Analyte post-column addition

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]. 

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... 

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]. 

Gjerde and Benson discovered that post-column addition of a suspension of sulfo-nated polystyrene particles may be used to reduce the background conductance of basic eluents used in anion chromatography [7]. The eluent cation (typically Na" ) is also replaced in the analyte ion bands by the more highly conducting as the counterion to a sample anion. Since the added reagent is a solid, it is invisible to detectors that respond only to the liquid phase, for example, conductivity and potentio-metric detectors. 

Figure 6.4. Detailed diagram of hardware configuration for post-column addition of SPR. (1 = Conductivity detector Waters 431 detector, four electrode cell design 2 = waste line 4 x 0.009 in. stainless connected to 431 + 24 X 1/16 X 0.060 in PTFE tubing 3 = tee to 431 15 x 1/16 x 0.010 in PTFE to 431 inlet 4 = column to lee shortest 1/16 x 0.010 in PTFE from column to tee 5 = tee Unmount tee from check valve block for shortest path length 6 = analytical colunm Waters 1C PAK A or 1C PAK A HR 7 = check valve to tee 2 x 1/8 in o.d. PTFE 8 = check valve 9 = polisher column to check valve 3 x 1/8 in o.d. PTFE 10 = polisher column 8 x 25 mm containing AGI x 8, 200 mesh 11 = reservoir to polisher column 12 x 1/8 in. o.d. PTFE 12 = air supply minimum of 90 p.s.i. compressed air supply 13 = reservoir for SPR reconfigure with outlet on left side. From Ret [9] with permission.)...

The determination of phenols was preferentially performed using GC-MS with analytes in underivatized or derivatized form, but LC-MS methods were also developed. API methods for the analysis of phenols in aqueous matrices were applied [315, 316, 317]. APCI-LC-MS was found to be more sensitive than ESI application despite the possibility of improving ESI-sensitivity by a post-column addition of diethyla-mine [317]. Detection limits were observed with 0.02-20 ng injected onto the column. The determination of alkylphenols and bisphenol A as compounds with endocrine disrupter potential was also performed by ESI-LC-MS from aqueous [318, 319] and sediment samples with detection limits in the low pg L range [346]. 

In negative ion mode, deprotonated molecules are the only folate species detected however, acidification of the mobile phase, which is essential for retention of folate on reversed-phase HPLC columns, suppresses the formation of deprotonated molecules [48]. As a consequence, negative ion ESI requires post column addition of organic bases, such as triethylamine [47,48], to improve ionization efficiency when low pH buffers are used in the mobile phase. This adds unnecessary complexity to the analytical system. 

Besides the addition of add or base after the separation column, many analytes are converted to cations or anions by the formation of adducts. For example, compounds bearing OH functional groups tend to form adducts with alkali metal cations. The recommended alkali concentration is below 1 mmol L (i.e., soditun acetate) in order to avoid ion suppression effects, ft is important to achieve the cation adduct formation by post-column addition and under no circrunstances by addition to the HPLC buffer. This would cause a long-lasting contamination of the HPLC system. Carbohydrates can be detected as chloride adducts in negative-ion electrospray mode by adding 50 mM HCl (2 mmol L to the ESI ion source). 

Figure 2.3. Electrospray mass spectrum of sucrose obtained with a single quadrupole mass spectrometer (Thermo Electron) operated in the positive-ion mode. Fragmentation was achieved by in-source collisionally induced dissociation. The post-column addition of submillimolar LiCl to the analyte solution facilitated the formation of lithium adducts. (Reprinted from Ref. 28, with permission.)...

Chromatographic methods including thin-layer, hplc, and gc methods have been developed. In addition to developments ia the types of columns and eluents for hplc appHcations, a significant amount of work has been done ia the kiads of detectioa methods for the vitamin. These detectioa methods iaclude direct detectioa by uv, fluoresceace after post-column reduction of the quiaone to the hydroquinone, and electrochemical detection. Quantitative gc methods have been developed for the vitamin but have found limited appHcations. However, gc methods coupled with highly sensitive detection methods such as gc/ms do represent a powerful analytical tool (20). 

Involatile inorganic buffers, when used as mobile-phase additives, are the prime cause of blocking of the pinhole. The situation can be alleviated either by replacing them by a more volatile alternative, such as ammonium acetate, or by using post-column extraction to separate the analytes from the buffer, with the analytes, dissolved in an appropriate organic solvent, being introduced into the mass spectrometer. 

Likewise, the luminescence properties of many analytes can be altered in the presenoe of surfactant aggregates (4,7.,8.). Consequently, addition of micelle-forming surfactants (present either in the LC mobile phase or added post-column) can improve the sensitivity of fluorimetric LC detectors (49,482). Micellar spray reagents have been utilized to enhance the fluorescence densitometric detection of dansylamino acids or polycyclic aromatic hydrocarbons (483). The effect was observed for TLC performed on cellulose or polyamide stationary phases with the micellar spray reagent being either CTAC, SB-12, or NaC (483). More recently, use of nonionic Triton X-100 has been found to improve the HPLC detection of morphine by fluorescence determination after post-column derivatization (486) as well as improve the N-chlorination procedure for the detection of amines, amides, and related compounds on thin-layer chromatograms (488). 

The CUSAL-HPLC couple has been combined additionally with pre- or post-column derivatization. Thus, pre-column derivatization was used for the determination of colistin A and B in feeds following USAL, the analytes were derivatized with o-phthaldialdehyde/2-mercaptoethanol and separated by HPLC for fluorimetric detection [48]. The experimental set-up used is depicted in Fig. 4.1 OA. Another application of CUSAL-HPLC is the determination of A/-methylcarbamates in soils and food [49] (see Fig. 4.10B), where the analytes were also derivatized with o-phthaldialdehyde after separation for fluorescence-based monitoring. A number of steps of the process including leaching, filtration, solid-phase extraction, liquid chromatographic separation, post-column derivatization and fluorescence detection were performed on-line, all in an automated manner. 

Desai and Armstrong [139] discussed the optimization of mobile-phase conditions for enantioselective separations in combination with LC-MS. Their conclusions can be summarized as (a) polar organic mobile phases as used with Chirobiotic T and V columns are best compatible with ESl-MS, (b) normal-phase mobile phases require post-colunm addition, (c) high-aqueous mobile phases compromise ESl-MS sensitivity, and (d) ammonium tiifluoroacetate provides response enhancement for analytes with amine or amide groups. 

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