seilc primesep method development

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2 Resolution Systems

Resolution Systems is a distributor and manufacturer of HPLC, SPE and Flash Chromatography consumables and hardware systems to support these consumables.

Resolution Systems has been providing technical and customer support for over 10 years. We provide consolidation of your procurement process by offering serveral HPLC and SPE suppliers through one souce. Our Close relationships with our manufactorers allow us to accelerate the delivery process.

We also offer on-site and online HPLC and SPE training seminars; hands-on assistance with instrumen-tation maintenance, and personalized technical support for the manufactureres which we represent. Resolution Systems, Inc serves the important role of bridging scientists to current technical products and procedures and providing educational and training services.

Transform your

Chromatography

If you are struggling

with any of the following

Resolution Systems 3

• Insufficient retention of polar compounds • Lack of selectivity for closely related compounds

• Poor peak shape for strong basic analytes • Long analysis time for samples containing vastly different compounds

will transform your Chromatography from this....

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If you are struggling

with any of the following

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C18

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Mixed-Mode Technology

Why is Mixed-Mode technology better?

Good retention and selectivity is the key to good chromatography. Reversed phase chromatography has retention and selectivity properties for hydrophobic compounds which differ in their hydrophobicity. Ion Exchange chromatography has retention for ionizable compounds and selectivity for compounds with different ionic properties. Neither can do both.

Alkyl Reversed PhaseStrengths WeaknessesPredictable retention mechanism Limited retention for polar compounds

High efficientcy Limited selectivity for polar compounds

Many choices Single mode of interaction

Unstable in highly aqueous environments

Water Wettable Phases (AQ Phases)Strengths WeaknessesStable in 100% aqueous mobile phase Small retention improvements

Increased retention for polar compounds Not ideal for LC-MS

Highly aqueous mobile phase

Polar Embedded PhasesStrengths WeaknessesImproved selectivity Small retention improvments

Stable in 100% aqueous mobile phase Significant phase bleed with LC-MS

Ion PairStrengths WeaknessesSignificant retention improvements Not compatible with LC-MS

Retention for non-ionic species Requires dedicated columns

Complex mobile phases

Ion ExchangeStrengths WeaknessesRetention for ionic compounds No retention for non-ionic species

Low efficiency


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Mixed-Mode, like having two columns in one!

The unique bi-modal bonding chemistry of Primesep retains polar-ionizable compounds by ionic interactions while maintaining reversed-phase properties for interaction with neutral species. The separation is controlled by both ionic strength and organic modifier strength, allowing near infinite control of retention and selectivity. Unlike conventional ion exchange phases, the patented mixed-mode ligand allows the use of LC-MS friendly mobile phases.

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Primesep™Strengths

Retention for polar ionic compounds

Retention for neutral compounds

Independent control of ion-exchange and hydrophobic mechanisms

Compatible with LC-MS

Cation Exchange/ Reversed Phase Anion Exchange/Reversed Phase

Hydrophobic Control

Organic Modifiercontrols hydropobic retention

Percent IonizationControl

Mobile phase pH effects % ionization of column and analyte

Ionic Control

Mobile phase ionic strength controls ion exchange.

Cation Exchange functionality for retention of polar-ionic species. Retention is controlled by buffer strength.

Hydrophobic tail for retention of neutral compounds, controlled by mobile phase organic concentrations.

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Each Primesep cation exchange phase differs by its embedded carboxylic acid moiety. Each acidic moiety has a distinct pKa which controls the ionic properties of the phase, in other words the ionic interactions can be “switched” on and off.

• The hydrophobic properties of all Primesep phases are similar. • The lower the column’s pKa the stronger the ionic interactions and more retention for basic analytes. • Mobile phase pH determines the ionic state of the column

• Mobile phase pH > Column pKa- - column is ionized • Mobile phase pH = Column pKa- -column is 50% ionized • Mobile phase pH < Column pKa- -column is neutral and behaves like a polar embedded column

Switch Phase™Technology

Column Type Transition Point = pKaPrimesep C pH= 3.5

Primesep 200 pH=2

Primesep 100 pH=1

Primesep A Ionized at all working pH

+H

_COOH

COOH

pH above transition

On Off

COO

COO

COO

pH below transition

COOH


Tech Tip

Retention is Proportional to % Ionization

Effect of Mobile Phase pH on Column Ionization

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Greatest retentionfor Ionizable compounds

Mobile phase pH can be used to control retention

Least retention for ionizable compounds. Column behaves like a polar embedded phase

Primesep 100

Primesep 200

Primesep C

Neutral Guefenesin is not affected by pH or ionic strength changes

Changing pH from 4-3.5 changes ionization of column from 100% to 50% for LC-MS friendly analysis

Protocol # 2: This method is designed to offer the most optimized method.

• Ion Exchange retention is first set by adjusting ionic-strength

• Hydrophobic retention is than adjust by organic concentration

Applications:

• Analytical R&D • Simple Samples

Analyte Types:

• Strong Bases

• Medium Bases

• Weak Bases • Neutrals

• All Acids

• Column: 50x2.1mm or 50x4.6mm, 5um

– Acidic Sample: Primesep B2

– Basic Sample: Primesep 200 or Primesep C • If not enough retention on Primesep C or very weak base, then use Primesep 100 or A

• Mobile Phase:

– A=H2O

– B=ACN

– C=100mM (TFA, Formic Acid, Ammonium Formate, H2SO4, etc.) • Control Buffer Composition Accuracy

• Start with 50/50/0.3% or 30mM

– This achieved by

• 20%A

• 50%B

• 30%C • Continue decreasing acid/buffer until desired retention is achieved for ionic species

– Try for instance 50/50/0.1% if ionic compound is not retained.

• 40% A

• 50% B

• 10% C

– If using Formic acid, up to 3.0% maybe necessary, or weaker column • Adjust organic contration until desired retention/selectivity for neutral and non-polar compounds is achieved

• If too much ionic strength is required for elution, try lowering mobile phase pH to “dim down” the ion properties of the

column or try a weaker column.

• Remember that retention for polar compounds will increase at very high organic.

• If compounds vary significantly hydrophobically, it maybe necessary to employ an organic gradient. • Once desirable conditions are found, a simply binary mobile phase system maybe employed for routine use.

• Please see the Troubleshooting section below for more method development ideas.

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Column

Primesep A

Our strongest CationExchange Phase. Only select for very weak basic analytes; pyridines, anilines and some heterocyclyes.

Column

Primesep 100

For basic analytes too well retained on Primesep™ A or not retained enough on Primesep™200.

Column

Primesep 200

For samples containing a wide variety of bases including strong bases weak bases, zwitterions, and amino acids.

Column

Primesep C

Our most universal phase for LC-MS. pH can be used to control retention. Select for strong to moderately basic analytes and compounds containing more than one basic functionality. Note: Benzylamine is not retained under these conditions because the ionic interactions are swtiched off at this pH.

Column Introduction

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23


Column

Primesep D

For samples containing weak acids such as carboxylic acids with pKa>4.

Column

Primesep B2

For medium to strong acids with pKa 2-4.

Column

Common C18

Offers no retention for polar ionizablecompounds, maleic acid and benzylamine under these conditions.

Columns: 150 x 4.6mmMobile Phase: H2O/ACN/TFA 60/40/0.1%Flow: 1.0 mL/min

1. Maleic Acid2. Benzonitrile3. Benzylamine

Tech Tip:Due to the mixed mode nature of Primesep, most samples can be analyxed on a 50mm column. To minimize method development time, always start with a 50 mm column.

Note: Neutral compound benzonitrile has the same retention time on C18 and all Primesep phases.


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Problem: Poor Retention for Polar Compounds.To achieve good chromatographic retention and selectivity an analyte must by attracted to and partition into the bonded phase. Very polar compounds are not attracted to greasy, hydrophobic phases like C18 and therefore do not partition into the bonded phase. This is similar to trying to mix oil and water; the two prefer to remain in two separate layers. Without interaction, the compound is not retained. However if that same compound is ionized, and the phase bares an opposite charge, the compound will be strongly attracted to the phase and will be retained.

Problem Solved

Primesep for Retention of Polar Compounds

• Polar organic molecules retain poorly on RP columns

• The ionizable amine groups on the polar compound act as an extreme case of polarity making the compounds even more polar

• The amine functionalities of the polar compounds interact with the acidic functionality of the Primesep 200 phase.

Primesep technology easily retains creatine and creatinine which are unretained on C18 & polar embedded phase.

Solution

Column: Primesep 200Column size:50 x 3.0 mm Mobile Phase: HCOOH0.1% with MeCN-20%Flow: 0.5 ml/minDetection: UV 270 nm

1. Creatine2. Creatinine3. Histidine

Column: Primesep 200Column size: 250 x 4.6 mm x 5umDetection: UV 270 nmMobile Phase: Water/MeCN/TFA-85/15/0.075%

Column: Common C18Column size: 250 x 4.6 mm x 5umDetection: UV 270 nmMobile Phase: Water/MeCN/TFA- 90/10/0.1%

1. Deoxyuridine2. Deoxyguanosine3.Deoxycytidine4.Deosyadenoxine

• Retention

• Selectivity • Simple, LC-MS

mobile phases

1

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C18 Primesep 200Primesep 200


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Problem: Easily replace existing ion-pairing methods for use with LC-MS. Ion-pairing has been the most effective tool for retaining polar compounds in reversed-phase HPLC. This technique has not been without problems, such as requiring dedicated columns and complex moble phases. Unfortunately with the rising popularity of LC-MS, this is no longer a viable tool as ion-pairing regents are not volatile for use with mass spec. Although compatible with mass spec, other phases such as water wetable and polar embedded phases do not offer the same retention and selectivity benefits as ion-pairing. What if the ion-pairing reagent is permanently bonded to the phase and never comes into contact with the mass spec?

Problem Solved

• Embedded ion-exchange functionality acts as an ion-pair

• Mobile phases are LC-MS compatible

• Retention is a comparible to Ion-paring

• Does not require dedicated columns.

Solution Primesep for Existing Ion-Paring Methods

Column: C18, 300 x 3.9 mmMobile Phase: ACN/H2O with 1%AcOH, 0.1%; Octanesulfonic Acid-13/87Flow rate: 1.5 mL/minDctector: UV 280

Column: Primesep 100, 150 x 4.6 mmMobile Phase: H2O/MeCN/TFA-80/20/0.1Flow rate: 1.0 mL/minDctector: UV 280

C18 Primesep

1. Dopamine

Similar Retention

11

Primesep for Retention of Polar Compounds


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Problem: Separate methods required for inorganic and organic components in drug formulations. In many instances, ionizable compounds exist as salts of organic molecules with inorganic counter ions; both of which must be quantitated. Typically two independent methods are created for each part: reversed-phase HPLC for the organic part, ion chromatography or titration of the inorganic part. Two methods require two validations,two systems, and possibly two analysts costing time and money.

Problem Solved

• Single analysis for inorganic and organic compounds

• Primesep columns offer a unique ability to analyze both parts of such salts simultaneously

- The inorganic ion is retained by ion-exchange

- The organic ion is retained by ion exchange

• Ultimately decreases cost of analyses while increasing throughput

Solution Primesep for simultaneousanalysis of Organic and Inorganic Compounds

1. Procainamide2. CI

Primesep B 150 x 4.6 mm x 5 umFlow rate: 1.0 mL/min.Mobile phase: Water/MeCN/AmAc–40/60/0.2Detector: ELSD , UV 210 nm

Procainamide is retained by hydrophobic interactions.

Chloride is retained by ionic interactions.

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Problem: Insufficient selectivity for closely related compounds. Reversed-phase columns have been engineered to minimize secondary interactions, resulting in a homogeneous surface and purely hydrophobic retention mechanism. Unfortunately, positional isomers tend to have very minuscule differences in hydrophobic character resulting in co-eluting or closely eluting peaks. However, as the position of the ionizable groups change in the molecule, their pKa values tend to change significantly. So when the miniscule differences in hydrophobicity are coupled with the noticeable changes in pKa value,the dual retention mechanism of Primesep allows these compounds to be easily resolved.

Problem Solved

Primesep for Closely Eluted or Co-eluted CompoundsSolution

These isomers have such similar hydrophobocity that they co-elute on reversed-phase, Primesep’s bi-modal technology easily separates them

• Two dimensional control of selectivity• Ionizable compounds interact with the stationary phase by : • Reversed-phase

• Ion-exchange or ion-exclusion• Amount of acid/buffer in the mobile phase influences ion-exchange• Amount of organic modifier influences hydrophobic interactions

Primesep B150x4.6 mmFlow rate: 1.0mL/min.Mobile phas: H2O/MeCN/TFA-100-50/0-50/0.03-0.1 in 15 minSample: 0.2 mg/ml of each inMeCN/waterInjection: 5 µlTemperature: 40° CDetector: UV-270 nm

1. 3,4-Dihydroxybenzoic Acid2. 3,5-Dihydroxybenzoic Acid3. 2,3-Dihydroxybenzoic Acid4. 2,5 Dihydroxybenzoic Acid5. 2,4 Dihydroxybenzoic Acid

1 α- aminobuyric acid pka=2.552 β- aminobutyric acid pKa= 4.233 γ- aminobutyric acid

Primesep’s bi-modaltechnology easily separates these isomers, which co-elute on reverse phase

Primesep C150x3.2 mmFlow rate: 0.5mL/min.Detection: ELSDMobile Phase: Water/MeCN-90/10 withNH, Acetate 10mMol pH 4.1

Column C18

Primesep for simultaneousanalysis of Organic and Inorganic Compounds


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Problem: Long analysis time for samples containing vastly different compounds.The separation of vastly different compounds by HPLC, although often necessary, is challenging and complex, requiring either gradient elution or multiple assays for adequate analysis. When reversed-phase techniques are employed for such samples the very polar compounds are often difficult to retain even in highly aqueous mobile phases while the very non-polar compounds are retained indefinitely under these conditions, requiring a gradient to higher percent organic to elute. It is often difficult to quantitate the early eluting compounds due to lack of selectivity while much time is lost waiting for the late eluting compounds.

Problem Solved

• Retain and “park polar compounds by ionic interactions • Control with pH and ionic strength

•Retain non-polar compounds by hydrophobic interactions • Control with organic modifier

Solution Primesep for Vastly Different Comounds

Polar compound, histimine is not affected by organic con-centration and is “parked”.

Column: C18 150x4.6 mmFlow rate: 1.0 mL/minUV: 205 nmInjection: 5 ulMobile phase: TEA 25mM Phosphate pH 3.0/MeCN-80/20

Column: Primesep C150x4.6 mmFlow rate: 1.0 mL/minUV: 205 nmInjection: 5 ulMobile phase: TEA 50mM Phosphate pH 3.0/MeCN-40/60

1. Succinate2. Bromate3. Acetaminophen4.Pseudoephendrine5. Doxylamine6. Dextromethorphan

1. 2.

3.

4.

5.

6.

�15 minutes wasted waiting forDextromethorphan to elute.

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Primesep retains and resolves polar compounds. Cuts

analysis time in half!

Column: Primesep C 50 x3.6 mmMobile phase: AmFormate25 mM pH 3.5Flow rate: 0.5 mL/minDetector:UV-210nm

Hyrdophobic compound, phenethylamine is affected by organic concentration, allowing for short analysis time.

1. Histamine2. Phenethylamine


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Primesep for Vastly Different Comounds

Problem: Poor peak shape for basic analytes. Since the beginning of reversed-phase HPLC, chromatographers have strugged with poor peak shape for basic analytes. Chromatographers have added strong buffers like phosphate to our mobile phases, column manufacturers have eliminated most of the metal impurities from their silica surface, and added secondary end-capping; all attempts to minimize interactions of basic compounds with residual silanol groups on the silica surface. With the growing popularity of mass-spec detection, strong buffers cannot be used and no matter how well manufacturers bond and end-cap their phases, somehow the bases still find the free silanols to interact with. What if the basic analyte is never able to come close to the silanols?

Problem Solved

• Near perfect peak shape for quaternary amines

• Basic analyte is repelled from the silica surface: unable to come in contact with the silanol groups

• Retention is due to hydrophobic interactions

Solution Primesep for Strong Basic Analytes

8 Fold increase in efficiency.

PrimesepC18

C18 Column150x4.6mm x 3.5umMobile Phase: H20/MeCN/TFA-70/30/0.1Flow rate: 1.0 mL/minDetector: ELSDPeak plate count 640Peak symmetry 0.37

Primesep B150x4.6mmMobile Phase: H20/MeCN/TFA-70/30/0.1Flow rate: 1.0 mL/minDetector: ELSDPeak plate count 5200Peak symmetry .70

TetrabutylammoniumHydroxide

Hydrophobic interactions for retention

Repel!

The positive charge on the phase repels the positively charged analyte, effectively shielding the analyte from the silanol group.

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Primesep’s patented bi-modal retention mechanism offers almost limitless control of retention and selectivity. In-order take complete advantage of this superior control the following conditions must be met.

• Very polar compounds must be ionized • Primesep column must be ionized • Buffer System must be Compatible with your detection method

General Assay Considerations

Ask yourself the following questions to streamline your method development process.

Type of Method Development • Protocol #1: Validated long term assay • Protocol #2: Generic screening method many different types of compounds

Type of Mobile Phase • LC-MS or ELSD • UV

Type of Column and Mobile Phase • Analyte Properties

- pKa values if available - acidic, basic, zwitterionic, neutral - Hydrophobic or Hydrophilic

Method DevelopmentGuide

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Analyte ConsiderationsUnlike conventional reversed-phase chromatography, mobile phase pH has dramatic effects on analyte retention. Almost all ionizable organic compounds are weak acids or bases which have at least one pKa.

• When mobile phase pH is equal to the analyte’s pKa the analyte is 50% ionized.

• For bases as the mobile phase pH drops below the analyte’s pKa, the compound becomes more ionized, approaching 100% ionized at mobile phase pH 1.5 units below the analyte’s pKa. ( See figure on right)

• For acids , as the mobile phase pH increases above the an analyte’s pKa, the compound becomes more ionized, approaching 100% ionized at mobile phase pH 1.5 units above the analyte’s pKa .

• In an ideal world the analyte’s should have at least one ionizable group 100% ionized; significantly improved retention can be achieved if the analyte is at least 30% ionized.

Simplified pH Effect on Analyte Ionization



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100

0 1 2 3 4 5 6 7 8Mobile Phase pH

CreatineCreatinineHistidine

Analyte Percent Ioniztion vs. Mobile PhaseMaximum retention

Minimum ionization required for appreciable increase in retention

% Ionization = 100

1+10 (charge) (pH-pKa)

Charge= (+1) for BasesCharge+ (-1) for Acids

Creatine, pKa=2.63Creatinine, pKa=4.83Histidine, pKa=6.04

% Io

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Considerations for Column Selection • Each Primesep phase differs by the ionic moiety

• Each ionic moiety has a distinct pKa

• For ion exchange interactions to occur, column and analyte must be charged

• Select the weakest column which will be ionized for your application

• 50x2.1mm for method development. Retention and selectivity will be much greater than conventional Reversed Phase.


Column Type Transition Point

Cation Exchange-Reversed Phase

Weak

Strong

Primesep C 3.5

Primesep 200 2

Primesep 1001

Primesep A Ionized at all working pH

Anion Exchange-Reversed Phase

Weak

Strong

Primesep B2 Ionized at all working pH

Primesep D Ionized at all working pH

_

H

COO

COO

COO

_

_

COOH

COOH

COOH

+

pH above transition pH below transition


Analyte Type Column Comments

Strong Bases: pKa>9 Quaternary Amines, more than one basic group, inorganic cations

Primesep C Select for LC-MS. pH Can be used to control retention

Primesep 200 Select if more retention is required than on Primesep C

Primesep B Use in ion-exclusion mode if compound is hydrophobic and shows poor peak shape on RP columns

Medium Bases: pKa=7-9 Amines, amino sugars, phosphines

Primesep C Primesep 200

Weak Bases: pKa<7 Pyridines, anilines, some heterocycles

Primesep 100 Primesep A

Strong Acids: pKa<3 Phosphates, Sulfates, polyacids, inorganic anions

Primesep B2

Medium Acids: pKa= 2-4 Diacids, polyacids

Primesep B2

Weak Acids: pKa>4 Carboxcylic acids, aromatic acids, phenols

Primesep B2 Primesep D

Zwitterions: pKa4 Amino acids, peptides

Primesep 200 Use Mobile phase pH ≈ 2, 0.1% Formic acid

Zwitterions with one basic and 2 acidic groups Aspartic acid, glutamic acid

Primesep 200 Use Mobile phase pH≈ 2, 0.1% Formic acid

Primesep D Use mobile phase pH >5

Neutral: All Similar behavior on all columns

Select Column Based on Analyte Type

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Considerations forMobile Phase Buffer Selection • Mobile phase pH must be selected so that analytes are ionized.

– For Basic analytes, pH must be < than analyte’s pKa

– For Acidic analytes, pH must be > than analytes pKa

•Mobile phase pH must be selected so that column is ionized

•Mobile phase must be compatible with detection technique

•Acetonitrile is always preferred organic modifier

• Buffer must have buffering capacity at the desired pH.

– This is designated by the white regions in the chart below

– Buffer has no buffering capacity in the colored regions

Acid Approximate pH

0.1% TFA 2

0.1% Formic Acid

3

0.1% Acetic Acid 4

Method Development


Now you are ready to take total controlof your separation.


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Organic Modifier • Always use Acetonitrile

•Polar ionizable and non-ionizable compounds have maximum retention at high and low organic concentrations

•Very polar ionizable compounds will not be appreciably affected by organic concentration…The ionic interactions will dominate their retention

•Retention for non-polar compounds will decrease will increasing organic strength

Method Development

0

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10 20 30 40 50 60 70

% Water

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0.0 0.1 0.1 0.2% Acid

Re

ten

tio

n t

ime

, m

inEffect of Acid Concentration on Retention: Primesep

Your Control Panel

Step 4: Mobile Phase Ionic Strength: � Retention for ionizable compounds and non-

ionizable polar compounds will decrease with

increasing ionic strength

� Retention for non-ionic, non-polar compounds will be

unaffected by ionic strength � “Park” Retention for ion species with ionic

strength

� In ion-exclusion mode, retention for ionizable

compounds will increase with increasing ionic

strength

Step 5: Organic Modifier: � Always use Acetonitrile

� Polar ionizable and non-ionizable compounds have maximum retention at high and low organic

concentrations

o Very polar ionizable compounds will not be

apprecialby affected by organic

concentration…The ionic interactions will dominate their retention

� Retention for non-polar compounds will decrease

will increasing organic strength

Basic Compounds Polar Neutral Compounds Non-Polar Neutral Compounds Acidic Compounds


Effect of Water Concentration on Hydrophobic Retention on Primesep 100

Mobile Phase Ionic Strength: • Retention for ionizable compounds and non-



• Retention for non-ionic, non-polar compounds will be unaffected by ionic strength

•“Park” Retention for ion species with ionic strength

• In ion-exclusion mode, retention for ionizable compounds will increase with increasing ionic strength

Your Control Panel

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% Water

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0.0 0.1 0.1 0.2% Acid

Re

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Effect of Acid Concentration on Retention: Primesep

Your Control Panel

Step 4: Mobile Phase Ionic Strength: � Retention for ionizable compounds and non-



� Retention for non-ionic, non-polar compounds will be

unaffected by ionic strength � “Park” Retention for ion species with ionic

strength

� In ion-exclusion mode, retention for ionizable

compounds will increase with increasing ionic

strength

Step 5: Organic Modifier: � Always use Acetonitrile

� Polar ionizable and non-ionizable compounds have maximum retention at high and low organic

concentrations

o Very polar ionizable compounds will not be

apprecialby affected by organic

concentration…The ionic interactions will dominate their retention

� Retention for non-polar compounds will decrease

will increasing organic strength



Effect of Water Concentration on Hydrophobic Retention on Primesep 100

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Mobile PhaseIonic Strength

Organic Modifier

pH Control

Effect of Acid Concentration on Retention:

Primesep 100

Effect of Water Concentration on Hydrophobic Retention:

Primesep 100


pH Control, your “Dimmer Switch” on Primesep C • At pH approaching 5, 100% of potential ionizable sites on column are charged (i.e. turned on).

Therefore column has maximum amount of ion-exchange retention. (See “On” this page)

• At pH<3.5 less than 50% of potential ionizable sites on column are charged (i.e. Dimmed). Therefore column has minimum amount of ion-exchange retention. (See “Dimmed” this page)

• This is similar to a light on a dimmer switch, at pH 5 the light is completely turned on, at pH 3.5 we have very dim, mood lighting.

• At higher pH the weaker bases will elute and at lower pH the stronger bases will elute.

• This allows control of ion-exchange properties without increasing ionic strength.

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Primesep 100

Primesep 200

Primesep C

Maximum IonExchange

“Dimmer Switch” Region

Polar Embedded/HyrodphobicInteraction

1.Caffeine2.Benzonitrile3.Dopamine4.Procainamide5.Benzylamine6.Aminopyridine7.Clorfeniramine

On Dimmed

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Method Development

Fine Tune Selectivity WithMobile Phase Counter Ion Effect•Cation Exchange columns have (-) charge•Mobile phase contains H(+) from dissociated acid or another (+) ion such as NH4

(+) from a dissociated salt

–These ions will form ion-pair with cation exchange group•Each ion-pair will offer slightly different selectivity due to differing electronic environment

interacting with analyte •Different selectivity can be obtained by changing from: -Formic acid to ammonium formate -Acetic acid to ammonium acetate

One Final Tweak: Mobile Phase Counter Ion Effect • Cation Exchange columns have (-) charge • Mobile phase contains H(+) from dissociated acid or another (+) ion such as NH4(+) from a

dissociated salt – These ions will form ion-pair with cation exchange group

• Each ion-pair will offer slightly different selectivity due to differing electronic environment interacting with analyte

– Different selectivity can be obtained by changing from: • Formic acid to ammonium formate • Acetic acid to ammonium acetate

If counter-ion remains the same column • Electronic environment remains similar • requires just 2-3 column volumes to equilibrate

Column inner diameter

Column length

Amount of silica

Column ion-exchange capacity

Amount of buffer in mL withconcentration mMol requires to replace column contra-ions

mm mm g mMol10mMol

20mMol

50 mMol

4.6 250 3.7 1.9 225 115 45

4.6 150 2.2 1.1 135 70 30

4.6 50 0.7 0.4 45 25 10

3.0 250 1.6 0.8 100 50 20

3.0 150 1.0 0.5 60 30 15

3.0 50 0.3 0.2 20 10 5

2.0 250 0.7 0.4 45 25 10

2.0 150 0.4 0.2 30 15 10

2.0 50 0.1 0.1 10 5 5

If counter-ion changes � Electronic environment changes � Selectivity changes � Column Equilibration can be lengthy and the table

below should be followed.

One Final Tweak: Mobile Phase Counter Ion Effect • Cation Exchange columns have (-) charge • Mobile phase contains H(+) from dissociated acid or another (+) ion such as NH4(+) from a

dissociated salt – These ions will form ion-pair with cation exchange group

• Each ion-pair will offer slightly different selectivity due to differing electronic environment interacting with analyte

– Different selectivity can be obtained by changing from: • Formic acid to ammonium formate • Acetic acid to ammonium acetate

If counter-ion remains the same column • Electronic environment remains similar • requires just 2-3 column volumes to equilibrate

Column inner diameter

Column length

Amount of silica

Column ion-exchange capacity

Amount of buffer in mL withconcentration mMol requires to replace column contra-ions

mm mm g mMol10mMol

20mMol

50 mMol

4.6 250 3.7 1.9 225 115 45

4.6 150 2.2 1.1 135 70 30

4.6 50 0.7 0.4 45 25 10

3.0 250 1.6 0.8 100 50 20

3.0 150 1.0 0.5 60 30 15

3.0 50 0.3 0.2 20 10 5

2.0 250 0.7 0.4 45 25 10

2.0 150 0.4 0.2 30 15 10

2.0 50 0.1 0.1 10 5 5

If counter-ion changes � Electronic environment changes � Selectivity changes � Column Equilibration can be lengthy and the table

below should be followed.

If counter-ion changes -Electronic environment change -Selectivity changes -Column Equilibration can be lengthy and

the table (on right) followed.

If counter-ion remains the same column •Electronic environment remains similar•requires just 2-3 column volumes to equilibrate


Column Inner Diameter

mm

ColumnLength

mmAmount of Silica

Column ion-exchange

capacitymMol

Amount of buffer in mL with concentration mMol required to replace column counter-ions

10 mMol 20 mMol 50mMol

4.6 250 3.7 1.9 225 115 454.6 150 2.2 1.1 135 70 304.6 50 0.7 0.4 45 25 103.0 250 1.6 0.8 100 50 203.0 150 1.0 0.5 60 30 153.0 50 0.3 0.2 20 10 52.0 250 0.7 0.4 45 25 102.0 150 0.4 0.2 30 15 102.0 50 0.1 0.1 10 5 5

Selectivity Can Be Changed by Changing Acid

1.Theobromine2. 1, 7 Dimethylxanthine3. Theophylline

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Protocol 1: This method is designed to offer the most optimized method.

• Ion exchange retention is first set by adjusting ionic strength• Hydrophobic retention is then adjusted by organic concentration

Applications: • Analytical R&D •Quality control •Stability

• Column: 50x2.1mm or 50x4.6mm, 5um – Acidic Sample: Primesep B2 – Basic Sample: Primesep 200 or Primesep C • If not enough retention on Primesep C or very weak

base, then use Primesep 100 or A • Mobile Phase: – A=H2O – B=ACN – C=100mM (TFA, Formic Acid,

Ammonium Formate, H2SO4, etc.) • Control buffer composition accurately • Start with 50/50/0.3% or 30mM – This achieved by 20% A 50% B 30% C • Continue decreasing acid/buffer until desired

retention is achieved for ionic species – Try for instance 50/50/0.1% if ionic compound is not retained. 40% A 50% B 10% C – If using Formic acid, up to 3.0% may be necessary, or weaker column • Adjust organic concentration until desired retention/selectivity for neutral and non-polar

compounds is achieved •If too much ionic strength is required for elution, try lowering mobile phase pH to “dim

down” the ionic properties of the column or try a weaker column. • Remember that retention for polar compounds will increase at very high organic. • If compounds vary significantly hydrophobically, it maybe necessary to employ an organic

gradient. • Once desirable conditions are found, a simply binary mobile phase system maybe

employed for routine use. • Please see the Troubleshooting section below for more method development ideas.

Analyte Types: • Strong Bases • Medium Bases • Weak Bases • Neutrals • All Acids

Putting it All Togeather


Protocol 2: This method is designed to offer the most selectivity, retention and best peak shape with the minimum amount of method development.

• Here we capatalize on the unique cationexchange properties of Primesep C. • Ion-Exchange is controlled pH gradient.• Hydrophobic retention is affected by the organic gradient

Applications• Generic Screening• High-Throughput Bio-analytical• High-Throughput Cominatorial• LC-MS• Complex Samples• Vastly different compounds

Column: Primesep C, 5 um, 50x2.1 or 50x4.6

Mobile Phase: (Adjust pH of aqueous portion before adding organic)A= 90% 10-25mM NH4 Acetate, pH 5 10% ACNB= 30% 10-25mM NH4 Acetate, pH 3.5 70% ACN

Gradient:

Flow: 2mL/min

Time (Min.) %A

%B

0 100 0 4 0 100

4.1 100 0 5.0 100 0

Analyte Types• Strong bases• Medium Bases• Weak Hydrophobic Bases• Neutrals• Hydrophobic Acids

Peak shape improves with faster gradient

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Too Much Retention •Increase mobile phase ionic strength •Use stronger buffer ( See Figure A)• Increase % organic

- Above 70% organic, retention time will increase for polar ionic and neutral compounds due to polar organic mode interactions

•Try weaker column (See Figure B)

Not Enough Retention •Decrease mobile phase ionic strength •Use weaker buffer (See Figure A)•Decrease % organic

•Above 70% organic, retention time will increase for polar ionic and neutral compounds due to polar organic mode interactions

•Try stronger column (See Figure B)• Increase column length • Columns are available as long as 250mm • Primesep C, increase mobile phase pH

Cation Exchange Anion

Exchange

Primesep A Primesep D Primesep 100

Primesep 200

Primesep C Primesep B2

Buffer

H2SO4, H3PO4, phosphate Strong

Weak

TFA

Ammonium Formate

Formic Acid

Ammonium Acetate

Acetic Acid

Troubleshooting

• Decrease column length - Columns are available as short as 10mm

•Primesep C, decrease mobile phase pH

Figure A Figure B

Strong

Weak


Not Enough Selectivity Independantly adjust organic and ionic strength • Adjust mobile phase pH • Try different buffer

Poor Peak Shape • For di- and poly-basic analytes try Primesep C with pH Gradient as described in

strategy 1 -Increase flow rate -Increase speed of pH gradient -Start with lower pH, (i.e. 4.5 or 4 instead of 5) -Try shorter column •For hydrophobic, strong bases use ion-exclusion mode on Primesep B or B2 column where surface has (+) charge and analyte has (+) charge

Need MS Conditions • Try Primesep C with pH gradient as described in protocol 1 • Try Primesep 200 with formic acid or ammonium formate • Try Primesep C with high organic, >70% with 10mM ammonium acetate, pH 4

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Bonded Phase

Part Number Description Price

Primesep A

A-21.050.0510 5um, 50x2.1mm Primesep A HPLC Column $495.00


A-46.050.0510 5um, 50x4.6mm Primesep A HPLC Column $495.00A-220.050-0510 5um, 50x22mm Primesep A HPLC Column $1750.00

A-21.100.0510 5um, 100x2.1mm Primesep A HPLC Column $520.00A-32.100.0510 5um, 100x3.2mm Primesep A HPLC Column $525.00

A-46.100.0510 5um, 100x4.6mm Primesep A HPLC Column $555.00A-220.100-0510 5um, 100x22mm Primesep A HPLC Column $2850.00


A-46.150.0510 5um, 150x4.6mm Primesep A HPLC Column $595.00A-220.150.0510 5um, 150x22mm Primesep A HPLC Column $3180.00



A-220.250-0510 5um, 250x22mm Primesep A HPLC Column $4850.00

Ordering Information

Phone: (616) 392-8001Toll Free: (800) 572-6653Fax: (616) 396-7520

email: [email protected] ordering: www.resolutionsys.com


Primesep 100

100-21.050.0510 5um, 50x2.1mm Primesep 100 HPLC Column $495.00


100-46.050.0510 5um, 50x4.6mm Primesep 100 HPLC Column $495.00100-220.050-0510 5um, 50x22mm Primesep 100 HPLC Column $1750.00

100-21.100.0510 5um, 100x2.1mm Primesep 100 HPLC Column $520.00100-32.100.0510 5um, 100x3.2mm Primesep 100 HPLC Column $525.00



100-46.150.0510 5um, 150x4.6mm Primesep 100 HPLC Column $595.00100-220.150.0510 5um, 150x22mm Primesep 100 HPLC Column $3180.00



100-220.250-0510 5um, 250x22mm Primesep 100 HPLC Column $4850.00

Primesep 200





200-46.100.0510 5um, 100x4.6mm Primesep 200 HPLC Column $555.00200-220.100.0510 5um, 100x22mm Primesep 200 HPLC Column $2850.00





200-220.250-0510 5um, 250x22mm Primesep 200 HPLC Column $4850.00

Bonded Phase


Phone: (616) 392-8001 Toll Free: (800) 572-6653 Fax: (616) 396-7520 email: [email protected] web ordering: www.resolutionsys.com

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Primesep B2

B2-21.050.0510 5um, 50x2.1mm Primesep B2 HPLC Column $495.00


B2-46.050.0510 5um, 50x4.6mm Primesep B2 HPLC Column $495.00B2-220.050-0510 5um, 50x22mm Primesep B2 HPLC Column $1750.00

B2-21.100.0510 5um, 100x2.1mm Primesep B2 HPLC Column $520.00B2-32.100.0510 5um, 100x3.2mm Primesep B2 HPLC Column $525.00

B2-46.100.0510 5um, 100x4.6mm Primesep B2 HPLC Column $555.00B2-220.100.0510 5um, 100x22mm Primesep B2 HPLC Column $2850.00





B2-220.250-0510 5um, 250x22mm Primesep B2 HPLC Column $4850.00

Primesep D

D-21.050.0510 5um, 50x2.1mm Primesep D HPLC Column $495.00


D-46.050.0510 5um, 50x4.6mm Primesep D HPLC Column $495.00D-220.050-0510 5um, 50x22mm Primesep D HPLC Column $1750.00

D-21.100.0510 5um, 100x2.1mm Primesep D HPLC Column $520.00D-32.100.0510 5um, 100x3.2mm Primesep D HPLC Column $525.00

D-46.100.0510 5um, 100x4.6mm Primesep D HPLC Column $555.00D-220.100.0510 5um, 100x22mm Primesep D HPLC Column $2850.00





D-220.250-0510 5um, 250x22mm Primesep D HPLC Column $4850.00

Bonded Phase



Ordering Information


Primesep B2





B2-46.100.0510 5um, 100x4.6mm Primesep B2 HPLC Column $555.00B2-220.100.0510 5um, 100x22mm Primesep B2 HPLC Column $2850.00





B2-220.250-0510 5um, 250x22mm Primesep B2 HPLC Column $4850.00

Primesep D





D-46.100.0510 5um, 100x4.6mm Primesep D HPLC Column $555.00D-220.100.0510 5um, 100x22mm Primesep D HPLC Column $2850.00





D-220.250-0510 5um, 250x22mm Primesep D HPLC Column $4850.00

Primesep C

C-21.050.0510 5um, 50x2.1mm Primesep C HPLC Column $495.00


C-46.050.0510 5um, 50x4.6mm Primesep C HPLC Column $495.00C-220.050-0510 5um, 50x22mm Primesep C HPLC Column $1750.00

C-21.100.0510 5um, 100x2.1mm Primesep C HPLC Column $520.00C-32.100.0510 5um, 100x3.2mm Primesep C HPLC Column $525.00

C-46.100.0510 5um, 100x4.6mm Primesep C HPLC Column $555.00C-220.100.0510 5um, 100x22mm Primesep C HPLC Column $2850.00


C-46.150.0510 5um, 150x4.6mm Primesep C HPLC Column $595.00C-220.150-0510 5um, 150x22mm Primesep C HPLC Column $3180.00



C-220.250-0510 5um, 250x22mm Primesep C HPLC Column $4850.00

Bonded Phase



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Notes


Primesep and all the other trademark of SIELC. Comparisons shown may not be representative of every application. Copyright 2005 Resolution Systems, Inc. Specifications, terms,

and pricing are subject to change.

Primesepmixed-modecolumnswereengineeredbychemistswhoneededtosolvethedifficultsep-arations theyencounteredwhileworking in thepharmaceutical industry. The two founders,whoworkedindifferentcapacitiesatapharmaceuticalcompany,alsohappenedtobegoodfriends:Oneisasyntheticchemistwhomadethecompounds,andtheotherisananalyticalchemistwhodevelopedtheLC/MSmethodstoanalyzethecompounds.

Theanalyticalchemiststruggledwithhisfriend’scompoundsbecausetheywerejusttoopolartoberetainedonC1�,AQphasesandpolarembeddedphases,withoutusingionpairing.Unfortunately,thiswasn’tanoptionbecauseionpairingisnotcompatiblewithMassSpectrometry.Doesthisstorysoundfamiliar?Ifso,readon...

Fortunately,theanalyticalchemistsharedhisstrugglewithhisfriend,thesyntheticchemist.Realizingthatmostpolarcompoundsareeasilyionized,thetwobegandevelopingwaysinwhichtocapital-izeonthisinteraction,whilemaintainingcompatibilitywithLC-MS.Theresultwasaseriesofcontrolledmixed-modeionexchange/reversedphasecolumnstheycalledPrimesep.Thesecolumnsallowin-dependentcontrolofpolarionicandhydrophobicinteractions,resultinginanalmostinfinitecontrolofretentionandselectivity.Thus,SIELCTechnologieswasstarted-acompanygearedtowardsolvingchromatography’smostdifficultproblems.

SIELCTechnologies isaprivatelyheldcompany thatunderstands theneed foracustomer-drivenapproachtochromatography.Wedevelopcolumnstosolveyourproblems.Weprovidedirectsup-portandsuggestions–fromonechemisttoanotherchemst.We’veputthe“friendly”backinLC-MSfriendlyconditions.

The Primesep Story:

1-616-396-7520

seilc primesep method development

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