Chemistry Reference
In-Depth Information
preserving the integrity of samples.
The analysis time for protein hydrolysates is 85 min using standard columns. For extra-
high resolution a high-resolution lithium cation exchange column is recommended which
achieves baseline separation of virtually all 40 amino acids (Fig. 1.4).
Reversed-phase chromatography
The most commonly used chromatographic mode in high performance liquid
chromatography is reversed-phase chromatography. Reversed-phase chromatography is
used for the analysis of a wide range of neutral compounds such as carbohydrates and
polar organic compounds. Most common reversed-phase chromatography is performed
using bonded silica-based columns, thus inherently limiting the operating pH range to
2.0-7.5. The wide pH range (0-14) of some columns (eg Dionex Ion Pac NSI and NS 1-
5 µcolumns) removes this limitation, and consequently they are ideally suited for ion-
pairing and ion-suppression reversed-phase chromatography, the two techniques which
have helped extend reverse-phase chromatography to ionisable compounds.
High-sensitivity detection of non-chromophoric organic ions can be achieved by
combining the power of suppressed conductivity detection with these columns.
Suppressed conductivity is usually a superior approach to using refractive index or low-
ultraviolet-wavelength detection.
Reversed-phase ion-pairing chromatography
Typically, reversed-phase ion-pairing chromatography is carried out using the same
stationary phase as reversed-phase chromatography. A hydrophobic ion of opposite
charge to the solute of interest is added to the mobile phase. Samples which are
determined by reversed-phase ion-pairing chromatography are ionic and thus capable of
forming an ion pair with the added counter ion. This form of reversed-phase
chromatography can be used for anion and cation separations and for the separation of
surfactants and other ionic types of organic molecules. An unfortunate drawback to using
silica-based columns is that ion-pairing reagents increase the solubility of silica in water,
leading to loss of bead integrity and drastically reducing column life. Some
manufacturers (eg Dionex) employ neutral macroporous resins, instead of silica, in an
attempt to widen the usable pH range and eliminate the effect of ion-pairing reagents.
The technique has been applied to the analysis of phosphorus-containing insecticides in
water. This technique has been used for the determination of free cyanide in non saline
waters.
Ion exclusion chromatography
The following anions have been determined by this technique.
Non saline
waters:
arsenate, arsenite, chloride, condensed phosphates, sulphate, sulphide, sulphite,
nitrite, phosphate. Also tartrate, glycolate, malate, malonate, carboxylates, citrate,
formate and fumarate.
Sea water:
silicate.
Potable
waters:
bicarbonate.
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