Corrected Reveleris App Notebook 25-6-151).pdf · A Full Spectrum of Puriﬁcation Solutions...

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


12 gram Reveleris cartridge

Flow Rate: 15mL/min

Cartridge equilibration: 4 min.

Slope detection: High

ELSD Threshold: 3mV

UV Threshold: 0.02AU



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


12 gram Reveleris RP C18 cartridge

Flow Rate: 30mL/min

Cartridge Equilibration: 3 min.

Slope Detection: Low

ELSD Threshold: 3mV





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


Cartridge: Reveleris® silica 12g

Solvent A: Hexane

Solvent B: Ethyl acetate

Flow Rate: 25mL/min




Cartridge Equilibration: 5.0 min


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


12 gram Reveleris® RP C18 cartridge

Flow Rate: 30mL/min

Cartridge Equilibration: 3 min.

Slope Detection: Low

ELSD Threshold: 10mV




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


®

Flow Rate:

Cartridge Equilibration:

Slope Detection:

ELSD Threshold: 3mV

UV Threshold:

UV1 Wavelength:


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



6

6



Weight: 6g




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



Solvent A: Hexane


Flow Rate: 25mL/min





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.


Extract type: Pulverized

Weight: 2g






Solvent A: Hexane


Flow Rate: 18mL/min





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



Weight: 2g

Extraction Solvent: Ether



Figure: Separation of Rhizoma chuanxiong extract shows the detection of peaks using the UV and the evaporative light scattering detectors.



Solvent A: Hexane


Flow Rate: 25mL/min





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:


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


Extract Type: Oil

™


Cartridge: Reveleris® C18 media 12g

Solvent A: Water

Solvent B: Methanol

Flow Rate: 30mL/min





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.


Cartridge: Reveleris® C18 and Amino 12g

Solvent A: Acetonitrile

Solvent B: Methylene chloride

Flow Rate: 30mL/min





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


Cartridge: Reveleris® amino 12g

Flow Rate: 36mL/min

Equilibration: 10.0 min

Run Length: 15.0 min



Solvent A: Water with 0.1% TFA


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


Extract type: Powder

Weight: 3g

Extraction Solvent: Methanol


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.



Solvent A: Water

Solvent B: Methanol

Flow Rate: 30mL/min





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



Solvent A: Water


Flow Rate: 36mL/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





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.


resolved.

™

™

17

15

Fast and Efficient Purification of Fatty Acid Methyl Esters (FAMEs)



Solvent A: Methanol


Flow Rate: 30mL/min





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



Flow Rate: 36mL/min

Equilibration:

Run Length:



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


Cartridge: Reveleris® amino media 40g

Flow Rate: 40mL/min





Solvent A: Water


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


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


Cartridge: Reveleris® C18 12 g

Solvent A: Water

Solvent B: Methanol

Flow Rate: 36mL/min





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.


Cartridge: Reveleris® C18 40 g

Solvent A: Water

Solvent B: Methanol

Flow Rate: 40mL/min





(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


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)


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


Cartridge: Reveleris® C18 12g

Solvent A: Water

Solvent B: Methanol

Flow Rate: 36mL/min



Cartridge Equilibration: 5.0 min.


2

3

™ ®

Conclusion

Using the RevealX detection technology, separation of both chromophoric and non-chromophoric compounds can improve sample recovery and ™

References


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


Cartridge: Reveleris ® C18 12g

Flow Rate: 36mL/min




UV2 Wavelength:



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


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



Solvent A: Water


Flow Rate: 36mL/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



Solvent A: Water with 0.1 TFA


Flow Rate: 36mL/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



Solvent A: Water

Solvent B: Methanol

Flow Rate: 36mL/min





®

™

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.

®

®




Solvent B: Acetonitrile with 0.05 TFA

Flow Rate: 25mL/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


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%


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%


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



Solvent A: Water

Solvent B: Methanol

Flow Rate: 30mL/min





®

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.

™ ®

™

®

®

®



Solvent A: Water

Solvent B: Methanol

Flow Rate: 40mL/min






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.

®


Cartridge: Reveleris® C18 WP 12g



Flow Rate: 20mL/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.




Solvent B: Acetonitrile with 0.05% TFA

Flow Rate: 30mL/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 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)


50 mg Load

300 mg Load


600 mg Load


Cartridge: Reveleris ® C18 WP flash

cartridges, 12g

Flow Rate: 20 mL/min



UV1 Wavelength: 254 nm





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









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








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








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


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


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


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


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 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 B: 20% EtOAc in Petroleum ether

Flow Rate: 15mL/min




Gradient Table

Step Time (min.) %B

1 0.0 1

2 8.0 18

3 0.5 100

4 3.0 100



Sample: Lemongrass Oil


®

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


Flow Rate: 15mL/min


Prep Conditions

Sample: Lemongrass oil


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


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.




®

®

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:




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 B: 20% EtOAc in Petroleum ether

Flow Rate: 25mL/min




Gradient Table

Step Time (min.) %B

1 0.0 2

2 4.0 20

3 2.0 20

4 1.0 60



Sample: Clove oil


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


Flow Rate: 15mL/min


Prep Conditions

Sample: Clove oil


®

®

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


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.




®

®

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)


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


Injection Type: Dry sample

®Reveleris Prep Purification System:


Sample: Red chilli skin extract


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%






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® ®

®


Extract Type: Hot Percolation

Weight: 50g

Extraction Solvent:


30 min

Ethanol 70%

Extraction Time:

HPLC Conditions:

Column: ™

Mobile Phase A:



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:



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



Solvent A:Acetonitrile

Solvent B: Water

Flow Rate: 30mL/min

UV@ 210,254,280nm and ELSD

Sample:

Injection Type:Dry sample (3g loader)


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 B: Water

Flow Rate: 25mL/min


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.




Solvent B: Water

Flow Rate: 30mL/min


Sample:

Injection Type:Dry sample (3g loader)


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 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.

®

®

®


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


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:



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:


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:



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


Cartridge: Reveleris®C18 12g

Solvent A:Water

Solvent B: MeOH

Flow Rate: 15mL/min


Sample:

Injection Type:


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%


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)


Cartridge: Reveleris®C18-WP 12g

Solvent A:ACN : MeOH (60:40)

Solvent B: Water

Flow Rate: 15mL/min


Sample:

Injection Type:


Detection:

Red chili skin powder (3g mixed with 6g celite)

Gradient Table

Step Time (min.) %B

1 0.0 40

2 15.0 40


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


Capsaicin

Purity 89.79%

Recovery: 4.48 mg/g


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:

®


Cartridge: Reveleris®C18 40g

Solvent A:Water

Solvent B: ACN

Flow Rate: 20mL/min


Sample:

Injection Type:


Detection:

P. kuroa Powder (3g mixed with 6g celite)


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.®


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


Picroside II

Purity 47.67%


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


Picroside I

Purity 97.53%


53


Cartridge: Reveleris® Silica 12g

Solvent A:DCM

Solvent B: MeOH

Flow Rate: 15mL/min


Sample:

Injection Type:


Detection:

Flash fraction 17-23 (fromstep 1) on celite


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


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

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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

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