Calibración de los modelos lluvia

Because of the similarities in the theory and practice of these two procedures, they will be considered together. Both are examples of partition chromatogra-phy. In paper chromatography, the cellulose support is extensively hydrated, so distribution of the analyte occurs between the immobilized water (sorbent) and the mobile developing solvent. The initial stationary liquid phase in thin-layer chromatography (TLC) is the solvent used to prepare the thin thin-layer of adsorbent. However, as developing solvent molecules move through the sorbent, polar solvent molecules may bind to the immobilized support and become the sorbent.

Preparation of the Sorbent

The support medium may be a sheet of cellulose or a glass or plastic plate cov-ered with a thin coating of silica gel, alumina, or cellulose. Large sheets of cellu-lose chromatography paper are available in different porosities. These may be cut to the appropriate size and used without further treatment. The paper should never be handled with bare fingers. Although thin-layer plates can easily be pre-pared, it is much more convenient to purchase ready-made plates. These are available in a variety of sizes, materials, and thicknesses of stationary support.

They are relatively inexpensive and have a more uniform support thickness than handmade plates.

Figure 5.2 outlines the application procedure. The sample to be analyzed is usually dissolved in a volatile solvent. A very small drop of solution is spotted onto the plate with a disposable microcapillary pipet and allowed to dry; then the spotting process is repeated by superimposing more drops on the original spot. The exact amount of sample applied is critical. There must be enough sam-ple so the developed spots can be detected, but overloading will lead to “tailing”

and lack of resolution. Finding the proper sample size is a matter of trial and error. It is usually recommended that two or three spots of different concentra-tions be applied for each sample tested. Spots should be applied along a very faint line drawn with a pencil and ruler. TLC plates should not be heavily scratched or marked. Identifying marks may be made on the top of the chro-matogram, where solvent does not reach.

D C

A B

TLC plate

Origin

Detection

Solvent development

Solvent front Solvent

C

B

w x

y For B, R_f = w

y For C, R_f = x y

FIGURE 5.2 The procedure of paper and thin-layer chromatography. A Application of the sample. B Setting plate in solvent chamber. C Movement of solvent by capillary action.

D Detection of separated components and calculation of Rf.

Solvent Development

A wide selection of solvent systems is available in the biochemical literature. If a new solvent system must be developed, a preliminary analysis must be done on the sample with a series of solvents. Solvents can be rapidly screened by develop-ing several small chromatograms in small sealed bottles containing the solvents. For the actual analysis, the sample should be run on a larger plate with appropriate standards in a development chamber (Figure 5.3). The chamber must be airtight and saturated with solvent vapors. Filter paper on two sides of the chamber, as shown in Figure 5.3, enhances vaporization of the solvent.

Paper chromatograms may be developed in either of two types of arrangements—ascending or descending solvent flow. Descending solvent flow leads to faster development because of assistance by gravity, and it can offer better resolution for compounds with small values because the solvent can be allowed to run off the paper. values cannot be determined under these conditions, but it is useful for qualitative separations.

Rf

Rf

12 * 6 cm2

Cover

Filter paper

Plate

Solvent FIGURE 5.3 A

typical chamber for paper and thin-layer chromatography.

Two-dimensional chromatography is used for especially difficult separa-tions. The chromatogram is developed in one direction by a solvent system, air dried, turned and developed in a second solvent system.

Detection and Measurement of Components

Unless the components in the sample are colored, their location on a chro-matogram will not be obvious after solvent development. Several methods can be used to locate the spots, including fluorescence, radioactivity, and treatment with chemicals that develop colors. Substances that are highly conjugated may be detected by fluorescence under a UV lamp. Chromatograms may be treated with different types of reagents to develop a color. Universal reagents produce a colored spot with any organic compound. When a solvent-developed plate is sprayed with concentrated and heated at for a few minutes, all organic substances appear as black spots. A more convenient universal reagent is The solvent-developed chromatogram is placed in an enclosed chamber containing a few crystals of The vapor reacts with most organic substances on the plate to produce brown spots. The spots are more intense with unsaturated compounds.

Specific reagentsreact with a particular class of compound. For example, rhodamine B is often used for visualization of lipids, ninhydrin for amino acids, and aniline phthalate for carbohydrates.

The position of each component of a mixture is quantified by calculating the distance traveled by the component relative to the distance traveled by the solvent. This is called relative mobility and symbolized by In Figure 5.2D, the values for components B and C are calculated. The for a substance is a constant for a certain set of experimental conditions. However, it varies with

R_f R_f

R_f. I₂

I₂. I₂.

100°C H₂SO₄

90°,

solvent, type of stationary support (paper, alumina, silica gel), temperature, humidity, and other environmental factors. values are always reported along with solvent and temperature.

Applications of Planar Chromatography

Thin-layer chromatography is now more widely used than paper chromatography.

In addition to its greater resolving power, TLC is faster and plates are available with several sorbents (cellulose, alumina, silica gel).

Partition chromatography as described in this section may be applied to two major types of problems: (1) identification of unknown samples, and (2) isolation of the components of a mixture. The first application is, by far, the more widely used.

Paper chromatography and TLC require only a minute sample size, the analysis is fast and inexpensive, and detection is straightforward. Unknown samples are applied to a plate along with appropriate standards, and the chromatogram is developed as a single experiment. In this way, any changes in experimental condi-tions (temperature, humidity, etc.) affect standards and unknowns to the same extent. It is then possible to compare the values directly.

Purified substances can be isolated from developed chromatograms; how-ever, only tiny amounts are present. In paper chromatography, the spot may be cut out with scissors and the piece of paper extracted with an appropriate sol-vent. Isolation of a substance from a TLC plate is accomplished by scraping the solid support from the region of the spot with a knife edge or razor blade and ex-tracting the sorbent with a solvent. “Preparative” thin-layer plates with a thick coating of sorbent (up to 2 mm) are especially useful because they have higher sample capacity.

R_f R_f

STUDY EXERCISE 5.1

A mixture containing five amino acids (Ala, Asp, Gly, Phe, Pro) was analyzed using two methods of planar chromatography, paper and cellulose-coated thin layer. The sol-vent system was n-propanol/water (70/30 v/v). Predict the order of the mobility of the amino acids (low to high ) on the chromatograms.

Solution: In cellulose planar chromatography, the amino acids interact with two phases, the sorbent (extensively hydrated cellulose, which is very polar) and the mobile phase (n-propanol/water, which is less polar than the sorbent). The more polar the amino acid, the stronger it will interact with the hydrated cellulose, thus the slower it will move with solvent during development (lower ). The order may be predicted by looking at the polarity of each amino acid side chain and arranging the amino acids in order of decreasing polarity. The correct order of migration (low to high ) is: Asp, Gly, Ala, Pro, Phe.

Rf

Rf

Rf

Rf

Advanced Planar Chromatography

The applications of planar chromatography listed above require only minimal equipment and supplies that are relatively inexpensive. It is likely that every un-dergraduate student majoring in biochemistry or molecular biology has

completed such an experiment in a science lab. If more complex, sophisticated chromatographic analyses are needed, such as in an academic or biotech research laboratory, then advanced equipment and specialized techniques must be used. Most new advances in planar chromatography have focused on TLC, as paper chromatography is very limited in its applications and flexibility. The major characteristics in TLC that have been improved include standardized methodology, instrumentation, and more effective stationary phases. These changes now make possible the advent of high-performance TLC (HPTLC).

One of the biggest problems with TLC analysis is that experimental condi-tions are difficult to duplicate. Separation of the components of a mixture is dependent on environmental conditions such as temperature, humidity, and extent of solvent saturation in the chamber. These conditions can be controlled by running the chromatography experiment in a specialized, commercially available enclosure called a plate development chamber. This leads to much more reproducible and standardized results.

Improvements are also being made in the development of new stationary phases. The most widely used stationary phase in TLC is silica, which separates molecules on the basis of polarity. (The more polar the component, the stronger it interacts with the very polar silica; hence, it migrates more slowly than a nonpo-lar component.) As silica separates primarily by pononpo-larity, this limits the types of molecules that can be separated. There is now strong interest in the development of reverse stationary phases such as C-18 functionalized silica to separate nonpo-lar molecules. Another limitation of silica is that it can be used only to separate biomolecules less than 2000-3000 in molecular weight. New and more porous stationary phases are being developed using photopolymerization techniques.

These new stationary phases are able to separate protein mixtures containing

insulin cytochrome lysozyme

and myoglobin