Indirect Absorbance

In this detection mode, an eluent anion is chosen that absorbs in the visible or UV spectral region. The elution of sample anions is monitored by measuring the decrease in absorbance at the detection wavelength as transparent sample ions replace a fraction of the absorbing eluent anions. Under conditions where the solute anion (S ) is fully dissociated, which is the usual case for IC, the change in absorbance (AA) is given by 

The concentration of the eluent anion must be high enough to elute the sample ions within a reasonable time and to exceed the concentration of any sample anion at its peak maximum. Howevei if the concentration of E greatly exceeds that of S , the background absorbance will be relatively high and the noise of peak detection will be poor. So while a reasonable eluent concentration is needed to promote ion exchange with the column, the concentration should still be as low as possible to reduce noise. To achieve the best sensitivity, should be as high as possible so that the difference in molar absorptivities in Eq. (6.14) will 

Miura and Fritz investigated the use of polycarboxylic acid salts as eluents in anion chromatography [26]. At alkaline pH values, 1,3,5-benzenetricarboxylic acid (BTA) exists as the 3- anion and pyromellitic add (1,2,4,5-benzenetetracarboxylic acid) can exist as the 4— anion. 

Potentiometric detection of anions is feasible when an electrode is available that responds quickly, reversibly and reprodudbly to the concentration (or more precisely to the activity) of sample ions. It is often possible to detect a given ion or class of ions with excellent selectivity. For example, soUd-state or crystalline ion selective electrodes have been used in IC to detect halide anions. The fluoride ion-selective electrode is particularly selective [27]. A copper wire electrode has been used to detect anions such as iodate, bromide and oxalate [28]. 

A metallic silver electrode responds rapidly and reproducibly to the activity of free silver ions in solution. At 25 C  

In summary, the eluting anion selected for indirect UV detection should have a strong affinity for the ion exchanger so that a relatively low concentration can be used, and it should have a high molar absorptivity. 

Several anions have proven to be effective for indirect photometric detection, figure 6.16 shows an excellent separation of conunon anions with 2.0 mM potassium phthalate, pH 6.0, with indirect detection at 280 nm. By switching to 4.0 mM p-hydro-xybenzoic acid, pH 8.5 with 2.5 % methanol, detection at 310 nm, and reducing the column length to 10 cm, the same anions plus phosphate could be separated in only 

A sodium molybdate eluent with indirect detection at 250 nm was found to provide an excellent separation and a very sensitive detection of inorganic anions. The separa- 

---

Similar to indirect absorbance detection, indirect fluorescence has been employed to detect MPA, EMPA, IMPA, and PMPA, using tetrakis(4-sul fopheny 1 iporphine (TSPP) as the indirect probe (19). A violet diode laser operating at 415nm was used for excitation. Using an electrolyte composed of 50 tM TSPP and 5mM [Bios(2-hydroxyethyl)-amino]tris-(hydroxymethyl)methane (Bistris) at pH 7.2 under normal polarity, baseline separation was achieved in less than 2 min. A limit of detection of 0.1 xM (9ppb) for MPA was achieved. 

Absorbance Detection in Capillary Electrophoresis Indirect Absorbance Detection... 

Problems encountered with CE include poor solubility of fatty acids in aqueous solutions and the unavailability of sensitive modes for aliphatic acids. This is often counteracted by nonaqueous capillary electrophoresis (NACE) with indirect detection. NACE-1A (indirect absorbance) is a simple, sensitive, and effective technique for the determination of fatty acids using i-PrOH and 40% acetonitrile (as organic modifier) and HIQSA... 

NACE with indirect detection has been applied to the determination of fatty acids (FAs) and ascorbic acid (AA), respectively. C2-C18 FAs have been separated in less than 12 min using 8-hydroxy-7-iodoquinoline sulfonic acid as chromophores in NACE with indirect absorbance. The dissociation constant (p/linear plot -log[(mu 0/mu) - 1] versus pH, using 20% isopropanol and 40% ACN as the organic modifier in NACE, are all above about two units than those obtained in aqueous solution. NACE with indirect laser-induced fluorescence, using merocyanine 540 MC540) asfluorophores, has been performed for the analysis of AA and its stereoisomer, isoascorbic acid (lAA), and the limits of detection of AA and lAA are 0.30 and 0.17 pM, respectively. This method has been applied to the determination of AA in a lemon juice spiked with lAAasthe internal standard in less than three minutes, and its concentration is 76.7 + 0.4 mM. 

Indirect absorbance 10 to 10 10 to 10 Small ions, amino acids, fatty acids, polymers... 

Figure 12 Electropherogram for a mixture of anions concentration is 50ng/ml. Separation carried out by CZE using an open silica capillary and detected by indirect absorbance. Peak identifications 1, Br 2, Cl 3, SO 4, NO2 5, NOf 6, F 7, PO4. (Electropherogram provided by Joanne Smith, GE-CRD.

The detection method most often used for the analysis of small inorganic ions by CE is indirect absorbance. Pyromellitic acid is a typical chro-mophore and can be purchased as a buffer solution from instrument manufacturers. The most common method of detection in IC analysis is suppressed conductivity. 

The same principle is used in indirect absorbance measurement when working with absorbing substances between 250 and 300 nm and TLC plates with phosphorescence indicator. As inorganic indicators absorb in the same range, the residual phosphorescence emission of the indicator is inhibited by the degree the substance absorbs the UV radiation exciting the indicator. 

Indirect absorbance measurement is generally not as sensitive as the direct measurement. The detection limits of absorbance measurement are from 0.01 to 0.2 pg of substance per chromatogram zone in the most favorable cases. 

---