Deviating retention times

Usually the problem comes from the pump or the gradient unit  

Check the flow accuracy with measuring flask and stop-watch. It may be necessary 

The more volatile component evaporates from the mobile phase reservoir  

Cool the reservoir. Cover it with an air-tight lid with valve (thus preventing 

Gradient profile or solvent composition deviates from the written method  

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Figure 10.13 shows the excellent performance of reversed-phase HPLC in biotechnological research. It represents the separation of the tryptic hydrolysate of the normal form and of a mutant of tissue-type plasminogen activator. This protein is built up of 527 amino acids and has a mass of approximately 67 000 Da. The mutant differs in a single amino acid, which leads to a deviating retention time of this specific fragment. 

Equation (3) merely sums the two peaks to produce a single envelope. Providing retention times can be measured precisely, the data can be used to determine the composition of a mixture of two substances that, although having finite retention differences, are eluted as a single peak. This can be achieved, providing the standard deviation of the measured retention time is small compared with the difference in retention times of the two solutes. Now, there is a direct relationship between retention volume measured in plate volumes and the equivalent times, which is depicted in Figure 6. 

Mathematical methods for determining the gas holdup tine are based on the linearity of the plot of adjusted retention time against carbon number for a homologous series of compounds. Large errors in this case can arise from the anomalous behavior of early members of the homologous series (deviation from linearity in the above relationship). The accuracy with which the gas holdup time is determined by using only well retained members of a homologous series can be compromised by instability in the column temperature and carrier gas flow rate [353,357]. The most accurate estimates... 

An automated procedure to measure peak widths for peak capacity measurements has been proposed.35 Since peak width varies through the separation, the peak capacity as conventionally measured depends on the sampling procedure. The integral of reciprocal base peak width vs. retention time provides a peak capacity independent of retention time, but requires an accurate calculation of peak width. Peak overlap complicates automation of calculation. Use of the second derivative in the magnitude-concavity method gives an accurate value of the standard deviation of the peak, from which the base peak width can be calculated. 

Chromatographic system (See Chromatography <621 >.) The liquid chromatograph is equipped with a 230 nm detector and a 4.6 mm x 30 cm column that contains packing L7. The flow rate is about 2 mL/min. Chromatograph the Resolution solution and the Standard preparation, and record the peak responses as directed under Procedure the resolution, R, between the dibutyl phthalate and miconazole peaks is not less than 5, the tailing factor for the miconazole peak is not more than 1.3, and the relative standard deviation for replicate injections of the Standard preparation is not more than 2%. The relative retention times are about 0.7 for dibutyl phthalate and 1 for miconazole. 

Chromatographic system. (Follow the method described in the general procedure <621 >.) The gas chromatograph is equipped with a flame ionization detector and a 1.2 m x 2 mm column packed with 3% phase G32 on support S1A. The injection port, detector, and column temperatures are maintained at about 250, 300, and 250 °C, respectively, and helium is used as the carrier gas, flowing at rate of about 50 mL/min. The relative retention times for cholestane and miconazole nitrate are about 0.44 and 1, respectively. Chromatograph the Standard preparation, and record the peak responses as directed for procedure The resolution, R, between cholestane and miconazole nitrate is not less than 2 and the relative standard deviation of replicate injections is not more than 3%. 

Chromatographic system. The gas chromatograph is equipped with a flamioniza-tion detector and a 2 mm x 1.8 m glass column packed with 10% phase G34 on 80- to 100-mesh support SI A. The column temperature is maintained at about 150 °C, and the injection port and the detector block temperatures are maintained at about 250 °C. Dry helium is used as the carrier gas at a flow rate of about 40 mL/min. Chromatograph the Standard preparation, measure the peak responses, and calculate the ratio, Rs, as directed for procedure the relative retention times are about 0.5 for valproic acid and 1.0 for biphenyl the resolution, R, between valproic acid and biphenyl is not less than 3.0 the relative standard deviation for replicate injections is not more than 2.0%. 

Efficiency, N, for column separations is related to retention time and peak width measured in terms of the standard deviation, assuming an ideally Gaussian-shaped peak (p. 16), i.e. 

Figure 6.18 presents example chromatograms obtained during the evaluation of retention time and peak area reproducibility across the 24 columns (within a single composite run). Table 6.1 lists the average retention time reproducibility and peak area reproducibility values obtained across 24 columns for a total of 24 composite runs (column to column within run). Reproducibility values are expressed as percentage relative standard deviation (%RSD). 

Initially the substance at Rt 19.95 was identified as 2-nonen-l-ol based on mass spectrum library search. The comparison with a commercial 2-nonen-l-ol standard indeed revealed a high degree of similarity between the mass spectra, but a distinct deviation regarding the retention time suggesting a similar molecule with a chain length greater than 2-nonen-l-ol. The substance Rt 20.95 was tentatively identified as 6,10-dimethyl-5,9-undecadien-2-one which corresponds with the authentic standard regarding mass spectra and retention time. 

Pigments were separated on a normal (150 X 4 mm i.d., particle size 5 /tin) and on a microbore ODS column (150 X 2 mm i.d., particle size 4 jttm) using gradient elution. The steps of gradient elution are shown in Table 2.7. Carotenoids were detected at 440 nm. Columns were not thermostated and separations were performed at room temperature (20 2°C). The mean and the relative standard deviation of retention time and peak area were computed from three parallel measurements. The carotenoids capsanthin, zeaxanthin and j0-carotenein and the extracts were tentatively identified comparing their retention time with those of authentic standards. 

Chemical compound Retention time (min) Standard deviation... 

Fenvalerate Data. Calibration data for the GC measurement of Fenvalerate were furnished by D. Kurtz (17). Average responses for five replicates at each of five standard concentrations are given in Table III. It should be noted that the stated responses are not raw observations, but rather on-line computer generated peak area estimates (cm ). (Had we started with the raw data [chromatograms], the problem would actually have been two-dimensional, including as variables retention time and concentration.) The stated uncertainties in the peak areas are based on a linear fit (o a+bx) of the replication standard deviations to concentration and the "local slopes" [first differences] in the last column of Table III are presented... 

As readily observed in most chromatograms, peaks tend to be Gaussian in shape and broaden with time, where W, becomes larger with longer This is caused by band-broadening effects inside the column, and is fundamental to all chromatographic processes.The term, plate number (N), is a quantitative measure of the efficiency of the column, and is related to the ratio of the retention time and the standard deviation of... 

Because of some peak tailing, the number of theoretical plates was based on peak width at one-half peak height Ns5.S4 he pooled standard deviation (all temperatures) of retention time measurements (dfs34) was t 0.007 minutes. 

The effect of the detector time constant on the apparent efficiency depends only on the time width of the bands. It has been shown by Sch-mauch 41) and by Me William and Bolton 42) that the profile recorded with a detector having a time constant r is wider than the actual profile by a factor (1 -f r/ert), where is the time standard deviation of the profile, provided this factor is less than about 1.2. Moreover, the peak heigh becomes smaller although the peak area remains unchanged. 1 he (list mu ment (retention time) of a peak increases by r and the retention time of the... 

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