column_selection_guide.pdf

7/30/2019 column_selection_guide.pdf

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Using Stationary Phase Selectivity to Assist Method Development

CSG 01-01

IntroductionSelecting an HPLC column presents a wide range of choices, as Thermo Hypersil-Keystone offers a variety of phases ancolumns, with a range of silica specifications and varying phase coverage, all of which influence selectivity. The columcharacteristics that define performance are also influenced by physical parameters such as pore size, surface area, carboload, and particle size.

Pore sizeThermo Hypersil-Keystone silica-based columns vary from60 to 300 in pore size. The strength of the interactionsbetween the analyte and bonded phase is influenced by theavailability of the bonded phase to the analyte. This in turnis controlled by pore size, as over 90% of the silica surfacearea occurs within the pores. When the silica pore diameteris smaller than the diameter of the analyte, the analyte isexcluded from the pore and interactions are minimized, whichleads to shorter retention and lower loading capacity. Largemolecules (MW>2000 Da) show limited penetration of smallpores (300 m2/gm) will increase thlikelihood of the analyte being retained beyond the column void volume.

Carbon loadThe degree of retention of a neutral hydrophobic analyte on a wholly alkyl phase such as C18 or C8 can be inferred from thcarbon load value, as this indicates the degree of bonded phase coverage. A theoretical maximum for phase loadinoccurs at 50 - 60% of the silica surface area. As the silica pore size increases, surface area decreases, and therefore tcapacity for bonded phase loading decreases. Hence, a 100 pore size silica typically shows higher carbon loading forC18 phase than a 300 pore size silica. When analytes are only moderately hydrophobic, selection of a column with higcarbon load (>10%) will increase the likelihood of the analyte being retained beyond the column void volume. High carboload hydrophobic phases (e.g. BetaMax Neutral) show the greatest degree of hydrophobic interaction, and therefore thmost retention.

Particle size3m and 5m particle sizes are typically used for analytic

separations, with larger particle sizes used for preparativseparations. Reducing the particle size to 3m increasecolumn efficiency by approximately 30%, but also increasecolumn backpressure. For this reason, columns longer tha150mm are not recommended when 3m particles are used

2/2002


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Selecting a bonded phaseThe most popular bonded phase in reversed-phase chromatography is C18, as it offers good retention for a wide range ocompounds. C18 bonded phases show a high degree of retention for analytes containing hydrocarbon groups, withhydrophobic (dispersive) interactions being the primary attractive force. Neutral compounds are retained primarily bdispersive interactions with the C18 bonded phase.

Figure 1

In reversed phase chromatography,water is a weak solvent, having limitedelution capability. Most mobile phasestherefore contain a proportion of astronger organic solvent (most typicallyacetonitrile or methanol). The ratio ofaqueous/organic components is thenused to either increase or decrease theretention of any given analyte. Typicallya lower organic solvent percentage isused to promote retention of the morepolar analytes, whereas an increasedproportion of the organic component isused to reduce retention of the morehydrophobic analytes.

Secondary interactions (including ionic,polar and chelating interactions) canoccur between the analyte and bondedphase when polar functional groups arepresent in either. The base silica also

plays an important role, as significantdifferences in selectivity result fromvarying degrees of silica purity and basedeactivation. Columns and phases withpolar and ionic selectivity are discussedlater in this Guide.

Figure 1 shows a hydrophobicity chart,comparing a selection of ThermoHypersil-Keystone and other columnsaccording to their retention ofphenylheptane, a neutral hydrophobiccompound. To identify a column giving

greater hydrophobic retention, selectfrom higher up the chart. Conversely,to select a column with less hydropho-bic retention, choose from lower downthe chart. Bonded phases that have avariety of interaction mechanisms (suchas ionic or polar interactions) show lesshydrophobic retention, but may providealternative selectivity, especially forpolar analytes.


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ADVANTAGES DISADVANTAGES

Type A versus Type B silica

In 1986, Kirkland and others1 popularized the term Type B to describe a new generation of highly purified, deactivatesilicas with improved performance for chromatography of basic compounds. Type B silica has replaced older Type A silicas the packing of choice in new LC methods. However, Type A silica continues to be used in many established method

Type A silica has a high concentration of metal impurities, resulting in a heterogeneous, acidic surface. This surface leadto non-uniform bonded phase coverage and active sites that interact strongly with sample components, causing poor peashapes. Type A silica can undergo a base deactivation process to remove many of the metal impurities, generating a mohomogeneous surface and essentially Type B silica performance. True Type B silica is synthesized from metal-free reagento ensure a very low total metal content and offer even greater performance advantages. Type A and Type B silica can b

compared as shown in Table 1.Thermo Hypersil-Keystone columns can be categorized according to their silica type (Table 2).

Less expensive Higher surface activity

Poor recovery of solutes at lowest levels

Poor peak shapes for basic solutes

Requires mobile phase additives to control peak shape

Less uniform bonding and lot to lot variability

TYPE B SILICA

Lower surface activity More costly

Good solute recovery at low levels Can be less rugged at high pH

Good peak shapes for basic compounds

Simple mobile phases can be used

More uniform dense bonding for better lot reproducibility

Table 1

Type A silica All classical Hypersil columns

Base deactivated Type A silica Hypersil BDS columns

Type B silica AQUASIL C18, BetaMax, BETASIL, BetaBasic, BioBasic,Fluophase, HyPURITY, PRISM columns

Silica Category Products

Table 2

ADVANTAGES DISADVANTAGES

TYPE A SILICA


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Hypersil BDS / ODS Comparison

H301-281 Comp1

2

2

1

0 2 4 6 MIN

Columns: 5m, 150x4.6mmEluent: 60% ACN / 40% 0.05M KH2PO4, pH 4.5

Flow: 1.0 mL/minDetector: UV @ 254Sample: 1. Pyridine

2. N-Methylaniline3. N,N-Dimethylaniline4. Toulene

Hypersil ODS

0 2 4 6 MIN

3

4

Hypersil BDS

3

4

Figure 5

Sample:1 . Uraci l2. Benzyl Alcohol3. 2,7-Dihydroxynaphthalene

4. 4-Nitrobenzoic Acid5. 2,3-Dihydroxynaphthalene6. Chlorocinnamic Acid

Acids and Chelators at pH 1.5

1

3

2

0 10 20 MIN

HyPURITY C18, 5m, 150x4.6mmEluent: 70% 0.1M H3PO4 / 30 % ACN

Flow: 1.0 mL/minDetector: UV @ 215

H221-10003

Sample:1. Capsaicin2. Impurity

Drugs at pH 12

1

2

BetaBasic 18, 5m, 150x4.6mmEluent: 60% MeOH / 37% 10mM Sodium Borate,

pH 12Flow: 1.0 mL/min

Detector: UV @ 280Data courtesy of Kristine Phillips, Dept. of Chemistry,

Univ of New Hampshire

715-084

Figure 2 Figure 3

4

5

6

0 5 10 15 MIN

Figure 4

High Purity Silica

Ultra pure silica provides exceptionalperformance under demanding HPLCconditions. HyPURITY silica containsexceptionally low metal content (40 ppbtotal levels), and HyPURITY phasesshow superior peak shapes for basicand chelating compounds (Figure 2).

BetaBasic phases, based on a Type B

high purity silica, have particularly denseand uniform bonding chemistry. Thismakes BetaBasic columns ideal forgeneral purpose chromatography, withexceptional peak shapes for basiccompounds. The BetaBasic 18 phasehas also demonstrated exceptionalstability and performance at both highand low pH extremes (Figure 3).

As shown in Figure 4, the surface ofType B silica is uniform, with almostideal bonding characteristics. Type B

silica shows only weakly acidic proper-ties compared to older Type A silica.Type B silica minimizes the interactionswith bases that can cause poor peakshape.

The advantages offered by the newersilicas over Type A silica are illustratedby a comparison of a Hypersil ODS col-umn (based on a Type A silica) againsta Hypersil BDS C18 column (made froma base-deactivated silica), as shown inFigure 5.


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Surface area Pore sizeColumn Bonded Phase Chemistry % Carbon (m2/gm) ()

Bonded Phase Interactions and Column Selectivity

The functional groups on an analyte determine its degree of neutrality or polarity. In selecting a bonded phase, it important to further categorize polar compounds as either acidic or basic, as different interactions can occur between thstationary phase and either an acidic polar compound or a basic polar compound. The behavior of neutral, polar acidic anpolar basic compounds with a variety of stationary phases has been explored to simplify the selection of bonded phaseand to reduce method development time.

Mass spectrometry is increasing in popularity as an HPLC detection method, which brings limitations to the HPLC methodevelopment process. Examples include a restricted choice of mobile phase composition, and the use of volatile buffers additives at minimum concentrations to maximize sensitivity and minimize instrument down time. The use of colum

switching techniques is becoming widely adopted, typically with a generic gradient to simplify analysis. This approach alsrequires a wide range of column functionalities to gain significant selectivity differences. Thus, a stationary phase wialternative functionality may be necessary to achieve the required selectivity for the sample.

For compounds with significant polar or ionic character, an alternative retention mechanism to hydrophobicity may brequired. Bonded phases that employ ionic and polar interactions provide unique selectivity options to achieve retentioand separation of polar compounds.

Several approaches to bonded phase development have beenused by Thermo Hypersil-Keystone to increase retention of polarmolecules, including:

1) Embedding polar groups into the alkyl chain to create amore polar phase, as shown in Figure 6 (HyPURITY

ADVANCE, BetaMax Acid, and PRISM RP phases).2) Creating a more wettable C18 chain with polar endcapping

groups (AQUASIL C18 phase).

3) Fluorination of alkyl chains and benzene rings to impartpolarity (Fluophase RP & Fluophase PFP packings).

4) Development of mixed-mode phases with multiple ligands(BETASIL Phenyl/Hexyl phase)

5) Graphitization of carbon to create a hydrophobic yet highlypolarizable surface (Hypercarb packing).

These stationary phases are compared in Table 3.

*BetaBasic 18 is selected as a hydrophobic reference column, as it is representative of a traditional C18 phase bonded onto type B silica.

AQUASIL C18 Polar end-capped C18 12 300 100

BetaBasic 18 * Traditional C18 13 100 150

BetaMax Acid Polar embedded C12 15 540 60

BetaMax Base Cyano 9 540 60

BETASIL Phenyl/Hexyl Phenyl & C6 7 350 100Fluophase PFP Perfluorinated phenyl 12 330 100

Fluophase RP Perfluorinated C6 11 330 100

Hypercarb 100% porous graphitic carbon 100 120 250

HyPURITY ADVANCE Polar embedded C8 7 190 180

PRISM RP Polar embedded C12 12 330 100

Figure 6

Table 3Thermo Hypersil-Keystone Column Comparison


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Test 1 - Neutral Compounds

The n-alkylbenzene series was selected to characterize the hydrophobic interaction associated with each phase. Thehomologous series has a carbon chain length which extends by n+1 through the series.

Propylbenzene Butylbenzene

0 4 8 12 16 MIN

1

2 34

5 6 7 8

BetaBasic 18

AQUASIL C18

BetaMax Acid

PRISM RP

BETASIL Phenyl-Hexyl

Fluophase PFP

Fluophase RP

BetaMax Base

HyPURITY ADVANCEAlkyben-X

Columns: 5m, 150x4.6mmEluent: 25% H2O / 75% ACN

Flow: 1.25 mL/minDetector: UV @ 254Sample: 1.Uracil

2. Benzene3. Ethylbenzene4. Propylbenzene5. Butylbenzene6. Pentylbenzene7. Hexylbenzene8. Heptylbenzene

Neutral Compound Comparison

The retention of alkylbenezenes by

dispersive interactions is influenced bythe carbon load of the bonded phaseHigh carbon load phases show greate

retention and columns with lower carbonload show less retention for neutra

analytes.

The hydrophobic reference column (theBetaBasic18 column) shows the long

est retention of heptylbenzene (14 minutes), whereas the BetaMax Acid and

HyPURITY ADVANCE columns show

the shortest retention (4 minutes). Although the HyPURITY ADVANCE andBetaMax Base columns show little reten-tion, the C8-chain component of the

HyPURITY ADVANCE bonded phaseallows it to give significantly better se-

lectivity than the non-hydrophobic cyanogroups present on BetaMax Base.

The Hypercarb column is not included

in this test, as under these conditions themore hydrophobic compounds do no

elute. Please refer to the Hypercarb

Technical Guide for method developmenguidelines using Hypercarb columns.

Figure 7

Column Characterization

To differentiate between various columns with polar character, and to help with column selection, we have characterizedthis series of Thermo Hypersil-Keystone phases using a series of sensitive analytical probes. These analytes demonstratdifferences in retention behavior towards a) hydrophobic neutral compounds, b) polar basic compounds and c) polar acidiccompounds, as shown in Figures 7-9.


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Test 2 - Basic/Polar CompoundsProcainamides are used to compare the selectivity of basic compounds, plus caffeine and phenol as additionmarkers of comparative performance.

0 4 8 12 16 MIN

12

34

5

6

BetaBasic 18

AQUASIL C18

BetaMax Acid

PRISM RP


Fluophase PFP

Fluophase RP

BetaMax Base

HyPURITY ADVANCE

procain5

Polar Basic Compound Comparison Polar embedded phases can show different elution profile when compareto traditional C18 columns. This is duto amide and urea groups in the bondephase that shield analytes from silanointeractions, and also to a repulsion frothe silica surface for basic compoundsThe variety and combination of interactions displayed by polar embeddegroup phases (dispersive, ionic, polacan also result in alternative selectivity

The degree of selectivity change seewith a polar embedded group will vardepending on the phase chemistrySelectivity remains similar to an alkC18 with the PRISM RP column; however, the elution profile alters significantwith the BetaMax Acid and HyPURITYADVANCE columns. Where the pola

character of the column is from hydroxgroups, such as with the AQUASIL C1column, retention is increased due tpolar and hydrophobic interactions.

Both Fluophase chemistries showincreased retention and selectivity fothe basic compounds tested. Thselectivity of the RP (fluorinated C6) anPFP (fluorinated phenyl) phases are diferent, due to the capability of the PFphase to induce pi-pi interactions witsolutes from the phenyl ring structure

In this case the Fluophase RP phasshowed ideal selectivity under thesconditions.

The Hypercarb column is not includein this test, as under these conditionthe compounds do not elute. Pleasrefer to the Hypercarb Technical Guidfor method development guidelineusing Hypercarb columns.

2

1

5

4 Reversed elution order

2

1,3 54

Reversed elution order

Columns: 5m, 150x4.6mm

(250x4.6mm - HyPURITY ADVANCE)Eluent: 90% 50mM KH2PO

4, pH 3.5 / 10% ACN

Flow: 1.25 mL/minDetector: UV @ 254

Sample:1 . Uraci l2. Procainamide3. N-Acetylprocainamide4. Caffeine

5. N-Propionylprocainamide6. Pheno l

Figure 8

Uracil Procainamide N-Acetylprocainamide Caffeine N-Propionylprocainamide Phenol


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Test 3 - Acidic/Polar Compounds

Compounds that are very polar show very low log P values. Such compounds prove difficult to retain on a traditional C18column, and commonly elute at the column void volume. In these circumstances a phase with polar character, such aspolar embedded group phases, are needed to gain sufficient retention. A phenolic test mix represents polar compoundswith decreasing log P values, and can be used to compare the polar embedded phases for selectivity and retention.

0 4 8 12 16 MIN

12

34

BetaBasic 18

AQUASIL C18

BetaMax Acid

PRISM RP


Fluophase PFP

Fluophase RP

BetaMax Base

HyPURITY ADVANCE

phenols-2

Polar Acidic Compound Comparison

Columns: 5m, 150x4.6mmEluent: 80% 0.1% Formic acid / 20% ACN

Flow: 1.0 mL/minDetector: UV @ 254Sample: 1.Uracil

2. Phloroglucinol3. Resorcinol4. Phenol

All columns except the Hypercarb

coumn showed the same elution ordeofPhloroglucinol < Resorcinol < Phenol

although there are significant selectivitydifferences between the phases.

Of the silica columns, the BetaMaxAcid

and HyPURITY ADVANCE columnsshow the greatest retention for phloro-

glucinol, the most polar compoundtested. The AQUASIL C18 column givesincreased retention of both moderately

polar and very polar solutes compareddirectly to a traditional alkyl C18 (the

BetaBasic18 column), due to increasedinteractions from the AQUASIL C18

columns polar endcapping.

The unique nature of the Hypercarbphase produces a polar retention effect

whereby analyte retention increases asthe polarity of the analyte increases. This

polar retention effect results in a dramatically different elution order, wherePhenol < Resorcinol < Phloroglucinol

Retention is also influenced by analyteshape, as the flat surface of the

Hypercarb phase allows stronger interactions, and hence stronger retention, fo

analytes that have a planar structure anda greater molecular surface area.

Hypercarb

43 2

Reversed elution order

Phenol log P = 1.47 Resorcinol log P = 0.81 Phloroglucinol log P = 0.14

Figure 9


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Figure 10

Neutral Compound Retention

Figure 12

Polar Acidic Compound Retention

Figure 11

Polar Basic Compound Retention

No elution of the test compound occured with Hypercarb under the conditions used

Summary of Comparative ColumnRetention

To aid in HPLC column selection, theresults of these chromatographic testshave been summarized in order ofdecreasing retention, for neutral, polaracidic and polar basic compounds(Figures 10-12).

The BetaBasic 18 column is a typical

type B silica, traditionally endcappedC18 phase, showing strong retention forhydrophobic compounds and lessretention for polar compounds. OtherThermo Hypersil-Keystone phases withsimilar performance are the Hypersil

BDS C18, HyPURITY C18, BETASIL

C18, BioBasic 18 and BetaMax Neu-tral columns.

The AQUASIL C18 column demonstratesexcellent retention for acidic, basic, andneutral compounds in comparison to

the other polar phases, making it anideal choice for a wide range of sampletypes. The AQUASIL C18 column alsoshows alternative selectivity for polarcompounds as a result of its polar end-capping. This polar character alsoallows the AQUASIL C18 column toequilibrate rapidly in 0 to 100 percentorganic gradients.

Fluorinated phases are a good choicefor halogenated samples, or whereshape selectivity will assist resolution.The Fluophase PFP column shows

stronger retention than many of theother columns studied for polar basiccompounds, and often gives a differentpeak elution profile than other phases.

The HyPURITY ADVANCE, BetaMaxAcid and PRISM RP phases all employpolar embedded groups to achieveunique sample-phase interactions. Ingeneral, these columns exhibit reducedretention for bases combined withincreased retention for the most polaracidic compounds, and give the most

apparent changes in selectivity.

References1. J. J. Kirkland, Practical HPLC Method

Devleopment, 178-182

For Trademark information, please refer to page 6of the 2002 Thermo Hypersil-Keystone ColumnTechnologies for HPLC and LC-MS catalog.

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