Chapter 5 - Introduction - Page 141

Ever since the beginning of the 20th century, when Tswett first described the separation of chlorophylls in a liquid phase using a device filled with porous inorganic particles, and named the method chromatography (Tswett, 1906), chromatographers have been using columns packed with stationary phases in the shape of particles. Obviously, the column technology has been perfected during the following more than 110 years, and columns packed with particles are the “workhorse” of current liquid chromatography. The concept is simple: The mobile phase is pumped through the packed column and flows through the interstitial spaces that are, by default, always present between the particles.
The vast majority of chromatographic packings are porous, with most of the interactive sites being presented on the pore surface, not at the external surface of the particles. The interactive sites are instrumental for attaining the separation, and the separated compounds have to reach them to achieve the separation. The pores inside the particle are filled with the mobile phase, which does not move and is stagnant. After injection of the sample in the stream of the mobile phase that is pushed through the voids between the packed particles, the difference between concentration of the separated compounds in the mobile phase and in the stagnant liquid in the pores is the driving force for transport of thes compounds in the pores. Once the compound interacts with the interactive sites, its concentration in the stagnant liquid decreases and the concentration gradient drives more compounds to enter the pores.
The mechanism of this transport is diffusion, which is described by Fick’s second law:
(5.1)
where 4 is the concentration of the substance, t is the time, x is the position, and D is the diffusion
coefficient defined by the StokeseEinstein equation:
(5.2)
where kB is the Boltzmann constant, T is the temperature, h is the viscosity, and r is the radius of the diffusing entity. Eq. (5.1) indicates that the rate of diffusion depends on the diffusion coefficient D, which, according to Eq. (5.2), is specific for each compound and can only be enhanced by an increase in temperature or a decrease in viscosity of the mobile phase. These two effects are often interrelated because the viscosity of many liquids decreases with an increase in temperature. The size of the separated compound is given. The larger the molecule, the smaller the diffusion constant and the lower the overall diffusion rate. Hence, the diffusion of large molecules, such as proteins, nucleic acids, viruses, and synthetic polymers, is significantly slower than that observed for much smaller molecules including gases, ions, and small organic compounds.

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Figure 5.1

Differential pore size distribution plots of the poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads and monolith prepared from the same polymerization mixture consisting of glycidyl methacrylate (24%), ethylene dimethacrylate (15%), cyclohexanol (48%), dodecanol (12%), and azo-bis-isobutyronitrile (1% with respect to monomers) at a temperature of 70^oC.

Figure 5.2

Fast reversed-phase separation of proteins. Conditions: Poly(styrene-co-divinylbenzene) monolithic column 50 x 4.6 mm I.D., mobile phase gradient 42%-90% acetonitrile in 0.15% aqueous trifluoroacetic acid in 0.35 min, flow rate 10 mL/min, UV detection at 280 nm. Peaks: ribonuclease (1), cytochrome c (2), bovine serum albumin (3), carbonic anhydrase (4), and ovalbumin (5).

Figure 5.3

The separation of crude products from synthesis of triazole (m/z 176) using the mobile phase ethyl acetate n-hexane (1:2 v/v) with 2% acetic acid and presented as ultrathin layer chromatography (UTLC)-UV densitogram of the synthesis sample (top). Identification of peaks was achieved using atmospheric pressure matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) spectra of the by-product A (m/z 369) (center) and the product (m/z 198 [M+Na]⁺ and m/z 107) (bottom). The main matrix ions are marked with asterisks.

Figure 5.4

Scanning electron microcope images illustrating polyacrylonitrile layer prepared “conventional” electrospinning (A) and aligned electrospun polyacrylonitrile nanofibers generated on the rotating collector at rotational speeds of 1250 rpm (B).

Figure 5.5

Comparison of chromatograms showing the separation of (1) benzo[a]pyrene, (2) chrysene, (3) pyrene, (4) fluoranthene, and (5) phenanthrene using 0.5% multiwall carbon nanotubes-polyacrylonitrile plates, (A) 0.5% edge-plane carbon-polyacrylonitrile plates (B) using mobile phase 60:40 acetonitrile-water, and pure polyacrylonitrile plates (C) using mobile phase 70:30 acetonitrile-water mobile phase.

Figure 5.6

Matrix enhanced surface-assisted laser desorption/ ionization time-of-flight mass spectrum of angiotensin I using the poly(vinyl alcohol) substrate. An amount of 800 amol of angiotensin I was applied, and the CHCA matrix concentration was 0.1 mg/mL. The spectrum is the sum of 100 laser shots.

Figure 5.7

Mask used to prepare monoliths with a circular shape via photopolymerization of butyl methacrylate and ethylene dimethacrylate on the top face of a matrix-assisted laser desorption/ionization (MALDI) plate (A), top view of the MALDI plate with monoliths (B), optical micrograph of two adjacent monolithic spots (C), and scanning electron microscope micrograph of macroporous structure of monolith C (D).

Figure 5.8

Mold consisting of two glass plates separated by Teflon strips located along the long side used for the preparation of monolithic layer (A), the mold containing the white monolithic layer (B), scanning electron microscope image of the cross section of the monolith (C), and detailed view of the morphology of the monolithic layer (D).

Overview of the Contents:

The Handbook of Advanced Chromatography /Mass Spectrometry Techniques is a compendium of new and advanced analytical techniques that have been developed in recent years for analysis of all types of molecules in a variety of complex matrices, from foods to fuel to pharmaceuticals and more. Focusing on areas that are becoming widely used or growing rapidly, this is a comprehensive volume that describes both theoretical and practical aspects of advanced methods for analysis. Written by authors who have published the foundational works in the field, the chapters have an emphasis on lipids, but reach a broader audience by including advanced analytical techniques applied to a variety of fields.

Chapter 5 - Introduction - Page 141

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