**4.6. Organic compounds**

### *4.6.1. Hippuric acid*

IC can also be used in detection of some acids. Zhao et al. [25] proposed a simple and ecofriendly ion chromatographic method for the determination of Hippuric acid (HA) in human urine (see Figure 12). Hippuric acid is a kind of metabolite of toluene in human body, therefore, HA is a physiological component of human urine if toluene was inhaled. The content of HA in human urine actually is confirmed as a diagnostic marker of exposure to toluene [26]. It has been reported that exposure to high concentrations of volatile organic compounds such as toluene lead to a series of diseases such as acute and chronic respiratory effects, functional alterations of the central nervous system, mucous and dermal irritations, and chromosome aberrations.. In order to diagnose patients who are suffering from a series of diseases caused by elevated HA levels, the determination of HA in human urine is necessary. Comparing with other chromatographic methods such as GC and HPLC, the proposed IC method used ecofriendly mobile phase (not containing organic solvent), and avoided complicated sample pretreatment. The separation was carried out on an anion exchange column with 2.0 mmol/L NaHCO3 as mobile phase at the flow-rate 0.7mL/min. A suppressed conductivity detector was used and the detection limit was 1.0 µg/L (S/N = 3) for hippuric acid. The analysis time for one run was 30 min under the optimized IC condition. The recovery of hippuric acid was 93.2– 98.0% while the relative standard deviation (RSD) was 1.4–2.3% by seven measurements. Furthermore the results shown that the proposed method has the advantages of easy operation, high sensitivity and accuracy. This method is suitable for routine clinical analysis of HA.

**Figure 12.** Chromatogram of a standard solution of HA (10mg/L) [25].

*4.6.2. Amines and its derivatives*

content of absorption solution can be directly determined by the optimized IC-PAD method without any pretreatments. The linear range is 0.0147–2.45µg/mL with R2 value of 0.9997. The limit of the detection is 3µg/L for a 25µL injection loop. The overall relative standard deviation of the method is less than 5.20% and the recovery range from 94.3% to 101.0%. This developed method proves to be advantageous, due to expanded detection range with greater accuracy

**Figure 11.** Typical chromatograms obtained under the following eluent composition: A: 0.2M NaOH, 0.2M NaAC; B: 0.4M NaOH, 0.2M NaAC; C: 0.6M NaOH; D: 0.6M NaOH, 0.3M NaAC; E: 0.6M NaOH, 0.2M NaAC. Flow rate: 1.0 mL/

IC can also be used in detection of some acids. Zhao et al. [25] proposed a simple and ecofriendly ion chromatographic method for the determination of Hippuric acid (HA) in human urine (see Figure 12). Hippuric acid is a kind of metabolite of toluene in human body, therefore, HA is a physiological component of human urine if toluene was inhaled. The content of HA in human urine actually is confirmed as a diagnostic marker of exposure to toluene [26]. It has been reported that exposure to high concentrations of volatile organic compounds such as

min, injection volume: 25 µL, column temperature 30oC. Peak 1: unidentified, peak 2: cyanide [24].

**4.6. Organic compounds**

*4.6.1. Hippuric acid*

22 Column Chromatography

and is thus highly anticipated to find wide applications in cigarette smoke analysis.

Erupe et al. [27] developed an ion chromatography method with non-suppressed conduc‐ tivity detection for the simultaneous determination of methylamines (methylamine, dimethylamine, trimethylamine) and trimethylamine-N-oxide (TMAO) in particulate matter air samples. The method can be used to detect, quantify and determine whether TMAO and methylamines are quantitatively significant components of organic nitrogen aerosol in the atmosphere. This was done using aerosol collected from smog chamber reactions of trimethylamine with ozone and/or nitrogen oxide. The method was tested using a solution of laboratory-generated aerosol containing a mixture of the analytes. The analytes were well separated by means of cation-exchange chromatography using a 3 mM nitric acid / 3.5% acetonitrile (v/v) eluent solution and a Metrosep C 2 250 (250mm×4mm i.d.) separation column. The composition of the mobile phase was optimized and effi‐

cient separations between the analytes were achieved (Figure 13 and 14). Detection limits

of methylamine, dimethylamine, trimethylamine, and trimethylamine-N-oxide were 43, 46,

76 and 72 µg/L, respectively. The method described is simple and has low detection limits

suitable for analysis of aerosols generated in smog chamber experiments and in ambient

**Figure 14.** Chromatogram of smog chamber filter analysis from reaction of trimethylamine with ozone. Analytes: 1 sodium, 2-ammonium, 3-potassium, 4-dimethylamine (1.72 µg/m3), 5-trimethylamine-N-oxide (0.25 µg/m3), 6-mag‐ nesium, and 7- trimethylamine (0.57 µg/m3). The inset is a magnification of the trimethylamine-N-oxide peak (5) from

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Phenolic compounds have attracted great concern in recent years due to their high toxicity and bio-recalcitrant effect in the ecosystem water cycling process. Numerous techniques have been studied and developed to determine phenols. However, most of these detection techniques focuses on high performance liquid chromatography (HPLC) equipped with various kinds of detectors such as UV, electrochemical, fluorescence, and mass spectroscopy [28,29]. Among these detection techniques, fluorescence detector is a better choice in terms of selectivity and sensitivity. HPLC combined with fluorescence detector (HPLC/FD) have been used in numerous applications in trace analysis. However, some phenols have weak fluorescent property and post-column derivatization is often required to convert these compounds into strong fluorescent substances that can then be efficiently detected by the fluorescence detector [30]. Using on line electrochemical derivatization, Karst et al. [30] presented a method to determine mono-substituted phenols via HPLC equipped with fluorescence detector (HPLC/ ED/FD). This method addressed the problems on phenols that could not be detected via fluorescence detector. However, the separation was performed by common silica-based C18 separation column. Unfortunately, the silica column works well only in the pH range of 2–8 (pH < 3), whereas the optimum pH for producing the fluorescence of oxidized phenols is basic (pH ~10). Obviously, the separation condition could not match well with that of downstream detection. Therefore, buffer solution of NH3/NH4Cl at pH 9.5 had to be added to the effluent

the chromatogram [27].

*4.6.3. Phenolic compounds*

air where the concentration of these species is expected to be high.

**Figure 13.** Separation of methylamines and methylamine-N-oxides from standard solutions. Analytes: 1-sodium, 2 ammonium, 3-methylamine (195 µg/L), 4-dimethylamine (390 µg/L), 5-trimethylamine-N-oxide (465 µg/L), and 6-tri‐ methylamine (615 µg/L) [27].

**Figure 14.** Chromatogram of smog chamber filter analysis from reaction of trimethylamine with ozone. Analytes: 1 sodium, 2-ammonium, 3-potassium, 4-dimethylamine (1.72 µg/m3), 5-trimethylamine-N-oxide (0.25 µg/m3), 6-mag‐ nesium, and 7- trimethylamine (0.57 µg/m3). The inset is a magnification of the trimethylamine-N-oxide peak (5) from the chromatogram [27].

### *4.6.3. Phenolic compounds*

i.d.) separation column. The composition of the mobile phase was optimized and effi‐

cient separations between the analytes were achieved (Figure 13 and 14). Detection limits

of methylamine, dimethylamine, trimethylamine, and trimethylamine-N-oxide were 43, 46,

76 and 72 µg/L, respectively. The method described is simple and has low detection limits

suitable for analysis of aerosols generated in smog chamber experiments and in ambient

**Figure 13.** Separation of methylamines and methylamine-N-oxides from standard solutions. Analytes: 1-sodium, 2 ammonium, 3-methylamine (195 µg/L), 4-dimethylamine (390 µg/L), 5-trimethylamine-N-oxide (465 µg/L), and 6-tri‐

methylamine (615 µg/L) [27].

24 Column Chromatography

air where the concentration of these species is expected to be high.

Phenolic compounds have attracted great concern in recent years due to their high toxicity and bio-recalcitrant effect in the ecosystem water cycling process. Numerous techniques have been studied and developed to determine phenols. However, most of these detection techniques focuses on high performance liquid chromatography (HPLC) equipped with various kinds of detectors such as UV, electrochemical, fluorescence, and mass spectroscopy [28,29]. Among these detection techniques, fluorescence detector is a better choice in terms of selectivity and sensitivity. HPLC combined with fluorescence detector (HPLC/FD) have been used in numerous applications in trace analysis. However, some phenols have weak fluorescent property and post-column derivatization is often required to convert these compounds into strong fluorescent substances that can then be efficiently detected by the fluorescence detector [30]. Using on line electrochemical derivatization, Karst et al. [30] presented a method to determine mono-substituted phenols via HPLC equipped with fluorescence detector (HPLC/ ED/FD). This method addressed the problems on phenols that could not be detected via fluorescence detector. However, the separation was performed by common silica-based C18 separation column. Unfortunately, the silica column works well only in the pH range of 2–8 (pH < 3), whereas the optimum pH for producing the fluorescence of oxidized phenols is basic (pH ~10). Obviously, the separation condition could not match well with that of downstream detection. Therefore, buffer solution of NH3/NH4Cl at pH 9.5 had to be added to the effluent from the column to perform the electrochemical conversion to enhance the fluorescence signal. Polymer-based stationary phases (e.g. divinylbenzene/ ethylvinylbenzene, DVB/EVB) in IC dominate most of the applications due to their wide pH tolerance (0–14). Since the polymerbased column can work well in alkaline solution (e.g., pH ~10). The choice of alkaline eluent matching with the downstream fluorescence detection will not be a barrier if the phenols could be well separated by IC. Based on these considerations, a method to determine phenols, where their separation is performed using IC combined with online post-column, electrochemical derivatization and fluorescence detection (IC/ED/FD), has been developed [31] Six model phenols including 4-methylphenol (pMP), 2,4-dimethylphenol (DMP), 4-tert-butylphenol (TBP), 4-hydroxylphenolacetic acid (pHPA), 4-acetamidophenol (pAAP), and phenol (P) were well separated on an anion-exchange column under ion exchange mode using NaOH with small amount of acetonitrile added as eluent (as shown in Figure 15). The separation of phenols was carried out in the anion exchange column with basic eluent and the electro-oxidation of phenols is performed using a laboratory-made electrolytic cell (EC) consisting of porous titanium electrode and cation exchange membrane (CEM) which allows the oxidation products that are strongly fluorescent to be detected by the fluorescence detector. NaOH eluent used in the present method matches well with the maximal fluorescence intensity obtained at alkaline condition for oxidized phenols, thus the addition of specific buffer solution after oxidation could be eliminated. This method leads to a simplified procedure and eliminates the use of additional setup and greatly simplifies the operating procedures. The proposed method was sensitive to the limits of detection in the range of 0.4 µg/L and 3.8 µg/L and the limits of quantification between 1.2 µg/L and 13 µg/L due to the strong electro-oxidation capacity of porous titanium electrode, as well as the implementation of time-programmed potential over EC. The linear ranges were 2.0–1.0 × 104 µg/L for pAAP and DMP, and 10–1.0 × 104 µg/L for P, pMP, pHPA, and TBP, respectively. The relative standard deviations range from 0.9% to 4.8%. The utilization of the method was demonstrated by the analysis of real samples.

**Figure 15.** Chromatograms of phenolic compounds (~10 mg/L) at different potential [31].

This chapter deals with ion exchange chromatography, IC, as a subset of liquid chromatogra‐ phy. Due to the continuous growth, chromatography became one of the most widely used methods in different branches of science encompassing chemistry, physical chemistry, chemical engineering, biochemistry and cutting through different fields of analytical proposes.

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Discovery and historical background on IC were mentioned. Steps of ion chromatography process were intensively discussed in addition to instrumental components of typical IC instrument including: pump, injector, column, suppressor, detector and recorder or data

The chapter emphasizes the superior analytical power of ion chromatography so that it can be used for qualitative and quantitative analysis of common cations, anions and halides in their different forms and matrices in trace and ultra-trace concentrations. Heavy metals separation and detection was also mentioned as well as hydrogen cyanide as an example of inorganic

**5. Conclusion**

system.

**Figure 15.** Chromatograms of phenolic compounds (~10 mg/L) at different potential [31].
