Eugene M. Fujinari

Nitrogen-specific liquid chromatography detector based on chemiluminescence: Application to the analysis of ammonium nitrogen in waste water

Abstract A novel high-performance liquid chromatography-chemiluminescent nitrogen detection (HPLC-CLND) system is described. The analysis of ammonium nitrogen in metropolitan waste water by a new ion chromatographic method is presented. The significance of this novel nitrogen-specific detector is the capability for a routine and continuous operation. HPLC-CLND applications for anion and particularly reversed-phase techniques are also amenable. A nitrogen-specific detector for liquid chromatography opens a new dimension of analytical chemistry.

Peptide content determination of crude synthetic peptides by reversed-phase liquid chromatography and nitrogen-specific detection with a chemiluminescent nitrogen detector

Abstract The need for a rapid and accurate method to assess peptide content is driven by the recent surge in demand for pharmaceutical-grade synthetic peptides manufactured by solid-phase chemistry. Routinely, the purity of synthetic peptides is analyzed by reversed-phase HPLC with UV detection, and peptide content is typically determined from amino acid analysis of synthetic peptides. Recently, we developed a new method to assess the peptide content of synthetic peptides in one simple technique: simultaneous reversed-phase HPLC-chemiluminescent nitrogen detection and UV detection. In this paper we present the simplicity of this method and its universal application to analyzing crude synthetic peptides produced by solid-phase synthesis, an important first step in manufacturing bioactive peptides.

Nitrogen-specific detection of peptides in liquid chromatography with a chemiluminescent nitrogen detector

Abstract High-performance liquid chromatography with chemiluminescent nitrogen detection (HPLC-CLND) in the reversed-phase mode was used to quantitate peptides that were isolated from casein hydrolysate. When CLND is used simultaneously with a UV detector in peptide mapping, unique quantitative information about the nitrogen distribution of the sample is obtained. Nitrogenous compounds without UV chromophores are easily detected by CLND without pre- or post-column derivatization. Of further significance, the non-nitrogenous compounds in the sample matrix are transparent to the detector. This paper will focus primarily on the analysis of two peptides, identified as 1 and 2 . The UV peptide map showed peptide 2 as the largest component. On the other hand, the CLND results indicated that peptide 1 was the major peak. RP-HPLC amino acid analysis of the peptides 1 and 2 confirmed the CLND results. This analysis shows that peptide 1 was the major component and did not contain aromatic amino acid residues. Peptide 2 however, contained aromatic groups with strong chromophores, thereby explaining the UV response.

Chemiluminescence nitrogen detection for packed-column supercritical fluid chromatography with methanol modified carbon dioxide

The novel interface of a chemiluminescent nitrogen detector (CLND) with a packed column chromatographic system, utilizing supercritical methanol modified carbon dioxide, has been achieved. Both pressure gradients and mobile phase gradients have been successfully demonstrated. Detector optimization in terms of sensitivity and performance is described. Polymeric materials such as nitrogen containing cyclic oligomers and triazine herbicides were successfully analyzed. Pharmaceutical applications are also demonstrated using a combination of SFC-UV and SFC-CLND.

Determination of molecular-mass distribution of food-grade protein hydrolyzates by size-exclusion chromatography and chemiluminescent nitrogen detection

Size-exclusion chromatography (SEC) and chemiluminescent nitrogen detection (CLND) were used to estimate the molecular-mass distribution of food-grade protein hydrolyzates. Simultaneous CLND and UV (214 nm) detection is demonstrated for analytical SEC of an experimental casein hydrolyzate. In order to validate the estimated average M(r) values derived from the SEC column data, a preparative SEC separation of an extensive casein hydrolyzate was pursued. Fractions were collected on a time basis and analyzed by time-of-flight (TOF) mass spectrometry. A plot of TOF M(r) vs. SEC M(r) indicated that the peptides below M(r) of 1200 were eluted as estimated by the calibrated preparative SEC column. This paper demonstrates the power of using a dual CLND and UV detection system for analytical SEC analysis of protein hydrolyzates with a calibrated column.

Feasibility of supercritical fluid chromatography-chemiluminescent nitrogen detection with open tubular columns

Abstract A supercritical fluid chromatography-chemiluminescence nitrogen detection (SFC-CLND) system for open tubular columns is described. The chemistry for the post-restrictor detector is based on the chemiluminescent reaction between ozone generated from oxygen and nitric oxide via high-temperature pyrolysis of nitrogen-containing compounds. The minimum detectable quantity is found to be 60 pg nitrogen of indole by flow injection analysis. Detector linearity was at least three orders of magnitude, and a selectivity of 10 5 was obtained. Several applications of SFC-CLND in the analysis of compounds containing nitrogen are presented.

Sulfur-selective chemiluminescence detection with packed column supercritical fluid chromatography

Abstract A new generation sulfur chemiluminescence detection (SCLD) system was interfaced and tested for supercritical fluid chromatography (SFC) with packed columns using 100% SF-CO 2 and methanol modified CO 2 as the mobile phase. The detection chemistry for the SCLD is based on ozone-induced chemiluminescence following a two-step combustion process of consecutive oxidation and reduction of sulfur-containing compounds. A seven-day evaluation study showed excellent sensitivity, selectivity and linearity, as well as day-to-day repeatability. The minimum detectable quantity was determined to be 3 pg sulfur (0.2 pg S/s) at the detector. Equimolar response of SCLD to sulfur compounds with different bonding environments was also observed. Unique applications and capabilities of the SFC-SCLD system for sulfur speciation and detection are presented for a petroleum product, thermally labile pesticides and herbicides which are difficult or impossible to analyze by GC techniques.

Simulated distillation-chemiluminescent nitrogen detection: SimDis-CLND

Publisher Summary The nitrogen boiling point distribution of refinery streams can be studied by simulated distillation with a chemiluminescent nitrogen detector (SimDis-CLND) to improve the production of fuels and petrochemicals. Some of the valued features of the CLND for SimDis are: high sensitivity and selectivity of the detector for nitrogen containing compounds. Simulated distillation is often used for estimating the hydrocarbon boiling range distribution of the petroleum fractions in order to control the efficiency of the plant refining processes. The application using multi-element simulated distillation software for the atomic emission detector (AED) was reported by Quimby and Dryden. The AED was used to first calibrate the hydrocarbon (standards) boiling point (-89 to 522oC) distribution from C2 to C40, using both carbon and hydrogen detection modes. A simulated distillation method with the CLND is then developed using matched capillary columns (as AED) in the same GC oven. Simultaneous automated sample injections (one injection to CLND and the other to AED) are accomplished using two separate HP7673A systems mounted on a single HP5890 GC. The CLND is used to calibrate the nitrogen boiling point distribution from 171 to 349oC.

Nitrogen-specific liquid chromatography detection of nucleotides and nucleosides by HPLC-CLND

Abstract Chemiluminescence detection is emerging as an important chromatographic tool in food science. HPLC analyses with a chemiluminescent nitrogen detector (HPLC-CLND), can simplify complex analyses by peering through non-nitrogenous components in sample matrices. By using this detector for LC analyses, the chromatographer can quantitate the nitrogen content of nucleotides, nucleosides, and their corresponding bases. Because these classes of compounds each contain an aromatic UV chromophore, a simultaneous CLND and UV detection can be advantageous in comparing the nitrogen content of these analytes with the corresponding UV responses. Since these molecules play a paramount role in food, flavor, pharmaceutical, and bioanalytical chemistry, a preliminary study of column, mobile phase, and the HPLC-CLND optimization is presented.

Gas chromatography-chemiluminescent nitrogen detection: GC-CLND

Publisher Summary The chemiluminescent nitrogen detection (CLND) mechanism for chromatography is the same as the total nitrogen detection. For GC-CLND, sample components are eluted from the column and then oxidized at high temperatures (1000o–1100oC). The ٠NO and O 3 chemiluminescence detected by the PMT is proportional to the amount of each nitrogen containing compound eluting from the chromatographic column. In order to obtain the maximum benefit of the CLND, the chromatographer must quantitatively get the nitrogen containing analytes to the detector. Therefore, it is very important that (1) sample injection system, such as split/splitless, cool on-column, or valve (sample loop) injection and transfer lines etc., (2) capillary column (also consider: stationary phase, film thickness, column I,D., and length etc.), and (3) interface to the detector (CLND pyro-furnace and transfer line) are clean and fully operational for the type of sample to be analyzed. Although this is very basic, it is the criteria for every successful chromatographic application using any detector and certainly applies to the CLND.