Illustrative Examples

The performance of correlation LC, compared with conventional LC, can be illustrated with a calibration graph as shown in Figure 3(A). Phenol was measured

Figure 3 (A) Calibration graph with fluorimetric detection for five concentrations of phenol (0.01 ^g to 100 ^g L~1); s and c indicate single injection and correlation, respectively. For each point the chromatogram length or the correlation time (min) is given. The bars indicate $ 3a (standard deviation of the peak area). (B) Correlogram of a 0.01 ^g L phenol sample. The detection limit is about 0.001 ^g L~1; 80 min correlation time.

Figure 3 (A) Calibration graph with fluorimetric detection for five concentrations of phenol (0.01 ^g to 100 ^g L~1); s and c indicate single injection and correlation, respectively. For each point the chromatogram length or the correlation time (min) is given. The bars indicate $ 3a (standard deviation of the peak area). (B) Correlogram of a 0.01 ^g L phenol sample. The detection limit is about 0.001 ^g L~1; 80 min correlation time.

Figure 4 LC analysis of PAH samples by single-injection (SI) and correlation (CC 511 - the number of clock periods in the Pseudo Random Binary Sequence) chromatography under similar separation conditions.

Table 2 Concentrations of the compounds in the samples used in the SCC experiment

Concentration (mg L 1)

Naphthalene

Anthracene

1,2-Benzanthracene

Sample 1

7.690

0.320

2.152

Sample 2

3.845

0.320

1.076

Sample 3

1.922

0.320

0.538

Figure 4 LC analysis of PAH samples by single-injection (SI) and correlation (CC 511 - the number of clock periods in the Pseudo Random Binary Sequence) chromatography under similar separation conditions.

at a five concentrations: 0.01 to 100 igL-1. The three higher concentrations (1-100 ^gL_1) were determined by conventional reversed-phase chro-matography, the three lower concentrations (0.01-1 ^gL_1) by CLC. Measurements at the 1 level were done by both techniques. The bars indicate the peak area + 3^ (arbitrary units), where U\ is the standard deviation of the integrated noise. The correlation time is 1, 3 and 16 sequences or chromatogram lengths, corresponding to 5, 15 and 80 minutes, respectively.

Figure 3(B) shows the correlogram of a very low concentration of phenol for 80 minutes of correlation. The detection limit in this case is approximately 0.003 |gL_1. For a comparison with conventional LC it must be noted that the injection volume injected in one clock period of the PRBS is 48 |L, a factor of 2.4 more than the 20 |L single injection.

Another example of the possibilities of CLC is shown in Figure 4. Here a diluted rather complex standard material was analysed, containing a number of compounds at different known concentrations. The separation is not optimal, but the condi-

tions are effective for examining the behaviour of CC in the case of more complicated mixtures.

Table 1 gives the composition of the samples, consisting of a mixture of polynuclear aromatic hydrocarbons (PAHs), prepared from standard reference material SRM 1647 (National Bureau of Standards). The improvement by application of CC, particularly in case of the diluted sample, is considerable.

A typical application of simultaneous correlation chromatography (SCC) is accurate calibration in LC, as is shown in the following experiment. Three different samples, each composed of naphthalene, anthracene and 1,2-benzanthracene, were prepared (Table 2). The concentration of anthracene was kept constant. Anthracene was used as an internal standard to correct for variations in the injected volumes of the different samples. The samples were injected according to a PRBS of 127 clock periods; the starting points of the injection patterns of the different samples were shifted over one-third of the sequence length. The clock period was divided into three subperiods, one for the injection of each sample.

Figure 5 shows the simultaneous correlogram. The peak areas were determined and corrected for systematic errors with the internal standard anthracene. The calibration graph is shown in Figure 6; the calculated correlation coefficient of the linear fit was 0.999 97 - a very good fit.

In principle SCC allows excellent quantification. Deterministic disturbances and drift influence

Table 1 Composition of the PAH sample

Compound

Concentration

Compound

Concentration

Compound

Concentration

(ML'1)

(ugL-)

(ug L -)

Naphthalene

18.0

Fluoranthene

8.08

Benzo[^]fluoranthene

4.02

Acenaphthylene

15.3

Pyrene

7.87

Benzo[a]pyrene

4.24

Acenaphthene

16.8

Benz[a]anthracene

4.02

Benzogh/perylene

3.21

Fluorene

3.94

Chrysene

3.74

Dibenz[a,h]anthracene

2.94

Phenanthrene

4.05

Benzo[b]fluoranthene

4.09

Indeno[ 1,2,3-cd]pyrene

3.25

Anthracene

2.63

Figure 5 Simultaneous chromatogram of three samples of mixtures of naphthalene (n), anthracene (a), and 1,2-benzanthracene (b) with different concentration ratios.

measurement and calibration samples in exactly the same way. Also, the noise-reducing property of CC is maintained. A comparison with sequential calibration can be made by successively performing independent experiments. Each calibration experiment yields an almost perfectly fitting linear calibration plot, but the points for the same concentration, measured successively, are distributed with rather large standard deviations. The bars in Figure 6 indicate the standard deviations of the measurements.

An illustrative example of the application of chemical concentration modulation correlation chromatography is the selective determination of traces of phenol. An electrochemical modulation cell (EMC) and a fluorescence detector are used. This combination, together with a suitable column, is both selective and sensitive to phenol.

Figure 7 shows log-log calibration graphs for conventional loop injection and EMC-CC, respectively. The signal-to-noise enhancement of EMC-CC is a factor of 11 higher at the most, equal to the theoretically predicted value.

Figure 6 1,2-Benzanthracene calibration graph. The bars indicate the confidence interval for successive independent measurements.

Figure 7 Log-log calibration graphs (solid lines) using loop injection (A), and electrochemical concentration modulation CC (B), both with fluorescence detection. The dashed lines represent the 3cbaseline noise curves. Solid symbols were from 63 clock period (cp) injection sequences (11 min); open symbols were from 511 cp sequences (80 min).

Figure 6 1,2-Benzanthracene calibration graph. The bars indicate the confidence interval for successive independent measurements.

Figure 7 Log-log calibration graphs (solid lines) using loop injection (A), and electrochemical concentration modulation CC (B), both with fluorescence detection. The dashed lines represent the 3cbaseline noise curves. Solid symbols were from 63 clock period (cp) injection sequences (11 min); open symbols were from 511 cp sequences (80 min).

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