2157
Anal. Chem. 1981, 53,2157-2158
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CYCLED MOBllI PHASE
Flgure 1. Comparison of chromatography with fresh (A) vs. cycled samples containing quinidine (A and B) or zero standard (C). Conditions: detection, UV 254 nm, 0.005 AUFS; injection, 80 pL; column, Whatman PXS C-8,25 cm; flow 2.0 mL/min; mobile phase, 40% CH&N in1 4.3% acetic acid-water pH 4.0. (B and C) mobile phase serum
phase pool. The sample components eluted from the column are diluted in the mobile phase pool, and cause a slow and uniform increase in detector offset. We have injected more than 400 samples over a ‘7-day period using cycled mobile phase with satisfactory reproducibility and sensitivity. The method has worked well with both UV and electrochemical detection for drug analyses using reversed-phase columns. Another group has reported the use of cycled mobile phases for carbohydrate analyses, on silica columns with a radial compression system ( I ) . We prepare the mobile phase pool by adding aqueous buffer (filtered through a 0.45 pm membrane) to the organic solvents and degassing under vacuum 3-5 min. During chromatography, the mobile phase is continually mixed and capped, and we begin cycling only after the column is equilibrated. The amount of mobile phase we use, 500 mL to 4 L, depends on the concentration of the peaks of interest, or the rate of increase in detector offset due to other components to which the detector responds. We do not allow the concentration of components of interest in the mobile phase to exceed our lower limit of detection nor the detector offset to be high enough to adversely affect signal to noise ratio. We continuously pump the mobile phase for about 1 week, during which approximately 500 trace drug analyses are performed. We then clean the column with acetonitrile/water: (75/25, v/v) for
1-2 h. Chromatograms of extracted serum samples containing quinidine and an internal standard are shown in Figure 1. Figure 1 A represents a sample chromatographed using fresh mobile phase. Figure 1B,C represents samples chromatographed using a 4-L pool of mobile phase which had been cycled for 21/2days, during which 200 injections were made. The UV offset increased from 0.015 to 0.300 during the week. With an autozero on our integrator/recorder, the base line exceeded the autozero limit after 20-30 samples (every 3-4 h). The detector offset was simply adjusted to keep chromatograms on scale. Linearity and precision of samples chromatographed using cycled mobile phase were comparable to values obtained using fresh mobile phase. Day-to-day precision over a 5-week period was typically 2-6% for all of our HPLC trace analyses. We observed no spurious peaks nor alteration in column life. A typical reversed-phase column yielded about 2000-3000 chromatographic runs. The retention times varied by less than 10% over 3-4 weeks, due to evaporation of organic solvents from the mobile phase and gradual changes in the stationary phase. We have used cycled mobile phases with six different assays of serum and urine extracts. Continuous cycling of mobile phase from an HPLC system has many advantages over the current practice of replacing mobile phase. It results in a saving in preparation time, solvent use, and disposal. In addition, the mobile phase is filtered by the column; therefore new contaminants do not enter the column. The HPLC system can be run at a low flow continuously, avoiding nightly cleanout of the apparatus. By cycling the entire mobile phase rather than only the “blank portions” of the chromatogram, we eliminate the necessity for switching valves or technician assistance. Precautions must be taken when cycling mobile phase that linearity is maintained. Blank samples should be run periodically to test for ghost peaks and signal to noise ratio should be analyzed. Every method in which cycled mobile phase is used should undergo ntandard validation procedures. We use cycled mobile phases for routine isocratic analyses of extracted samples and in methods development work. It is particularly useful for analyses with high flow rates or long run times and when the chromatographic system is used 12-24 h/day. We have also used it for analyses with mobile phases which are difficult to equilibrate, such as paired ion reagents. LITERATURE CITED (1) Hendrix, C. L.; Lee, R. E., Jr.; Baust, J. G.; James, H. J . Chromatogr. 1981, 210, 45-53.
RECEIVED for review May 26,1981. Accepted August 11,1981.
Determination of Dissolved Organic Carbon in Water Ronald A. van Steenderen” and Jlunn-Shyh Lin’ National Institute for Water Research of the Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, Republic of Sooth Africa Credit must be given to Goulden and Brooksbank ( I ) for developing a highly successful method for determining dissolved organic carbon (DOC) in water. We do however, from a cost point of view, contest the superfluous amount of ul1 Present address: Department of Bimhemistr National Defence Medical Centre, Taipei, Taiwan, Republic of Ckna.
traviolet (UV) irradiation (power of UV lamp) and the excessive length of silica coil used to attain complete oxidation of certain organic compounds (in this case, EDTA). Furthermore, Figure l illustrates that the potassium persulfate catalyst used in their method is at the extreme lower limit of oxidation effectivenless leaving little, if any, scope for internal system fluctuations. Therefore, although their method
0003-2700/81/0353-2157$01.25/00 1981 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
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Final concentration of K2S208in digestion stage (plus UV irradiation). Potasslum persulfate concentratlons In the methods described by Goulden et al. ( 7 ) , Van Steenderen et al. (3), and GravelBt-Blondin et al. ( 4 ) as indlcated. Flgure 1.
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Flgure 2. Modified TOC flow diagram: smc = short mixing coil; 1.8 m sllica coil, i.d. 3 mm, UV lamp 150 W; system for removal of air bubble formed during activation of sample arm.
worked, certain discrepancies gave rise to suspicion concerning the evaluation and optimization of the method described. In an attempt to seek optimal analytical conditions for the determination of DOC in seawater, Collins and Williams (2) prescribe a UV lamp relatively similar to that employed by Goulden and Brooksband ( I ) but admit (although they did not test them) that low-pressure lamps of considerably lesser wattage may also be effective. Their extreme length of silica coil used to attain complete oxidation as compared with that of Goulden and Brooksbank ( I ) is puzzling. We investigated various oxidation techniques and their effectiveness for the salts potassium biphthalate, DL-valine, and EDTA up to concentrations of 20 mg/L C, modifying the basic method described by Van Steenderen et al. (3). The method which evolved from these investigations is set out in Figure 2. Complete oxidation of these three salts was confirmed by means of a Beckman 915A TOC Analyzer. After considerable experimentation with various catalysts and their concentrations, UV intensities, varying lengths of silica coils, increased digestion temperatures, and the influence of pH on the digestion stage, it became evident that there were really only two criteria responsible for the effective conversion of
Figure 3. Influence of acid concentration on digestion stage Incorporating K2S208and UV irradiation. Acid concentration in the methods described by Goulden et al. ( 7 ) , Van Steenderen et ai. (3), and GravelBt-Blondin et 81. ( 4 ) as indicated.
DOC to COZ and these were the concentration of KzS208in the sample prior to the digestion stage combined with UV irradiation. The optimum effective oxidant conditions for the three salts tested were accomplished by passing the sample containing 0.25% K,S2O8 through a 3 mm i.d. (not critical) silica tube of 1.8 m length which was coiled around a 150-W UV lamp (retention time in the 20 mm diameter silica coil was 1.5 min). No heat, except that received from the W lamp was applied to the digestion stage. Increasing the digestion temperature, length of silica coil, UV lamp intensity, or adding Ag+ or Hg' salts as catalysts did not improve further oxidation efficiency. The conditions set out above were primarily directed toward the amino acid and EDTA since we found that potassium biphthalate, a widely accepted primary standard for standardizing of TOC Analyzers, decomposed relatively easily even without the use of UV treatment (3). Although potassium biphthalate is therefore a suitable calibration standard, it does not indicate oxidation efficiency and should at all times be used in conjunction with compounds such as those mentioned which are known to be more resistant. Acidification of the digesting liquid served no purpose but excessive acidification did have adverse effects on DOC recoveries (Figure 3). The latter findings are of significance since DOC analyses of natural waters require the prior removal of inorganic carbon by means of sample acidification. The unjustified use of excessive amounts of acid in a method described by Gravel6bBlondin et al. (4) is illustrated in Figure 3, and since these authors do not give any reasons for this excessive use of acid, we presume that they were unaware of its effects. ACKNOWLEDGMENT This paper is published with the approval of the Director of the National Institute for Water Research. LITERATURE CITED (1) GouMen, P. D.;Brooksbank, P. Anal. Chem. 1975, 47, 1943-1946. (2) Collins, K. J.; Le B. Williams, P. J. Mar. Chem. 1977, 5 , 123-141. (3) van Steenderen, R. A,; Basson, W. D.; Van Duuren, F. A. Water Res. 1879, 13, 539-543. (4) GraveiBt-Blondln, L. R.; Van VIM, H. R.; Mynhardt, P. A. Water SA 1980, 6, 138-143.
RECEIVED for review May 22, 1981. Accepted July 20, 1981.
