Pseudo-chromatograms

It is essential to be able to quantify the spots in order to use TLC as a quantitative method of analysis (Fig. 5.5). This is done by scanning the plate with a densitometer that can measure either absorption or fluorescence at one or many wavelengths. This instrument produces a pseudo-chromatogram that contains peaks whose areas can be measured. This corresponds to an isochronic image at the end of the separation. 

Figure 5.5—A pseudo-chromatogram obtained by scanning a TLC plate.

Figure 5.6 Separation of three steroids by TLC on a phase of reversed polarity. The migration distance increases with the polarity of the compound. This pseudo-chromatogram has heen obtained by scanning the TLC plate. The same separation effected by HPLC would lead to a chromatogram in which the order of the peaks would be reversed, a compound strongly retained having the longest elution time.

Correlation of the code with itself (autocorrelation) yields only one correlation point in the time domain defined by the sequence and the unit code interval (see Figure 5c) and an otherwise clean baseline. Since the detector in our chromatogram just follows what the sample valve is doing, it also should be a pseudo random sequence and the cross-correlation of input and output is really an autocorrelation and thus yields the single pulse correlogram with an otherwise clean baseline. 

Figure 6.29 shows mass spectra recorded during elution reduced to a two-dimensional contour plot. Each point is produced from pseudo-molecular ions, cluster formation or fragmentation. AU ions eluting in parallel with respect to time, at c. 29 min are assumed to belong to the main component, but there are some points clearly seen on the front edge of the main peak that indicate the presence of an impurity. This was confirmed by the production of a mass chromatogram of miz 486. 

Figure 6.11 shows the pseudo-on-flow NMR chromatograms of a soil sample extracted with water. The soil sample was prepared by a Polish laboratory as part of the sixth proficiency test. It was spiked with five compounds, with two of them (compounds 3 and 4) being related to chemical weapons. 

The H NMR spectra of the soil extract were run in the time-slice mode, i.e. the chromatographic run was stopped every 20 s and the NMR spectrum measured twice, with (a) and without (b) 31P decoupling. Processing of the data files led to the pseudo-on-flow NMR chromatograms. 

Comparison of the two pseudo-on-flow NMR chromatograms reveals that compounds 1,2 and 3 are organophosphorus compounds (Table 6.5), but only compounds 2 and 3 are phosphonates and related to chemical weapons. In fact, compound 2 is a degradation product of compound 3... 

Figure 6.11 Pseudo-on-flow 1 H NMR chromatograms of a spiked soil sample (a) with and (b) without31P decoupling. Conditions column, Purospher RP18, 250 x 4 mm, 5 pm eluents, A - acetonitrile, B - 1 % formic acid in D20 gradient, t = Omin A/B (1/99), t = 5 min A/B (10/90), t = 10min A/B (90/10) flow, l.Oml/min spectrometer, Bruker Avance 500MHz probe head, TX1 H/ C/31 4 mm LC probe 64 scans were acquired per row...

Chromatograms of degraded aspartame solutions also showed irregular, low intensity, strongly retained peaks which were more prominent in solutions with higher initial aspartame concentrations [26]. These may be due to polymerization products from pseudo-second order intermolecular self-aminolysis of aspartame. 

Figure 5.37 An example of a spectro-chromatogram recorded with a multichannel UV absorbance detector in LC. The sample contains a series of dipeptides, (a) (top) pseudo-isomeric three-dimensional plot dimensions are time, wavelength and absorption, (b) (bottom) contour plot with constant absorption lines. Figure taken from ref. [588]. Reprinted with permission.

This effect and similar ones are sometimes responsible for unwanted, unexpected peaks in chromatograms, and then they are called ghost peaks or pseudo peaks. A recent review of this entire subject55 calls them system peaks, and it is a good source of further details and references. 

The on-flow experiment was carried out on a mixture of eight flavonoids (Fig. 2) (20 pg each). MS and NMR data were obtained during this on-flow experiment. The UV chromatogram is depicted in Fig. 3. Table 1 and Fig. 4 show the pseudo-molecular ion information, where M is the molecular weight with all the hydroxyl protons deuterated, in negative mode, for the eight flavonoids obtained in this on-flow experiment. Fig. 5 is the 2-D data set (time vs. chemical shift) where each NMR spectrum was acquired for 16 scans and decreasing the delays (total time per spectrum of 20 s). Fig. 6 depicts the NMR traces of each flavonoid extracted from the 2-D data set. It is notable that catechin... 

Using gas chromatography of the N-oxide of the pseudo series of the alkaloids, decomposition of the alkaloids was observed in all cases, giving strychnine as the main peak on the chromatograms. The authors presumed that this decomposition might be due to the use of a... 

Suppose that in second-order calibration a standard Xi contains one analyte (hence, there is one chemical source of variation) and this standard is measured in such a way that the pseudo-rank of Xi equals one. The mixture X2, measured under the same experimental circumstances, contains the analyte and one unknown interferent. If the instrumental profiles (e.g. spectra and chromatograms) of the analyte and interferent are different, then the three-way array X having Xi and X2 as its two individual slices has two chemical sources of variation. This equals the number of PARAFAC components needed to model the systematic part of the data, which is the three-way rank of X, the systematic part of X. 

Fig. 2.2 [64] shows chromatograms of commercial-grade 1,3-pentadiene, obtained on two columns connected in series, the first column containing a saturated solution of silver nitrate in ethylene glycol and the second containing chloromaleic anhydride reacting with a trans isomer. As a result of the reaction the relative content of the irons isomer decreases. The pseudo-first-order rate constant varied from 2.0-10 to 1.4 10 sec (the half-time of transformation changed from 30 to 100 sec) [64]. 

Reobservation of the HPLC chromatogram of N. navis-varingica isolates from several areas in the Philippines showed that only isolates from Bnlacan estuary produced isodomoic acids A (lA) and B (IB) instead of DA, and all of the isolates from other areas prodnced DA and IB. It seemed to be interesting to solve whether N. navis-varingica isolated from other areas ontside the Philippines also produces lA or IB and DA. And it also seemed to be interesting to know whether P. multiseries or some other Pseudo-nitzschia could produce DA derivatives such as lA and IB. 

Figure 9. A chromatogram of the separation of some common anions monitored by an electrical conductivity detector. Anions l=pseudo-peak, 2=chloride, 3=nitrate, 4=bromide, 5=nitrate, 6=phosphate, 7=(Aiosphite, 8=sulfate, and 9=iodide.

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