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ChromJournalInnovative products for chromatography

vwr.comIssue 9 Autumn 2010

editorial

table of contents

EditorVWR International Europe bvbaHaasrode Researchpark Zone 3Geldenaaksebaan 4643001 LeuvenBelgium

CopywritingVWR International Europe bvba

Layout and typesettingMarketing Services VWR

PrintingStork, Bruchsal, Germany

No part of this publication may be reproduced or copied without prior permission by writing of VWR International Europe.

Run46.000 copiesPublication date: September 2010

Due to the high sales volume of promoted articles some items may be temporarily out of stock - VWR Terms and Conditions of Sale apply.

Validation Manager 3 .......................................................................................................................3-5

The new VWR Collection LINEA 10 gas generators ...........................................................................6-7

ChromSword® Auto 4.0 ..................................................................................................................8-11

Parameters of Planar Chromatography .........................................................................................12-13

ChromaScan - The affordable system for TLC plate capture and analysis ...........................................14

Lab Water Gets “Ultra, Ultra-Pure” ....................................................................................................15

Rapid high sensitivity LC/MS analysis of sugars using ZIC®-HILIC HPLC columns .........................16-17

Built-in solutions for the safe disposal of liquid waste .......................................................................18

HD plunger for headspace gas chromatography ................................................................................19

SilTite™ FingerTite .............................................................................................................................20

eVol® – Everyone’s an expert .............................................................................................................21

A method for the quality control analysis of Methylsalicylate in oral care products ..........................22

New VWR ELSD 85 ........................................................................................................................23-25

Purospher® STAR RP-18e UHPLC columns ....................................................................................26-27

Monolithic silica capillaries ................................................................................................................28

LiChroTest® ........................................................................................................................................29

Regenerated cellulose ........................................................................................................................30

Pall Life Sciences and Sotax deliver cost-effective automation certified solutions for automated

pharmaceutical testing systems .........................................................................................................31

PESTINORM: High purity solvents for capillary GC analysis ...............................................................32

Dear Chromatographer,

Welcome to the autumn edition of the VWR ChromJournal. As usual the magazine is

packed with application notes, new product innovations and technical updates to help

you save time, save money and improve results.

We’ve been getting some great feedback about the updated VWR.com - there is

a complete microsite dedicated to chromatography and this alongside our new

search and recommendation tools are helping customers to find the products they

want quickly and easily. The great thing to is that we are now able to present the

promotions alongside the technique so you don’t have to waste time going through

everything that is available to see if there is anything applicable to you!

If you haven’t had a look yet, why not check it out!

Very best regards

the VWR Chromatography team

VWR ChromJournal Issue 9 September 20102

VWR ChromJournal Issue 9 September 2010 3

For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website http://eu.vwr.com/chromSoftware

Validation Manager 3Compliance and ease of operation in the validation of analytical methods

When operating within a quality management systems (e.g. according to GMP, GLP or ISO guidelines), validation of the procedures used is vital. However, this process including the compilation of the validation report is often tedious and time consuming.

Validation Manager 3 is an invaluable, time saving aid, which checks whether your analytical methods are suitable for the intended use and automatically produces a validation report.

• Universally accepted Since its first release in 1992, Validation Manager

has continuously been developed in line with international regulations and recommendations on validation of assay methods. Validation Manager 3 is based on the ICH recommendations for method validation (ICH Q2(R1)), EMEA, FDA, USP, EP guidelines and the relevant ISO standards.

• Versatile configuration for all analytical

techniques Validation Manager 3 is a configurable software

designed for validation of any kind of analytical method. It contains templates relating to the terminology of the respective analytical techniques and to the configuration of the statistical tests and types of assay for validation. For ease of use these templates are pre-configured or the user can create and save new templates. This high degree of versatility enables Validation Manager 3 to be

used for the validation of all analytical procedures irrespectve of the industry or application.

• Easy creation of the validation project Validation Manager 3 is extremely simple to use.

The wizard guides the user during the creation of the validation project. First the user is asked for selection of the validation type (drug product, impurity assay, assay on raw material or finished product, trace amount assay, inter-laboratory trial etc…), then for the selection of the template relating to the analytical technique and one relating to the calculations and statistical tests to be applied. Finally, the validation project and document is created with a push of a button.

• Easy creation of sample preparation worksheets and injection tables

Another programming assistant helps the user to create sample preparation worksheets relevant to the method used. For HPLC techniques the injection tables in the EZChrom Elite™ chromatography data system can be created automatically.

• Easy data input After data acquisition, chromatography data

can be imported from various chromatography data systems (EZChrom Elite™ or D-7000 HPLC System Manager). Moreover, data can be copied from Microsoft™ Excel™ or Word™ tables. Alternatively data can be manually input.

• Easy project management Once the validation project has been created, a

navigation pane (Figure 1) provides the user with an easy way to navigate through the different characteristics to be assessed, the data tables, the configuration of the statistical tests and the results screens. Each characteristic can be reviewed or modified separately before calculation.

• Automatic calculation After data input, method parameters are

automatically calculated and statistically checked according to the configuration. The final conclusion on each characteristic is proposed to the user. In addition the software provides large spaces for any comment or decision with regards to the results. According to the profile of the user rights, the final conclusion on each characteristic can be modified and justified separately.

Figure 1: Validation project navigation pane


• Automatic report compilation After entering your comments and decisions, the

entire method validation report is created with a single mouse click. For the report, different contents and sizes are selectable by the user. This report can contain a fully detailed description of the statistical procedures used, the data and calculation results and/or the summarised results. Validation Manager provides the user with three different ways to edit the validation report as a:

•ProtectedstandardValidationManager3report, •Adobe® Acrobat™ pdf file •AutomatictransferintoaMicrosoft® Word™

document.

• Compliant with international standards Whatever is being assessed, Validation Manager

3 has been designed for full compliance with international standards, and industry recommendations and regulations.• Specificity Defined in Validation Manager 3 according to the ICH Q2(R1) recommendations. Assessment of this characteristic is done with analytical recordings (e.g. chromatograms), directly imported from the chromatography data systems EZChrom Elite™ or D-7000 HPLC System Manager, or graphics imported as JPEG files (.jpg), Windows® Metafiles (.wmf) or bitmap files (.bmp), produced by the data acquisition software used. • Precision Calculated according to ISO 5725 standard: It includes an optional test for outlier values and series. Method repeatability is calculated for each of the data series and for the set of series.

Intermediate Precision or Reproducibility are derived from repeatability and the contribution of the means. The final results are given as calculated variances and corresponding coefficients of variation (CV %). • LOD and LOQ Estimated according to ICH Q2(R1) recommendations and ISO 11843 standard: Signal to noise ratio, signal to noise standard deviation ratio and residual variance to slope ratio. Once LOQ is estimated, it is validated by Validation Manager 3 separately or included in the characteristic Accuracy Over The Method Range. • Linearity Assessed in Validation Manager 3 by means of the least square regression method. In order to help the user make a decision, the results include a Student t-test for compatibility of the y-intercept with zero and a graph of the residual values. An optional Analysis of Variance (ANOVA), based on ISO 11095 and/or ISO 8466 standards, gives full confidence in the linearity assessment.• Accuracy Assessed in Validation Manager 3 either at any chosen target concentration, and/or over the method range and/or specifically at the LOQ level. Method Trueness is first validated by means of the calculated mean value of the concentrations and its confidence interval, at each concentration level or over the full concentration range. Once Trueness is verified, Validation Manager 3 applies the Total Error concept, using Trueness and Precision results, to assess method accuracy by means of calculation of the tolerance interval, according to ISO 16269-6 standard, or the Confidence interval, according to ISO 5725 standard. The Tolerance interval is defined as the interval which contains more than a chosen proportion (e.g. 90%) of the population of the results. If the Tolerance interval contains the true value, method Accuracy is verified. Figure 2 gives an example of the usefulness of this concept in the case of an Accuracy study including LOQ assessment and Figure 3 shows the use of the Confidence intervals in the case of a large scale method transfer.

• Validated and fully FDA 21CFR part 11 compliant

Validation Manager 3 is validated and the validation certificate is included in the software package. In addition to the operating system security layer, Validation Manager 3 provides all functions necessary for full FDA 21 CFR part 11 note compliance:

Figure 2: Use of the Tolerance Interval,

according to ISO 16269-6 standard, for assessment of method LOQ (method

specifications set to 90-110% of the true value).

Result: For the lowest sample amount studied

(60 ng) the tolerance interval is out of

specifications. LOQ must be defined according to

the sample amount corresponding to the

second lowest concentration (120 ng).


For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website http://eu.vwr.com/chrom

New VWR BrochuresVWR International has recently launched three new brochures on the range of special software tools for method development and validation:

ChromSword®, ChromSword Auto®

& AutoRobust

Intelligent software tools for automatic HPLC method

development and robustness testing.

UncertaintyManager®

For the rapid and reliable calculation of measurement uncertainty in your

analytical results.

Validation ManagerSaves you days or even weeks of work in validation of

analytical methods.

Get your copy by contacting your local VWR sales office

• User administration, based on defined user profiles (user rights)

• Audittrials• Validationprojectsmanagementbyrevision• Electronicsignature• Archiving• Writeprotectionofthesoftwarefunctionsand

calculation algorithms• Writeprotectionofthevalidationreport

• Substantial time savings As a comprehensive tool for computer assisted

method validation, Validation Manager 3 saves users lots of time by automatically compiling of the validation report.

Validation Manager 3 - can save days or even weeks of work. So if you have the need to validate analytical methods please contact our specialists and learn more about Validation Manager 3.

Figure 3: Use of the Confidence Intervals,

according to ISO 5725 standard, for method

transfer assessment (transfer specifications set

to 97-103% of the true value, data from ISO

5725-4).Result: the results of

laboratory no. 2, 4, 15, 16 and 18 are out of

specifications, mainly due to poor repeatability,

while laboratories no. 1, 7 and 13 show out of

specification results due to an important bias.

Software


VWR Collection

The new LINEA 10 gas generators

LINEA 10 Hydrogen GeneratorsIn addition to their high safety (production of hydrogen from pure water only when required), the new VWR Collection LINEA 10 hydrogen gas generators have been designed to meet highest quality and reliability standards. The hydrogen generators employ the newest Proton Exchange Membrane technology available for electrolytic production of pure hydrogen gas, including exclusive no maintenance auto-drying technology.• “No Maintenance” systems• Very compact• External water tank• Safe• Cost effective• Cascading option: combination and control of

several hydrogen generators in parallel• Remote control option

Applications:

H2 FID model series:• Fuel gas for GC-FID detectors• Reactor gas for other types of detectors in gas

chromatography

Why choose a gas generator?The main reasons why more and more laboratories are replacing their traditional gas supply (cylinders) with gas generators are:

For greater purity• No risk of contamination from cylinder changes etc• Gas generators control the purity of the gases permanently• No risk of contaminating systems (GC, LCMS) with gas of poor quality from a centralised

gas supply

For more flexibilityUnits can be easily moved around the lab.• Free from the constraints of a centralised gas supply • By means of the “Cascading” function, several hydrogen generators can be combined for

generation of larger gas flow rates or for back-up (in case of failure)

To reduce costsGas generators continuously produce gas directly in or near the lab:• No cost for rental, transport and storage of gas

cylinders• No expensive installation of tubing or manometers

etc.

To increase safetyGases are produced on demand with small buffer volumes:• No need for storage or transportion of high

pressure gas cylinders• Gas generators stop gas production automatically

in case of leaks and errors

H2 CARRIER model series:• Hydrogen as carrier gas and fuel gas in GC• Total hydrocarbon analysers• Sulphur analysers • Air pollution monitoring systems

Hydrogen + Zero Air Station (requires external source of compressed air):• Designed to feed GC-FID detectors

with hydrocarbon-free hydrogen and hydrocarbon-free Zero Air at the same time

• Small footprint (unit itself + external water tank)



LINEA 10 Zero Air and Ultra - Zero Air GeneratorsZero Air Generators:• High quality hydrocarbon-free air for GC FID detectors• Requires an external compressed air source

Ultra - Zero Air Generators:• Supply hydrocarbon-free air• Remove particles, moisture, CO2, CO, NOx, SO2 and O3 contaminants to less

than 0,1 ppm• Requires an external compressed air source

Zero Air and Ultra - Zero Air Stations:• Complete with a built-in air compressor

LINEA 10 Nitrogen Generators

The N2 Bora series is ideal for all kinds of laboratory and chromatography applications where high purity nitrogen is required. Other typical applications are ICP, ELSD, PID or incubators.

• Small units• PSA (Pressure Swing Adsorption) technology• N2 with a purity of up to 99,999%.• Optional internal oil-free compressor

N2 Sirocco large flow rate and high purity nitrogen generators:• DS-PSA (Double Stage PSA) technology• Most N2-Sirocco models include an internal oil-free compressor• N2 with a purity of up to 99,999%

Mistral-LCMS:• Specially designed to aliment LC-MS systems• PSA (Pressure Swing Adsorption) technology• Includes an internal compressor• Up to 35 l/min nitrogen with a purity of 98,5.

N2-Mistral-0:• Designed for LCMS applications• Based on hollow fibre membrane technology• Requires external compressed air supply• Works pneumatically, no power supply is needed• Completely silent• No moving parts, resulting in reliable and trouble-free

operation with virtually no maintenance

LINEA 10 Air CompressorsThis series of air compressors provides ideal air supply for Zero Air or Nitrogen generators and other applications.

Equipment


ChromSword Auto® 4.0 Save time and costs while improving your HPLC methods!

Your professional expert for automatic HPLC optimisation

All you have to do is place your sample in the autosampler, enter your data and press the “Start” button! Your methods are then

developed and optimised in an automatic overnight or weekend run. This is how easy HPLC method development is with ChromSword Auto, recognising the best available software for fully automatic HPLC method development.The intelligent ChromSword Auto software is simply connected to the HPLC. It is then capable of optimising reversed-phase chromatography fully automatically using the following parameters:

• Composition of the mobile phase (isocratic, linear and step-gradients)

• pH of the mobile phase• Column temperature

Objectives of the automatic optimisation are:• Separation of the maximum number of peaks• Optimal peak resolution• Minimal analysis time

ChromSword Auto, with its sophisticated and intelligent calculating processes and the huge computer capacity available, takes into account far

more method development possibilities than could possibly be coped with manually.On completion of the optimisation process, the system presents the chromatograms that can be obtained using the predicted optimal conditions.

Screening, fast optimisation and fine optimisation

These three stages of the professional method development process can be carried out by ChromSword Auto automatically.

ScreeningTo rapidly develop a new separation method, a selection of columns and solvents are tested using a rapid solvent gradient. The HPLC system in question is fitted with the appropriate column and solvent switching valves. ChromSword Auto is then programmed with the required gradient and subsequently carries out the complete screening process automatically within a short period of time. The chromatograms produced can be observed by means of the Report Viewer. The best column/solvent combination can then be selected for further optimisation.

Fast optimisationUsing this function, a suitable method can be developed within a very short period of time. Initially, the system performs an overview gradient run which enables it to obtain information on the peaks and the distribution of analyte retention times. Utilising this data, the software calculates optimal linear and step gradients. A suitable separation method can be developed in this way in a maximum of 3 hours. The

Fine optimisation and separation of a pharmaceutical sample containing impurities.

In combination with an HPLC system ChromSword Auto provides fully automatic unattended method development in reversed-phase chromatography

Automatic Method Development with ChromSword AutoSuitable HPLC system configurations for screening and fine optimisation

Standard system

with 1 column Standard system

with 2-3 columns Professional system with 8-10 columns + 16 aqueous solvents



Software

period depends on the column length and sorbent used, the flow rate, the polarity of the analytes and the number of peaks.Fine optimisationIn this mode, the software collects and evaluates further data and calculates, as closely as possible, appropriate retention models (dependence of analyte

retention time on optimisation parameter). Using this information, a number of alternative optimal HPLC conditions (isocratic, linear and step gradients) are calculated.

How does it work?

Retention models and gradient optimisationThe more information the software is given concerning analyte and separating system (column/solvent), the better it can predict the optimal separation conditions. Thus, in the course of the optimisation process, the system carries out chromatographic runs under varying conditions and then calculates the retention models. These are then used to predict the optimal conditions. These functions, together with the super-fast Monte Carlo gradient optimisation system, are included both in the off-line version of ChromSword and in ChromSword Auto.Further expert functions render the HPLC method development process fully automatic:

Automation moduleA special software interface connects ChromSword Auto with the chromatography data system of the HPLC in use.

Artificial intelligenceA software module equipped with self-learning artificial intelligence, exercises control over the optimisation process and makes all the necessary decisions pertaining to method development.

Peak tracking for target substancesThe somewhat difficult task of unambiguous peak tracking can be solved by simply using pure substances during the experimental phase of method development. This procedure is employed by ChromSword Auto for the separation optimisation of analytes if these are available as individual standards.

Intelligent peak allocation for substance mixtures containing impuritiesIn the case of samples where there may be no pure substances available, ChromSword Auto is equipped with a novel function for intelligent peak tracking. This enables peak tracking and assignment to be carried out reliably when separating mixtures of substances, even without having recourse to spectral data. However, should such be available, DAD and MS data can additionally be applied for peak tracking as well. All that is required is to place the sample vial in the autosampler. By identifying and tracking the peaks produced under different chromatographic conditions, the system optimises the method to produce the maximum number of peaks with optimal peak resolution in the shortest possible period of time.

Fast optimisation and separation of 11 standards of drugs of abuse:1. Overview gradient2. Optimal linear gradient3. Optimal step gradient


Typical samples for automatic HPLC method development with ChromSword Auto®

Column and solvent switchingIf the HPLC system is equipped with a column selection valve, the ChromSword Auto software performs screening and HPLC optimisation with up to 10 columns and three organic solvents automatically. For pH optimisation, the gradient system can be fitted with an additional solvent valve containing up to 16 channels for connecting buffers of different pH values. In this way, rapid screening of columns, solvents and pH can be carried out.

Easy to programme ...Via a Programming Assistant, the necessary data

are requested by very few screen prompts. The optimisation process is then initiated by activating the “GO” key. On completion, the HPLC system can be switched automatically to standby mode. ... and to generate reportsChromSword Auto documents the parameters entered and all the optimisation runs along with the corresponding chromatograms. Using the Report Viewer, all the chromatograms and data collected can be displayed and assessed. Using the export function based on Microsoft® Word™, a tailor-made method optimisation report with chromatograms, gradient profiles etc. can be generated.

Fast and fine optimisation and separation of a pharmaceutical samplecontaining degradation products.1. Overview gradient2. Optimal linear gradient3. Optimal step gradient4. Optimal step gradient subsequent to fine optimisation

• Active ingredients, new compounds (including chiral separations)

• Pharmaceutical formulations• Major products and

unknown impurities, by-products, degradation products

• Reaction mixtures• Biological extracts

Fine optimisation and separation of a

pharmaceuticalsample containing

impurities.



Software

• Separation of more substances and also with lowest concentrations of impurities (> 50 peaks, < 0.05%)

• Better peak resolution• Higher degree of robustness and hence more

reliable• Faster separations, shorter analysis times• Saving of time and effort in method development

(fully automatic, optimisation overnight)

• Savings of solvents in method development and routine analysis (cost, disposal, protection of the environment)

• Easy conversion of methods to more favourable solvents

• Increase in productivity and reliability in HPLC analysis

• Amortisation of software costs after only few method optimisation runs

• Chiral substances/enantiomers

• Achiral isomers and diastereomers

• Inorganic ions • Proteins and peptides• Carbohydrates • Extremely hydrophilic

substances • Extremely hydrophobic

substances • Highly charged substances • Very low concentrations

(< 0.05%)• Samples with more than

50 peaks

Even the separation of complex samples can be developed and optimised with ChromSword Auto®:

• VWR-Hitachi• Agilent• Waters• Dionex• Knauer

ChromSword® Auto controls the following HPLC and UHPLC systems

• EZChromElite™• Agilent ChemStation® • Waters Empower™• Dionex Chromeleon®

• Knauer ChromGate®

Using the following chromatography data systems (stand-alone and client/server)

Further software products, upgrades, installation and training services available on request.

Fine optimisation and separation of 16 standards of polyaromatic hydrocarbons (PAH).

Fine optimisation and separation of 16 standards of

polyaromatic hydrocarbons (PAH).

9 good reasons for Automatic Method Development using ChromSword® Auto

Description Cat. No.ChromSword Auto® 4.0 Professional with column and solvent switching (up to 10 columns, up to 16 solvents) and many further special functions

908-0055

ChromSword Auto® 4.0 Standard with column and solvent switching (up to 3 columns, up to 4 solvents)

908-0056

ChromSword Auto® 4.0 Basic without column and solvent switching (with 1 column and 2 solvents)

908-0057

ChromSword® 2.0 off-line 908-0058AutoRobust 1.1 software for automated robustness testing

908-0017


Parameters of Planar Chromatography

An inherent advantage of planar chromatography is its flexiblity due to the number of variable parameters that can be selectively optimised. That advantage can be undermined by misunderstanding the importance of the individual parameters. When this occurs a technique that is fundamentally simple and stable can appear unpredictable and a matter of chance.The articles in this series are dedicated to the various steps of planar chromatography and understanding the parameters in these steps that influence the chromatographic result. Hints for optimisation are given. The article aims to help the reader avoiding the common pitfalls and problems that can lead to frustration with this technique.

Planar chromatography differs from all other chromatographic techniques in the fact that a gas phase is present in addition to stationary

and mobile phase. This gas phase can significantly influence the result of the separation.

Processes in the developing chamber The ”classical” way of developing a chromatogram is to place the plate in a chamber which contains a sufficient amount of developing solvent. The lower end of the plate should be immersed several millimeters. Driven by capillary action the developing solvent moves up the layer until the desired running distance is reached and chromatography is interrupted.The following considerations primarily concern silica gel as the stationary phase and developments which can be described as adsorption processes.Providing the chamber is closed and reasonably tight, four partially competing processes occur:

1. Between the components of the developing solvent and their vapour phase, equilibrium is being established eventually (1). Depending on the vapour pressure of the individual com-ponents the composition of the gas phase can differ significantly from that of the developing solvent.

2. While still dry, the stationary phase adsorbs molecules from the gas phase. This process is also approaching an equilibrium called adsorp-tive saturation. In this particular way the polar components will be withdrawn from the gas phase and loaded onto the surface of the sta-tionary phase (2).

3. Simultaneously the part of the layer which is already wet with mobile phase interacts with the gas phase. Thereby the especially less polar components of the liquid are given off in the gas phase (3. Unlike (1) this process is more governed by vapour pressure than by adsorption forces.

4.During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts.

The following considerations should also be applied to chromatogram development: With the exception of single component liquids (neat solvents), developing solvent and mobile phase are, strictly speaking, not the same. Their composition changes with progressing chromatography. Unfortunately the terms ”developing solvent” and ”mobile phase” are often used as synonyms. In the true sense only the liquid in the chamber should be called developing solvent, while the liquid moving through the layer constitutes the mobile phase. Only the composition of the developing solvent at the time when it is placed into the chamber is completely known.Processes (1) and (2) can be experimentally affected by: • Lining the chamber as fully as possible with filter

paper soaked with developing solvent• Applying an ’equilibration’ time between the

introduction of developing solvent into the chamber and beginning of chromatography to allow saturation of the chamber

• Allowing the plate to interact with the gas phase prior to chromatographic development, i.e. without contact to the developing solvent so called preconditioning

Part 1: Chromatogram development – chamber type and chamber saturation

Number 2

Number 3

Number 1



Analytical TLC

An interaction relating to (2) and (3) can be effectively prevented by placing a counter plate at a distance of one or a few millimeters to the chromatographic layer. This is called a ”sandwich configuration”. The greater the equilibration between (1) and/or (2) is, the less different the components of the mobile phase are in respect to their adsorption behaviour, the more we reduce secondary fronts resulting from (4). In well saturated chambers and on pre-conditioned layers, secondary fronts are often not observed. In sandwich configurations and particularly in OPLC, secondary fronts are very likely. During chromatography, components of the developing solvent which have been loaded onto the dry layer via the gas phase as in 2), are pushed ahead of the true (but invisible) solvent front. Exceptions are very polar components such as water, methanol, acids, or bases. This results in Rf values being lower in saturated chambers and particularly on pre-conditioned layers, than in unsaturated chambers and sandwich configurations.

Consequences:

Planar chromatography, in most cases, proceeds in a non-equilibrium between stationary, mobile, and gas phase. That is why it is very difficult to describe the conditions in a developing chamber as mathematically as they are inherantly unstable.

Reproducible chromatographic results can only be expected when all parameters are kept as constant as possible. Chamber form and chamber saturation play a predominant role in this regard. Unfortunately this means that the chromatographic result is different in each chamber!

Choosing a chamber

There are no “good” or “poor” chambers. However, in some chambers the parameters can be better controlled, i.e. reproduced, than in others.

Typically the user will select a chamber based on a number of ’practical’ considerations: convenience; familiarity; or consistency (using the same chamber for ’comparative purposes). A better process would be to focus on obtaining a chamber that minimised solvent consumption, reduced the time required for separations, and eliminated as many variables as possible.The CAMAG Horizontal Developing Chamber

has proven to be exceptionally economical, flexible and reproducible.

1) HPTLC plate (layer facing down) 2) Glass plate for sandwich configuration 3) Reservoir for developing solvent 4) Glass strip 5) Cover plate 6) Conditioning tray Principle: The HPTLC plate is placed into the chamber with the layer facing down. The reservoir (3) is charged with developing solvent – the plate can be developed horizontally either from one side only or from opposite sides simultaneously, this way doubling the number of samples per plate. Chromatography is started when the glass strip (4) is brought into a vertical position.Unsaturated configuration: The conditioning tray (6) is empty; the glass plate (2) is removed.Saturated configuration: The conditioning tray (6) contains developing solvent; the glass plate (2) is removed.Pre-conditioning: The conditioning tray (6) contains conditioning liquid; the glass plate (2) is removed. Development is started after pre-conditioning is completed.Sandwich configuration: The conditioning tray (6) is empty; the glass plate (2) is in place.

Separation of two plant extracts (track 1 Schisandra chinensis, track 2 Schisandra spenanthera) on HPTLC silica gel 60 F 254 (Merck) with toluene – ethyl acetate – acetic acid (70:30:3). Documentation with VideoStore under UV 254 (upper row) and after derivatisation with sulfuric acid reagent (10% H2SO4 in methanol). a) Twin trough chamber (TTC) saturated b) TTC unsaturated, c) Horizontal-Developing Chamber (HDC)

saturated d) HDC unsaturated e) HDC saturated and plate preconditioned 10

min with developing solvent f) HDC sandwich configuration under white light

(lower row)

The CAMAG Horizontal Developing Chamber (HDC) is available for the plate formats of 10 x 10 cm and 20 x 10 cm


ChromaScanThe affordable system for TLC plate capture and analysis.

The ChromaScan offers a compact and affordable system for the capture and analysis of TLC plates. Featuring a powerful 12 bit colour digital camera with outstanding colour rendition, the system will give perfect images of TLC plates quickly and conveniently.

Most importantly, the ChromaScan allows to acquire a digital image of a plate using a range of wavelength options and then to

archive it. This gives the ability to view both sample and standard adjacent to each other and which is a very powerful tool.The ChromaScan combines an exceptionally versatile software module with a robust and easy-to-use imaging system making an outstanding instrument for all TLC plate applications. Built to the highest standards, but at an economic cost, the ChromaScan is the ideal choice for any laboratory.Features:• Easy-to-use• Low cost system, but with powerful features

normally only found on more expensive systems• A range of illumination options• High specification 12 bit CCD digital camera• Powerful analysis software module• Image browser• Profile comparisons

Darkroom and illuminationThe ChromaScan darkroom provides a safe and secure environment in which to illuminate TLC plates. The illumination options are: direct UV (254 nm and 365 nm) and visible light and also transmitted visible light. It is also possible to use a UV transilluminator to provide 302 nm transmitted illumination. The large opening door provides easy access to the sample area. A safety interlock on the door protects against accidental exposure to UV.Features:• Compact darkroom• Safety interlock• 254 nm direct light• 365 nm direct light• Visible light direct and transmitted• Optional transilluminator 302 nm• Filter slider

Colour CCD cameraThe ChromaScan camera is a 12 bit CCD colour model with exceptional low noise. The dynamic range can be further extended using Syngene EDR technology which boasts image output up to 16 bit. Reproducibility is an important consideration but with the ChromaScan the performance of the camera assures this.The system uses a motorised zoom lens with feedback.Features:• 12 bit colour CCD camera• 16 bit Extended Dynamic range• Adjustable exposure times• Software controlled

Analysis softwareThe Analysis software enables the user to perform a quantitative evaluation of the captured images. It also allows for the chromatogram images and the analysed data to be saved and printed.Features: • Provides quantitative peak data from the

chromatogram images• Can perform quantitative analysis of samples when

compared to calibration standards• Displays individual chromatogram profiles• Chromatogram comparisons• Quantitative evaluations• Choice of calibration mode• Report generation



Equipment

Lab Water Gets “Ultra, Ultra-Pure” New polisher removes trace organics detected by the most sensitive chromatography instruments with C18 reverse phase silica technology

Recent advances in analytical techniques such as UPLC, LC-MS, LC-MS/MS have considerably improved the sensitivity of organic and biochemical molecule detection. As a result, these techniques now require improved water quality for the production of mobile phase, buffers, blanks, standards preparation, sample dilution, glassware rinsing or extraction.

To answer this emerging need, Millipore has developed a new application-Pak, designed to produce ultra-pure water with an organics contamination level below most water sources available today, and at a fraction of the cost.

LC-Pak™ point-of-use polisher Key Benefits

• Final purification step uses efficient C18 reverse phase silica technology to provide fresh ultra-pure water with low traces of organics at high flow rate, when you need it

• The LC-Pak™ cartridge is easily connected to the outlet of all Millipore type I water systems (Milli-Q®, Direct-Q®, Synergy® and Simplicity®)

• Designed to answer the demands of UPLC, LC-MS and LC-MS/MS analytical techniques used for organic trace and ultra-trace analysis

• Validated for the production of water whose quality equals or exceeds the specifications of bottled water for LC-MS

• Delivers a minimum of 500 litres of organic-free ultra-pure water with a TOC level below 5 ppb

• Each LC-Pak™ cartridge is delivered with a certificate of quality

LC-Pak™ cartridge specifications

Parameter Specification of LC-Pak™ ultra-pure water

Additional notes

HPLC Gradient Test - Absorbance of highest eluted peak

At 210 nm < 0,006 AU Concentration of 60 ml water at 1 ml/min prior to elution

At 254 nm < 0,002 AU HPLC Gradient Test - Absorbance of highest eluted peak

At 210 nm < 0,003 AU No water pre-concentration At 254 nm < 0,001 AU

Optical properties: absorbance in UV range

UV 200 nm < 0,05 AU UV 205 nm < 0,01 AU UV 210 nm < 0,01 AU UV 254 nm < 0,005 AU

Fluorescence as quinine At 254 nm < 1 ppb At 365 nm < 1 ppb

Compliance with suitability for LC/MS: Reserpine test

No peak higher than 10 ppb Reserpine at 609 m/z in ESI +

Residue after evaporation < 0,0001% w/w Test performed as specified in ISO 3696 procedure

Description Cat. No.LC-Pak™ cartridge (1/pk) delivered hermetically sealed, with a certificate of quality

171-0333

Cartridge installation and conditioning kit including the following reusable parts: Polyethylene ¼ Gaz F-Hose barb connection with O-ring Polyethylene ¼ GF – 1/4 Gaz F connectors for connection to Millipak

171-0244

Millipak 0,22 µm end filter 172-0009


Rapid high sensitivity LC/MS analysis of sugars using ZIC®-HILIC HPLC columns

Petrus Hemström and Patrik Appelblad, MerckSeQuant AB, Umeå, Sweden

Analysis of sugars, sugar alcohols and other carbohydrates in different type of formulation and matrices is important to the food and beverage, pharmaceutical and biotech industries. Over time, many different analytical approaches have been used, such as ion chromatography, reversed phase chromatography, or gas chromatography, however, new and more attractive approaches are now available.

Hydrophilic interaction liquid chromatography (HILIC) is the technique of choice and an attractive separation mode for polar, hydrophilic compounds like carbohydrates. HILIC is easy-to-use and works well where traditional reverse phase methodology fails, and is more intuitive and with a greater flexibility than ion chromatography. The elution order is typically the opposite of RPC, with the most polar compounds eluting after the non-polar compounds, resulting in an alternative selectivity. A bonded zwitterionic stationary phase like the SeQuant™ ZIC®-HILIC columns (Figure 1) allow chromatographers to maximise analyte sensitivity with evaporative detection techniques, by eluting polar compounds under high percentage organic mobile phase conditions. This is ideally suited for analyte ionization and increases sensitivity for many analytes in electrospray ionisation mass spectrometry (ESI-MS). These bonded zwitterionic stationary phases are available either with silica or a polymer core base particles, an advantage for carbohydrate analysis, as many analytes of interest are pH dependent. Using a silica core particle, the working pH range is typically 3-8, while a polymeric material tolerates both more acidic and more basic conditions.

Three minute baseline separation With a polymer-based SeQuant™ ZIC®-pHILIC column and a gradient elution protocol, baseline separation of all four sugar analytes was achieved within 3 minutes, see Figure 2. The use of a basic buffer component, like ammonium hydroxide (NH4OH) in the mobile phase is a key component to alleviate any dual peaks from anomers of reducing sugars and aid in the separation. In this work, simple carbohydrates were separated at an elevated pH using direct ESI-MS detection. The model compounds, fructose and sucrose easily separate at any pH, whilst glucose and lactose mutarotate at neutral pH, at a time scale slower than that of the separation, causing peak splitting, and potentially eluting as isomeric pairs. Applying a basic mobile

phase and adjustment of sample pH, effectively elutes the anomers as a single peak on a polymeric ZIC®-pHILIC column, which allows for separation of more complex analytes. Gradient elution was chosen to sharpen peaks and thus maximise sensitivity and while also keeping chromatographic run time short. Column temperature was kept at 55 °C to reduce eluent viscosity and minimise effects of changes in column pressure due to altered eluent composition during the gradient profile.

Remarkably high efficiency and sensitivityLinear calibration curves, based on peak area evaluations, were constructed from duplicate analysis of each standard at five concentration levels. All analytes produced linear calibration curves with a correlation coefficient of 0.997 or better, and the detection limits (LOD) were better than 0,2 ppm overall. This is better than evaporative light scattering (ELSD) and refractive index (RI) detection would produce. These results are also better than what has been reported on a new composite ethylene bridged sub-2 micron material with amide functionality. It is quite remarkable, and worth pointing out, that a 5 micron polymeric material produces better detection limits than silica-based material with a much smaller particle size. However, the chromatographic

Figure 1. Schematic structure of

SeQuant™ ZIC®-HILIC bonded

stationary phase.


For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website http://eu.vwr.com/chromApplication

selectivity between these columns is different and different experimental conditions were used, which somewhat limits direct comparability.

ConclusionThe chemical stability of the polymer-based SeQuant™ ZIC®-pHILIC columns allow for direct HPLC ESI-MS quantitation of simple and complex carbohydrates. Using high-pH mobile phases, it is possible to increase mutaroation rates and avoid peaks from anomers, thus simplifying identification

of carbohydrates in different types of samples even with difficult matrices. Combined with simple sample preparation procedures like protein precipitation or liquid-liquid extraction, efficient and cost efficient analytical work schemes can be developed and used for monitoring of sugar, sugar alcohols and other carbohydrates in different type of formulation and matrices.

References1. www.sequant.com/sugars

Figure 2. Rapid high pH gradient separation of fructose, sucrose, lactose and glucose, detected by ESI-MS. Column: ZIC®-pHILIC 100 x 2.1 mm, 5 µm. Temperature: 55 °C. Pressure drop: 6.5 MPa (936 psi). Mobile Phase: A: 100% Acetonitrile; B: 100% Ammonium Hydroxide 1% (257 mM). Initial composition: 73% A and 27% B. Linear gradient from 0-3 min with 4.33% increase of B/min, followed with 4 min re-equilibration. Flow-rate: 0.35 ml/min. Injection: 2 µl of a mixture containing 0,5 ppm each of Fructose, Glucose, Sucrose and Lactose in mobile phase. Detector: Shimadzu LC-2010 Evolution, detector voltage: 1,6 kV, heat block temp: 200 °C; CDL temp: 200 °C; scan range: m/z 150-450; SIM mode: m/z 159 (Lactose), 177 (Fructose and Glucose) and 341 (Sucrose).

A global site in local language

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For the latest innovations, product updates and promotions check out our new look

0 1 2 3

Retention Time (min)


Built-in solutions for the safe disposal of liquid waste

People working in a lab are aware of the problem of liquid waste disposal. Ordinarily there is no space in the extractor hood as it is usually full of different solvent waste containers. The funnel in the container is often not locked and the drainpipe left open, contravening most laboratory guidelines. For example, HPLC systems waste containers are either placed on top of the counter, on the floor, or beside the extractor hood. Not really safe for the lab personnel!

SCAT Europe specialises in safety in the laboratory and provides a solution. A flexible fully integrated system, complies with all safety

requirements, is space-saving and can be customised to changing work requirements.

A table duct is the core element of this electro-conductive system. Only the screw thread for a normal GL45 screw cap is visible above the bench top. This connection has specifically been conceived for this very common thread type. SCAT Europe offers electro-conductive funnels as a feature of this interface through which large quantities of liquid waste can be disposed of. In addition the well known SCAT SafetyWasteCaps can be used for connecting tubes and capillaries. Different multiway adapters are available to make the system more efficient and flexible.

As only the filling unit is mounted on the bench top and all other components are fitted below, more space is created in the work area. If the system is temporarily not in use, the system can be closed with a normal GL45 screw cap. If the method of operation changes, it is possible to change the filling unit on the table-mount. Yesterday’s safety funnel is today’s SafetyWasteCap for HPLC waste.

Various conduit systems are constructed below the bench top for conducting liquid waste safely into the containers. SCAT Europe also provides a pipe system for longer distances.

Level control systems prevent spilling while filling the waste container. The level indicator can be displayed by a SCAT signal box or a system already integrated in the lab. For applications in an ex-zone, signal cables can be fused via an isolating switch amplifier. The container is ventilated through the connection of an exhaust filter, (for non-aspirated floor units) or through a flexible and electro-conductive tube, which conducts the exhaust air to the drainpipe.

Besides the standard solutions, SCAT Europe offers tailor-made modules for every requirement.

Features:

• Suitable for solvent wastes such as acids and alkaline solutions

• Can be used in ex-zones• Space saving compared to waste containers in the

extractor hood• Flexible applications and design: Funnel today -

capillaries tomorrow (via safety waste cap through GL 45 screw thread

• Electro-conductive material prevents anti-static discharge

• Principle of building blocks• Level control monitoring of waste container for

preventing overfill of liquids• Optional stopcock for preventing the spillage of

liquid remaining in the tube when changing the container

• Suitable in the planning phase for new labs or when upgrading.



Accessories

HD plunger for headspace gas chromatographyWork at the temperatures you need for your headspace GC analysis.

Hamilton has introduced a headspace syringe with a new plunger designed for the CTC PAL autosampler system. This High Dynamic (HD) plunger has been optimised for higher throughput in headspace techniques. Hamilton has set new standards for headspace syringes with this design.

Highlights/Key benefits:• New sealing system based on a novel metal spring

allows working with enhanced tightness • Excellent performance over a large temperature

range and temperature gradients• Increased lifetime compared to traditional

headspace syringes on the market • Increased accuracy and reproducibility of

headspace GC analysis

The problem

Modern GC headspace analysis requires injecting over large temperature ranges (e.g. ITEX analysis). Traditional headspace syringes are composed of rubber O-ring sealed plungers which have a limited sealing performance at high temperatures.

How does the HD plunger work?The key element of the new High Dynamic (HD) plunger is a novel spring supported sealing. This metal spring compensates the thermal expansion of the PTFE tip which is in contact with the glass barrel. Conventional headspace syringes are composed of rubber O-ring sealed plungers which lack such compensation properties.The sealing performance of these new HD plungers was compared to traditional plungers for headspace GC on the market. This was determined by measuring the force needed to move the plunger in the glass

barrel. The force was monitored for a temperature ramp from 25 °C to 150 °C and back to 25 °C. Test results are shown in the figure below illustrating that the traditional plunger loosens its sealing properties at high temperatures, when cooling down (no force is needed to move the plunger). The new HD plunger, however, dynamically reacts to the temperature change and compensates for the thermal expansion of the PTFE tip. The HD syringe allows working at various temperatures including temperature ramps with a perfectly sealing plunger. This is a clear benefit over traditional syringes

Models Volume (ml)

Needle options (gauge)

Cat. No.

1001 1,0 23 549-132426 549-1327

1002 2,5 23 549-132526 549-1328

1005 5,0 23 549-132626 549-1329


SGE Analytical Science has launched SilTite™ FingerTite the next generation ferrule system for gas chromatography systems that delivers an easy, leak-free installation for capillary columns – no tools needed! SilTite™ FingerTite simplifies column installation allowing more time for chromatography.

SilTite™ FingerTite A new innovation for gas chromatographers.

SilTite™ FingerTite is a GC ferrule that has been designed to be installed only with the force of your fingers. It has been developed with the unique, leak- and air-free properties of SilTite™ ferrules, resulting in superior air tightness. This is especially useful in helping to reduce background noise in sensitive MS

applications, allowing you to use SilTite™ FingerTite on your MS interface with confidence.

Advantages of SilTite™ FingerTite:

• No practical temperature limit, is chemically inert and won’t deteriorate

• Doesn’t need re-tightening after installation• Cannot break the capillary column with over-

tightening• Ideal for GC-MS applications due to minimal

porosity

SilTite™ FingerTite GC Ferrules the smart alternative.

SilTite™ FingerTite requires an adapter to enable the finger force connection. Once you have a starter kit with the corresponding adapter for your instrument, you can complete the one-off 5 minute install and you are ready to go. There are no permanent instrument modifications, so if for any reason you need to switch back to conventional connections, you can do this quickly and easily.


For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website http://eu.vwr.com/chromGC

eVol® – Everyone’s an expertThere’s a new buzz in liquid handling – eVol® has arrived! eVol® is the coupling of a digitally controlled electronic drive and an XCHANGE® (patent pending) enabled analytical syringe. The result is a digitally controlled positive displacement dispensing system that is programmable to reproducibly and accurately perform a wide variety of liquid handling procedures. More importantly, eVol® will improve your laboratory workflow and reporting confidence.eVol® features a familiar touch wheel user interface and full colour screen. The logical menu allows all functions to be accessed quickly and programming functions are intuitive with help screens and prompts. Only three XCHANGE® syringes are required to dispense liquid volumes from 200 to 500 µl.

Customer experience

People who have already bought eVol® are successfully using it in a range of laboratory processes with varying reagents as listed in the table below.

Benefits of eVol®

• Liquid handling procedures become user independent creating efficiencies in work flow scheduling

• XCHANGE® syringes are easily and quickly changed to allow for dedication to one particular liquid

• Can be used with aqueous and non-aqueous liquids

• Can be calibrated• Calibration factors are stored for up to

ten XCHANGE® syringes

With eVol®, everyone’s an expert.

Process ReagentsStandard preparation Acetone, methanol, DCM, hexane, pentane, IPA, chloroform, benzene, ethanol, gaseous mercuryCalibrations DCM, hexane, methanol, water, pyridine, pentane, heptane, acetoneImpurity profiling TolueneChromatography DCM, methanol, toluene, ethanolAddition of derivatisation reagents MSTFA, hexaneRepeat dispense Water, acetonitrile, ethyl acetate, hexane, acetone, ethanol, tolueneSample dilution Acetone, ethyl acetate, hexane, methanolPrecise low volume measurement Hexane, methanol, chloroformMethod development MethanolQuantitative NMR Chloroform


The flavour compound was suspected to be unstable in the formulation. To confirm this a method was developed for the quantification of

this target compound.

Sample Preparation:

Liquid samples were diluted in the initial HPLC eluent and filtered through a 20 µm pore sized filter. The filtrate was injected and analysed by HPLC.

Results and Discussion:

On a conventional column (5 µm, C-18) the run length was 25 minutes. Under the same elution conditions the run time could be reduced to 5 minutes by using a monolith silica column (Chromolith® FastGradient RP-18 Endcapped (50 x 2 mm)

The elution of the 3 relevant peaks at 254 nm is shown in the chromatogram below. In figure 1. we can observe the advantages of reducing the run time by a factor of 5; peak widths are reduced enhancing the signal to noise ratios and improving sensitivity; peak tailing is reduced significanly improving the integration reliability and reproducibilty of the method.

Conclusion:

The retention time of the compound (Methylsalicylate) could be reduced from 15.78 minutes to 2.15 minutes. The reduced peak width improved the sensitivity by a factor of 10. This provides a faster more stable quantification of the target compound significantly enhancing the abilty to screen products for stability purposes.

Figure 1: Chromatogram on a conventional 5 µm particle column compared to the shorter monolithic column (upper trace) with a C18 stationary phase. Elution conditions were identical.

A method for the quality control analysis of Methylsalicylate in oral care products

Michael Rothaupt, Fragance Research Dübendorf Switzerland

The aim was to develop a fast method for the analysis of Methylsalicylate in oral care products.

Signal/Noise:

LC conditions on Agilent 1100 (Binary Pump, Autosampler with (1.4 µl loop), Column Oven, Diode-Array-Detector)Buffer composition

Channel Solvent A Water B Methanol

Flow 0,8 ml/minTemperature 40 °CInjection volume 1 µlGradient Time Channel A Channel B

0 65% 35% 1 60% 40% 15 0% 100%

Column S/N ratio150 x 3 mm, 3 µm C18 AQ 430:01:00Monolith 50 x 2 mm 4280:01:00



Evaporative light scattering detection provides a universal detection method for HPLC. It is not dependant on the presence of chromophores in the compound, therefore it is a very useful technique for ‘difficult‘ or unknown compounds. Typical applications include proteomics, combinatorial chemistry, lipids, surfactants, high-throughput screening, and biofuels.

New VWR ELSD 85Evaporative light scattering detector

byEZChromEliteandtheLaChromEliteTMsystem

The unique design of VWR ELSD 85 nebulisation cell allows the selection of the droplets as a function of the size. Large droplets, (which

are more difficult to evaporate) are responsible for increased noise level. In the glass nebulisation cell, the largest droplets are simply eliminated, this design allows the temperature to be lowered without compromising the signal to noise ratio (sensitivity). Control of the new VWR ELSD 85 with EZChrom EliteTM

The VWR ELSD 85 can be fully controlled by the EZChrom EliteTM chromatography data system. All functions of the detector can be controlled from your PC.

Applications of the VWR ELSD 85The VWR ELSD 85 typically has ten times the sensitivity than an refractive index (RI) detector. ELSD

is immune to the solvent effects seen in RI detection and is therefore compatible with gradients. Faster runs can be achieved as the ELSD is not limited to isocratic methodolgy.

Summary: The VWR ELSD 85 includes a newly designed high performance optical detection system and enhanced digital signal treatment providing a dramatic improvement in sensitivity (<200 pg). It can be fitted with four different nebulisers (including UHPLC nebuliser) to optimise its performance at flow rates from 5 µl to 5 ml/min. This wide flow range fits perfectly to the LaChrom EliteTM system and EZChrom EliteTM software, which now allows a full control of the VWR ELSD 85. This new detector has advantages over both refractive index detectors and competitive detectors in terms of both sensitivity and gradient compatibility.

Fig.1 LaChrom Elite system with the Sedex 85 LT-ELSDTM detector

VWR ELSD 85


Figure 5EZChrom EliteTM control: Instrument Status of the VWR ELSD 85

Figure 6EZChrom EliteTM control: Instrument Setup of the VWR ELSD 85

Figure 7Sensitivity comparison between the VWR ELSD 85 and the VWR-Hitachi L-2490 refractive index detectorSample: Fructose, Glucose, Saccharose, Maltose, Lactose standardsRed: 100 ppm standard with the VWR-Hitachi L-2490 RI detectorBlue: 12,5 ppm standard with the VWR ELSD 85.

Eluent: 20% water/80% acetonitrile, pre-mixed and vacuum degassedFlow rate: 1,0 ml/min

Figure 2In the nebulizer the eluent from the HPLC column is transformed into a fine mist of droplets. The design of the VWR ELSD 85 selects optimally sized droplets, and excludes large ones from the evaporation tube. (Large droplets are responsible for increased background noise, as they are more difficult to evaporate.)

Figure 3Solute molecules are separated from the vapour cloud in a heated evaporation tube using low temperature (LT) technology. All ELSD detectors are designed to evaporate high boiling mobile phases at very low tem-peratures. This unique feature minimises the potential for thermal decomposition of the compound of interest, this makes the ELSD method both robust and reliable.

Figure 4The solute molecules pass throgh the drift tube and are then focussed into an optical head using gas supported focusing (GSF).

The effect of GSF is to concentrate the mol-ecules into the optical head and so enhance detection.

L-2490

VWR ELSD 85



Figure 8Simultaneous separation of anions and cationsBlack Trace: Standard with nitrate, bromide, chlo-ride, potassium and sodium Blue Trace: Tap water, 1:1 diluted with acetonitrileRed Trace: Beer, 1:10 diluted with 50% acetonitrileColumn: Merck ZIC-HILIC, PEEK 150 x 2,1 mm, 3,5 µm, 200A Eluent A: acetonitrile; Eluent B: 20 mM ammonium formate pH 3, Isocratic: 82% A/18% B, mixed by the pump and vacuum degassedFlow rate: 0.3 ml/minELSD conditions: 40 °C, 3,5 barTemperature: 40 °C

Figure 9aSample: Amino acid standard 1. Asp, 2. Pro, 3. Thr, 4. Ala, 5. His, 6. Lys, 7. Val, 8. Met, 9. Arg, 10. Tyr, 11. Ile, 12. Leu, 13. Nle (Nor-Leucin), 14. Phe Column: Merck LiChroCart® Purospher® STAR RP-18e, (5 µm) 150-4,6 mm Flow rate: 1,0 ml/minColumn temperature: 40 °CEluent A: 0,1% HFBA (Heptafluoro butyric acid, pH 2,2)Eluent B: Acetonitrile flow rate: 1,0 ml/minGradient: 0-3 min 0% acetonitrile (B), from 0-25% B in 20 min/hold 25% B up to 25 min, re-equilibration with 0% B from 25.1 up to 35 min. Injection volume: 10 µlELSD conditions: 40 °C, 3,5 bar, Gain 8

Figure 9bOverlay of the amino acid standard and a human plasma sample that was spiked the amino acids Val, Arg, Ile, Leu and PheAn ELSD detector allows the detection of primary and secondary amino acids without any derivatisation

Equipment


Analytical HPLC

Purospher® STAR RP-18 endcapped 2 µm UHPLC columns accelerate separations by 10 times – and provide a decrease in solvent consumption of up to 84.5%.

Purospher® STAR RP-18 endcapped 2 µm UHPLC columns are available in 30, 50, 100 and 150 mm length for fast and high resolution separations of most complex samples.

Purospher® STAR RP-18 endcapped (2 µm) UHPLC columns are ideal for ultra-fast applications where resolution, sensitivity and sample throughput are crucial. They are the first choice for high-throughput screening, QC analysis, process monitoring, method development, and LC/MS applications. Due to its balanced selectivity, Purospher® STAR RP-18 endcapped UHPLC columns cover almost all demanding separations with tailing-free separations.

Obtain better resolution with ultra-fast HPLCPurospher® STAR RP-18e UHPLC columns

Separation of Lamotrigine and 9 related compounds using Hibar® HR 50-2.1 and 150-2.1 Purospher® STAR RP-18e (2 µm) UHPLC columns

Figure 1 Lamotrigine is an anti-convulsant drug used in the treatment of epilepsy and bipolar disorder. The mixture of Lamotrigine (Fig. 1) and 9 related compounds 2-Chloro-Lamotrigine, 3-Chloro-Lamotrigine, 4-Chloro-Lamotrigine, 2.5-Dichloro-Lamotrigine, 2.4-Dichloro-Lamotrigine, 3.5-Dichloro-Lamotrigine, 3.4-Dichloro-Lamotrigine, 2.3.5-Trichloro-Lamotrigine, Lamotrigine (open form) were separated on Purospher® STAR RP-18 endcapped 2 µm Hibar® HR 50-2.1 in 6 min (Figure 2). A baseline separation of Peak 7 (3.5-Dichloro-Lamotrigine) and Peak 8 (3.4-Dichloro-Lamotrigine) was not possible (Resolution factor (USP) 1,5) on this short column, although the column provides a very

N

NNH2 NH2

N

Cl

Cl

Lamotrigine

high efficiency of 200 000 N/m.To improve the separation of the critical peak pair (Peak 7 and 8) a column length of 150 mm was used (Figure 3). The resolution of Peak 7 and 8 obtain to a baseline separation (Resolution factor (USP) 1,9).



Figure 2Column: Purospher® STAR RP-18 endcapped, 2 µm Hibar® HR 50-2.1 mm Column temperature: 40 °CEluent A: Buffer (14 ml Trieethyl-amine ad add 1 l of water, adjusted to pH 1,9 with perchloric acid)Eluent B: AcetonitrileFlow rate: 0,8 ml/minPressure: 495 barGradient: 0 min 12% Acetonitrile, from 12-27% B in 7.1 min, re-equilibration with 12% B from 7,2 up to 12 min. Injection volume: 2 µl

Sample: wLamotrigine & relat. related substances standard 1. 2-Chloro-Lamotrigine, 2. 3-Chloro-Lamotrigine, 3. 4-Chloro-Lamotrigine, 4. 2.5-Dichloro-Lamotrigine, 5. Lamotrigine, 6. 2.4-Dichloro-Lamotrigine, 7. 3.5-Dichloro-Lamotrigine, 8. 3.4-Dichloro-Lamotrigine, 9. 2.3.5-Trichloro-Lamotrigine, 10. Lamotrigine – open form

Figure 3Column: Purospher® STAR RP-18 endcapped, 2 µm Hibar® HR 150-2.1 mm Column temperature: 40 °CEluent A: Buffer (14 ml Trieethyl-amine ad add 1 l of water, adjusted to pH 1.9 with perchloric acid)Eluent B: AcetonitrileFlow rate: 0.38 ml/minPressure: 530 barGradient: 0 min 17% Acetonitrile, from 17-34% B in 16 min, re-equilibration with 17% B from 16.1 up to 25 min. Injection volume: 2 µlSample: Lamotrigine & relat. related substances standard 1. 2-Chloro-Lamotrigine, 2. 3-Chloro-Lamotrigine, 3. 4-Chloro-Lamotrigine, 4. 2.5-Dichloro-Lamotrigine, 5. Lamotrigine, 6. 2.4-Dichloro-Lamotrigine, 7. 3.5-Dichloro-Lamotrigine, 8. 3.4-Dichloro-Lamotrigine, 9. 2.3.5-Trichloro-Lamotrigine, 10. Lamotrigine – open form

Purospher® STAR RP-18 endcapped, Hibar® HR Particle size (µm) Ø x l (mm) Cat. No.2 30 - 2,1 1.50645.0001 2 50 - 2,1 1.50646.0001 2 100 - 2,1 1.50648.0001 2 150 - 2,1 1.50649.0001 3 30 - 2,1 1.50650.0001 3 50 - 2,1 1.50651.0001 3 100 - 2,1 1.50653.0001 3 150 - 2,1 1.50654.0001 3 250 - 2,1 1.50655.0001


Capillary chromatography

The macropores are equivalent to the interstitial volume of particle packed columns, which determines its permeability.

High permeability and porosity of the silica skeleton and the resulting low back pressure allow for more flexible flow rates compared to particulate columns and enable high throughput analysis without loss of separation efficiency and peak capacity. The mesopores are located either in the silica skeleton of monoliths or on the silica particle surface. These porous structures give high surface areas for monolithic and micro-particulate silicas, which are necessary for efficient separations in adsorption chromatography.Compared to particulate capillary columns, Chromolith® CapRod® capillaries show a better performance as shown by optimal resolution (narrow peak widths), increased productivity like sample throughput and prolonged column lifetime. Finally, column length is less limited as compared to any other type of column. The capillaries can even be bent to a certain degree in order to optimally fit in any LC configuration and instrument.Chromolith® CapRod® monolithic columns are designed to work with various Nano or Capillary LC systems, providing highest efficiency and performance when coupled to mass spectrometers, both online (ESI, nanospray) and off-line (MALDI).A trapping capillary is also offered in order to protect the precious separation column and to optimise the separation efficiency when using complex biological samples.Monolithic capillary columns have become increasingly important in the separation of biomolecules, especially in combination with mass spectrometry. Compared to particulate columns, monolithic capillaries do not require frits and have a much lower tendency to clog,

thus allowing higher flow rates improving speed and quality of biomolecule characterisation (see Figure 1).The strong growing interest for HPLC mediated protein and peptide separations now covered with our wide range of monolithic silica capillaries differing in inner diameter (50 µm – 200 µm), bonded phase (C8, C18), pore structure (“HR” products) and length (Trap) (see Table below).

Figure 1. Analysis of a tryptic digest of ß-galactosidase (100 fmol) with sequence coverage >40%. Column: monolithic silica capillary 100 µm; mobile phase: A (water with 0,1% formic acid) B (acetonitrile with 0,1% formic acid); gradient: 5% B to 100% B within 35min; flow rate: 2 µl/min. Courtesy of Prof. U. Tallarek.

Monolithic silica-based columns have proven to be a useful and in many respects advantageous tool for capillary and nano-LC/MS analysis. In contrast to conventional particle packed capillary columns, monolithic silica columns are made of a continuous piece of porous silica, in-situ utilising a sol-gel process leading to rod columns, which possess a defined bimodal pore structure with macro and meso pores in the micro and nanometre range.

Monolithic silica capillaries: High efficiency, resolution and flexibility for LC analysis

0 5 10 15 200

500

1000

Intensity[cps]

Time [min]

Recommended use

RP-18e RP-8e RP-18e RP-18e RP-18e RP-18e RP-18e RP-18e RP-18e 150 x 0,05

mm 150 x 0,1

mm 50 x 0,1 mm

Trap 150 x 0,1

mm 300 x 0,1

mm 150 x 0,1 mm HR

50 x 0,2 mm Trap

150 x 0,2 mm

150 x 0,2 mm HR

Separation of small molecules

X X X X X X X X

- of Peptides X X X X X X X X X

- of Proteins X

Micro ESI X X X X X X

Nano ESI X X X X X X

High Resolution X X

Flow rates (µl/min) 0,2 - 0,8 0,4 - 3 1 - 10 0,4 - 3 0,2 - 1.5 0,1 - 0,4 10 - 50 5 - 20 0.5 - 2

Max backpressure (bar)

200 200 200 200 200 218 218 218 218

Cat. No. 1.50403.0001 1.50400.0001 1.50426.0001 1.50402.0001 1.50424.0001 1.50404.0001 1.50409.0001 1.50405.0001 1.50407.0001



LiChroTest® Certified test samples for Operational Qualification and Performance Qualification ensure that your HPLC system yields the correct results.

Before starting a series of analyses, you first should establish whether your HPLC system meets with your requirements.

These “Operational Qualification” (OQ) and “Performance Qualification” (PQ) steps involve tests of the different modules on their specifications, and a check on the entire system using a real application with regard to laboratory specific requirements. In order to facilitate this instrument qualification in the HPLC laboratory, Merck KGaA and VWR International have developed the LiChroTest® range of products. This time saving operational and performance qualification can be performed routinely and according to standardised methods.

For both, Operational Qualification and Performance Qualification, different test samples for checking precision, accuracy and linearity of the different HPLC modules or the complete system are available. Each set contains several ampoules of sample accompanied by a Certificate of Analysis which guarantees uniform quality.

Qualification

For Performance Qualification, the LiChroTest® PQ, is ideal. It is a complete test kit for carrying out 8 different system tests. It comprises of test methods, an HPLC column, test samples and a description of the qualification process, including a sample test report., This will enable you to rapidly and routinely carry out fully automatic performance qualification of your HPLC systems. The documentation completed in the course of the qualification procedure is highly useful for passing the next audit.

LiChroTest® Standard Samples for HPLC System Performance Qualification (PQ)Description Cat. No.LiChroTest® (PQ)Complete kit for HPLC System Performance Qualification for all kinds of HPLC systems

909-0001

Set 1A : Precision and Linearity (PQ)Refill pack for the LiChroTest PQ kit 909-0001 Dilution series of methyl paraben in methanol/water (50/50) (concentrations 50, 100, 150, 200 mg/l)

909-0002

Set 1: Precision and Linearity (PQ)Refill pack for the old LiChroTest PQ kit 1.15958 Dilution series of methyl paraben in methanol/water (50/50) (concentrations 1, 10, 100, 200 mg/l)

909-0003

Pack Set 2: Precision (PQ)5 Standard samples of 100 mg/l methyl paraben in methanol/water (50/50) Refill pack for the LiChroTest® PQ Kit

909-0004

Set 3: Separation (Parabens) (PQ)5 Standard samples with 3 different parabens + t0-marker in methanol/water (50/50)

909-0005

LiChroTest® standard samples for HPLC system Operational Qualification (OQ)Description Cat. No.Set 5: Autosampler Test (OQ)5 Standard samples with perylene in methanol for checking injection precision (OQ) of LaChrom autosamplers

909-0006

Set 7: Precision (OQ)60 mg/l methyl paraben in methanol

909-0007

Set 8: Linearity (OQ)Dilution series of methyl paraben in methanol (concentrations: 1,5, 7,5, 15, 75, 150 mg/l)

909-0008

Caffeine solution for gradient test (OQ)20 mg/l caffeine in water (0,5 l)

909-0009

Caffeine solution for gradient test (OQ)20 mg/l caffeine in methanol (0,5 l)

909-0011


Regenerated celluloseSuperior performance, great flexibility, great choice

The hydrophilic filter media is based on cellulose acetate, and additional production steps create a highly resistant, pure membrane that

is compatible with a number of solvents, including commonly used solutions such as acetonitrile, methanol, and tetrahydrofuran. RC Membrane can be used in place of hydrophobic Teflon™ membranes for the filtration of organic solvents. It can also replace hydrophilic media, such as cellulose acetate, PVDF, and nylon membranes for the filtration of aqueous solutions. Samples are the main source of particle contamination in HPLC and UHPLC columns. To maximise column lifetime, it is important to ensure that both the sample and the mobile phase being used are degassed and free of particulates. Elimination of solid materials is important, as those particles can interfere with the compound of interest and can very easily clog separation columns. This has an impact on the separation performance (i.e. back pressure, retention time, peak size and peak shape). In the worst case, the column is irreversibly blocked and has to be replaced.As a precaution, the last preparation step before sample injection into an HPLC instrument, is to remove small particles from the sample by filtration. There are several Whatman™ products, featuring the RC membrane, that are suitable for chromatography sample and solvent preparation.Ready-to-use Spartan™ syringe filters are well suited for HPLC sample preparation. Equipped with a hydrophilic Whatman™ RC Membrane, it is both chemically resistant and free of interfering extractables, which ensures reproducible results. The

housing is made of HPLC grade polypropylene to ensure the highest possible performance. Each batch is tested for UV absorbing extractables; this data is documented and available to download.High quality disposable GD/X™ syringe filters, equipped with WhatmanTM RC Membrane, and a glass microfibre pre-filter, allows more samples to be filtered in less time, whether it is an aqueous sample or a sample based on organic solvents. The pre-filter layer allows high particulate samples to be filtered with less hand force, and it reduces blockage and the requirement to replace the filter in the middle of a preparation.Mini-Uniprep™ syringeless sample preparation device, equipped with a WhatmanTM RC Membrane, allows samples for HPLC and UHPLC to be processed 60% faster than traditional syringe filter sample preparations.Volumesofmaximum400μlcanbeeasily processed, allowing laboratory technicians to prepare smaller volume samples, hence reducing solvent usage. The vial shape fits easily into most standard autosamplers and the slit septum cap version is suitable for HPLC instruments with sensitive needles. This allows for high throughput analysis and enhances laboratory productivity. The additional choice of amber coloured vials helps prevent photo degradation of light sensitive samples; the amber vials meet USP specifications for light resistance.Samples removed from a dissolution test apparatus not only contain the dissolved active pharmaceutical ingredient (API), but may also contain particulates of the undissolved dosage form. This can result in a false high rate of release. In addition, these particulates may block HPLC columns or interfere with spectrophotometric determinations.Filtration is an essential step in the dissolution process. The Roby™ syringe filter with integrated Whatman™ RC Membrane helps to avoid such interfering factors and fits automated robot systems from Sotax™, Caliper, Erweka™, and Varian™ for sample preparation during dissolution testing. The housing is made from mechanically stable polypropylene and the geometry meet the requirements for dissolution instruments.In most cases, RC Membrane can be as used as a universal media for both organic and aqueous solutions. It is available in several formats to provide a high degree of flexibility and excellent performance to meet your sample preparation needs

Flexibility is one of the key criteria required in filter media used for chromatography sample and solvent preparation. Whatman™ Regenerated Cellulose (RC) Membrane, from GE Healthcare, combines solvent compatibility, low protein binding, filtration efficiency, purity, and a choice of pore sizes to meet all chromatography sample and solvent preparation needs.



Pall Life Sciences and Sotax delivercost-effective automation certified solutions for automated pharmaceutical testing systems

Pall Acrodisc® PSF syringe filters are used by research and analytical laboratories to help expedite automated sample processing. PSF

Acrodisc® syringe filters provide improved retention efficiency to help extend HPLC column life by as much as 46 times. The multi-layered pre-filter provides two to four times the throughput of standard glass fibre pre-filter devices to allow quick and easy filtration of samples that are difficult to filter. The PSF housing has been designed for smooth operation in automated systems and operating pressures, eliminating filter burst.Pall Life Sciences has announced a long-term joint marketing agreement with the SOTAX Group that will include the Acrodisc® PSF syringe filters in all SOTAX dissolution systems for solid dosage forms. The exclusive agreement is the initial step that allows the companies to begin delivering complete certification solutions that save time and testing costs for quality control laboratories. SOTAX is a Swiss-based provider of high quality dissolution testing systems, composite assay and content uniformity workstations along with physical tablet testing instruments for the pharmaceutical industry. Dissolution testing is used by the pharmaceutical industry to characterise the dissolution properties of an active drug, an active drug’s release and the dissolution from a dosage formulation. SOTAX

systems formulate the drug dosage form and develop quality control specifications for the manufacturing process. “By exclusively recommending Pall Acrodisc® PSF technology as part of our testing systems, we can ensure we are providing the pharmaceutical industry with a significantly improved automated dissolution process for multiple unattended tests. This includes applications for media preparation and delivery, automated dosage introduction, automated sampling, filtering and sample analysis,” said Gilles Devidts, Head of Product Management, SOTAX. At the start of this agreement, all new SOTAX AT 70smart Dissolution systems and SOTAX CTS Content Uniformity test systems will include the Acrodisc® PSF syringe filters. Filter refills will be available exclusively through VWR, Pall Life Sciences exclusive distribution partner“This marketing partnership with SOTAX advances the performance of the automated laboratory analysis platform,” said Larry O’Connell, Senior Vice President, Global Lab Products for Pall Life Sciences. “Customers can now obtain the dual benefits associated with SOTAX and Pall technology to achieve superior analysis and performance.” To learn more about Acrodisc PSF syringe filters, visit www.pall.com/lab or contact your local VWR sales office.

Over thirty years ago, Pall Life Sciences revolutionised sample preparation for analytical chemists with the development of the Acrodisc® syringe filter.

Sample preparation

KC-VTD2010Go to www.vwr.com for the latest news, special offers and details of your local VWR distribution partner.

BDH Prolabo chemicals - The Chemical Brand from VWR International

PESTINORM: High purity solvents for capillary GC analysis The detection of trace organic substances in the environment requires the use of highly purified solvents for all stages of analysis, starting with sample preparation.

The BDH Prolabo PESTINORM capillary GC grade solvents, including a very high purity purge and trap grade methanol have extremely low organic and halogenated derivatives, to prevent contamination.• Evaporation residue less than 5 ppm• Impurities causing interfering peaks on gas chromatogram are not greater than:

Capillary GC grade solvents• 5 ng/l of Lindane by electron capture detection • 10 ng/ml of Octanol by flame ionisation detection Purge and trap grade methanol • 0,5 ppm of 2-Butanone by GC-MS

Certificates of analysis are readily available on the VWR website.

Combining the best quality and price competitive

* Stabilised with 50 ppm of 2-methyl-2-butene

Your European Distribution Partner

AustriaVWR International GmbHGraumanngasse 71150 WienTel.: 01 97 002 0Fax: 01 97 002 600E-mail: [email protected]

BelgiumVWR International bvbaResearchpark Haasrode 2020Geldenaaksebaan 4643001 LeuvenTel.: 016 385 011Fax: 016 385 385E-mail: [email protected]

DenmarkVWR - Bie & BerntsenTransformervej 82730 HerlevTel.: 43 86 87 88Fax: 43 86 87 90E-mail: [email protected]

FinlandVWR International OyValimotie 900380 HelsinkiTel.: 09 80 45 51Fax: 09 80 45 52 00E-mail: [email protected]

FranceVWR International S.A.S.Le Périgares – Bâtiment B201, rue Carnot94126 Fontenay-sous-Bois cedexTel.: 0 825 02 30 30 (0,15 EUR TTC/min)Fax: 0 825 02 30 35 (0,15 EUR TTC/min)E-mail: [email protected]

GermanyVWR International GmbHHilpertstraße 20aD - 64295 DarmstadtTel.: 0180 570 20 00*Fax: 0180 570 22 22*E-Mail: [email protected](*0,14 €/Min. aus d. dt. Festnetz,Mobilfunk max. 0,42 €/Min.)

HungarySpektrum-3D LtdA VWR International CompanySimon László u. 4.4034 DebrecenTel.: (52) 521 131Fax: (52) 470 069E-mail: [email protected]

Ireland / Northern IrelandVWR International Ltd. / VWR International (Northern Ireland) Ltd.Orion Business CampusNorthwest Business ParkBallycoolinDublin 15IrelandTel: 01 88 22 222Fax: 01 88 22 333Email:[email protected]

ItalyVWR International s.r.l.Via Stephenson 9420157 Milano (MI)Tel.: 02 332 03 11Fax: 800 152 999E-mail: [email protected]

The NetherlandsVWR International B.V.Postbus 81981005 AD AmsterdamTel.: 020 4808 400Fax: 020 4808 480E-mail: [email protected]

NorwayVWR International ASHaavard Martinsens vei 30 NO-0978 Oslo Tel.: 0 2290Fax: 815 00 940E-mail: [email protected]

PortugalVWR International - Material de Laboratório, LdaEdifício NeoparkRua Tomás Ribeiro, 43- 3 D2790-221 CarnaxideTel.: 21 3600 770Fax: 21 3600 798/9E-mail: [email protected]

SpainVWR International Eurolab S.L.Ronda Can Fatjó, nº 11 Edifici Tecnopark, III Parc Tecnològic del Vallès08290 Cerdanyola del VallèsBarcelonaTel.: 902 222 897Fax: 902 430 657E-mail: [email protected]

SwedenVWR International ABFagerstagatan 18a163 94 StockholmTel.: 08 621 34 00Fax: 08 621 34 66E-mail: [email protected]

SwitzerlandVWR International AGLerzenstrasse 16/188953 DietikonTel.: 044 745 13 13Fax: 044 745 13 10E-mail: [email protected]

UKVWR International LtdCustomer Service CentreHunter BoulevardMagna ParkLutterworthLeicestershireLE17 4XNTel.: 0800 22 33 44Fax: 01455 55 85 86E-mail: [email protected]

Capillary GC grade Pack size (l) Standard box quantity (2,5 l)

Cat. No.

Acetone 2,5 4 83960.320Dichloromethane (*stab.MB) 2,5 4 83961.320N-Hexane 2,5 4 83962.320Ethyl acetate 2,5 4 83963.320N-Pentane 2,5 4 83964.320Petroleum spirit, boiling 40-60 °C 2,5 4 83965.320Methanol 2,5 4 83966.320Methanol purge and trap grade 2,5 4 83967.320

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