Interpretation of the Chromatogram

The appearance of the peaks on chromatograms can also provide information about the quality of the resolution. Thus, if the compounds are well separated, the second peak will emerge only after the detector has completely 

CONCEPTS AND PRINCIPLES OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

The resolution of any two components, therefore, is a ratio relating the distance between the apex of the peaks and the distance between their bases. With baseline separation, the bases of the peaks do not overlap. In the absence of baseline separation, however, the apex of each peak may be separate while the bases overlap. A mathematical expression can be written to describe this 

The symmetry of each peak can provide information about the sample. Tailing (Fig. 2.9A) suggests some heterogeneity in the sample—either real or introduced by the chromatographic conditions. Flat-topped peaks (Fig. 2.9B) suggest that the capacity of the column has been exceeded. 

Of course, the magnitude of the signal from the detector can be used as a measure of the relative amount of each sample. While arbitrary units of area 

---

Water-soluble polymers obtained through a radical polymerization [e.g., poly(acrylic acid) PAA] often contain sodium sulfate Na2S04 as a decomposition product of the initiator. The peak of Na2S04 is eluted before the dimer. In the interpretation of the chromatogram, a typical GPC program has to be truncated before the Na2S04 peak, or at a Mpaa value of about 200. The calibration curve in this region can be flattened by an additive small pore column as well, but the principle problem remains unsolved. 

Note With the mobile phase described a pale colored B-front appeared at h/ f 5-10, but it did not affect the interpretation of the chromatogram. 

Aromatic amines that have been used include o-toluidine, p-aminosali-cylic acid, p-aminobenzoic acid, diphenylamine and p-aminophenol. Their ability to react preferentially with a particular carbohydrate or class of carbohydrate is often useful, e.g. p-aminophenol, which shows some specificity for ketoses compared with aldoses and is useful for measuring fructose. These reagents have proved particularly useful for the visualization and identification of carbohydrates after separation of mixtures by paper or thin-layer chromatography, when colour variations and the presence or absence of a reaction aid the interpretation of the chromatogram. 

Note Some lots of diethyl ether contain foreign residues that interfere with the analysis and/or the interpretation of the chromatograms. If the ether quality is unknown or suspect, concentrate 50 mL to a volume of about 1 mL in the concentrator, and then chromatograph a 2.0-pL portion using the conditions outlined under the Procedure. If the chromatogram is excessively noisy and contains signal peaks that overlap or interfere in the measurement of the peaks produced by the propylene chlorohydrin isomers, the ether should be redistilled. 

For quantitative interpretations of the chromatogram the detected compounds must be identified, and response factors have to be apphed to transform peak areas into weights. Automatic identification of peaks may be accomplished by numbering the peaks, on the basis of absolute retention time bands or by retention times relative to an internal time standard. Since retention times relative to one standard are not constant enough for reliable identification especially in long chromatograms, multistandard... 

The most appropriate IS needs to be selected from the above list of candidates. Consult with your laboratory instructor and proceed to inject one or more ISs and base your decision on an interpretation of the chromatogram. 

Saponification of the oil or fat, then separation of the unsaponifiable matter. Isolation of the sterols from the unsaponifiable matter by TLC. Analysis by GLC of the sterol fraction so isolated (or of the trimethyl-silyl ethers prepared from the sterol fraction) and interpretation of the chromatograms. 

It is also possible to perform GLC on the trimethyl-silyl ethers of the sterols. In that case, the residue is gently heated with a mixture of dimethylformamide, hexamethylsilazane and trimethylchlorosilane to prepare the trimethylsilyl ethers. The gas chromatographic method described provides a qualitative analysis of sterols in oils and fats. However, it can be employed for quantitative analysis within certain limitations (a) the response of each sterol is a function of the type of detector used (b) an accurate quantitative interpretation of the chromatogram... 

---