TOP

Introduction to HPLC and Ion Chromatography

HPLC and Ion Chromatography are a mature and established techniques, with inbuilt flexibilities to isolate, detect and measure billions of compounds (analytes) in a wide variety of sample matrices.  These techniques are used within a wide variety of analytical chemistry laboratories, such as within the following sectors:-





Theory


HPLC (high performance liquid chromatography) and Ion Chromatography (IC) are accomplished by the injection of a small amount of a liquid mixture into a moving stream of liquid (called the mobile phase), which passes through a column packed with particles of stationary phase, under high pressure.


In a simple example, component A has the same interaction with the stationary phase as the mobile phase.  Component B has a stronger interaction with the stationary phase than it has with the mobile phase.  After a mixture of both A and B has been injected into the column, these components will be forced through the column by the flow of the mobile phase.  Molecules of component A will interact with the stationary phase and adsorb to it in the same way that the mobile phase does.  Hence component A will move through or elute through the column with the same speed as the mobile phase.  


Because molecules of the component B are strongly adsorbed onto the stationary phase they will interact with the stationary phase for much longer than component A does.  Hence, component B will move through the column at a slower rate than the mobile phase.


The presence of each component is detected by a signal to a detector, after they have passed (eluted) through the column.  A detector is a device that senses the presence of components different from the liquid mobile phase and converts that information to an electrical signal.  Different types of detectors can be used in HPLC/IC, the type depends on which physical/chemical property of the analytes may be taken advantage of.


Hence compounds A and B, will exit the column at different times i.e., they have different retention times.  The retention time is the time between injection and detection.


The detector will pass this signal information to PC or Mac chromatography software, or to a printer plotter or to an integrator.


A plot of the analyte signal detected against retention time is a chromatogram.


The figure below represents the general shape of the chromatogram for the mixture of A and B.



Qualitative analysis is achieved by comparing the retention time of peaks in a sample to those of a known standard. Quantitative analysis is achieved by measurement of the areas or heights of the peaks in the chromatogram.  


The analyses of mixtures of widely varying composition frequently leads to very wide retention time ranges.  Sometimes the retention time is too long, or the components do not separate effectively from each other.  Hence different types of elution have been established, these include isocratic and gradient.


In isocratic elution, compounds are eluted using constant single mobile phase.


In gradient elution, components are eluted by increasing the strength of the mobile phase.  Two (binary gradient), three (ternary gradient) or four (quaternary gradient) different mobile phases can be used.  In practice most analytical methods use a maximum of two different mobile phases, a few, use three and only a very rare number use four.


With gradient elution, successively eluted compounds emerge from a column more rapidly than they would under conditions in which the solvent was not varied.


The interaction of the analyte with mobile and stationary phases can be manipulated through different choices of both mobile and stationary phases.  As a result, HPLC/IC acquires a high degree of versatility, and thus has the ability to easily separate a wide variety of chemical mixtures.


A typical HPLC/IC system consists of:-
































Samples


Samples must be in a liquid form.  So solids may need to be dissolved or melted, and gas samples need to be condensed or solubilised into liquid states.


In addition, samples may need pre-treated by a variety of techniques to isolate the analyte of interest from the remainder of the sample, or to reduce the levels of interference from other analytes.  Derivatisation may also be required to render the analyte of interest, detectable to the required detector.



Columns


There is a bewildering array of thousands of different HPLC/IC columns available, but it is helpful to remember that most columns fall into the following main category types:-



There are some column categories which will use a combination of separation modes, such as HILIC, for use with highly hydrophilic analytes.


The column on which the intended separation is performed is the analytical column.  In order to preserve the life of the relatively expensive analytical column, a cheaper guard column is often used, so that the injected sample may first pass through the guard column, which will filter out extraneous material, before the sample reaches the analytical column.



Column Heater/Chillers


Maintaining analytical columns at constant temperatures, helps to ensure that retention times are reproducible.  Constant temperatures cannot usually be maintained if the column is kept at ambient temperatures.  Column Heater/Chillers or ovens provide constant and reproducible temperature control.  In addition, in general, faster separations will occur when the temperature of the analytical column is increased, and vice versa at lower temperatures.  


There are some HPLC analyses, where temperature programming over the course of each elution is desirable.



Autosamplers


Large numbers of samples can be analysed by HPLC/IC without user intervention by the use of automatic sample injectors or autosamplers.  Autosamplers are also very useful as their injection volume and technique is exactly the same for each required injection, hence they provide a large degree of injection volume precision.



Detectors


There are many types of detectors that can be used with HPLC/IC.  The more common detectors include Refractive Index (RI), Ultra-Violet/Visible (UV/Vis), Conductivity, Fluorescence, Radiochemical, Electrochemical, Near-Infra Red (Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR), and Light Scattering (LS).


If the analytes are expected to fluoresce, then a fluorescence detector may be considered.  In the case of analytes which have an ionic charge, a conductivity detector may be useful.  If the analytes are able to undergo significant oxidation/reduction reactions in an electrical current, an electrochemical detector may be used.  If the analytes have a UV/Visible chromophore or if a chromophore can be added, a UV/Visible detector may be of enormous value.  If each analyte molecule is large, a refractive index detector or a light scattering detector may be considered.  Use of a charged aerosol detector may be considered for non-volatile analytes.  The use of a mass spectrometer may also be considered for a wide range of analytes.


The choice of mobile phase is a factor which must always be considered when selecting a detector type.



Anion suppression


In the case of the conductivity detection of anions, using a conductivity detector, an anion suppressor is often used.  Many mobile phases used in the conductivity detection of anions contain sodium bicarbonate and sodium carbonate.  Sodium bicarbonate and sodium carbonate themselves contain anions groups, so will interfere with a chromatogram by increasing the level of the baseline.  An anion suppressor, removes sodium bicarbonate and sodium carbonate from the mobile phase, before the mobile phase enters the conductivity detector.  Hence the baseline of chromatograms remains at a relatively low level of conductivity.  Anion suppression is necessary, when performing high sensitivity measurements, using sodium bicarbonate and sodium carbonate mobile phases.



Pumps


In HPLC/IC the pump which is used to deliver the mobile phase at a uniform rate, operates at very high pressures to around 40 MPa.  Different pumps will operate at different ranges of flow rate capabilities, of a few microlitres per minute at the capillary scale, to the preparative of litres per minute.


Gradient elutions may be achieved by use of high pressure mixing systems, where individual isocratic high pressure pumps are used to provide the proportions of each mobile phase.  All the mobile phases are then blended under high pressure, into a homogenous mixture, before being introduced to the column.


As an alternative, low pressure mixing systems, may be used, where a single pump, with up to four channels, will blend proportions of up to four different mobile phases under low pressure and then pump that homogenous mixture onto a column.


The main advantage of low pressure mixing is that it offers the ability to blend two or more mobile phases with one pump, hence a lower purchase price than for high pressure mixing.


However, high pressure mixing does offer rapid proportion response and a more precise creation and control of gradients.



Degassing


Mobile phase needs to be free of air before it reaches the pump/column.  Users have three main options for degassing the mobile phase:-


Sonication, by placing containers of mobile phase into ultrasonic water baths and sonicating until the gas is deemed to have escaped the container.  This method is off-line and time consuming.


Sparging with an Inert Gas, here the mobile phase is sparged with an inert gas such as helium  This off-line method is very time consuming


On-line degassers, these are machines which will remove gases by vacuum, from the mobile phase just before the mobile phase enters the pump.  They are very efficient.  The mobile phase is degassed as and when is required.



Software


This should be able to acquire, manipulate and process chromatograms.  From the data, calibration curves and automatic calculation of unknown quantities in samples should be possible.


Often users require traceability of the acquisition and any modification changes to a chromatogram.  FDA 21 CFR part 11 compliance of the software, is used to this end.


Sometimes, automatic transfer of the reported chromatographic data to a LIMS system is required.


Software should be as easy to use and as robust as possible.