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The Application Notebook-09-01-2008

The Application Notebook

Simultaneous Separation of an Acidic Drug Substance and its Counterion on a Weak Cation-Exchange/Reversed-Phase Mixed-Mode HPLC Column

September 01, 2008

Pharmaceutical

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The separation and quantitation of drug substances and counterions are two important determinations in the pharmaceutical industry (1). Drug substances and counterions are determined by HPLC (often on reversed-phase columns) and by ion chromatography (IC), respectively. IC is the preferred method for selective and sensitive screening of both cationic and anionic pharmaceutical counterions (2). To increase the analysis throughput, it is desirable that for analysis of drug formulation both drug substance and counterions can be determined within a single run. Naproxen is a non-steroidal anti-inflammatory drug commonly used for treating moderate to severe pain, fever, inflammation, and stiffness. Naproxen sodium was approved by the U.S. Food and Drug Administration (FDA) as an over-the-counter drug. No reports were found describing one technique for the simultaneous determinations of both naproxen and the counterion (Na+).

Analysis of Melamine and Cyanuric Acid in Food Matrices by LC–MS-MS

September 01, 2008

Food and Beverage

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In March 2007, several North American manufacturers of pet food voluntarily issued nationwide recall notices for some of their products that were reportedly associated with renal failure in pets. The raw material wheat gluten, used to manufacture the pet food, was imported from China and was identified as the source of contamination.

Additional Studies in the Separation of PEGylated Proteins by Reversed Phase Chromatography

September 01, 2008

Biological

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Additional studies were undertaken to better understand the chromatographic behavior of PEGylated proteins in an effort to improve purification and characterization techniques of such proteins. Proteins were PEGylated using larger (20 KDa and 40 KDa) PEGylation reagents that are commonly used in pharmaceutical drug development. Generated PEGylated proteins were separated from unmodified proteins using different reversed phase medias (Jupiter® C4 and Jupiter® C18). In these studies it was found that the Jupiter C18 media provided the best separation of PEGylated proteins from their unmodified counterparts. Such results further clarify good method starting points for developing analytical and preparative separations of PEGylated proteins.

From the Publisher: Timing is Everything

September 01, 2008

From the Publisher

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They say that timing is everything, and though this saying may have originally been applied to Broadway and Vaudeville in the early twentieth century, it is just as true when it comes to the field of modern-day separation science. For this September issue of The Application Notebook should be reaching our audience of analytical chemists, lab managers, and other industry professionals just as the busiest time of the year ramps up in the world of chromatography.

Agilent J&W Ultra Inert GC Columns: A New Tool to Battle Challenging Active Analytes

September 01, 2008

General

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QC test probes serve a vital function in ensuring the reproducibility of modern GC columns. These probes ensure that the columns have been properly deactivated, contain the correct amount of stationary phase, and have the same relative retention as the last column purchased. The choice of individual compounds in these test mixes varies widely and can have profound consequences on the performance of a column in the users' applications.

Multi-Residue Analysis of Pesticides in Fruits Using DisQuE, a Dispersive Solid-Phase Extraction Kit

September 01, 2008

Environmental

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A new technique, QuEChERS, standing for Quick, Easy, Cheap, Effective, Rugged, and Safe, is readily accepted by both the AOAC International and the Committee of European Normalization (CEN) for the pesticide residues in foods and agriculture products. Waters DisQuEâ„¢ Dispersive Sample Preparation Kit contains conveniently-packaged centrifuge tubes with pre-weighed sorbents and buffers designed for use with the AOAC official QuEChERS methods.

Techniques for Reducing Purge-and-Trap Cycle Times in VOC Analysis

September 01, 2008

Environmental

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The purge-and-trap (P&T) technique for analysis of volatile organic compounds (VOCs) was pioneered in the 1970s at the United States Environmental Protection Agency (USEPA) research laboratory in Cincinnati. Many of the operational parameters developed during this time period are still included in USEPA methods. While these parameters still produce good analytical results, they do not take advantage of advances in instrumentation that enable analysis of emerging contaminants such as fuel oxygenates, and increased sample throughput.

Removal of Endogenous Matrix Components Using EVOLUTE&® CX Mixed-Mode Cation Exchange Solid Phase Extraction

September 01, 2008

Pharmaceutical

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Sample preparation is an essential technique to remove unwanted matrix components prior to LC–MS-MS analysis of drugs in biological fluids. Plasma matrix components whether endogenous (salts, proteins, and phospholipids) or exogenous (dosing vehicles, e.g. PEG 400), can interfere with compounds of interest leading to regions of ion suppression or enhancement. This can lead to inaccurate quantitation and have adverse effects on sensitivity. Mixed-mode SPE provides cleaner extracts as a result of rigorous interference wash steps, afforded by the dual retention mechanism of the sorbents.

Synthetic Fused Silica Capillary Tubing: A Discussion of Diffusion

September 01, 2008

General

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Synthetic fused silica capillary tubing is commonly used as the preferred substrate for chromatographic separation columns. In limited instances the absorption and diffusion of molecules into and through the fused silica deserves consideration.

Fast Gradient LC Using Your Existing Instrumentation

September 01, 2008

General

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Transforming "standard" gradient HPLC systems into extremely fast gradient systems is readily achievable with proper application of chromatographic principles, particularly temperature control, combined with utilization of advanced HPLC columns. Bottom line: You don't have to buy a new LC to achieve ultra high quality and speed!

Fast Analysis of Vanilla Extracts on the Acclaim RSLC 2-mm C18 Column

September 01, 2008

Food and Beverage

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The recent development of ultra-HPLC (UHPLC) has provided a great potential for high throughput analysis achieved using small (sub-2 μm) particle size columns at increased linear velocities. The advantage of UHPLC over conventional HPLC is increased throughput without sacrificing resolution. Despite their popularity, sub-2 μm particle columns impose practical difficulties, such as high backpressure (which often requires a UHPLC system) and susceptibility to column fouling. Thus difficulty can be overcome using 2.0 to 2.5 μm particles. This study describes an example (analysis of vanilla extract) of transferring a conventional LC method to a high-throughput method using newly developed Acclaim® RSLC 2-μm columns that are based on spherical, porous, high-purity silica particles (dp = 2 μm, pore size = 120 Å, surface area = 320 m2/g).

Determination of Cefepime and Related Compounds Using HPLC with UV Detection

September 01, 2008

Pharmaceutical

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Cephalosporins contain a four-member β-lactam ring that is inherently strained and prone to hydrolysis and photolysis, limiting stability and leading to degradation products that may be toxic (1). In addition, synthetic byproducts are generated and persist during production of these antibiotics including cefepime. Analysis of cefepime purity is particularly challenging due to the presence of such isomeric synthetic impurities. The Acclaim® 120 C18, 3 μm can be used to meet and exceed the criteria set by the USP for determining related substances and assaying the purity of cefepime.

Ascentis® Express RP-Amide Expands the Selectivity of Fused-Core ™ Particle Technology HPLC Columns

September 01, 2008

General

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The Fused-Core particle consists of a 1.7 micron solid core and a 0.5 micron porous shell yielding a 2.7 micron diameter. One of the benefits of the Fused-Core particle is the small diffusion path (0.5 microns) compared to conventional fully porous particles. The shorter diffusion path minimizes peak broadening. In fact, there have been many reports on the vast improvements in efficiency provided by Fused-Core particles versus conventional particles. These improvements provide sub-2 micron like performance at half of the backpressure allowing Ascentis Express columns to be used in conventional HPLC as well as UHPLC systems.

Fraction Collection and Simultaneous Concentration for Chiral Purifications Using Supercritical Fluid Chromatography (SFC)

September 01, 2008

Chiral

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A novel centrifugal fraction collector CFCâ„¢ enables SFC users to simultaneously collect and concentrate fractions. The technique is particularly useful for chiral separations where large volumes of enantiomers are being purified by stacked injection methods.

Accelerating Beverage Quality Control Using HRes Fast-LC

September 01, 2008

Food and Beverage

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Recent developments in HRes Fast-LC systems have enabled the use of sub-3 μm stationary phases at higher flow rates and elevated pressures. HRes Fast-LC can provide significant improvements in both resolution and throughput.

Semi-Micro Columns for Size-Exclusion Chromatography in Organic Solvent Systems: TSK-GEL SuperMultiporeHZ Series Columns

September 01, 2008

General

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In size exclusion chromatography, obtaining calibration curves over a wide range of molecular weights is a difficulty investigators often encounter when analyzing polymers with a broad molar mass distribution. To overcome this problem two procedures are typically used. One option is to use multiple columns of different pore sizes linked together in series. A second is to use a column packed with a mixed bed resin of different pore sizes at an optimized mix ratio. However, problems can occur with both of these methods, which include distortion of the chromatogram or deviations between the actual calibration curve and the calibration curve approximated from data obtained from the molecular weight standards.

Investigation of Branched Polydienes with GPC/MALS

September 01, 2008

General

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Our work focuses on the synthesis of branched (functionalized) polydienes via a facile two-step synthesis. Despite the numerous strategies developed for the preparation of branched polymers in recent years, there is still a limited number of simple synthetic strategies based on common vinyl monomers.

Performance Gains Using Hydrogen Carrier Gas in Refinery Gas Analysis

September 01, 2008

Industrial

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Escalating costs and increasing demands for helium, coupled with diminishing helium supply, have given rise to the investigation and use of hydrogen as an alternate choice of carrier gas for use in gas chromatography (GC) applications. This application note illustrates that the use of hydrogen as a carrier gas as an alternative to helium in refinery gas applications is not only possible, but also results in improved performance and higher sample throughput.

IC–MS Analysis of Low Molecular Mass Organic Acids in Beverages

September 01, 2008

Food and Beverage

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Organic acids are present in many matrices and play crucial roles. Extensive research involving low molecular mass organic acids (LMMOA) has been performed in food chemistry since these acids contribute to the organoleptic properties of food and beverages, including flavor, color, aroma, taste, shelf-life, and health effects. Developing a profiling method to monitor LMMOA levels in raw materials and final products is very desirable. Many reported methods focus on limited numbers of LMMOAs and are incapable of providing a complete LMMOA profile.

Rapid Separation of Rhodamines

September 01, 2008

Biological

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This application note describes a rapid U-HPLC method for the separation of sulphorhodamine 101 (Texas Red®) and its three water-soluble derivatives using a Hypersil GOLD™ column packed with 1.9 μm particles.

On-Site Sample Preparation Using MEPS for Waste Water Analysis

September 01, 2008

Environmental

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MEPS uses a barrel insert and needle (BIN) device to reduce Solid-Phase Extraction (SPE) to a micro-scale suitable for small volume samples and for the online adaptation of conventional SPE techniques. Because the SPE cartridge (BIN) is incorporated into the needle assembly of a gas-tight syringe, MEPS is also a simple field-portable SPE device that may be operated manually without need for sampling pumps or, alternatively, may be incorporated into robotic samplers. MEPS devices are of glass and stainless steel construction allowing them to be fully immersed for sampling at depth or, alternatively, used at needle depth to avoid perturbing the stream from which the sample was drawn. An extension pole allowed MEPS to be used to sample back along pipes or down inspection vents. When sampling from drainage pits and open sumps, there was minimal requirement to remove grates to gain access. An extension pole also allowed sampling from outflows that were offensive and could be readily adapted for safe sampling of..

CUSTODION ™ SPME Syringe for Rapid Sample Collection and Sample Preparation of Drinking Water for the GC–MS Determination of Trihalomethanes

September 01, 2008

Environmental

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The CUSTODION™ SPME Syringes are a series of novel solid phase micro extraction syringes that incorporate Supelco Analytical's® (Bellefonte, Pennsylvania) SPME fiber technology. The SPME syringes are fabricated with injection-molded components and the device resembles a ball-point pen. The Supelco Analytical® SPME fiber assembly is housed inside the syringe.

Pesticide Analysis in Dietary Supplements Using Gas Chromatography Time-of-Flight Mass Spectrometry

September 01, 2008

Food and Beverage

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Screening for pesticide content is a common analysis that is done in laboratories where many samples need to be analyzed over a short period of time. The desire for a high-speed analysis is due to the need for a fast result from a potentially large volume of samples. A fast analysis is typically achieved through the use of high carrier gas flow rates in combination with temperature programming. A fast detector is needed in order to fully characterize the narrow chromatographic peaks generated from these experiments. TOFMS is ideal for detecting these narrow peaks because of its fast acquisition rate over a full mass range at all times during the experiment. In addition, TOFMS provides for non-skewed mass spectra making peak deconvolution possible.

Analysis of Deoxynivalenol in Beer

September 01, 2008

Food and Beverage

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Mycotoxins, toxic secondary metabolites of several fungal species, represent food safety issues of high concern. Deoxynivalenol, the most abundant trichothecene mycotoxin, can be found worldwide as a contaminant of wheat, barley, maize and other cereals (1,2). The transmission of deoxynivalenol from barley into beer has been reported in several studies (3,4). Therefore, its levels should be controlled.

Determination of Carbohydrates in Foodstuffs by Ion Chromatography with Pulsed Amperometric Detection

September 01, 2008

Food and Beverage

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This article describes a straightforward ion chromatographic method that uses isocratic elution and pulsed amperometric detection (PAD) to sensitively determine water-soluble polyols and sugar alcohols as well as mono-, di- and oligosaccharides in essential and nonessential foodstuffs. While carbohydrate determination of most foodstuffs requires only minimal sample pretreatment such as dilution and filtration, samples with interfering matrices such as protein-containing dairy products have to be dialyzed prior to injection.

Analysis of Voglibose in Pharmaceutical Formulations by HPLC with Post-Column Derivatization

September 01, 2008

Pharmaceutical

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Voglibose is an Alpha-Glucosidase inhibitor widely used for the treatment of diabetes. Alpha-glucosidase inhibitors are agents that delay the glucose absorption at the intestinal level and thereby prevent sudden surge of glucose after a meal. Voglibose is the safest and most effective drug of its class.

A Case Study of Using Thar SFC-MS Prep 30® to Purify Polar, Basic Pharmaceutical Relevant Compounds

September 01, 2008

Pharmaceutical

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In the past decade, supercritical fluid chromatography (SFC) has experienced a striking resurgence and exponential growth in acceptance, particularly in pharmaceutical and chemical laboratories. In SFC, "supercritical" CO2, in combination with one or more polar organic solvents, most commonly alcohols, are used as mobile phase. The polarity of CO2 is similar to that of hexane, and thereby making SFC a normal phase chromatographic technique. SFC has readily lent itself as an attractive complement to reversed phase HPLC (RPLC). For instance, in separating polar compounds that have little retention, and/or selectivity, even with special polar group embedded columns, SFC holds a unique advantage over RPLC due to its normal phase separation mechanism.

Semivolatile Analysis Using an Inertness-Performance-Tested Agilent J&W DB-5ms Ultra Inert Column

September 01, 2008

Environmental

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Semivolatile analyses using methods similar to US EPA method 8270 (1) are important in environmental laboratories worldwide. A number of acidic compounds such as benzoic acid or 2,4-dinitrophenol and strong bases such as pyridine or benzidine are active species found in the semivolatile sample set. These highly polar species are particularly susceptible to adsorption into active surfaces in the sample flow path, including the column itself. System and column inertness are critical for effective analysis of these active chemical species.

Determination of N-Methylpyrrolidine in Cefepime Using Reagent-FreeTM Ion Chromatography

September 01, 2008

Pharmaceutical

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Cefepime is a fourth generation cephalosporin (1). During preparation and storage, cefepime degrades by release of the N-methylpyrrolidine (NMP) side chain and opening of the beta-lactam ring. An NMP concentration increase will directly affect the potency of the active component of the drug. Therefore, it is critical to determine the amount of NMP in cefepime. The US Pharmacopeia (USP) monograph specifies the limit of NMP to

Reversed-Phase HPLC Column at Extreme High Temperature (150°C or Higher)

September 01, 2008

General

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In general, polymer-based columns have a broad pH range (pH 2 to 13), and some have high temperature tolerance (up to 150°C or higher). Considerably large selectivity changes can be obtained by varying analysis temperature and mobile phase pH. Having control on these two parameters over wide ranges can be especially useful in method development.

Real-Time Response to Bacteria Infection of Bioethanol Fermentation Using a Short Rezex™ ROA Column

September 01, 2008

Industrial

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Political priorities as well as economic interests have fueled a dramatic growth in the biofuel industry, due much in part to research funding and tax incentives. Currently the world's ethanol production is estimated to be over 16 billion gallons a year. This number is expected to increase in the next few years, reaching an estimated yearly production of 20 billion gallons by 2012. Today, many ethanol producers add fermentors to expand their production capacity. In order to continue using existing HPLC equipment for the increased monitoring, increased analytical throughput is needed.

SELS - Solvent Enhanced Light Scattering

September 01, 2008

Industrial

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Viscotek has been a strong advocate of good chromatography as a prerequisite for GPC (Gel Permeation Chromatography) data accuracy. Our recent work in application development has been driven by extremely difficult samples from industrial, biopharmaceutical, and academic sources. These samples present challenges ranging from sample solubility, column adsorption, as well as detection issues. This report will attempt to highlight a new approach that could be very helpful in certain advanced GPC applications.

Quick Start-Up for a Modified QuEChERS Multi-residue Pesticide Analysis in Lettuce by GC–MSn

September 01, 2008

Environmental

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Use of a modified QuEChERS sample preparation procedure was fully evaluated for identification and quantitation of pesticides in lettuce using gas chromatography and ion trap mass spectrometry. Final results compared favorably to the reporting and detection limits offered by several worldwide agencies, demonstrating a robust and reliable solution for analyzing pesticide residues in lettuce.

Determination of Selected Pesticides and Flame Retardants in Drinking Water by SPE for UCMR2 and EPA Method 527 Using the Atlantic 527 Disk

September 01, 2008

Environmental

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The second unregulated contaminant monitoring regulation (UCMR2) program was developed to monitor US drinking water sources for currently unregulated compounds. EPA Method 527 is categorized under List 1; Assessment Monitoring in the UCMR2 program. EPA Method 527 focuses on a wide range of semi volatile organic contaminants, including pesticides that were deferred during the first UCMR, flame retardants, and pyrethroid pesticides. This application for EPA Method 527 employs SPE with analysis by gas chromatography–mass spectrometry (GC–MS).

High Speed Analysis of Paracetamol and its Process Impurities

September 01, 2008

Pharmaceutical

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Paracetamol is a major ingredient in numerous medications due to its analgesic and antipyretic properties. During its synthesis (Figure 1), a total of ten process-related impurities are observed. Several HPLC applications have been developed for the monitoring of these impurities (1, 2), including the European Pharmacopoeia which has adopted an isocratic HPLC method using a silica-based C8 column with 5 μm particle size, requiring a run time of 45 min (3). By using a gradient method and standard HPLC instrumentation, the analysis can be reduced to 7 min (4).

Chiral HPLC Separation of Enatiomers of Racemic Drugs Used in the Pharmaceutical Industry

September 01, 2008

Chiral

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A large percentage of commercial and investigational pharmaceutical compounds are enantiomers and many of them show significant enantioselective differences in their pharmacokinetics and pharmacodynamics. The importance of chirality of drugs has been increasingly recognized, and the consequences of using them as racemates or as enantiomers have been frequently discussed in the pharmaceutical literature during recent years. With increasing evidence of problems related to stereoselectivity in drug action, enantioselective analysis by chromatographic methods has become the focus of intensive research of separation scientists. Most of the pharmaceutical and pharmacological studies of stereoselectivity of chiral drugs before the mid eighties involved pre-column derivatization of the enantiomers with chiral reagents forming diastereomers.

Multi-Mode Water Soluble Vitamin Separation Using ZirChrom®-SAX

September 01, 2008

General

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Traditionally the analysis of water-soluble vitamins by reversed-phase HPLC has been complicated by the lack of retention for these compounds on conventional silica C18 columns. Here we demonstrate efficient, multi-mode, baseline resolution of six water-soluble vitamins in 6 min using a ZirChrom®-SAX column.

Branching in High Molar Mass Synthetic Amyloses

September 01, 2008

Biological

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Amylose is an occasionally-branched biopolymer and, together with amylopectin, the hyper-branched component, a constituent of starch. Determination of branching in amylopectin on the basis of amyloses may be performed with the help of synthetic amyloses. Synthetic amyloses from enzymatic (phosphorolytic) reaction were checked for their linearity.