Analytical Protocols

7.4.1 Chromatographic Separation Techniques An Overview

Chromatography is a process of separating gases, liquids, or solids in a mixture or solution by adsorption. For example, this is done by selective adsorption on particle clay, silica gel, fine powdered sugar, alumina, or on paper. As an extraction mixture flows through the adsorbent medium or phase, often in a column, each substance in the sample is separated on the basis of differences in hydro-phobicity and/or ionic charges. Consequently, each substance appears in the adsorbent medium eluting from the column at a different time. This time difference is called the retention time (Rt); it represents the relative position of a compound(s) with respect to the origin (top) and the bottom of a column.

FIGURE 7.12A Small table-model gas chromatograph (GC) in Dr. Akira Okubo's laboratory at the University of Tokyo.

Primary methods of chromatography in use today include paper chroma-tography (PC), thin-layer chromatography (TLC), liquid column chromatogra-phy (LC), gas chromatography (GC) (Figure 7.12A-C), HPLC, fast protein liquid chromatography (FPLC), immobilized metal ion affinity chromatography, and antibody affinity chromatography.3-7 Colored products or bands are obtained which can be measured with a spectrophotometer, as with an enzyme reaction. Compounds eluting at particular times during HPLC can also be read spectro-

FIGURE 7.12B Hitachi gas chromatograph (GC)/mass spectrometer (MS) in Dr. Akira Okubo's laboratory at the University of Tokyo.

FIGURE 7.12C JEOL gas chromatograph (GC)/mass spectrometer (MS) in Dr. Akira Okubo's laboratory at the University of Tokyo.

photometrically at wavelengths equivalent to the maximum absorption peaks for such compounds. Compounds appearing at a particular Rt value during TLC can be quantified densitometrically or spectrophotometrically after scraping the compound(s) off the adsorbent phase. Highly volatile compounds (e.g., ethylene, ethane) that exist primarily in the gas phase can be subjected to gas chromatography using a flame ionization detector (FID). GC's also have other types of detectors for selective ion monitoring such as a thermal conductivity detector, or even a mass spectrometer (Figure 7.12B and C). Chromatographic Separation of Organic Molecules

Chromatography refers to the separation of chemical compounds by partitioning them between two phases, one of which is stationary and the other is in motion. In this process, the compounds to be separated are distributed between the stationary and mobile phases. This technique is very powerful because it allows one to separate very similar compounds, within a given extract, which may be isomers of each other. It is also faster, easier, and more economical.

There are three types of chromatography: (1) adsorption chromatography, (2) partition chromatography, and (3) gel permeation chromatography. TLC, gas-liquid chromatography (GLC), affinity chromatography (AC), HPLC, and gel-filtration chromatography are adaptations of these three basic types of chro-matography, explained in the following sections. Adsorption Chromatography

In adsorption chromatography, the compounds of interest are separated by allowing them to adsorb (bind) to the surface of a solid phase, such as DEAE cellulose (diethylaminoethyl cellulose) or charcoal. The compounds are des-orbed (removed) from the solid phase by an eluting solvent such as NaCl of varying concentrations (e.g., a linear gradient of increasing NaCl concentrations from 0 to 1.0 M) or by shifting the pH of the mobile phase to lower or higher pH's. The solid phase is poured as a slurry into a column fitted with sintered glass at its base. The column typically has a length-to-width ratio of 10:1 (of the actual poured solid phase). A filter paper disk is added to the top of the buffer-covered (ca. 1 cm of buffer) solid phase, over which the compounds of interest are gently layered without disturbing the solid phase. Elution is accomplished with the help of gravity and a gradient maker. A fraction collector can be used to collect 1-, 2-, 5-, or 10-ml fractions for later analysis of enzyme activity (using enzyme assays), salt composition and concentrations (using a conductivity meter or a spectrophotometer), and protein composition and concentrations (using Bradford7a or Lowry et al.7b protein assays in conjunction with a spectrophotometer).

One specialized type of adsorption chromatography is AC.8 In this case, a receptor, such as an antibody, is linked by covalent bonds to an inert solid support phase. The receptor has a high binding affinity for one of the compounds (its ligand) in the mixture of compounds from the prepared extract. Such binding is both specific and reversible. Inert solid support phases include cross-linked dextrans, cross-linked polyacrylamide, cellulose, and agarose. Due to its great selectivity, AC offers a very powerful means of achieving excellent separation and purification of biological molecules in as little as one step. Applications include purification of proteins (including antibodies and enzymes), nucleic acids, or any compound that acts as a ligand for a given bindable receptor. For example, some drugs, such as taxol, can be purified by using its monoclonal antibody as the bound receptor. After eluting unwanted compounds from the column, the bound ligand is then easily eluted from the column by shifting the mobile phase to a low, or in some cases, a high pH. This procedure can be done by immobilizing the ligand or by immobilizing the ligand's receptor to purify the ligand.

One hybrid modification of AC and ion exchange chromatography is high performance immobilized metal-ion affinity chromatography (HPIMAC) used to separate peptides and other organic molecules.7 It utilizes several types of stationary phases: synthetic polymers, silica, or cross-linked agarose. Basically, chromatographic separation involves a metal-ion chelator bound to the solid support phase. This in turn binds positively charged metal ions. The metal ions frequently used are Zn2+, Mg2+, and Cu2+. Negatively charged side groups on a given protein (or other negatively charged species such as polysaccharides, nucleotides, or nucleic acids) bind to the positively charged metal ions via ionic interactions. Nonbound compounds simply pass through the column and elute off, whereas the bound compounds stick to the solid phase until they are eluted off by use of pH shifts or a selective change in ionic strength.

Another variation of adsorption chromatography is ion-exchange chroma-tography. Here, the solid adsorbent phase has charged groups that are linked chemically to an inert solid matrix. What happens during the chromatography is that ions become electrostatically bound to the charged groups of the solid adsorbent. These ions may then be exchanged for ions in the mobile aqueous phase. This is accomplished by changing the ionic strength, or pH, of the eluting solvent. Two types of ion exchangers are used in ion exchange chromatography: (1) cation exchangers, which are exchangers with chemically bound negative charges, and (2) anion exchangers, which are exchangers with chemically bound positive charges. On the exchangers, the charges are balanced by counterions. For this purpose, chloride ions (Cl) are used for anion exchangers and positively charged metal ions are used for cation exchangers. To elute the molecule of interest from such ion exchange columns, one can use (1) changes in pH of the eluting buffer, (2) increasing ionic strength of salt (e.g., [NaCl] or [KCl] in solution, and (3) affinity selection, which depends on both charge (opposite to that of the bound macromolecule) and specific affinity for the bound macromol-ecule. Partition Chromatography

Partition chromatography, often called liquid-liquid partition chromatogra-phy, involves two liquid mobile phases. The substances to be analyzed are separated based on their different solubilities in the two liquid phases. An inert support is used in this type of chromatography. Examples of such inert supports include sheets of paper (cellulose) as used in paper chromatography, or a thin layer of silica gel (SiO2 • nH2O) or powdered alumina on a glass plate, as used in TLC.

Paper chromatography is usually carried out in a large glass tank or cabinet and involves either ascending or descending flow of the mobile phase solvents. Descending paper chromatography is faster due to gravity facilitating the flow of solvents. Large sheets of Whatman #1 or #2 filter paper (the latter is thicker) are cut into long strips (e.g., 22 x 56 cm long) for use in descending paper chromatography or a wide strip of paper (e.g., 25 cm wide) of variable height is used for ascending paper chromatography.

For descending liquid-paper chromatography, substances to be separated are applied as spots (e.g., 25 mm apart) along a horizontal pencil line placed down from the V-trough folded top of the paper. The V-trough folded paper is placed in a glass trough, held down by a glass rod, and when the tank has been equilibrated (vapor-saturated) with "running solvents" (mobile phase), the same solvent is added to the trough via a hole (later, stoppered shut) in the lid covering the chromatography tank. The lid is sealed onto the chamber with stopcock grease in order to make the chamber air-tight. After the mobile phase trails to the base of and off the paper sheet, the paper is hung to dry in a fume hood where it can then be sprayed with reagents (e.g., Ninhydrin reagent for amino acids) that give color to the separated compounds of interest in white or UV light. Some compounds of interest have their own distinctive colors, e.g., chlorophylls, and hence can be purified using this technique. In other cases, the dyes used to stain the location of the compound or protein causes irreversible covalent changes to the compound. In these cases, purification is not possible.

In ascending paper chromatography, the same basic set-up and principles apply with the exception that the mobile phase is placed at the bottom of the tank. Separation is achieved when the mobile phase travels up the paper via capillary action.

Another type of liquid-liquid partition chromatography is TLC which has several advantages over paper chromatography: (1) greater resolving power, (2) faster speed of separation, and (3) availability of a diverse array of adsorbents. The first two of these advantages are attributed to the fine particle size of the solid support adsorbent (less than 0.1 mm diameter particles) which allows more contact of this solid support with the compounds of interest as they travel up the plate. The adsorbents (e.g., silica gel, alumina, cellulose, and derivatives of cellulose) are available commercially on glass plates of various sizes. TLC plates are used in glass tanks, using ascending, or in some cases, descending chroma-tography. For the former, 1- to 10-pl samples of interest are spotted at 2- to 3-cm intervals across a line 15 to 20 mm from the base of the plate. The spots are allowed to dry. Then the plate is placed in the glass chromatography tank with the solvent previously placed in the bottom of the tank to a depth of 10 mm. Often it is necessary to equilibrate the vapor in the tank by placing filter paper around the sides of the tank. Next, a lid is sealed to the top of the tank with stopcock grease and the solvent is allowed to rise by capillary flow to the top of the plate. Once the mobile phase reaches the top of the plate, the plate is removed and allowed to dry. The spots are then developed with appropriate reagents for the types of compounds being separated and assessed. However, this procedure, as in paper chromatography, may result in sample destruction.

TLC can be run in two dimensions, using different solvent systems, as can paper chromatography, in order to allow for better separation of compounds. This procedure is very similar to 2-D electrophoresis. TLC is widely used to separate lipids, fructans, sugars, and hormones.

GLC is another type of partition chromatography where a high boiling point liquid is the stationary phase and an inert gas is the mobile phase. There is also an inert solid packing used in columns where these two phases are separated. Separation of compounds of interest is achieved due to differential solubility of the compounds in the mobile and stationary phases. Thus, as the carrier gas passes through the column, the compounds in the sample come off the column at different times (Rt's). A GLC apparatus basically consists of a tank of carrier gas (e.g., helium or nitrogen), an oven containing a coiled metal or glass chromatography column, a sample injection port, a detector (e.g., FID or thermal conductivity detector), and a recorder.

With FIDs, hydrogen gas is used to provide fuel for the flame. This is coupled to a flow of air to the detector to provide oxygen that allows the hydrogen to burn. A wire loop is positioned above the flame to detect compounds that pass from the column to the flame which, in turn, is connected to the recorder. FIDs are very sensitive to most organic compounds, but not to water, carbon monoxide, carbon dioxide, or the inert gases. Obviously, samples are destroyed when using this type of detector. Thus, GC is usually not used for purification of compounds. Thermal conductivity detectors, on the other hand, are less sensitive than an FID. However, they are nondestructive to the samples which allows one to completely recover a sample.

HPLC can also be grouped into this section on partition chromatography because it functions using the same principles previously discussed. It has two main advantages. First, it uses a pump to force the mobile phase through a given type of column at high pressure. The column can be made (or usually purchased) using any of the above-discussed solid absorbent phases. Hence, HPLC is commonly used to shorten the running times of any of the above types of chromatography which are usually time-restricted by gravity or capillary action. Second, because high pressure is used, much smaller adsorbent solid support particles can be used in the columns in conjunction with much smaller column volumes. Together, these two factors allow much better resolution of the compounds passing through the column due to the increased contact of the compound with the solid support adsorbent. FPLC works in the same manner as HPLC, but it makes use of specialized columns for use in protein purifications. The main disadvantage of HPLC or FPLC is the high cost of the specialized equipment. There is also the disadvantage of the adsorbent only being able to detect substituents on the surface of the compounds or proteins of interest, but this is a disadvantage in all forms of chromatog-raphy. Gel Filtration or Permeation Chromatography This type of chromatography is often called gel filtration chromatography. It involves the use of porous gel molecules of agarose, cross-linked dextran, or polymers of acrylamide, allowing the separation of compounds based on their molecular sizes/weights. One commercial series called Sephadex (Pharmacia Fine Chemicals, Inc.) is used for this purpose. These types of column packings must be hydrated before they are functional as a separation medium. The hydration process causes the pores in the Sephadex to swell to the appropriate size for the given Sephadex type. For example, G-10 Sephadex, during hydration, gains 1 ml of water per 1 g of dry gel; G-200 Sephadex gains 20 ml of water per 1 g of dry gel. Bio-gels from Bio-Rad laboratories consist of long polymers of acrylamide that are cross-linked to N,N'-ethylene-bis-acrylamide. These gels have a larger range of pore sizes than the Sephadex G series. Still another porous gel with an even wider pore size is agarose. It is made up of the neutral polysaccharide fraction from agar. Agarose and polyacrylamide are used to separate viruses, ribosomes, nucleic acids, and proteins. Sephadex is widely used in purification of proteins and in determining their molecular weights.

The general rationale for separation is as follows: (1) a gel having an appropriate pore size is chosen in relation to the size of the molecule of interest; (2) samples are added to the top of the gel column and are washed through using an appropriate mobile phase that is based on the solubility of the molecule of interest; (3) molecules that are too big to fit in the pores of the solid support will travel around the gel particles and hence elute first from the column; (4) molecules that fit in the pores will elute at different times according to their mobility through the gel pores.

Very fast, efficient separation of macromolecules is now possible by a technique termed capillary zone electrophoresis.9 This is not a form of chromatog-raphy, but deserves recognition as an extremely powerful technique for separating compounds of interest via electrophoresis. Basically, capillary electrophoresis (Figure 7.13A and B) utilizes small-bore open capillary tubes (e.g., 200 pm internal diameter) in a system equipped with a grounded highvoltage power supply, solvent reservoir in a Plexiglas® box connected to the capillary tube, a detector, a solvent reservoir after the detector, and a power supply for current flow to ground. Once a very small sample (e.g., nanogram quantity) is loaded into the capillary tube at one end, negatively charged species, such as the negatively charged side groups of proteins, are separated by the same mechanism as ordinary electrophoresis. The major advantage here is that the capillary tube has large surface-to-volume ratio allowing rapid dissipation of the heat produced by the electric current. Consequently, much higher voltages can be used in capillary zone electrophoresis than can be used in normal elec-trophoresis. High voltages in normal electrophoresis tend to cause heat convection within the gel. This results in distortions and blurring of the separation bands. High voltages in capillary zone electrophoresis allow much better resolution of related species of compounds as well as much faster running times. In addition, the capillary tube's inner surface is negatively charged and, thus, attracts positively charged species of molecules. As the buffer travels through the capillary tube via the electrical current, there is an electro-osmotic flow produced which carries these positively charged species of molecules in the same direction as the negatively charged species. Hence, both negatively and positively charged species can be separated and analyzed at the same time. Capillary zone electrophoresis utilizes several types of detectors including spectrophotometers, mass spectrometers (MS), electrochemical detectors, and radiometric detectors instead of the cumbersome stains used in ordinary electrophoresis.

FIGURE 7.13A Waters capillary electrophoresis apparatus in Dr. Akira Okubo's laboratory at the University of Tokyo.

FIGURE 7.13B JASCO capillary electrophoresis apparatus in Dr. Akira Okubo's laboratory at the University of Tokyo. Use of Mass Spectrometry to Identify Biologically Important Molecules

In order to analyze the compound(s) or protein(s) of interest, one must be able to unambiguously identify their positions or retention times (Rts) on or through the adsorbent phase used in any given type of chromatography. For example, HPLC chart recordings produced by a chart recorder monitoring a spectropho-tometer during a sample run will produce a number of peaks whose identities are not necessarily known. In order to determine which peak is the compound or protein of interest, known standards are run on HPLC prior to running the collected sample. This will determine the Rt of the compound or protein of interest. The Rt is the time, in minutes, on the chart recording where a given peak of interest occurs. Unknown compounds whose Rt's are similar to those of the standards can be tentatively identified using multiple forms of chroma-tography, but MS (Figure 7.14A and B) or nuclear magnetic resonance (NMR) analysis of the collected unknown peak must be performed in order to unambiguously identify the compound of interest. A cogent application is the characterization of taxol by MS by McClure et al. (1992)10 and NMR by Falzone et al. (1992).11

FIGURE 7.14A Bruker TOF-mass spectrometer (MS) in Dr. Akira Okubo's laboratory at the University of Tokyo.

FIGURE 7.14B Hitachi liquid chromatography (LC)-mass spectrometer (MS) in Dr. Akira Okubo's laboratory at the University of Tokyo.

The amount of the compound of interest under a given peak on a chromato-gram is determined by measuring the area under the peak for this compound. Several methods can be used.

1. Measure one-half the peak height times its width at the base and compare with the areas of standard peaks analyzed in the same manner to produce a standard curve (see Figure 7.11). The advantage of this technique is that it is very easy. However, only very crude estimations of the area, and hence concentration, are obtainable using this method.

2. Cut out the curve on the chart paper and weigh it in comparison to the weight of known standard peaks. This procedure is slightly more accurate than method 1 — but only if the paper is of uniform density.

3. Integrate the area under the peak using an electronic integrator/scanner (see Figure 7.10A for a view of the Shimadzu C-R4A Chromatopac electronic integrator used in our lab). This is by far the most accurate of these procedures because computer-controlled integrators can implement very complicated mathematical algorithms for integration, giving very accurate results. This procedure also has the advantage of requiring no mathematical manipulations by the researcher.

It is very difficult to say exactly what precise compound is contained in a given peak from a purified extract that shows up from HPLC. MS is very useful for clarifying such ambiguities. MS functions by bombarding a compound with high-energy electrons (or other particles), causing the loss of an electron from each molecule, yielding molecular ions. If the energy of the electron beam is high enough, the molecular ions will have enough excess vibrational and electronic energy to break the molecules into various positive and negative ion fragments. These ion fragments are then passed through a very strong magnetic field which, depending on the charge of the fragment, deflects the fragments at an angle into a detector. This results in a spectrum of the compound's ion fragments. Every compound has its own unique mass spectrum. A good example is the mass spectrum for taxol which has been determined by fast atom bombardment (FAB) MS.10 In this study, three ion series are observed: (1) the M-series which are characteristic of the intact taxol molecule; (2) the T-series whose ion fragments are derived from the taxane ring, and (3) the S-series that represents the C-13 side chain of taxol (see Figure 7.15 derived from McClure et al. [1992]).10

GC can be combined with MS to first separate the compounds of interest in an extract according to their boiling points. Then, as each compound comes through the MS, it is broken up by the particle beam and produces a new mass spectrum. The mass spectrum for each compound is then compared to the known library of mass spectra that can be easily accessed from a computer to tell you exactly what your compound is with no ambiguity.


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