Principles of HPLC and Instrumentation

Liquid Chromatography (Figure 2.3) is an analytical technique that is used to separate a mixture in solution into its individual components. The separation relies on the use of two different phases or immiscible layers, one of which is held stationary while the other moves over it. The mobile phase is liquid. The separation occurs because, under an optimum set of conditions, each component in a mixture will interact with the two phases differently relative to the other components in the mixture. HPLC is the term used to describe liquid chromatography in which the liquid mobile phase is mechanically pumped through a column that contain the stationery phase. An HPLC instrument consists of: an injector, a pump, a column, a detector [48]

A recording of detectors’ response with time forms a chromatogram.

Figure 2.3: HPLC Instrumentation

The chromatogram contains the analytical data for the components of a mixture. Qualitative information appears in the characteristic retention time of each component. Quantitative information is contained in peak area.

Depending on the mechanism of separation there are the following types of Chromatography:

64

adsorption chromatography: separation is due to a series of adsorption/desorption steps. The stationary phase is solid.

ion-exchange chromatography: separation of ionic components of the mixture because they are electrostatically retained in different strength with converse ionic static phase groups

molecular exclusion chromatography: separation based on molecule size (molecular weight)

affinity chromatography: separation based on the highly selective interaction of a molecule of the mixture with a molecule chemically bounded (immobilized) to the solid static phase

partition chromatography: separation is based on solute partitioning between two liquid phases

Partition chromatography can divide into:

 Normal Phase: when liquid stationary phase more polar than liquid mobile phase, and

 Reversed Phase: when mobile phase is more polar than liquid stationary phase [17].

2.3.1.1 RP-UPLC

In this work we used a chromatographic system of Reversed Phase Ultra Performance Liquid Chromatography (RP-UPLC). UPLC enhance mainly in three areas: “speed, resolution and sensitivity. UPLC applicable for particle less than 2μm in diameter to acquire better resolution, speed, and sensitivity compared with HPLC. The separation and quantification in UPLC is done under very high pressure (up to 100M Pa). As compare to HPLC, under high pressure it is observed that not any negative influence on analytical column and also other components like time and solvent consumption is less in UPLC [49].

In RPLC the stationary phase (Figure 2.4) is less polar than the mobile phase and the interaction between analyte and the stationary phase has a predominantly hydrophobic (apolar) character. The most commonly used stationary phase in RPLC is silica gel in which octadecyl silica chains are

65

covalently bound to the free hydroxyl groups, indicated as a C18 phase. Other commonly used stationary phases are silica gels modified using octyl (indicated, e.g., as a C8 phase), hexyl, butyl, or ethyl groups. Occasionally organic polymer-based phases are also used. Modified silica gels may be used up to several hundred bars pressure and across a pH range of 2–8.

Care must be taken to select the right pH, as the chemically bound groups begin to hydrolyze at pH below 2 and the silica gel begins to dissolve at pH higher than 8–9. Retention of compounds occurs by apolar interaction between the analyte and the immobilized octadecyl silica chain. Most compounds exhibit hydrophobic character to some extent and thus they can be analyzed by RPLC. Even strongly polar or ionic substances can be analyzed by RPLC if the pH is adjusted so that the analyte will be in neutral form. The ability of RPLC to separate apolar and very polar analytes in one run makes it possible to determine pesticides simultaneously with their usually distinctly more polar metabolites [48]. The surface of C18 phases always contains unreacted silanol groups, which may form secondary polar interactions with the analyte. This is generally disadvantageous in RPLC as it often causes peak broadening. An important improvement is the introduction of the so-called end-capping procedure: The residual silanol groups in the C18 phase are reacted with monofunctional chlorosilane, which decreases surface polarity. This very popular stationary phase is called C18ec, where the notation “ec” stands for end-capped [50].

Mobile phases in RPLC are mostly polar solvents such as water, acetonitrile, methanol, and isopropanol. In RPLC apolar solvents have high solvent strength. Accordingly, the order of solvent strength is water<

acetonitrile<methanol<ethanol<acetone (from weak to strong). The most commonly used solvent mixture is a water–acetonitrile gradient, in which the amount of acetonitrile is increased during a chromatographic run to elute first the polar components and then the more strongly bound apolar compounds.

Mixtures containing a wide range of compounds may be studied by a fast gradient starting from high water content and ending at high acetonitrile content. RPLC is widely applicable, although pH control must often be

66

applied. Most important application areas include peptide and protein analysis (proteomics), drugs and their metabolites, fatty acids, and also volatile compounds such as aldehydes and ketones, although these require derivatization [50].

Figure 2.4: Typical RP stationary phase