The Theory of HPLC. Ion Pair Chromatography

Transcription

1 The Theory of HPLC Ion Pair Chromatography i Wherever you see this symbol, it is important to access the on-line course as there is interactive material that cannot be fully shown in this reference manual.

2 Aims and bjectives Aims and bjectives Aims To explain the principles of Ion Pair Chromatography (IPHPLC) and to demonstrate the circumstances under which this approach might be used To investigate common reagents used for IPHPLC and to explain how to choose the appropriate reagents To investigate the effects of the important parameters associated with IPHPLC, concentrating on Ion Reagent choice and concentration bjectives At the end of this Section you should be able to: To illustrate how to practically optimise IPHPLC separations To demonstrate applications of IPHPLC including the use of volatile reagents for Liquid Chromatography Mass Spectrometry (LC-MS)

3 Content undamental Mechanism 3 Reagents 4 Mechanisms of Ion Pairing 5 Retention & Selectivity 5 ptimising Ion Pair Concentration 6 Important Parameters in Ion Pair Chromatography 8 Applications and Ion Pairing for LC-MS 10 Practical Ion Pair Chromatography 12 Getting Started with Ion Pair HPLC 13 Crawford Scientific 2

4 undamental Mechanism The technique of using ph to suppress ionisation, and therefore gain retention, for ionisable analytes in reverse phase HPLC is applicable to weakly acidic or basic compounds only. If a separation of MIXTURES of weak acids and bases (or analytes that are amphoteric) is required, then ion suppression is of limited use. When both acidic and basic functional groups are present, as the ionisation of one functional group is suppressed, that of the other may be promoted, and a compromise is often very difficult to reach which results in non-robust methodology. Difficulty in finding a suitable mobile phase ph for Ion Suppression with Amphoteric Analytes Addition of an ion-pairing reagent (IP reagent) to the mobile phase will increase the retention of ionised species. or acids a mobile phase ph of 7 is recommended to ensure analytes and IP reagent are fully ionised. or the analysis of basic molecules a ph of 3.5 is recommended. i You should have noticed there is no ph value at which both functional groups are sufficiently ion suppressed (>~80%)to give good retention in reverse phase HPLC It is possible to adjust the mobile phase ph so that at least one functional group is ion-suppressed This gives the possibility of ionsuppression for one functional group whilst the other is rendered less polar (ionic) with an ion-pair reagent added to the mobile phase This molecule is particularly difficult to deal with as the ph required to ion suppress either of the functional groups is outside the working ph range for traditional silica based columns. The analyst would need to employ an HPLC column capable of operating at more extreme mobile phase ph values At ph 1 the acidic functional group will be non-ionised (ion-suppressed), and the basic group will be fully ionised allowing the use of an anionic ion pairing reagent At ph 11 the basic functional group will be non-ionised (ion-suppressed), and the acidic group will be fully ionised allowing the use of a cationic ion pairing reagent Similarly, if stronger acids or bases are to be analysed, then the mobile phase ph at which the functional group is suppressed, may lie outside the working range of traditional silica columns, precluding the use of ion-suppression HPLC. The analysis of mixtures of weak acids and bases or amphoteric (having characteristics of both an acid and a base and capable of reacting as either) analytes is possible however, using ion-pair chromatography. In this mode, a reagent is added to the mobile phase that has both a hydrophobic tail and a charged functional group. The hydrophobic section of the ion-pairing reagent interacts with the stationary phase whilst the charged functional group undergoes an ion exchange or ion-pairing interaction with the analyte. Crawford Scientific 3

5 Reagents A cationic analyte molecule is paired in solution with hexane sulphonic acid (Sodium Hexane Sulphonate). Depending upon the ion pair hydrophobicity and concentration in the mobile phase, two mechanisms dominate: 1. The analyte is paired in solution with the IP reagent and the neutral complex undergoes partition interactions with the stationary phase 2. The IP reagent populates the hydrophobic surface and the analyte molecules undergo an ion exchange interaction At low concentrations both mechanisms are apparent, however the first is more dominant. As ion pair concentration is increased, the second mechanism begins to dominate. In each case the analyte is competing with background buffer ions and the counter ion of the IP reagent to interact with the reagent itself. Table 1. Commonly used ion paired reagents Separate Bases with: Separate Acids with: Trifluoroacetic acid (TA) Quaternary Alkyltriethylamines Heptafluorobutyric acid (HTBA) Triethylamine (TEA) Hexanesulphonic acid Tetramethylammonium phosphate (TMA) Tetrabuthylammonium phosphate (TBA) Tetraethylammonium acetate (TEAA) C C Triflouroacetic acid (TA) H C C C C Heptafluorobutyric acid (HTBA) S Hexanesulfonic acid P N H N H P N H Tetrabutylammonium phosphate (TBA) Triethylammonium acetate (TEAA) Tetramethylammonium phosphate (TMA) N NH Quaternary Alkyltriethylamines Triethylamine (TEA) Crawford Scientific 4

6 Mechanisms of Ion Pairing i The dual mechanism of ion-pairing in Reverse Phase HPLC Ion Pairing in Solution and Ion Exchange with Adsorbed Reagent Retention & Selectivity The size of the hydrophobic portion the ion-pair reagent controls retention (k ), but would rarely induce any change in elution order, in changing from a hexane to a heptane sulphonic acid. f course, the selectivity between ionic and neutral analytes will change after adding ion pair reagent to the mobile phase. Higher concentrations of ion-pair reagent typically mean higher retention for samples with opposite charge. This holds up to approximately 50 mm. At higher ion-pair reagent concentrations, neutral samples may have decreased retention times due to more limited access to the stationary phase (which is saturated with ion-pair reagent and effectively becomes an ion exchange phase). Shifts in selectivity may be found when changing the composition or organic modifier used in the mobile phase. Methanol is often used in ion-pair chromatography for its good ionpair solubility. Ion-pair separations are more temperature sensitive than typical reversed-phase separations. Before clicking on the two active areas of the curve of IP reagent concentration against analyte capacity factor, it is important that you consider why each curve has a plateau and why the decyl sulphate reagent curve reaches a maximum analyte retention prior to the analyte retention factor then decreasing. Crawford Scientific 5

7 Relationship between analyte capacity factor and Ion Pair Concentration in the Mobile Phase for four typical reagents ptimising Ion Pair Concentration It is interesting to note that each of the ion-pair vs. analyte retention curves either has a maximum value or a plateau. The more hydrophobic an IP reagent, the more readily it is taken up by the stationary phase (and larger reagents occupy more space on the silica surface). This leads to the concept that lower concentrations of IP reagent are required as the hydrophobicity increases. The plateau or maxima in analyte retention indicates that no further increase in analyte retention is possible with increasing IP reagent concentration. Essentially, the silica surface is saturated and the main retention mechanism is ion exchange between the analyte and the IP reagent on the silica surface. It is IMPRTANT to remember that the mobile phase ph needs to be adjusted so that6 bot IP reagent and analyte are charged. As IP reagents are usually strong acids or bases, this essentially means adjusting mobile phase ph for analyte ionisation (approx. ph 7 for weak acids and ph 3.5 for weak bases). Crawford Scientific 6

8 i Limiting factors in increasing analyte capacity factor using Ion Pair Reagents The maxima seen in the decyl sulphate curve is of great interest. The reduction in retention of the analyte above an IP concentration of around 23mM corresponds to saturation of the silica surface with IP reagent. At this point the main mechanism of interaction is ion exchange between the analyte and the adsorbed IP reagent. If more IP reagent is then added to the mobile phase, it will remain in the mobile phase and will tend to associate with the analyte molecule. This will render the analyte a neutral complex. As the silica surface is now an ion exchange surface, the neutral complex formed will not be so highly retained and analyte retention will shorten. It is therefore of great importance that IP reagent concentrations in solutions are optimised. i Limiting actors for the Maximum Ion Pairing Reagent Concentrations in Reverse Phase HPLC Crawford Scientific 7

9 Important Parameters in Ion Pair Chromatography The important parameters in Ion Pair HPLC are shown in the table. It is important to understand how to make changes to ion pair chromatography in order to achieve desired effects. Table 2. Important Variables In Ion Pair Chromatography Variable Controls %rganic Retention (k) Ion Pair Reagent type Retention Ion Pair Concentration Retention, Selectivity (α) ph Retention, Selectivity (α) Buffer concentration Retention (k) % rganic - As usual in reverse phase HPLC, the percentage organic modifier controls retention. Increasing the amount of organic modifier decreases the retention time even though the analyte may be charged, the mechanism is still one of hydrophobic partitioning. Ion Pair Reagent Type - The more hydrophobic the IP reagent, the more highly retained will be the analyte molecules. Some may argue that selectivity may also be altered, this will only happen in rare cases for charged analyte molecules. Ion Pair Concentration - As the concentration of IP reagent is increased, the selectivity between neutral and charged analytes, and between charged analytes will alter. f course, as the IP concentration is increased, the retention factor will also increase. It should be noted however, that as IP reagent concentrations increase, neutral compound retention may decrease, due to limited access to the hydrophobic portion of the stationary phase. ph - As ph is altered, the selectivity between ionised and neutral compounds will alter, particularly due to the extent of ionisation of the charged analytes. The selectivity between charged analytes may also alter. It is important to remember that the mobile phase ph should be adjusted for a charged stationary phase (via the IP reagent) as well as a charged analyte the reverse situation to ion suppression HPLC. Adjusting mobile phase ph for an acidic analyte in ion pair HPLC Buffer Concentration - The mobile phase buffer ions will compete with the analyte for ion pair counter ions. Therefore, the high the buffer ion concentration the shorter the retention time of the analyte. Crawford Scientific 8

10 The mobile phase ph can be used to affect retention (extent of analyte ionisation) or selectivity, by altering the relative extent of ionisation of the analytes. Increasing buffer concentration will increase the competition between the buffer ions and analyte ions with the ion pair reagent by mass action the higher the buffer concentration the shorter the retention time of the analyte species as it is displaced from the ion pair. The chromatogram shown, is a typical example of the situation in which ion pair reagents are best employed. Compounds 4-7 are neutrals and are well separated on this C18 column ( mm, 3 μm), in a 48% MeH mobile phase at ph7.1. However, the ionised strong basic analytes (peaks 1-3 min. pka ~10.1), are very poorly retained. It would not be possible to remedy this problem using changes in organic modifier, and adjusting ph to gain retention of the bases would require a ph of 12 or above out of the range of most silica based columns. i Column: C18 150x4.6mm, 3um low: 1mL/min. Mobile Phase: 52% 0.01M NaH 2P 4 (ph 7, Sodium ctyl Sulphonate) : 48% MeH Interactive Experiment on ptimising Ion Pair Reagent It is often necessary to add ion-pairing reagents to the eluent in order to gain satisfactory retention of all analytes when dealing with strong acids or bases, mixtures of weak acids and bases, or when analysing amphoteric analytes The choice and concentration of ion pair reagent is critical in obtaining an optimised separation Altering the ion pair reagent concentration changes the selectivity and resolution of the separation therefore empirical optimisation of ion pair concentration may be necessary The retention times of species that are charged in solution will undergo a greater change than those of neutral species in solution It is often necessary to alter mobile phase ph to ensure only one functional group is ionised in solution i.e. suppress acidic moieties by lowering the ph - this of course will promote the ionisation of basic analytes In complex chromatograms such as this one, it may be difficult to obtain an ion pair concentration that results in a robust method. This is mainly due to the number of analytes present. It is important to investigate method robustness and to be aware of the mobile phase ion pair reagent concentration accuracy necessary when making up eluent solutions Crawford Scientific 9

11 Applications and Ion Pairing for LC-MS In the example below, synthetic dyes were analysed using ion-pair, reversed phase chromatography on a base-deactivated column. This separation mechanism was chosen to gain retention and reduce tailing effects of highly polar dyes. Use of Ion Pair Chromatography to improve chromatographic performance during a dyestuff separation During method development, four different mobile phases were evaluated. Two mobile phases were simple buffer systems (C and D) and the other two phases contained ionpairing reagents (A and B). Mobile phases C and D (buffer only) show tailing and reduced retention for compounds with sulphonic groups. Using ion-pairing chromatography, A and B, the separation of dyes with different functional groups and different chemical structures was achieved with minimum or no tailing. Crawford Scientific 10

12 The use of ion pairing reagents with Electrospray Ionisation (ESI) LC-MS detection can be particularly problematical. The ESI mechanism of ion production requires analytes to be ionised in the mobile phase - these being subsequently librated from charged eluent droplets sprayed in the spectrometer interface. Traditional ion pairing reagents cause a great reduction in signal intensity, as the neutral complex formed is not amenable to spraying, ion evaporation or detection processes. Therefore, volatile ion pairing reagents must be used which aid in chromatographic retention but which dissociate in the LC-MS interface to produce gas phase analyte ions. The example shows the use of simple amines as effective IP reagents in ESI LC-MS of acidic analytes. Volatile anionic IP reagents such as HBA (heptafluorobutyric acid) are also available. With DBA as ion-pairing reagent and propan-2-ol as mobile phase modifier, a water sample spiked with ng/ml of nine HAAs could be determined by LC-ESI-MS using timescheduled selected ion monitoring. The correlation coefficients for all target analytes were higher than The detection limits ranged from 24 to 118 pg/ml. The repeatability and reproducibility were in the range % and %, respectively. DMBA N,N-dimethyl dibutylamine DBA Dibutylamine TBA Tributylamine MCAA, DCAA, TCAA mon-, di, & tri-chloroacetic acid, MBAA, DBAA, TBAA mon-, di, & tri-bromoacetic acid DBCAA dibromochloroacetic acid DCBAA di chlorobromomacetic acid Use of Ion Pair Chromatography with MS Detection systems is possible but requires empirical optimisation Crawford Scientific 11

13 Practical Ion Pair Chromatography It is generally true that Ion Pair separations are more efficient than Ion Exchange separations Shifts in selectivity may be found when changing the composition of the organic Methanol is often used in ion-pair for its retention and ion-pair solubility reasons Ion-pair separations are more temperature sensitive than typical reversed-phase separations Shifts in Selectivity encountered when altering the nature of the organic modifier in Ion Pair Chromatography Equilibration time for ion-pair chromatography can be 20 column volumes and longer. ften, it is difficult to remove the ion-pair reagent from the column entirely. Therefore, a common recommendation is that once a column has been used in ion-pair mode, it always be used in the ion pair mode. High molecular weight ion pair reagents are more difficult to remove from the column. Use endcapped columns to eliminate residual silanol effects. Triethylamine (20mM) can be added to the mobile phase to improve peak shape as has been discussed. Peak shape can be improved if ion-pairing reagent is added to the sample diluent solution. Impurities in ion-pair reagents may cause unstable baselines. Excess ion-pair reagent can elute - resulting in spurious peaks. Columns may have to be cleaned with high percentage organic solvents to remove strongly retained sample components. Crawford Scientific 12

14 Getting Started with Ion Pair HPLC The conditions for starting an investigation into an Ion Pair HPLC separation are very similar to those for reverse phase HPLC using ionisable samples with a few notable exceptions. Methanol is preferred to acetonitrile as the organic modifier for solubility reasons Lower Buffer concentrations are preferred so that the effects of the ion pair reagents are not countered Cations are analysed using acidic reagents and the hexane homolog is chosen for initial investigation or cations the mobile phase ph is adjusted between 2 and 3 to ensure that analyte is fully ionised or anionic analytes tetrabutyl ammonium phosphate is added to the mobile phase which is adjusted to ph 6-7 in order to ensure the acidic analyte is fully ionised Temperature can cause large differences separation selectivity and this parameter should be strictly controlled Table 3. Suggested Starting Conditions for Ion Pair Chromatographic Separations Variable Initial Choice Column Dimensions cm Particle 5 or 3.5μm Bonded C18 or C8 Mobile Phase Solvents %B Buffer Additives low Rate Aqueous buffer/methanol Variable (k=2-20) 25mM Potassium Phosphate 20mM TEA if needed with bases 1-2mL/min Cations mM sodium hexane sulfonate (ph 2-3) Anions 10-40mM tetrabuthyl ammonium phosphate (ph 6-7) Temperature o C Sample Volume 50μL Mass < 25μg The next figure presents a practical application of ion pairing chromatography Suggested Starting Conditions for Ion Pair Chromatographic Separations Crawford Scientific 13

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