hplc columns for the retention of very polar · pdf filehplc columns for the retention of very...

By Paul Ross

HPLC Columns for the Retention of Very Polar Molecules

Retention of Basic Analytes on C18, PRISM and HyPURITY ADVANCE

BetaBasic® 18Part No.: 71505-154630

HyPURITY ADVANCEPart No.: 21005-154630

Figure 1.

In this technical bulletin, we review columns made with several packings that have beentailored to go beyond the limitations of the traditional C18 packing materials. The columnsthat we review are listed as follows and are discussed individually throughout the bulletin:

! AQUASIL C18 - Wettable reversed phase ! PRISM® - Imbedded polar group phase

! HyPURITY®ADVANCE - Imbedded ! Fluofix® and Fluophase

® - Perfluorinated

polar group phase phases

! Hypercarb® – Porous Graphitic Carbon

Without exception, the phases outlined above achieve quite different chromatographicbehavior to the more traditional type C18 packings in that they provide:

! Alternative selectivity ! The ability to run in highly aqueousmobile phase

! Increased retention of polar compounds ! The ability to be used at reduced bufferconcentrations

To achieve these chromatographic char-acteristics a certain degree of polar char-acter is incorporated into the phasechemistry of the packing. Interactionsthat take place between the analyte andthe stationary phase are then a mixtureof dispersive interactions (as for tradi-tional type C18 packings) and also dipole/hydrogen bonding interactions. The ex-ception is porous graphite Hypercarb col-umns, where a quite unique mechanismof retention provides a surface that canretain both polar and non-polar analytes.

Figure 1 gives an indication of how twoof the phases, the PRISM RPN columnand the HyPURITY ADVANCE column,can give rise to alternative selectivity forthe more polar basic compounds in a testmixture compared to C18. These phasesare discussed in greater detail later in thebulletin.

0 2 4 6 8 MIN 0 2 4 6 8 10 MIN 0 2 4 MIN

715-169

1

2

3 4 5

1

2

3

5

4

320-012 HyPAdv-3

1

23

5

4

PRISM RPNPart No.: 32005-154630

Dimensions: 5µm, 150x4.6mmEluent: 10% ACN / 90% 0.05M KH2PO4, pH 3.5

Flow: 1.25 mL/minDetector: UV @ 254

Sample:1. Procainamide2. Uracil3. n-Acetylprocainamide

TB0008IN

4. Caffeine5. n-Propionylprocainamide

INTRODUCTION

The requirement to retain and analyze polarmolecules by HPLC is one that has grownsteadily over the last few years, and has beenthe driving force behind the generation of arange of new stationary phases dedicated tothis purpose. The new packings offer an al-ternative mechanism of interaction that takesplace in addition to dispersive interactionsgenerally associated with the more traditionalkyl silane type packings. Such interactionsallow for increased retention and improvedpeak shape of analytes with polar function-ality whether basic or acid in nature. Suchpackings are not only targeted at the sepa-ration of polar analytes but often encompassthe application range of the traditional alkylC18 packings also.

1/2002

SelectivityPack ings that o f fe r add i t iona lmodes of interaction give rise toquite different retention behaviorand selectivity. In general analyteswith the greatest polar functional-i ty wi l l typical ly show greatestchanges in selectivity and retention.

Highly Aqueous Mobile Phases– Increased RetentionThe inclusion of polar functionalityto the stationary phase also in-creases the wetting characteristicsof the packing in highly aqueousmobile phases (100% aq). Severalof the packings outlined in this bul-letin can be run in 100% aqueousmobile phase conditions and showno tendency towards phase col-lapse or folding. Phase collapse isoften seen for alkyl C18 packingswhere a small amount of organicsolvent (1-5%) is generally requiredin the mobile phase to help wet theC18 surface and prevent phase col-lapse. See separate technical bul-letin #TB99-01 for further explana-tion of phase collapse/folding.

The requirement to analyze com-pounds that contain increasing po-lar functionality is common to thepharmaceutical, environmental andfood industries. This trend is drivenby the need to identify not only drugcompounds, pesticides or foodsubstances but also the impuritiesand derivatives of the parent com-pounds that may be present. Oftenthis requires investigating the effectthese compounds have on thehuman body, and therefore mayrequire detecting and measuringextremely smal l quantit ies of acompound in complex matrices.Alternatively, water samples may betaken from the environment whereonly trace levels of a pesticide maybe found in our rivers and oceans.In both cases complex break downproducts in the environment orglycosilation of the compound inthe body can lead to more com-plex and difficult HPLC analysisthan first might have been envis-aged for a new chemical.Further to this, the coupling of MassSpectrometry to an HPLC Systemhas become common place in manylaboratories. For specif icity thecompounds are analyzed in theirionic state. In this form moleculesare at their most polar and we seeagain the drive towards analyzingincreasingly polar molecules byHPLC.

The bulletin outlines the propertiesfor each of the phases listed abovethat make them quite different totraditional C18 packings. Particularemphasis is placed on the follow-ing characteristics:

Reduced Buffer Concentra-tions – Increased MS SensitivitySeveral of the phases outlined inth is repor t a re a lso shown tomaintain chromatographic perfor-mance when very low concentra-tions of buffers are used. Low bufferconcentrations offer the reward ofincreased sensitivity for MS. This isan important consideration whentrying to identify trace quantities ofa drug compound or impurity thatmay normally disappear into thenoise of the base line of the MSresponse.

Further InformationA brief review of typical questionsthat are often raised concerningthese stationary phases is given inthe fo l lowing pages. For morein-depth in format ion on eachproduct please request theindividual Product Bulletins (ashighlighted for each product) fromThermo Hypersil-Keystone TechnicalSupport.

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AQUASIL C18 Columns

How does retention of polar analytescompare with that of tradition C18 alkylbonded phase?

The AQUASIL C18 phase was designedfor the reversed phase separation of polarmolecules. It has a high concentration ofC18 groups as well as hydrophilic sitesthat help to provide retention of highlypolar water soluble compounds (Figure 2),but offers the added benefit of nearlytwice the retention of polar compoundswhen compared to BetaBasic® 18.

AQUASIL C18 retention is comparable toa traditional C18 when run in mobile phasewith high organic.

Sample:1. Uracil2. Procainamide3. n-acetylprocainamide4. Caffeine5. n-propionylprocainamide

715-036

1

BetaBasic 18, 5µm, 150x4.6mmPart No.: 71505-154630

Eluent: 10% ACN / 90% 0.05MKH2PO4, pH 3.5


AQUASIL C18, 5µm, 150x4.6mmPart No.: 77505-154630

Eluent: 10% ACN / 90% 0.05MKH2PO4, pH 3.5


0 2 4 6 8 10 12 14 16 MIN0 2 4 6 8 MIN

2

3

4

5 1

2

3

4

5

775-016

The AQUASIL C18column is twice as retentive

AQUASIL C18with polarfunctionality

Can I use the AQUASIL C18 in 100%aqueous mobile phase?

Yes! The extra polar character associatedwith AQUASIL C18 allows the use ofmobile phases without any organiccomponent, i.e. 100% aqueous (Figure3).Traditional C18 packings with highcarbon loads require at least 3 to 5%organic component in the mobile phaseto prevent phase collapse (folding).During this process, the retention andselectivity of the phase is lost and thecolumn must be regenerated using a pureorganic solvent wash. AQUASIL C18 isimmune to this folding due to its uniquepolar functionality.

Can I run low pH applications withreduced buffer concentration?

When used in high concentrations,buffers (e.g. trifluoroacetic acid) can causeMS ion source supression and conse-quently can reduce sensitivity. The choiceof column used is of key importance forLC/MS applications since the quality of theC18 packing and underlying silica canstrongly influence the concentration ofbuffer required. In this example, we showthat buffer concentration (TFA) can bereduced to zero concentration without lossin performance when using AQUASIL C18(Figure 4). It is generally good practice tobuffer your mobile phase, but with theAQUASIL C18 column, very low concen-trations can be used.

0 2 4 6 MIN

AQUASIL C18, 5µm, 150x4.6mmPart No.: 77505-154630

Eluent: 0.05M KH2PO4

+0.03M H3PO4


Sample:1. Tartaric Acid2. Malonic Acid3. Fumaric Acid4. Succinic Acid

775-009

1

Immune to Folding

AQUASIL C18, 5µm, 150x4.6mmPart No.: 77505-154630Gradient: A: TFA in H2O

B: TFA in ACN10% B to 50% in 20 min, linear to10% B at 21 min, hold to 30 min


Sample: Tryptic Digest of β-Lactoglobulin

775-068

Run withReduced Buffer Concentration

Retains Polar Samples

0 2 4 6 MIN

Initial Run After 113 Hoursof Flushing with Mobile Phase

No Phase Collapse in 100% aqueous buffer

2

3

4

1

2

3

4

775-008

0 10 MIN

775-069

0 10 MIN

0 10 MIN

775-070

0.1% TFA

0.01% TFA

0.00% TFA

Figure 2.

Figure 3. Figure 4.

For more information, please request Product Bulletin 99-03

3

Sample:1. Angiotensin III2. Angiotensin II3. Angiotensin I

1,2

PRISM RP, 5µm, 150x4.6mmPart No.: 32105-154630Gradient: A: TFA in H2O

B: TFA in ACN10% B to 50% in 20 min


0 5 10 15 MIN

3

321-053

Effect of TFA Concentration on Resolution

0 4 8 12 MIN

Dimensions: 5µm, 150x4.6mmPart No.: 32105-154630

Eluent: 25% ACN / 75% 0.05MFlow: 1.25 mL/min

Detector: UV @ 254

Sample:1. Doxepin2. Nortriptyline

Antidepressants

Dimensions: 5µm, 150x4.6mmPart No.: 32005-154630

Eluent: 0.05M KH2PO4

+0.03M H3PO4


psmrpn05

PRISM RPN Phase Immune to Folding

PRISM RP column PRISM RPN column

2

1

2

1

Doxnor01D

0 2 4 6 MIN

0 4 8 12 MINDoxnor10D

0 2 4 6 MIN

psmrpn06

1

2

3

4

1

2

3

4

PRISM RPN(not end-capped)

Sample:1. Tartaric Acid2. Malonic Acid3. Succinic Acid4. Fumaric Acid

After storagein 100% aqueous

mobile phase overnight

321-051

0 5 10 15 MIN 0 5 10 15 MIN

1

2

3

321-052

1

2

3

0.1% TFA 0.05% TFA 0.01% TFA

Figure 5. Figure 6.

Figure 7.

For more information, please request Product Bulletin PB01-18.

PRISM® RP and PRISM RPN Columns

Are PRISM columns available in bothendcapped and non-endcapped versions?

Yes. Both columns offer alternative selectivityand excellent peak shape for basic compounds.The non-endcapped PRISM RPN phase givesdifferent selectivity for moderately polarand basic analytes such as tricyclicantidepressants (Figure 5).

The endcapped PRISM RP phase gives slightlydifferent selectivity and offers improved peakshape for acidic analytes in particular.

Can the PRISM RP and PRISM RPNcolumns be run in 100% aqueousmobile phase?

Yes. In order to maximize retention of many verypolar compounds, it is usual practice to reducethe percentage of the organic component in themobile phase. Packings, such as PRISM RPand PRISM RPN, which contain imbedded po-lar groups near the surface of the silica, allowthe organic component of the mobile phase tobe reduced to zero, therefore maximizing thepossibility of retention of highly solvated polarmolecules (Figure 6).

How does trifluoracetic acid (TFA)concentration affect resolution?

Figure 7 shows the separation of three simpleAngiotensin peptides. The resolution is shownto improve significantly for peaks 1 & 2 as theconcentration of TFA decreases to 0.01%.

Increasing the TFA concentration is generallythought to increase the hydrophobic propertiesof basic analytes when in ionic state. It doesthis by displacement of water molecules withTFA counterions. An increase in retention is ob-served as the concentration of TFA is increasedbut also a loss of resolution. Even at low con-centrations of TFA, the polar PRISM packing caninteract with the analyte to provide increasedresolution. Changes in pH of the different con-centrations can also affect resolution.

PRISM RP & PRISM RPNPolar imbedded groupswith C12 chemistry

PRISM was developed to offer alternativeselectivity to traditional alkyl C8 or C18packings. It has unique chemistry that in-volves the incorporation of polar functionalgroups near the silica surface. These polarfunctional groups also form part of the alkylchain that is responsible for the primarymode of interaction between the stationaryphase and the analyte. Imbedded polargroups allow for a secondary or mixedmode type of interaction to take place,which in turn leads to a quite different re-tention behavior for polar analytes.

4

HyPURITY ADVANCERetention Mechanisms

HyPURITY ADVANCE was developed to offeralternative selectivity to the traditional alkyl C8or C18 packings. It has unique chemistry thatinvolves the incorporation of polar functionalgroups (different chemistry than PRISM® phases)near the silica surface. An alkyl C8 group is re-sponsible for the primary mode of interactionbetween the stationary phase and the analyte.The imbedded polar groups allow a secondaryor mixed mode type of interaction to take place,which leads to quite a different retention behav-ior and improved peak shape for many of themore polar analytes.

Figure 8.

HyPURITY ADVANCE, 5µm, 250x4.6mmPart No.: 21005-254630

Eluent: 100% 20mM K2HPO4, pH 7.5Flow: 1.0 mL/min

Detector: UV @ 270Temp: 25°C

HyPAdv-2

Catecholamines in100% Aqueous Mobile Phase

0 2 4 6 8 10 MIN

1

2

3

Sample:1. L-DOPA2. Adrenaline3. Dopamine

HyPURITY ADVANCE PhasePolar embeddedgroup withC8 chemistry

Should I expect increased or decreasedretention of my polar analytes?

Basic compounds generally show reduced re-tention on HyPURITY ADVANCE columns, es-pecially at pH 2-5 where the imbedded polargroup carries a positive charge and can repelapproaching similarly charged groups (Figure 8).

Acidic compounds conversely are retainedslightly longer. Both effects contribute to quitedifferent retention behavior compared totraditional C18 silicas with the added advan-tage that excellent peak shapes are obtainedfor acidic, basic and neutral compounds.Where ionization of the analyte has beensuppressed, interaction takes place viadispersive interactions with the C8 group, andalso by dipole-dipole interactions betweenpolar groups on the stationary phase and thepolar groups on the analyte. This gives riseonce again to alternative selectivity (Figure 9).

Can I run HyPURITY ADVANCE columnsin 100% aqueous mobile phase?

Yes. The HyPURITY ADVANCE phase shows acomplete absence of folding or phasecollapse when run in 100% aqueous conditions.

Figure 10 shows an example where theHyPURITY ADVANCE column has been usedto analyze several polar catecholamines. Themobile phase consists of 20mM phosphatebuffer, pH 7.5. The analysis is run in completeabsence of any organic modifier.

Are there other applications availablethat have been run on the HyPURITYADVANCE column?

Yes. Please request the HyPURITYAppl icat ions Booklet and also theHyPURITY ADVANCE Product Guide forfurther information on select iv i ty,stabi l i ty, compatibi l i ty with MS andspeed of analysis.

HyPAdv-1

Alternative Selectivity

1

23

HyPC18-1

1

2

3

0 5 10 15 20 MIN

4

0 5 10 15 MIN

4

HyPURITY C18 ColumnPart No.: 22105-154630

HyPURITY ADVANCE ColumnPart No.: 21005-154630

Dimensions: 150x4.6mmEluent: 72% Heptanesulfonic acid / 28% MeOH

Detector: UV @ 254

Sample:1. Sulfathiazole

2. Sulfanethiazole

3. Sulfadimethoxine4. Sulfaquinoxaline

Figure 10.

Figure 9.

HyPURITY® ADVANCE Columns

For more information, please requestthe HyPURITY Applications Booklet and HyPURITY Product Guide.

5

Fluofix and FluophasePerfluorinated Phases

Fluofix 120E, 150x4.6mmPart No.: 21131

Eluent: 0.02M KH2PO4, pH 3Flow: 1.0 mL/min

Detector: UV @ 210

11131-19

Resistant to Folding

0 2 4 6 MIN

1

2 3

Sample:1. Tartaric Acid2. Malonic Acid3. Fumaric Acid4. Succinic Acid

Fluorinated stationary phases offer a newapproach to stationary phase selectivity.Fluor ine atoms with their highlyelectronegative character offer alternativemolecular interactions by which polaranalyte retention can take place. Threedifferent packings are available; seeFigure 11 to compare selectivity.

Does the fluorine chemistry give rise toalternative chromatographic selectivity?

The fluorine chemistry often behaves ina similar manner to a traditional alkylbonded packing. Where an analyte hassome polar functionality, fluorinatedpackings can often give quite differentselectivity. This is particularly the case forfluorinated or chlorinated compounds.Fluofix and Fluophase columns have alsobeen shown to give excellent results onnon-halogenated compounds such as lip-ids, surfactants, taxanes, catechins andmany other polar compounds with car-boxyl or nitro groups.

Figure 11 shows how improved resolu-tion can be obtained using Fluophase RPand Fluofix when compared to analysisof the same compounds using BetaBasic18. Similar reports have been observedfor isomers or compounds closely relatedin structure.

Can I use 100% aqueous conditionswith fluorinated phases?

Resistance to folding is observed for boththe Fluofix and Fluophase RP. FluophasePFP has shown some tendency to foldunder 100% aqueous conditions and forthis reason it is recommended that atleast 5% of the mobile phase composi-tion should contain organic solvent(Figure 12).

What different fluorinated phases areavailable?

Fluofix 120E is a fluorinated branched-chain hexyl phase on 120Å si l ica.Fluophase RP and Fluophase WP arefluorinated straight-chain hexyl phaseson 100Å and 300Å silica, respectively.Fluophase PFP is a pentafluorylphenylphase on 100Å silica.

Improved Selectivity of Fluorinated Compounds

1

2

3

715-143

1

2

0 2 4 6 MIN

4

BetaBasic®18 ColumnPart No.: 71505-154630

Fluofix ®120E ColumnPart No.: 21131

Dimensions: 5µm, 150x4.6mmEluent: 60% ACN / 40% H2O


Sample:1. Uracil2. 1,3 Difluorobenzene

0 2 4 6 MIN

850-003

1

2

3

4

0 2 4 6 MIN

825-003

3

4

Fluophase® RP ColumnPart No.: 82505-154630

4

Figure 11.

Figure 12.

Fluorinated Phase Columns

3. 1,3,5 Trifluorobenzene4. 1,2,4,5 Tetrafluorobenzene

For more information, please request Product Bulletin PB01-11.

6

Hypercarb – Porous GraphiticCarbon ColumnsPorous graphitic carbon has unique prop-erties as a stationary phase and now oftenprovides solutions to what might be con-sidered ‘problem HPLC separations’. Twosuch problem areas are:

(1) the retention and separation of verypolar analytes not normally retainedon C18 packings.

(2) the separation of structurally similarcompounds, such as geometricisomers and diastereomers , notalways separated on C18 silica.

HypDraw-2

Polyethoxylated Alcohols and Phenols

0 10 20 30 40 MIN

Polar Retention Effect on Hypercarb®

Compounds of increasing polarity areretained more strongly on Hypercarb

The unique mechanism by which analytes areretained at the graphite surface gives rise to manynew and attractive opportunities for the reten-tion of polar molecules.

Figure 13 shows how retention behaviorchanges for phenol and some polyhydroxyben-zenes versus percent organic modifier in themobile phase. Note how the retention order dif-fers for both Hypercarb and Hamilton PRP:

Hypercarb: 1,3,5 trihydroxybenzene >1,2dihydroxybenzene > phenol.

PRP-1: Phenol>1,2 dihydroxybenzene >1,3,5 trihydroxybenzene

(Note: under these conditions the twopolyhydroxylbenzene analytes are typically not re-tained on C18 packings.)

Figure 14 shows retention of several verypolar pharmaceutical compounds not nor-mally retained on alkyl C18 silica.

What other benefits does Hypercarboffer over traditional alkyl bonded silicapackings?

! Enhanced selectivity for closely relatedcompounds

! Retain highly polar compounds verystrongly

! Stable across the pH range 1-14

! Can be used with a wide range of solventsfrom 100% aqueous to 100% hexane ormethylene chloride

The flexibility of solvent choice is demonstratedin Figure 15 where polyethoxylated alcohols andphenols are separated on a single Hypercarbcolumn. It is common practice to have to usetwo columns for this analysis – a C18 for thereverse phase separation of the more polaranalytes and a normal phase column for the veryhydrophobic analytes.

Hamilton® PRP-1 Polymeric Column Hypercarb Column

Courtesy of V.Cocquart & M.C. Hennion, J.Chrom, 1992

Figure 14.

Figure 13.

Hypercarb, 5µm, 100x4.6mmPart No.: 35005-104630Gradient: A: H2O B: ACN C: Dichloromethane

From 20% A - 100% B during 15 min then 0% C to80% C in 25 min


Courtesy P.Chambault et al. J.Chromatogr A 797 (1998)

Non-Ionic Surfactants

Figure 15.

Polar Pharmaceutical Compounds

Sample: Morphine and Metabolites1. Normorphine2. Morphine-3-glucuronide3. Morphine-6-glucuronide4. Morphine5. Codeine

0 10 20 30 MIN

HypCarb-2

12

3

4

5

Hypercarb, 5µm, 100x4.6mmPart No.: 35005-104630

Eluent: 60% MeOH / 40% Ammonium Acetate, pH 9Flow: 1.0 mL/min

Detector: UV @ 220Courtesy of Wan, Shaw, Davies and Barrett, Nottingham Univ.

Hypercarb porousgraphitic carbon

For more information, please request the Hypercarb Product Guide.

BetaBasic, Fluophase, Hypercarb, HyPURITY and PRISM are registered Trademarksof Thermo Hypersil-Keystone.©2002 Thermo Hypersil-Keystone. All Rights Reserved.

AquaSil™Siliconizing Fluid for treating glass surfaces is sold by Pierce Chemcial Co., Rockford, IL.Fluofix is a registered Trademark of Neos Corp.Hamilton is a registered Trademark of Hamilton Co.

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