PROCEDURES AND APPLICATIONS
Modes of HPLC Almost any type of solute mixture can be separated by HPLC because of the wide range of stationary phases available, and the additional selectivity provided by varying the mobile phase composition. Both normal- and reversed-phaseseparations are possible, depending on the relative polarities of the two phases. Although these are sometimes referred to as modes of HPLC, the nature of the stationary phase and/or the solute sorption mechanism provide a more specific means of classification, and modes based on these and the types of solutes to which they are best suited are summarized below.
● Adsorption chromatography. Separations are usually normal-phase with a silica gel stationary phase and a mobile phase of a nonpolar solvent blended with additions of a more polar solvent to adjust the overall polarity or eluting power, e.g. n-hexane + dichloromethane or di-ethyl ether. The choice of solvent is limited if a UV absorbance detector is to be used. Traces of water in the solvents must be controlled, otherwise solute retention will not be reproducible. Solutes are retained by surface adsorption; they compete with solvent molecules for active silanol sites (Si-OH), and are eluted in
Key Notes
Modes of HPLC are defined by the nature of the stationary phase, the mechanism of interaction with solutes, and the relative polarities of the stationary and mobile phases.
After selection of an appropriate mode, column and detector for the solutes to be separated, the composition of the mobile phase must be optimized to achieve the required separation. A trial and error approach or a computer aided investigation can be adopted.
Unknown solutes can be identified by comparisons of retention factors or times, spiking samples with known substances or through spectrometric data.
Quantitative information is obtained from peak area or peak height measurements and calibration graphs using internal or external standards, or by standard addition or internal normalization.
Related topics Principles of chromatography (D2) High-performance liquid chromatography: principles and instrumentation (D6) Modes of HPLC
Optimization of separations
Qualitative analysis
Quantitative analysis
order of increasing polarity. This mode is not used extensively, but is suitable for mixtures of structural isomers and solutes with differing functional groups. Members of a homologous series can not be separated by adsorption chromatography because the nonpolar parts of a solute do not interact with the polar adsorbent surface.
● Modified partition or bonded-phase chromatography (BPC). Most HPLC stationary phases are chemically-modified silicas, or bonded phases, by far the most widely used being those modified with nonpolar hydrocarbons. The solute sorption mechanism is described as modified partition, because, although the bonded hydrocarbons are not true liquids, organic solvent molecules from the mobile phase form a liquid layer on the surface.
The most popular phase is octadecyl (C18 or ODS), and most separations are reversed-phase, the mobile phase being a blend of methanol or aceto-nitrile with water or an aqueous buffer. For weakly acidic or basic solutes, the role of pH is crucial because the ionized or protonated forms have a much lower affinity for the ODS than the corresponding neutral species, and are therefore eluted more quickly. The dissociation of weak acids and the protonation of weak bases are shown by the following equations
RCOOH Æ RCOO-+ H+ RNH2+ H+Æ RNH3+
Thus, at low pH, bases are eluted more quickly than at high pH, whilst the opposite holds for weak acids (Fig. 1).
D7 – HPLC: modes, procedures and applications 167
16
14
12
10
8
6
4
2
0
0 2 4 6
pH
8 10 12 14
pH range for polymeric supports
pH range for silica supports
Weak
acid Ampholyte
Weak base
k
Fig. 1. Relation between retention factor, k, and the pH of the mobile phase for weak acids, bases and ampholytes in reversed-phase separations.
ODS and other hydrocarbon stationary phases will separate many mixtures, and are invariably a first choice in developing new HPLC methods.
They are particularly suited to the separation of moderately polar to polar solutes (Fig. 2(a)).
Aminoalkyl and cyanoalkyl (nitrile) bonded phases (the alkyl group is usually propyl) are moderately polar. The former is particularly useful in separating mixtures of sugars and other carbohydrates (Fig. 2(b)), whilst the latter is used as a substitute for unmodified silica, giving more reproducible retention factors and less tailing, especially with basic solutes. Both normal-phase and reversed-normal-phase chromatography is possible by appropriate choice of eluents.
● Ion-exchange chromatography (IEC). Stationary phases for the separation of mixtures of ionic solutes, such as inorganic cations and anions, amino acids and proteins, are based either on microparticulate ion-exchange resins, which are crosslinked co-polymers of styrene and divinyl benzene, or on bonded phase silicas. Both types have either sulfonic acid cation-exchange sites (-SO3
-H+) or quaternary ammonium anion-exchange sites (-N+R3OH-) incor-porated into their structures.
1 2 Packing : Spherisorb 5 ODS Flow rate : 1 ml/min.
Eluent : Solvent A:
methanol/water 20 : 80 Solvent B: acetonitril Gradient : 17.5% B 45% B
in 15 minutes Detector : UV 210 nm
Instrument : Du Pont HPLC
(8800 series)
Column : Zorbax™ NH2 25 cm × 4.6 mm i.d.
Flow rate : 4 cm3/min Mobile phase : Acetonitrile/water
(75:25)
Fig. 2. Separations of solutes of different polarities on bonded-phases. (a) Pharmaceuticals separated on ODS; UV absorbance detection; (b) sugars separated on aminopropyl silica; RI detection.
Ion-exchange chromatography is not that widely used. Inorganic ions and some cations are better separated by a related mode known as ion chro-matography (vide infra), whilst for organic ions, ion-pair chromatography is generally preferred because of its superior efficiency, resolution and selectivity.
● Ion chromatography (IC). This is a form of ion-exchange chromatography for the separation of inorganic and some organic cations and anions with conductometric detection after suppressing (removing) the mobile phase electrolyte (Fig. 3(a)).
The stationary phase is a pellicular material (porous-layer beads), the particles consisting of an impervious central core surrounded by a thin porous outer layer (~2 mm thick) incorporating cation- or anion-exchange sites. The thin layer results in much faster rates of exchange (mass transfer) than is normally the case with ion-exchange and therefore higher efficiencies.
Mobile phases are electrolytes such as NaOH, NaCO3 or NaHCO3 for the separation of anions, and HCl or CH3SO3H for the separation of cations. The detection of low levels of ionic solutes in the presence of high levels of an eluting electrolyte is not feasible unless the latter can be removed. This is accomplished by a suppressor cartridge that essentially converts the electrolyte into water, leaving the solute ions as the only ionic species in the mobile phase.
The following equations summarize the reactions for the separation of inorganic anions on an anion-exchange column in the HCO3-form using a sodium hydrogen carbonate mobile phase:
D7 – HPLC: modes, procedures and applications 169
Delivery mode
Fig. 3. Ion chromatography. (a) Schematic diagram of an ion chromatograph. (b) Anions in water separated on an anion-exchange column. Reproduced from Dionex UK Ltd with permission.
(a) (b)
Column reaction:
Similar reactions form the basis of the separation of cations. An example of the separation of inorganic anions at the ppm level is shown in Figure 3(b).
● Size exclusion chromatography (SEC). This is suitable for mixtures of solutes with relative molecular masses (RMM) in the range 102–108 Da.
Stationary phases are either microparticulate cross-linked co-polymers of styrene and divinyl benzene with a narrow distribution of pore sizes, or controlled-porosity silica gels, usually end-capped with a short alkyl chain reagent to prevent adsorptive interactions with solutes. Exclusion is not a true sorption mechanism because solutes do not interact with the stationary phase (Topic D2). They can be divided into three groups:
(i) Those larger than the largest pores are excluded completely, and are eluted in the same volume as the interstitial space in the column, Vo. (ii) Those smaller than the smallest pores, can diffuse throughout the entire
network and are eluted in a total volume, Vtot.
(iii) Those of an intermediate size separate according to the extent to which they diffuse through the network of pores, of volume Vpand are eluted in volumes between Voand Vtot.
Only those solutes in the third group will be separated from one another, and their retention volumes are directly proportional to the logarithm of their relative molecular mass (RMM; molecular weight). Columns can be calibrated with standards of known RMM before analyzing unknowns.
Figure 4 shows a typical plot of elution volume against log (RMM) and a chromatogram of a mixture with a range of solutes of differing molecular mass. SEC is of particular value in characterizing polymer mixtures and in separating biological macromolecules such as peptides and proteins. It is also used for preliminary separations prior to further analysis by other more efficient modes of HPLC.
● Chiral chromatography. Chiral stationary phases (CSP) enable enantiomers (mirror image forms) of a solute to be separated. Several types of these stereoselective materials have been investigated and marketed commercially, some of the most useful being cyclodextrins bonded to silica. The cyclodex-trins are cyclic chiral carbohydrates with barrel-shaped cavities into which solutes can fit and be bound by H-bonding, p-p and dipolar interactions.
Where the total adsorptive binding energies of two enantiomers differ, they will have different retention factors and can be resolved. Steric repulsion and the pH, ionic strength and temperature of the mobile phase all affect the resolution. Although of great interest to the pharmaceutical industry for the
nNa+
separation of enantiomeric forms of drugs having different pharmacological activities, chiral columns are expensive and most have very limited working lives. Capillary electrophoresis (Topic D8) provides a cheaper alternative.
The optimum conditions for an HPLC separation are those which give the required resolution in the minimum time. A stepwise approach based on the characteristics of the solutes to be separated, and trial chromatograms with different mobile phase compositions is usually adopted. The following is an outline of the procedure for a reversed-phase separation where a hydrocarbon stationary phase, usually C18 (ODS), is the first choice.
● The mode of HPLC most suited to the structures and properties of the solutes to be separated is selected, having regard to their relative molecular mass, polarity, ionic or ionizable character, and solubility in organic and aqueous solvents.
● The stationary phase and column are selected (Topic D6, Tables 2 and 3). The shortest column and the smallest particle size of stationary phase consistent with adequate resolution should be used.
● The detector, subject to availability, should match the solute characteristics.
UV-visible absorbancedetectors are suitable for many solutes except those that are fully saturated. Fluorescence and electrochemical detectors should be considered where high sensitivity is required.
● Mobile phase composition is optimized by obtaining and evaluating a number of trial chromatograms, often with the aid of computer optimization software packages. A typical series of chromatograms for a reversed-phase separation on a hydrocarbon bonded phase column is shown in Figure 5.
Optimization of separations
D7 – HPLC: modes, procedures and applications 171
Exclusion
Log relative molecular mass (RMM)
Retention volume (VR)
5 10 15 20 25 30 35 40 1. Thyroglobulin 670K
2. IgA 300K 3. IgG 150K 4. LDH 143K 5. Oralbumin 44K 6. Trypsin inhibitor 20.1K
1
Retention volume (cm3)
Fig. 4. A typical size exclusion calibration curve and chromatogram of the separation of a protein mixture. Column:
BIOSEP-SEC-S3000. Mobile phase: pH 6.8 phosphate buffer. Detector: UV abs. at 280 nm. Reproduced from W.J.
Lough & I.W. Wainer (eds), High Performance Liquid Chromatography, 1996, first published by Blackie Academic &
Professional.
1 3 2
6 4
5 Methanol (Meth) 7.5%
Tetrahydrofuran (THF) 42.5%
Water 50%
Meth 4.5%
THF 25.5%
Water 70%
1
2 3
6 5
4 Meth 10%
THF 25%
Water 65%
1 2
3
6
5 4
Meth 31.8%
THF 21.2%
Water 47%
1 2
3 64
5
Meth 0%
THF 35%
Water 65%
1 32 6
5 4
Fig. 5. Optimizing an HPLC separation using ternary mobile phases. Solutes: 1. benzyl alcohol; 2. phenol; 3. 3-phenyl-propanol; 4. 2,4-dimethylphenol; 5. benzene; 6. diethyl o-phthalate.
Note how the elution order of the six components in the mixture alters with the mobile phase composition.
Methods of identifying unknown solutes separated by chromatographic techniques are described in Topic D2. In the case of HPLC, there are four alternatives:
● Comparisons of retention factors (k) or retention times (tR) with those of known solutes under identical conditions, preferably on two columns of differing selectivity to reduce the chances of ambiguous identifications.
● Comparisons of chromatograms of samples spiked with known solutes with the chromatogram of the unspiked sample.
● Comparisons of UV-visible spectra recorded by a diode-array detector with those of known solutes. This is of limited value because most spectra have only two or three broad peaks so many solutes have very similar spectral features.
● Interfacing a high-performance liquid chromatograph with a mass spectro-meter. This enables spectral information for an unknown solute to be recorded and interpreted. Identifications are facilitated by searching libraries of computerized spectra (Topics F3 and F4).
Methods used in quantitative chromatography are decribed in Topic D2, and alternative calibration procedures are described in Topic A5. Peak areas are more reliable than peak heights as they are directly proportional to the quantity of a solute injected when working within the linear range of the detector. Most HPLC detectors have a wide linear dynamic range (Topic D6, Table 4), but response factors must be established for each analyte as these can vary consider-ably. Calibration is normally with external standards chromatographed sepa-rately from the samples, or by standard addition. Constant volume loops for sample injection give very good reproducibility (about 0.5% relative precision), making internal standards unnecessary, cf gas chromatography (Topic D5), and auto-injectors are frequently employed for routine work. An overall relative precision of between 1 and 3% can be expected.
Quantitative analysis Qualitative analysis
D7 – HPLC: modes, procedures and applications 173