2 - Chrom Journal 2 EN178.250.165.144/ex/downloads/chrom/journal0701/en/... · GC Headspace...

vwr.com

Incubation of the sample forGC Headspace analysis requiresvials with very different criteriato normal sample vials.

pages 16-17

High throughput PreparativeHPLC with the VWR LaPrep®

system and Chromolith Prepcolumns.

pages 20-21

ISSUE 2 - FEBRUARY 2007Products for chromatography

ChromJournal

vwr.com

Your HPLC applicationguaranteed with the newgeneration LaChrom Elite® -Application Inside!

pages 10-11

Time saving software tools from VWR.

pages 12

Fast purification of peptides using Chromolith® SemiPrep.

pages 22-23Find ChromJournal on vwr.com with all our latest news and offers

A - Cover Chrom Journal 2_EN.qxd 24/01/07 22:03 Page 2

Happy New year and welcome to VWR's latest ChromJournal. As usual

it is packed full of information on new products and applications

specifically for chromatographers.

This issue includes:

• Pall's Acrodisc® and Whatman's Mini-UniPrep™ - essential consumables

for effective sample preparation

• The new LaChrom Elite® HPLC system from VWR along with some

powerful software tools to make light work of method development

and validation

• Customer application stories demonstrating the power of the mighty

Chromolith®

• Plus new products for GC users

We hope that you enjoy this magazine - we are delighted to hear your

comments on this issue and your ideas and suggestions for future ones

through our dedicated email [email protected]

Sample preparation Analytical Acrodisc® syringe filters 3

Remove unwanted protein 3 times faster - Mini-UniPrep™ 4-5

Analytical HPLC Fast analysis with a standard HPLC system 6-7

Rapid analysis of low binding peptides 8-9

LaChrom Elite® A new generation 10-11of the reliable HPLC system

VWR special software: A unique toolbox for the analyst 12

Superior Amino Acid Analysis with the VWR-Hitachi L-8900 13

TLC Avoid mistakes with Merck silica gel GLP plates 14

Gas Chromatography The new generation of HAMILTON syringes for CTC Analytics 15

Chromacol Headspace Vials and Seals 16-17

SGE extends lifetimes for forte™ columns 18

BioChromatography Essential technologies for the purification of biomolecules 19

Preparative HPLC Fast preparative chromatography with the VWR 20-21LaPrep system and Chromolith® Prep columns

Fast purification of polar compounds with 22-23Chromolith® SemiPrep RP-18

EditorVWR International Europe bvbaHaasrode Researchpark Zone 3Geldenaaksebaan 4643001 LeuvenBelgium

CopywritingVWR International Europe bvba

Layout and typesettingMarketing Services VWR

PrintingCentral Production, Paris, FranceNo part of this publication may be reproduced or copied without priorpermission by writing of VWR International Europe.

Run45.000 copiesPublication date: February 2007

Chrom Journal

table of contents

editorial

VWR C h r o m J o u r n a l Issue 2 F e b r u a r y 2 0 0 72

2 - Chrom Journal 2_EN.qxd 26/01/07 0:30

What confidence can you have in Pall's HPLC certification?

VWR C h r o m J o u r n a l Issue 2 F e b r u a r y 2 0 0 7 3

Over thirty years ago, Pall Life

Sciences revolutionised sample

preparation for analytical chemists

with the development of the

Acrodisc® syringe filter.

Today Pall produce high-quality

premium syringe filters for sample

preparation and dissolution testing.

Because High Performance Liquid

Chromatography (HPLC) and other

analytical methods are very

sensitive and serve as qualitative

and quantitative techniques,

it is important to preserve the

integrity of the sample.

A filter for HPLC applications is designed toincrease accuracy by removing unwantedparticles. However, the wrong filter can be asource of contaminants in the form ofextractables that elute into the sample fromthe filter device. Extractables can jeopardiseanalytical results causing false quantitationand extraneous peaks.

Extractable material may be a membrane orhousing formulation component, or acomponent introduced during themanufacturing process. The appearance ofextractable materials from a syringe filterdepends on the solubility of device componentsin the sample fluid, and to choose the“cleanest” possible devices for your samplepreparation.

Solubility relates to chemical compatibility.As membrane and/or housing componentsbecome more soluble with the sample,extractable materials will increase. It istherefore important to consider the syringefilter compatibility with the sample fluid.

Membrane manufacturers use proprietaryformulations and different manufacturingtechniques. For this reason, Pall Life Sciences’

membranes, Acrodisc and Acrodisc PSF syringefilters are not the same as other look-a-likeproducts. The specific quality of raw materials,amount of quality control, membrane washingprocedures and post treatments all affect theamount of extractable materials.

Pall Life Sciences specifically selects the highestgrade of materials and performs rigorouscleaning methods on their membrane productsto eliminate the occurrence of undesiredextractables. Additionally, their polypropylenehousing material, also used in Pall Life Sciencesmedical devices, is the highest grade of plasticwith minimal additives, and passes UnitedStates Pharmacopeia (USP) BiologicalReactivity Test, In Vivo <88> plastics testing.

Pall Life Sciences HPLC certification ensuresthat analytical results will not be compromisedby extractable filter materials.

Our membranes have been tested forcompatibility with common HPLC solvents(water, acetonitrile and methanol) usingestablished HPLC procedures. In addition, toverify low levels of UV-detectable extractables,samples of the entire HPLC Acrodisc® syringefilter line are evaluated prior to release.

Analytical Acrodisc® syringe filters – Quality + Performance

Extractables

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

Order the New Analytical Sample PreparationTechnical guide for comprehensive details of Pall’s HPLC certification methods,and results of extractable experiments that demonstrate the quality of the Acrodisc®

and Acrodisc® PSF syringe filter product lines.

For more details of our Analytical SamplePreparation products contact your local VWRcustomer service team or visitwww.pall.com/lab

Order PALL literature piece PN33454.

Sample Preparation

Extractables canjeopardiseanalytical resultscausing falsequantitationand extraneouspeaks.


>


The reproducibility of protein removal fromplasma samples was measured by applying theMUP-PPT procedure to 40 replicates of normalhuman plasma. Filtrate was removed from theMini-UniPrep™ and dried down in a microfuge

tube prior to saline resuspension and micro-BCA protein quantitation. Percentprotein removal was calculated by comparingthe protein concentrations of pre- and post-filtration plasma/ACN mixtures.

Reproducibility of protein removal

Efficient protein removal for different sample types

Remove unwanted protein 3 times faster

The effectiveness of removing protein fromdifferent sample types was evaluated. Plasma,serum and whole blood (diluted 1:1 in H2O)were run through the Mini-UniPrep™ PPTprocedure. The same samples were also runthrough a conventional spin methodologywhich utilizes a 1.5 ml microcentrifuge tube in

place of the Mini-UniPrep™, and a 5 minutecentrifuge @ 10.000 x g to bring the proteinsdown. Supernatant was aspirated into aseparate tube for analysis. Micro-BCA was used to quantify proteins.All results are reported in % reduction inprotein concentration.

Whatman Mini-UniPrep™ (MUP)

Syringeless Filters offer a highly

efficient, simple alternative for

removing unwanted protein prior

to HPLC/MS analysis. Ideal for

performing analytical

characterisation in drug research,

the method utilises acetonitrile

precipitation and filtration by

compression to remove protein

from plasma, serum, whole blood

and other biological fluids. It is a

single tube method that saves time

and eliminates the manual transfer

steps which make spin clarification

problematic.Sample type

Sample Preparation



Enables up to six Mini-UniPrep™ filter devicesto be processed simultaneously. This handy toolwill speed sample prep processing and reducerisk of carpal tunnel syndrome.

The Mini-UniPrep™ compressor


Features & benefits

Ordering information

• Simple, one step, single tube method

Eliminates centrifugation plus aspiration issues and transfer steps.

• Process samples 3 times faster

Purify 6 samples in 3 minutes, 48 samples in <30 minutes

• >99% protein removal from plasma samples

Enables compound quantification and extends column life

• Self-sealing septa allows for repeated injections

Prevents evaporation and maintains sample integrity

• Compatible with all major autosamplers

Cat. No. Pore Size Media Quantity

514-8075 0,45 µm PTFE 100/box516-6356 0,45 µm PTFE 1.000/box516-6351 Compressor 6-position 1



Analytical HPLC

Stable retention times at high flow rates

The flow range of the LaChrom Elite® system up to 10 ml/min is ideal for rapid chromatography withmonolithic columns having internal diameters of 3 or 4,6 mm. To ensure that peak retention timesand areas remain as stable as possible, the system provides very high precision in flow and solventgradients even at high flow rates.

Even faster with flow gradientsThe low back-pressure provided by monolithic columns enables different flow speeds to be usedduring a chromatogram. For example, the flow can be increased at the points where no peaks orwell-resolved peaks appear, or also for purging and re-equilibrating the column. Again, in order toachieve stable retention times it must be possible to control these flow gradients very precisely(see figs. 3+4).

Eluent pre-heatingWith a column that is operated at 1 ml/min and 60 °C in a column thermostat with eluent pre-heating, the linear temperature on the column is still 7,6 °C. This can degrade the separationperformance and the shape of the peak. Therefore, it is particularly important to maintain thetemperature of the eluent at higher flow rates. The pre-heating zone in LaChrom Elite® columnthermostats enables the length of the pre-heated connection capillary to be adjusted to a givenflow speed.

Fast analysis with a standard HPLC system

Monolithic columns with a silica gel base are now standard equipment in

research and routine analysis because of their superb properties as an

excellent alternative to columns with particulate sorbents. One advantage in

particular that is associated with the outstanding permeability of these

columns is the ability to operate them at higher flow rates and still retain

very good separation, thereby shortening analysis times considerably.

For example, Chromolith® columns have an internal diameter of 4,6 mm and

can be operated at flow rates of up to 9 ml/min, Chromolith® columns have

an internal diameter of 3 mm, and can be operated at flow rates up to

4 ml/min, enabling analysis to be completed between 3 and 10 times faster.

Despite these high flow rates, the high permeability of these columns means

that back-pressure is relatively low, so that they can be used in conjunction

with standard HPLC systems.

Even so, the HPLC system must fulfil certain special requirements before it is

possible to take advantage of all the application possibilities offered by using

monolithic columns (see below). The LaChrom Elite® HPLC system by VWR-

Hitachi was deliberately designed with these special functions so that it could

be used with Chromolith® columns.

>Figure 1.Fast HPLC analysis of sweeteners in a soda drinkHPLC system: VWR-Hitachi LaChrom Elite®

Column: Chromolith® Performance 100 mm x 4,6 mmEluent: Buffer / Acetonitrile gradientFlow rate: 4 ml/minWavelength: 220 nmTemperature: 30 °C

New generationLaChrom Elite®




Rapid detector response

Modern HPLC detectors have variable response times, which can be adapted to the respectivechromatogram to optimise results. In this way, most high-frequency noise can be suppressedcompared with the chromatographic peaks by selecting the appropriate detector response time,and this in turn increases detection sensitivity. However, rapidly eluting peaks need the detectorsused to have very short response times in order for the peak shapes to be recorded correctly andwith maximum sensitivity. The LaChrom Elite® detectors can be adjusted for very short responsetimes for fast chromatography.

ConclusionFast chromatography can be carried out extremely successfully using standard HPLC systems inconjunction with monolithic silica gel columns, which permit higher flow rates because of their lowback-presssure. In order to be able to exploit all these advantages, the LaChrom Elite® HPLC systemhas been equipped with the following functions and specifications:• Flow range up to 10 ml/min• High flow and solvent gradient precision even at high flow rates• HIghly precise flow gradients• Eluent temperature control• Very short detector response times.

Figure 3.Fast chromatography with an extremelyprecisely controlled combined solvent andflow rate gradient, 10 cycles superimposedSample: 7 phenolsHPLC system: VWR-Hitachi LaChrom Elite®

Column: Chromolith® Performance 100 mm x 4,6 mmEluent: Water + 0,1% phosphoric acid /Acetonitrile gradientFlow rate: 4 ml/min, programme, see figure 4.Detection: L-2450 DAD detectorTemperature: 40 °C

Figure 2.Rapid HPLC separation of 8 sulphonamideswith a 3 mm Chromolith® column followingautomatic separation optimisation withChromSword® Auto SoftwareHPLC system: VWR-Hitachi LaChrom Elite®

Column: Chromolith® Performance RP-18e 100-3 mm

Eluent: Water / Acetonitrile (+ 0,1%TFA) gradientFlow rate: 2 ml/minDetection: L-2455 DAD detectorTemperature: 45 °C.

Figure 4. Programming of the LaChrom Elite® pumpprogram with the EZChrom Elite®

chromatography data system.

Wolf-Dieter Beinert and Hans-Peter ScholzVWR International GmbHScientific InstrumentsHilpertstr. 20AD-64295 [email protected]



Application from the Advanced Biotechnology Centre at Imperial College London

Ian MOSS - Peptide Synthesis Manager, ABC

The Advanced Biotechnology Centre (ABC) at Imperial College London is

a core facility providing peptide synthesis, DNA sequencing and GeneScan

services to the College and researchers world-wide.

The peptide synthesis service utilises reversephase HPLC as part of its quality control andpurification of peptide products. Thethroughput requirements of the service and thevariable physico-chemical properties ofpeptides put demands on column performance.Two important factors for our service withrespect to reverse phase columns are: 1) abilityto operate at high flow rates in order tominimise analysis time without loss ofperformance and 2) ability to bind and elutepeptides over a wide range of size andhydrophobicity.

The specification of Chromolith® reverse phasecolumns suggested that they would be suitablefor our requirements. We were particularlyinterested in achieving an analysis time of less than five minutes. A Chromolith® RP C18 50 mm x 4,6 mm column was investigated as follows.

In all cases solvent A was 0,1% TFA in water,solvent B 90% acetonitrile, 10% water,0,085 % TFA. Analyses were performed on aPerseptive Biosystems BioCad Sprintinstrument.

A mixture of two unrelated peptides A and B(OVA 323-339 and mMOG35-55 respectively) was analysed at four different flow rates usingconstant volumes of eluant. The analyticalconditions were:• Equilibration 94,5% A, 5,5% B, 4 ml• Sample size 5 µl approx 1 mg/ml• Sample loading 94,5% A, 5,5% B, 4 ml• Gradient 94,5% A, 5,5% B to 100% B

in 16 ml.• Clean column 100% B, 4 ml• Flow rates: 1,6 ml/min, 4 ml/min, 8 ml/min,

16 ml/min.• UV detection 220 nm.

Figures 1 to 4 display the analytical traces.These show that the analytical profiles did notsignificantly differ at the different flow rates;resolution and peak shape were bothmaintained. At the highest flow rate themaximum back pressure was recorded at 1900 psi (131 bar), well within the operatinglimit of the column (2,500 psi / 172 bar).

The overall analysis time at a flow rate of 1,6 mlper minute (approximately equivalent to 2 columnvolumes (CV) per minute) was 17,5 minutes.At 16 ml per minute (about 20 CV/min) theoverall analysis time was 1,75 minutes, a savingof 15,75 minutes per sample. For routineapplications a flow rate of 8 ml/min (about 10 CV/min) was adopted giving an overallanalysis time of 3,5 min and a maximumcolumn pressure of about 1000 psi (69 bar).

Analytical HPLC Application

Analysis of peptide mixture A + B at different flow rates.

Experiment 1:

To investigate the effects of varying flow rate

Fig 4: 16ml/min

Fig 3: 8ml/min

Fig 2: 4,0ml/min

Fig 1: 1,6ml/min

The ABC team, from left to right; Rachel Emerson, Wanda Stowe, Alex Montoya and Ian Moss


In summary, we found that theChromolith® column enabled us toperform rapid analyses of underfour minutes total time.With little modification of thecolumn conditions we were able to analyse a peptide with lowbinding characteristics. Differenteluting conditions can be rapidlyexplored because of the short runtimes facilitated by Chromolith®.We anticipate translating theseproperties of Chromolith®

to semi-preparative scale.



Analysis of peptide C under different equilibration and loading conditions.

Fig 8: Conditions (d)

Fig 6: Conditions (b)

Fig 7: Conditions (c)

Fig 5: Conditions (a)

Some small or hydrophilic peptides will fail tobind to a reverse phase column under standardequilibration conditions. Generally aconcentration of 5% acetonitrile is consideredoptimal to equilibrate a column prior to sampleloading. In this experiment we investigatedthe ability of the Chromolith® column to bind apeptide after equilibration at 1% acetonitrile.

Initially we attempted to analyse peptide C (anunpurified synthetic product) under theconditions (a) used in Experiment 1 with a flowrate of 8 ml/min. The resulting profile is shownin Fig 5. This shows the peptide eluting withina few seconds indicating poor binding of thepeptide to the column. The analysis wasrepeated under the following modifiedconditions:

(b) The concentration of acetonitrilewas reduced to 1% for theequilibration phase, see Fig 6.

(c) As (b) but with 1% acetonitrilefor the sample loading phase, seeFig 7.

(d) As (c) but with the equilibrationvolume increased to 8 ml, see Fig 8.

Under conditions (b) and (c) noimprovement in binding of thepeptide to the column wasobserved. Under conditions (d),when the equilibration volume wasdoubled, a normal analytical profilewas obtained showingpredominantly a single peptide andtraces of by-products as would beexpected with an unpurified sample.

Experiment 2:

To investigate binding of peptide C (Ac-ASQVRPSQR-nh2).



New generation LaChrom Elite®

The reliable HPLC system for highest demands andlowest running costs

The new generation LaChrom Elite® system from VWR-Hitachi is a robust,

versatile, long-lasting, high-performing HPLC instrument with excellent

precision and sensitivity as well as minimum sample carry-over. Designed

for easy maintenance and service all operational parts are accessible from

the front. Together with the comprehensive and FDA compliant

chromatography data system EZChrom Elite®, it offers a very high degree

of versatility in data processing and chromatography system control.

Unique detector sensitivityThe new generation LaChrom Elite® system extends the LaChrom Elite® detector family by two new instruments offering unique sensitivity. The new L-2455 diode array detector with an advancedreference wavelength function has extremely high sensitivity specifications, comparable to those of a top quality UV detector. It can also be used as a multi-wavelength detector. The new L-2485fluorescence detector shows superb sensitivity, which allows analyses at the lowest trace levels.

From Micro to Standard HPLCLaChrom Elite® provides optimal performance in all flow rate ranges from 50 µl/min up to 10ml/min applied with 1, 2, 3, 4 and 4,6 mm i.d. columns as well as for high-speed chromatographyusing Chromolith® columns.

Fast chromatographyThe flow rate range 1-10 ml/min is ideal for fast chromatography using 3 or 4.6 mm i.d.Chromolith® columns. Using Chromolith® columns the speed of analysis can be increased by afactor of 3-10 compared to chromatography using conventional particulate columns, saving timeand money in routine analysis. Their low back-pressure means less chance of leaks and longlifetime of seals throughout the whole system, thus increasing up-time and reliability whilereducing cost of analysis.

LaChrom Elite®

>>new

Analytical HPLC




Fast automated method development

LaChrom Elite® combined with the powerful ChromSword® Auto software performs completelyautomatic, unattended HPLC method development and optimisation within hours. The optimisationtargets is highest resolution with minimum analysis time so even existing methods can be optimisedand analysis time further reduced. In this way substantial time and cost savings can be achieved.

Superior column temperature control for reliable resultsIn addition to the well proven L-2300 oven, the new generation LaChrom Elite® system offers thenew high performance column thermostat L-2350 with excellent temperature homogeneity and anextended temperature range. It accommodates up to 8 columns. The increased temperature rangeup to 85 °C adds more versatility for high temperature applications. It can also be equipped withup to two valves for column switching, sample preparation by pre-column switching and otherapplications.

LC-MSThe LaChrom Elite® system can be easily combined with the esquire™ series ion trap MS ormicrOTOF™ series MS systems of Bruker Daltonics, which offer exciting possibilities in massdetection, structural elucidation and substance identification in research and routine analysis.

Operational Qualification (OQ) automatedLaChrom Elite® with Auto Validation software performs completely automated OQ of the systemand provides a comprehensive GMP compliant qualification report saving a lot of qualificationwork in the laboratory.

>

Special offer:LaChrom Elite® -Application inside!We guaranteeyour HPLCapplication. Speak to us andsee what we cando for you.

LaChrom Elite® -Robust Hitachitechnology forcutting down lab costs

Separation of a all impurities of a pharmaceutical sample with a multi-stepgradient, automatically optimised using the LaChrom Elite System®

and ChromSword Auto®.



Software

Special software from VWR - A unique toolbox for the analyst

ChromSword® Auto Version 3.5The unique ChromSword® Auto software performs completely automatic unattended HPLC methoddevelopment and optimisation within hours. Even existing methods can be optimised and analysistime considerably reduced. After some initial inputs ChromSword® Auto predicts methodconditions and controls the connected VWR-Hitachi LaChrom Elite®, Agilent or Waters HPLC systemto acquire the necessary chromatograms that are then analysed by the software to find the finaloptimum conditions. The rapid optimisation mode gives an overview of the chromatographicperformance of the column/solvent system selected while the detailed optimisation mode acquiresand analyses more data to predict and execute several alternative final optimum conditions. Bothmodes can be combined with column and solvent switching procedures in order to try a variety ofcolumn/solvent combinations automatically. Both in method development and routine analysisChromSword® Auto can achieve substantial time savings as well as improvements in methodperformance.

AutoRobustFor newly developed or existing methods robustness studies should be carried out in order to assess the tolerance limits for small deviations from method parameters. These tolerance limitsshould be described in the method SOP. AutoRobust software allows easy programming of therequired experimental matrix and is able to carry out robustness studies for HPLC methodsautomatically. AutoRobust software controls the VWR-Hitachi LaChrom Elite®, Agilent or WatersHPLC systems and acquires the chromatograms with the desired small variations in HPLC conditions.The chromatograms can easily be viewed and a robustness test report is generated automatically.Thus method robustness studies can be completed in a few hours without user intervention.

Validation ManagerValidation Manager checks whether your analytical method is suitable for the intended use andautomatically produces the validation report you need. Only a few questions must be answered ina wizard dialogue, then the validation plan is created with a push of a button. You are then shownimmediately how many samples have to be prepared and how many injections must be performedto calculate the method characteristics. Even the autosampler injection tables for EZChrom Elite®

are created automatically and are ready for data acquisition. From the data the required methodcharacteristics are automatically calculated and statistically checked according to reliable andacknowledged procedures. After entering your comments and decisions, the method validationreport is created with only a mouse click. Your virtual Validation Manager can save you days oreven weeks of validation work for any analytical technique.

VWR offers a variety of special software tools which can save you days or even weeks of work.

UncertaintyManagerIn order to be able to evaluate analytical results properly, one has to know the degree ofuncertainty involved. Therefore, measurement uncertainty of the analytical results is a parametermore and more required, especially from accredited laboratories. UncertaintyManager® is a newsoftware tool for the rapid determination of the measurement uncertainty of analytical resultsaccording to international standards Eurachem / CITAC Guide "Quantifying Uncertainty inAnalytical Measurement" and "ISO Guide to the Expression of Uncertainty in Measurement"(GUM). The software, based on a substantial database, comprises numerous functions thatfacilitate data entry, enable critical parameters to be visualised, calculate the measurementuncertainty and to compile a suitable report. Using UncertaintyManager® software allows tocalculate measurement uncertainty in very short time, taking into account the complete analyticalprocedure.

HPLC analytique


For more information or a demonstration please contact your localVWR Instrument SalesSpecialists or [email protected]


For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comAnalysis

VWR-Hitachi L-8900 Amino Acid Analyser

The new flexible automatic Amino Acid Analyzer L-8900 combinesthe advantages of the classical ion exchange separation methodwith modern HPLC techniques. The complete AAA solution fromVWR offers sophisticated instrumentation, a wide variety of pre-packed columns, a well developed gradient method combinedwith optimised ready-to-use buffers, creating a solution for anyanalytical lab which is dealing with amino acid analysis. The systemis fully controlled by the proven and FDA compliant EZChrom Elite®

Chromatography data system.

The unique features are:• Superior sensitivity of 3 pmol ( Asp )• Extremely good repeatability of peak areas and retention times.

- Fastest separation for protein hydrolysates and physiological fluids.- Lowest analysis costs.

>Autosampler Column Oven

Detection Cell

Reactor

Ninhydrin

Reagent and washing solution

containers

N2 gas supply

Pumps 1/2

Degasser

Waste

• Superior resolution

• Shortest

analysis time

• Lowest analysis costs

• FDA compliant



Merck GLP plates

From 2004, the English edition of the Europeanpharmacopoeia requires the documentation ofthin-layer chromatograms by photograph or animage datafile. Many laboratories follow goodlaboratory practice and have the requirementfor documented results to be free of errors and/ or falsifications. For this purpose the usage oflaser coded Thin Layer Chromatography platesis absolutely necessary. Merck GLP glassbacked plates are based on the proven Mercksilica available as TLC and HPTLC plates andprovide the same unsurpassed separationperformance as the corresponding TLC andHPTLC plates.The laser code consists of the name of themanufacturer, the item number, the batchnumber and a serial number of the plate.With this unique feature each and every plateis fully traceable.

Avoid mistakes and fakes in Thin Layer Chromatography by using Merck silica gel GLP plates

Sorbent Format (cm) Content (plates) Cat.No.

TLC GLP silica gel 60 F254 20 x 20 25 1.05566.000110 x 20 25 1.05702.0001

HPTLC GLP silica gel 60 20 x 10 25 1.13326.0001HPTLC GLP silica gel 60 F254 20 x 10 25 1.05613.0001

10 x 10 25 1.05564.0001

Figure 1: Birch leaf dry extract, exposuredsamples, HPTLC-silica gel 60 F254, 20 x 10cm, photograph taken under UV-light 254nm, black and white

Figure 4: Identification of real Saffronsamples, which have been bought inEgypt, Turkey, Spain and Iran, HPTLC-silicagel 60 F254, 10 x 10 cm, photographtaken under white light.

Figure 3: Dry extracts of flavonoid-containing phytopharmaceuticals,HPTLC-silica gel 60 F254, 20 x 10 cm, photograph taken underUV-light 365 nm. A narrow strip ofwhite paper has been placedunderneath the code

Figure 2: Active pharmaceuticalingredient, exposured samples, TLC-silicagel 60 F254, 20 x 20 cm, photographtaken under UV-light 254 nm, black andwhite

Figures of the plates no. 11 and 12 are takenfrom Elke Hahn-Deinstrop`s TLC-book. The 2nd, revised and extended edition was published in autumn 2006 (ISBN 3-527-31553-5). For more information send an email [email protected]

TLC




Hamilton Hamilton Syringe CTC P/N Cat. No.P/N description

203185 7701.2 CTC SyrC L1.2-26P-AS 549-1307203189 75 N CTC (26S/AS) SyrC L5-26S-AS 549-1311203072 701 N CTC (26S/2) SyrC L10-26S-2 549-1310203205 701 N CTC (26S/AS) SyrC L10-26S-AS 549-1312203361 701 N CTC (23S/AS) SyrC L10-23-AS 549-1309203362 701 N CTC (23S/26S/AS) SyrC L10-23S/26S-AS 549-0525203043 1702 N CTC (26S/AS) SyrC G25-26S-AS 549-1313203076 1710 N CTC (26S/AS) SyrC G100-26S-AS 549-1314203078 1725 N CTC (26/AS) SyrC G250-26-AS 549-1315

>

Gas Chromatography

Together with HamiltonBonaduz (Switzerland) CTCAnalytics has developed a newMicroliter syringe. The so-calledC- Line is the next generation of the CTC Microliter syringe.The new C - Line has thefollowing main features:

• Zero carry over

• New Teflon/polymer mixtureas sealing material- Better temperature stability- Surface is less porous

• Flange with colour codeindicating the needle gaugeand internal diameter- gauge 22 blue

• Adjustable plunger protectsplunger tip from squeezing

• New Teflon/polymer mixtureas sealing material for theplunger tip

• Needle attached to barrel.No sample contact with glueor cement.

The new generation of HAMILTON syringes for CTC ANALYTICS

Hamilton Hamilton Syringe CTC P/N Cat. No.P/N description

203245 1702 Plunger CTC 10pcs/pack Plg G25 549-0526203246 1710 Plunger CTC 10pcs/pack Plg G100 549-0527203249 1725 Plunger CTC 10pcs/pack Plg G250 549-0528

Syringes for GC PAL (C- Line)

Replacement plungers for GC PAL (C- Line)



Chromacol headspace vials and seals for GC instruments

The increasing need for

monitoring of the environment for

the presence of pollutants and

other organic material has made

headspace GC analysis an

important tool in the

environmental and analytical

laboratories. The introduction of

dedicated instruments for

particular applications in the food,

fragrance and security industries

has also used this GC headspace

sampling technology. One of the

important factors in the success of

the technique is the correct and

convenient treatment of the

sample before and during the

generation of the headspace

sample.

The solid or liquid sample has first to release the volatile components into the headspace withinthe sealed container. This has to be brought to a state of equilibrium so that the analysis of theheadspace is representative of the sample as a whole. The equilibrium is usually established byincubating the sample in a heated oven chamber at a temperature capable of releasing the volatilecomponents from their sample matrix.

This means that vials used for headspace analysis have to meet very different criteria comparedwith more conventional sample vials.

Requirements for GC headspace vials and seals• Large volume and wide opening to allow sufficient sample matrix to be added.• Strong construction allows pressure to build up within the vial without cracking or strain lines.• Glass is neutral borosilicate to USP class 1 for durability, heat resistance and low extraction.• Seals with a variety of compositions to cope with temperatures from sub-zero to 250 °C• Caps to be able to give robust sealing during incubation.• Compatibility with the transport mechanism required transporting the sealed vials to the

equilibration ovens and sampling ports.

Vials and sealsGas Chromatography


Chromacol headspace vials features:

• Rounded bases and shoulders for high-pressure strength.• The 20 mm crimp finish has a beveled top for optimal sealing with crimp caps.• The vials come in a range of volumes and compatibility with a wide range of instrument types.• Amber 10 ml and 20 ml vials with the same features are available for use with more light

sensitive samples.• The vials are compatible with magnetic caps to allow use with the CTC CombiPAL series of

autosamplers.

Chromacol sealsThe seals used with the headspace systems are also a key part in the assurance of results.Manufactured from high quality elastomers with fluoropolymer surfaces to minimise extraction andinterference during equilibration these seals may be selected from a range of formulations givingdifferent seal permeability, temperature ranges and hardness. The standard thickness of 3 mm isrecommended for most headspace instruments and applications. For convenience and to reducesources of contamination many seals are pre-assembled. The use of the 20 mm crimp closureallows the use of standard aluminium crimp caps which give secure closures.

Chromacol high temperature headspace seals Seals for high temperature applications have a special formulation that removes possibleinterfering components from the elastomer. These may be used at equilibration temperatures of220 ºC.

Magnetic crimp capsThe increasing popularity of the CTC Analytics platform in the form of the CombiPAL gives arequirement for a magnetic closure. Chromacol in addition to standard magnetic caps supplycomposite aluminium/steel caps that are more easily crimped and have been extensively evaluatedon these systems.

New screw top headspace vialsWhen using headspace analysis the normal means of closure has been the use of a 20 mm crimp.This gives a permanent and tamper proof closure but it does mean that the addition of material tothe sample vial after crimping has required de-capping and re-crimping. With the advent ofsample preparation techniques such as SBSE (Stir Bar Sorbent Extraction) and the use of suchproducts in the electronic nose instruments from a range of companies such as Alpha MOS andCyrano Systems, the application of a screw top closure becomes more necessary.

Magnetic capsThe screw caps are nickel plated metal with magnetic properties making them suitable for the vialtransport used in the popular range of CTC autosamplers.

New screw top headspace vials



>

The new Chromacol screw topheadspace vials and theirassociated caps are designedfor use in both headspace andsample extraction processes.They are truly universal as theyhave caps that will support theuse of thick headspace sealswhile also using thinner sealswhere more delicate SPMEfibres may be used.



Columns

The technology behind SilGuard™ will benefityour analysis by reducing the amount of non-volatile material that accumulates on thecolumn. As a result, forte™ analytical columnsoffer further enhanced endurance with no lossto sensitivity.

SilGuard™ also affords additional analyticalflexibility to the advanced user. For example, itallows different injection techniques to be usedwith less setup, and it can be used as a postcolumn restrictor or transfer line.

A revolutionary new integrated guard column system

What makes SilGuard™ special?

SGE extends lifetimes for forte™ columns

SGE's unique SilGuard™ integrated guardcolumn technology is now included as standardwith the most popular BPX5 columns, toprotect the analytical column fromcontamination from non-volatile materials.

In addition, the design of SilGuard™ allowsthe guard and analytical column to havedifferent ID, enabling a wide range of

applications not possible with other integratedguard columns. Additionally the new SilGuard™technology is also now available on any SGEpolyimide clad column. All you need to do isspecify that you would like your column to befitted with a 5 metre SilGuard™ as a specialwhen you place your order.

SGE have always believed in

providing maximum performance

for all chromatographers. This is

seen to optimum effect in the

forte™ range of capillary GC

columns. Manufactured to provide

high performance with maximum

endurance, the forte™ columns

will help you meet your analytical

objectives with confidence -

time after time.

>SilGuard™ is a unique integrated guardcolumn system, developed using SGE'srenowned SilTite™ ferrule technology.SilGuard™ provides significant benefitscompared to other integrated guard columns for the use of conventional unions:• Chromatographic performance — the

union is specially designed for low thermalmass to retain excellent peak shape, evenwhen using fast temperature programming

• Perfect connections — SGE factory-fits theSilGuard™ to ensure a perfect, void-freeconnection, resulting in no impact on peak shapes

• Flexibility — Your analytical column andguard column can have different internaldiameters, ideal for on-column injectionusing a wide-bore guard column.

• Ease of use — The guard column is factoryfitted to ensure a permanent leak-freeconnection, with no nuts to re-tighten,and no need to use polyimide sealing resin

• High visibility — the junction of theanalytical and guard column is clearlymarked by the SilGuard™ union, so thatthere can be no confusion as to what lengthof guard column is left on the column

Forte TM analyticalcolumnsoffer furtherenhancedendurance with no lossof sensitivity.



>


Bio Chromatography

Essential technologies for the purification of biomolecules

Whether your application

challenges are in research, scale-up

or polishing, Pall offers an

extensive portfolio of media for

affinity, ion exchange, size

exclusion, hydrophobic interaction

and hydroxyapitite

chromatography.

The Pall portfolio includes both

sorbent- and membrane-based

chromatography tools. Pall’s

portfolio features an array of

products covering the most

common and some very unique

modes of chromatography.

Pall chromatography media can be used forpurification of biomolecules (nucleic acids,proteins) and compounds on the research andprocess scale. Pall BioSepra® chromatographysorbents are useful in proteomic samplepreparation, laboratory bioprocessing andscale-up work.

Unique sorbent design for high performanceresults

When performing chromatography-basedpurification, the choice of sorbent is crucial forensuring effective capture (bindingselectivity/capacity) and sufficient separation(resolution). Pall offers a variety of basematerials ranging from soft beads (Trisacryl®,Ultrogel®, Hypercel™ ) to rigid beads, to aunique hybrid bead (HyperD® and HyperZ®).Pall’s BioSepra® HyperD and HyperZchromatography sorbents are created using thepatented “gel-in-a-shell” technology. This gel-in-a-shell bead is constructed of a high-capacity hydrogel polymerized within thegigapores of a rigid ceramic bead. The capturechemistry or ligand is attached to the hydrogelwithin the bead pores. The advantages of thisstructure are many, including higher than usualcapacity, tolerance to fast chromatography runswithout a concomitant increase in backpressure, and greater salt tolerance for ionexchangers.

Gel-in-a-Shell Design

Pall offers the BioSepra® line ofchromatography sorbents bottled in bulk (1 to100 ml) quantities. Pall also offerschromatography-based kits for abundantprotein removal. These ready-to-use kitsinclude all components necessary for depletionor selection of albumin and/or IgG frommultiple species.

The Pall BioSepra® chromatography sorbentscan be used in traditional chromatographycolumns or, for quick separations, can be usedin centrifugal spin columns (e.g. Pall Nanosep®

devices). For high throughput separations orscouting assays, the sorbents can be combinedwith the AcroPrep™ filter plates with up to 96mini-columns that can be processedsimultaneously.

For further information on thisproduct visit www.vwr.com orcontact your VWR representative.



Fast preparative chromatography with VWR LaPPrreepp system and Chromolith® Prep RP-18e columns

The VWR LaPrep system together with MerckChromolith® columns covers the wholeapplication range from analytical topreparative HPLC in the laboratory.LaPrep stands not only for modules, but alsofor many additional components, designed tocreate a complete separation system. Thus, avariety of individual solutions are possible. Thesystem is completed by the special EZPrepSoftware which is based on the EZChromElite® platform.

The Chromolith® prep columns with the specialbimodal pore structure increase speed,efficiency and productivity of the separation.With this monolithic silica rod technology backpressure problems are a thing of the past.

Preparative HPLC

The main features of theLaPPrreepp System are:

• Specially adapted software for preparativeHPLC. All functions important for prepchromatography such as peak recognition,fraction collection, sample recycling andsolvent recovery are available. Directcontrol during the run as well as a teachfunction (i.e. online updating of methods) is included.

• Pumps with exchangeable pump headsallow flow rates from 0.1 up to 1000ml/min, whilst guaranteeing highestreproducibility.

• A wide bandwidth of different detectorse.g. a filter photometer, a variablewavelength photometer, a photodiodedetector as well as a refractive indexdetector is available. All LaPrep detectorsare time programmable and can bedelivered with an optical fibre connection to the detector flow cell, if required.

• A broad range of different valves forinjection, column switching andfractionation is available.

Analytical Conditions:Column: Chromolith® Performance RP-18e 100-4,6Mobile Phase: 93% Methanol / 7% Water (v/v)Flow Rate: 3 ml/minWavelength: 280 nmSample: 4 mg natural - Tocopherol Rixen e oil/ ml Methanol

with 1% iso-Propanol dissolution mediator Injection: 100 µl

Typical analyticalchromatogram

Purification of natural Tocopherol oil with the aim topurify ��-Tocopherol

Analytical chromatography using the LaPrep system at 3 ml/min

LaPrep system




Preparative Chromatography

Analytical Conditions:Column: Chromolith® Prep RP-18e 100-25Mobile Phase: 93% Methanol/ 7% Water (v/v)Flow Rate: 60 ml/minWavelength: 280 nmSample.: 10 mg natural – Tocopherol Rixen e oil / ml Methanol with 1% iso-Propanol

dissolution mediatorInjection: 2 ml each 3,2 min - i.e. 3,2 min apart

Multi – injection (5x) of natural - TocopherolRixen e oil during a 20 minutes run. The benefit is the high productivity: - more than 300 mg product per hour

Conclusions:The technique of multi-injection during a run shows the high productivity for purification of theproduct. More than 300 mg of product per hour is obtained. This allows a higher throughput ofsample and decreased solvent consumption, compared to the use of a conventional particularpreparative column.

Inj.1 Inj.2 Inj.3 Inj.4 Inj.5 Inj.6



Report on the purification of samples with Chromolith®

SemiPrep 100-10 mm RP-18 Pascal VERDIE, Yann BROUILLETTE, Jean MARTINEZ

Within the LAPP*, research

activities are at the interface of

chemistry and biology. Chemists,

biologists, immunologists and

clinicians work in close cooperation

to design and obtain biologically

active compounds. One of our

activities concerns the synthesis,

purification and analysis by mass

spectrometry of neuropeptides and

heterocycles combinatorial

libraries.

*Laboratoire des Aminoacides, Peptides et Protéines,UMR 5810 - Faculté de Pharmacie, Université deMontpellier I et II, France.

Part of our work consists of the high-throughput purification of combinatorial libraries. With thisintention, we use an automated system of preparative liquid chromatography-mass spectrometry(prep LC/MS). This technique is recognised as an efficient and effective means for meeting thehigh-throughput purification needs of parallel synthesis programs. The fractionation of the samplesis directed by mass detection. The use of the Chromolith® SemiPrep 100-10 mm RP-18 columnenables us to carry out fast (gradient of 10 min) and effective purifications on peptides andheterocyclic compounds. Recently, we had to purify a family of polar compounds, benzodiazepinesbearing an imidazol subunit. We tested the effectiveness of several columns for the separation ofa representative example of the library.

The crude mixture to purify contained 5 compounds (noted from 1 to 5): the product of interest ofmass [M+H ]+ m/z = 263,4, a second product of the same mass and three other side-reactionsproducts. If the use of standard particulate C-18 silica columns does not allow the separation ofpolar compounds, the use of the “monolith” column enabled us to purify the sample.

Sample purification

Analytical LC conditionsColumn: Chromolith® SpeedRod RP-18 50-4,6 mm Mobile phase: A: H2O 0,1%TFA - B: AcN 0,1%TFA Gradient: from 0 to 100% of B in 5 min Flow: 3 ml/min

Preparative LC conditionsColumn: Chromolith® SemiPrep 100-10 mm Mobile phase: A: H2O 0,1%TFA - B: AcN 0,1%TFA Gradient: 0% of B for 4 min then 0 to 5% in 5 min Flow: 20 ml/min Sample: 80 mg of mixture solubilized in A

A - Analytical chromatogram of the mixture to be purified.

B - Analytical chromatogram of the pure product obtained after purification.

A

B

1

23

4

5

Preparative HPLC



Conclusion

The use of the Chromolith® SemiPrep100-10 mm RP-18 column makes itpossible to carry out fast andeffective purifications even on polarcompounds difficult to purify onstandard particulate C-18 columns.


C - SIR chromatogram of the five products contained in the mixture. In green is represented the collected fraction of the desired product.

D - SIR chromatogram of products 3 and 5 (same molecular weight m/z = 263,4)obtained on the Chromolith column and a standard silica column. In the second case,the two products mainly coeluted and no pure fractions were recovered.

1

2

3 5

3

3

5

5

D

C4


Go to http://www.vwr.com for the latest news, special offers and full specifications of our complete product range

Your European Distribution PartnerAustriaVWR International GmbHGraumanngasse 71150 WienTel.: 01 97 002 0Fax: 01 97 002 600E-mail: [email protected]

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NorwayVWR International ASKakkelovnskroken 1P.B. 45, Kalbakken0901 OsloTel.: 02290Fax: 0 22 90 00 40E-mail: [email protected]

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

> For more information on these products, contact your local VWR sales office or send an e-mail to [email protected]

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