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Continuous Flow Alternating Current Polarographic Detection of. Nitrazepam in Liquid Chromatography. Herman B. Hanekamp, Willem H. Voogt, Roland W. Fr...
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Anal. Chem. 1981, 53, 1362-1365

Continuous Flow Alternating Current Polarographic Detection of Nitrazepam in Liquid Chromatography Herman B. Hanekamp, Willem H. Voogt, Roland W. Frei,’ and Pieter Bos Department of Analytical Chemistry, Free University, De Boelelaan 1083, 7081 HV Amsterdam, The Netherlands

The potential of alternating current polarography for detection of reducible organics in continuous flow systems is explored. Systematic studies in batch and in dynamic flow conditions have been carried out with the model compound nitrazepam. Parameters such as influence of mobile phase composition, applied frequency, amplitude and phase angle on the signal were studied. Although striking differences were found between batch and flow condttions for the influence of the adjusted phase angles, a phase angle of 0’ seems to offer the best measurement condition in both instances. The detection limit Is in the order of 5-20-fold lower than for previously reported DME detectors. The AC polarographic cell assembly for continuous flow conditions was tested for the detection of four benzodiazepines after reversed-phase chromatographic separation. The enhanced selectivity of this detection mode makes it promising for organic trace analysis in complex matrices.

The usefulness of electrochemical detectors in high-performance liquid chromatography (HPLC) has already been recognized (1-3). Especially solid-state electrode detectors are able to detect quantities a t the subnanogram level (I, 4-13). Mercury possesses several advantages as an electrode material. However, the relatively low sensitivity of polarographic detectors obstructed the introduction of this type of detector. Some of the problems have been overcome by the construction of a fast dropping mercury electrode (DME) detector with a conically ground horizontal capillary (14). With direct current amperometry good results can be obtained with such a detector (15).The fast DME system, however, does not allow the application of techniques with enhanced sensitivity and selectivity such as (differential) pulse techniques, which require a synchronization between the electronics and the drop lifetime. This necessary synchronization has recently been achieved by means of a potential pulse supplied to a horizontal DME with a partially conical glass capillary (16). The advantages of pulse techniques become apparent only if the content of electrochemically active impurities (oxygen, trace metals, etc.) in the eluent stream is very low. These impurities cause a background current and are actually determining the noise in the base line and hence the attainable detection limit (11, 16-19). As purging with nitrogen is not sufficient, the impurities have to be removed by reduction in a flow-through cell with porous silver electrodes (19). Thus, a favorable detection limit is obtainable with normal pulse amperometry (18).

An other approach can be the use of sampled alternating current (AC) polarography (20). In AC polarography a sinusoidal voltage of small amplitude is superimposed on the usual DC potential. The resulting polarogram is peak shaped and consequently possesses a better selectivity than DC measurements. The total measured alternating current exhibits a shift in phase angle relative to the superimposed 0003-2700/81/0353-1362$01.25/0

voltage. By means of a phase-sensitive readout the faradaic component of the current can be distinguished from the capacitive current. The latter is primarily responsible for the noise, and its elimination results in an improved detection limit (21). It is also possible to measure the change in the capacity at the mercury drop. Thus, adsorbing but nonelectroactive compounds can be determined too. This tensiometric type of detection has been demonstrated by Lankelma and Poppe (22) and Kemula and Kutner (23). In addition AC measurements could be very useful to investigate electrode kinetics (24, 25). In the present work it was our aim to examine to what extent employment of this technique can improve the sensitivity and selectivity of a DME detector in continuous-flow systems.

EXPERIMENTAL SECTION The first experiments were performed in batch and compared to the corresponding measurements under flow conditions. Nitrazepam was chosen as test compound as used in earlier work (18).

The detector used in this work was identical with the one developed for the application of pulse techniques (16, 18). As the drop lifetime and electronics can be synchronized by using this cell, a sampled AC measurement was applied. All measurements in the chromatographic system were made with the electrochemical eluent scrubber as an indispensable part of the instrumentation (19). Apparatus. In all experiments a Bruker modular polarograph (E 310 M, Bruker Spectrospin,N.V., Brussels, Belgium) was used. The chromatographic system consisted of a PE 601 pump (Perkin-Elmer), the electrochemical eluent scrubber, an injection valve (25 fiL loop) (Valco Instruments Co., Houston, TX) and a column (ss, 10 cm X 4.6 mm id.) packed with ODS hypersil5 pm (Shandon, Rumcorn, U.K.). The electrochemical scrubber and the polarographic detector were constructed in our workshop and have been extensively described elsewhere (16, 19). The electrical currents were measured with the Bruker and recorded on a XY recorder (BD 30, Kipp en Zonen, Delft, The Netherlands). All potentials and electrical currents were checked on a Tektronix 5103 N oscilloscope (Tektronix, Beverton, OR). The applied potential was always measured vs. an AglAgCll 1M LiCl, methanol-water (50/50% v/v) reference electrode. Chemicals. In all measurements water-methanol mixtures, M HN03or 0.02 M HaOd, which contained 0.1 M KNOBand were used as eluent. The stock solution was deaerated for several hours by purging with nitrogen (A 28). Nitrazepam was supplied by Nogefa (Haarlem, The Netherlands), All other chemicals were analytical-reagent grade (Baker “Analyzed”or Merck P.A.) and used without further purification. Water was demineralized and distilled. Samples were deaerated by purging with nitrogen for 10 min.

RESULTS AND DISCUSSION Batch Experiments, These experiments were performed in a classical polarographic setup. The DC polarogram of nitrazepam (1,3-dihydro-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one, mol w t 281.26) exhibits two waves. The more positive one is the reduction of the nitro group and the more negative one that of the azomethine function. On comparison 0 1981 American Chemical Society






310 290

270 0.4

250 1230





Flgwe 3. The peak height vs. the adjusted phase angle; for conditions see Figure 1.


Figure 1. The peak height and summit potential as a function of the percentage of methanol: lo-' M nitrazepam, f = 35 Hz, amplitude = 7 mV eff, scan rate = 2 mV s-', drop time = 1 s, hHo= 50 cm,

6 = oo.




0.6 0




Flgure 4. The peak height vs. the root of the applied frequency in flow Conditions: lo-' M nitrazepam, amplitude = 7 mV eff, scan rate = 2 mV s-', cycle time = 0.4 s, h y = 50 cm, 4 = Oo, flow rate = 1


mL min-'.





30 AEoc (mVoff)

Flgure 2. The peak height vs. the applied amplitude: ( 0 )f = 11.6 Hz, ( X ) f = 35 Hz,(V)f = 116 Hz. For further conditions see Figure 1.

of the DC and AC polarograms, the half-wave and summit potentials for the nitro group are equal within 5 mV. For the azomethine function the summit potential is 25 mV more negative than the half-wave potential. However, in this work we paid attention mainly to the behavior of the nitro group as most interesting functional group for flow-through detection. In order to establish the influence of the eluent as used in reversed-phase chromatography, we measured the peak height and summit potential as a function of the percentage methanol (see Figure 1). From this plot it is obvious, that the highest peak currents occur in the low methanol percentage range and that the summit potential is dependent on the eluent composition. Further, the summit potential depends on the pH. In a p H range of 2-6.5 (Britton-Robinson buffers) a linear relation of -58 mV per p H unit was found. From a logarithmic plot of (id - i)iP vs. the potential in the rising part of a DC polarogram, a value of 2.0 can be calculated for an. As this is a four-electron electrode reaction, it appears that the reaction is not reversible and a value for a of 0.5 can be assumed. The plot of the peak height vs. the root of the applied frequency indicates a fairly irreversible behavior in AC polarography. A constant value for the peak height is reached already at a rather low frequency. As there is no gain in using high frequencies, it is attractive to apply a low frequency, where the capacitive current will be small.

The dependence of the peak height upon the applied amplitude has been measured at three frequencies. The response is actually the same for all frequencies (see Figure 2). Up to 15 mV the peak height increases linearly with the amplitude, but up to 35 mV an important gain in signal can be achieved. Another interesting feature of AC measurements is the phase angle between the applied voltage and the resulting current. In Figure 3 a vectorial representation is given of the current for nitrazepam as function of the adjusted phase angle. In this batch experiment an expected pattern is found and a phase angle of 0" offers the best ratio between faradaic and capacitive currents. For a concentration range of 10-4-104 M a calibration line was computed via the method of least squares. A sensitivity of 6.99 X A L mol-' was found with a regression coefficient of 0.9977. At higher concentrations the sensitivity is slowly decreasing and the response is no longer linear. A concentration of lo4 M can be estimated as the lower detection limit. Experiments in a Flow System. These experiments were carried out with the DME detector, a t a flow rate of 1 mL min-'. The eluent contained a constant level of M nitrazepam and the electrochemical scrubber cannot be used. In the AC polarogram the summit potential for the nitro group has shifted 30 mV in cathodic direction compared to the half-wave potential measured under the same conditions. The plot of the peak height as function of the root of the applied frequency exhibits the same form as in the corresponding batch experiments. Only a t very low frequencies does the peak height increase with the frequency, but a plateau is soon reached (see Figure 4). Again the dependence of the peak height upon the applied amplitude was measured at two frequencies (see Figure 5). In contrast to the behavior in the batch experiment there is a difference in response for the two





AEOC ( m V Q ' f )


T -

Flgure 5. The peak height vs. the applied amplitude in flow conditions: ( 0 )f = 10 Hz, (X) f = 40 Hz. For further conditions see Figure 4.






Figure 7. Determination of nitrazepani near the detection limit: flow rate = 1 mL min-', eluent water-methanol (50150% (v/v)), 0.1 M KN03, lo3 M HN03, f = 11.7 Hz,amplitude = 7 mV eff, 4 = Oo, cycle time = 0.4 s, hHB= 50 cm. 2




0 25pA .925mV

Figure 6. The peak height vs. the applied phase angle in flow conditions, f = 10 Hz. For further conditions see Figure 4. frequencies used. But equally, the peak height increases linearly with the amplitude up to 15 mV, while up to 35 mV there is a favorable gain in signal. In the measurement of the peak height as a function of the adjusted phase angle the difference between batch and flow conditions is striking. In Figure 6 a vectorial representation of the total, capacitive and faradaic currents is given. Especially the relation for the capacitive current differs greatly with that under nonflow conditions. Probably a phase angle shift due to the geometry and construction of the cell could be held responsible for this phenomenon. However, a phase angle of Oo still offers the best distinction of the faradaic component of the total current from the capacitive current. Performance as Detector for HPLC. For the measurement of the linearity of response, sensitivity, and detection limit an actual chromatographic system was adapted. The oxygen present in the sample can easily be separated chromatographically from nitrazepam. The indication potential was fixed at the summit potential of the nitro group. At a flow rate of 1mL min" (water-methanol, 50/50% (v/v), 0.1 M KN03, M HNOJ the capacity factor for nitrazepam was 4.5 and the effective plate number of the coIumn was 1500. A linear dynamic range of 4 orders of magnitude was found (10-4-4 x M). The calibration line was computed via linear regression with the method of least squares; a sensitivity of 5.1 x 10" A L mol-' (or 200 A mol-' injected) with a regression coefficient of 0.9995 was found. With a noise of 0.05 nA (peak-to-peak variation in the base line), the detection limit (3X noise) was 0.7 pmol (or 0.2 ng) per injection of 25 pL (see Figure 7). Calculated from the chromatographic data this would represent a least detectable amount (for an ideal plug mol (or 0.3 pg). This is a 5-fold injection of 25 ILL)of 1 x improvement in detection limit in comparison to pulse PO-



. 1 r -








Flgure 8. Chromatograms of four benzodiazepines at two detection potentials: (1)bromazepam, (2) nitrazepam, (3) diazepam, (4) clonazepam; 1 gg of each injected. H N 0 3 replaced by 0.02 M H2S04. For further conditions see Figure 7. larographic detection and about 20-fold compared to the fast DME system (18). To demonstrate the selectivity of the detector, another well-known advantage of AC measurement, we chromatographed a mixture of four benzodiazepines: bromazepam, nitrazepam, diazepam, and clonazepam. Two of them contain a nitro group (nitrazepam and clonazepam) and all four possess, of course, the azomethine function. If the potential is fixed at a certain value, only compounds having a summit potential within the window d e f i e d by the AC amplitude will be detected. So, the first two compounds can be selectivity detected a t the summit potential of the nitro group. All of the benzodiazepines can be recorded a t the summit potential of the azomethine group (see Figure 8) without interference from the nitro groups or other compounds with summit potentials different from the one for the azomethine poup.

CONCLUSION Phase-sensitive sampled alternating current measurement appears to be a powerful analytical technique. Especially when used in polarographic detection for HPLC a very low detection

Anal. Chern. 1981, 53,

limit for nitrazepam can be found. The added selectivity of this type of detection can be advantageous in separations of complex mixtures. Also the possibility of electrokinetic investigations makes AC measurement highly interesting.

ACKNOWLEDGMENT We wish to thank H. G. de Jong for technical assistance. LITERATURE CITED (1) Kissinger, P. T. Anal. Chem. 1977, 49, 448. (2) Brunt, K . Pharm. Weekbl. 1978, 773, 689. (3) Rucki. R. J . Talanfa 1980, 2 7 , 147. (4) Kissinger, P. 1.;Refshauge, C.; Dreiling, R.; Adams, R . N . Anal. Left. 1973, 6, 259. (5) Boilet, C.; Oliva, P.; Caude, M. J . Chromatogr. 1977, 749, 625. (6) Cox, J. A.; Przyjazny, A. Anal. Left. 1977, 70, 869. (7) McCrehan, W. A.; Durst. R. A.; Bellama, J . M. Anal. Lett. 1977, 10, 1175. (8) Sasa, S.; Leroy Blank, C. Anal. Chem. 1977, 49, 354. (9) Lores, E. M.; Bristol, D. W.; Moseman, R. F. J . Chromfogr. Sci. 1978, 16, 358. (10) Carr, R. S.; Neff, J . M . Anal. Chem. 1980, 5 2 , 2428. (11) McCrehan, W. A. Anal. Chem. 1981, 53, 74.



(12) Blaedel, W. J . ; Wang, J . Anal. Chem. 1981, 53, 78. (13) Beauchamp, R.; Boinay, P.; Fombon, J. J.; Tacussei, J.; Breant, M.; Georges,J.; Porthault, M.; Vittori 0. J . Chromafcgr. 1981, 204, 123. (14) Hanekamp, H. B.; Bos, P.; Brinkman, U. A. Th.; Frei, R. W. Fresenius’ 2.Anal. Chem. 1979, 297, 404. (15) Hanekamp, H. B.; Bos, P.; Frei, R. W . J . Chromatogr. 1979, 786, 489. (16) Hanekamp, H. B.; Voogt, W. H.; Bos, P. Anal. Chim. Acta 1980, 778, 73. (17) (ireenberg, M. S.; Mayer, W. J . J . Chromatogr. 1979, 769, 321. (18) Hanekamp, H. 6.; Voogt, W. H.; Bos, P.; Frei, R. W. J . Llq. Chromatogr. 1980, 3, 1205. (19) Hanekamp, H. B.; Voogt, W. H.; BOS, P.; Frei, R. W. Anal. Chlm. Acta 1980, f78, 81. (20) submitted for Dublicatbn in Anal. . . HanekamD. H. B.; Bos, P.: Vittorl. 0.. Chlm Acta. (21) Jee, R. D. J . Elecfroanal. Chem. 1978, 69, 109. (22) Lankelma, J.; Poppe, H. J . Chromatogr. Sci. 1978. 74, 310. (23) Kemula, W.; Kutner, W. J . Chromafogr. 1981, 204, 131. (24) Smith, D. E. Elecfroanal. Chem. 1986, 7 . (25) Sluyters-Rebach, M.; Sluyters, J . H. Necfroanal. Chem. 1970, 4 .


RECEIVED for review March 3,1981. Accepted May 11,1981. Sandoz (Basle) is thanked for financial support.

Correlation between the Retention Behavior of Mono- and Difunctional Solutes in Binary Solvent-Silica Gel Liquid Ck romatography Shoji Hara’ and Sayuri Miyamoto Tokyo College of Pharmacy, Horinouchi, Hachioji, Tokyo 192-03, Japan

To systematize the optimization of the binary solvent for multifunctional solutes in Ilquld-solld chromatography, we determined the correlation between the logartthms of capacity ratio vs. molar fraction of the stronger eluent by employing 3- and l’ir-mOnO- and 3,17-disubstituted androstanes containing the same functions at the same positions of the steroid nucleus. According to the linear relationship, It became possible to estimate the retention of difunctional solutes on the baas of the retention of two corresponding monofunctional solutes.

In the liquid chromatographic separation of a given sample mixture, optimization of the stationary and mobile phases is the major and most important process. The technique of separating mixtures by solvent system selectivity in liquidsolid chromatography has been widely utilized, especially in the field of organic chemistry, and much data related to the optimization of chromatographic systems has been accumulated. Snyder has determined the parameters for the adsorption energies of functional groups of solute molecules and the solvent strength parameters for the mobile phase on the basis of the theoretical and experimental investigations ( I ) . The quantitative relationship between the retentions and the binary solvent compositions has been exploited by Sonewinski and found to be useful for the selection of the mobile phase (2, 3). In these studies, silica gel-binary solvent systems containing a diluent and a stronger solvent have been the most commonly employed. Consequently, the retention behavior of a given sample in a binary solvent for silica gel chromatography has come to be predictable if the hydrogen bonding of the functional groups in the solute molecules with the silanol groups on the silica gel surface is evaluated (4-6). However, when the number of functional groups present in the solute increases, difficulty arises in applying directly

such procedures. In order to predict the retention behavior of a multifunctional compound and to optimize the chromatographic system based on the data obtained from the corresponding monofunctional solutes, a series of model compounds having typical substituents on the same nucleus was selected and their retention behaviors were compared. In this paper, three series of mono- and disubstituted solutes were prepared. In the sample compounds, acetoxyl, keto carbonyl, and hydroxyl groups, chosen as the most common functions in organic compounds having medium polarity, were located on the 3 and/or 17 position of a steroid nucleus. Various binary solvent systems were adapted as mobile phases and capacity ratios of the solutes related with the solvent composition were quantitatively determined.

EXPERIMENTAL SECTION Samples. 3- and 17-Monosubstituted and 3J7-disubstituted steroids were prepared by a standard procedure from 17phydroxy-5~~-androstar1-3-one and 3P-hydroxy-5-androsten-17-one which were commercially available. Monosubstituted androstane derivatives were obtained by deoxygenation of one of the oxygen functions in the disubstituted androstanes. A keto group in hydroxyandrostanone was converted to the methylene group via tosylhydrazone by Wolff-Kishner reduction. The remaining hydroxyl function was modified to the acyloxy group by acylation or to the keto group by chromium oxide or Oppenauer oxidation. Isolation and purification of the products were carried out by employing a direct fractionation procedure previously proposed by Hara (7, 8). Compounds were characterized and identified according to spectral data and compared directly with standard samples. Adsorbent and Columns. Irregular-shaped silica gel, with a pore size of 70 A, and particle diameter of 10 wm, Wakogel LCH-10,obtained from Wako Pure Chemicals Co., Osaka, was packed into glass tubes, 4 mm i.d. X 20 cm length. A CIG column system described in a previous report (9) was utilized. Chromatographic Procedure. Flow rate of the mobile phase

0003-2700/81/0353-1365$01.25/0 0 1981 American Chemical Society