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Anal. Chem. 1901, 53, 2156-2157
Table I. Accuracy and Precision of NTA Determinations in Disodium EDTA added
NTA content, % found av
1A 1B
0.000 0.000 0.000
0.000 0.000 0.000
2A
3A
0.050 0.050 0.050 0,100
3B 3c
0.100 0.100
4A
0.200 0.200 0.200 0.500 0.500 0.500 1.000
0.053 0.054 0.052 0.103 0.102 0.102 0.193 0.194 0.192 0.510 0.507 0.509 1.022 1.013 1.023
sample no.
1c 2B 2C
4B 4C 5A
5B 5C 6A 6B 6C a
1.000
1.000
Table 111. Determination of NTA in EDTA Based Products std devu
product
1A
EDTA freeacid
1B
0.000
0.053
sample no.
1c 0.001
1D 1E
0.001
0.193
0.001
0.509
0.002
1.019
0.006
2A
2B 2c
magnesium EDTA
2E 2F 3B 3c 3D
calcium EDTA
4A
Table 11. Comparison of HPLC t o Polarography NTA content, % sample polarproduct no. ography HPLC 1 edetate disodium USP 0.05 0.04 2 edetate disodium USP 0.02 0.01 3 edetate disodium USP 0.06 0.07 4 EDTA (free acid) 0.04 0.05 EDTA (free acid) 5 0.28 0.29 EDTA (free acid) 6 0.17 0.16 7 Na,EDTA 0.08 0.07 8 Na,EDTA 0.22 0.21 9 Na,EDTA 0.24 0.24 with from 0.5 to 10 mg (0.05-1.00%) of NTA and subsequently analyzed by HPLC as described above. In all cases the amount of NTA found was within *470 of that added. The relative standard deviation from the mean was within f 2 % . A comparison was made between the analytical results obtained utilizing the HPLC technique described above and the standard polarographic method. The results of this comparison are shown in Table 11. In general, good agreement was found. The results obtained with the HPLC method are somewhat lower than those obtained with the polarographic method. However, this was expected because of the inherent specificity of the HPLC technique. Copper complexes of NTA and EDTA are very stable (stability constants = 1012.68for copper NTA and 101s~so for copper EDTA) ( 4 ) . Therefore, in the presence of excess copper, the formation of the copper complexes should be favored over those of other metals. It should thus be possible to analyze NTA in other EDTA-based chelates. In order to
4B 4c
0.0 0.0 0.0
0.2 0.2 0.2
3E
3F
std dev = standard deviation.
0.0 0.0 0.0
0.2 0.2 0.2
2D
3A
0.0 0.0 0.0
0.2 0.2 0.2
1F
0.102
NTA content, % added found av std dev
iron(II1) EDTA
4D 4E
4F
0.0 0.0 0.0
0.2 0.2 0.2
0.173 0.177 0.195 0.376 0.366 0.352 0.305 0.313 0.308 0.502 0.506 0.506 0.057 0.054 0.049 0.253 0.254 0.255 0.006 0.005 0.005
0.182
0.012
0.365
0.012
0.309
0.004
0.505
0.002
0.053
0.004
0.254
0.001
0.005
0.001
0.181
0.006
0.178
0.177 0.187
test this hypothesis, 1g portions of EDTA (free acid), magnesium EDTA, calcium EDTA, and iron(II1) EDTA were “spiked with 2 mg (0.2%) of NTA and analyzed by the HPLC technique. The results of the analyses are presented in Table 111. The amount of NTA found ranged from 88 to 100% of that added. Lowest recovery was obtained with the iron(II1) complex. The above data show that the HPLC method may be useful for the quantitation of NTA in other EDTA based chelates besides the disodium salt. In addition, we have found it applicable to the analysis of NTA in other non-EDTA chelating agents, as well as in other matrices (Le., detergents).
ACKNOWLEDGMENT The authors wish to acknowledge the technical assistance of Emily Owens and thank the personnel in the Analytical Department at Ciba-Geigy, McIntosh, AL, who performed the polarographic analyses. (1)
LITERATURE CITED Clba-Geigy Product Bulletin “Sequestrene Na, Food Grade and Ne2 Ca Food Grade EDTA Derivative “N””; Clba-Gelgy Corp., Dyestuffs &
Chemicals Division: Greensboro, NC, 1973. (2) “US Pharmacopoela XX: The National Formulary XV”; United States Pharmacopoeial Convention Inc.: Rockville, MD, 1979; p 273. (3) Perfettl, G. A,; Warner, C. A. J . Assoc. Off. Anal. Chem. 1979, 62, p 1092. (4) Chaberek, Stanley; Martell, Arthur E. “Organic Sequestering Agents”; Wlley: New York, 1959; pp 564, 572.
RECEIVED for review May 1,1981. Accepted August 7,1981.
Continuous Cycling of Liquid Chromatographic Eluents Joy R. Miksic Bidecision Laboratories, 3 126 Forbes Avenue, Pittsburgh, Pennsylvania 152 13
We save approximately 80% in solvent use and in preparation and disposal expense by cycling our mobile phase for
our HPLC systems. Our mobile phase is continually replenished by pumping the column effluent directly into the mobile
0003-2700/81/0353-2156$01.25/0 0 1981 Amerlcan Chemical Society
Anal. Chem. 1981, 53,2157-2158
i
..-a
i
.-a .-
CYCLED MOBllI PHASE
Flgure 1. Comparison of chromatography with fresh (A) vs. cycled serum 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
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
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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. (1)
LITERATURE CITED 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
