
ELSD
7
2
HO
UV1 Wavelength:t254nm
+
NH CO2- Na
OH
HO
Solvent Volume: H
10mL
(3)
0
UV Threshold: 0.02AU
O
H
H
Extraction Solvent: Methanol
H
H
H
OH
H
Melamine
16
Cat’s claw, a tropical vine, is considered a valuable medicinal resource for scientific research. The active substances are alkaloids, tannins, Cartridge Equilibration: 3 min.
and several other phytochemicals that may have the potential to boost the immune system.1 The presence of alkaloids provides anti1) Separation sodium glycocholate from cholic acid and and glycine on a 12g
Slope Detection:tLow
1) Separation ofof sodium glycocholate from cholic acid glycine on a cholesterol; contribute anti-inflammatory, antioxidant, and anticancer properties.2
hypertensive effects and may lower
Reveleris C18 flash cartridge
12g Reveleris® C18 flash cartridge
ELSD Threshold: 3mV
Solvent B: Methanol
N
H2N
Flow Rate: 30mL/mint
Results
N
12
12 gram Reveleris™ RP C18 cartridge
Results:
Flow Rate: 36mL/min (12g),
40mL/min (40g)
Milk, infant formula and other dairy products were recently found contaminated with melamine, following an earlier melamine
contamination outbreak in pet food in 2007.
NH2
Flash Chromatography Conditions:
Introduction
Flash Chromatography Conditions:
0.0
3.0
9.0
1.0
30
30
100
100
0
y = 1.2924x - 1.8252
R2 = 1.000
1200
Area (mAU x sec.)
Visit www.discoverysciences.com/FlashChromatography/ApplicationNotes.aspx
to access the latest applications utilizing Reveleris® flash cartridges
Introduction
Flash Chromatography Conditions:
Introduction
Column: Reveleris C18
Bile acids, such as cholic acid, are steroidal acids secretedWaterthe gastrointestinal tract to emulsify fats and encourage digestion.1
Solvent A: into
®
Hepatic conversion of cholesterol into bile acids increases the breakdown of LDL cholesterol, adding value in treatment of
Solvent B: Methanol
Flow the 36ml/min (12g), of other (40g)
coronary patients. Bile acids are starting materials forrate: semi-synthesis40ml/min medicinal steroids due to their availability as raw
™
materials. Conjugation of cholic acid with glycineUV1 Wavelength: 254nm water solubility for drug development.
or taurine increases its
UV2 Wavelength: 220nm
Using the RevealX™ technology of the Reveleris®ELSD carrier: Iso-propanol system and the Reveleris® reversed-phase cartridge,
iES flash chromatography
Column Equilibration: 3.0 min
chromophoric and non-chromophoric target compounds can be separated and isolated in the presence of small impurities present
Injection
in a chemical reaction by a single chromatography run. Type: Liquid
Conc.
0.5
1
1.5
2
2.5
3 Min.
Inj.
(µL)
50µg/mL
800
0.1
50µg/mL
1000
0.5
50µg/mL
600
1
Weight
(ng)
5
25
50
0
200
400
600
800
1000
7.3
32
63
5
10
500
641
50µg/mL
0
250
Peak
Area
50µg/mL
50µg/mL
400
200
0
UHPLC Column: VisionHT™ HILIC, 1.5µm, 2 x 50mm (Part No. 5141919)
Mobile Phase: Acetonitrile:Water(20mM Ammonium Formate) (90:10)
Flow Rate: 0.2mL/min
Detection: UV@240nm
Column Temperature: 30ºC
Injection: 50µg/mL x 0.1µL
20
1000
1293
318
1200
Sample Weight (ng)
This method exhibits excellent linear response between 5ng and 1000ng for
accurate quantitation.
