1 Presented by: Bill Ciccone MicroSolv Technology Corporation Using Cogent TYPE-C Silica™ Where...

1

Presented by: Bill Ciccone

MicroSolv Technology Corporation

Using Cogent TYPE-C Silica™

Where Tradition Meets Tomorrow

Eatontown, NJ USA

2

MicroSolv’s Mission Statement

Our mission is to encourage, develop, promote, assist, and aid in the improvement and development of chemistry and biological technologies. To participate in the discovery of new technologies that will improve the living condition of people and animals all over the world.

It is within the scope of our mission that one day one of our customers using our technology will make a major improvement to the human condition.

Also, our mission is to provide an easy, effective and efficient way to purvey information and to become an exchange of ideas that promotes science in both the professional and amateur fields.

3

About MicroSolv

MicroSolv started in 1992 Formed to work with technology transfer

departments at universities Role MicroSolv plays in product development

Tech Transfer Product Development and Marketing R&D Production Design QC Marketing and Sales Distribution of Wide Array of Products Education and Technical Support Product Line Extension

4

Cogent TYPE-C™ SilicaHPLC Columns

First Introduced at Pittcon 2003

5

1.What are TYPE-C™ Silica HPLC columns?

2. What do they do?

3. Why are they recommended for all chromatographers? Even for Reverse Phase?

4. Exciting Real life examples where problems were solved. To give you an idea why these columns are useful for your lab.

Agenda:

6

1. What are TYPE-C™ Silica based HPLC columns?

Unique Selectivity These are different from all Others.

EfficiencyStability-Superior

VersatilityReproducible

7

Type-C Silica Columns are

“Silica-Hydride” Based

HPLC Stationary Phases:

’

8

Ordinary SilicaStandard; Well Known by all

9

Silica Hydride

O Si OH

O

OOHSiO

O

O

OHSiO

O Si O

O

OOSiO

O

O

OSiO

Si H

O

OHSi

O

O

HSi

Ordinary Silica M TYPE-C™ Silica

10

Silica Hydride

O Si OH

O

OOHSiO

O

O

OHSiO

O Si O

O

OOSiO

O

O

OSiO

Si H

O

OHSi

O

O

HSi

Ordinary Silica

How Does this structural difference impact the performance of the columns?

TYPE-C™ Silica

11

How Does this structural difference impact the performance of the columns?

12

“Hydride” Silica

O Si OH

O

OOHSiO

O

O

OHSiO

O Si O

O

OOSiO

O

O

OSiO

Si H

O

OHSi

O

O

HSi

Ordinary Silica

Stationary Phases Solvated by aqueous Mobile Phase

Water Shell “Water Layer”

13

“Hydride” Silica

O Si OH

O

OOHSiO

O

O

OHSiO

O Si O

O

OOSiO

O

O

OSiO

Si H

O

OHSi

O

O

HSi

Ordinary Silica

In High Organic The “Shells” Can Differ

Water Shell Organic Layer

14

(CH2)6 CH3CHO

CHSiOO

HSiOOSiO

O Si

O

O

O Si H

O

OO Si

(CH2)6 CH3

Bidentate C8Cogent BDC8

(CH2)16 CH3CHO

CHSiOO

HSiOOSiO

O Si

O

O

O Si H

O

OO Si

(CH2)16 CH3

(CH2)16 CH3CHO

CHSiOO

HSiOOSiO

O Si

O

O

O Si H

O

OO Si

(CH2)16 CH3

Cogent BDC18

Bidentate C18 and C8 Moieties Bonded To Hydride Silica

Some Examples of TYPE-C Silica Phases

M

15

16

Bidentate C18 Direct Silicon-Carbon Bonds TYPE-C SilicaM

17

Bidentate C18 Direct Silicon-Carbon Bonds TYPE-C Silica

M

Two Points of Attachment

18

Bidentate C18 Direct Silicon-Carbon Bonds TYPE-C Silica

Not Susceptible to Hydrolysis.

Two Points of Attachment

19

The Cholesterol Column

O Si

O

OHSiO

OSiOO

O Si H

O

OO Si

OHSiO

CH3H

C

CH3

HO

H

H

O

CH2 (CH2)9

UDC Cholesterol

CH2 (CH2)9 C

CH3

HO

H

H

O

CH3H

H

20

(CH2)6 CH3CHO

CHSiOO

HSiOOSiO

O Si

O

O

O Si H

O

OO Si

(CH2)6 CH3

Bidentate C8Cogent BDC8

(CH2)16 CH3CHO

CHSiOO

HSiOOSiO

O Si

O

O

O Si H

O

OO Si

(CH2)16 CH3

(CH2)16 CH3CHO

CHSiOO

HSiOOSiO

O Si

O

O

O Si H

O

OO Si

(CH2)16 CH3

Cogent BDC18

Bidentate C18 and C8 Moieties Bonded To

Adsorbed Mobile Phase on

TYPE-C Silica

21

Stationary Phases•Cogent Silica-C™ –unmodified Silica-Hydride-Most likely to retain polar compounds in ANP.

•Cogent Diamond Hydride ™ -small amounts of carbon in the silica. Excellent for amino acids Patent Applied for.

•Cogent Bidentate C8 ™ - less hydrophobic than C18. Often best choice for complex mixtures.

•Cogent UDC-Cholesterol ™ - liquid crystal phase. Shape selectivity as well as 3 modes of HPLC

•Cogent Bidentate C18 ™ -Excellent for RP and ANP.

22

Are these Columns Stable?M

23

Are these Columns Stable?

What about in the presence of Water?

YES, These columns are VERY Stable…even in Water.YES, These columns are VERY Stable…even in Water.

24

TYPE-C BASED COLUMN AFTER MORE THAN 2 YEARS OF USE IN 0.05% Acidic MOBILE PHASE

A: initial conditions, B: after 2 years

uracil + pyridine

phenol

phenol

uracil + pyridine

25

TYPE-C Silica

Ordinary Silica

Nonendcapped NormalC18 Bonded Phase

26

TYPE-C Silica

Ordinary Silica

Nonendcapped NormalC18 Bonded Phase

27

2. What do TYPE-C™ Silica HPLC Columns do?:

Unique SelectivityEfficiency

Stability-SuperiorVersatility

Reproducible

28

TYPE-C Silica

Ordinary Silica

Non-endcapped OrdinaryC18 Bonded Phase

30

Isn’t this HILIC?

Developed for Bioanalytical Applications

Hydrophilic Interaction Chromatography

31

NO!

Two Different Mechanisms for Different Applications

32

DIFFERENCES BETWEEN AQUEOUS NORMAL PHASE AND HILIC

• HILIC Requires a “water rich” environment to work

• Retains polar compounds by a normal phase mechanism

• Does not retain nonpolar compounds

• Cannot usually separate samples having both polar and nonpolar compounds

• Does not require water to work• Retains nonpolar compounds

by reversed phase mechanism• Retains polar compounds by

normal phase mechanism• Both reversed phase and

normal phase mechanisms can operate simultaneously

• Can separate samples with both polar and nonpolar compounds

Aqueous Normal PhaseSilica Hydride-Based Column

Hydrophilic Interaction Chromatography (HILIC) uses ordinary Silica-Based Column

33

Comparison of the Retention of Acidic CompoundsComparison of the Retention of Acidic Compounds Ordinary Silica-Based (HILIC) and TYPE-C Silica-Based ColumnsOrdinary Silica-Based (HILIC) and TYPE-C Silica-Based Columns

Flow Rate: 3ml/minWavelength: 254nm

Solvents A: Buffer B: 100%ACN

TYPE-B PHASE pH 8

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80 90 100

% Acetonitrile

Ret

enti

on

tim

e (m

in.)

4-Hydroxybenzoic acid

Acetylsalicylic acid

2-Hydroxybenzoic acid

SILICA HYDRIDE PHASEpH 8

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80 90 100

% Acetonitrile

Ret

enti

on

tim

e (m

in.)

Acetylsalicylic acid

4 Hydroxybenzoic acid

2 Hydroxybenzoic acid

Aqueous Normal Phase

34

Tech Note:ANP Retention is a Function of the

Compound

35

ANP Retention is a Function of the Compound

&Some Compounds can be Retained in

both ANP and RP

36

Some Compounds can Display Both Reversed Phase and ANP Behavior on the Same Column

Retention Map w ith Acetonitrile/Water (Formic Acid)

0.5

1

1.5

2

2.5

55 60 65 70 75 80 85 90 95

% Acetonitrile

Ret

enti

on

Fac

tor

(k)

Cytidine-R1

Cytidine-R2

Cytidine-R3

B

Nucleosides with Alkyl Chains

No RP Retention

M

37

Some Compounds can Display Both Reversed Phase and ANP Behavior on the Same Column

Retention Map w ith Acetonitrile/Water (Formic Acid)

0.5

1

1.5

2

2.5

55 60 65 70 75 80 85 90 95

% Acetonitrile

Ret

enti

on

Fac

tor

(k)

Cytidine-R1

Cytidine-R2

Cytidine-R3

B

Nucleosides with Alkyl Chains

Both RP and ANP Retention

38

In Addition to ANP

What Other Capabilities to TYPE-C Columns Have?

39

Only Cogent TYPE-C Silica™ based columns operate in

3 chromatographic modes.Without hysteresis or damage to columns

Reverse PhaseAqueous Normal Phase

Normal Phase with Non Polar Solvents

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TYPE-C Based HPLC Columns are Excellent

Reversed Phase Columns!

41

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Time (min)

Steroid Mixture on Cogent UDC-Cholesterol Column

Solutes: 1 = adrenosterone; 2 = corticosterone; 3 = 4-androstene-3,17-dione; 4= 11-acetoxyprogesterone; 5 = estrone; 6 = estradiol.

MOBILE PHASE: 50:50 MeOH/waterColumn Dimensions:

4.6 x 150mm

42

Use Bidentate C18 Columns with 100% Water

No Loss of Rt over Time

43

Using 100% Aqueous Mobile Phase On Cogent Bidentate C18 HPLC Column

1- OXALIC ACID2- FORMIC ACID3- ACETIC ACID4- SODIUM AZIDE5- URACIL6- FUMARIC ACID7- PROPIONIC ACID

100% 0.05% v/v H3PO4

Flow Rate:1mL/minColumn: 75 x 4.6mm idUV: 215 nm

After 3 hours of 100% aqueous exposure, results are the same as with the first injection

44

A Very Useful and Unique Feature Of Cogent TYPE-C™ HPLC Columns

Organic Normal Phase Chromatography without Hysteresis or

Damage to The Column.

45

- APCI+ Column: Bidentate C18, mobile phase: 95:5 Hexane/Ethyl AcetateFlow rate: 1.0 mL/min.Samples: 1 – phenol with aldehyde, 2 – parent phenol, 3 – phenol with ketone, 4 – phenol with acid

Normal Phase Separation of Substituted Phenolic Compounds on a C18 HPLC Column

46

Columns:

A: Bidentate C18

B: Silica-C

Cogent TYPE-C Columns for Analytical or Prep

Normal Phase Gradients

A: 0-0.5 min 100% hexane; 0.5-7 min to 50:50 hexane/ dicholoromethane; 50:50 hexane/ dichoromethane to 10 min.

B: 0.0 to 1.0 min 100% dichloromethane; 1.0 to 3.0 min to 100% ethyl acetate

Analytical

Preparative

47

Cogent Silica-C™ Column in Normal Phase Chromatography- Selectivity Advantage

A: HYDRIDE COLUMN

B: COMMERCIAL SILICA COLUMN

Mobile phase: 10% Diethyl Ether in Hexane

Stationary phase is un-modified silica hydride

4848

Where do I start?Where do I start?

Generic Strategy is Very Easy and FastGeneric Strategy is Very Easy and Fast 1. Mobile Phase to Start with.1. Mobile Phase to Start with.

• A. Water w/0.1% Formic Acid/.05% TFAA. Water w/0.1% Formic Acid/.05% TFA• B. MeCN w/0.1% Formic AcidB. MeCN w/0.1% Formic Acid2. Gradient starting at 95% A to 40% A over 20minutes 2. Gradient starting at 95% A to 40% A over 20minutes

for a 75mm column.for a 75mm column.3. Equilibrate for one column volume.3. Equilibrate for one column volume.4.Inverse Gradient starting at 95% B to 40% B over 20 4.Inverse Gradient starting at 95% B to 40% B over 20

minutes for a 75mm column.minutes for a 75mm column.5. Optimize depending on your results.5. Optimize depending on your results.

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3. Why are TYPE-C™ Silica HPLC columns recommended for all chromatography labs?

Unique SelectivityEfficiency

Stability-SuperiorVersatility (3 Modes)

Reproducibility

& much more….

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Fast LC?

Equilibrate Between Gradientsin very little Time

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Rapid Equilibration of Bidentate C18 HPLC Column

Reversed Phase Mode - Gradient Elution

Retention Time (min)

Solute/Equilibration Time 25 min 10 min 1 minBenzene 7.30 7.35 7.25 Naphthalene 11.10 11.07 11.01Phenanthrene 14.39 14.37 14.37Anthracene 14.81 14.80 14.80Pyrene 16.52 16.51 16.56

Gradient program: 0-3 min ACN/water (50:50)3-18 min to 100% ACN 18-23 min 100% ACN Equilibration to 50:50 ACN/water

52

Reproducibility

53

Peptides: Simple isocratic RP – HPLC analysis

An HPLC peptide standard mixture was resolved in under 5 minutes using a short (4.6 x 75 mm) Cogent BD C18 column and a simple isocratic RP-HPLC method. The separation was very reproducible. To achieve the separation presented on a conventional HPLC column, 4.6 x 250 mm, a gradient method is required. If higher resolution is desired a longer column should be used. Columns from leading brands of manufacturers were evaluated using the same conditions and compounds 3 and 4 were never separated under RP-HPLC isocratic conditions.

1. Gly –Tyr; 2. Val – Tyr – Val; 3. Met-enkephalin4. Leu-enkephalin; 5. Angiotensin II

Mobile Phase: 25% acetonitrile/75% DI water + 0.1% formic acid

Flow rate: 1 mL/minute

Detection UV 214 nm

Chromatogram is a composite of 8 consecutive injections

54

Reproducibility of Cogent Silica-C Column in a real life example

Six Injections-OverlaidExcellent Run to Run Precision

%RSD-0.3%

55

Retain and Separate Sugars in Reverse Phase

56

SEPARATION OF CARBOHYDRATE STRUCTUAL ISOMERS

OHC

O

O

O

OH

OHOH

OH

OH

O

OH

OH

OH

OH

OH

HO

HHS R

R

R

R

R R

S

R SRR

R

R

CHOO

OOH

OH

HO

OHHO

HOO

OH O

OH

OH

OH

OH

HR

R

R

R

R

R

RS

S S

R

R

S

S

OHC

O

O

O

OH OH

OH

HO

OH

O

HO

OH

OH

OH

OH

HO H

HS

S

R

R

R

R

R S

S

S

RR

R

R

CHOO

OOHHO

OH

HO

OH

O

OH

OH

O

OH

OH

OH

OH

S

R

R

R

RS

S

S

R

SR

R

S

S

maltotriose 1,4

panose 1,4 + 1,6

isomaltotriose 1,6

cellotriose 1,4

Mobile Phase: 100% water

All compounds have MW = 504

Each compound detected by MS in APCI+ mode with single ion monitoring (SIM) using a specific fragment ion

57

You Can and Should Avoid high pH when Possible

Why Damage Your instrument when you do not have to?

58

Every Chromatographer should have a Suite of the Cogent TYPE-C Columns for:

1.Problem Selectivity...Polar Compounds2.Rapid Equilibration Ballistic Gradients

3. LCMS Increase Sensitivity 4. Non Robust Methods 100% Water

5. To Challenge Every Method with RP and Quickly convert to ANP without purging your system

6. Impurity Profiling. Orthogonality 7. Complex Mixtures/Metabolites Natural Products8. When ordinary columns are not stabile enough

9. When samples adsorb to ordinary silica columns10. When samples hyrdolize on ordinary silica columns

59

Suite of Columns for Scouting & Method Development:

1 of each Phase in the Same Column Configuration

60

4. Real Life Examples of Problems Solved with:

Unique SelectivityEfficiency

Stability-SuperiorVersatility

Reproducibility

61

Glucosamine on Bidentate C18

0

1

2

3

4

5

6

7

8

40 50 60 70 80 90 100

% Acetonitrile in DI Water + 0.5% FA

Ret

en

tio

n T

ime

(m

in.)

Glucosamine

GLUCOSAMINE on

A Bidentate C18

62

Carbohydrate Retention on Bidentate C18

0

5

10

15

20

25

30

35

40

45

50

70 75 80 85 90 95 100

% Acetonitrile in DI Water + 0.5% FA

Re

ten

tio

n T

ime

(m

in.)

Glucose (monosaccharide)

Lactose(disaccharide)

Raffinose (trisaccharide)

Retention of Smaller Carbohydrates on a Cogent Bidentate C18™ Column

Detection by MS in the APCI+ mode

Separation based on size

63

Method Conditions

Column: Cogent Bidentate C18, 4m, 100A.Catalog No.: 40018-75PDimensions: 4.6 x 75 mm Mobile phase: A. 90:10 Acetonitrile/DI Water +0.5% formic acid B. 85:15 Acetonitrile/DI Water + 0.5 % formic acidFlow rate: 0.5 mL/min.Injection Volume: 10 LSamples:1. Triacetylnormetanephrine (m/z 166.2)2. Triacetylmetanephrine (m/z 180.2)200 ng of each sample was dissolved in 1 mL of reverse osmosis waterDetection: APCI+ Single Ion Monitoring

In detection single ion monitoring (SIM) was used. Mass transition of m/z 310.2 to m/z 166.2 (triacetylnormetanephrine) and m/z 324.2 to m/z 180.2 (triacetylmetanephrine) that correspond to the fragmentation of the (M+H+) ions were monitored.

Clinical Application 1

64

Clinical Application 2The powerful anticancer drug, methotrexate (4-amino-N10-methylpteroyl glutamic acid) acts as an antimetabolite and is used for the treatment of many neoplastic diseases including acute leukemia, osteosarcoma, non-Hodgkins lymphoma, and breast cancer. There is a great interest in pharmacological studies and clinical monitoring of methotrexate.

A quadrupole mass spectrometer operating in the positive – ion mode and an atmospheric pressure ionization (API) source was used for selective detection and assured that no interfering peaks affect the quantitative results. A bidentate C18 column was the column of choice for the ANP gradient analysis of the drug. The retention of the methotrexate is more than sufficient. The LC-MS method developed assures both high specificity and sensitivity.

Inverse GradientA: DI Water + 0.5% Formic Acid

B: AcetonitrileTime (min.) %A %B

0.00 10.0 90.0 1.00 10.0 90.0 5.00 80.0 20.0 10.00 80.0 20.0 10.01 10.0 90.0 12.00 10.0 90.0

65

Exploiting the Separation Power of TYPE-C Based Stationary Phases

METFORMIN

Aqueous Normal Phase

GLYBURIDE Reversed Phase

66

Hydride based BD C18, 4.6x75 mm, METFORMIN & GLYBURIDE

0

5

10

15

20

25

40 50 60 70 80 90 100

% organic in DI water, + 0.5% FA

Ret

enti

on

tim

e (m

in.)

Metformin - Acetonitrile

Glyburide - Acetonitrile

LUNA C18, 4.6x150 mm

0

5

10

15

20

25

30

40 50 60 70 80 90 100

% Acetonitrile in DI water + 0.5% FA

Ret

enti

on

tim

e (m

in.)

metformin

glyburide

Agilent Zorbax C18, 4.6x150 mm

0

2

4

6

8

10

12

14

16

18

20

40 50 60 70 80 90 100

% Acetonitrile in DI water + 0.5% FA

Ret

enti

on

tim

e (m

in.)

metformin

glyburide

Comparison of Bidentate C18 and Ordinary C18 Columns

RP and Aqueous Normal Phase Mode

67

FAST SEPARATION

Metformin/Glyburide

On Cogent Bidentate C18 WITH

UV DETECTIONColumn dimensions: 2.1 x 20 mm

Mobile Phase:

A: 50:50 acetonitrile, DI water + 0.5% formic acid

B: 80:20 acetonitrile, DI water + 0.5% formic acid

C: 85:15 acetonitrile, DI water + 0.5% formic acid

Flow rate: 0.3 mL/minute

Injection Volume: 1 µL

Samples:

1. Metformin

2. Glyburide

100 µg/mL of each in the mobile phase

Detection: UV 254nm

68

FAST SEPARATION

Metformin/Glyburide

On Cogent Bidentate C18 WITH

UV DETECTIONColumn dimensions: 2.1 x 20 mm

Mobile Phase:

A: 50:50 acetonitrile, DI water + 0.5% formic acid

B: 80:20 acetonitrile, DI water + 0.5% formic acid

C: 85:15 acetonitrile, DI water + 0.5% formic acid

Flow rate: 0.3 mL/minute

Injection Volume: 1 µL

Samples:

1. Metformin

2. Glyburide

100 µg/mL of each in the mobile phase

Detection: UV 254nm

69

Compound No. Compound Type Mol Wt. ApKa BpKa Log P

1 Cytidine-R1 397 12.12 3.73 1.54 2 Cytidine-R2 454 12.07 3.73 3.66 3 Cytidine-R3 425 12.09 3.73 2.66 4 Quinolinedione-R1 536 8.65, 8.68 - 3.46 5 Tetramic acid 536 7.91, 10.98 5.36 2.96 6 Quinolinedione-R2 520 8.64 - 2.74 7 Benzopyran 396 - - 2.80

Mobile Phase: 60:40 acetonitrile/water

Separation of a Mixture of Polar and Non Polar Compounds

70

2.1 x 20 mm Column 92% ACN+ 8% DI water with 0.5% Formic Acid

SEPARATION OF ACETYLCHOLINE/CHOLINE ON STANDARD And Short Cholesterol Columns

4.6x75 mm column (standard ID)90% ACN + 10% DI water 0.5% Formic Acid

Challenging quaternary amine compounds

71

Hydride Based BD C18: Choline/Acetocholine

0

5

10

15

20

25

40 50 60 70 80 90 100

% Acetonitrile in DI water + 0.5% FA

Ret

enti

on

tim

e (m

in.)

Choline

Acetylcholine

Hydride Based Cholesterol: Choline/Acetylcholine

0

5

10

15

20

25

30

40 50 60 70 80 90 100

% Acetonitrile in DI water + 0.5% FA

Ret

enti

on

tim

e (m

in.)

Acetylcholine

Choline

Comparison of Choline and Acetylcholine Retention

Cogent UDC-Cholesterol & BD C18 Columns ANP Conditions

72

Nucleoside Isomer Drug Separation

0

1

2

3

4

5

6

50 55 60 65 70 75 80 85 90 95 100

% Acetonitrile in DI water + 0.5% FA

Re

ten

tio

n T

ime

(m

in.)

Nucleoside 1

Nucleoside 2

Nucleoside Retention

0

2

4

6

8

10

12

14

16

18

50 55 60 65 70 75 80 85 90 95

% Acetonitrile in 0.5% FA

Re

ten

tio

n T

ime

(m

in.)

Cyt Dev 1

Cyt Dev 2

Column: Cogent UDC Cholesterol 4.6 x 150 mm

Nucleoside Analog MW BpKa LogP

Compound 1 494.5 3.73 3.184

Compound 2 494.5 3.73 3.184

Column: Cogent UDC Cholesterol 4.6 x 150 mm

Nucleoside Analog MW BpKa LogP

Cytidine Derivative 1 358.4 3.73 -0.163

Cytidine Derivative 2 356.4 3.73 -0.952

Retention of Nucleosides on a Cogent UDC-Cholesterol HPLC Column

Same pKa and LogPShape Recognition

Same pKa and Different LogPIncreased ANP

73

Retention of the Basic Drug “Tobramycin” in Acid Conditions

74

Comparison of Peak Shapes on TYPE-C Columns-

And Ordinary HPLC Columns

Tobramycin - High Efficiency on TC Column

Cholesterol Column 4.6 x 75 mm

End-capped Commercial C18 Column 4.6 x 150 mm

75

Separating Tetramic Acid derivative and a Hydrophilic Peptide

with similar Log P and acid dissociation constants

on the cholesterol column

ANP Retention

0

2

4

6

8

10

12

14

50 60 70 80 90 100

Percent Acetonitrile in Mobile Phase

Ret

enti

on

Fac

tor

(k)

Tetramic Acid Derivative

Peptide

OH

NH

O-R

Tetramic Acid Derivative

76

Retention can be Observed for Other

Hydrophilic Peptides

General Peptide Structure: Ac-AXEXAHKAY-NH2

77

Phenylalanine Retention on a Cogent Silica-C™ Column:

70:30 ACN/water + 0.1% FA

tR = 2.96 min t0 = 0.95 min

80:20 ACN/water + 0.1% FA

tR = 5.22 min

90:10 ACN/water + 0.1% FA

tR = 18.27 min

PhHO 2C CH 2CH

NH 2

Detection: TOF-MS m/z = 166

78

Succinic Acid Retention on a Cogent Silica-C™ Column

t0

HO 2C CO 2 HCH 2 CH 2

Mobile Phase: 95:5 ACN/water + 0.1% FA

Detection: TOF-MS @ m/z = 117

79

Complex Amino Acid Mixture on a Cogent Diamond Hydride™

Column: 2.1 x 150mm DH Gradient: 95%B – 60% B

Flow Rate: 0.4ml/min Column Temp: 20°C

Detection: Agilent TOF-MS Mobile Phase: A:0.1% Formic Acid B:MeCN 0.1% FA

8080

Column: Cogent Diamond Hydride™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 75mm packed Mobile phase: 85:15 acetonitrile/DI water + 0.1% fa + 0.001% TFA Flow rate: 1.0 mL/min.

Polar-Alkaloids on Diamond Hydride™Tryptamine HCL

8181

Column: Cogent Diamond Hydride™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 75mm packed Mobile phase: 60:40 acetonitrile/DI Water + 0.1% acetic Acid 70:30 acetonitrile/DI Water + 0.1% acetic AcidFlow rate: 1.0 mL/min.

Metabolite of Cyromazine on Diamond Hydride™Melamine

8282

Column: Cogent Diamond Hydride™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 75mm packed Mobile phase: A: DI water + 0.1% formic acid

B: acetonitrile + 0.1% formic acidFlow rate: 1.0 mL/min. Inverse Gradient

Biogenic Amine on Diamond Hydride™Serotonin & Analogs

5-hydroxy-3-indole acetic acid(5-HIAA), metabolite of serotonin 192 m/z

3,4-dihydroxyphenylacetic acid (DOPAC) 169 m/z Serotonin 177 m/z

Epinephrine 184 m/

8383

Column: Cogent Diamond Hydride™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 150mm Mobile phase: A: DI water + 0.1% acetic acid

B: acetonitrile + 0.1% acetic acidFlow rate: 0.4 mL/min. Inverse Gradient

Carbohydrates on Diamond Hydride™Glucose

8484

Column: Cogent Diamond Hydride™ HPLC Column, 4 m, 100 Å Dimensions: 2.1mm i.d. x 150mm Mobile phase: A: DI water + 0.1% amm acetate

B: acetonitrile + 0.1% amm acetateFlow rate: 0.4 mL/min. Inverse Gradient

Organic Acids on Diamond Hydride™Citric, Maleic & Trans-Aconitic Acid

8585

Column: Cogent Bidentate C18™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 75mm Mobile phase: A: DI water + 0.1% formic acid

B: acetonitrileFlow rate: 0.4 mL/min. RP Gradient

Sulfonamide on C18Sulfonamide

8686

Column: Cogent Bidentate C18™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 75mm Mobile phase: A: DI water + 0.1% formic acid

B: acetonitrileFlow rate: 0.4 mL/min. Fast RP Gradient

Carbohydrates on C18Sulfonamide Retention

8787

Column: Cogent Bidentate C18™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 150mm Mobile phase: A: 100% DI water + 0.1% Phos acid + 1.5g/L pentane sulfonic acidFlow rate: 1.0 mL/min. Isocratic, no loss of RT over time.

Strong Bases on C18™Cleaning Validation for Guanidine

8888

Column: Cogent Bidentate C8™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 150mm Mobile phase: 30% Acetonitrile, 70% DI Water w/Phos Acid, SDSFlow rate: 1.5 mL/min. Isocratic 7 minute separation

USP Method on C8Guanfacine HCl & Degradants

min0 1 2 3 4 5 6 7 8 9

mAU

0

100

200

300

400

500

600

8989

Column: Cogent Diamond Hydride™ HPLC Column, 4 m, 100 Å Dimensions: 2.1mm i.d. x 150mm Mobile phase: A: DI water + 0.1% amm formate, pH 7.2

B: 90% acetonitrile + 10% DI Water + 0.1% amm formate, pH 6Flow rate: 0.3 mL/min. Inverse Gradient

Phosphorylated Sugars on Diamond Hydride™Nucleotides by LCMS

1. ADP Glucose

2. Proprietary Sugar Nucleotide

3. Proprietary Sugar Nucleotide

4. CDP Glucose

5. UDP Hexanolamine

9090

Column: Cogent Diamond Hydride™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 150mm Mobile phase: A: DI water + 0.1% acetic acid

B: acetonitrile + 0.1% acetic acidFlow rate: 0.4 mL/min. Inverse Gradient

Antibiotics on C18Furazolidone: API & Related Compounds

9191

Column: Cogent Bidentate C18™ HPLC Column, 4 m, 100 Å Dimensions: 4.6mm i.d. x 150mm Mobile phase: 2% Acetonitrile, 88% DI Water w/ 10ml/L acetic acidFlow rate: 2.0 mL/min. Isocratic

Antibiotics on C18Furazolidone: API & Related Compounds

92

Benefits of Silica Hydride Based Stationary Phases

1. Operate in 3 different modes of chromatography: normal phase, aqueous normal phase and reversed phases. Versatile and very convenient and useful.

2. Use in 100% aqueous mobile phases in reversed phase with no loss of Rt over time. More robust methods.

3. Bases are well retained at low pH so high pH mobile phases may not be necessary or useful as orthogonal.

4. Surface absorbs very little water so there is reproducible retention in the normal phase without drying mobile phases and rapid equilibration for gradient separations.

5. Use over a broad range of pH and can be used at high temperatures.

6. Increase MS Sensitivity with high organic MP and use TFA in low concentrations for MS Compatibility.

7. Expand your Reach without exotic solvents or additives.

93

Where can I get More Data?

• www.mtc-usa.com• Look under “Useful Information” in Top Menu Bar

– Application Notes• HPLC

– Cogent TYPE-C

94

You Can See there are MANY uses for the Cogent TYPE-C™ Silica Based

HPLC Columns.

These shown are only the beginning.

95

You Can See there are MANY uses for the Cogent TYPE-C™ Silica Based

HPLC Columns.

These shown are only the beginning.

96

Thank You.

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