Chromatography Theory and Applications

Chromatography Theory
u Overview u Separation Modes
F Anion Exchange Chromatography
F

Cation Exchange Chromatography F Ion Exclusion Chromatography F Ligand Exchange Chromatography F Reversed Phase Chromatography

u Chemical Suppression u Detection Modes

Overview
Chromatography (LC) is the separation of species by various separation modes followed by suppressed conductivity, UV or some other type of detection

Separator Column
Injector

Suppressor
Detector

Separation Modes
Chromatography (LC) is the separation of species by various separation modes followed by suppressed conductivity, UV or some other type of detection

Separator Column
Injector

Suppressor
Detector

Substrates: Polymers Used

Most common Polymer used is a co-polymer of polystyrene and divinylbenzene (PS/DVB)
CH2 CH CH2 CH CH2 CH

CH2

CH2

CH2

CH

CH2

CH

CH2

CH2

CH

Features of PS/DVB
u Rugged Cross-linked polymer
F pH range 0-14 F solvent compatible with many solvents F Temperature stable F Able to control shrink/swell with X-linking

u Control particle size and porosity u Well defined polymerization and functionalization chemistries

Separation Modes
u Ion Exchange
F Anion Exchange F Cation Exchange

u Ion Exclusion u Ligand Exchange u Reversed Phase (Ion Pairing)

Anion Exchange
u Transgenomic Bead Characteristics F Cross-linked PS/DVB core F Coating F Quaternary Ammonium Exchange Groups u Common Buffers F Sodium Hydroxide F Sodium Carbonate/Bicarbonate F Sodium Chloride

Mechanism for Anion Exchange

u Common Applications F Inorganic Anions F Organic Acids F Oxyhalides F Amino Acids F Proteins/Peptides F DNA/Oligos F Transition Metals
+ OH- +

Resin-NR3

A-

Keq

Resin-NR3+ A- + OH-

Anion Exchange IC Applications

1. Fluoride 2. Chloride
2 3 1 4 5 6 7

3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

Conductivity
0

10

Minutes

Oxyhalides by Anion Exchange

4

COMPONENT 1. 2. 3. 4. 5. 6. 7. 8. 9.
7 6 8 9 10

Conductivit

Fluoride Chlorite Bromate Chloride Nitrite Chlorate Nitrate Bromide Phosphate

10. Sulfate

10

15

20

25

30

Time

Cation Exchange
u Transgenomic Bead Characteristics F Cross-linked PS/DVB core F Sulfonic, Phosphonic, Carboxylic Acid Functional Groups u Common Eluants F Organic Acids (Citrate, Oxalate) F Methane Sulfonic Acid F Buffers F NaCl

Mechanism for Cation Exchange

u Common Applications F Grp I & II Cations F Ammonium Species F Transition Metals F Amino Acids F Proteins/Peptides

Resin-SO3

- H+ +

A+

Keq

Resin-SO3- A+ + H+

Cation Exchange Applications

Hydrolysate Amino Acid Analysis using a Beckman Sodium AAA Column
ala thr ser asp glu gly val met leu iso-leu lys phe arg tyr his

Cation IC Applications
1

1. Copper 2. Nickel 3. Zinc 4. Iron

6 2 3 4 5 7

5. Manganese 6. Cadmium 7. Lead

10

15 Minutes

20

25

30

35

Ion Exclusion Chromatography

u Bead Characteristics F Cross-linked PS/DVB core F Macroporous F Highly Sulfonated u Common Eluants F Strong Acids (HCL, Sulfuric) F Organic Acids (OSA, Perchloric, Perfluorobutyric Acid)

Mechanism for Ion Exclusion

u Common Applications F Organic Acids F Borate/Carbonate F Alcohols

RCOOH (retained)

pKa

RCOO- (excluded)

Ion Exclusion Applications

Krebs Cycle Acids
2 1 3 4 6 5 7

1. Citric 2. Tartaric 3. Glucose 4. Malic 5. Fructose 6. Lactic 7. Glycerol

8 10 9

8. Acetic 9. Methanol 10. Ethanol

0 5

10

15 Minutes

20

25

30

35

Reversed Phase (Ion Pairing) Chromatography

u Transgenomic Bead Characteristics F Cross-linked PS/DVB core F Macroporous or Nonporous F May be alkylated (-CO, -C18) u Common Mobile Phases F Acetonitrile/Water F Methanol/Water uCommon Ion Pairing Reagents F TEAA F TMAOH F TBAOH

Mechanism for Ion Pairing

u Common Applications F Surfactants F DNA F ethylamines F Aliphatic Acids F Transition Metals

TBA+A- (retained)

TBA+ + A- (moves)

Ion Pairing Reversed Phase

Reversed Phase Applications

3 2 5
1. Physostigmine 2. Acetophenone 3. Ethyl Phenone 4. Butyl Phenone 1 5. Valerphenone

6 8 Minutes

10

12

14

20-mer on an OLIGOSep Column

Separation of 20-mer from n-1, n-2 failures
19mer
15.0

18mer

10.0

20mer

UV Response

5.0

0.0

-5.0 0 5 10 15 20

Ligand Exchange Chromatography

u Bead Characteristics F Cross-linked PS/DVB core F Macroporous F Highly Sulfonated F Metal Loaded (Pb, Ca, etc.) u Common Eluants F Water F Sodium Hydroxide

Mechanism for Ligand Exchange

u Common Applications F Alcohols F Carbohydrates F Weak Acids

Resin-SO3

-M ++H

2O +

A-

Keq

Resin-SO3- M + + A- + H2O

Ligand Exchange Application

Monosaccharides on a CARBOSep CHO-620

suc raf sta glu gal fru man sor

0 min

12

16

Chemical Suppression
Used Only In IC: Ion Chromatography (IC) is the separation of ionic species by ion exchange chromatography coupled with suppressed conductivity detection

Separator Column
Injector

Suppressor
Detector

Chemical Suppression
u Purpose
Reduce background conductivity from eluant and increase signal from analytes to make high sensitivity ion analysis possible

u Types of Suppressors Available

F Dionex: self regenerating suppressor, membrane suppressors, fiber suppressors, packed bed suppressors F Altech: packed bed suppressor

How Chemical Suppression Works

Waste
Na+ Na+ H+ Na+ H+ H+ H+

Eluant
Na+ Na+ OHNa+ Na+ OH+ Na H+ OHH+ OHH+ H+

Waste

H+

H2O

Regenerant
H2O H O 2 H2O

Regenerant

Chemical Suppression Reactions

u Anion IC
H+

Na+ + OHH+

Na+

H 2O

Na+ + HCO3H+

Na+

H2CO3 H+ + A -

H2O + CO2

Na+ + A-

Na+

Chemical Suppression Reactions

u Cation IC
OH-

H+ + MSAMSAOH-

H 2O

A+ + Cl-

Cl-

A+ + OH-

LC Detection Modes
Chromatography (LC) is the separation of species by various separation modes followed by suppressed conductivity, UV or some other type of detection

Separator Column
Injector

Suppressor
Detector

LC Detection Modes
u UV/Vis - Most common for LS
F DIrect UV (diode array) F Indirect UV - Refractive Index F Direct UV w/ Post Column Reaction

u Conductivity - Most Common for IC u Amperometry - For Carbohydrates u Other (Mass Sepc, ELS, etc.)

Agenda
u Introduction u Chromatography Theory u Cetac IC Products u Transgenomic Life Science Products u Competitive Analysis u Conclusion

Transgenomic Ion Chromatography Products

ICSep Ion Exchange Columns

u ICSep Columns for Anion Analysis
l ICSep AN1 l ICSep AN1-SC l ICSep AN2 l ICSep AN300 l ICSep AN300b l ICSep ANSC

u ICSep Columns for Cation Analysis

l ICSep CN2

Features and Benefits of Cetac IC Columns

Features of the ICSep AN1 Column

u Separates 7 Standard Ions in 15 minutes u Fluoride separated from water dip with Carb/Bicarb Eluant u Runs in both suppressed and nonsuppressed formats u High Capacity for dirty samples u Direct replacement for the Dionex AS11 u Works with hydroxide, PHBA or Carbonate eluants

Conditions Eluant: 1.8mM Carb/1.7mM Bicarb Flow 1.0mL/min Detect: Suppressed Cond

Features of the ICSep AN300 Column

u Separates 7 Standard Ions in 8 minutes u Designed and Verified equivalent for EPA method 300.0(a) u Fluoride completely resolved from water dip u Works with hydroxide or Carbonate eluants u Direct Replacement for AS-4A column running anions in water

Conditions Eluant: 1.8mM Carb/1.7mM Bicarb Flow 2.0mL/min Detect: Suppressed Cond

Features of the ICSep AN300b Column

u Designed to separate 7 Standard Ions as well as oxyhalides and organic acids u Designed and Verified equivalent for EPA method 300.0(b) u Solvent compatible, can clean with even organic solvents (i.e. methanol) u Works with hydroxide or Carbonate eluants u Very High Capacity - Direct Replacement for AS9HC column running disinfection agents in water

Features of the ICSep ANSC Column

u Same selectivity as the Dionex AS-4ASC u Compatible with Organic Solvents u 7 Standard Anions in 11 minutes u Compatible with both hydroxide and carbonate-based eluants

Conditions Eluant: 1.8mM Carb/1.7mM Bicarb Flow 2.0mL/min Detect: Suppressed Cond

Features of the ICSep AN1-SC

u Same features as the AN1 u Direct Replacement for systems using the Dionex AS11/AS12 u Now Compatible with up to 100% Organic Solvents***

Conditions Eluant: 1.8mM Carb/1.7mM Bicarb Flow 1.0mL/min Detect: Suppressed Cond

Features of the New ICSep AN2

Very high Capacity Separates organic acids and ions in same run Compatible with Organic Solvents Direct replacement for systems running Dionex AS14 columns (1mM HCO3/3.5mM CO3) Compatible with both hydroxide and carbonatebased eluants

ICSep AN2

COMPONENTS: 1. FLUORIDE 2. ACETATE 3. CHLORIDE 4. NITRITE 5. BROMIDE 6. NITRATE 7. PHOSPHATE 8. SULFATE

Features of the ICSep CN2 Column

u Separates Group I and Group II Cations as well as Transition series metals u Separates Ammonium Species u Compatible with all common Cation exchange eluants u Works in both suppressed and nonsuppressed modes

OEM Vendors for IC Columns

u Latchatt - Special AN300 to Latchatt specs u Metrohm F Metrosupp 1 : AN300b F Metrosupp 2 : AN1 F Metrosupp 3 : AN1-SC

Symptoms of Column Contamination

u Inorganic Contamination - peaks tail, retention time decreases, pressure increases u Metals - Phosphate disappears u Organic Contamination - increase pressure, nitrate tails, etc. u Notes: Always recommend guard column, if pressure high change frit.

Care of Transgenomic IC Columns

u Long Term Storage - either water or 10mM NaOH u Inorganic Contamination
F 40mM EDTA @ 0.5mL/min for 1 hour (for metals) F 0.1M NaOH @ 1.0mL/min for 3 hours

u Organic Contamination
F Solvent Compatible Columns flush 90% ACN or MeOH @ 1.0mL/min for 2 hours (ANSC, AN1-SC, AN300b, AN2) F Non-SC columns flush with 10% MeOH @ 0.5mL/min overnight (AN1, AN300) F Follow with Inorganic Cleanup

Agenda
u Introduction u Chromatography Theory u Cetac IC Products u Transgenomic Life Science Products u Competitive Analysis u Conclusion

Transgenomic Chromatography Products for Life Science Applications

Amino Acid Columns

Amino Acid Analysis Columns

u AMINOSep Columns for Amino Acid Analysis
l AA 911 l AA 903 l AA 511

u Transgenomic Columns for the 63/7300

l Transgenomic Lithium

l Transgenomic Sodium

l Transgenomic Sodium AA column for System Gold

Features of Transgenomic AAA Columns

u Both Sodium and Lithium Cation Exchange Forms u Separates both hydrolysates and physiological fluid amino acids u Very tight particle size range for high efficiency u Standard for Beckman and Pharmacia AAA Systems (we supply 95% of the worlds use for IEX amino acid analysis columns/resin) u Rugged, long-lasting design

Transgenomic Lithium Column for 63/7300

Transgenomic Sodium Column for 63/7300

Sodium Column for System Gold

OEM Vendors for AAA Columns

u Beckman - System Gold, Na+ and Li+ u Waters - Na+ Cation Exchange u Merck - Na+ Cation Exchange u Phamacia - Resin u Pickering - Resin

Symptoms of Column Contamination

u Peaks Co-Elute u Retention Times Increase u Irreproducible Ammonia Peak u Peaks begin to tail u Notes: this is a really complex analysis and columns may need to be regenerated frequently

Care of Transgenomic AAA Columns

u Long Term Storage - either C Buffer or 10mM NaOH u Column Contamination
F First flush with 20% IPA or MeOH @ 0.5mL/min for 30 minutes F Next, If Lithium form column use 0.1M LiOH if Sodium form column use 0.1M NaOH F Flush @ 0.5mL/min for 1 - 3 hours F Flush with DI Water for 15 minutes F Reequilibrate on A Buffer Follow with Inorganic Cleanup

F NOTE: Column regeneration buffer available

Carbohydrate Analysis Columns

Carbohydrate Analysis Columns

u CARBOSep Columns for Carbohydrate Analysis
l CHO-411 l CHO-611 (Na) l CHO-620 (Ca) l CHO-682 (Pb) l CHO-820 l CHO-882 l COREGEL 87C l COREGEL 87H l COREGEL 87K l COREGEL 87N l COREGEL 87P

Features of Transgenomic Carbohydrate Columns

u Separates sugars and alcohols u Compatible with water and hydroxide mobile phases u Longest history with ligand-exchange gels

u Separates both mono and oligosaccharides up to DP7 u Compatible with USP methods u Very rugged, long lasting design

u Highest efficiency ligand-exchange columns available

87C

87P

87K

COREGEL 87H

Stachyose Raffinose Maltotriose Sucrose Cellobiose Trehalose Maltose Melibiose Lactose Lactulose Maltitol Glucose Sorbose Xylose Rhamnose Mannose Fructose Fucose Arabinose Mannitol Arabitol Sorbitol Xylitol

5.94 6.56 6.68 7.48 7.36 7.32 7.59 7.67 7.84 8.53 9.15 9.36 10.22 10.31 10.41 10.51 11.14 11.33 11.63 12.76 13.23 14.91 15.06

11.84 12.01 12.63 13.51 13.53 NA 14.43 15.25 15.09 18.93 NA 16.09 19.45 17.96 19.53 20.39 22.59 27.93 21.73 34.51 33.98 50.76 44.76

8.46 9.01 9.16 9.94 9.79 NA 10.06 NA 10.49 11.49 NA 12.09 NA NA NA 14.06 15.61 15.41 15.76 20.54 20.94 25.61 25.03

7.85 8.31 8.35 9.18 9.01 9.14 9.24 9.43 9.51 10.24 12.29 11.22 12.90 12.37 12.93 12.83 13.68 13.89 14.00 17.89 18.43 21.41 22.03

11.35 14.41 15.24 15.77 15.65 16.05 16.68 17.70 17.44 20.77 30.45 19.21 22.45 20.71 22.63 25.57 25.90 24.23 24.02 40.07 39.80 55.56 51.14

6.32 6.96 7.36 8.08 NA 8.22 8.56 NA 8.72 NA 8.16 11.20 13.16 12.24 13.37 12.48 12.16 11.98 13.44 10.08 10.80 10.64 11.36

6.94 7.65 7.18 ND 7.76 8.00 7.78 7.88 8.13 NA NA 10.11 9.90 10.33 11.20 9.98 10.39 12.05 11.23 NA NA 8.32 8.35

CARBOSep CHO-620 (Calcium Form)

CARBOSep CHO-682 (Lead Form)

CARBOSep CHO-820 (Calcium Form)

CARBOSep CHO-611 (Sodium Form)

Features of COREGEL Columns

u 9 micron particle size u 8% crosslinked u Very tight particle size range for highest efficiency u Available in Calcium, Sodium, Lead, Hydrogen, Silver and Potassium u Direct substitution for BioRad AMINEX columns and any other AMINEX equivalent

CARBOSep COREGEL 87C (Ca Form)

CARBOSep COREGEL 87K (K Form)

CARBOSep COREGEL 87N (Na Form)

CARBOSep COREGEL 87P (Pb Form)

OEM Vendors for Carbohydrate Columns

u Waters - SugarPak u Merck - Polysphere u MetaChem - Metacarb u Machery Nagel u Varian Chrompack

Symptoms of Column Contamination

u Peaks Broaden u Retention Time Shifts u Pressure Increases u Irrepudicible Retention Times u Note: Always recommend a guard column with carbohydrate columns

Care of Transgenomic Carbo Columns

u Long Term Storage - DI Water u Column Contamination
F These columns are very hard to clean and usually require replacement. F For Organic Contamination: NEVER EXCEED 10% ACN or MeOH because these resins swell very easily, flush at 0.3mL/m for several hours, then flush with DI Water. Watch column pressure.

Care of Transgenomic Carbo Columns

u Column Contamination - Inorganics and Biocontamination
F Select cleaning solvent very carefully - it should match the ionic form of the column.
F Use 0.1M NaOH for CHO-611/OH and COREGEL 87N F Use 0.1M CaOH for CHO-820 and COREGEL 87C F Use 0.1M KOH for COREGEL 87K

F Flush column at 0.3mL/min overnight then flush with water for several hours F For CHO-682 or COREGEL 87P use 0.1M NaOH overnight followed by 0.1M PbCl for 3 hours @

Ion Exclusion Columns

Ion Exclusion Columns

u ICSep Columns for Organic Acid, Carbonate and Alcohol Analysis
l ICSep ICE-ION-300 l ICSep ICE-ION-310 l ICSep ICE-ARH-601 l ICSep ICE-ORH-801 l ICSep ICE-COREGEL 64H l ICSep ICE-COREGEL 87H

Features of the ICSep ICE-ION 300

u Separates Organic Acids, Sugars and Alcohols with VERY high resolution u Small Particle Size provide high efficiency u Designed for use with normal matrices u Recommend for Wine Analysis u Designed for High Resolution Applications

Features of the ICSep ICE-ORH 801

u Separates Organic Acids, Sugars and Alcohols with good resolution u Intermediate resolution between ION-300 and Coregel 87H u Blended to provide optimum resolution with good ruggedness u Can also run inorganic anions such as sulfite, azide, fluoride and arsenic species u Designed as general purpose column

Features of the ICSep ICE-COREGEL 87H

u The most rugged column (8% cross linked) u Larger bed volume, bead size, lower pressure allows the column to be run at fast flow rates u Separates Sugars, Alcohols and Organic Acids all on one column in same run u Designed for use with tough matrices u Direct replacement for BioRad Aminex Columns

Features of the ICSep ICE-ION 310

u Designed for fast run organic acid analysis u High sensitivity, rapid analysis of borate and bicarbonate u Rugged column u Designed for use with tough matrices u Choose this column when speed is the primary concern

Features of the ICSep ICE-ARH 601

u Designed for analysis of Aromatic Organic Acids u Eluants employ only Sulfuric Acid and Water, no organic modifiers are required u Only ICE column available designed especially for Aromatic Organic Acids

Features of the ICSep ICE-COREGEL 64H

u Larger bed volume, bead size, lower pressure allows the column to be run at fast flow rates u Separates Sugars, Alcohols and Organic Acids all on one column in same run u Direct replacement for BioRad Aminex Column

OEM Vendors for Ion Exclusion Columns

u Metachem - MetaCarb u Alltech u Merck u Machery Nagel

Symptoms of Column Contamination

u Inorganic Contamination - peaks tail, retention time decreases, pressure increases u Metals - Irreproducible results u Organic Contamination - increased pressure u Notes: Always recommend guard column this will help protect the separator.

Care of Transgenomic Ion Exclusion Columns

u Long Term Storage - 10mM Sulfuric Acid u Inorganic Contamination
F 40mM EDTA @ 0.4mL/min for 1 hour (for metals) F 0.1M Sulfuric Acid @ 0.4mL/min for 6 hours F flush with DI-water then reequilibrate

u Organic Contamination
F NEVER EXCEED 10% ACN or MeOH and watch pressure carefully, flush at 0.4mL/min for 3 hours then flush thoroughly with water.

Reversed Phase Columns for Ion Pairing Chromatography

RPSep Reversed Phase/Ion Pairing Columns

u RPSep Columns for Reversed Phase and Ion Pairing Chromatography
l RPSep PRX-1 l RPSep ACT-1 C18 l RPSep PolyRP CO l OLIGOSep - 1

Column Options
Two Options: non-functionalized or alkylated PS/DVB
Non-Functionalized OLIGOSep , PRX, PolyRP C0 C18 Alkylated 5m PS/DVB ACT-1 5m PS/DVB

Features of the RPSep PolyRP-CO Column

u CO functionalization provides hydrophillic surface for separation of less polar compounds u Proprietary, patented, alkylation technology u Small particle size with tight particle size range for high efficiency

Features of the RPSep PRX-1 Column

u Nonfunctionalized PS/DVB ideal for separation of both polar and nonpolar species u Fine particle size range for high efficiency u Exceptionally rugged design u Direct replacement for Dionex NS1 or Hamilton PRP-1 columns

Features of the RPSep ACT-1 C18 Column

u Alkylated with C18 groups, provides hydrophobic surface ideal for the separation of polar species u pH Stable over range of 0-14 u Proprietary, patented, technology u Very high efficiency

Care of Transgenomic Reversed Phase Columns

u Long Term Storage - 40% ACN and Water u Inorganic Contamination
F 40mM EDTA @ 1.0mL/min for 1/2 hour (for metals) F 0.1M Sodium Hydroxide @ 1.0mL/min for 1 hours F flush with DI-water then reequilibrate

u Organic Contamination
F Flush with 95% ACN or MeOH @ 1.0mL/minute for 3 hours

Agenda
u Introduction u Chromatography Theory u Cetac IC Product Launch u Transgenomic Life Science Products u Competitive Analysis u Conclusion

Competitive Positioning and Analysis IC Products

Goals for Transgenomic IC Products

Product Positioning
Cetac Technologies Offers a Complete Line of Chromatography Columns that Provide Superior Solutions for the Analysis of Ionic Species at a lower cost than Competitive Alternatives

Summary:
Make Better Columns at a Lower Cost

Key Competitor in the IC Market-Dionex Corporation

Dionex - Background
u Founded 1975 u Recognized leader in Ion Chromatography u > 20,000 IC Systems Installed u ~$165M in Annual Sales u ~$37M Annual Sales in IC Columns

Dionex - Advantages
u Corporate IC Focus u Large Installed Base u Many Applications Developed u Approved Methods (E.P.A.) u Complete IC Product Offering u Proprietary Suppressor Technology u Protected Market

Dionex - Disadvantages
u Gouge Customers u Single Expertise - Not good at other fields u Not very flexible u Poor Service

Transgenomic Strategy
Offer the IC customer a compatible replacement for his Dionex column at a lower cost

Cetac Column

Dionex Compatible

ICSep AN2 ICSep AN1 ICSep AN1-SC ICSep AN300 ICSep AN300B ICSep ANSC ICSep CN2

IonPac AS14 IonPac AS11/AS12 IonPac AS11/AS12 IonPac AS4A IonPac AS9-HC IonPac AS4A-SC IonPac CS3

Transgenomic - Advantages
u Compatible Selectivities u Superior Fluoride Retention u Covalently Bound Surface Chemistries - Long Life u Lower Cost u Employ Guard Disc to provide added protection

Covalent and Pellicular Anion Exchangers

Covalent Anion Exchangers

May Have Pores ( holes ) or be NonPorous All ion exchange groups attached covalently to the surface of the bead

Porous

NonPorous

Characteristics of Covalent IC Resins

u Larger surface area for porous u Higher Capacity u Covalently bound (grafted) functional groups u Can control exclusionof molecules by pore size u Lower column pressures u Can be used for both anion and cation columns

Pellicular Anion Exchangers

u Latex microspheres ionically attached to sulfonated core u Ion exchange groups on the surface of the latex microbead
Latex Microsphere
NR3+ NR3+ NR + 3 NR3+ NR3+
+

PS/DVB

NR3

NR3

NR3+ NR + NR3+ NR + 3 3

Characteristics of Pellicular IC Resins

u All interactions are on the surface of the microbead u High efficiency from small microspheres u Use larger beads for less backpressure u Less secondary interactions u Core bead sulfonated, latex ionically attached

Benefits of Covalent and Pellicular Anion Exchangers

Covalent Anion Exchangers

Advantages of Covalent Ion Exchangers

u Higher capacity - can handle very dirty samples u More rugged and will last longer - covalently bound chemistries won t come off u Easily cleaned u Fluoride can be retained u Can take advantage of surface interactions u Less hardware requirements - lower running pressures

Disadvantages of Covalent Ion Exchanger

u Materials can hidein pores u More surface interactions - nitrate/iodide tails u Must use smaller particles for high efficiency

Anions on a Cetac AN1-SC

ICSep AN1SC Column
45.0
3

40.0

Fluoride Retained

Nitrate Tails

35.0

30.0
1

5 7

Response (mV)

25.0
6

20.0

15.0

10.0

5.0 0.0 4.0 8.0 12.0

Time

Pellicular Anion Exchangers

Advantages of Pellicular Ion Exchangers

u High Efficiency - Sharper Peaks u Less surface interaction - Nitrate tailing minimal u Use larger core bead for less backpressure

Disadvantages of Pellicular Ion Exchangers

u Fluoride not easily retained u Susceptible to metals contamination phosphate results inconsistent u Lower capacities - trouble with dirty samples u Ionically bound chemistries - shorter column lifetime u Can contaminate irreversibly

Anions on a Dionex AS4A-SC

7 Anions on a Dionex AS4A Fluoride not retained
14.0 2

12.0

Less Nitrate Tailing

10.0 3 4 5 8.0

6.0 6 4.0

2.0

2.00

4.00

6.00

8.00

10.00

Comparison of Efficiency Loss from Metal Contamination

6000

5500

5000

4500

4000
Efficiency

3500

AN1-SC AS6

3000

2500

2000

1500

1000 0 20 40 60 80 100

Reason
u Latex microspheres ionically attached to sulfonated core u Metals bind Ion Exchange Sites Irreversibly
Cu+2

SO3-

PS/DVB

+ NR NR + + NR3+ 3 3 NR 3 SO3NR3+ NR3+ SO3NR3+ NR3+ NR + NR3+ Cu+2 NR3+ 3

SO3

+2 Cu -

Selling Hints
u Target Dionex customers with Cloneapproach u ALWAYS recommend a guard cartridge u Cetac Columns designed to Last Longer u Promote compatible separation at lower cost u Focus on Cetac specialty - Inorganic Analysis u Highlight New Chemistries provide better separation

Comparison Cetac AN2 & the Dionex AS14

200.0 180.0 160.0
1

4 8 5 6 7

140.0 120.0 100.0 80.0 60.0 0.0 5.0 10.0 15.0 2

1. Fluoride 2. Acetate 3. Chloride 4. Nitrite 5. Bromide 6. Nitrate 7. Phosphate 8. Sulfate

Response

20.0

Time
2 8.00 1 6.00 3 4 7 5 6

4.00

1. Fluoride 2. Chloride 3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

2.00

0 0 2.00 4.00 6.00 8.00 10.00

Minutes

Comparison Cetac AN1 & the Dionex AS12

45.0
2 3

40.0

Response (mV)

35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 4.0 8.0 12.0
6 4 5

1. Fluoride 2. Chloride 3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

Time
2.50 2.00 1.50 2

3 1.00 0.50 0 -0.50 4 5 6

1. Fluoride 2. Chloride 3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

2.00

4.00

6.00

8.00

10.00

12.00

14.00

Comparison Cetac ANSC & Dionex AS4A-SC

2 7 3 4 5

Conductivity

1. Fluoride 2. Chloride 3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

0.0 14.0 12.0 10.0 8.0 6.0

2.0 2 1

4.0

6.0

8.0

10.0

12.0

14.0

16.0

Time 1. Fluoride 2. Chloride 3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

5 7 6

4.0 2.0 0 0 2.00 4.00 6.00 8.00 10.00

Comparison Cetac AN300 & Dionex AS4A

2 1 3 6 4 5 7

Conductivity

1. Fluoride 2. Chloride 3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

10

Minutes
14.0 12.0 10.0 8.0 6.0 6 4.0 2.0 0 0 2.00 4.00 6.00 8.00 10.00 1 2

5 7

1. Fluoride 2. Chloride 3. Nitrite 4. Bromide 5. Nitrate 6. Phosphate 7. Sulfate

Comparison Cetac AN300B & Dionex A9HC

4 5 1

Conductivity

1. Fluoride 2. Chlorite 3. Bromate 4. Chloride 5. Nitrite

7 6

6. Chlorate 7. Nitrate 8. Bromide 9. Phosphate 10. Sulfate

2 3

8 9

10

0
4.00 3.50 3.00 2.50

5
1

10

15 Time

20

25

30 1. Fluoride 2. Chlorite 3. Bromate 4. Chloride 5. Nitrite 6. Phosphate 7. Bromide 8. Chlorate 9. Nitrate 10. Sulfate

4 5 3 2 8 6 7 9

10

2.00 1.50 1.00 0.50 0

Competitive Positioning and Analysis Life Science Products

Life Science Position

Transgenomic supplies polymeric chromatography products which provide superior solutions for target biological applications

Summary:
Focus on the development of superior columns for niche applications for the life science market place

Conclusion

IC Product Positioning
Cetac Technologies Offers a Complete Line of Chromatography Columns that Provide Superior Solutions for the Analysis of Ionic Species at a lower cost than Competitive Alternatives

Life Science Product Position

Transgenomic supplies polymeric chromatography products which provide superior solutions for target biological applications