The Theory of HPLC Normal Phase (Absorption) 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.
Aims and Objectives Aims and Objectives Aims To give an overview of the mechanism of Normal Phase Chromatography (NPHPLC) and explain the basis of the retention mechanism To highlight typical NPHPLC Applications To explain retention order in NPHPLC and demonstrate the influence of mobile phase composition on retention To explain how the mobile phase composition and constituents might be manipulated to optimise chromatographic separations in NPHPLC To illustrate the principles which are used to select appropriate stationary phases and column geometry in NPHPLC Objectives At the end of this Section you should be able to: To outline the advantages and limitations of NPLHPLC compared to RPHPLC To outline the issues with water in NPHPLC mobile phases and give strategies to practically overcome problems To explain the best way to get started with NPHPLC and to optimise the chances of a successful separation
Content Mechanism of Normal Phase Chromatography 3 Applications of Normal Phase Chromatography 4 Retention and Selectivity in Normal Phase Chromatography 6 Separation of Isomers using Normal Phase Chromatography 7 Mechanism of Isomer Recognition in Normal phase HPLC 7 Stationary Phases for Normal Phase HPLC 8 Typical Mobile Phases HPLC 11 Controlling Retention 12 Mobile Phase Optimisation 13 Problems with Water in the Mobile Phase 14 Getting Started with Normal Phase HPLC 15 Glossary 16 Crawford Scientific www.chromacademy.com 2
Mechanism of Normal Phase Chromatography Normal phase chromatography was the first Liquid Chromatographic technique, chronologically. As we have seen, Tswett used this mode to separate plant pigments using a calcium carbonate stationary phase with a petroleum ether mobile phase. By definition, normal-phase HPLC utilises a stationary phase that is more polar than the mobile phase. Typical stationary phases include bare silica as well as cyano, diol, and amino bonded phases. Typical mobile phase constituents include organic solvents such as hexane and ethyl acetate. The retention mechanism in normal phase HPLC is based on polar adsorption of either the solvent molecules or the analyte onto the polar stationary phase surface. If the solvent molecules are localising they will be adsorbed onto the stationary phase surface. If the analyte molecule contains highly polar functional groups, it may also be capable of localising onto the stationary phase surface essentially displacing the solvent molecule and gaining retention. Mass action will then displace the analyte from the stationary phase surface back into the mobile phase, where it will be transported down, and eventually elute from, the column. i Initially the acetonitrile solvent molecules (a localising solvent), are adsorbed to polar retention sites on the silica surface (silanol groups). The analyte molecule will compete for retention sites with the solvent molecule. The nature and concentration of localising solvent in the mobile phase will have a large effect on normal phase retention characteristics. In normal phase chromatography less polar (hydrophobic) compounds elute first, whilst more polar (hydrophilic) compounds elute later. As can be seen in the example the hydrocarbon portion of the analyte is only weakly attracted to the stationary phase, whereas the polar hydroxyl functional group is strongly attracted. The polar phenol molecule localises onto the stationary phase and displaces the acetonitrile molecules. Crawford Scientific www.chromacademy.com 3
In normal phase chromatography less polar (hydrophobic) compounds elute first, whilst more polar (hydrophilic) compounds elute later. Vitamin molecules generally show poor water solubility and may be analysed using normal phase chromatography. The order of elution is least polar first, followed by increasingly polar (less hydrophobic) analytes. This separation uses a bonded phase column. Hexane is a non-localising (non-polar, weak) solvent and Ethanol is the localising (strong) solvent used to displace the analyte from the silica surface. As the vitamin molecules are relatively non-polar, only a very small amount of strong solvent is required for elution. Applications of Normal Phase Chromatography In normal phase chromatography, polar (hydrophilic) analytes are retained longer than less polar (hydrophobic) analytes. Normal phase chromatography has been used for the separation of both neutral and ionisable compounds, although neutral sample separations predominate the literature. Reverse phase chromatography is usually attempted first, if the required retention or selectivity is not obtained using the strategies outlined, then normal phase chromatography is used as a second choice. Some samples are only sparingly soluble, or insoluble in aqueous media. This renders them unsuitable for reverse phase HPLC. Whilst it is possible to introduce samples into reverse phase HPLC systems using 100% organic solvent diluents, peak shapes are often very poor. Normal phase is often a good alternative as samples are much more soluble in the organic solvent systems used. Crawford Scientific www.chromacademy.com 4
Table 1. Advantages and Disadvantages of Normal Phase Chromatography Normal Phase Advantages Normal Phase Disadvantages Separation selectivity can be greatly influenced by altering the mobile phase constituents and ratio of solvents Organic compounds are highly soluble in the solvent systems used a big Most amenable to low and mid polarity samples ionic samples are best analysed by reverse phase (although addition of triethylamine to the mobile phase assists with analysis of bases in advantage for preparative normal phase) chromatography Controlling solvent strength can be Solvent viscosity is lower therefore higher flow rates can be used to achieve improved sample throughput unpredictable Solvents are more prone to air bubble formation giving rise to instrument problems and noisy baselines Care must be taken to exclude mobile phase water with non-bonded stationary phases Gradient elution is often not feasible due to solvent de-mixing Solvents used have a much higher cost of disposal and environmental impact Due to the localising behaviour on the stationary phase, normal phase systems are excellent at discriminating between compounds whose spatial geometry differs. Hence, normal phase systems are popular for the separation of chiral enantiomers as well as positional isomers. If large amounts of analyte need to be recovered from solution using preparative chromatography normal phase systems are usually employed due to the ease of solvent removal. Normal phase solvent fractions are more easily evaporated to dryness than the highly aqueous systems encountered with reverse phase chromatography. In the next example a 21.2 mm i.d. preparative HPLC column is used to separate phospholipid analytes using normal phase chromatography with a fairly complex mobile phase system. Normal phase preparative separation of Soy Phospholipids Crawford Scientific www.chromacademy.com 5
Normal phase has several advantages for this separation: High volatility solvents for easy fraction (analyte) recovery Low viscosity phases for high sample throughput at increased flow rate Enhanced selectivity via the adsorption mechanism and better selectivity control using normal phase localising solvents Lack of analyte chromophore means alternative detection mechanism the volatile solvent systems are highly compatible with evaporative light scattering detectors Retention and Selectivity Stationary Phases The retention order in normal-phase HPLC is generally the opposite of reversed phase HPLC. The stationary phase is very selective for the number, type, and orientation of polar functional groups. A general elution order is shown opposite. Adding more polar functional groups to a molecule increases the retention. As a general rule it is the most polar functional group that determines retention. General Retention Trends in Normal Phase HPLC The retention factor data shows tremendous selectivity for polar functionalities in normalphase mode. The only difference in the molecules on the left is the polar functionality, yet their k values range from 0.6 to 5.5. Crawford Scientific www.chromacademy.com 6
Separation of Isomers using Normal Phase Chromatography The example on the right illustrates the selectivity for structural isomers on bare silica. The compounds differ only in the location of the polar constituents, one in the meta position and one in the para position. The selectivity for structural isomers is primarily restricted to the bare silica columns. Separation of Positional Isomers of Baythroid using Normal Phase CHromatography Mechanism of Isomer Recognition in Normal phase HPLC The selectivity movie indicates the primary reason for the ability to discriminate between such closely related compounds localisation onto the silica surface. Depending upon the geometry of the analyte molecule, and the relative strengths of the dipoles or hydrogen bonding capability, the analytes will bind more or less well to the stationary phase surface resulting in excellent selectivity. Crawford Scientific www.chromacademy.com 7
i Mechanism of Positional Isomer Separation in Normal Phase HPLC Stationary Phases for Normal Phase HPLC Bonded stationary phases offer several advantages over bare silica in normal phase HPLC applications. They equilibrate more rapidly than silica columns; therefore gradient elution is possible. Strong (localising) solvents tend to bind very strongly to bare silica and equilibration can take 20 column volumes or more. In gradient elution the strong solvent being introduced tends to be irreversibly adsorbed to the silica surface. Once the surface is saturated, a sudden increase in the modifier (strong solvent) eluting from the column is seen, and some compounds may elute with low retention and inadequate separation. This problem is much less apparent when using bonded phase columns and as such gradient elution is possible. Bonded phase columns for Normal phase chromatography are available in a wide variety of polarities for better selectivity. These columns also have a higher sample capacity than bare silica columns. The mobile phase water content does not have to be strictly controlled as it does with silica columns (more on this later). Crawford Scientific www.chromacademy.com 8
Advantages of Bonded Phases: Mobile phase trace water does not have to be controlled Gradient elution possible Column equilibrates rapidly Wide variety of polarities, selectivity High capacity Peaks don t tail as with bare silica Silica first choice for preparative separations as bonded phases have higher cost, lower stability and lower loadability Silica Amino Diol Cyano Typical Bonded Phase Stationary Phases used in Reverse Phase HPLC As a result of all these advantages, it is recommended that method development in normal-phase mode is initially carried out using bonded phase columns. In particular, with cyano columns, which have intermediate polarity and good stability. Diol columns are the most polar and silica like. They however, like amino columns are not as stable. Cyano: Most popular phase to begin normal phase method development. Dipolar compounds such as chloro, nitro and nitrile substituents are more strongly retained on cyano columns relative to amino or diol columns. Diol: The most polar of the bonded phases. Basic compounds such as amines, ethers, esters and ketones are preferentially retained on amino and diol columns relative to cyano columns. Silica: The use of silica phases is less convenient for analytical applications due to problems with adsorption of trace water and solvent de-mixing affecting reproducibility and the ability to use solvent gradients. Silica is the phase of choice for many isomer separations and for large-scale preparative chromatography applications. Amino: Basic compounds such as amines, ethers, esters and ketones are preferentially retained on amino and diol columns relative to cyano columns. Amino columns should not be used with adehydes and ketones as they can form Schiff bases. Amino columns have been useful for the separation of vitamins A and D in normal phase mode. If the selectivity obtained is not appropriate on the bonded-phase columns, then switch to bare silica. Bare silica is recommended as the starting point for the separation of structural isomers. Crawford Scientific www.chromacademy.com 9
Separation of herbicides using different normal phase stationary phases Differences in stationary phase selectivity are demonstrated in this application of preparative and analytical scale chromatography of herbicide analytes from a sample of natural oats. Separation a) is carried out on a cyano column due to the large number of sample components the separation is impossible in a single analysis. The area of the chromatogram indicated by the arrow is diverted via column switching onto a diol column using the same mobile phase this is separation b), which showed improved separation characteristics and the herbicide is seen but is heavily interfered. The collected fraction from this chromatogram was re-analysed using a silica column this is separation c) and again showed a markedly different selectivity to the two previous stationary phases. Potential for poor peak shape in Normal Phase HPLC when using bare silica stationary phases Care should be taken when working with silica stationary phases. Surface acidity (related to the silanol conformation, metal ion content etc.) can cause poor peak shape with some polar compounds Zorbax Rx-Sil has lower surface acidity and improved peak shape. Crawford Scientific www.chromacademy.com 10
Typical Mobile Phases HPLC Common normal-phase solvents along with their elution strengths can be seen behind the solvent strength button opposite. The data shown is for solvents on bare silica columns and all strengths are relative to n-pentane. Weak solvents such as fluoroalkanes and n-hexane have negative or low elution strengths. The stronger solvents can be divided into three groups: non-localising, basic localising, and non-basic localising, referring to the solvents ability to compete with various analyte types. Remember that localising refers to the ability to interact with the stationary phase surface through dipole or hydrogen bonding interactions. Solvent Selectivity Triangle for Normal Phase Solvents When developing a normal-phase method, select a weak solvent, such as hexane or 1,1,2-trifluoro-1,2,2-trichloroethane, and one of the stronger solvents such as methylene chloride or ethyl acetate and vary the concentration from strongest to weakest mobile phase composition. This example illustrates the nature of the normal-phase mechanism. The three sample components are xylene (0.24), toluene (0.28), and benzene (0.20). The methylene chloride is stronger than all three sample components with elution strength of 0.32. Therefore, the sample components are not strong enough to displace the methylene chloride from the active sites. All sample components elute as one peak in the dead volume. When the mobile phase is changed to npentane, the samples are found to be stronger than the mobile phase and retention takes place. Separation of Benzologs by Normal Phase Chromatography illustrating the elution strengths of various normal phase solvents Crawford Scientific www.chromacademy.com 11
Take note that analyte retention will not vary in a linear fashion with changes in mobile phase composition as with reversed-phase HPLC. If the solvent combination does not provide the desired selectivity, switch to one of the other categories (e.g. basic to nonbasic) for strong solvent and test combinations. It may be necessary to use all three strong solvent types (non-localizing, basic localizing, and non-basic localizing) in combination with the weak solvent to achieve the desired selectivity. Hexane is good for low UV adsorption, but is not miscible with all strong solvents and care should be taken in this respect. Add methylene chloride to the mobile phase to ensure miscibility. Controlling Retention Use the slider to see how the chromatogram changes at different mobile phase strengths. i 1-nitronaftalene 1,7- dimetoxynaftalene You should particularly notice the shape of the plots for the retention of peaks 1 and 2 and notice how large retention changes occur at lower %B concentrations and changes at higher concentrations have little effect on retention -this is a general observation about all normal phase separations You should also notice how the separation selectivity changes as the modifier concentration is increased this is also a facet of normal phase chromatography. It is more useful to introduce a different type of strong solvent than to vary the modifier concentration over a wide range Optimising Mobile Phase Strength in Normal Phase HPLC Crawford Scientific www.chromacademy.com 12
Mobile Phase Optimisation As we have seen, different solvents may be employed to change the selectivity in normal phase chromatography. There are many charts and graphs to help you in this regard, however the solvent optimisation process is much more empirical than with reverse phase HPLC, usually involving a good deal of trial and error. Perhaps all the components in your sample elute within the correct k range when you use, for example, 92% n-pentane with 8% methyl acetate. Some of your chromatographic peaks, however, are not well separated. You can refer to a chart or graph found in a text or paper and find solvent combinations of equivalent elution strength. The chart of isoeluotropic mobile phase combinations indicates that in this case you may try 62% n- pentane with 38% methylchloride to achieve similar overall analysis time, but with altered selectivity. i Nomographic Relationships between mobile phase systems for Normal Phase HPLC Where Table 2. List of compounds Compound Name MTBE Methyl tertiarybutyl ether EtOAc Ethyl Acetate MC Methylene Chloride PrOH 1-propanol Nomomgraphs of the type shown opposite are also available in the literature and may be used to ensure isoeluotropic behaviour, whilst changing solvents to adjust selectivity. You can use the slider to investigate isoeluotropic compositions for various solvents Crawford Scientific www.chromacademy.com 13
Problems with Water in the Mobile Phase Bare silica columns exhibit a number of problems not associated with bonded phase columns including peak tailing, irreproducible retention times, and long equilibration times. The problems are caused by silanol groups, which have varying strengths on the stationary phase surface and trace water in solvents. Due to ambient humidity, trace water will be taken up by the mobile phase. This dissolved water is then taken up by the column, which can lead to chromatographic variability. The trace water level is not easily controlled leading to different water concentrations at different times. Water can be picked up from glass surfaces and the air. The trace water will adsorb to the strongest of the silanol groups, leading to reduced (and variable) retention of analyte components. Table 3. Effect on Retention Factor of some typical analytes for wet and dry solvents in Normal Phase HPLC Compound type Dry solvent 50% H 2 O st. Solvent Aromatics 0.05 0.25-0.2 0.25 Halides 0.0 0.3-0.2 0.1 Mercaptans 0.0-0.2 Ethers 0.1 0.0 Nitros, esters, nitrites, carbonyls 0.2 0.3 0.1 Alcohols 0.3 0.2 Phenols 0.3 0.2 Amides 0.2 0.6 0.0 0.4 Acids 0.4 0.3 Amides 0.4 0.6 0.3 0.5 Researchers have also published data indicating the approximate elution strength (ε o ) necessary to separate a given class of compounds. An example appears above. The values can be used in conjunction with the solvent strength values given earlier. Snyder and Kirkland have proposed that the mobile phase is equilibrated with an intermediate ( 50% saturation ) amount of water. A portion of mobile phase is saturated with water, then this portion is blended with an equal volume of dry (over molecular sieve) solvent which has not been treated with water. This can often result in much improved retention time reproducibility and column equilibration times can be shortened from many hundreds of column volumes to only a few. In some cases, the effect of varying mobile phase water concentrations on sample retention may be minimised by adding 0.1 to 0.5% methanol or propanol to the mobile phase. Crawford Scientific www.chromacademy.com 14
Getting Started with Normal Phase HPLC Some suggested starting conditions for normal phase HPLC are shown. The mobile phase compositions are recommendations only and, as has been discussed, the optimisation of the solvent system will be very application dependant. Switching between basic and non-basic localising solvents is recommended to investigate selectivity in the early stages of method optimisation. The temperature does not have a marked effect on selectivity in normal phase chromatography. However, it does alter retention characteristics, and as such, it is important that temperature is controlled. A list of critical issues in normal phase chromatography is shown. Note that the addition of a sacrificial base such as Triethyamine (TEA) or acid such as acetic acid can markedly improve peak shape in normal phase HPLC. This is analogous to the situation found in reversed phase HPLC. Table 4. Some suggested starting conditions and critical issues for Normal Phase HPLC Bonded Silica CN Silica SIL Column Packed in normal phase solvents ZORBIX Rx-Sil-basic solutes Mobile phase Hexane with 1- or 2- propanal Methylen chloride with 0.05% - 0.5% methanol Temperature Ambient 60 o C Ambient 60 o C Table 5. Critical Issues in Normal Phase HPLC Poor peak shape Injection solvent stronger that mobile phase Basic samples give better peak shape using high purity silica Basic samples may require 20mM TEA in the mobile phase Basic samples may require 20mM acetic acid in the mobile phase Strongly retained polar materials can build on the column Reproducibility Silica requires addition of water or methanol to maintain reproducibility Use columns packed in normal-phase solvents It is important to match the solvent strength of the sample diluent with the mobile phase in order to avoid poor peak shape if necessary a weaker diluent strength is acceptable otherwise use a sample concentration as high as possible in strong solvent and inject under 10μL. It is often necessary to flush both bonded and non-bonded phase with 100% strong solvent to remove adsorbed sample components this will restore column performance. Crawford Scientific www.chromacademy.com 15
Glossary Localising refers to the ability of an analyte or solvent to interact, via polar functional groups, and be adsorbed onto the stationary phase surface. A basic representation of this process might be: Mass action a concentration effect in which a species in vast excess of another is able to displace the species which is dilute. Preparative chromatography - a mode of chromatography in which the large columns (21 50 mm i.d. are common), are overloaded using large volumes or masses of analyte. The mobile phase eluting for the column around the retention time of the peak of interest is collect, with the intention of drying down the solvent to recover the purified analyte for further characterisation or use a standard material or use in a further reaction etc. Snyder and Kirkland - L.R. Snyder and J.J. Kirkland, Introduction to Modern Liquid Chromatography, 2 nd. Ed., Wiley-Interscience, New York, 1979, pp. 374-383 Crawford Scientific www.chromacademy.com 16