- Email: [email protected]
Microcolumn Solid-phase Extraction Coupled Online to Capillary Liquid Chromatography Using a Valve Switching Technique
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 34, Issue 6, June 2006 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2006, 34(6), 759−763.
RESEARCH PAPER
Microcolumn Solid-phase Extraction Coupled Online to Capillary Liquid Chromatography Using a Valve Switching Technique Tian Hongzhe, Yang Bingcheng, Guan Wenna, Guan Yafeng* Department of Analytical Chemistry and Micro-Instrumentation, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
Abstract:
A packed microcolumn solid-phase extraction coupled online to a capillary liquid chromatography (CapLC) was
described and evaluated. A short packed capillary column (65 mm×0.45 mm i.d., 5 μm C18) was used as the extraction cartridge and was mounted on a 10-port switching valve. A given volume of sample in a sample loop mounted on an injection valve passed through the cartridge, which retained the target compounds of the sample. The compounds were then desorbed by mobile phase, and were separated on an analytical column. Anisaldehyde water sample was used as the standard sample to evaluate the system. The calibration curve was linear (r > 0.998) over the concentration range of 0.01–0.5 mg l–1. The relative standard deviations of the retention time, the peak height, and the peak area of anisaldehyde in the linear ranges were 1.4%–2.2%, 4.2%–5.7%, and 6.0%–10.1%, respectively. The detection limit of anisaldehyde using this device was 6.0×10–3 μg/L (S/N=3). In addition, the sensitivity of some apolar aromatic hydrocarbon compounds could be improved by 30–100 folds. The method was applied for the determination of traces of naphthalene in seawater, with a detection limit of 0.13 μg l–1 (S/N=3). Key Words:
Microcolumn solid-phase extraction; Capillary liquid chromatography; Anisaldehyde; Polycyclic aromatic
hydrocarbons; Sample preparation; Sea water
1
Introduction
Green chemistry is an important concept in the field of analytical chemistry that requires considerably reduction of consumption of detrimental solvents both in the sample preparation steps and in the analytical procedure, enhancing the sample utility, or even solvent-free methodology in sample preparation. The sample volumes in trace analysis by high performance liquid chromatography (HPLC) method was reduced from liters to milliliters, or even microliters by using new technologies. The solid-phase microextraction (SPME)[1] coupled offline to liquid chromatography (LC)[2,3] and the in-tube solid-phase microextraction (in-tube SPME) coupled online to LC or gas chromatography (GC)[4–6] are examples of successful applications of the concept. For online coupling of in-tube SPME-capillary LC, the trap column should have a thin coating extraction phase, short length, and small diameter Received 23 May 2005; accepted 14 December 2005 * Corresponding author. Email: [email protected]; Tel: +86 411-84379590
because of the limitation of sample load in capillary LC. The drawbacks of such trap columns are a relatively small volume of the extraction phase and low extraction efficiencies. In 1981, Erni[7] achieved online sample concentration and separation with conventional HPLC columns, using a valve switching technique. Subsequently, the valve switching and online concentration techniques have been applied to the biological sample analysis in a large variety of complex matrices such as serum, urine, and animal feed[8–10]. More attention is focused on the use of CapLC to reduce the solvent consumption in recent years. The mobile phase consumption of CapLC was 1/500–1/50 of the conventional LC, and the sample load on the capillary columns is about 0.05–0.5 μl, which was 1/400–1/20 of that of the conventional columns. Because the effective path length in the detector cell decreased from 10 mm in conventional LC to 0.2–3 mm in CapLC, and the concentration sensitivity was reduced accordingly. The
TIAN Hongzhe et al. / Chinese Journal of Analytical Chemistry, 2006, 34(6): 759–763
detection limit of CapLC was about 20–100 folds higher than that of the conventional LC, that could hardly meet the requirement of routine analysis. Feng and Shintani[11,12] reported the use of monolithic column as extraction cartridge, which was mounted as a sample loop on an injection valve for extracting the sample. Higher enrichment factor was achieved compared with in-tube-SPME because of the relatively large surface area of the monolithic column. Similar work had also been reported by the authors of this study[13]. However, the reproducibility of the preparation of monolithic column was not satisfactory, and the variety of the commercial monolithic column is rather limited compared with the stationary phases for packed HPLC columns. The conventional packing materials have better reproducibility and considerably more variety for use in practical applications. In this study, a C18 packed capillary column was employed as the trap column. The target analytes were desorbed with the mobile phase and were then separated on the separation column. The solvent consumption of the whole analytical system was only 3 ml/day.
2 2.1
the procedure by Guan[14]. The two ends of the trap columns were connected to fused-silica capillaries with standard peek unions (Upchurch Inc, USA). The trap column was eluted with pure acetonitrile for 1 hour and then with pure water for 2 hours when it was used for the first time. 2.3
Combination of the trap column with LC
The overall system configuration for online extraction is presented in Fig. 1. The solid-phase extraction includes two steps: sample load and extraction (position A in Fig. 1) and the desorption of the target compounds (position B in Fig. 1), which is injected into the separation column.
Experimental Instrument and reagents Fig. 1 Diagram of the extraction system
An Ultra-Plus II model consisting of quaternary pumps, a pump controller, two mixers, a ten-port automated valve and a six-port automated valve, and a Linear UVIS 200 detector were obtained from Micro-Tech Scientific (Vista, CA, USA). The detection wavelength was 254 nm. Chrom Perfect (Justice Innovations, Mountain View, CA, USA) was used for data acquisition and processing. Methanol and acetonitrile were of HPLC grade (Spectro, USA). The water used in the experiment was prepared from the Wahaha purified water (Wahaha Group, Hangzhou, China). The polycyclic aromatic hydrocarbon (PAH) compounds: biphenyl, fluorene, and anthracene were of analytical grade. Anisaldehyde (purity 99%) was obtained from Sigma (Beijing, China). The stock solutions of the compounds mentioned above were prepared by dissolving a precise weight of each compound in pure methanol to the concentration of 1000 mg l–1. The standard solutions were obtained by gradually diluting the stock solutions with 1:1 (v/v) water/acetonitrile to the concentration of 20 mg/l. The aqueous solutions were prepared by further diluting the standard solutions with purified water to form spiked water samples. 2.2
Chromatographic conditions
A 150 mm × 0.32 mm i.d., 5 μm ODS (Micro-Tech Scientific Inc, USA) and a homemade column 300 mm × 0.45 mm i.d., 5 μm C18 were used for the separation. Five trap columns of 65 mm × 0.45 mm i.d. C18 were made according to
Real line: loading and extraction of the sample; dashed line: desorption and injection
The aqueous sample was injected into the sample loop on a six-port valve with a 50-μl syringe and then swept into a trap column on a ten-port valve by the aqueous mobile phase after switching the six-port valve. The unretained compositions and the solvent were eluted as waste. The target solutes were retained in the trap column during the process; the ten-port valve was then switched, and the target solutes were desorbed by the mobile phase, separated by the analytical column, and detected by the UV detector.
3 3.1
Results and discussion Extraction capacity of the trap column
The sample capacity of the trap column was measured according to literature [12]. The aqueous sample of anisaldehyde (concentration 5 mg l–1) was passed continuously through the trap column by the eluent of pump 1, and the outlet of the trap column was directly connected to the detector. The abrupt increase in the absorbance on the detector suggested the breakthrough of anisaldehyde from the trap column. The breakthrough volume was the extraction capacity under conditions of sample concentration and flow rate. Different extraction capacities were obtained at different flow rates (Table 1).
TIAN Hongzhe et al. / Chinese Journal of Analytical Chemistry, 2006, 34(6): 759–763
Table 1 Extraction capacity of the extraction column No 1
–1
Flow rate (μl min ) 1
Time (min) 3381
Extraction amount (ng) 3381
2
5
661
3305
3
10
326
3260
The results showed that the extraction capacity changed slightly under different flow rates, revealing that rapid partition equilibrium could be achieved. In the following experiment, concentration of the samples was low, and the analytes were completely concentrated in the trap column because a considerably smaller injection volume was used. 3.2
Characteristics of the trap column coupled with LC
3.2.1 Correlation between injection volume and peak height or peak area The effect of the extraction volume over the amount of extraction was studied with different injection volumes of 50, 100, 200, 300, 400, 500, 600, and 700 μl of the standard anisaldehyde water solution (0.05 mg l–1) under similar separation conditions. Table 2 shows the relationship between the peak areas and the peak heights versus the injection volumes. As seen in Table 2, the amount of extraction on the trap column was directly proportional to the extraction volume. Therefore, the detection limit was considerably reduced when the injection volume was large (less than the breakthrough volume of the target compounds). 3.2.2
Validation of the analytical method
The analyses of the spiked water sample of standard anisaldehyde at concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg l–1 and injection volume of 5 μl-samples were carried out to evaluate the performance of the extraction method. To evaluate the precision of the measurements, six parallel analyses of the spiked samples at different concentrations were carried out. The RSDs of the retention time, the peak height, and the peak area were 1.4%–2.2%, 4.2%–5.7%, and 6.0%–10.1%, respectively. The calibration curve of the peak areas of the spiked samples to the sample concentrations showed good linearity with a correlation coefficient (r) of more than 0.998. A regression equation is established: Y = A + BX, where A = 0.0330 and B = 50.6044, Y represents the peak area, and X represents the concentration of the compounds. The detection limit of anisaldehyde was calculated to be 6 ng l–1 (S/N=3) at an injection volume of 700 μl (chromatogram not shown), whereas detection limit of anisaldehyde was estimated to be 9.80 μg l–1 (S/N=3) by direct injection of 0.2 μl sample. The gain on sensitivity was about
1633 folds by the trap column instead of the direct injection in CapLC. The stability of the trap column was tested after 120 injections, the mean deviation of the peak area being 9.4%. The variations of enrichment factor for six trap columns packed with the same stationary phase was less than 1.6% RSD on the peak area under identical conditions. Table 2 Effect of the extraction volumes on the peak heights and the peak areas of the samples No
Extraction
Retention time
Peak height
Peak area
volume (μl)
(min)
(mV)
(mV s)
1
50
5.34
95.1
779
2
100
5.56
184.9
1515
3
200
5.53
351.9
2934
4
300
5.55
510.5
4359
5
400
5.41
622.9
6379
6
500
5.69
776.8
7316
7
600
5.66
926.9
8948
8
700
5.64
999.7
10807
3.3
Desorption carryover
The desorption carryover of the trap column was examined using the ratio of the peak areas of the target compound from the first desorption and the second desorption. The results indicated that the carryover effect was negligible at a sample concentration less than 25 μg/l. However, when the sample concentration was over 0.1 mg/l, a carryover was observed in the range of 1.0%–6.1% because of the presence of a relative large amount of the extracted analytes on the trap column and incomplete desorption of the analytes by the mobile phase. To eliminate the carryover effect, the trap column was conditioned with pure acetonitrile for 15 minutes, followed with pure water for 10 minutes after each extraction. Using this procedure, the extraction capacity of the trap column was recovered, and no carryover was observed. The separation column was conditioned using 80/20 (v/v) acetonitrile/water until the next run during this period. 3.4
Analysis of PAHs
Fig. 2 illustrates the CapLC chromatograms of PAHs in water samples (10–25 μg/l each) using different injection methods. Fig. 2A is the chromatogram of PAHs spiked sample by direct injection of 0.1 μl sample; Fig. 2B shows the chromatogram with injection of 5 μl of sample on the trap column followed by desorption. The extraction technique exhibited higher detection sensitivities (biphenyl, 32 times; fluorene, 34 times; anthracene, 95 times) than those obtained by direct injection. The capacities of the three analytes (biphenyl, fluorene, and anthracene) on the trap column were 10.3, 11.0 and 28.4 μg, respectively. The retention times of
TIAN Hongzhe et al. / Chinese Journal of Analytical Chemistry, 2006, 34(6): 759–763
the trap method were prolonged by about 0.44–0.83 min when compared with the direct injection method because of the internal volume of the trap and the desorption process. An excellent peak shape was achieved by the sharp difference in elution strength between the sample matrix (water) and mobile phase. The injection volume for trap column was still smaller than that of conventional HPLC.
Fig. 2
commercial phases are available to meet the requirements of the samples from different sources and different target analytes. (2) It is suitable for trace analysis when a limited volume of samples are available. (3) The device is easy to operate and simple in structure. (4) Good reproducibility is obtained. The system has great potential for application to environmental, plasma, and urine samples.
Typical chromatograms obtained by different injection
methods A: direct injection; B: extraction-sampling. Conditions: extraction volume: 5 l; isocratic elution: 90/10 (v/v) ACN/H2O; flow rate: 8 l min–1; separation column: 300 mm×0.45 mm i.d. (C18, 5 μm); 1.25 g l–1 biphenyl; 2.25 g l–1 fluorene; 3.10 g–1 anthracene
3.5
Real-sample analysis
Seawater samples from Xinghaiwan and Xianglujiao (Dalian, China) were analyzed at optimized conditions as described above (shown in Fig. 3). The seawater samples were filtered through a 0.45-μm-pore filter and were then injected into the trap column. A strong background signal was observed with UV detection because of the matrix effect of the seawater samples. The identification of analytes in the seawater samples was accomplished using the retention time of the spiked samples and the seawater samples. Naphthalene was detected in the Xianglujiao seawater sample, whereas PAHs was not detected in the Xinghaiwan samples. The detection limit of naphthalene in the seawater samples was 0.13 μg/l (S/N = 3), and the recovery of naphthalene in seawater was in the range of 88.1%–95.2%.
Fig. 3 Chromatograms of sea water samples A: blank sea water; B: blank sea water spiked with 50g l–1 naphthalene; C: Xianglujiao sea water. Conditions: gradient elution: 0–8 min 40%–95% ACN; 8–20 min, 95% ACN; flow rate, 5 l min–1; separation column: 150 mm×0.32 mm i.d. (ODS, 5 μm); B: extraction volume, 1.0 ml; C: extraction volume, 4.0 ml
References [1]
Arthur C L, Pawliszyn J. Anal. Chem., 1990, 62(19): 2145–2148
[2]
Chen S, Han Z X, Quan X, Lin G X, Yang F L. Chinese J. Anal. Chem., 2003, 31(2): 171–174
4
Conclusions
[3]
Tao J Q, Wang C Y, Li B F, Li G K. Chinese J. Chromatogr., 2003, 21(6): 599–602
Microcolumn solid-phase extraction coupled online with CapLC has many advantages: (1) Preparation of trap columns is relatively simple and reproducible. Large varieties of
[4]
Lord H, Pawliszyn J. J Chromatogr. A, 2000, 885(1–2): 153–193
[5]
Eisert R, Pawliszyn J. Anal. Chem., 1997, 69(16): 3140–3147
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[6]
Wang H W, Liu W M, Guan Y F. LC-GC North America, 2004, 22(1): 16–24
[7]
Erni F, Keller H P, Morin C, Schmitt M. J Chromatogr., 1981, 204: 65–76
[8]
Little C J, Tompkins D J, Stahel O, Frei R W, Werkhoven G C E. J. Chromatogr., 1983, 264: 183–196
[9]
Ramsteiner K A, Böhm K H. J. Chromatogr., 1983, 260: 33–40
[10] Imai H, Masujima T, Morita-Wada I, Tamai G. Analytical Sciences, 1989, 5(4): 389–393
[11] Fan Y, Feng Y Q, Da S L, Gao X P. Analyst, 2004, 129(11): 1065–1069 [12] Shintani Y, Zhou X J, Furuno M, Minakuchi H, Nakanishi K. J. Chromatogr. A , 2003, 985(1–2): 351–357 [13] Shu X, Yang B C, Tan F, Guan Y F. Chinese Journal of Analysis Laboratory, 2005, 24(2): 41–43 [14] Guan Y F, Zhou L M, Shang Z H. J. High Resolut. Chromatogr., 1992, 15(7): 434–436
Cite this article as: Chin J Anal Chem, 2006, 34(6), 759−763.
RESEARCH PAPER
Microcolumn Solid-phase Extraction Coupled Online to Capillary Liquid Chromatography Using a Valve Switching Technique Tian Hongzhe, Yang Bingcheng, Guan Wenna, Guan Yafeng* Department of Analytical Chemistry and Micro-Instrumentation, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
Abstract:
A packed microcolumn solid-phase extraction coupled online to a capillary liquid chromatography (CapLC) was
described and evaluated. A short packed capillary column (65 mm×0.45 mm i.d., 5 μm C18) was used as the extraction cartridge and was mounted on a 10-port switching valve. A given volume of sample in a sample loop mounted on an injection valve passed through the cartridge, which retained the target compounds of the sample. The compounds were then desorbed by mobile phase, and were separated on an analytical column. Anisaldehyde water sample was used as the standard sample to evaluate the system. The calibration curve was linear (r > 0.998) over the concentration range of 0.01–0.5 mg l–1. The relative standard deviations of the retention time, the peak height, and the peak area of anisaldehyde in the linear ranges were 1.4%–2.2%, 4.2%–5.7%, and 6.0%–10.1%, respectively. The detection limit of anisaldehyde using this device was 6.0×10–3 μg/L (S/N=3). In addition, the sensitivity of some apolar aromatic hydrocarbon compounds could be improved by 30–100 folds. The method was applied for the determination of traces of naphthalene in seawater, with a detection limit of 0.13 μg l–1 (S/N=3). Key Words:
Microcolumn solid-phase extraction; Capillary liquid chromatography; Anisaldehyde; Polycyclic aromatic
hydrocarbons; Sample preparation; Sea water
1
Introduction
Green chemistry is an important concept in the field of analytical chemistry that requires considerably reduction of consumption of detrimental solvents both in the sample preparation steps and in the analytical procedure, enhancing the sample utility, or even solvent-free methodology in sample preparation. The sample volumes in trace analysis by high performance liquid chromatography (HPLC) method was reduced from liters to milliliters, or even microliters by using new technologies. The solid-phase microextraction (SPME)[1] coupled offline to liquid chromatography (LC)[2,3] and the in-tube solid-phase microextraction (in-tube SPME) coupled online to LC or gas chromatography (GC)[4–6] are examples of successful applications of the concept. For online coupling of in-tube SPME-capillary LC, the trap column should have a thin coating extraction phase, short length, and small diameter Received 23 May 2005; accepted 14 December 2005 * Corresponding author. Email: [email protected]; Tel: +86 411-84379590
because of the limitation of sample load in capillary LC. The drawbacks of such trap columns are a relatively small volume of the extraction phase and low extraction efficiencies. In 1981, Erni[7] achieved online sample concentration and separation with conventional HPLC columns, using a valve switching technique. Subsequently, the valve switching and online concentration techniques have been applied to the biological sample analysis in a large variety of complex matrices such as serum, urine, and animal feed[8–10]. More attention is focused on the use of CapLC to reduce the solvent consumption in recent years. The mobile phase consumption of CapLC was 1/500–1/50 of the conventional LC, and the sample load on the capillary columns is about 0.05–0.5 μl, which was 1/400–1/20 of that of the conventional columns. Because the effective path length in the detector cell decreased from 10 mm in conventional LC to 0.2–3 mm in CapLC, and the concentration sensitivity was reduced accordingly. The
TIAN Hongzhe et al. / Chinese Journal of Analytical Chemistry, 2006, 34(6): 759–763
detection limit of CapLC was about 20–100 folds higher than that of the conventional LC, that could hardly meet the requirement of routine analysis. Feng and Shintani[11,12] reported the use of monolithic column as extraction cartridge, which was mounted as a sample loop on an injection valve for extracting the sample. Higher enrichment factor was achieved compared with in-tube-SPME because of the relatively large surface area of the monolithic column. Similar work had also been reported by the authors of this study[13]. However, the reproducibility of the preparation of monolithic column was not satisfactory, and the variety of the commercial monolithic column is rather limited compared with the stationary phases for packed HPLC columns. The conventional packing materials have better reproducibility and considerably more variety for use in practical applications. In this study, a C18 packed capillary column was employed as the trap column. The target analytes were desorbed with the mobile phase and were then separated on the separation column. The solvent consumption of the whole analytical system was only 3 ml/day.
2 2.1
the procedure by Guan[14]. The two ends of the trap columns were connected to fused-silica capillaries with standard peek unions (Upchurch Inc, USA). The trap column was eluted with pure acetonitrile for 1 hour and then with pure water for 2 hours when it was used for the first time. 2.3
Combination of the trap column with LC
The overall system configuration for online extraction is presented in Fig. 1. The solid-phase extraction includes two steps: sample load and extraction (position A in Fig. 1) and the desorption of the target compounds (position B in Fig. 1), which is injected into the separation column.
Experimental Instrument and reagents Fig. 1 Diagram of the extraction system
An Ultra-Plus II model consisting of quaternary pumps, a pump controller, two mixers, a ten-port automated valve and a six-port automated valve, and a Linear UVIS 200 detector were obtained from Micro-Tech Scientific (Vista, CA, USA). The detection wavelength was 254 nm. Chrom Perfect (Justice Innovations, Mountain View, CA, USA) was used for data acquisition and processing. Methanol and acetonitrile were of HPLC grade (Spectro, USA). The water used in the experiment was prepared from the Wahaha purified water (Wahaha Group, Hangzhou, China). The polycyclic aromatic hydrocarbon (PAH) compounds: biphenyl, fluorene, and anthracene were of analytical grade. Anisaldehyde (purity 99%) was obtained from Sigma (Beijing, China). The stock solutions of the compounds mentioned above were prepared by dissolving a precise weight of each compound in pure methanol to the concentration of 1000 mg l–1. The standard solutions were obtained by gradually diluting the stock solutions with 1:1 (v/v) water/acetonitrile to the concentration of 20 mg/l. The aqueous solutions were prepared by further diluting the standard solutions with purified water to form spiked water samples. 2.2
Chromatographic conditions
A 150 mm × 0.32 mm i.d., 5 μm ODS (Micro-Tech Scientific Inc, USA) and a homemade column 300 mm × 0.45 mm i.d., 5 μm C18 were used for the separation. Five trap columns of 65 mm × 0.45 mm i.d. C18 were made according to
Real line: loading and extraction of the sample; dashed line: desorption and injection
The aqueous sample was injected into the sample loop on a six-port valve with a 50-μl syringe and then swept into a trap column on a ten-port valve by the aqueous mobile phase after switching the six-port valve. The unretained compositions and the solvent were eluted as waste. The target solutes were retained in the trap column during the process; the ten-port valve was then switched, and the target solutes were desorbed by the mobile phase, separated by the analytical column, and detected by the UV detector.
3 3.1
Results and discussion Extraction capacity of the trap column
The sample capacity of the trap column was measured according to literature [12]. The aqueous sample of anisaldehyde (concentration 5 mg l–1) was passed continuously through the trap column by the eluent of pump 1, and the outlet of the trap column was directly connected to the detector. The abrupt increase in the absorbance on the detector suggested the breakthrough of anisaldehyde from the trap column. The breakthrough volume was the extraction capacity under conditions of sample concentration and flow rate. Different extraction capacities were obtained at different flow rates (Table 1).
TIAN Hongzhe et al. / Chinese Journal of Analytical Chemistry, 2006, 34(6): 759–763
Table 1 Extraction capacity of the extraction column No 1
–1
Flow rate (μl min ) 1
Time (min) 3381
Extraction amount (ng) 3381
2
5
661
3305
3
10
326
3260
The results showed that the extraction capacity changed slightly under different flow rates, revealing that rapid partition equilibrium could be achieved. In the following experiment, concentration of the samples was low, and the analytes were completely concentrated in the trap column because a considerably smaller injection volume was used. 3.2
Characteristics of the trap column coupled with LC
3.2.1 Correlation between injection volume and peak height or peak area The effect of the extraction volume over the amount of extraction was studied with different injection volumes of 50, 100, 200, 300, 400, 500, 600, and 700 μl of the standard anisaldehyde water solution (0.05 mg l–1) under similar separation conditions. Table 2 shows the relationship between the peak areas and the peak heights versus the injection volumes. As seen in Table 2, the amount of extraction on the trap column was directly proportional to the extraction volume. Therefore, the detection limit was considerably reduced when the injection volume was large (less than the breakthrough volume of the target compounds). 3.2.2
Validation of the analytical method
The analyses of the spiked water sample of standard anisaldehyde at concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg l–1 and injection volume of 5 μl-samples were carried out to evaluate the performance of the extraction method. To evaluate the precision of the measurements, six parallel analyses of the spiked samples at different concentrations were carried out. The RSDs of the retention time, the peak height, and the peak area were 1.4%–2.2%, 4.2%–5.7%, and 6.0%–10.1%, respectively. The calibration curve of the peak areas of the spiked samples to the sample concentrations showed good linearity with a correlation coefficient (r) of more than 0.998. A regression equation is established: Y = A + BX, where A = 0.0330 and B = 50.6044, Y represents the peak area, and X represents the concentration of the compounds. The detection limit of anisaldehyde was calculated to be 6 ng l–1 (S/N=3) at an injection volume of 700 μl (chromatogram not shown), whereas detection limit of anisaldehyde was estimated to be 9.80 μg l–1 (S/N=3) by direct injection of 0.2 μl sample. The gain on sensitivity was about
1633 folds by the trap column instead of the direct injection in CapLC. The stability of the trap column was tested after 120 injections, the mean deviation of the peak area being 9.4%. The variations of enrichment factor for six trap columns packed with the same stationary phase was less than 1.6% RSD on the peak area under identical conditions. Table 2 Effect of the extraction volumes on the peak heights and the peak areas of the samples No
Extraction
Retention time
Peak height
Peak area
volume (μl)
(min)
(mV)
(mV s)
1
50
5.34
95.1
779
2
100
5.56
184.9
1515
3
200
5.53
351.9
2934
4
300
5.55
510.5
4359
5
400
5.41
622.9
6379
6
500
5.69
776.8
7316
7
600
5.66
926.9
8948
8
700
5.64
999.7
10807
3.3
Desorption carryover
The desorption carryover of the trap column was examined using the ratio of the peak areas of the target compound from the first desorption and the second desorption. The results indicated that the carryover effect was negligible at a sample concentration less than 25 μg/l. However, when the sample concentration was over 0.1 mg/l, a carryover was observed in the range of 1.0%–6.1% because of the presence of a relative large amount of the extracted analytes on the trap column and incomplete desorption of the analytes by the mobile phase. To eliminate the carryover effect, the trap column was conditioned with pure acetonitrile for 15 minutes, followed with pure water for 10 minutes after each extraction. Using this procedure, the extraction capacity of the trap column was recovered, and no carryover was observed. The separation column was conditioned using 80/20 (v/v) acetonitrile/water until the next run during this period. 3.4
Analysis of PAHs
Fig. 2 illustrates the CapLC chromatograms of PAHs in water samples (10–25 μg/l each) using different injection methods. Fig. 2A is the chromatogram of PAHs spiked sample by direct injection of 0.1 μl sample; Fig. 2B shows the chromatogram with injection of 5 μl of sample on the trap column followed by desorption. The extraction technique exhibited higher detection sensitivities (biphenyl, 32 times; fluorene, 34 times; anthracene, 95 times) than those obtained by direct injection. The capacities of the three analytes (biphenyl, fluorene, and anthracene) on the trap column were 10.3, 11.0 and 28.4 μg, respectively. The retention times of
TIAN Hongzhe et al. / Chinese Journal of Analytical Chemistry, 2006, 34(6): 759–763
the trap method were prolonged by about 0.44–0.83 min when compared with the direct injection method because of the internal volume of the trap and the desorption process. An excellent peak shape was achieved by the sharp difference in elution strength between the sample matrix (water) and mobile phase. The injection volume for trap column was still smaller than that of conventional HPLC.
Fig. 2
commercial phases are available to meet the requirements of the samples from different sources and different target analytes. (2) It is suitable for trace analysis when a limited volume of samples are available. (3) The device is easy to operate and simple in structure. (4) Good reproducibility is obtained. The system has great potential for application to environmental, plasma, and urine samples.
Typical chromatograms obtained by different injection
methods A: direct injection; B: extraction-sampling. Conditions: extraction volume: 5 l; isocratic elution: 90/10 (v/v) ACN/H2O; flow rate: 8 l min–1; separation column: 300 mm×0.45 mm i.d. (C18, 5 μm); 1.25 g l–1 biphenyl; 2.25 g l–1 fluorene; 3.10 g–1 anthracene
3.5
Real-sample analysis
Seawater samples from Xinghaiwan and Xianglujiao (Dalian, China) were analyzed at optimized conditions as described above (shown in Fig. 3). The seawater samples were filtered through a 0.45-μm-pore filter and were then injected into the trap column. A strong background signal was observed with UV detection because of the matrix effect of the seawater samples. The identification of analytes in the seawater samples was accomplished using the retention time of the spiked samples and the seawater samples. Naphthalene was detected in the Xianglujiao seawater sample, whereas PAHs was not detected in the Xinghaiwan samples. The detection limit of naphthalene in the seawater samples was 0.13 μg/l (S/N = 3), and the recovery of naphthalene in seawater was in the range of 88.1%–95.2%.
Fig. 3 Chromatograms of sea water samples A: blank sea water; B: blank sea water spiked with 50g l–1 naphthalene; C: Xianglujiao sea water. Conditions: gradient elution: 0–8 min 40%–95% ACN; 8–20 min, 95% ACN; flow rate, 5 l min–1; separation column: 150 mm×0.32 mm i.d. (ODS, 5 μm); B: extraction volume, 1.0 ml; C: extraction volume, 4.0 ml
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