Reduced, chromatogram

Fig. 203. Comparison of TLC with PC. Two-dimensional chromatogram reduced from original size by the factor 0.57...

A satisfactory chromatographic analysis demands, a priori, on an adequate separation of the constituents of the sample that will permit the accurate quantitative evaluation of each component of interest. To achieve this, an appropriate phase system must be chosen so that the individual components of the mixture will be moved apart from one another in the column. In addition, their dispersion must be constrained sufficiently to allow all the solutes of interest to be eluted discretely. At this stage it is necessary to introduce the concept of the Reduced Chromatogram. 

Any chromatogram that represents the separation of a complex mixture of solutes can be reduced to a relatively simple separation that will concisely and accurately represent the limits and extent of the separation problem. The simple separation can be depicted in the form of a reduced chromatogram, an example of which is given in Figure 2. 

The reduced chromatogram contains four peaks the first will be the dead volume peak (which, as has been shown previously, must be the fully excluded peak determined from the retention volume of a salt or solute of large molecular weight). 

The selection of the mobile phase and the conditions of development. Having chosen the solvent(s) the following are defined from the reduced chromatogram,... 

Column design involves the application of a number of specific equations (most of which have been previously derived and/or discussed) to determine the column parameters and operating conditions that will provide the analytical specifications necessary to achieve a specific separation. The characteristics of the separation will be defined by the reduced chromatogram of the particular sample of interest. First, it is necessary to calculate the efficiency required to separate the critical pair of the reduced chromatogram of the sample. This requires a knowledge of the capacity ratio of the first eluted peak of the critical pair and their separation ratio. Employing the Purnell equation (chapter 6, equation (16)). 

The emission of the indicator is reduced in places where there are substance zones that absorb at 2 = 254 nm present in the chromatogram. This produces dark zones (Fig 4A), whose intensity (or rather lack of it) is dependent on the amount of substance applied. If the plate background is set to 100% emission the phosphorescence is reduced appropriately in the region of the substance zones. When the chromatogram is scanned peaks are produced, whose position with respect to the start can be used to calculate Rf values and whose area or height can be used to construct cahbration curves as a function of the amount applied (Fig. 25). 

The same applies to the use of spray pistols (spray guns and aerosol cans), the frequency of whose use ought probably to be reduced on account of the propellant gas (chlorofluorohydrocarbons) employed Manual depression of the button valve of the vertically held spray can shoots the propellant gas through a fine jet and drags the sucked-up reagent solution with it onto the vertically held chromatogram (water pump pnnciple)... 

Note The dipping solution can also be employed as spray reagent. The detection limits per chromatogram zone are reported to be 1 — 5 pg substance [1] for the oxyacids of halogens and ca. 10 pg substance for reducing sugars [4]. 

Note Note that the diazotization of primary aromatic amines can also be achieved by placing the chromatogram for 3 — 5 min in a twin-trough chamber containing nitrous fumes (fume cupboard ). The fumes are produced in the empty trough of the chamber by addition of 25% hydrochloric acid to a 20% sodium nitrite solution [2, 4], iV-(l-Naphthyl)ethylenediamine can be replaced in the reagent by a- or -naphthol [10, 14], but this reduces the sensitivity of detection [2]. Spray solutions Ila and lib can also be used as dipping solutions. 

It is often possible to increase the detection sensitivity in visible light by exposing the dipped or sprayed chromatogram to ammonia vapors it can also be sprayed with caustic soda or potash solution. When this is done the fluorescence intensity is reduced on silica gel layers and increased on RP ones. 

Further development of the chromatogram with 0.1N ammonia afforded fractions positive to ninhydrin test. From the fractions, 249 mg. of a colorless material was obtained. It was dissolved in 19 ml. of water and the solution was adjusted to pH 4.0 with dilute hydrochloric acid. A colorless material, after condensing under a reduced pressure and lyophilization, was recrystallized from aqueous methanol with a small amount... 

The chromatographic challenge associated with a particular sample can be summarized by the reduced chromatogram. The reduced chromatogram consists of three peaks and is represented in figure 6. 

All samples can be reduced to such a chromatogram and if the reduced chromatogram can be resolved then, almost without exception, so can the sample. In the following discussion it is assumed that all the components of the mixture have equal importance and must be isolated and quantitatively estimated. The analyst will, at times, be presented with samples for which a full analysis is not required and such samples will be discussed subsequently. 

The time that the last peak is eluted in the reduced chromatogram represents the total analysis time after which, the analysis can be terminated. The two peaks that are eluted closest together are the most difficult to separate and which, for obvious reasons, are called the "critical pair". 

The columns must be designed or chosen such that the critical pair are separated and, as a second priority, the last peak must be eluted in a reasonable time. The first peak in the chromatogram is not considered part of the reduced chromatogram and is included as the dead volume marker from which the capacity factors of each solute can be calculated, together with the separation ratio of the critical pair. 

The above columns were calculated for a sample where the first peak of the critical pair in the reduced chromatogram was eluted at a (k ) of 2.5 and the last peak was eluted at a (k ) of 5.0. The method of calculation is given in (1). 

It is seen that, although the dimensions and particle sizes may not be precisely matched, all three columns are of a size closely similar to those commercially available with, perhaps, the exception of the long high efficiency column. The small 3 cm column is excellent for the preliminary assessment of a sample. As a result of its size it does not use large volumes of solvent and can be quickly reconditioned after a separation in readiness for the next run. It is very convenient for choosing the best phase system in method development. The other columns would be chosen on a basis of the efficiency required to separate the critical pair in the reduced chromatogram of the sample for analysis. 

Reactions can be exploited more speciHcally if it is known that particular functional groups are present [cf. Chapter 2]. They still do not allow direct identification, but they increase the specificity of the evidence. The chromatographic separation carried out before detection also contributes to this. This reduces the number of potential components. However, this does not exclude the possibility that there might be several substances in the particular part of the chromatogram involved. This not only applies to thin-layer chromatography but also applies with equal force to other microanalytical separation methods (GC, HPLC). 

Nitrates and nitrites are first reduced to nitrosyl chloride with thionyl chloride. The volatile nitrosyl chloride then reacts with 4-aminobenzenesulfonic acid to yield a diazonium salt that then couples with 8-hydroxyquinoline to form a colored azo compound. Hence, the coupling reagent is applied to the chromatogram first. 

Primary and secondary amines and amides are first chlorinated at nitrogen by the chlorine released by the gradually decomposing calcium hypochlorite. Excess chlorine gas is then selectively reduced in the TLC layer by gaseous formaldehyde. The reactive chloramines produced in the chromatogram zones then oxidize iodide to iodine, which reacts with the starch to yield an intense blue iodine-starch inclusion complex. 

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