PRINCIPLES AND INSTRUMENTATION
Key Notes
High-performance liquid chromatography (HPLC) is a technique for the separation of components of mixtures by differential migration through a column containing a microparticulate solid stationary phase. Solutes are transported through the column by a pressurized flow of liquid mobile phase, and are detected as they are eluted.
The mobile phase is either a single solvent or a blend of two or more having the appropriate eluting power for the sample components. It ranges from a nonpolar liquid to aqueous buffers mixed with an organic solvent.
The solvent delivery system comprises a means of degassing, filtering and blending up to four solvents which are then delivered to the top of the column under pressure by a constant flow pump.
Liquid samples or solutions are introduced into the flowing mobile phase at the top of the column through a constant or variable volume loop and valve injector that is loaded with a syringe.
Columns are straight lengths of stainless steel tubing tightly packed with a microparticulate stationary phase. The column packings are chemically-modified silicas, unchemically-modified silica or polymeric resins or gels.
Solutes are detected in the mobile phase as they are eluted from the end of the column. The detector generates an electrical signal that can be amplified and presented in the form of a chromatogram of solute concentration as a function of time.
A dedicated microcomputer is an integral part of a modern high-performance liquid chromatograph. Software packages facilitate the control and monitoring of instrumental parameters, and the display and processing of data.
Related topics Principles of chromatography (D2) High-performance liquid chromatography: modes, procedures and applications (D7)
Principles
Mobile phase
Solvent delivery system
Sample injection
Column and stationary phase
Solute detection
Instrument control and data processing
Principles High-performance liquid chromatography (HPLC) is a separation technique where solutes migrate through a column containing a microparticulate stationary phase at rates dependent on their distribution ratios (Topic D2).
These are functions of the relative affinities of the solutes for the mobile and stationary phases, the elution order depending on the chemical nature of the solutes and the overall polarity of the two phases. Very small particles of stationary phase are essential for satisfactory chromatographic efficiency and resolution, and the mobile phase must consequently be pumped through the column, resulting in the generation of a considerable back-pressure. The compo-sition of the mobile phase is adjusted to elute all the sample components reason-ably quickly. Solutes eluted from the end of the column pass through a detector that responds to each one. There are a number of modes of HPLC enabling an extremely wide range of solute mixtures to be separated. The modes (Topic D7) are defined by the type of stationary phase and associated sorption mechanism.
A schematic diagram of a high-performance liquid chromatograph is shown in Figure 1. It consists of five major components:
● solvent delivery system;
● sample injection valve;
● column;
● detection and recording system;
● microcomputer with control and data-processing software.
These are described in the following sections.
Mobile phase The mobile phase, or eluent, is most frequently a blend of two miscible solvents that together provide adequate eluting power and resolution. These are
deter-He He
Fig. 1. Schematic diagram of a high-performance liquid chromatograph. Reproduced from A. Braithwaite & F.J. Smith, Chromatographic Methods, 5th edn, 1996, first published by Blackie Academic & Professional.
mined by its overall polarity, the polarity of the stationary phase and the nature of the sample components. Unlike a GC carrier gas, which plays no part in chromatographic retention and selectivity, the composition of an HPLC mobile phase is crucial in both respects. For normal-phase separations (stationary phase more polar than mobile phase), eluting power increases with increasing solvent polarity, whilst for reversed-phase separations (stationary phase less polar than mobile phase), eluting power decreases with increasing solvent polarity. An eluotropic series of solvents, which lists them in order of increasing polarity, is a useful guide to solvent selection for HPLC separations.
Table 1 is an example that also includes UV cut-off wavelengths as UV absorbancedetectors are the most widely used (vide infra). Elution can be under isocratic conditions (constant mobile phase composition) or a composition gradient can be generated by a gradient former to improve the resolution of complex mixtures, especially if the sample components have a wide range of polarities. The most widely used mobile phases for reversed-phase separations are mixtures of aqueous buffers with methanol, or water with acetonitrile. For normal-phase separations, which are less common, hydrocarbons blended with chlorinated solvents or alcohols are typical.
Table 1. An eluotropic series of solvents for HPLC
Solvent Solvent strength Solvent strength UV cut-off
parameter, e° parameter, p¢ (nm) (adsorption) (partition)
Ethanoic acid >1 4.4 255
Water >1 10.2 170
The mobile phase is either a single solvent or a blend of two to four solvents delivered at pressures of up to about 5000 psi (350 bar) with a constant and reproducible flow rate of <0.01–5 cm3 min-1. The solvent delivery system comprises the following components:
● A mechanical pump designed to deliver a pulse-free flow of mobile phase.
Most are single or dual piston reciprocating pumps (Fig. 2) with specially designed cams and pulse dampers to minimize or eliminate inherent flow variations, or one-shot pulseless syringe pumps used primarily with micro-bore columns (vide infra) requiring low flow rates. The wetted parts of the pump should be inert to all solvents (stainless steel, titanium, industrial sapphire, ruby, and Teflon being the principal choices) with minimal volume pumping chambers to facilitate rapid changes of mobile phase composition.
Solvent delivery system
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● Solvent reservoirs with in-line filters (2 mm porosity or less) for each solvent to remove dust and other particulate material. This reduces pump wear and protects the column from becoming clogged which results in increased back-pressures.
● A means of de-gassing the solvents to remove dissolved air. Air interferes with the detector response by forming bubbles in the flow-through cell as the pressure reduces to atmospheric at the end of the column. De-gassing is normally accomplished by bubbling helium through each solvent to displace the air, or by passing them through a commercial permeable-membrane de-gassing unit.
● A gradient former to generate binary, ternary or quaternary mixtures of solvents with a pre-programmed composition profile during a separation (gradient elution).
Sample injection Liquid samples and solutions are injected directly into the pressurized flowing mobile phase just ahead of the column using a stainless steel and Teflon valve fitted with an internal or external sample loop (Fig. 3). The loop, generally of between 0.5 and 20 ml capacity, is first filled or partially filled with sample from a syringe while the mobile phase flows directly to the column. By turning a handle to rotate the body of the valve, the mobile phase is diverted through the loop thus injecting the sample onto the top of the column without stopping the flow. A disposable guard column is sometimes positioned between the injector and the analytical column to protect the latter from a buildup of particulate matter and strongly retained matrix components from injected samples. It consists of a short length of column tubing, or a cartridge, packed with the same stationary phase as is in the analytical column.
Valve injection can easily be automated, controlled by computer software and used with autosamplers. For quantitative analysis, filled-loop injection has a relative precision of about 0.5%.
The column is where the separation process occurs and it is, therefore, the central component of a high-performance liquid chromatograph. There are two Column and
stationary phase
Common inlet
Common outlet
Fig. 2. A typical twin-headed reciprocating pump. Reproduced from W.J. Lough & I.W.
Wainer, High Performance Liquid Chromatography, 1996, first published by Blackie Academic
& Professional.
types of HPLC column, conventional and microbore, and a comparative summary is given in Table 2.
Microbore columns have three principal advantages over conventional columns, i.e.:
● solvent consumption is about 80% less because of the much lower mobile phase flow rate (10–100 ml min-1)
● the low volume flow rate makes them ideal for interfacing with a mass spectrometer (Topics F3 and F4)
● sensitivity is increased because solutes are more concentrated, which is especially useful if sample size is limited, e.g. for clinical specimens.
However, in practice, they are not as robust as conventional columns and are not necessary for many routine applications.
Columns are connected to the sample injection valve and the detector using short lengths of very narrow bore (~0.15 mm internal diameter) stainless steel or PEEK (polyether ether ketone) tubing to minimize dead-volume which contributes to band spreading in the mobile phase by diffusion.
HPLC stationary phases are predominantly chemically-modified silicas, unmodified silica or cross-linked co-polymers of styrene and divinyl benzene.
The surface of silica is polar and slightly acidic due to the presence of silanol (Si-OH) groups. It can be chemically modified with reagents, such as chlorosilanes, which react with the silanol groups replacing them with a range of other func-tionalities (Fig. 4(a)). The resulting bonded phases, which are hydrolytically stable through the formation of siloxane (SiæOæSiæC) bonds, have different chromatographic characteristics and selectivities to unmodified silica.
Octadecyl silica(ODS or C18) is the most widely used of all the stationary phases, being able to separate solutes of low, intermediate and high polarities.
Octyl and shorter alkyl chains are considered to be more suitable for polar solutes. Aminopropyl and cyanopropyl (nitrile) silicas are good replacements for unmodified silica, which can give variable retention times due to traces of water in the solvents. Polar, and especially basic solutes, tend to give tailing
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Injection
Fig. 3. Sample-injection valve and loop. (a) Sample-loading position; (b) sample-injection position.
peaks on bonded phase silicas because of adsorptive interactions with residual silanols and metallic impurities in the silica. The problem is reduced by end-capping, a process of blocking the sites with trimethylsilyl ((CH3)3-Si-) groups (Fig. 4(b)), and by using highly purified silica (< 1 ppm metals).
Size exclusion and ion-exchange stationary phases are either silica or polymer based. Sulphonic acid or quaternary ammonium groups provide cation and anion-exchange capabilities respectively, but slow rates of exchange leading to poor efficiencies and low sample capacities have limited their use, except for ion chromatography(Topic D7).
Chiralstationary phases have been developed for the separation of mixtures of enantiomers but are expensive and have a very limited working life.
The availability of a wide range of bonded phases together with polymeric materials, has resulted in the development of a number of modes of HPLC (Topic D7). The more important stationary phases and their characteristics are summarized in Table 3.
Table 2. A comparison of conventional and microbore HPLC columns
Conventional columns Microbore columns
Tubing Tubing
Stainless steel Stainless steel
Lengths 3, 10, 15, 20 and 25 cm Lengths 25 and 50 cm Coupled lengths 1 m or more
1⁄4≤ outside diameter 1⁄4≤ outside diameter
Internal diameter 4.6 mm Internal diameter 1 or 2 mm
Stationary phase (packing) Stationary phase (packing)
Porous, microparticulate silica, chemically-modified Porous, microparticulate silica, chemically-modified silica silica (bonded phases) or styrene/divinyl benzene (bonded phases) or styrene/divinyl benzene co-polymers co-polymers
Mean particle diameters 3, 5 or 10 mm with a narrow Mean particle diameters 3, 5 or 10mm with a narrow
range of particle sizes range of particle sizes
Operating pressures Operating pressures
500–3000 psi (35–215 bar) 1000–5000 psi (70–350 bar)
Typical mobile phases Typical mobile phases
Hydrocarbons + chlorinated solvents or alcohols for Hydrocarbons + chlorinated solvents or alcohols for normal-phase; methanol or acetonitrile + water or normal-phase; methanol or acetonitrile + water or aqueous buffers for reversed-phase aqueous buffers for reversed-phase
Flow rate 1–3 cm3 min-1 Flow rate 10–100ml min-1 Modified instrumentation
Solvent delivery system capable of accurate flow control down to 10ml min-1or less
Small volume sample injection valves Small volume detector cells
Performance Performance
Efficiency increases with diminishing particle size, Very efficient and sensitive, but slow but column life for 3 mm particles is shorter
Separations on 3 cm fast columns in less than Solvent consumption only a quarter that of conventional
1 minute columns
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Fig. 4. Formation of bonded-phase silicas. (a) Monomeric bonded phases; (b) End-capping of residual silanols. R = alkyl, aminoalkyl, ion-exchange groups.
Table 3. Stationary phases for HPLC
Stationary phase Sorption mechanism Characteristics
Unmodified silica, SiO2 Adsorption, normal-phase Polar, retention times variable due to adsorbed water
Bonded phases
Octadecyl silica, -C18H37(ODS or C18) Modified partition, reversed- Nonpolar, but unreacted silanol
Octyl silica, -C8H17 phase groups cause polar solutes,
Propyl silica, -C3H7 especially bases, to tail, pH range
limited to 2.5–7.5
All separate a very wide range of solutes
Aminopropyl, -C3H6NH2 Modified partition, normal or Polar, separates carbohydrates reversed phase pH range limited to 2.5–7.5
Sulphonic acid, -(CH2)nSO3H Cation-exchange Slow mass transfer broadens peaks, limited sample capacity, pH range limited to 2.5–7.5 for silica-based materials
Quaternary amine, -(CH2)nNR3OH Anion-exchange
Controlled-porosity silicas (some with Size exclusion Compatible with both organic and
–Si(CH3)3groups) aqueous solvents, pH range limited
to 2.5–10
a-, b-, g-cyclodextrin silicas Chiral selectivity based on Expensive, limited life, resolution adsorptive interactions sensitive to mobile phase
composition Polymer phases
Cross-linked styrene/divinyl benzene Modified partition, exclusion Nonpolar if unmodified, co-polymers, unmodified or with or ion-exchange stable over pH range 1–13 ion-exchange groups
Solute detection Detectors are based on a selective response for the solute, such as UV-absorbanceor fluorescence, or on a bulk property of the mobile phase which is modified by the solute, such as refractive index. Ideally, detectors should have the following characteristics:
● a rapid and reproducible response to solutes;
● high sensitivity, i.e. able to detect very low levels of solutes;
● stability in operation;
● a small volume cell to minimize band broadening, i.e. 8 ml or less for a conventional column, 1 ml or less for a microbore column;
● a signal directly proportional to solute concentration or mass over a wide range (linear dynamic range);
● insensitivity to changes in temperature and flow rate;
● a cell design that does not entrap air bubbles that outgas from the mobile phase at the end of the column.
Many types of detector have been investigated, and the most widely used are summarized below and in Table 4.
● UV-visible absorbance detector. This type, which is the most widely used, is based on the absorbance of UV or visible radiation in the range 190–800 nm by solute species containing chromophoric groups or structures (Topics E8 and E9). Detector cells are generally 1 mm diameter tubes with a 10 mm optical path length and designed so as to eliminate refractive index effects which can alter the measured absorbance. There are three types of UV-visible absorbance detector:
(i) Fixed-wavelength filter-photometers, which are the simplest, employing mercury-vapor lamp sources and optical filters to select a limited number of wavelengths, e.g. 254, 280, 334 and 436 nm, and a phototube detector.
They have a limited use, lacking versatility, but they are cheap.
(ii) Variable-wavelength spectrophotometers (Fig. 5) are much more
Table 4. Characteristics of HPLC detectors
Detector Sensitivity g cm-3 Linear range Characteristics
UV-visible absorbance Good sensitivity, most widely used, selective Filter-photometer 5 ¥ 10-10 104 for unsaturated groups and structures. Not Spectrophotometer 5 ¥ 10-10 105 significantly flow or temperature sensitive.
Diode-array Can be used with gradient elution.
Spectrometer >2 ¥ 10-10 105
Fluorescence 10-12 104 Excellent sensitivity, selective, including fluorescent derivatives. Not flow or temperature sensitive.
Refractive index 5 ¥ 10-7 104 Almost universal, but only moderate sensitivity.
Very temperature sensitive (control to ±0.001°C).
Cannot be used with gradient elution.
Electrochemical
Conductimetric 10-8 104 Flow and moderately temperature sensitive.
Amperometric 10-12 105 Cannot be used with gradient elution. Detects only ionic solutes. Excellent sensitivity, selective but problems with electrode contamination.
versatile as they allow monitoring at any wavelength within the working range of the detector to give the optimum response for each solute. They employ deuterium and tungsten lamp sources for the UV and visible regions, respectively, a diffraction grating monochromator for wave-length selection and a photomultiplier detector. Many are computer-controlled for programmable wavelength switching during a separation to optimize sensitivity and selectivity.
(iii) Photodiode-array detectors are spectrometers with fixed optics and a detection system consisting of one or two arrays of photodiodes on a silicon chip positioned to receive radiation dispersed by a diffraction grating (Fig. 6). Electronic scanning, digitizing and processing of the signals by a microcomputer enables ‘snapshots’ of the complete spec-trum of the flowing eluent to be collected and stored every 0.1 s. The spectra and the developing chromatogram at any wavelength can be displayed on a VDU screen in real time and subsequently shown as a 3-D color plot of absorbance, wavelength and time (Fig. 7). The data can be manipulated and re-plotted on the screen, and comparisons made with library spectra for identification purposes.
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Mirror
Reference photo diode
Flow cell Sample photo diode
Mirror
Entrance slit
Beam splitter Mirror Grating
Prealigned lamp
Fig. 5. UV-visible variable-wavelength spectrophotometric detector.
Concave holographic grating
Slit
Flow cell Aperture D2 lamp Lens
2 & 3
Shutter Lens 1
Diode array Spectrum
190–800 nm
Fig. 6. Diode-array detector (DAD).
● Fluorescence detectors are based on filter-fluorimeters or spectrofluori-meters. They are more selective and can be up to three orders of magnitude more sensitive than UV absorbance detectors. The detector responds selec-tively to naturally fluorescing solutes such as polynuclear aromatics, quino-lines, steroids and alkaloids, and to fluorescing derivatives of amines, amino acids and phenols with fluorogenic reagents such as dansyl chloride (5-(dimethylamino)-1-naphthalene sulfonic acid).
● Refractive index (RI) monitors are the closest to being universal HPLC detectors, as nearly all dissolved solutes alter the refractive index of the mobile phase. They are differential detectors, generating a signal that depends on the difference between the RI of the pure mobile phase and the modified value caused by the dissolved solute, which can, therefore, be positive or negative.
They are several orders of magnitude less sensitive than UV absorbance detectors, but are invaluable in the separation of saturated solutes such as carbohydrates, sugars and alkanes. They are highly temperature sensitive and are very difficult to use with gradient elution because the sample and reference cells cannot be continuously matched.
● Electrochemical detectors are based on measuring either the conductance of an aqueous mobile phase containing ionic solutes, or the current generated by the electrochemical reduction or oxidation of solutes at a fixed applied potential (amperometry) (Topic C9 ).
0.20
Fig. 7. 3-D display mode for a diode-array detector (DAD).
Conductance monitorsare used in ion chromatography, a mode of HPLC useful for separating low levels of inorganic and organic anions and cations by ion-exchange (Topic D7).
These aspects of HPLC closely parallel those described for GC (Topic D4).
Additional instrument parameters to be set and monitored include:
● solvent composition, flow rate and pressure limit;
● gradient elution programs;
● wavelength(s) and wavelength-switching for UV-visible absorbance detec-tors;
● wavelength range, sampling frequency and mode of display for a DAD.
Instrument control and data processing
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