Liquid chromatography mobility separation

Fig. 9. Conjoint Liquid Chromatography (CLC). Separation of proteins from mouse ascites and isolation of monoclonal antibody IgG in one step obtained by a combination of CIM QA and CIM Protein A Disks. Conditions Separation mode CLC (first disk CIM QA, 12 x 3 mm ID, 0.34 ml second disk - CIM Protein A, 12 x 3 mm ID, 0.34 ml, inserted in monolithic column housing) Instrumentation Gradient HPLC system with extra low dead volume mixing chamber Sample Mouse ascites Injection volume 20 pL Mobile Phase Buffer A 20 mM Tris-HCl, pH 7.4 Buffer B Buffer A + 1 M NaCl Buffer C 0.1 M Acetic acid Conditions Gradient 0 - 50 % B in 50 s, 100% A for 40 s, 100% C for 30 s Flow Rate 4 ml/min Detection UV at 280 nm... 

HPLC is a form of liquid chromatography, where separation (or partition) occurs between a mobile phase (the solvent) and a stationary phase (the column packing). It is the ability with which the sample constituents will distribute themselves between the two phases that will effect the separation. Depending on the nature of the stationary phase, the separation process can be of four different modes ... 

A wide range of stabilizers in plastics is identified commercially using high performance liquid chromatography, a separation technique based on the distrihu-tion of compounds between two phases known as the stationary phase and mohUe phase. The stationary phase comprises a thin layer created on the surface of fine particles and the mobile phase comprises the liquid flowing over the particles. Each component in a sample has a different distribution equilibrium depending on its solubility in the phases and/or molecular size. As a result, the components move at different speeds over the stationary phase and are thereby separated from each other. The sample is dissolved using a suitable solvent, a non solvent such as methanol is added, and the extract presented to the HPLC. 

The occurence of the complete adsorption of macromolecules forms a base of the full adsorption-desorption liquid chromatography-like separation method, FAD (Berek and Nguyen, 1998). A very weak mobile phase is employed, which acts as an adsorb for n-1 sample constituents. They are completely retained within the appropriate full adsorption-desorption column packed with nonporous particles. The unretained sample constituent is directly forwarded to an online SEC column for its molecular characterization. In the next stage, eluent strength is stepwise increased and sample constituents are successively one-by-one desorbed and forwarded into the SEC column. Successful separation of up to six distinct polymers with help of FAD method was reported. In the FAD method, solvents are chosen so that they always act as a desorb just for one sample constituent. 

Capillary electrochromatography (CEC) is a hybrid of CE and liquid chromatography. The separation is carried out in capillaries that are either fully packed with octadecyT(ODS)-silica particles or partially packed having an open segment. The separation of the analytes is based on the difference in the magnitude of the distribution between the mobile and stationary phase. The flow of mobile phases is generated... 

However, to increase separation efficiency, high velocities and small-diameter (<10pm) particles are essential as the stationary phase. Unfortunately, this leads to an increase in back-pressure. To overcome this back-pressure, a pump is necessary for transport of the mobile phase (high-pressure- or high-performance liquid chromatography, HPLC). Separation is performed in a closed system. 

Analytical separations may be classified in three ways by the physical state of the mobile phase and stationary phase by the method of contact between the mobile phase and stationary phase or by the chemical or physical mechanism responsible for separating the sample s constituents. The mobile phase is usually a liquid or a gas, and the stationary phase, when present, is a solid or a liquid film coated on a solid surface. Chromatographic techniques are often named by listing the type of mobile phase, followed by the type of stationary phase. Thus, in gas-liquid chromatography the mobile phase is a gas and the stationary phase is a liquid. If only one phase is indicated, as in gas chromatography, it is assumed to be the mobile phase. 

For most samples liquid-solid chromatography does not offer any special advantages over liquid-liquid chromatography (LLC). One exception is for the analysis of isomers, where LLC excels. Figure 12.32 shows a typical LSC separation of two amphetamines on a silica column using an 80 20 mixture of methylene chloride and methanol containing 1% NH4OH as a mobile phase. Nonpolar stationary phases, such as charcoal-based absorbents, also may be used. 

Liquid chromatography is complementary to gas chromatography because samples that cannot be easily handled in the gas phase, such as nonvolatile compounds or thermally unstable ones, eg, many natural products, pharmaceuticals, and biomacromolecules, are separable by partitioning between a Hquid mobile phase and a stationary phase, often at ambient temperature. Developments in the technology of Ic have led to many separations, done by gc in the past, to be carried out by Hquid chromatography. 

The model was tested by the micellar liquid chromatography separ ation of the five rarbornicin derivatives and four ethers of hydroxybenzoic acid. Micellar mobile phases were made with the sodium dodecylsulfate and 1-pentanol or isopentanol as modifier. In all cases the negative signs of the coefficients x and y indicate that at transition of the sorbat from the mobile on the stationar y phase the number of surfactant monomers as well as the number of modifier molecules increases in its microenvironment. 

The application of pressure to the liquid phase in liquid chromatography generally increases the separation (see HPLC). Also in PIC improved efficiency of the column is observed if pressure is applied to the mobile phase (Wittmer, Nuessle and Haney Anal Chem 47 1422 1975). 

D. E. Martire, Unified Approach to the Theory of Chromatography Incompressible Binary Mobile Phase (Liquid Chromatography) in Theoretical Advancement in Chromatography and Related Separation Techniques (Ed. F. Dondi, G. Guiochon, IGuwer, Academic Publishers, Dordrecht, The Netherlands,(l993)261. 

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