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thermoThe CHROMacademy Essential Guide:
Mixed Mode Chromatography – the answer to everything?

28th March 2013 11am EST / 3pm GMT

In this session, Dr Xiaodong Liu (R&D Manager, Thermo Fisher Scientific) and Scott Fletcher (Technical Manager, Crawford Scientific) consider the technique of mixed mode chromatography and the application areas in which this technique can help
chromatographers. The mechanisms of mixed-mode chromatography are discussed alongside the parameters which can be
used to optimize selectivity and retention. A series of useful illustrative example are presented with strategies for colum
selection and screening to allow this technique to quickly become part of the analytical armory!

desolvation zoneTopics covered include:

  • What is mixed mode chromatography? - evolution and fundamental retention mechanisms
  • Mixed mode separations – comparison with reversed phase, normal phase ion exchange and HILIC chromatography
  • Suitable analytes / applications for mixed mode chromatography
  • Column Selection
  • Retention and selectivity control using organic modifier, buffer strength and pH
  • Screening and initial method development
  • Interesting illustrative examples
  • The future for mixed – mode phases

Who Should Attend:

  • Anyone working with highly polar or ionized compounds who wish to gain good selectivity and retention
  • Anyone working regularly with Electrospray mass spectrometry
  • Anyone wishing to replace ion-pair chromatography methods with more MS friendly or robust methods

Find out more about this Month's Essential Guide Webcast »


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The CHROMacademy Essential Guide Tutorial
Mixed Mode Chromatography – the answer to everything?

In this session, Dr Xiaodong Liu (R&D Manager, Thermo Fisher Scientific) and Scott Fletcher (Technical Manager, Crawford Scientific) consider the technique of mixed mode chromatography and the application areas in which this technique can help chromatographers. The mechanisms of mixed-mode chromatography are discussed alongside the parameters which can be used to optimize selectivity and retention. A series of useful illustrative example are presented with strategies for colum selection and screening to allow this technique to quickly become part of the analytical armory!

  ask the CHROMacademy experts

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Although reversed-phase columns (e.g. C18) are used in a wide range of applications, they often fail to retain highly polar molecules (e.g. counter ions), and offer limited selectivity.
 HILIC is often used to analyze polar analytes, but it encounters challenges such as poor solubility of analytes (in highly organic media), retention severely affected by sample matrix, and less reliable method.
Ion Exchange (IEX) can be used to retain charged molecules, but it fails to retain neutral analytes and poor selectivity for analytes of same charge.

  • Analytes in HPLC are highly diverse: hydrophobicity, charge, size, etc.
  • Many are highly polar; many have poor chromophore.
  • Reversed-phase columns (e.g. C18); the “workhorse”
    • Not suited for highly hydrophilic analytes
  • Hydrophilic interaction (HILIC)/normal phase (NP); the solution?
    • Solubility challenge
    • Sample matrix effect
    • Limited to hydrophilic molecules
  • Ion exchange (IEX) chromatography; good for charged analytes
    • Lack of retention for neutral analytes
  • Rough breakdown of HPLC mode usage: RP ~ 60+%; HILIC/NP ~18%; IEX ~ 5%

To better understand these challenges, let’s take a look at this well-known equation showing the dependency of resolution (Rs) upon column efficiency (N), selectivity (a), and retention factor (k). The objective of a separation is to separate analytes, which is expressed as resolution Rs.
This dependency is also illustrated with the plot below.

 
 
 

This plot is basically saying that out of all three parameters that affect the quality of separation, the selectivity has the largest impact. In other words, the key factor for a separation is Selectivity. Selectivity is mainly determined by the column chemistry.
As we mentioned in the previous slide, while many HPLC applications are developed on RP columns such as C18, C8, polar-embedded phase, phenyl, and others, the selectivity options are rather limited, especially for those highly hydrophilic molecules for example, pharmaceutical related counterions.

 
 

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Mixed-mode chromatography provides a viable solution to most of these challenges.

  • Mixed-Mode chromatography is a method that utilizes more than one separation modes; mainly RP combined with IEX interactions
  • The biggest benefit of this approach is that selectivity can be optimized by adjusting mobile phase ionic strength, pH and/or organic solvent. As the result, not only are their selectivity complementary to RP columns, but also complementary to themselves under different conditions
  • In addition, mixed-mode chromatography requires no ion-pairing agents in the mobile phase for separating highly hydrophilic charged analytes, which simplifies the mobile phase and is compatible with MS
  • With adjustable selectivity, it is also possible to separate analytes with dramatically different hydrophobicity and charge state, such as simultaneous separation of pharmaceutical API and counter ion, in a single analysis

Before we dig into the details, let us be clear on why we need mixed-mode columns.

Retention
  • Highly hydrophilic analytes, e.g. pharmaceutical counter ions - suitable for both neutral and charged, both hydrophobic and hydrophilic.
Selectivity
  • Adjustable selectivity allows for easy method optimization
Throughput
  • Determining analytes of various charges and hydrophobicity with one single injection.  Because of adjustable selectivity, these types of column can separation challenging analytes with a single injection, which otherwise would require different columns and methods.
MS-compatibility
  • No need for ion-pairing reagent for hydrophilic charged analytes

Method robustness

More resistant to sample matrix effect than HILIC

 
 

By chemistry design, RP/IEX bimodal mixed-mode stationary phases can be grouped into four types as shown here.

  • The 1st is a blend of two different stationary phases into a column, here the RP (in black) and the other is ion-exchange (in red).
  • The 2nd type involves the use of a mixture of two different silyl ligands in the bonding step, one for RP, and one for IEX. As a result, the bonded silica is modified by both RP and IEX ligands.
  • Type 3 material involves functionalizing silica with IEX-embedded alkyl silyl ligands, which highlights the RP characteristic, and can be viewed as “IEX modified RP column.”
  • By comparison, Type 4 material involves bonding IEX-tipped alkyl silane ligand to the silica substrate, which emphasizes more on its IEX property, and features  a “RP modified IEX column.”
 

1&2 : + straightforward; - selectivity drifting (mainly due to the different hydrolytic stability RP ligand bonded sites and IEX ligand functionalized sites).
To overcome this problem, more modern mixed-mode stationary phases were developed using functional silyl ligands bearing both RP and IEX.


3&4: + greatly improve the selectivity ruggedness

 
 
Here are some mixed mode ligands which operate in two modes – i.e. hydrophobic and anion OR cation exchange mode
 
 
 
 
 
 
 

As for the RP/AEX/CEX trimodal columns, to our knowledge, there are three types of commercial products in the marketplace:

  1. Mixed-beads
  2. Bonded phase with amphoteric silane chemistry
  3. Nanopolymer silica hybrid technology
 

The Acclaim Trinity P1 represents the latest advancement of column technology of mixed-mode chromatography. It is a trimodal functional column, designed for the separation of charged analytes, such as drug molecules and their counterions. Its column chemistry along with other product information is shown here.
Acclaim Trinity P1 is based on a high-purity porous spherical silica particle whose inner-pore area is covalently modified with silyl ligands containing both RP and anion-exchange moieties while the outer surface is coated with negatively charged nano-polymer beads by electrostatic interactions. This chemistry design creates a distinctive spatial separation of the anion-exchange and cation-exchange regions, and allows reversed-phase, cation-exchange and anion-exchange retention mechanisms to function simultaneously and be controlled independently.

 
 
 

Bonded phase with amphoteric silane chemistry

 
 
 
 
 

Mixed-bead type phases

 
 
 
 

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In the following discussion, we will see how to develop analytical methods using this trimode phase.

The most important feature of the mixed mode columns is “Adjustable Selectivity.”

To make it easier to understand, two probe molecules were used to make the point: one neutral and one acidic.

As shown here, at pH6 when increasing mobile phase ionic strength, from 20 to 100 mM while keeping mobile phase pH and organic content constant, the negatively charged acid elutes earlier, but virtually has no effect on retention of the neutral molecule. As the result, the elution order is reversed.

 
 
 

Selectivity can also be “tuned” by mobile phase pH

At pH6 the acid is negatively charged. The retention is mainly governed by both electro-static interaction and hydrophobic interaction. On the other hand, the neutral probe is retained solely by hydrophobic interaction. As the result, 4-hydroxybenzoic acid elutes after butylbenzene.

When lowering the pH to 2.6 the acid is protonated to a mostly neutral form, and the retention is mainly caused by hydrophobic interaction. In this case, the retention of both neutral and acidic molecules are mostly governed by hydrophobic interaction. Because 4-hydroxybenzoic acid is more hydrophilic than butylbenzene, it elutes first, causing selectivity change.

 
 
 

Mobile phase organic content mainly affect hydrophobic retention.
As shown here, increasing acetonitrile content from 45 to 50%, the retention of the neutral molecule is affected more dramatically than the acidic molecule as pH6, thus the elution order reversal is observed.
To sum up, the mixed-mode column provides adjustable selectivity, which can be “tuned” by changing mobile phase ionic strength, pH, and organic content either independently or collaboratively.

 
 
 
Method Development Step 1
 

The first step for any method development is to understand the application itself including analytes, sample matrix and method requirement.

  • Analytes of interest
    • Chemical Structure
    • Size
    • Charge
    • Hydrophobicity
    • Chromophore etc.
  • Sample matrix
  • Method requirement
    • Sensitivity
    • Resolution
    • Run time etc.
 
Method Development Step 2 - Column Selection
 

Choose between C18 and a mixed-mode column.

Always consider reverse-phase column (e.g., C18) first, because of its broadest acceptance, well-documented method development strategy, and familiarity.

However, in many cases, reverse-phase columns fail to provide desirable results due to their limitation in selectivity and inability of retaining hydrophilic charged molecules. For example, no reverse-phase can retain sodium and chloride – two ions that are most often used as pharmaceutical counterions.

When no reverse-phase column is proved suitable, consider use a mixed-mode column, because of adjustable selectivity and adequate retention for charged hydrophilic analytes.

  • Choose between C18 and a mixed-mode column
    • Generally consider an C18 first
    • This will not always provide the required selectivity
    • Other options will need to be considered for polar, charged  and complex mixtures
    • Choose the most suitable mixed-mode for your given requirements
  • Selection of a appropriate mixed-mode column is application-specific
    • Anionic and neutral (hydrophobic) analytes → RP/AX
    • Cationic and neutral (hydrophobic) analytes → RP/CX
    • Anionic, cationic and neutral (hydrophobic) analytes → RP/CX/AX
    • Anionic, cationic and neutral (polar) analytes → HILIC/CX/AX
    • Neutral (hydrophobic and polar) analytes → RP/HILIC
    • Application-specific columns
 
Method Development Step 3 - Get Ready
 

After potential columns are selected, we need to make sure we have all gears for the method development.

Most mixed-mode columns must be stored and used in buffered mobile phases. In general, ammonium acetate and ammonium formate are the most commonly used buffers for method development using a mixed-mode column. For the application that involves nonvolatile analytes with poor chromophore, it is important to use high-purity buffer salt to achieve satisfactory limit of detection using CAD or ELSD.

Before starting the experiment, read the column manual carefully. Different mixed-mode columns have different surface chemistry and design philosophy. Thus it is very helpful to understand the column chemistry and familiarize example applications provided by the column manufacturer.

  • LC system
    • HPLC system equipped with an gradient pump, auto sampler, column oven, and PDA-detector
    • For nonvolatile analytes with no or weak chromophore, consider an aerosol based detector, such as ELSD or Corona CAD.
  • Mobile phase
    • During method development, it is more convenient and flexible to generate desired mobile phase by proportioning acetonitrile (HPLC grade), ammonium acetate or formate buffer, and D.I. water (>18 MΩ-1)
    • The buffer should be made from 99.99+% ammonium acetate, ammonium formate, acetic acid or formic acid. The pH should be with ±0.5 unit of the pKa of corresponding acid
  • Know the column
    • Read the column manual thoroughly
    • Perform the column performance test (described in the column certificate) to ensure it is in good condition
    • Use guard columns for real-life samples
 
Method Development Step 4
 

After all that reading and preparation, the next step is to start the method development.

Depending on the complexity of the applications, different approaches can be used: when number of analytes < 4, such as API and counter ion, it is often not difficult to choose a starting condition to ensure adequate condition and hopefully reasonable analysis time. It seems to be kind of guess-work, but for those who have good understanding of the column chemistry, it is a rational thinking process. Based on the result from the initial condition, further modification(s) can be implemented by adjusting buffer concentration, pH and/or organic solvent.

For the application that involves a larger number of analytes, it makes more sense to take the screening approach. The screening protocol consists of several dimensions: solvent content, buffer concentration and pH, which should be done. In fact, this approach can be used for any of method development. Based on the result from initial screening, further optimization can be performed within a more targeted development space. Again, good understanding of mixed-mode chromatography will help this process.

When the number of analytes < 4
  • Choose a starting condition
    • Adequate retention
    • Fast analysis
  • Method optimization
    • Buffer concentration
    • pH
    • Organic solvent
When the number of analytes > 4
  • Set up the screening protocol
    • Organic solvent (e.g., 10 to 90% v/v with 10% intervals)
    • Buffer concentration (e.g., 5 to 100 mM)
    • pH (e.g., 2 or 3 pH levels between pH3 and 6)
  • Further optimization
    • More targeted screen range (buffer concentration, pH, organic solvent)
 
 

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The Acclaim Trinity P1 column is a novel reversed-phase/anion-exchange/cation-exchange trimodal silica-based stationary phase developed using Nanopolymer Silica Hybrid (NSH) technology. The new material consists of high-purity porous spherical silica particles coated with nano-polymer beads via an electrostatically driven self-assembly process that results in a distinctive spatial separation of the anion-exchange and cation-exchange regions, and allows both retention mechanisms to function simultaneously and be controlled independently.

The ZIC-HILIC is a highly polar zwitterionic silica-based stationary phase, specially designed for separating polar analytes under HILIC conditions. Its zwitterionic nature results in the fact that selectivity cannot be controlled by adjusting the pH and that it retains ions via salt-exchange rather than via ion-exchange. Consequently, this phase fails to provide “controllable” selectivity for both anions and cations. Moreover, the low hydrophobicity prevents it from broad use, such as less hydrophilic APIs.

The column chemistry of both stationary phases is depicted below.

 

Comparison of Mixed-Mode vs. HILIC - Overview

 
 
 

Pharmaceutical counterions:

The separation below demonstrates that the Acclaim Trinity P1 column provides ideal selectivity for separating pharmaceutical counterions (including both cations and anions) using acetonitrile/ammonium acetate mobile phase. Baseline separation of five cations and five anions is achieved within a single run. The column selectivity is designed such that cations elute before anions. This is the first and only column available that separates both cations and anions easily and reliably.

We also tested the same application using a ZIC-HILIC column. To obtain the best possible chromatographic condition, various mobile phase acetonitrile contents (20 to 80% with 10% intervals) and buffer concentrations (10, 15 and 20 mM) were tested. The optimal separation was achieved at 80% acetonitrile with 15 mM ammonium acetate. As shown in Figure B, however, compared to the Trinity P1, the ZIC-HILIC column gives the inferior separation, consumes more organic solvent and requires longer analysis time.

 

Comparison of Mixed-Mode vs. HILIC - Separation of Pharmaceutical Counterions

 
 
 

Hydrophilic acidic API and counterion: Penicillin G is an antibiotic compound belonging to the b-lactam antibiotic family and is often formulated in the potassium salt form. Because of the highly hydrophilic nature of both the API and the counterion, it is impossible to assay both components within the same analysis on any RP column. The Trinity P1column provides baseline separation of both penicillin G and K+ ion in either RP/IEX mode or HILIC mode, with excellent peak shape and adequate retention (Figures A and B). Note that elution order can be reversed by changing acetonitrile content in the mobile phase. By comparison, the ZIC-HILIC column was unable to provide adequate retention (k’ = 0.5) for this drug molecule under HILIC conditions while the retention of the counterion (K+) is excessively long (k’ = 15.0), as shown in the separation below.

 

Comparison of Mixed-Mode vs. HILIC - Hydrophilic Acidic API & Counterion – Penicillin G Potassium

 
 
 
Hydrophilic basic API and counterion: 1,1-Dimethylbiguanide hydrochloride (Metformin), a highly hydrophilic basic drug formulated in the chloride salt form, is an antidiabetic agent that reduces blood glucose levels and improves insulin sensitivity. Figure A illustrates separations of the API and its counterion using the Trinity P1 at four acetonitrile levels. Due to both the hydrophilic nature of the analytes and the multiple retention mechanisms facilitated by this column, baseline separations can be achieved in both RP/IEX mode and HILIC mode (not shown) with good peak shape and adequate retention. As shown in Figure B, the ZIC-HILIC column also provides well for this application under HILIC conditions, but not under RPLC conditions (not shown).
 

Comparison of Mixed-Mode vs. HILIC - Hydrophilic Basic API & Counterion – Metformin▪HCl

 
 
 

Hydrophobic acidic API and counterion: Naproxen, often formulated in its sodium form, is a non-steroidal anti-inflammatory drug (NSAID) commonly used for the reduction of moderate to severe pain, fever, inflammation. While the API is a highly hydrophobic acidic molecule, the counterion – Na+ can’t be retained on any RP columns. Because of the co-existence of anion-exchange and cation-exchange properties on the Trinity P1, both hydrophobic API and hydrophilic Na+ ion can be retained and separated on a 50-mm long Trinity P1 column with excellent resolution, peak shape, and retention ( 5 > k’ > 2) within merely 3 minutes (Figure A). On the other hand, the ZIC-HILIC column fails to provide adequate retention for naproxen under either HILIC (70 to 90% acetonitirle, result at 85% acetonitrile shown in Figure B) or RPLC (10% acetonitrile, not shown) conditions.

 

Comparison of Mixed-Mode vs. HILIC - Hydrophobic Acidic API & Counterion – Naproxen Sodium

 
 
 

Hydrophobic basic API and counterion: Trimipramine is a tricyclic antidepressant (TCA) and often formulated as a maleate salt. It has antidepressant, anxiolytic, antipsychotic, sedative, and analgesic effects. While trimipramine is a highly hydrophobic basic compound, its counterion – maleate is very hydrophilic.  As a result, the ZIC-HILIC cannot provide adequate retention for the API under either HILIC (Figures B and C) or RPLC (not shown) conditions. By comparison, the Trinity P1 column demonstrates excellent separation capability, with baseline resolution and fast analysis (less than 3-min run time), as shown in Figure A.

 

Comparison of Mixed-Mode vs. HILIC - Hydrophobic Basic API & Counterion – Trimipramine Maleate

 
 
 
 

Comparison of Mixed-Mode vs. HILIC - Summary

 
 
 

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In this study, we evaluated three commercial columns as shown here. All of them have anion-exchange, cation-exchange, and reversed-phase functional groups.

The first type uses uniformly blended packing material consisting of two types of porous silica particles: silica modified with C18 and cation-exchange silyl ligands and silica modified with C18 and anion-exchange silyl ligands.

Another trimodal phase is based on silica particles covalently functionalized with a silane containing reversed-phase, cationic and anionic groups on the same ligand. The cationic group is close to the silica surface separated from anionic group by a hydrophobic chain.

The third trimodal phase, prepared from an electrostatically driven self-assembly process, consists of high-purity porous spherical silica particles whose inner-pore area is covalently modified with silyl ligands containing both RP and anion-exchange moeities while the outer surface is coated with negatively charged nano-polymer beads by electrostatic interactions. This chemistry design creates a distinctive spatial separation of the anion-exchange and cation-exchange regions, and allows reversed-phase, cation-exchange and anion-exchange retention mechanisms to function simultaneously and be controlled independently.

 

Commercially Available Trimodal Columns - Overview

 
 
 

Here is a visual comparison among three different trimode phases with respect to reversed-phase, cation-exchange and anion-exchange retentions. The condition is specified on the slide.
The blue bar represents RP retention. As we can see, Scherzo SM-C18 exhibits the highest RP retention – more than 7 times and 12 times compared to Obelisc R and Trinity P1, respectively.
The cation-exchange capacity (represented in red) follows the order of Trinity P1 > Obelisc R > Scherzo SM-C18.
The anion-exchange capacity (represented in green) follows the same trend as cation-exchange.
The different combinations of RP, CEX and IEX capacities result in different selectivities:

  1. Trinity P1 provides strong anion-exchange, slightly smaller cation-exchange and rather weak RP retention. Thus it can be characterized as a “RP modified cation and anion-exchange phase”.
  2. Scherzo SM-C18 shows strong RP retention, but very little cation-exchange and anion-exchange capacities. Thus it can be viewed as an “ion-exchange modified RP material”.
  3. The RP and IEX retentions of Obelisc R are somewhere between Trinity P1 and Scherzo SM-C18, and clearly a different type of trimode phase.
 

Commercially Available Trimodal Columns - Comparison of RP and IEX Interactions

 

Mobile Phase: 60/40 v/v MeCN/NH4OAc (15 mM total), pH5.0

 
 

Our first example is simultaneous separation of pharmaceutical counterions:

The top trace shows the separation of 5 cations and 5 anions on the Acclaim Trinity P1 column, with ideal selectivity and symmetrical peaks. The column is designed such that cations elute before anions.

The blue trace is the same separation performed on Obelisc R. Under this optimized condition, five anions can be separated but cations co-elute close to the void.

The result on the Scherzo SM-C18 is shown in the green trace. Clearly, this phase is not suitable for counterion analysis.

We also tested the same application using a ZIC-HILIC column. To obtain the best possible chromatographic condition, various mobile phase.

 

Commercially Available Trimodal Columns - Separation of Pharmaceutical Counterions

 
 
 

Hydrophilic acidic API and counterion:

Penicillin G is an antibiotic compound and is often formulated in the potassium salt form. Because of the highly hydrophilic nature of both the API and the counterion, it is impossible to assay both components within the same analysis on any RP column. Here are the results obtained on three different trimode phases, under respective optimized conditions:

Both Trinity P1 and Obelisc phases are capable of providing adequate retention and resolution. The difference is the workable window for Obelisc R is quite narrow – to ensure the k’ for the first peak greater than 1.5, and the 2nd peak is not excessively long, as well as both peaks are sufficiently resolved, the condition is almost locked. On the other hand, on the Trinity P1, multiple methods with different retention, different elution order and different spacing, can be easily developed to meet these requirements. Thus, this phase provide more flexibility in method development.

The green trace suggests that the Scherzo SM-C18 is not suitable for this application.

 

Commercially Available Trimodal Columns - Hydrophilic Acidic API & Counterion – Penicillin G Potassium

 
 
 

Hydrophilic basic API and counterion:

1,1-Dimethylbiguanide hydrochloride (Metformin), a highly hydrophilic basic drug formulated in the chloride salt. The simultaneous determination of both API and counterion is not possible on any RP columns.

As shown here, Trinity P1 and Obelisc phases can do the job while Obelisc R provides faster analysis.
While Scherzo SM-C18 retains the API fairly well, the counterion Cl- elutes next to the void due to its inadequate anion-exchange retention.

 

Commercially Available Trimodal Columns - Hydrophilic Basic API & Counterion – Metformin▪HCl

 
 
 

Hydrophobic acidic API and counterion:

Naproxen is a non-steroidal anti-inflammatory drug (NSAID), often formulated in its sodium form. While the API is a highly hydrophobic acidic molecule, the counterion – Na+ is too hydrophilic to be retained on any RP columns.

Both Trinity P1 and Obelisc phases provide adequate retention and resolution. The difference is the workable window for Obelisc R is quite narrow – to ensure the k’ for the first eluting analyte greater than 1.5, and the other analyte is not excessively long, as well as both peaks are sufficiently resolved, the condition is almost locked.

On the other hand, on the Trinity P1, multiple methods with different retention, different elution order and different spacing, can be easily developed to meet these requirements. Thus, the Trinity P1 provides more flexibility in method development.

While Scherzo SM-C18 retains the API well, the counterion na+ elutes next to the void due to its inadequate cation-exchange retention.

 

Commercially Available Trimodal Columns - Hydrophobic Acidic API & Counterion – Naproxen Sodium

 
 
 

Hydrophobic basic API and counterion

Trimipramine is a tricyclic antidepressant (TCA) and often formulated as a maleate salt. While trimipramine is a highly hydrophobic basic compound, its counterion – maleate is very hydrophilic.

As shown here, both Trinity P1 and Obelisc phases provide adequate retention and resolution while Obelisc R gives better peak shape for the basic drug. Scherzo SM-C18 retains the API well, the counterion maleate elutes next to the void due to its inadequate anion-exchange retention.

 

Commercially Available Trimodal Columns - Hydrophobic Basic API & Counterion – Trimipramine Maleate

 
 

Commercially Available Trimodal Columns - Review

 
  • All three commercially available trimodal columns are different
  • The show different selectivities based in differing propensities for reversed phase, cation-exchange and anion-exchange interactions
  • They also differ in their targeted applications
 
 
 

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In this session, Dr Xiaodong Liu (R&D Manager, Thermo Fisher Scientific) and Scott Fletcher (Technical Manager, Crawford Scientific) consider the technique of mixed mode chromatography and the application areas in which this technique can help chromatographers. The mechanisms of mixed-mode chromatography are discussed alongside the parameters which can be used to optimize selectivity and retention. A series of useful illustrative example are presented with strategies for column selection and screening to allow this technique to quickly become part of the analytical armory!

Dr Xiaodong Liu
R&D Manager
Thermo Fisher Scientific

Scott Fletcher
Technical Manager
Crawford Scientific

Key Learning Objectives:

  • Understand the nature of mixed-mode chromatography ligands and the fundamental separation mechanisms in mixed mode chromatography
  • Appreciate the primary variables and how these may be adjusted to change retention and selectivity
  • Understand how to select the most appropriate column type and to screen eluent / column combinations during initial method development
  • Appreciate when to use mixed mode chromatography and, through a series of illustrative examples, how this might apply to your separations