Ion Chromatography is the separation of positively and negatively charged ions by HPLC. Positively charge ions are known as Cations, and negatively charged ions are known as Anions.
To achieve separation, ions of the opposite charge are bonded to the column packing material, and the various ions in the sample travel through the column at different speeds, causing a separation to occur. Hence it follows that separate columns are required for anions and for cations.
Ions are detected using a conductivity detector. Since the eluent must be conducting, we are measuing a change in conductivity when a peak is eluted. Typically we are looking at very small changes against a very large background signal. However because there is very little noise, sensitivity is very high, and detection down to femtogram levels may be possible. Cations analysis is usually more sensitive than anion analysis.
Multivalent ions may need a complexing agent, such as dipicolinic acid, to allow them to be analysed in the same chromatogram as monovalent ions (eg sodium(+) and magnesium (2+))
All ions have an Equivalent Ionic Conductivity. This is a measure of how much they conduct electricity at a given concentration.
Ionic conductivities usually fall in the range 35-80 S cm2 /mol, with the exceptions of H+ (350 Scm2mol-1) and OH- (198Scm2/mol). Amongst the lowest, and hence the best eluents, are salicylate (30), benzoate (32) H2PO4- (33) (cf HPO42- present at higher pH (57)) N(Et)4+ (33) propionate (36) phthalate (38) and acetate (41).
Anions are usually analysed using a very low conducting eluent, while cations are almost invariably analysed using an acid eluent (containing H+, hence very high conductivity). For cation analysis, all peaks are therefore negative, and it is necessary to invert the detector signal to get positive peaks. We must be careful though with cation analysis. Sometimes the eluent concentration required for good elution times, gives rise to a conductivity that can exceed the maximum detectable by the detector.
Temperature does not make a huge difference to the elution times in ion chromatography, affecting monovalent ions much more than divalent or multivalent ions. However it is also very important to maintain very stable temperature control because the conductivity can vary by +/-2% per degree centigrade. Hence the effect of temperature change is primarily to affect the quantitation of the peaks.
Eluent choice in ion chromatography. Apart from the considerations of Equivalent Ionic Conductivity, the higher the eluent buffer concentration, the faster the peaks elute. Different eluents will give different selectivity, so for cations for example, it may be worth trying different acids to optimise selectivity. If organic species might also be present, or for analysing organic ions, it may help to add a low percentage of acetonitrile to the eluent to help with solubility and to reduce any hydrophobic interaction with the column.
Column loading capacity is much lower than with reversed phase HPLC. Normal HPLC samples will require at least a 1:25 dilution for IC. Column overload is indicated by triangular peaks looking like a sharks fin.The injection volume should be 10ul or less, and the concentration around 50ppm or less.
Dual column technology. There are two methods of reducing the background conductivity. The conventional method is Electronic Suppression, which is effectively a switch which acts like the Autozero button on a UV detector. The baseline is allowed to become totally stable and the the switch is activated, reducing the conductivity reading to zero. This is the method of choice for cation analysis. However the dual column technique offers significant advantages for anions. A second column is fitted between the analytical column and the detector, which exchanges certain ions in the eluent for those of lower conductivity, thus increasing sensitivity. To use this technique, it is necessary to rethink the eluent that is to be used, making it compatible with such a post column exchange process. A common example is to use (for example) sodium hydrogen carbonate as eluent. In the suppressor column, the sodium is exchanged for H+, forming carbonic acid (present mainly as CO2 and H2O) and which gives rise to very low conductivity. If using a suppressor system, a column should be selected which has been optimised for use with this type of eluent. Note that suppressor columns need to be periodically regenerated, and automated systems are available, using two suppressor columns, so that one can be regenerated while the other is in use.
Calibration curves in IC are almost invariably linear using electronic suppression, so single point calibration is normal. However with a suppressor column, a quadratic calibration curve is to be expected, and multipoint calibration is therefore required.
Column lifetime should be in excess of 1000 injections if using clean samples. To achieve the best lifetime for IC columns, remove traces of protein, remove hydrophobic contaminants, filter to remove particles, and use a guard column.
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