CSG 01-01

Using Stationary Phase Selectivity to Assist Method Development

Introduction Selecting an HPLC column presents a wide range of choices, as Thermo Hypersil-Keystone offers a variety of phases and columns, with a range of silica specifications and varying phase coverage, all of which influence selectivity. The column characteristics that define performance are also influenced by physical parameters such as pore size, surface area, carbon load, and particle size. Pore size Thermo Hypersil-Keystone silica-based columns vary from 60 to 300 in pore size. The strength of the interactions between the analyte and bonded phase is influenced by the availability of the bonded phase to the analyte. This in turn is controlled by pore size, as over 90% of the silica surface area occurs within the pores. When the silica pore diameter is smaller than the diameter of the analyte, the analyte is excluded from the pore and interactions are minimized, which leads to shorter retention and lower loading capacity. Large molecules (MW>2000 Da) show limited penetration of small pores (<100). A pore size of 60 may limit the penetration of solutes having molecular weights as low as 300 Da. Surface area Thermo Hypersil-Keystone silica-based columns vary from 100 to 540 m2/gm in surface area. Surface area dictates bonded phase load, and therefore high surface area columns have high phase loading and typically give higher retention. When analytes are only moderately hydrophobic, selection of a column with high surface area (>300 m2/gm) will increase the likelihood of the analyte being retained beyond the column void volume. Carbon load The degree of retention of a neutral hydrophobic analyte on a wholly alkyl phase such as C18 or C8 can be inferred from the carbon load value, as this indicates the degree of bonded phase coverage. A theoretical maximum for phase loading occurs at 50 - 60% of the silica surface area. As the silica pore size increases, surface area decreases, and therefore the capacity for bonded phase loading decreases. Hence, a 100 pore size silica typically shows higher carbon loading for a C18 phase than a 300 pore size silica. When analytes are only moderately hydrophobic, selection of a column with high carbon load (>10%) will increase the likelihood of the analyte being retained beyond the column void volume. High carbon load hydrophobic phases (e.g. BetaMax Neutral) show the greatest degree of hydrophobic interaction, and therefore the most retention. Particle size 3m and 5m particle sizes are typically used for analytical separations, with larger particle sizes used for preparative separations. Reducing the particle size to 3m increases column efficiency by approximately 30%, but also increases column backpressure. For this reason, columns longer than 150mm are not recommended when 3m particles are used.

2/2002

Selecting a bonded phase The most popular bonded phase in reversed-phase chromatography is C18, as it offers good retention for a wide range of compounds. C18 bonded phases show a high degree of retention for analytes containing hydrocarbon groups, with hydrophobic (dispersive) interactions being the primary attractive force. Neutral compounds are retained primarily by dispersive interactions with the C18 bonded phase. Figure 1

In reversed phase chromatography, water is a weak solvent, having limited elution capability. Most mobile phases therefore contain a proportion of a stronger organic solvent (most typically acetonitrile or methanol). The ratio of aqueous/organic components is then used to either increase or decrease the retention of any given analyte. Typically a lower organic solvent percentage is used to promote retention of the more polar analytes, whereas an increased proportion of the organic component is used to reduce retention of the more hydrophobic analytes. Secondary interactions (including ionic, polar and chelating interactions) can occur between the analyte and bonded phase when polar functional groups are present in either. The base silica also plays an important role, as significant differences in selectivity result from varying degrees of silica purity and base deactivation. Columns and phases with polar and ionic selectivity are discussed later in this Guide. Figure 1 shows a hydrophobicity chart, comparing a selection of Thermo Hypersil-Keystone and other columns according to their retention of phenylheptane, a neutral hydrophobic compound. To identify a column giving greater hydrophobic retention, select from higher up the chart. Conversely, to select a column with less hydrophobic retention, choose from lower down the chart. Bonded phases that have a variety of interaction mechanisms (such as ionic or polar interactions) show less hydrophobic retention, but may provide alternative selectivity, especially for polar analytes. 2

Type A versus Type B silica In 1986, Kirkland and others1 popularized the term Type B to describe a new generation of highly purified, deactivated silicas with improved performance for chromatography of basic compounds. Type B silica has replaced older Type A silica as the packing of choice in new LC methods. However, Type A silica continues to be used in many established methods. Type A silica has a high concentration of metal impurities, resulting in a heterogeneous, acidic surface. This surface leads to non-uniform bonded phase coverage and active sites that interact strongly with sample components, causing poor peak shapes. Type A silica can undergo a base deactivation process to remove many of the metal impurities, generating a more homogeneous surface and essentially Type B silica performance. True Type B silica is synthesized from metal-free reagents to ensure a very low total metal content and offer even greater performance advantages. Type A and Type B silica can be compared as shown in Table 1. Thermo Hypersil-Keystone columns can be categorized according to their silica type (Table 2).

Table 1
TYPE A SILICA ADVANTAGES Less expensive DISADVANTAGES 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 ADVANTAGES Lower surface activity Good solute recovery at low levels Good peak shapes for basic compounds Simple mobile phases can be used More uniform dense bonding for better lot reproducibility DISADVANTAGES More costly Can be less rugged at high pH

Table 2
Silica Category Type A silica Base deactivated Type A silica Type B silica Products All classical Hypersil columns Hypersil BDS columns AQUASIL C18, BetaMax, BETASIL, BetaBasic, BioBasic, Fluophase, HyPURITY, PRISM columns

High Purity Silica Ultra pure silica provides exceptional performance under demanding HPLC conditions. HyPURITY silica contains exceptionally low metal content (40 ppb total levels), and HyPURITY phases show superior peak shapes for basic and chelating compounds (Figure 2). BetaBasic phases, based on a Type B high purity silica, have particularly dense and uniform bonding chemistry. This makes BetaBasic columns ideal for general purpose chromatography, with exceptional peak shapes for basic compounds. The BetaBasic 18 phase has also demonstrated exceptional stability and performance at both high and low pH extremes (Figure 3). As shown in Figure 4, the surface of Type B silica is uniform, with almost ideal bonding characteristics. Type B silica shows only weakly acidic properties compared to older Type A silica. Type B silica minimizes the interactions with bases that can cause poor peak shape. The advantages offered by the newer silicas over Type A silica are illustrated by a comparison of a Hypersil ODS column (based on a Type A silica) against a Hypersil BDS C18 column (made from a base-deactivated silica), as shown in Figure 5.

Figure 2
Acids and Chelators at pH 1.5
HyPURITY Eluent: Flow: Detector: C18, 5m, 150x4.6mm 70% 0.1M H3PO4 / 30 % ACN 1.0 mL/min UV @ 215

Figure 3
Drugs at pH 12
BetaBasic 18, 5m, 150x4.6mm Eluent: 60% MeOH / 37% 10mM Sodium Borate, pH 12 Flow: 1.0 mL/min Detector: UV @ 280 Data courtesy of Kristine Phillips, Dept. of Chemistry, Univ of New Hampshire 1 Sample: 1. Capsaicin 2. Impurity

4 Sample: 1. Uracil 2. Benzyl Alcohol 3. 2,7-Dihydroxynaphthalene 4. 4-Nitrobenzoic Acid 5. 2,3-Dihydroxynaphthalene 6. Chlorocinnamic Acid 3

2 5

2 6 H221-10003 715-084 0 5 10 15 MIN

10

20

MIN

Figure 4

Figure 5
Hypersil BDS / ODS Comparison
1 Columns: Eluent: Flow: Detector: Sample: 5m, 150x4.6mm 60% ACN / 40% 0.05M KH2PO4, pH 4.5 1.0 mL/min UV @ 254 1. Pyridine 2. N-Methylaniline 3. N,N-Dimethylaniline 4. Toulene

Hypersil ODS
3

Hypersil BDS

1 H301-281 Comp

MIN

MIN

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 is important to further categorize polar compounds as either acidic or basic, as different interactions can occur between the stationary phase and either an acidic polar compound or a basic polar compound. The behavior of neutral, polar acidic and polar basic compounds with a variety of stationary phases has been explored to simplify the selection of bonded phases and to reduce method development time. Mass spectrometry is increasing in popularity as an HPLC detection method, which brings limitations to the HPLC method development process. Examples include a restricted choice of mobile phase composition, and the use of volatile buffers or additives at minimum concentrations to maximize sensitivity and minimize instrument down time. The use of column switching techniques is becoming widely adopted, typically with a generic gradient to simplify analysis. This approach also requires a wide range of column functionalities to gain significant selectivity differences. Thus, a stationary phase with alternative 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 be required. Bonded phases that employ ionic and polar interactions provide unique selectivity options to achieve retention and separation of polar compounds. Several approaches to bonded phase development have been used by Thermo Hypersil-Keystone to increase retention of polar molecules, including: 1) Embedding polar groups into the alkyl chain to create a more 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 impart polarity (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 highly polarizable surface (Hypercarb packing). These stationary phases are compared in Table 3. Figure 6

Table 3
Thermo Hypersil-Keystone Column Comparison
Column AQUASIL C18 BetaBasic 18 * BetaMax Acid BetaMax Base BETASIL Phenyl/Hexyl Fluophase PFP Fluophase RP Hypercarb HyPURITY ADVANCE PRISM RP Bonded Phase Chemistry Polar end-capped C18 Traditional C18 Polar embedded C12 Cyano Phenyl & C6 Perfluorinated phenyl Perfluorinated C6 100% porous graphitic carbon Polar embedded C8 Polar embedded C12 % Carbon 12 13 15 9 7 12 11 100 7 12 Surface area (m2/gm) 300 100 540 540 350 330 330 120 190 330 Pore size () 100 150 60 60 100 100 100 250 180 100

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

Column Characterization To differentiate between various columns with polar character, and to help with column selection, we have characterized this series of Thermo Hypersil-Keystone phases using a series of sensitive analytical probes. These analytes demonstrate differences in retention behavior towards a) hydrophobic neutral compounds, b) polar basic compounds and c) polar acidic compounds, as shown in Figures 7-9. Test 1 - Neutral Compounds The n-alkylbenzene series was selected to characterize the hydrophobic interaction associated with each phase. The homologous series has a carbon chain length which extends by n+1 through the series.

Figure 7
Neutral Compound Comparison
Propylbenzene Butylbenzene

3 4 5 6 7 8 BetaBasic 18

AQUASIL C18

BetaMax Acid

Columns: Eluent: Flow: Detector: Sample:

PRISM RP

5m, 150x4.6mm 25% H2O / 75% ACN 1.25 mL/min UV @ 254 1. Uracil 2. Benzene 3. Ethylbenzene 4. Propylbenzene 5. Butylbenzene 6. Pentylbenzene 7. Hexylbenzene 8. Heptylbenzene

The retention of alkylbenezenes by dispersive interactions is influenced by the carbon load of the bonded phase. High carbon load phases show greater retention and columns with lower carbon load show less retention for neutral analytes. The hydrophobic reference column (the BetaBasic 18 column) shows the longest retention of heptylbenzene (14 minutes), whereas the BetaMax Acid and HyPURITY ADVANCE columns show the shortest retention (4 minutes). Although the HyPURITY ADVANCE and BetaMax Base columns show little retention, the C8-chain component of the HyPURITY ADVANCE bonded phase allows it to give significantly better selectivity than the non-hydrophobic cyano groups present on BetaMax Base. The Hypercarb column is not included in this test, as under these conditions the more hydrophobic compounds do not elute. Please refer to the Hypercarb Technical Guide for method development guidelines using Hypercarb columns.

BETASIL Phenyl-Hexyl

Fluophase PFP

Fluophase RP

BetaMax Base

HyPURITY ADVANCE
Alkyben-X 0 4 8 12 16 MIN

Test 2 - Basic/Polar Compounds Procainamides are used to compare the selectivity of basic compounds, plus caffeine and phenol as additional markers of comparative performance.
Uracil Procainamide N-Acetylprocainamide Caffeine N-Propionylprocainamide Phenol

Figure 8
Polar Basic Compound Comparison
2 1 3 4 6 BetaBasic 18 5

AQUASIL C18 2 1 5 4 Reversed elution order BetaMax Acid

Polar embedded phases can show a different elution profile when compared to traditional C18 columns. This is due to amide and urea groups in the bonded phase that shield analytes from silanol interactions, and also to a repulsion from the silica surface for basic compounds. The variety and combination of interactions displayed by polar embedded group phases (dispersive, ionic, polar) can also result in alternative selectivity. The degree of selectivity change seen with a polar embedded group will vary depending on the phase chemistry. Selectivity remains similar to an alkyl C18 with the PRISM RP column; however, the elution profile alters significantly with the BetaMax Acid and HyPURITY ADVANCE columns. Where the polar character of the column is from hydroxyl groups, such as with the AQUASIL C18 column, retention is increased due to polar and hydrophobic interactions. Both Fluophase chemistries show increased retention and selectivity for the basic compounds tested. The selectivity of the RP (fluorinated C6) and PFP (fluorinated phenyl) phases are different, due to the capability of the PFP phase to induce pi-pi interactions with solutes from the phenyl ring structure. In this case the Fluophase RP phase showed ideal selectivity under these conditions. The Hypercarb column is not included in this test, as under these conditions the compounds do not elute. Please refer to the Hypercarb Technical Guide for method development guidelines using Hypercarb columns. 7

PRISM RP

Sample: 1. 2. 3. 4. 5. 6.

Uracil Procainamide N-Acetylprocainamide Caffeine N-Propionylprocainamide Phenol

BETASIL Phenyl-Hexyl

Fluophase PFP

Fluophase RP

Columns: 5m, 150x4.6mm (250x4.6mm - HyPURITY ADVANCE) Eluent: 90% 50mM KH2PO4, pH 3.5 / 10% ACN Flow: 1.25 mL/min Detector: UV @ 254

BetaMax Base 1,3 2 5 4 Reversed elution order HyPURITY ADVANCE

procain5 0 4 8 12 16 MIN

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 C18 column, and commonly elute at the column void volume. In these circumstances a phase with polar character, such as polar embedded group phases, are needed to gain sufficient retention. A phenolic test mix represents polar compounds with decreasing log P values, and can be used to compare the polar embedded phases for selectivity and retention.
Phenol log P = 1.47 Resorcinol log P = 0.81 Phloroglucinol log P = 0.14

Figure 9
Polar Acidic Compound Comparison
Columns: Eluent: Flow: Detector: Sample: 5m, 150x4.6mm 80% 0.1% Formic acid / 20% ACN 1.0 mL/min UV @ 254 1. Uracil 2. Phloroglucinol 3. Resorcinol 4. Phenol

All columns except the Hypercarb coumn showed the same elution order ofPhloroglucinol < Resorcinol < Phenol, although there are significant selectivity differences between the phases. Of the silica columns, the BetaMax Acid and HyPURITY ADVANCE columns show the greatest retention for phloroglucinol, the most polar compound tested. The AQUASIL C18 column gives increased retention of both moderately polar and very polar solutes compared directly to a traditional alkyl C18 (the BetaBasic 18 column), due to increased interactions from the AQUASIL C18 columns polar endcapping. The unique nature of the Hypercarb phase produces a polar retention effect, whereby analyte retention increases as the polarity of the analyte increases. This polar retention effect results in a dramatically different elution order, where Phenol < Resorcinol < Phloroglucinol. Retention is also influenced by analyte shape, as the flat surface of the Hypercarb phase allows stronger interactions, and hence stronger retention, for analytes that have a planar structure and a greater molecular surface area.

2 3 4

BetaBasic 18

AQUASIL C18

BetaMax Acid

BetaMax Base

BETASIL Phenyl-Hexyl

Fluophase PFP

Fluophase RP

HyPURITY ADVANCE

PRISM RP 3 4 2 Reversed elution order Hypercarb

phenols-2 0 4 8 12 16 MIN

Summary of Comparative Column Retention To aid in HPLC column selection, the results of these chromatographic tests have been summarized in order of decreasing retention, for neutral, polar acidic and polar basic compounds (Figures 10-12). The BetaBasic 18 column is a typical type B silica, traditionally endcapped C18 phase, showing strong retention for hydrophobic compounds and less retention for polar compounds. Other Thermo Hypersil-Keystone phases with similar performance are the Hypersil BDS C18, HyPURITY C18, BETASIL C18, BioBasic 18 and BetaMax Neutral columns. The AQUASIL C18 column demonstrates excellent retention for acidic, basic, and neutral compounds in comparison to the other polar phases, making it an ideal choice for a wide range of sample types. The AQUASIL C18 column also shows alternative selectivity for polar compounds as a result of its polar endcapping. This polar character also allows the AQUASIL C18 column to equilibrate rapidly in 0 to 100 percent organic gradients. Fluorinated phases are a good choice for halogenated samples, or where shape selectivity will assist resolution. The Fluophase PFP column shows stronger retention than many of the other columns studied for polar basic compounds, and often gives a different peak elution profile than other phases. The HyPURITY ADVANCE, BetaMax Acid and PRISM RP phases all employ polar embedded groups to achieve unique sample-phase interactions. In general, these columns exhibit reduced retention for bases combined with increased retention for the most polar acidic compounds, and give the most apparent changes in selectivity. References 1. J. J. Kirkland, Practical HPLC Method Devleopment, 178-182
For Trademark information, please refer to page 6 of the 2002 Thermo Hypersil-Keystone Column Technologies for HPLC and LC-MS catalog.

Figure 10
Neutral Compound Retention

Figure 11
Polar Basic Compound Retention

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

Figure 12
Polar Acidic Compound Retention