CAPÍTULO 1: CONTEXTO HISTÓRICO
1.4 Comparación de la improvisación de Hank Mobley y John Coltrane
Chem. Listy 104, s634s639 (2010) ACP 2010 Súčasný stav a perspektívy analytickej chémie v praxi Posters %), and 3-NFt (99 %) were prepared by dissolving the
substances in 100 ml of methanol (gradient grade purity, Merck, Prague, CZ), the stock solution of 5-NQ (99 %) was prepared by dissolving the substance in 100 ml of deionized water. The concentration of all stock solutions was 1·10–3 mol l–1, the pure substances were supplied by
Sigma-Aldrich. All stock solutions were stored in glass vessels in the dark. It followed from a spectrophotometric study of the stability of all stock solutions that they are stable for at least 90 days.
Britton – Robinson (BR) buffers were prepared in a usual way, i.e. by mixing a solution of 0.04 mol l–1 in
phosphoric acid, 0.04 mol l–1 in acetic acid and 0.04 mol l–1
in boric acid with the appropriate amount of 0.2 mol l–1
sodium hydroxide solution. Borate buffer was prepared by mixing 0.05 mol l–1 of sodium tetraborate with appropriate
amount of 0.1 mol l–1 hydrochlorid acid to obtain pH 9.0.
Phosphate buffer was prepared by mixing 0.01 mol l–1
sodium dihydrogen phosphatewith appropriate amount of 0.2 mo L–1 sodium hydroxide solutionto obtain pH 7.0 (all
chemicals Lachema, Brno, CZ). Methanol (Merck, Prague, CZ) of gradient grade purity was used for mobile phase preparation. De-ionized water was produced by Milli-Qplus
system (Millipore, USA). Apparatus
Electrochemical detector used in FIA and HPLC measurements consisted of a platinum wire auxiliary elec- trode, silver/silver chloride (1 mol l–1 KCl) reference elec-
trode (both Monokrystaly, Turnov, CZ) and the working electrode, m AgSAE with the disc diameter 2.47 mm in three electrode arrangement used in “wall-jet” arrange- ment. m-AgSAE was prepared from a drawn-out glass tube, its narrow end was filled with a fine silver powder, amalgamated by liquid mercury and connected to an elec- tric contact16. The exact volume of mercury (0.4 μl if not
stated otherwise) to form the meniscus of m-AgSAE was applied by Hamilton syringe.
The FIA system consisted of linear high-pressure pump HPP 5001 (Laboratorní přístroje, Prague) and elec- trochemical and spectrometric detector. ED in “wall-jet” arrangement described earlier18 was used. Electrode sys-
tem was driven by potentiostat ADLC 2 (Laboratorní přístroje, Prague). The working m-AgSAE was kept at a constant potential so that the electrochemical cell served as an amperometric detector. The distance of electrode surface from the end of capillary was 0.5 mm. Samples were injected using six-way valve D (Ecom, Prague). Fol- lowing parameters for FIA determination were used: de- tection potential 1.6 V, flow rate 4 ml min–1 and injected
sample volume 0.1 ml.
Volume of spectrophotometric cell was 10 μl and optical path length was 5 mm. Output voltage corre- sponded to 5 V per 1 A.U. (1 mV = 2.10–4 A.U.). The sig-
nal of spectrophotometric detection was measured at con- stant wavelength 220 nm.
Program CSW32 version 1.4.11.06 (DataApex, Pra-
gue), driven under operational system Microsoft Windows 98 (Microsoft Corp.), was used for data collection.
The HPLC system consisted of high-pressure pump L-2130 HTA (Hitachi, Japan) governed by the software EZChrom Elite (Agilent Technologies, USA) working in Windows XP (Microsoft Corporation, USA). The column KROMASIL (250 4.6 mm, 7 μm) with reversed C18
phase (Phenomenex, Torrance, CA, USA) was used. Man- ual injection of samples degassed for 3 min by passing of nitrogen (purity 4.0, Linde, Prague, CZ) using 20 μl Rheo- dyne (IDEX Health & Science, Rohnert Park, CA, USA) injection valve was used. The diode array detector L-2450 (LaChrom Elite, Hitachi, USA) for measurement from 220 nm to 400 nm was used. The mobile phase was de- gassed by ultrasonic bath PS 02000A (Powersonic, USA) followed by passing nitrogen (purity 4.0, Linde, Prague, CZ) continuously for the whole period of measurement. The measurements were carried out at ambient laboratory temperature (22 °C).
The pH measurements were carried out by pH meter Jenway 4330 (Jenway, UK) with combined glass electrode (Ag/AgCl/3 mol l–1 KCl (type: 924005)). The pH meter
was calibrated with standard pH buffers (Sevac, Prague, CZ). pH values refer to those of the aqueous phase, pHf
values refer to those of the resulting pH of the mixtures of the aqueous phase with the organic solvent.
Procedures
In the case of flow injection analysis, 0.05 mol l–1
borate buffer, pH 9.0 was used as run buffer. More diluted analyte solutions were prepared by diluting of exact vol- ume of 5-NQ stock solution with run buffer. All samples were deaerated with N2 (5 min) before injection into flow
of run buffer. The m-AgSAE was activated before starting the work, as well as after every pause longer than one hour. The electrochemical activation of m-AgSAE was carried out by insert potential –2200 mV for 300 s in the stirred solution of 0.2 mol l–1 KCl and then washed by
distilled water. No other electrochemical pretreatment was performed prior each injection.
In the case of HPLC-ED, the mixture of 0.01 mol l–1
phosphate buffer, pH 7.0 : methanol (15:85, v/v) was used as mobile phase. More diluted analyte solutions were pre- pared by dilution of exact volume of the stock solutions with mobile phase. The flow rate Fm was set at 1 ml min–1
and the injected sample volume Vinj was 20 μl. The m-
AgSAE in HPLC experiments was activated by the same way as in FIA measurements. No other electrochemical pretreatment was performed prior each injection.
All calibration curves were measured in triplicate. The statistical parameters of calibration curves were calcu- lated using statistic software OriginPro 6.0 (OriginLab Corporation, USA). The significance of the intercepts of linear calibration dependences was tested by statistic soft- ware ADSTAT24, which was also used to calculate the
limit of quantitation in FIA measurements.
quantitation (LQ) were calculated from the peaks heights as
the concentration of an analyte which gave a signal three and ten times the background noise (S/N = 3; S/N = 10, respectively). All the statistical data are calculated for the level of significance α = 0.05.
Results and discussion
Flow injection analysis using m-AgSAE as amperometric sensor
First, the conditions for determination 5-NQ were optimized. 0.05 mol l–1 borate buffer pH 9.0 medium was
used as a run buffer for FIA determination of 5-NQ. The UV detector was also serially arranged prior to the electro- chemical detector for the sake of comparison. Optimal detection potential (Edet) inserted on m-AgSAE, flow rate
(Fm) of the run buffer and injection volume (Vinj) were
found during the process of optimization. Firstly, the influ- ence of the Edet on electrochemical signal was investigated.
Edet = –1.6 V was selected as optimum potential as it pro-
vides the highest signals. Further, the optimum flow rate was investigated (Fig. 1). Fm = 4 ml min–1 was set as the
maximum of peak heights achieved by electrochemical detector (and optimum for UV detector has the same value). The injected sample volume (Fig. 2) was set at 0.1 ml, which provides the highest signal, further not increas- ing when higher volumes are injected. This optimum value was reached for both detectors.
Calibration dependences were measured under opti- mized conditions in the range from 2·106 mol l1 to 1·104 mol l1 using ED and UV detector, they are linear within the whole concentration range. The peak heights were evaluated from the negative peak height obtained after injection of blank solution into FIA system. The record
peak of the ED is shown in Fig. 3. In Figure 3B detecting the lowest attainable concentration range from 2·106 mol l1 to 1·105 mol l1, there is a negative peak which is related to the presence of oxygen in the run buffer. The system is probably not totally hermetic and the freshly deaerated blank solution (and the samples) has lower content of oxy- gen than run buffer. This phenomenon was not observed for UV detector. The LQ achieved with UV (6·106 mol l1) and
ED (3·106 mol l1) are comparable.
The HPLC-ED method for the determination of 1-NP, 2-NF, 3-NFt, and 5-NQ
Firstly, the separation of 1-NP, 2-NF, 3-NFt, and 5- NQ was optimized using reversed C18 phase. Mobile phase
with high content of organic phase has to be used due to the extended aromatic system of studied NPAHs. Using 0.01 mol l–1 phosphate buffer, pH 7.0 : methanol (15:85, v/
v) mobile phase, baseline separation of tested analytes was achieved in fifteen minutes.
The detection potential Edet = –1.5 V was chosen
based on hydrodynamic voltammograms (Fig. 4) of tested analytes and the highest signal to noise ratio. The conse- quent decrease of peak currents Ip after the height maxi-
mum at Edet is reached is caused by an increase of the
background current due to the cathodic decomposition (i.e., hydrogen evolution) of the mobile phase. It proceeds at relatively negative potentials due to the high content of the organic phase in mobile phase. The noise of the system is independent of Edet and it is about 50 nA.
Calibration dependences were measured under opti- mized conditions within the range from 2·10–5 to 1·10–4
mol l–1 using electrochemical detector and within the range
from 3·10–7 to 1·10–4 mol l–1 using diode array detector
(DAD).
Parameters for the electrochemical detector are sum- marized in the Table I, limit of quantitation (10 S/N), is
0 4 8 -15000 -30000 -45000 IP , n A Fm, mL min-1 Fig. 1. Dependence of the peak heights Ip of 5-NQ (c = 1·10−4 mol l−1) on flow rate F
m (Edet = −1.6 V) for FIA-ED determina- tion of 5-NQ. Run buffer 0.05 mol l−1 borate buffer, pH 9.0;
injected volume Vinj = 100 l. Evaluated from peak heights
0 200 400 0 -20000 -40000 -60000 IP , n A Vinj, L
Fig. 2. Dependence of the peak heights Ip of 5-NQ (c = 1·10−4 mol l−1) on injected volume V
inj (Edet = −1.6 V) for FIA-ED determination of 5-NQ. Run buffer 0.05 mol l−1 borate buffer,
Chem. Listy 104, s634s639 (2010) ACP 2010 Súčasný stav a perspektívy analytickej chémie v praxi Posters
always 3.3 times higher than LD. The achieved LQs (from
3.0·10–5 mol l–1 for 5-NQ to 1.0·10–4 mol l–1 for 3-NFt) are
more than one order of magnitude than LQ reported above
for FIA-ED for 5-NQ (3·10–6 mol l–1). This may be caused
by higher noise using the high-pressure pump working in pulse regime in HPLC rather than the linear pump used for FIA.
Using DAD the optimum wavelength was found for each test substance. They are listed in the Table II together with the parameters of calibration dependences evaluated from both, peak areas and peak heights. The noise is inde- pendent on the wavelength and its value is about 8·10–5
A.U. The differences of LD calculated at optimum λ for
each compounds listed in Table II (from 0.13·10–6 mol l–1
for 5-NQ to 0.39·10–6 mol l–1 for 3-NFt) and for the com-
promise wavelenght of λ = 227 (from 0.32·10–6 mol l–1 for
5-NQ to 0.59·10–6 mol l–1 for 3-NFt) are about compara-
ble. The LDs were evaluated from peak heights.
0 3 6 9 0 -15000 -30000 0 3 6 9 -150 -300 t, min 1 2 3 4 5 A I, nA 2 1 I, nA t, min 3 4 5 6 B
Fig. 3. Peaks of 5-NQ recorded with FIA-ED with m-AgSAE detector in “wall-jet” arrangement for 5-NQ concentration: (A): 2
(1), 4 (2), 6 (3), 8 (4) and 10 (5) ·105 mol l–1, and (B): 0 (1), 2 (2), 4 (3), 6 (4), 8 (5) and 10 (6) ·106 mol l–1. Run electrolyte 0.05 mol l–1
borate buffer pH 9.0, Edet = –1.6 V, flow rate Fm = 4 ml min1, injected volume Vinj = 100 μl
-1,0 -1,2 -1,4 -1,6 -500 -1000 -1500 I p , nA Edet, V 1 2 3 4
Fig. 4. Dependence of peak heights Ip of 5-NQ (1), 2-NF (2), 1- NP (3), and 3-NFt (4) on detection potential Edet in HPLC-ED.
Concentration of each analyte 1·10–4 mol l–1. Mobile phase 0.01
mol l–1 phosphate buffer pH 7.0 : methanol (15:85; v/v),
Fm = 1 ml min–1, Vinj = 20 μl
Table I
Parameters of calibrations dependences for the determination of 5-NQ, 2-NF, 1-NP, and 3-NFt using HPLC-ED with “wall-jet” m-AgSAE detector. Mobile phase 0.01 mol l–1 phosphate buffer pH 7.0 : methanol (15:85; v/v), F
m = 1 ml min–1,
Vinj = 20 l, detection potential –1.5 V, evaluated from peak heights
Analyte Concentration range
[mol l–1] Slope[mA l mol–1] Correlationcoefficient [mol lLD –1] L[mol lQ –1]
5-NQ 20 – 100 –150 0.9917 9.2 30.4
2-NF 20 – 100 –99 0.9911 17.0 56.1
1-NP 20 – 100 –84 0.9952 28.0 92.4
Conclusion
In this study, we focused on the FIA-ED and HPLC- ED determination of selected nitrated aromatic compounds using an amperometric sensor in “wall-jet” detection cell.
FIA-ED was tested using 5-NQ. Using optimized conditions (run buffer 0.05 mol l1 borate buffer of pH 9.0, detection potential 1.6 V, flow rate 4 ml min1, injected
volume 0.1 ml), the LQ obtained for FIA-ED (3·106mol l1)
is comparable with UV detection (LQ = 6·106 mol l1).
Further, HPLC separation of 1-NP, 2-NF, 3-NFt, and 5-NQ using C18 phase was proposed in mobile phase con-
sisting of 0.01 mol l1 phosphate buffer pH 7.0: methanol 15:85 (v/v). Using electrochemical detection , LDs lying
mostly in the 105 mol l–1 concentration range were
achieved, which is about 2 orders of magnitude higher than using DAD when the optimum wavelengths for each analyte are used. In the following measurement we will focuse on the removal of the electronic noise from the measurement system, which contributes significantly to the relatively high LDs of electrochemical detection.
The project was supported by the Czech Ministry of Education, Youth and Sports (projects LC 06035, MSM 0021620857, and RP 14/63) and by Grant Agency of Charles University (project SVV 261 204).
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Table II
Parameters of calibrations dependences for the determination of 5-NQ, 2-NF, 1-NP, and 3-NFt using HPLC-DAD. Mobile phase 0.01 mol l–1 phosphate buffer pH 7.0 : methanol (15:85; v/v), F
m = 1 ml min–1, Vinj = 20 l Analyte λ [nm] Concentration range [mol l–1] Slope a Correlation coefficient LD [mol l–1]
A - evaluated for peak height
5-NQ 220 0.3 – 100 1790 0.9998 0.13
2-NF 335 0.3 – 100 656 0.9997 0.36
1-NP 238 0.3 – 100 1080 0.9996 0.22
3-NFt 233 0.3 – 100 621 0.9993 0.39
B- evaluated for peak area
5-NQ 220 0.3 – 100 10510 0.9999
2-NF 335 0.3 – 100 5620 0.9996
1-NP 238 0.3 – 100 13220 0.9999
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