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Presented by: Bill Ciccone
MicroSolv Technology Corporation
Using Cogent TYPE-C Silica™
Where Tradition Meets Tomorrow
Eatontown, NJ USA
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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.
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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
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Cogent TYPE-C™ SilicaHPLC Columns
First Introduced at Pittcon 2003
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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:
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1. What are TYPE-C™ Silica based HPLC columns?
Unique Selectivity These are different from all Others.
EfficiencyStability-Superior
VersatilityReproducible
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Type-C Silica Columns are
“Silica-Hydride” Based
HPLC Stationary Phases:
’
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Ordinary SilicaStandard; Well Known by all
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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
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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
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How Does this structural difference impact the performance of the columns?
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“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”
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“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
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(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
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Bidentate C18 Direct Silicon-Carbon Bonds TYPE-C SilicaM
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Bidentate C18 Direct Silicon-Carbon Bonds TYPE-C Silica
M
Two Points of Attachment
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Bidentate C18 Direct Silicon-Carbon Bonds TYPE-C Silica
Not Susceptible to Hydrolysis.
Two Points of Attachment
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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
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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
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TYPE-C Silica
Ordinary Silica
Nonendcapped NormalC18 Bonded Phase
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TYPE-C Silica
Ordinary Silica
Nonendcapped NormalC18 Bonded Phase
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2. What do TYPE-C™ Silica HPLC Columns do?:
Unique SelectivityEfficiency
Stability-SuperiorVersatility
Reproducible
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TYPE-C Silica
Ordinary Silica
Non-endcapped OrdinaryC18 Bonded Phase
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Isn’t this HILIC?
Developed for Bioanalytical Applications
Hydrophilic Interaction Chromatography
31
NO!
Two Different Mechanisms for Different Applications
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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
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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
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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!
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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
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Use Bidentate C18 Columns with 100% Water
No Loss of Rt over Time
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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.
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- 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
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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
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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
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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
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Suite of Columns for Scouting & Method Development:
1 of each Phase in the Same Column Configuration
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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
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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.
97
