Liquid chromatography, peak-area analyses

Fig. 3.5 Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of (A) rAHF and (B) rAHF-PFM following thrombin digestion. Samples of both products activated with thrombin show similar elution profiles and peak areas. The...

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

Ruiz-Sala et al. (129) described a reversed-phase HPLC method with a light-scattering detector for the analysis of TGs in milk fat. The identification of TGs was carried out by a combination of HPLC and gas-liquid chromatography (GLC), and was based on the equivalent carbon numbers and retention times of different standard TGs. Finally, quantitation of peak areas from HPLC chromatograms was carried out after applying a deconvolution program to the parts of chromatograms with poor resolution. 

High Performance Liquid Chromatography. Tissue extracts were analyzed with a Varian model 5020 liquid chromatograph equipped with a Rheo-dyne model 7120 loop injector valve, a Tracer 970 variable wavelength detector set at 257 nm, an automated Hewlett-Packard 3385A printer-plotter system for determining retention times and peak areas, and a Waters /x Bondapak column (3.9 mm i.d. X 300 mm) for carbohydrate analysis. The buffer was eluted isocratically at 1 mL/min with a 1 4 (v/v) mixture of 0.01 M monobasic sodium phosphate (pH 4.46) and methanol. The minimum amount detectable was 10 ng. 

Figure 4.12 Analysis by the external standard method. The precision of this basic method is improved when several solutions of varying concentrations are used in order to create a calibration curve. For trace analyses by liquid chromatography it is sometimes advisable to replace the areas of the peaks by their heights as they are less sensitive to variations in the mobile phase flow rate.

The procedure of Beroza and Bowman, Anal. Chem. 37,291, 1965, which follows, shows how it is done. Assume a hexane-acetonitrile system. A 5.0 mL aliquot of the upper phase containing a given pesticide is analyzed by gas-liquid chromatography. To a second 5.0 mL aliquot in a graduated, glass-stoppered, 10.0 mL centrifuge tube is added an equal volume of lower phase, the tube is shaken for about 1 minute, and the upper phase is analyzed exactly like the first 5.0 mL aliquot. The ratio of the quantitative results of the second analysis to those of the first (peak areas or heights) is the P-value. This is the amount of the pesticide in the upper phase (second analysis) divided by the total amount of pesticide (first analysis). Table 9-1, p. 104, shows how valuable this can be o,o -DDT and TDE have identical retention times. 

Analysis of Products. The three fractions collected from each sample were analyzed by gas chromatography. The noncondensable fraction, containing hydrogen and methane, was analyzed on silica gel at room temperature. The fraction containing C2-C4 hydrocarbons was analyzed at 75 °C. on silica gel treated with didecyl phthalate. Aliquots of the liquid fraction were analyzed on three columns of different selectivity Bentone-34-didecyl phthalate silicone SE-30 and m-polyphenyl ether (five-ring). Products were identified, and their yields were determined by comparison of retention volumes and peak areas with values for known amounts of authentic samples. 

Liquid chromatography can also be used for the quantitative analysis of the separated compounds. In column chromatography, the detector response is normally related to the amount of sample in the effluent. Thus, the area under a chromatographic peak is useful for quantitative analysis in Fig. 21.2, the darkened area under peak C represents the peak area of that component. The peak height (distance h in Fig. 21.2) can also be used. In thin-layer or paper chromatography, the area of the spot is related to the amount of substance. The separated component can also be eluted from the plate or paper and measured externally by another technique (for instance, spectrophotometry). 

Since its introduction at the end of the 1950s, gas chromatography (GC) has developed into a versatile tool in the analysis of natural products. Clear advantages of GC are the high resolution and high sensitivity of the most common detection method, the flame ionization detector (FID), and the fact that the detector response of similar compounds will be about the same, i.e., peak areas may be directly compared for quantification. This is in contrast to high performance liquid chromatography (HPLC), in which the detector response in the most common detection mode, ultra violet (UV)"absorption, may vary widely for different compounds since the molar extinction coefficients can be very different. 

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