Chrom Journal 3 IR:ChromMagazine EUR.qxduk.vwr-cmd.com/ex/downloads/magazine/chromjournal... · The...

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Chromolith® demonstratesspeed and accuracy for the Swedish University of Agricultural Sciences

pages 4 - 5

Hydrophilic InteractionChromatography for theanalysis of extremely polaranalytes

pages 10 - 11

ISSUE 3 - SEPTEMBER 2007Innovative products for chromatography

ChromJournal

vwr.com

VWR Prolabo solvents areguaranteed to performeverytime

page 13

A reusable closed systemfor HPLC solvents

page 13

NEW AcroSep™

Chromatography Columnsfor Ion Exchange from PallLife Sciences

pages 14 - 15

Get all the up to date news and special offerswith the VWR e-newsletter Register on vwr.com. Find ChromJournal too!

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EditorVWR International Europe bvbaHaasrode Researchpark Zone 3Geldenaaksebaan 4643001 LeuvenBelgium

CopywritingVWR International Europe bvba

Layout and typesettingMarketing Services VWR

PrintingPRT, Paris, FranceNo part of this publication may be reproduced or copied withoutprior permission by writing of VWR International Europe.

Run45.000 copiesPublication date: September 2007

In this issue

Welcome to the Autumn edition of the VWR ChromJournal.

After what we hope has been an excellent summer for everyone, as the

days get shorter and the weather a bit colder and wetter the ChromJournal

aims to brighten your day by bringing you news and application stories

from all types of chromatography. If you’d like to contribute to the journal

or have any comments or suggestions about topics you would like included

in future issues then please email us at [email protected]

SGE, Forte columns for abusive GC! - NEW 03

Rapid separation of berry anthrocyanins with Chromolith® columns 04

Chromolith® HPLC columns convert standard HPLC systems into racehorses 06

Purospher STAR® HPLC columns - for successful separations 08

YMC HILIC columns for extremely polar analytes 10

Hamilton PRP-h1 HPLC columns with extra sensitivity 12

Solvents purity, packaging and price - the choices 13

Pall AcroSep™ Chromatography Columns for Ion Exchange - NEW 14

VWR collection storage vials - NEW 16

Schott Duran® HPLC Laboratory bottle 16

VWR C h r o m J o u r n a l Issue 3 S e p t e m b e r 2 0 0 72

ChromJournal editorial

Don't miss out!Enclosed with this ChromJournal

is a very special offer flyer that is

packed full of offers for all

chromatographers. If your copy

is missing get in touch to make

sure that you benefit from our

special actions including free

gifts and money off.

Chromatography Consumables are the focus for Autumn at AGB. With 3

new exciting offers available; Receive an additional 10% discount plus a

free gift with any purchase from our extensive range of HPLC certified

syringe and membrane filters. And a bonus … a free Technical guide on the

use of Acrodisc Syringe Filters for analytical sample preparation comes with

each order.

Or what about a free pack of screw vials and lab timer with all €1000

orders from our wide range of HPLC columns including Hi-Chrom, YMC and

Macherey Nagel.

Plus fantastic discounts available on VWR Collection Vials. With an extra

15% discount off our range of vials for GC and HPLC, micro inserts,

closures, kits and tools!! With a wide opening, this allows for easy filling.

So contact AGB and place your call-off orders to get an additional Autumn

discount!

Until recently, subjecting a GCcolumn to crude plant extractswould be a sure way to shortenthe longevity of the column as theextracts are notorious fordamaging column phase.However, to test the robust phasechemistries of the new Forterange of GC columns we haveactively subjected the columns tothe “Green Tea Test”.

In the green tea experiment, a standard testmix was analysed to produce a controlchromatogram. Then the column was subjectedto 100 injections of green tea followed by arepeat analysis of the test mixture. Theresulting before and after chromatogramsshow an 'as new' performance, demonstratingthe lack of activity and inert nature of thehighly robust column phase (Figure 1).

Pushing the concept further, the green tea testwas carried out on more polar phases such asthe BPX50. Traditionally, polar phases are lessrobust than non-polar phases. Despite this, theBPX50 handled the green tea test and evenafter 200 injections of green tea there waslittle difference between before and afterchromatograms. The analysis of organochlorinepesticides shows that the highly robust phaseremained inert, as evident by the lack of on-column decomposition of any of thepesticides (Figure 2).

Similar robustness can be seen with the BPX35,which provided excellent separation of PAHs after 100 injections of the dreaded green tea (Figure 3).

Extreme column abuse like the green tea testmight seem drastic but, in a busy laboratory,time restraints and cost concerns can oftenlead to compromises in sample clean-up.Conventional wisdom would have it thatinadequate sample preparation is a trade offwith reduced column life, ultimately leading tomore frequent column replacement andinstrument downtime. However, even if yoursamples are not as dirty as green tea, the newForte range of columns will provide exceptionalcolumn durability and performance.

Taking the science a little further, weinvestigated absolute bleed measurements forthe Forte columns. With clean samples, columnlife is determined by gradual loss of phase(resulting in deteriorating resolution) that takesplace as a result of column bleed. Column bleedis simply phase leaving the column and, byquantifying bleed against a standard, the rate ofphase loss can be measured in ng/second. Sincethe initial amount of phase in the column isknown, the time taken for 1% of the phase tobe lost can be calculated. The absolute bleedresults can be seen in (Figure 4).

Not satisfied with subjecting our columns togreen tea, we decided to test the widely heldbelief that exposing a column to oxygen orrunning out of carrier gas, particularly atelevated temperatures (especially with polarphases), will quickly damage the column.Contrary to this belief, even the more polarphases of the Forte range coped with this typeof abuse. Figure 5 shows that after a brief re-conditioning the performance of the BPX50 wasrestored after no carrier gas for 90 minutes at250 °C. Even using 1% air mixture as a carriergas caused little damage as shown by analysingorganochlorine 8081 mix (Figure 6).

Although not wanting to encourage poorsample preparation and extended columnabuse, our experiments have demonstrated thedurability of the Forte range. More columnabuse to follow!

SGE’s new Forte column gets tough with green tea

by Dr. Hugh Malkin, Market Development Specialist

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

Gas Chromatography

VWR C h r o m J o u r n a l Issue 3 S e p t e m b e r 2 0 0 7 3


Rapid separation of berry anthocyanins withChromolith® columns Johanna Witzell & Marie Lundström

The phenolic anthocyanin pigments are widely distributed in the plantkingdom. The red, deep purple and black berries, such as bilberries(Vaccinium sp.) are particularly rich in anthocyanins and provide animportant source of anthocyanins in the human diet. Recent researchhas underlined the potential antioxidant function of anthocyanins,which increases the nutritional importance of berries. From theindustrial point of view, the anthocyanins of berries are of currentinterest because of their importance as natural food colourants. In orderto reliably and quickly analyse the anthocyanin content of rawmaterials, the analysis methods need to be continuously evaluated andoptimised. Here, we tested if a reversed-phase HPLC method, previouslydescribed for analysis of anthocyanins in berries and wine (Nyman andKumpulainen 2001), could be made faster with the help of a monolithiccolumn.

Material and methodsGlycosides were extracted from the bilberries that were first frozen, freeze dried and milled to afine powder, using 2M HCl in methanol (shaking at +4 °C for 20 minutes). Aglycones werereleased from the same extracts by hydrolysis (90 °C in a water bath for 25 min). All samples werefiltered using disposable filters (0,22 µm pore size) before injection to HPLC.

A Merck Hitachi LaChrom HPLC system consisting of an L-7100 pump, an L-7200 autosampler, anL-7360 column oven and an L-7455 diode array detector were used. The oven temperature was setat 30 °C. The compounds were separated on a monolithic column (Chromolith™, Merck KGaA,Darmstadt, Germany) using a method modified from Nyman and Kumpulainen (2001). In ouranalyses, gradient elution was performed using 100% methanol (solution A) and 10% formic acid(solution B) as follows: 5% A in B (0-14 min), 40% A in B (15-19 min), followed by short flushingwith methanol and equilibration to initial conditions. Flow rate was 2 ml/min. The injection volumewas 25 µl for all samples and standard solutions. Peak areas from the analysed compounds weremeasured at 525 nm and identified by comparing their retention times and spectral characters tothose of reference compounds. Cyanidin, delphinidin, malvidin, peonidin and petunidin (chloridesalts) and cyanidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-rutinoside and delphinidin-3-glucoside standards were purchased from Extrasynthese (Geney, France) and used as externalstandards.

>

Analytical HPLC

Anthocyanins are glycosylated

phenolic compounds, which

after hydrolysis release their

aglycon (anthocyanidin).

Because of their use as natural

colourants and health

promoting agents,

anthocyanidins and

anthocyanins are of

considerable current interest for

pharmaceutical and food

industries. We studied

anthocyanidin and anthocyanin

profiles in berries of the

Swedish bilberry (Vaccinium

myrtillus). By using a monolithic

Chromolith® column we

obtained an excellent

separation of 17 anthocyanin or

anthocyanidin peaks. The five

characteristic anthocyanidins of

bilberry eluted within 13

minutes, with retention times of

delphinidin and malvidin being

7,0 and 12,6 minutes,

respectively. Thus, the

monolithic column allowed

rapid and accurate analysis

of bilberry anthocyanins and

anthocyanidins.

Figure 1.HPLC chromatogram of hydrolyzedsamples of Vaccinium myrtillusberries. 1 = delphinidin; 2 = cyanidin; 3 = petunidin; 4 = peonidin; 5 = malvidin.


ResultsIn the hydrolysed V. myrtillus berries, five characteristic aglycones, delphinidin, cyanidin, peonidin,petunidin and malvidin were found (Figure 1). The HPLC-chromatogram of the non-hydrolysedsamples resulted in a total of 17 peaks (Figure 2). Among these peaks, delphinidin-3-glucoside,cyanidin-3-galactoside, cyanidin-3-glucoside and aglycones petunidin, peonidin and malvidin wereidentified.

ConclusionThe use of the monolithic column allowed good separation of 17 anthocyanins or anthocyanidins.The five bilberry anthocyanidins eluted within 13 minutes with retention times of delphinidin andmalvidin being 7,0 and 12,6 minutes, respectively, instead of about 10 and 20 min as reportedearlier (Nyman and Kumpulainen 2001). Thus, the HPLC conditions used here allowed a fastanalysis of bilberry chemicals, making a higher sample throughput possible. Moreover, it is likelythat by adjusting the gradient and flow rate, the speed of analysis over the monolith column couldbe further improved without negative effect on compound separation.


Columns

Johanna WitzellSouthern Swedish Forest ResearchCentre, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden

Figure 2.HPLC chromatogram of non-hydrolysed samples of berries. 2 = delphinidin-3-glucoside; 3 = cyanidin-3-galactoside; 5 = cyanidin-3-glucoside; 15 = petunidin; 16 = peonidin; 17 = malvidin. Other peaks areunidentified compounds.

Reference:Nyman N.A. and Kumpulainen J.T., 2001: Determinationof Anthocyanidins in Berries and Red Wine by High-Performance Liquid Chromatography. J. Agric. FoodChem. 49: 4183-4187.


Analytical HPLCsection line 2section line 1

The efficiency of analytical methods is moreand more important in times of high costpressure. Fast chromatographic separations andhigh sample throughput are frequently themain goal of method development.

One approach to achieve faster separations isto reduce the particle size of the silica used.However very small particles in HPLC columnsgenerate very high column backpressure and inmany cases (eg. with 1,7 µm particles) it isnecessary to use special HPLC systemsenabling pressures up to 1000 bar (15.000psi).

A better approach is to use Chromolith® HPLCcolumns, which enable ultra fastchromatography with standard HPLC systems.The recent publication by Bolivar and co-workers1) describes a time saving of 85% inone application using Chromolith® columns.Depending on the mode of calculation, thisconverts to a cost saving of 80.000 Euro peryear.

In contrast to conventional HPLC columns filledwith silica particles, monolithic HPLC columnsare made of one single piece of continuouslyporous silica with high mechanically stability.The combination of high permeability and highsurface area makes monolithic silica ideal forfast HPLC separations.2)

Figure 1 shows the structure of monolithic

silica under the electron microscope. It looksvery similar to the typical structure of coral. Thesilica forms a continuous skeletal structure andthe space around the silica is completely openand permeable. Liquids are able to flowthrough these open “macropores” with verylow resistance. The unique characteristic of HPLC columnsmade of monolithic silica, e.g. Chromolith®

HPLC columns, is that they provide optimalchromatographic performance withsignificantly lower backpressure thanconventional particulate HPLC columns.

Reduced requirement for sample preparation!A second important characteristic ofChromolith® HPLC columns is that they can beused with simpler sample preparationtechniques. Many samples contain tinyparticles in suspension, which cause blockingor plugging of conventional particulate HPLCcolumns. Such particles tend not to block themonolithic silica structure with its large 2 µmmacropores. Because of this, the columnlifetime is much longer. This opens the way tovery simple “dilute and shoot” samplepreparation techniques1,3,4). The sample issimply filtered through a 0,45 µm syringe filter,in some cases after dilution and centrifugation,

Chromolith® HPLC columns convert standard HPLCsystems into robust racehorses

Figure 1.

Figure 2.


Monolithic silica

columns (Chromolith®)

enable very fast HPLC

analysis with high

detection sensitivity.

These columns are

ideal for use with

standard HPLC

systems.

The requirements for

sample preparation

are reduced, thereby

further reducing

analysis time and cost.


Columns

and then injected directly onto the column withno further sample preparation. Even in theanalysis of foodstuffs or phytopharmaceutics,solid phase extraction steps can frequently beeliminated.

With Chromolith® HPLC columns, time savingsresult not only from faster separations. Owingto the reduced requirement for samplepreparation, total analysis time and costs aredramatically reduced. This is demonstrated bythe following examples:

1) For the determination of caffeine in coffee3)

the time of analysis was reduced from 18minutes using particulate columns to only 0,68minutes using a Chromolith® SpeedRodcolumn. The sample throughput increased to 60samples per hour using a completely standardHPLC system. No solid phase extraction stepwas required. The samples were simply filteredbefore injection.

2) For the determination of iso-α-acids in beer1)

a cost saving of 60% and time savings of 86%has been reported when using a Chromolith®

Performance column . The RSD was improvedfrom 1,6% to 0,9%. The samples of light beerwere injected directly onto the column afterfiltration. A solid phase extraction step waseliminated (this step was required when usingparticulate columns).

Ultra fast separationswith Chromolith® 3mm

Chromolith® HPLC columns with 3 mm internaldiameter instead of 4,6 mm enable very fastseparations at relatively low flow rates usingisocratic (Figure 2.) or gradient conditions (Figure 3.). Figure 3 shows the separation of 8 sulphonamides in only 1,4 minutes at 2 mlmin-1 6). The same separation using 3 µmparticulate columns typically requires about 6 minutes.

References:

1. A.Bolivar, M.Gasparri, C.Zufall,

J.Am.Soc.Brew.Chem. 2006,64(1),39-46

2. K.Cabrera, J.Sep.Sci, 2004, 27, 843-852

3. Paraskevas D. Tzanavaras, Demetrius G. Themelis -

Analytica Chimica Acta (2006),

doi:10.1016/j.aca.2006.07.081

4. V.Borges et al, J.Chromatography B, 2004, 804,

277-287

5. Hydroxybiphenyls - a class of compounds used as

fungicides and relevant for food and environmen-

tal analysis

6. LaChrom Elite Application Note 2006 -

Sulphonamides

Figure 3.


Purospher® STAR HPLC columns – the best choice for successful HPLC separations

Purospher® STAR RP-18 endcappedHPLC columns make it very simple tochoose the best column for mostapplications, as proven by the manyusers who appreciate their excellentproperties. The absence of metals in the silicamatrix, in combination with a completecoverage of the silica surface, enablesthis stationary phase to producetailing-free chromatography of acidic,basic and chelating compounds.Purospher® STAR provides the bestall-round retention characteristics. Theoutstanding pH stability up to pH 10,5allows the separation of strong basiccompounds with alkaline eluents. Inaddition, Purospher® STAR RP-18endcapped is suitable for use with100% aqueous mobile phases.The combination of thesecharacteristics makes Purospher® STARRP-18 endcapped an all-round topperformance column, almost universalin its range of applications. Experiencethe performance of Purospher® STARRP-18 endcapped HPLC columns – onecolumn for different demands.Purospher® STAR RP-18 endcapped isideally suited to the separation of, forexample, Aflatoxins in foodstuffs withvery high sensitivity.

Aflatoxins in foodstuffsAflatoxins B1, B2, G1 and G2 are the main toxins produced by Aspergillus flavus, A. parasiticus andA. nomius. These toxins are of relatively high molecular weight and contain one or moreoxygenated alicyclic rings. They can contaminate food products when storage conditions arefavourable to fungal growth. The main aflatoxin contaminants have been reported in maize,peanuts, brazil or pistachio nuts, copra and cotton seeds. Aflatoxins are carcinogenic, mutagenic,teratogenic and immunosuppressive to most animal species. The Internal Agency for Research onCancer (IARC) has classified all four aflatoxins as group one carcinogens. Confirmation of the presence of aflatoxins in a sample by HPLC requires derivatisation of theaflatoxins B1 and G1 in order to enhance their natural fluorescence under UV light and make themmore easily detected. Previously, the only options available for derivatising aflatoxins involved theuse of either trifluoroacetic acid (TFA) or iodine. Both of these methods are reliable, however theydo have some significant limitations, which can be overcome by use of a third method with theCoring Cell.

The European standard HPLC method EN 12955 for the determination of aflatoxin B1 and the sumof aflatoxin B1, B2, G1 and G2 in cereals, shell-fruits and derived products features a high-sensitivefluorescence detection by post column derivatisation with iodine and immunoaffinity column cleanup. Iodine derivatisation as a post column technique does have other disadvantages such as longerreaction time at higher temperatures (peak broadening), additional HPLC pump necessary anddaily preparation of the corrosive iodine solution. This European standard specifies a method forthe determination of aflatoxin content of greater than 8 µg/kg.

The official German method for measuring compounds in food products (§35 LMBG methods)features the clean-up of the aflatoxins B1, B2, G1 and G2 by means of an immunoaffinity columnand the following HPLC detection with an electrochemical cell (Coring Cell). The Coring Cell is anelectrochemical cell that generates the derivatising agent, bromine, from potassium bromidepresent in the mobile phase. The derivatisation of aflatoxins occurs rapidly (reaction time isapproximately 4 seconds) at ambient temperature. A daily preparation of derivatising reagent(iodine) is not necessary and the additional pump for addition of derivatising reagent is notneeded.

For both methods Purospher® STAR RP-18 endcapped shows excellent separation of the 4Aflatoxins with outstanding peak symmetry as the comparison of different HPLC columnsdemonstrates (Figure 1- Figure 3).

Analytical HPLC

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

20

60

40

80

100

Retention Time (min)

G2

G1

B2

B1

Chromatographic conditions:Column: EcoCART® 125-3 LiChrospher RP-18, 5 µmMobile phase: Water/Methanol/Acetonitrile (65:15:20)+ 100 µl HNO3 65%/l + 119 mg KBr/l

Flow rate: 0,6 ml min-1

Injection volume: 100 µl

Derivatisation: Coring cell

Figure 1Separation of Aflatoxins on LiChrospher® RP-18

59

0 2 4 6 8 10 12 14 16

58,5

58

57,5

57

56,5

56

55,5

55

54,5

B1

B2

G1

G2

Chromatographic conditions:Column: 250-4.6 RP-18 column, 5 µmMobile phase: Water/Methanol/Acetonitrile (6:3:2)+ 350 µl HNO3=4 mol/l + 120 mg KBr/lFlow rate: 1 ml min-1

Injection volume: 200 µl

Derivatisation: Coring cell

Figure 2Separation of Aflatoxins on RP-18 column ;Chromatogram from the § 35 LMBG method

0 2 4 6 8 10 12 1614 18 20-5

0

5

10

15

20

25

30

35

-5

0

5

10

15

20

25

30

35

Minutes

FLU

Time: 19,9796 Minutes - Amplitude: -0,40675 FLU

G2

G1

B2

B1

Chromatographic conditions:Column: Purospher® STAR RP-18 endcapped, 150 x 4,6 mm, 5 µm Pre column: Purospher® STAR RP-18 endcapped, 4 x 4 mm, 5 µm Mobile phase: Water + 183,1 mg KBr/l + 154 µlHNO3 65%/l / Methanol / Acetonitrile65 % A / 17.5 % B / 17.5 % C(V/V/V), IsocraticFlow Rate: 1 ml min-1

Temperature: 40 ºCInjection volume: 100 µlDerivatisation: Coring cell

Figure 3Separation of Aflatoxins on Purospher® STARRP-18 endcapped


Columns

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

-0,25

0,00

0,25

0,50

0,75

1,00

1,25

1,50

-0,25

0,00

0,25

0,50

0,75

1,00

1,25

1,50

Minutes

FLU


G2

G1

B2B1

Method 1: Aflatoxins with iodine derivatisation (EN 12955)

G2 - 30 pg/ml, G1 - 100 pg/ml, B2 - 30 pg/ml, B1 - 100 pg/ml

Chromatographic conditions:Column: Purospher® STAR RP-18 endcapped, 150 x 4,6 mm,

5 µm Pre-column: Purospher® STAR RP-18 endcapped, 4 x 4 mm, 5 µm Mobile phase A: Water/Acetonitrile/Methanol 65/17,5/17,5 (V/V/V)Mobile phase B: Rinse the system after the sequence for 30 minutes with

90/10 water/Methanol (V/V)Flow Rate: 1 ml/minDetection: Fluorescence EX 365 / EM 435Temperature Oven: 60 ºCInjection volume: 50 µl

Post column derivatisation:Derivatisation reagent: saturated iodine solutionReaction pump: The pump is stopped by the timer

of the L-2130 pump when the rinsing program is started. Never let this pump run when the L-2130 pump is stopped. The iodine will damage your column.

Derivatisation coil: PTFE knitted coil, 5 m x 0,5 mm I.D. or home made PEEK coil 3 m x 0,5 mm I.D.

Flow Reaction pump: 0,2 ml/min

0 2 4 6 8 10 12 1614 18 20

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

-0,5

0,0

0,5

1,0

1,5

2,0

2,5

Minutes

FLU


G2

G1

B2

B1

Method 2: Aflatoxins with Coring Cell (§ 35 LMBG)

B1 and G1: 10 pg/mL; B2 and G2: 2.5 pg/mL

Chromatographic conditions:Column Purospher® STAR RP-18 endcapped, 150 x 4,6 mm,

5 µm Pre-column Purospher ® STAR RP-18 endcapped, 4 x 4 mm, 5 µm Mobile phase: Water + 183,1 mg KBr/l + 154 µl HNO3 65%/l /

Methanol / Acetonitrile65 % A / 17,5 % B / 17,5 % C(V/V/V), Isocratic

Flow Rate: 1 ml min-1

Detection: Fluorescence EX 365 / EM 435Temperature: 40 ºCInjection volume: 100 µl

Post column derivatisation:Derivatisation coil: PEEK coil, 1,38 m x 0,25 mm I.D.

Full applications are available from your localVWR sales office

>VWR C h r o m J o u r n a l Issue 3 S e p t e m b e r 2 0 0 7 9


Analytical HPLC

YMC HILIC providesan ideal mechanismfor the analysis ofextremely polaranalytes offering awide variety of HILICphases to cover abroad range of polarselectivities. HILICeluents arecompatible withLC-MS systems

Over the years, YMC has increased its international exposure to innovative marketplaces. Productsand services have matured to a level of world-wide recognition, specifically in the area ofpharmaceutical and biotech compounds, where YMC is one of the few companies to fully supportnormal phase and reversed phase chromatography techniques from analytical to process scale,throughout the entire lifetime of a compound from R&D to scale-up, production and qualitycontrol.

Substantial investments into facilities and staff represent YMC’s ongoing commitment towardshigh quality products and technical support. In Komatsu/Japan, new premises provide equipmentfor professional industrial-scale custom purification services, for example, and organic synthesisfacilities under GMP-conditions, where applicable.

General

Hydrophilic Interaction Chromatography is a technique that has attracted more and more attentionsince it offers an alternative approach for the separation of highly polar compounds. The methoditself, although it has been known for more than 25 years, has become more popular recently dueto the introduction of several specialised HILIC selectivities onto the market. However, it is not wellknown that HILIC can be accomplished with any highly polar stationary phase. This opens up alarge range of materials, which are suitable for HILIC separations. In order to understand themechanism and the separation principle it is helpful to use a schematic drawing. As shown inFigure 1, HILIC uses a highly polar stationary phase and a non polar mobile phase e.g.functionalised silica with a hydrophilic coating and an acetonitrile/ water mixture (80:20). The polaror hydrophilic sample experiences more attraction towards the stationary phase or the more polar

and more stationary part of the mobile phase. Polar samples can be eluted usingisocratic or gradient methods, giving the mobile phase a more hydrophilic characteror even a higher ionic strength.

Advantages

In general, reliable applications in HPLC require a minimum retention factor (k’) of2. In reversed phase methods polar compounds such as peptides, organic acids orpyrimidines often exhibit little or no retention and therefore low resolution on aconventional C18 phase. The use of additives such as ionpair reagents is not analternative since it is not applicable to LC-MS. Also normal phase chromatographyfails due to poor solubility of the polar compounds in organic solvents. HILIC fillsthe gap between reversed phase and normal phase chromatography, because polarcompounds can be retained while using e.g. acetonitrile/water eluents and bufferssuitable for LC-MS.

>HPLC Columns for HILIC



Software

>

Conclusion

HILIC is a very interesting tool since a single reversed phase material hardly canretain polar, watersoluble compounds. An alternate mode of retention and separationneeds to be considered for very polar compounds such as actives, metabolites andpeptides. Hydrophilic Interaction Chromatography has also been referred to as“aqueous normal phase” or “reverse reversed phase” since elution is in the order ofincreasing hydrophilicity and the organic portion of the mobile phase (typicallyacetonitrile) is the weak solvent and the aqueous portion is the strong elutingsolvent. HILIC columns from YMC are rugged stationary phases, which provideimproved LC/ESI-MS response, direct SPE solvent compatibility and complementaryselectivity to reversed phase. This is important to R&D and drug metabolismscientists since the impurity or metabolite is frequently more polar and present atmuch lower concentrations than the parent compound. With YMC HILIC columns,these very polar compounds elute later than the higher hydrophobic parentcompound, thereby minimising the MS ion suppression that can occur at thebeginning of the chromatogram. Another very important field of application is theseparation and purification of polar compounds on a preparative to industrial scale.For this purpose YMC’s well known phases are fully scaleable from kg to multi-tonsupply and of course are available with particle sizes up to 150.

YMC HILIC PHASES:

Note: YMC-Pack PVA-Sil, YMC-Pack Polyamine II and YMC-Pack Diol are not availablein 3 µm particle size.

For more information contact:

Donal Farrell, Chromatography Specialist, AGB Scientific Ltd. Email: [email protected] orTel 01 88 22 234.

Particle size / µmPore size / nmCarbon content / %pH range

Silica 3; 5; 10; 15; 20; 506; 12; 20; 30n/a 2 - 7.5

PVA-Sil 5 12n/a 2 - 9.5

Amino3; 5; 1012 3 2 - 7

Polyamine II 5 3; 12n/a2 - 9

Cyano5; 10; 15 12; 307; 2.5 2 – 7.5

Diol5; 10; 156; 12; 20; 30n/a2 – 7.5


>Technical DataMaterial: Cross-linked polystyrene-co-divinylbenzene polymer

Particle size: 5 µm

Pore size: 100 Å

Ordering Information

0.0

2.0x10 6

4.0x10 6

6.0x10 6

8.0x10 6

1.0x10 7

1.2x10 7

1.4x10 7

20%

40%

60%

80%

100%

20 30 40 50 60 70 80 90

Data courtesy of S. Cohen and C. RitlandNevada Department of Agriculture

Column Bleed Characteristics PRP-h1 vs. Traditional Polymeric Column

Time [min]

MS

Sig

nal

[m

/z =

50-

2000

]

% A

ce

ton

itri

le

0.0

2.0x10 6

4.0x10 6

6.0x10 6

8.0x10 6

1.0x10 7

1.2x10 7

1.4x10 7

20%

40%

60%

80%

100%

20 30 40 50 60 70 80 90


0,0

2,0x10 6

4,0x10 6

6,0x10 6

8,0x10 6

1,0x10 7

1,2x10 7

1,4x10 7

20%

40%

60%

80%

100%

20 30 40 50 60 70 80 90


Column Bleed Characteristics PRP-h1 vs. Traditional Polymeric Column

Time [min]

MS

Sig

nal

[m

/z =

50-

2000

]

% A

ce

ton

itri

le

50 mm 100 mm 150 mm 250 mm

2,1 mm ID 554-1378 554-13814,6 mm ID 554-1377 554-1379 554-1382 554-138410 mm ID 554-1380 554-1383100 mm ID 554-1385

PRP-h1 Guard ColumnsAnalytical Guard Column Starter Kit (1 holder, 2 cartridges) 554-1386Analytical Replacement Cartridges (5/pk) 554-1387Semiprep/Prep Guard Column Starter Kit (1 holder, 2 cartridges) 554-1388Semiprep/Prep Replacement Cartridges (2/pk) 554-1389

PRP-h1 Bulk Resin12-20 µm Bulk Resin (1 g) 554-1390

HAMILTON’s new PRP-h1 HPLC-columns:higher sensitivity, half backpressure and no column bleed!

HAMILTON´s newly developedHPLC column phase based on PS-DVB retains the advantagesover silica usually associated withpolymeric columns and at thesame time minimises columnbleed.

In fact the PRP-h1 columns havevirtually no bleed, even at 100%acetonitrile and run at half thebackpressure of traditionalpolymeric columns.

The outstanding pH stability (pH0-14) completes theexceptional features of these newcolumns.

Analytical HPLC Columns

Determination of Naproxen and IbuprofenColumn: PRP-h1, 5 µm, 100 Å, 4,1 x 50 mm

Mobile Phase:A: 10 mmol/l Sodiumdihydrogenphosphate, pH=2B: Acetonitrile, Isocratic 50% A / 50% B

Flow Rate: 0,6 ml/min

Temperature: 60 °C

Detection: UV@ 230 nm

Sample: (1) Naproxen (110 µg/ml)(2) Ibuprofen (830 µg/ml)

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To maintain the high quality of

their solvents until you use them,

Merck have developed a practical

packaging concept for chemicals.

For LiChrosolv®, Perpsolv® (HPLC), Uvasol® (UV/VIS), SeccoSolv®, SupraSolv® and Unisolv® (GC) we offer a unique variety of differentpackaging sizes and types:• Glass bottles• Aluminium bottles• Stainless steel barrels• Other barrels and containers• Withdrawal systems

Ideal packaging and withdrawal systems for HPLC solvents


Solvents

BDH Prolabo chemicals The new Chemical Brand from VWR International

New HiPerSolvChromanormrange

• High purity solvents for HPLC applications

• Guaranteedspecification conformsto HiPerSolv solvents

• High performance andreliability

>

Combining the best qualityand price competitivecharacteristics

For a free copy of the new BDHProlabo Chemicals

brochure or furtherinformation, please

contact your local VWRInternationalorganisation or checkout vwr.com

Your benefits:

• Easy, safe and contamination –freesolvent handling

• Central storage and supply possible• Individual user installation or other

customised solutions possible• Direct connection to laboratory

equipment such as an HPLC isachievable

Merck’s expertise is recognised in theirauthorisation as an official inspection authorityby the Federal Institute for Material Researchand Testing of Germany (RAM).


NEW AcroSep™ Chromatography Columnsfor Ion Exchange from Pall Life Sciences

BioChromatography

Pall has recently extended their range ofchromatography products to include the newAcroSep™ Chromatography Columns for IonExchange. The columns are ideal foroptimisation studies of protein purificationschemes using small sample volumes prior toscale-up and offer an efficient and reliablemethod for sample preparation of proteins forstructural, functional and yield analysis.

The ion exchange columns contain Pall’sHyperD chromatography sorbents, which arecreated using patented “gel-in-a-shell”technology.

Gel-in-a-Shell Design

The gel-in-a-shell bead is constructed of ahigh-capacity hydrogel polymerised within thegigapores of a rigid ceramic bead. The capturechemistry is attached to the hydrogel withinthe bead pores. The advantages of thisstructure are many, including higher than usualcapacity, tolerance to fast chromatographyruns without a concomitant increase inbackpressure, and greater salt tolerance for ionexchangers.

Whether your application

challenges are in research,

scale-up or polishing, Pall offers

an extensive portfolio of bulk

resin chromatography media

for affinity, ion exchange,

size exclusion, hydrophobic

interaction and hydroxyapitite

chromatography.

For a complete listing ofPall’s Protein Purificationproduct range or toorder a new ProteinSample Preparation andAnalysis ApplicationManual please contactyour local VWR salesoffice

Bulk chromatography sorbents (bottled in 1 to1000 ml quantities) are also available fromPall. The chromatography sorbents can be usedin traditional chromatography columns or, forquick separations, can be used in centrifugalspin columns (e.g., Pall Nanosep® devices). Forhigh throughput separations or scoutingassays, the sorbents can be combined with theAcroPrep™ filter plates with up to 96 mini-columns that can be simultaneously processed.

Pall also offers chromatography-based kits forabundant protein removal. These ready-to-usekits include all components necessary fordepletion or selection of albumin and/or IgGfrom multiple species.

To assist with your Proteomics research Pall has developed a detailed application handbookcontaining a range of protocols for samplepreparation and analysis. The handbookcontains protocols for handling thechromatography sorbents and the use of Pall'sother proteomic products.


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Columns

Features:-

Gel-in-a-Shell Technology –Patented HyperD ion exchangechromatography resin is a highcapacity hydrogel polymerisedwithin the large pores of a rigidceramic bead

Rapid and Efficient – Rigidresin structure allows faster flowrates and higher pressures whilemaintaining high dynamic bindingcapacities.

High Resolution – Distinctseparation of proteins forcomplete isolation andpurification

High Recovery – Maximumrecovery yielding highly pureproteins

Versatile – Luer lock inlet andoutlet for convenient use withsyringe, pump or automatedchromatography system

Convenience – Hexagonalcollar, prevents column fromrolling when set down

User-friendly – Colour-codedand labelled by chemistry type

Ion Exchange ResinParticle Stability Cleaning Ion Exchange

Media Function Size (µm average) Range pH pH Capacity (1)

CM Ceramic Weak Cation 50 2-12 1-14 > 60 mg/ml (2)HyperD® F Exchanger

DEAE Ceramic Weak Anion 50 2-12 1-14 > 85 mg/ml (3)HyperD F Exchanger

Q Ceramic Strong Anion 50 2-12 1-14 > 85 mg/ml (3)HyperD F Exchanger

S Ceramic Strong Cation 50 2-12 1-14 > 75 mg/ml (3)HyperD F Exchanger

(1) Dynamic binding capacity determined at 10% breakthrough, 200 cm/h with7 ml resin packed in a column of 1,1cm ID and 7 cm height using:

(2) 5 mg/ml human IgG in 50 mM sodium acetate buffer, 100 mM NaCl, pH 4,7(3) 5 mg/ml BSA in 50 mM Tris-HCl buffer, pH 8,6(4) 5 mg/ml lysozyme in 50 mM sodium acetate buffer, pH 4,5

Description Qty/Pk Cat. No.

HyperD F CM 1ml Column 5 569-1000HyperD F S 1ml Column 5 569-1001HyperD F Q 1ml Column 5 569-1002HyperD F DEAE 1ml Column 5 569-1003

Your Irish Distribution Partner

A new range of screw neck vials withvolumes ranging from 2 – 60 ml are nowavailable to complement the VWRCollection wide range of chromatographyvials and accessories.

All vials are made of 1st class hydrolyticglass in clear or amber. As the screw neckvials require screw seals with differenttypes of thread, and as a broad selectionof various septa is available in the caps,closures should be asked for individually.The vials are in packs of 100.

For better identification of stored samplesbarcode labelling of the vials is alsopossible, however, a minimum orderquantity may be required.

Volume (ml) Type Thread Size (mm) Glass Cat. No.

4 Screw Neck 13-425 45 x 14.7 Clear 548-00514 Screw Neck 13-425 45 x 14.7 Amber 548-00528 Screw Neck 15-425 61 x 16.6 Clear 548-08218 Screw Neck 15-425 61 x 16.6 Amber 548-088912 Screw Neck 15-425 66 x 18.5 Clear 548-082012 Screw Neck 15-425 66 x 18.5 Amber 548-090320 Screw Neck 20-400 86 x 22.7 Clear 548-089020 EPA Screw Neck 57 x 27.5 Clear 548-015420 EPA Screw Neck 57 x 27.5 Amber 548-063830 EPA Screw Neck 72.5 x 27.5 Clear 548-015530 EPA Screw Neck 72.5 x 27.5 Amber 548-063740 EPA Screw Neck 95 x 27.5 Clear 548-015640 EPA Screw Neck 95 x 27.5 Amber 548-063960 EPA Screw Neck 140 x 27.5 Clear 548-064060 EPA Screw Neck 140 x 27.5 Amber 548-0641

New VWR Collection screw neck vials

For more information on the closures, septa or any other VWR Collection chromatography vialsplease contact your local VWR sales office.

Schott Duran® HPLC laboratory bottlesThe SCHOTT DURAN® pressure plus bottle is pressure resistant ( –1 to +1,5 bar) thanks to its special geometryand is TÜV-GS certified (TÜV ID: 0000020716 ) and safety tested to EN 1596. The bottle has a retrace code fordownloading a certificate of conformity from the Internet including batch, manufacturing, relevant standardsand USP/FDA conformity. The screw cap has four ports. The fourth port is for sterile pressure equalisation andthe other three are suitable for tube diameters of 1,6 and 3,2 mm. Unused ports can be closed with a siliconeseal:

• Hoses are tightly fixed to prevent them from moving and the HPLC column from running dry• The cap is made of PTFE and PP and can be completely autoclaved together with the bottle without taking it

apart! • Can be repeatedly reused• Working in the enclosed system retains toxic gases in the bottle• Spare parts available

A flexible modular system is also available, for further information please contact yourlocal VWR sales office

AGB Scientific LimitedA VWR InternationalCompanyOrion Business CampusNorthwest Business ParkBallycoolin, Dublin 15.Tel.: 01 8822222Fax: 01 8822333E-mail: [email protected]

Labkem Chemicals LimitedBaldoyle Industrial EstateDublin 13t 01839 1212f 01839 [email protected]

Lennox Lab supplies LtdJF Kennedy DriveNaas RoadDublin 12t 01 455 2201f 01 455 [email protected]

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