Introduction to Soil Chemistry
Introduction to Soil Chemistry
Introduction to Soil Chemistry
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190 chroma<strong>to</strong>graphy<br />
R f is applied <strong>to</strong> thin-layer chroma<strong>to</strong>graphy, while R t is applied <strong>to</strong> GC and<br />
HPLC. For identification purposes the R f values of pure samples of all the compounds<br />
<strong>to</strong> be found in the unknown are determined and are used <strong>to</strong> “identify”<br />
these same compounds in an unknown mixture. The Rf of a compound is<br />
determined by placing a spot of sample on a thin layer sheet slightly above<br />
the bot<strong>to</strong>m, placing the TLC sheet in the developing chamber with the appropriate<br />
elutant and allowing the elutant <strong>to</strong> raise the thin layer until it is close<br />
<strong>to</strong> the <strong>to</strong>p (but never at or over the <strong>to</strong>p). The distance from the site where the<br />
spots were placed on the plate <strong>to</strong> <strong>to</strong>p of the plate where the elutant s<strong>to</strong>pped<br />
is recorded.This number is then divided in<strong>to</strong> the distance the spot moved from<br />
the beginning as shown in Figure 9.8.<br />
The distances should always be measured from the starting point, which is<br />
where the original mixtures were spotted both from and <strong>to</strong> the midpoint of the<br />
spots. The determination of the R f values of five spots on a thin-layer chroma<strong>to</strong>gram<br />
is given in Figure 9.6. The R f values of the spots are listed in Table<br />
9.4.The material in spots A 2 and B 2 are expected <strong>to</strong> be the same since they have<br />
the same R f and the chroma<strong>to</strong>gram was run under exactly the same conditions.<br />
R t is used in the same manner as is R f except that it is the retention time of<br />
components in the GC or HPLC column (see Figure 9.6). The time can be<br />
measured from the time of injection <strong>to</strong> the time when the <strong>to</strong>p of the peak<br />
elutes from the column. Another approach is <strong>to</strong> include a compound that is<br />
not retained by the solid or liquid phase in the injection and its peak used are<br />
the starting time for measuring the time for each peak <strong>to</strong> exit the column. Two<br />
compounds exiting the column at the same R t will be assumed <strong>to</strong> be the same<br />
compound. Often R t values are used for identification, although only a spectroscopic<br />
method can truly be an identification method.<br />
Another method is <strong>to</strong> chroma<strong>to</strong>graph the unknown and determine the R f<br />
values of the components present. When a compound is “identified” by its R f,<br />
Start<br />
A1<br />
B3<br />
A2 B2<br />
A B<br />
B1<br />
Solvent (elutant) front<br />
Start<br />
2cm<br />
3cm<br />
12 cm<br />
9cm<br />
15 cm<br />
Figure 9.8. Thin layer at start (left) and after development (right). Right chroma<strong>to</strong>gram shows<br />
the distance that the elutant and spots moved during the development of the chroma<strong>to</strong>gram.