Transform your Chromatography

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 instrumentation 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.

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If you are struggling with any of the following

Insufcient 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.... ...to this.

C18

Resolution Systems

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 Phase

Strengths
Predictable retention mechanism High efcientcy Many choices

Weaknesses
Limited retention for polar compounds Limited selectivity for polar compounds Single mode of interaction Unstable in highly aqueous environments

Water Wettable Phases (AQ Phases)

Strengths
Stable in 100% aqueous mobile phase Increased retention for polar compounds

Weaknesses
Small retention improvements Not ideal for LC-MS Highly aqueous mobile phase

Polar Embedded Phases

Strengths
Improved selectivity Stable in 100% aqueous mobile phase

Weaknesses
Small retention improvments Signicant phase bleed with LC-MS

Ion Pair
Strengths
Signicant retention improvements Retention for non-ionic species

Weaknesses
Not compatible with LC-MS Requires dedicated columns Complex mobile phases

Ion Exchange
Strengths
Retention for ionic compounds

Weaknesses
No retention for non-ionic species Low efciency

Resolution Systems

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 modier strength, allowing near innite control of retention and selectivity. Unlike conventional ion exchange phases, the patented mixed-mode ligand allows the use of LC-MS friendly mobile phases.

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

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.

Hydrophobic Control
Organic Modier controls hydropobic retention

Percent Ionization Control

Ionic Control
Mobile phase ionic

Mobile phase pH effects % ionization of column and analyte

strength controls ion exchange.

Resolution Systems

Switch Phase Technology

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 columns 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

Column Type
Primesep C Primesep 200 Primesep 100 Primesep A

Transition Point = pKa

pH= 3.5 pH=2 pH=1 Ionized at all working pH

pH above transition
COO _

pH below transition
COOH

H
COO

+
COOH

COO

COOH

On
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Off

Strong Bases Medium Bases Weak Bases Neutrals All Acids

Tech Tip

Retention is Proportional to % Ionization

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, H2 SO4, 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 Changing pH from 4-3.5 Try for instance 50/50/0.1% if ionic compound is not retained. 40% A changes ionization of column 50% B from 100% to 50% for LC-MS 10% C friendly analysis 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 Neutral Guefenesin is not column or try a weaker column. Remember that retention for polar compounds will increase orvery high organic. affected by pH at ionic If compounds vary significantly hydrophobically, it changes strength 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.

Effect of Mobile Phase pH on Column Ionization

Mobile phase pH can be used to control retention

Greatest retention for Ionizable compounds

Primesep 100 Primesep 200 Primesep C

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

Resolution Systems 7

Column Introduction
Column Our strongest Cation Exchange Phase. Only select for very weak basic analytes; pyridines, anilines and some heterocyclyes.

Primesep A

Column For basic analytes too well retained on Primesep A or not retained enough on Primesep200.

Primesep 100

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

Primesep 200

Column
1

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.

Resolution Systems

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

Primesep D

Column For medium to strong acids with pKa 2-4. Primesep B2

Tech Tip:

Note:

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.

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

Columns: 150 x 4.6mm Mobile Phase: H2O/ACN/ TFA 60/40/0.1% Flow: 1.0 mL/min 1. Maleic Acid 2. Benzonitrile 3. Benzylamine

Column Offers no retention for polar ionizable compounds, maleic acid and benzylamine under these conditions.

Common C18

Resolution Systems

Problem Solved

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.

Solution
Primesep 200

Primesep for Retention of Polar Compounds

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

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.

C18

Retention Selectivity Simple, LC-MS mobile phases

Primesep 200
1. Creatine 2. Creatinine 3. Histidine

2 3
1. Deoxyuridine 2. Deoxyguanosine 3.Deoxycytidine 4.Deosyadenoxine

Column: Common C18 Column size: 250 x 4.6 mm x 5um Detection: UV 270 nm Mobile Phase: Water/MeCN/TFA90/10/0.1%

Column: Primesep 200 Column size: 250 x 4.6 mm x 5um Detection: UV 270 nm Mobile Phase: Water/MeCN/TFA85/15/0.075%

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Column: Primesep 200 Column size:50 x 3.0 mm Mobile Phase: HCOOH 0.1% with MeCN-20% Flow: 0.5 ml/min Detection: UV 270 nm

Problem Solved

Problem: Easily replace existing ion-pairing methods for use with LC-MS.

the phase and never comes into contact with the mass spec?

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 ionpairing 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 benets as ion-pairing. What if the ion-pairing reagent is permanently bonded to

Solution

Primesep for Existing Ion-Paring Methods

Similar Retention

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.

C18

Primesep

1. Dopamine

Column: C18, 300 x 3.9 mm Mobile Phase: ACN/H O with 1% AcOH, 0.1%; Octanesulfonic Acid-13/87 Flow rate: 1.5 mL/min Dctector: UV 280
2

Column: Primesep 100, 150 x 4.6 mm Mobile Phase: H O/MeCN/TFA-80/20/0.1 Flow rate: 1.0 mL/min Dctector: UV 280
2

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11

Problem Solved

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

Problem: Separate methods required for inorganic and organic components in drug formulations. In many instances, ionizable compounds exist as salts
systems, and possibly two analysts costing time and money.

Solution

Primesep for simultaneous analysis of Organic and Inorganic Compounds

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

Procainamide is retained by hydrophobic interactions.

1. Procainamide 2. CI

Chloride is retained by ionic interactions.

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Resolution Systems

Problem Solved

Problem: Insufcient 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 signicantly. 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.

Solution

Primesep for Closely Eluted or Co-eluted Compounds

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 inuences ion-exchange Amount of organic modier inuences hydrophobic interactions Primesep C
Primeseps bi-modal technology easily separates these isomers, which co-elute on reverse phase

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

150x3.2 mm Flow rate: 0.5mL/min. Detection: ELSD Mobile Phase: Water/MeCN-90/10 with NH, Acetate 10mMol pH 4.1

Primesep B

Column C18

150x4.6 mm Flow rate: 1.0mL/min. Mobile phas: H2O/MeCN/TFA100-50/0-50/0.03-0.1 in 15 min Sample: 0.2 mg/ml of each in MeCN/water Injection: 5 l Temperature: 40 C Detector: UV-270 nm

1 - aminobuyric acid pka=2.55 2 - aminobutyric acid pKa= 4.23 3 - aminobutyric acid

1. 3,4-Dihydroxybenzoic Acid 2. 3,5-Dihydroxybenzoic Acid 3. 2,3-Dihydroxybenzoic Acid 4. 2,5 Dihydroxybenzoic Acid 5. 2,4 Dihydroxybenzoic Acid

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13

Problem Solved

Problem: Long analysis time for samples containing vastly different compounds.

early eluting compounds due to lack of selectivity while much time is lost waiting for the late eluting 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 difcult to retain even in highly aqueous mobile phases while the very non-polar compounds are retained indenitely under these conditions, requiring a gradient to higher percent organic to elute. It is often difcult to quantitate the

Solution
4 3

Primesep for Vastly Different Comounds

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

Retain and park polar compounds by ionic interactions Control with pH and ionic strength Retain non-polar compounds by hydrophobic interactions Control with organic modier

2 1 5

15 minutes wasted waiting for Dextromethorphan to elute.

6

Column: C18

150x4.6 mm Flow rate: 1.0 mL/min UV: 205 nm Injection: 5 ul Mobile phase: TEA 25mM Phosphate pH 3.0/MeCN-80/20

Primesep retains and resolves polar compounds. Cuts analysis time in half!
3. 4.

1. Succinate 2. Bromate 3. Acetaminophen 4.Pseudoephendrine 5. Doxylamine 6. Dextromethorphan

6. 1. 2.

5.

Column: Primesep C
150x4.6 mm Flow rate: 1.0 mL/min UV: 205 nm Injection: 5 ul Mobile phase: TEA 50mM Phosphate pH 3.0/MeCN-40/60

Polar compound, histimine is not affected by organic concentration and is parked.

1. Histamine 2. Phenethylamine
Column: Primesep C 50 x3.6 mm Mobile phase: AmFormate 25 mM pH 3.5 Flow rate: 0.5 mL/min Detector:UV-210nm

14

Resolution Systems

Problem Solved

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 nd the free silanols to interact with. What if the basic analyte is never able to come close to the silanols?

Solution
Near perfect peak shape for quaternary amines Retention is due to hydrophobic interactions

Primesep for Strong Basic Analytes

Hydrophobic interactions for retention

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

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

Repel!

8 Fold increase in efciency.

Tetrabutylammonium Hydroxide
C18 Column 150x4.6mm x 3.5um Mobile Phase: H20/MeCN/TFA -70/30/0.1 Flow rate: 1.0 mL/min Detector: ELSD Peak plate count 640 Peak symmetry 0.37

C18

Primesep

Primesep B 150x4.6mm Mobile Phase: H20/MeCN/TFA -70/30/0.1 Flow rate: 1.0 mL/min Detector: ELSD Peak plate count 5200 Peak symmetry .70

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15

Method Development Guide

Primeseps 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

16

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17

Method Development Guide

Analyte Considerations

Unlike 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 analytes pKa the analyte is 50% ionized. For bases as the mobile phase pH drops below the analytes pKa, the compound becomes more ionized, approaching 100% ionized at mobile phase pH 1.5 units below the analytes pKa. ( See gure on right) For acids , as the mobile phase pH increases above the an analytes pKa, the compound becomes more ionized, approaching 100% ionized at mobile phase pH 1.5 units above the analytes pKa . In an ideal world the analytes should have at least one ionizable group 100% ionized; signicantly improved retention can be achieved if the analyte is at least 30% ionized.

Simplied pH Effect on Analyte Ionization

18

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Maximum retention

Analyte Percent Ioniztion vs. Mobile Phase

100 90 80 70 60 50 40 30 20 10 0
0 1 2

% Ionized

Creatine Creatinine Histidine

Minimum ionization required for appreciable increase in retention

Mobile Phase pH

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

% Ionization =

100 1+10
(charge) (pH-pKa)

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

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19

Method Development Guide

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.

pH above transition
COO

pH below transition
_
COOH

H
COO

+
COOH

_
_
Transition Point

COO

COOH

Column Type
Cation Exchange-Reversed Phase Primesep C Weak Primesep 200 Primesep 100 Strong Primesep A

3.5 2 1 Ionized at all working pH

Anion Exchange-Reversed Phase Ionized at all working pH

Weak

Primesep B2

Strong

Primesep D

Ionized at all working pH

20

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Select Column Based on Analyte Type

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

Column
Primesep C Primesep 200 Primesep B

Comments
Select for LC-MS. pH Can be used to control retention Select if more retention is required than on Primesep C 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 Weak Bases: pKa<7 Pyridines, anilines, some heterocycles Strong Acids: pKa<3 Phosphates, Sulfates, polyacids, inorganic anions Medium Acids: pKa= 2-4 Diacids, polyacids Weak Acids: pKa>4 Carboxcylic acids, aromatic acids, phenols Zwitterions: pKa4 Amino acids, peptides Zwitterions with one basic and 2 acidic groups Aspartic acid, glutamic acid

Primesep C Primesep 200 Primesep 100 Primesep A Primesep B2

Primesep B2 Primesep B2 Primesep D Primesep 200 Primesep 200 Primesep D Use Mobile phase pH 2, 0.1% Formic acid Use Mobile phase pH 2, 0.1% Formic acid Use mobile phase pH >5

Neutral:

All

Similar behavior on all columns

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21

Method Development

Considerations for Mobile Phase Buffer Selection

Mobile phase pH must be selected so that analytes are ionized. For Basic analytes, pH must be < than analytes 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 modier 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 0.1% TFA 0.1% Formic Acid 0.1% Acetic Acid Approximate pH 2 3 4

22

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Now

you are ready to take total control of your separation.

Resolution Systems

Resolution Systems

23

23

Method Development Your Control Panel

Mobile Phase Ionic Strength

Organic Modier

pH Control

Your Control Panel

Your Control Panel Step 4: Mobile Phase Ionic Strength:

Retention for ionizable compounds and non-

Mobile Phase Ionic Strength:

Retention time, min Retention time, min

increasing for ionizable compounds and nonRetention ionic strength Retention for non-ionic, non-polar compounds will be ionizable polar compounds will decrease with unaffected by ionic strength increasing ionic strength Retention for ionizable compounds non-polar with ionic will be Park Retention for ion and nonRetention for non-ionic, species compounds ionizable polar compounds will decrease with strength unaffected by ionic strength In ion-exclusion mode, retention for ionizable Park Retention for ion species with ionic increasing ionic strength will increase with increasing ionic compounds strength strength In ion-exclusion mode, retention for ionizable Retention for non-ionic, non-polar compoundsionic compounds will increase with increasing will strength be unaffected by ionic strength

Stepionizable polar compounds will decrease with 4: Mobile Phase Ionic Strength:

Effect of Acid Concentration on Retention: Primesep Effect of Acid Concentration on Retention: 5 Effect of Acid Concentration on Retention: Primesep Basic Compounds Primesep 100
5 4 4 3 3 2 2 1 1 0 0.0 0 0.0

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

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

0.1 0.1

% Acid % Acid

0.1 0.1

0.2 0.2

Organic Modier Step 5: Organic Modifier:

Effect of Water Concentration on Hydrophobic Retention: Effect of Water Concentration on

50 50 40

o concentrationThe ionic interactions will Very compounds will Very polar ionizable polar ionizable compounds will not be dominate their retention organic apprecialby affected by not be appreciably non-polar compoundsinteractions will affected by organic RetentionconcentrationThe ionic will decrease for dominate their retention concentrationThe ionic strength will increasing organic interactions will Retention for non-polar dominate their retention compounds will decrease
Retention for non-polar compounds will decrease will increasing organic strength
will increasing organic strength

concentrations apprecialby affected by organic

Retention time, min Retention time, min

Always use Acetonitrile Acetonitrile Always use StepPolar ionizable and non-ionizable compounds have 5: Organic Modifier: Polar ionizable and non-ionizable compounds maximum retention at high and low organic Always use Acetonitrile have maximum retention atand non-ionizable compounds have concentrations high and low Polar ionizable maximum polar ionizable compounds will o Very organic concentrations retention at high and low organic not be

HydrophobicPrimesep 100 Retention on Primesep 100 Effect of Water Concentration on Hydrophobic Retention on Primesep 100 Basic Compounds Polar Neutral Compounds Basic Compounds Non-Polar Neutral Compounds Polar Neutral Compounds Acidic Compounds Non-Polar Neutral Compounds Acidic Compounds

40 30 30 20 20 10 10

0
10 20 20 30 30 40 50 50 60 60 70 70

% Water
10 40

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% Water

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.

Maximum Ion Exchange

% Ionized

Polar Embedded/Hyrodphobic Interaction Dimmer Switch Region
Primesep C Primesep 200 Primesep 100

On

Dimmed
1.Caffeine 2.Benzonitrile 3.Dopamine 4.Procainamide 5.Benzylamine 6.Aminopyridine 7.Clorfeniramine

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25

Method Development
Fine Tune Selectivity With 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 Effect Phase Counter Ionsalt These ions will form ion-pair with cation exchange group -) charge Each ion-pair ion such slightly different dissociated acid or another (+) will offer as NH4(+) from a selectivity due to differing electronic environment interacting with analyte pair with cation exchange group offer slightly different selectivity due can be obtained by changing from: Different selectivity to differing electronic racting with analyte -Formic acid to ammonium formate be obtained by changing from: -Acetic acid to ammonium acetate mmonium formate monium 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 IfIfcounter-ion changes counter-ion changes environment interacting with analyte -Electronicenvironment changes Electronic environment change Different selectivity can be obtained by changing from: -Selectivitychanges Selectivity changes Formic can be lengthy the -Column Equilibrationcan be lengthy and formate Column Equilibration acid to ammoniumand table Acetic acid below should right) followed. the table (on be followed.to ammonium acetate

lumn emains similar volumes to equilibrate

Column Amount of buffer in mL with t ionconcentration mMol exchange requires to replace column If counter-ion remains the same column capacity If counter-ion remains the same column similar contra-ions environment remains Electronic Electronic environment remains similar requires just 2-3 column volumes to equilibrate requires just20 column volumes to equilibrate 10 2-3 mMol 50 mMol If counter-ion changes mMol mMol Electronic environment changes Selectivity changes 1.9 225 115 45 Column Equilibration can be lengthy and the table Resolution Systems below should be followed. 1.1 26 135 70 30 0.4 45 25 10 0.8 100 50 20

Column Inner Diameter mm

Column Length mm

Amount of Silica

Column ion-exchange capacity mMol

Amount of buffer in mL with concentration mMol required to replace column counter-ions 10 mMol 20 mMol 50mMol

4.6 4.6 4.6 3.0 3.0 3.0 2.0 2.0 2.0

250 150 50 250 150 50 250 150 50

3.7 2.2 0.7 1.6 1.0 0.3 0.7 0.4 0.1

1.9 1.1 0.4 0.8 0.5 0.2 0.4 0.2 0.1

225 135 45 100 60 20 45 30 10

115 70 25 50 30 10 25 15 5

45 30 10 20 15 5 10 10 5

Selectivity Can Be Changed by Changing Acid

1.Theobromine 2. 1, 7 Dimethylxanthine 3. Theophylline

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27

Putting it All Togeather

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

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

Applications:

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

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=H O 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 signicantly 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.
2

28

Resolution Systems

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 cation- exchange 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

Analyte Types

Strong bases Medium Bases Weak Hydrophobic Bases Neutrals Hydrophobic Acids

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% ACN B= 30% 10-25mM NH4 Acetate, pH 3.5 70% ACN Gradient:
Time (Min.) 0 4 4.1 5.0 %A 100 0 100 100 %B 0 100 0 0

Peak shape improves with faster gradient

Flow: 2mL/min

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29

Troubleshooting

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) Decrease column length - Columns are available as short as 10mm Primesep C, decrease mobile phase pH

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

Figure A
Buffer
H2SO4, H3PO4, phosphate TFA Ammonium Formate Formic Acid Ammonium Acetate Acetic Acid

Figure B
Cation Exchange Strong Primesep A Primesep 100 Primesep 200 Primesep C Weak Strong Anion Exchange Primesep D

Weak

Primesep B2

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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 ow 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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Ordering Information

Phone: (616) 392-8001 Toll Free: (800) 572-6653 Fax: (616) 396-7520 email: [email protected] web ordering: www.resolutionsys.com
Bonded Phase Part Number
A-21.050.0510 A-32.050.0510 A-46.050.0510 A-220.050-0510 A-21.100.0510 A-32.100.0510 A-46.100.0510 A-220.100-0510 A-21.150.0510 A-32.150.0510 A-46.150.0510 A-220.150.0510 A-21.250.0510 A-32.250.0510 A-46.250.0510 A-220.250-0510

Description
5um, 50x2.1mm Primesep A HPLC Column 5um, 50x3.2mm Primesep A HPLC Column 5um, 50x4.6mm Primesep A HPLC Column 5um, 50x22mm Primesep A HPLC Column 5um, 100x2.1mm Primesep A HPLC Column 5um, 100x3.2mm Primesep A HPLC Column 5um, 100x4.6mm Primesep A HPLC Column 5um, 100x22mm Primesep A HPLC Column 5um, 150x2.1mm Primesep A HPLC Column 5um, 150x3.2mm Primesep A HPLC Column 5um, 150x4.6mm Primesep A HPLC Column 5um, 150x22mm Primesep A HPLC Column 5um, 250x2.1mm Primesep A HPLC Column 5um, 250x3.2mm Primesep A HPLC Column 5um, 250x4.6mm Primesep A HPLC Column 5um, 250x22mm Primesep A HPLC Column

Price
$495.00 $495.00 $495.00 $1750.00 $520.00 $525.00 $555.00 $2850.00 $550.00 $560.00 $595.00 $3180.00 $575.00 $598.00 $698.00 $4850.00

Primesep A

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Bonded Phase

Part Number
100-21.050.0510 100-32.050.0510 100-46.050.0510 100-220.050-0510 100-21.100.0510 100-32.100.0510 100-46.100.0510 100-220.100-0510 100-21.150.0510 100-32.150.0510 100-46.150.0510 100-220.150.0510 100-21.250.0510 100-32.250.0510 100-46.250.0510 100-220.250-0510 200-21.050.0510 200-32.050.0510 200-46.050.0510 200-220.050-0510 200-21.100.0510 200-32.100.0510 200-46.100.0510 200-220.100.0510 200-21.150.0510 200-32.150.0510 200-46.150.0510 200-220.150-0510 200-21.250.0510 200-32.250.0510 200-46.250.0510 200-220.250-0510

Description
5um, 50x2.1mm Primesep 100 HPLC Column 5um, 50x3.2mm Primesep 100 HPLC Column 5um, 50x4.6mm Primesep 100 HPLC Column 5um, 50x22mm Primesep 100 HPLC Column 5um, 100x2.1mm Primesep 100 HPLC Column 5um, 100x3.2mm Primesep 100 HPLC Column 5um, 100x4.6mm Primesep 100 HPLC Column 5um, 100x22mm Primesep 100 HPLC Column 5um, 150x2.1mm Primesep 100 HPLC Column 5um, 150x3.2mm Primesep 100 HPLC Column 5um, 150x4.6mm Primesep 100 HPLC Column 5um, 150x22mm Primesep 100 HPLC Column 5um, 250x2.1mm Primesep 100 HPLC Column 5um, 250x3.2mm Primesep 100 HPLC Column 5um, 250x4.6mm Primesep 100 HPLC Column 5um, 250x22mm Primesep 100 HPLC Column 5um, 50x2.1mm Primesep 200 HPLC Column 5um, 50x3.2mm Primesep 200 HPLC Column 5um, 50x4.6mm Primesep 200 HPLC Column 5um, 50x22mm Primesep 200 HPLC Column 5um, 100x2.1mm Primesep 200 HPLC Column 5um, 100x3.2mm Primesep 200 HPLC Column 5um, 100x4.6mm Primesep 200 HPLC Column 5um, 100x22mm Primesep 200 HPLC Column 5um, 150x2.1mm Primesep 200 HPLC Column 5um, 150x3.2mm Primesep 200 HPLC Column 5um, 150x4.6mm Primesep 200 HPLC Column 5um, 150x22mm Primesep 200 HPLC Column 5um, 250x2.1mm Primesep 200 HPLC Column 5um, 250x3.2mm Primesep 200 HPLC Column 5um, 250x4.6mm Primesep 200 HPLC Column 5um, 250x22mm Primesep 200 HPLC Column

Price
$495.00 $495.00 $495.00 $1750.00 $520.00 $525.00 $555.00 $2850.00 $550.00 $560.00 $595.00 $3180.00 $575.00 $598.00 $698.00 $4850.00 $495.00 $495.00 $495.00 $1750.00 $520.00 $525.00 $555.00 $2850.00 $550.00 $560.00 $595.00 $3180.00 $575.00 $598.00 $698.00 $4850.00

Primesep 100

Primesep 200

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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33

Ordering Information

Bonded Phase

Part Number
B2-21.050.0510 B2-32.050.0510 B2-46.050.0510 B2-220.050-0510 B2-21.100.0510 B2-32.100.0510 B2-46.100.0510 B2-220.100.0510 B2-21.150.0510 B2-32.150.0510 B2-46.150.0510 B2-220.150-0510 B2-21.250.0510 B2-32.250.0510 B2-46.250.0510 B2-220.250-0510 D-21.050.0510 D-32.050.0510 D-46.050.0510 D-220.050-0510 D-21.100.0510 D-32.100.0510 D-46.100.0510 D-220.100.0510 D-21.150.0510 D-32.150.0510 D-46.150.0510 D-220.150-0510 D-21.250.0510 D-32.250.0510 D-46.250.0510 D-220.250-0510

Description
5um, 50x2.1mm Primesep B2 HPLC Column 5um, 50x3.2mm Primesep B2 HPLC Column 5um, 50x4.6mm Primesep B2 HPLC Column 5um, 50x22mm Primesep B2 HPLC Column 5um, 100x2.1mm Primesep B2 HPLC Column 5um, 100x3.2mm Primesep B2 HPLC Column 5um, 100x4.6mm Primesep B2 HPLC Column 5um, 100x22mm Primesep B2 HPLC Column 5um, 150x2.1mm Primesep B2 HPLC Column 5um, 150x3.2mm Primesep B2 HPLC Column 5um, 150x4.6mm Primesep B2 HPLC Column 5um, 150x22mm Primesep B2 HPLC Column 5um, 250x2.1mm Primesep B2 HPLC Column 5um, 250x3.2mm Primesep B2 HPLC Column 5um, 250x4.6mm Primesep B2 HPLC Column 5um, 250x22mm Primesep B2 HPLC Column 5um, 50x2.1mm Primesep D HPLC Column 5um, 50x3.2mm Primesep D HPLC Column 5um, 50x4.6mm Primesep D HPLC Column 5um, 50x22mm Primesep D HPLC Column 5um, 100x2.1mm Primesep D HPLC Column 5um, 100x3.2mm Primesep D HPLC Column 5um, 100x4.6mm Primesep D HPLC Column 5um, 100x22mm Primesep D HPLC Column 5um, 150x2.1mm Primesep D HPLC Column 5um, 150x3.2mm Primesep D HPLC Column 5um, 150x4.6mm Primesep D HPLC Column 5um, 150x22mm Primesep D HPLC Column 5um, 250x2.1mm Primesep D HPLC Column 5um, 250x3.2mm Primesep D HPLC Column 5um, 250x4.6mm Primesep D HPLC Column 5um, 250x22mm Primesep D HPLC Column

Price
$495.00 $495.00 $495.00 $1750.00 $520.00 $525.00 $555.00 $2850.00 $550.00 $560.00 $595.00 $3180.00 $575.00 $598.00 $698.00 $4850.00 $495.00 $495.00 $495.00 $1750.00 $520.00 $525.00 $555.00 $2850.00 $550.00 $560.00 $595.00 $3180.00 $575.00 $598.00 $698.00 $4850.00

Primesep B2

Primesep D

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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Bonded Phase

Part Number
C-21.050.0510 C-32.050.0510 C-46.050.0510 C-220.050-0510 C-21.100.0510 C-32.100.0510 C-46.100.0510 C-220.100.0510 C-21.150.0510 C-32.150.0510 C-46.150.0510 C-220.150-0510 C-21.250.0510 C-32.250.0510 C-46.250.0510 C-220.250-0510

Description
5um, 50x2.1mm Primesep C HPLC Column 5um, 50x3.2mm Primesep C HPLC Column 5um, 50x4.6mm Primesep C HPLC Column 5um, 50x22mm Primesep C HPLC Column 5um, 100x2.1mm Primesep C HPLC Column 5um, 100x3.2mm Primesep C HPLC Column 5um, 100x4.6mm Primesep C HPLC Column 5um, 100x22mm Primesep C HPLC Column 5um, 150x2.1mm Primesep C HPLC Column 5um, 150x3.2mm Primesep C HPLC Column 5um, 150x4.6mm Primesep C HPLC Column 5um, 150x22mm Primesep C HPLC Column 5um, 250x2.1mm Primesep C HPLC Column 5um, 250x3.2mm Primesep C HPLC Column 5um, 250x4.6mm Primesep C HPLC Column 5um, 250x22mm Primesep C HPLC Column

Price
$495.00 $495.00 $495.00 $1750.00 $520.00 $525.00 $555.00 $2850.00 $550.00 $560.00 $595.00 $3180.00 $575.00 $598.00 $698.00 $4850.00

Primesep C

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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35

Notes

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Primesep and all the other trademark of SIELC. Comparisons shown may not be representative of every application. Copyright 2005 Resolution Systems, Inc. Specications, terms, and pricing are subject to change.

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1-616-396-7520

The Primesep Story:

Primesep mixed-mode columns were engineered by chemists who needed to solve the difficult separations they encountered while working in the pharmaceutical industry. The two founders, who worked in different capacities at a pharmaceutical company, also happened to be good friends: One is a synthetic chemist who made the compounds, and the other is an analytical chemist who developed the LC/MS methods to analyze the compounds. The analytical chemist struggled with his friends compounds because they were just too polar to be retained on C1, AQ phases and polar embedded phases, without using ion pairing. Unfortunately, this wasnt an option because ion pairing is not compatible with Mass Spectrometry. Does this story sound familiar? If so, read on... Fortunately, the analytical chemist shared his struggle with his friend, the synthetic chemist. Realizing that most polar compounds are easily ionized, the two began developing ways in which to capitalize on this interaction, while maintaining compatibility with LC-MS. The result was a series of controlled mixed-mode ion exchange/reversed phase columns they called Primesep. These columns allow independent control of polar ionic and hydrophobic interactions, resulting in an almost infinite control of retention and selectivity. Thus, SIELC Technologies was started - a company geared toward solving chromatographys most difficult problems. SIELC Technologies is a privately held company that understands the need for a customer-driven approach to chromatography. We develop columns to solve your problems. We provide direct support and suggestions from one chemist to another chemst. Weve put the friendly back in LC-MS friendly conditions.