Paper chromatography mobile phases

Chromatography is one of the most powerful and widely used means for separating mixtures, because it is often inexpensive and it can be used to provide quantitative as well as qualitative information. The simplest method is paper chromatography. A drop of solution is placed near the bottom edge of the stationary phase, an absorbant support, such as a strip of paper. The mobile phase, a fluid solvent, is added below the spot and the solvent is absorbed on the support. As the mobile phase rises up the stationary phase by capillary... 

The principles of paper chromatography are similar to those of column chromatography discussed in Chapters 14 to 18. The main difference is that a piece of paper is used for the inert phase. The solution to be examined is deposited as a small spot on the paper. The mobile phase is allowed to move over the spot in a definite direction, and the substances are separated by their differences in solubility in the moving solvent and the stationary phase, which generally is considered to be the water normally present in paper. Isaac Asimov, the well-known science fiction writer. 

Paper chromatography is the simplest of the chromatographic techniques, and is often used to separate dyes in a mixture. In this technique the stationary phase is made up of water molecules that are trapped in the cellulose fibres of paper. The mobile phase is the aqueous or organic solvent that moves up the paper by capillary action. This capillary action is caused by the forces between the cellulose fibres of the paper and the solvent. Dyes that are more soluble in the solvent than they are in the water molecules of the stationary phase move rapidly up the paper, while those that are more soluble in the water are not carried as far up the paper (Figure 21.17). 

There is no other facet where thin-layer chromatography reveals its paper-chromatographic ancestry more clearly than in the question of development chambers (Fig. 56). Scaled-down paper-chromatographic chambers are still used for development to this day. From the beginning these possessed a vapor space, to allow an equilibration of the whole system for partition-chromatographic separations. The organic mobile phase was placed in the upper trough after the internal space of the chamber and, hence, the paper had been saturated, via the vapor phase, with the hydrophilic lower phase on the base of the chamber. 

In the case of thin-layer chromatography there is frequently no wait to establish complete equilibrium in the chamber before starting the development. The chamber is usually lined with a U-shaped piece of filter paper and tipped to each side after adding the mobile phase so that the filter paper is soaked with mobile phase and adheres to the wall of the chamber. As time goes on the mobile phase evaporates from the paper and would eventually saturate the inside of the chamber. 

While PLB were introduced first (14,15) more recently small PB 5-15 M diameter) have become of major interest. This is a result of the higher separation speeds found with such particles. Not only is the "stagnant" mobile phase mass transfer problem reduced, as in PLB, but solute mixing in the flowing stream is enhanced as a result of the smaller distance between the particles. The performances achieved with the small particle columns are equivalent to those obtained with capillary columns in gas chromatography (13), Examples illustrating the separation speed of such columns will be presented in the applications section of this paper. 

FIGURE 2.15 Concentration profiles of Sphenyl-l-pentanol, obtained on Whatman No. 3 chromatography paper with -octane as mobile phase. Concentrations of the analyte solutions in 2-propanol were (a) 0.5, (b) 1.0, (c) 1.5, and (d) 2.0 mol 1" [14,25]. 

Recently, Janjic et al. published some papers [33-36] on the influence of the stationary and mobile phase composition on the solvent strength parameter e° and SP, the system parameter (SP = log xjx, where and denote the mole fractions of the modiher in the stationary and the mobile phase, respectively) in normal phase and reversed-phase column chromatography. They established a linear dependence between SP and the Snyder s solvent strength parameters e° by performing experiments with binary solvent mixtures on silica and alumina layers. 

Males et al. [103] used aqueous mobile phase with formic acid for the separation of flavonoids and phenolic acids in the extract of Sambuci flos. In a cited paper, authors listed ten mobile phases with addition of acids used by other investigators for chromatography of polyphenolic material. For micropreparative separation and isolation of antraquinone derivatives (aloine and aloeemodine) from the hardened sap of aloe (Liliaceae family), Wawrzynowicz et al. used 0.5-mm silica precoated plates and isopropanol-methanol-acetic acid as the mobile phase [104]. The addition of small amounts of acid to the mobile phase suppressed the dissociation of acidic groups (phenolic, carboxylic) and thus prevented band diffusions. 

Paper chromatography using Whatman No. 1 paper was once used to determine the homogeneity of halcinonide. 60 Twenty percent formamide in methanol comprises one stationary phase and methylisobutyl ketone-formamide (20 1) is the mobile phase. A second solvent system uses 25% propylene glycol-chloroform as the stationary phase and toluene saturated with propylene glycol as the mobile phase. 

Even though it is sometimes not thought of as a chromatographic technique, we should also include electrophoresis. In this instance, paper or a gel is the stationary phase and electricity is the mobile phase. Although all types of chromatography are in extensive use in all kinds of investigations, electrophoresis has a particular prominence today because of DNA analysis [29],... 

The term liquid chromatography (LC) is applied to any chromatographic procedure in which the moving phase is liquid, as opposed to gas chromatography (GC) where a gas is utilized as a mobile phase (see discussion in Chapter 14). Classical column chromatography (see Section 15.1), paper chromatography—a forerunner of thin-layer chromatography (see Chapter 13), and HPLC are all examples of LC. This should clarify why it is inappropriate to further abbreviate HPLC to LC unfortunately, it is still commonly done. 

Paper chromatography and thin-layer chromatography (TLC) constitute the planar methods mentioned above. Paper chromatography makes use of a sheet of paper having the consistency of filter paper (cellulose) for the stationary phase. Since such paper is hydrophilic, the stationary phase is actually a thin film of water unintentionally adsorbed on the surface of the paper. Thus, paper chromatography represents a form of partition chromatography only. The mobile phase is always a liquid. 

TLC has similar applications to paper chromatography. The stationary phase is a coating, such as silica gel, on a glass or plastic plate. Depending on the TLC plate used, components may be separated based on differences in molecular weight, charge, or polarity (see Chapter 11). TLC with a 70% isopropyl alcohol mobile phase and a silica gel plate is an effective substitute for paper chromatography separation of amino acids. Nucleotides may be separated on a special silica gel plate and a 20% ethanol (in water) mobile phase. 

Obtain a paper chromatography developing chamber (similar to that shown in Figure 11.12). Pour 70% isopropyl alcohol (the mobile phase) into the chamber to a depth of 2 cm. Then, place the paper sheet in the chamber as shown in Figure 11.12. Cover the chamber. 

The efficiency of any chromatographic technique depends upon the number of sequential separations or equilibria that take place, which in the case of paper chromatography are due to the large number of compartments of cellulose-bound water. The test solutes are carried up the paper dissolved in the mobile phase and encounter successive compartments of water. At each one, rapid partition between the two phases occurs leaving the mobile phase to carry up the residual solute to the next water compartment and another partitioning effect. The solute, which is dissolved in the water and hence not carried up the paper, is now presented with fresh solvent rising up the paper and again is redistributed between the two phases. 

Cellulose is itself polar in nature and can cause some adsorption, which may result in the tailing of zones. However, this adsorptive effect may contribute to the separation process in some instances and the use of a polar mobile phase can enhance this effect further, e.g. the separation of amino acids using an aqueous solution of ammonia as the mobile phase. The combination of partition and adsorption generally influences separations on cellulose thin-layer plates, which have superseded paper chromatography in most instances and offer increased speed and resolution. 

Although the overwhelming majority of theoretical papers in liquid chromatography are dealing with the various aspects of RP-HPLC separation, theoretical advances have also been achieved in some other separation modes. Thus, a theoretical study on the relation between the kinetic and equilibrium quantities in size-exclusion chromatography has been published, hi adsorption chromatography the probability of adsorbing an analyte molecule in the mobile phase exactly r-times is described by... 

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