Chromatogram measurements

Standard additions—Standards are prepared by adding a constant known amount of the analyte to the unknown sample, with the chromatogram measured after each addition. Alternatively, a series of standards could be prepared with the unknown as the diluent. 

Figure 2.12 Ratio of peak area and height related to time constant of detector. Experimental conditions are the same as those in Figure 2.11. Peak height and area ratios are calculated from the data of peak nos. 1 and 4 as a %> of the maximum value. Lines A, peak height ratio of chromatograms measured at...

Procedure Separately inject suitable portions (about 5 p,L) of the Standard Preparation and the Assay Preparation into the gas chromatograph, and record the chromatograms. Measure the areas under the peaks for each isomer and the Internal Standard in each chromatogram, and calculate the quantity, (7), in milligrams, of each isomer in the sample taken by the equation... 

Procedure Inject a suitable portion of the Standard Preparation into the gas chromatograph, and record the chromatogram. Measure the areas under the peaks, and record the values of the sum of the areas under the derivatized monoglyceride peaks and of the area under the hexadecyl hexadecanoate peak as As and Ap, respectively. Calculate the response factor, F, taken by the formula... 

However, this method is not recommended for low-abundance proteins due to poor accuracy. Software to assist in label-free quantitation includes an ion intensity chromatogram measurement called MSI filtering. This increases peak selection accuracy by incorporating the peptide retention time... 

Dge in temperature may influence the chromatogram measurably. Also, may occur when the column length or diameter are changed. Endele sured an increase in temperature of about 10°C and a decrease of the opacity factor by about 20% when the pressure was increased from 1 to iMPa. 

If a minor peak is present in a chromatogram measured with a conventional GC detector such as a flame ionization detector (FID), but is not observable in the GS or FG chromatogram, it may be possible simply to inject a greater volume of the sample into the chromatograph. Even if the major components overload the GC column in this case, the minor components will not. However, sometimes the major peaks will broaden to the point that they start to overlap a neighboring minor peak. In this case, it may become necessary to subtract the spectrum of the major peak (linear in absorbance) from the spectrum measured in the region of the minor peak, to identify the minor component. This procedure is needed because of the relatively low sensitivity of light-pipe-based GC-IR instruments. Two other approaches have been described that have led to increased sensitivity for GC-IR measurements, and are described below. 

Subtraction of the analytical background. The average signal of the baseline of the chromatogram, measured before the elution of the peak, is subtracted from the signal of the peak of the species [13, 15]. 

Calculation. In the calibration and analysis chromatograms, measure the peak heights of the n-undecane and the aromatic hydrocarbons allowing for any attenuation factors. 

Figure 26.2 Liquid chromatograms, measured at the critical condition for linear polystyrene, of cyclization reaction products of dilute-solution ring closure of end-functionalized PS (solid lines) and their linear precursors (dotted lines) (Lee et al., 2000). The linear precursors elute at nearly identical times (5.4 min), independent of molecular weight. The major peak in the cyclic reaction product, marked Ring , elutes at times that increase with molecular weight. Note that all Ring products contain linear precursor impurities. (Reprinted with permission from H.C. Lee, H. Lee, W. Lee etal, Fractionation of cyclic polystyrene from linear precursor by HPLC at the chromatographic critical condition, Macw-molecules, 33, 8119-8121, 2000. 2000 American Chemical Society.)...

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