Post-column addition

An alternative way of eliminating water in the RPLC eluent is to introduce an SPE trapping column after the LC column (88, 99). After a post-column addition of water (to prevent breakthrough of the less retained compounds), the fraction that elutes from the RPLC column is trapped on to a short-column which is usually packed with polymeric sorbent. This system can use mobile phases containing salts, buffers or ion-pair reagents which can not be introduced directly into the GC unit. This system has been successfully applied, for example, to the analysis of polycyclic aromatic hydrocarbons (PAHs) in water samples (99). 

The effect of the buffer on the efficiency of electrospray ionization was mentioned earlier in Section 4.7.1. This is a good example of the dramatic effect that this may have - good chromatographic separation and ionization efficiency with formic, acetic and propionic acids, and good separation, although with complete suppression of ionization, with trifluoroacetic acid (TFA), the additive used for the protein application described previously. Post-column addition of propionic acid to the mobile phase containing TFA has been shown to reduce, or even... 

In effect, the composition of the mobile phase, and thus the selectivity of the chromatographic system, has been changed. As mentioned in the text, dynamic FAB operates effectively with lower concentrations of matrix than static FAB and although its effect may be minimal it should always be considered. Post-column addition of matrix overcomes potential problems of this nature. 

The amount of current that flows is dependent not only on the condition of the electrode, but also on temperature, pH, and ionic strength of the solvent. Therefore, careful control of the conditions of detection is essential. A reduction of the slope of the baseline in gradient elution is often performed by post-column addition of a solution of strong alkali. Flow is also an important variable,58 and pump fluctuations may be an important source of noise.59 At very high flow rates, amperometric response can decrease depending on... 

For the cationic surfactants, the available HPLC detection methods involve direct UV (for cationics with chromophores, such as benzylalkyl-dimethyl ammonium salts) or for compounds that lack UV absorbance, indirect photometry in conjunction with a post-column addition of bromophenol blue or other anionic dye [49], refractive index [50,51], conductivity detection [47,52] and fluorescence combined with postcolumn addition of the ion-pair [53] were used. These modes of detection, limited to isocratic elution, are not totally satisfactory for the separation of quaternary compounds with a wide range of molecular weights. Thus, to overcome the limitation of other detection systems, the ELS detector has been introduced as a universal detector compatible with gradient elution [45]. 

Fig. 2.147. UV (254) trace of a crude extract of Rubia tinctorum roots and mass spectra (single MS) of the chromatographic peaks for individual anthraquinones lucidin primeveroside (1), ruberythic acid (2), pseudopurpurin (5) and munjistin (6). Mass spectra (a-b) were obtained with NI-ESI with post-column addition of ammonia. Reprinted with permission from G. C. H. Derksen et al. [320].

Post-column introduction of a sheath liquid gives more flexibility to the selection of pH values for a mobile phase. Chiron et al. applied post-column addition of tripropylamine to reduce the acidicity of the mobile phase in their study of bentazone and chlorophenoxyacetic acids to enhance ESI negative ion signals. Apffel et al." on the other hand, introduced propionic acid/isopropanol to displace TEA in the mobile phase, and improved the ESI positive ion signals. 

It is used in IC systems when the amperometric process confers selectivity to the determination of the analytes. The operative modes employed in the amperometric techniques for detection in flow systems include those at (1) constant potential, where the current is measured in continuous mode, (2) at pulsed potential with sampling of the current at dehned periods of time (pulsed amperometry, PAD), or (3) at pulsed potential with integration of the current at defined periods of time (integrated pulsed amperometry, IPAD). Amperometric techniques are successfully employed for the determination of carbohydrates, catecholamines, phenols, cyanide, iodide, amines, etc., even if, for optimal detection, it is often required to change the mobile-phase conditions. This is the case of the detection of biogenic amines separated by cation-exchange in acidic eluent and detected by IPAD at the Au electrode after the post-column addition of a pH modiher (NaOH) [262]. 

Fig. 8.21. FAB mass spectra of steroids separated by CEC. Column, 350 x 0.05 mm i.d. packed with 3 pm Hypersil ODS eluent, 4 mmol/1 sodium tetraborate, 80% acetonitrile applied voltage, 21.5 kV detection, CFFAB-MS, 230-500 amu matrix, post column addition of 1% glycerol in 50% water-50% methanol, 5 pl/min sample, testosterone ([M+H]+ 209), hydrocortisone ([M+H]+ 363), aldosterone ([M+H]+ 361) (in order of elution). (Reproduced from ref. [25] with permission of John Wiley Sons).

Secondary products capable of further oxidation can also increase the unit response for certain compounds. The principle was recently demonstrated in the detection of catecholamines where a post-column addition was used to adjust effluent pH increasing the rate of the chemical step leading to electroactive secondary products. Oxidation of secondary products of phenolic oxidations probably occurs to a degree under all conditions. ... 

One of the widely used methods to qualitatively assess the matrix effect consists of post-column addition of analytes to the LC-eluent flowing from the column to the ESI interface of the mass spectrometer (Figure 13-11) [35]. Briefly, an analyte and the internal standard (IS) dissolved in the same EC eluent are infused (e.g., flow rate lOpL/min) using a syringe pump, through a tee-mixer, located between the column eluent (e.g., flow rate 200pL/min) and the ESI interface of the mass spectrometer. An extract (using LEE or SPE) or supernatant (if PPT is used) from an analyte-free matrix, such as blank or control plasma, is injected via the autosampler, while the test article and the internal standard are introduced, post-column, to the MS ionization... 

Colurrms like Chiralpak AD, Chiralpak AS, and Chiralcel OJ-R are used with normal-phase mobile phases of an alkane, e.g., hexane, Ao-hexane, or heptane, and a small amount of alcohol, e.g., methanol, ethanol, isopropanol. With ESI-MS, post-column addition of an alcohol in water or 5 rrrmol/1 aqueous ammonium acetate must be performed. 

TFA is frequently applied as an additive in LC-MS, for instance in the RPLC separation of peptides (Ch. 16.3.2). TFA is applied as an ion-pairing agent and to mask secondary retention effects of RPLC stationary phases. Without TFA in the mobile phase, the peptides would be almost irreversibly adsorbed. TFA restrlts in significant signal suppression due to both ion-pairing and sirrface-tension effects. The TFA anion more-or-less masks the positive charge on an analyte molecttle at the droplet surface and thereby prohibits lEV of that ion. In the TFA-fix , a post-column addition of propionic acid in 2-propanol (75 25, v/v) is used to counteract the suppression [103-104]. This approach has been found to be successful in some cases, but not in all. 

Mobile-phase additives can also influence the relative abundance of the various adduct ions. Karlsson [105] performed post-column addition of alkali cations to enhance ESI-MS of carbohydrates and other compormds without nitrogen atoms. For most analytes, the adduct formation increased with the size of the cation. Optimum concentration of the cation in the solution was ca. 5x10 mol/1. Alkali-metal affituties and alkali-metal influence on fragmentation in MS-MS have been studied by others as well [106-107]. 

Mass spectra for paraquat, diquat, mepiquat, chlormequat, and difenzoquat, obtained at two different cone voltages in both ESI and APCI, were tabulated by Castro et al. [57]. An acetonitrile in 15 mmol/1 aqueous heptafluorobutyric acid (HFB A) gradient was applied, with post-column addition of acetonitrile. Under these conditions, no doubly-charged ions were observed for paraquat, difenzoquat, and diquat. In ESI, the [Cation-H] -ion was most abundant for paraquat and diquat, while the [Cation] was most abundant for mepiquat, chlormequat, and difenzoquat. In APCI, the [Cation-CH3] -ion was most abundant for paraquat, the [Cation-H]" -ion for diquat, the [Cation] -ion for mepiquat, chlormequat and difenzoquat [57]. In a subsequent study, SIM and positive-ion ESI was applied using the [Cation]" -ion for mepiquat, chlormequat, and difenzoquat, and the [Cation-H]" -ion for diquat and paraqnat [58]. 

Taguchi et al. [56] reported a method based on SPE using ENVI-8 disk material, LC separation on a Ci-column using a mobile phase of 7% methanol in water, containing 25 mmol/1 TEA. A post-column addition of propionic acid in methanol was applied (TFA-fix, Ch. 6.6.3). Detection was based on the [Cation-H] -ion at m/z 183 for diquat, and the [Cation/TFA] -ion pair for paraquat. Detection limits were 0.1 and 0.2 pgA. 

Neutral corticosteroids are prone to the formation of acid adducts [M+RCOO] in negative-ion mode [20-21]. Abundant acetate adducts are observed for steroids with a relatively acidic hydroxyl group [22]. In negative-ion TSP ionization, Kim et al. [23] observed more abundant acid adducts with decreasing pK, of the acid. Marwah et al. [24] showed signal enhancement for a variety of steroids like dehydroepiandrosterone (DHEA) and related compounds due to the addition of low concentration of acid, i.e., typically 1-5 mmol/1 formic acid, 1-8 mmol/1 acetic acid, or 0.05-0.15 mmol/1 trifluoroacetic acid, while higher acid concentrations were found to compromise the response. Formic acid was the best choice for the neutral steroids, while acetic acid is preferred for sulfate conjugates. Post-column addition of 10 nmol/1 silver nitrate resulted in a ten-fold increase in the response for androst-5-ene-3p,17P-diol. [M+Ag] is observed instead of [M+H-HjO] [24]. 

Additionally, experiments have been reported with adduct formation of oligosaccharides with a variety of divalent metal ions, e.g., with Ca " and Mg " [15-17], with Co, Cu, and Mn " [16-17], and with [18], Post-column addition of metal-ion solution by means of a triaxial probe was also described [19], The rationale for the application of divalent cations is response enhancement and/or induction of specific fragmentation reactions. Co " and Ca appear to be most effective in this respect. 

In order to study lipid absorption, TAG were determined in lymph samples from rats, which either received a stmctured lipid (8 0/18 1/8 0 or 8 0/18 2/8 0) or safflower oil [30], Nonaqueous RPLC-APCI-MS was applied with a gradient of acetonitiile-isopropanol/hexane and post-column addition of 50 mmol/1 ammonium acetate. The TAG were detected as [M+NHJ and DAG fragment ions. 

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