Post-column hydrolysis

The increased use of IV-methyl carbamate insecticides in agriculture demands the development of selective and sensitive analytical procedures to determine trace level residues of these compounds in crops and other food products. HPLC is the technique most widely used to circumvent heat sensitivity of these pesticides. However, HPLC with UV detection lacks the selectivity and sensitivity needed for their analysis. In the late 1970s and early 1980s, HPLC using post-column hydrolysis and derivatization was developed and refined with fluorescence detection to overcome these problems. The technique relies on the post-column hydrolysis of the carbamate moiety to methylamine with subsequent derivatization to a fluorescent isoindole product. This technique is currently the most widely used HPLC method for the determination of carbamates in water" and in fruits and vegetables." " ... 

A complete post-column LC system for the analysis of oxime carbamates using this approach is commercially available (Pickering Laboratories). Alternative post-column hydrolysis conditions 50 X 4.0-mm i.d., 15 p.m, Aminex A-27 column (Bio-Rad), 120°C. 

Miscellaneous pesticides/insecticides Fruit and vegetables Extraction gel permeation chromatography, Nuchar-Celite clean-up HPLC with post-column hydrolysis and addition of o-phthaldehyde to produce fluorophores [129]... 

Inositol bis- and tris-phosphates and other sugar phosphates have been separated on an anion-exchange column and subjected to post-column hydrolysis by immobilized alkaline phosphatase with... 

R.T. Krause, High-performance liquid chromatographic determination of aiyl A-methylcarbamate residues using post-column hydrolysis electrochemical detection, J. Chromatogr., 1988, 442, 333-343. 

Post-column hydrolysis of carbamate esters, such as carbaryl, was discussed in Chapter 4, Section 2. Further consideration of anticholinesterase insecticides can be found in Chapter 7, Section 4. 

Application of the phosphorus-specific detection (see Section 3.8.2) described by Vaeth et al. [118] allows the use of the AS7 column for the analysis of higher condensed phosphates. A mixture of potassium chloride and EDTA [119] is used as an eluant. The potassium chloride concentration determines retention EDTA is only added for improving peak symmetry. However, post-column derivatization with ferric nitrate, as mentioned above, cannot be apphed in this case because the Fe(III) ions of the derivatization reagent form iron-chloro complexes with the chloride ions of the mobile phase, which leads to a high background absorbance. Conductivity detection is also impossible due to the high salt concentration in the mobile phase. The detection system developed by Vaeth et aL comprises a post-column hydrolysis of the polyphosphates to orthophosphate... 

A method to determine nabam by HPLC after acidic hydrolysis to ethylenediamine and post-column derivatization with o-phthalaldehyde-mercaptoethanol has also been reported. " ... 

Reagent pump (post-column) Merck-Hitachi 655A-13, equipped with one reactor containing a 10-m Teflon loop (thermostated at 95 °C) for hydrolysis which is connected to a 50-p.L Teflon loop (at ambient temperature) for fluorescence reagent addition... 

Femia, R. E. and Weinberger, R., Determination of reducing and non-reducing carbohydrates in food products by liquid chromatography with post-column catalytic hydrolysis and derivatization comparison with refractive index detection, /. Chromatogr., 402, 127, 1987. 

HPLC analysis of furosine (-peak II) in hydrolyzates of non-exposed- (bottom), buffer-exposed (middle), and glucose-exposed (top) dentin samples. Dentin was not reduced prior to hydrolysis. Only the relevant parts of the chromatograms are shown. Amino acids are visualized after post-column labelling with a fluorescent dye. I lysine, II furosine. III homoarginine (internal standard). Column Merck Polyspher AA-NA 120 x 4.6 mm flow 0.2 ml/min gradient pH 5.0 -10.2 postcolumn reagent 0.2 ml/min fluorescence Xgx 330 nm, 440 nm 100-yl injections in buffer pH 2. 

Carbamate type Crops C8 HPLC with post-column basic hydrolysis and electrochemical detection [65]... 

Figure 4.10. On-line coupling of USAL and detection with derivatization (A) in the pre-column mode for the determination of coiistin A and B in feeds (B) in the post-column mode for the determination of N-methyicarbamates in soil and foods, AC — analytical column, C — carrier, DC — derivatization coil, EL — elution loop, F — filter, FD — fiuorimetric detector, HC — hydrolysis coil, HPiV — high-pressure injection valve, HPP — high-pressure pump, IV — injection valve, L — leachant, LC — leaching chamber, MP — mobile phase, OPA — o-phthaldiaidehyde, PC — preconcentration column, PL — propagating liquid, PP — peristaltic pump, S / — switching valve, UP — ultrasonic probe, W — waste and WB — water bath. (Reproduced with permission of Elsevier, Refs. [48,49].)...

Amino acid analysis. There are some 20 amino acids found in proteins and these are released by overnight hydrolysis in 6M HCl. Plasma and urine contain an even larger number of amino acids or related compounds. At low pH, amino acids are cations and for 40 years have been separated by cation exchange column, chromatography. The problem with amino adds is that in general they possess no chromophores by which they can all be detected. In the traditional amino add analyser, their detection was accomplished by a post-column reaction with nin-hydrin which forms a purple colour on heating with an amino acid at pH 5.5. This colour, Ruhemann s purple, is formed with all primary amino acids and can be detected at 570 nm. Secondary amino acids such as proline form a yellow chromophore measurable at 440 nm. 

An important constituent in copper pyrophosphate baths is nitrate, which enhances the maximum permissible current density [31]. Fig. 8-30 shows the respective chromatogram with the separation of nitrate and orthophosphate. The latter is the hydrolysis product of pyrophosphate that is formed during the plating process. The main component pyrophosphate may also be separated on a latexed anion exchanger. It is detected after complexation with ferric nitrate in a post-column reaction by measuring the light absorption (see Section 3.3.5.2). 

Immobilized /3-glucosidase served for enzymatically catalyzed hydrolysis of benzene metabolites in urine. End analysis of phenol was by RP-HPLC with ELD at 0.85 V vi. Ag/AgCl electrode. ELD avoids interference from other componnds present in urine. LOD was 10 p.gL" (20 p,L injection, 0.2 ng), with RSD 1.16% and 3.38% for 1.2 ng and 2.0 ng, respectively. A stndy was carried out of two FIA systems for enzymatically catalyzed determination of dopamine (10a). Thus, a combination of a packed bed reactor containing immobilized tyrosinase followed by photometric detection was compared with ELD based on a graphite electrode with its surface covered by immobilized tyrosinase. The former configuration was linear up to 0.75 mM while the latter reached 1 mM. LC separation and post-column detection with the bioelectrode was applied to analysis of spiked serum samples. ... 

Enzymatic hydrolysis (protease, lipase) followed by AE separation (Hamilton PRP-XlOO column) with 5 niolL ammonium citrate in 2% (v/v) methanol and post-column IDA-ICP-MS with octapole reaction system... 

An important step for any analytical method is the sample extraction. The majority of these post-column derivatization HPLC methods apply, for shellfish samples, an extraction protocol based on the 959.08 AOAC Official Method [4] that uses hydrochloric acid with boiling. This leads to partial hydrolysis of certain PSP toxins (Cl, C2, and GTX5) into more toxic analogs (GTX2, GTX3, STX). 

After hydrolysis, the free amino acids are separated by ion exchange chromatography (section 2.3.2) or RP-HPLC (section 2.3.1). They can be identified according to their elution times and quantified according fo fheir elution volumes. To increase sensifivify, the amino acids are usually derivatised either pre- or post-column. Dansyl Chloride, Edman s reagent (Fig. 7.1) as well as ortho-phthalaldehyde (OPA) and 2-mercaptoethanol can be employed to form highly fluorescent adducts (Fig. 7.9) that can be detected easily. 

---