Applications and features of the Agilent 1100 Series micro fraction collector

Application

Edgar Nägele, Martin Vollmer

Abstract

To reduce the complexity of a sample it is often necessary to purify orfractionate samples prior to analysis. This holds especially true forapplications in proteomics, metabolomics, natural product research andlibrary generation where the sample is usually highly complex. If inaddition, only a limited amount of the sample is available, the problembecomes much more challenging. For this purpose the Agilent 1100Series micro fraction collector was developed. In combination with theAgilent 1100 Series capillary LC, the micro fraction collector offers ahigh performance solution for processing these special samples. ThisApplication Note describes the field of application for the Agilent 1100Series micro fraction collector and discusses the features and softwarecontrol of this device. In addition, the main modules of the micro frac-tion collection system are also discussed - the Agilent 1100 Seriescapillary pump, the Agilent 1100 Series diode-array detector with 80-nLcell and the Agilent 1100 Series micro well-plate autosampler .

Introduction

Life science researchers fromseveral disciplines are often facedwith the difficulty that they havesamples of very high complexity.On one hand, a large number ofcompounds might be of investiga-tional interest, or on the other handa few compounds in a large back-ground of undesired compoundsmight be the focus of the analysis.This is often accompanied withthe problem that only a limitedamount of sample is available.

These factors principally determinethe strategy for the analysis of thesample. In order to process samplesavailable in low concentrationsonly, the fractionation collectionor purification device must beadapted. Therefore, the Agilent 1100Series micro fraction collector1,which is best suited to the Agilent1100 Series capillary LC system,was developed.

Several strategies can be employedusing capillary LC separation incombination with micro fractiona-tion (figure 1). These include directanalysis after fractionation, chemi-cal modification of compounds inthe fractions with subsequentanalysis of the derivatives orfurther fractionation in a secondorthogonal separation dimension.An example of the last strategy isthe analysis of proteome samplesby off-line two-dimensional LC/MS.Here a digested proteome sampleof high complexity is fractionatedby SCX (strong cation exchange)chromatography in the firstdimension with the Agilent 1100Series micro fraction collectionsystem. The tryptic peptides con-tained in the fractions are subse-quently further separated withnano RP chromatography in thesecond dimension and analyzedby tandem mass spectrometry2.

Further useful applications for theAgilent 1100 Series micro fraction

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collection system, could be themultidimensional fractionation ofpeptide samples in one LC system,for example, the purification ofbiomarkers, the isolation of meta-bolic compounds out of a complexbiological matrix, compound iso-lation from combinatorial librariesand MALDI-TOF analysis3 as wellas purification of a small amountof a natural product for biologicalscreening or fractionation of avery complex naturally occurringor synthetically gained mixtureinto a chemical library for screen-ing of active compounds in drugdiscovery4. In addition, microfraction collection allows storageof fractions for repeated analysesor running parallel analyticaltechniques like mass spectrometryin combination with other analyticaltechniques further downstream(figure 2). It is also possible todeposit small fractions on a MALDI-plate concurrent to an electro-spray mass spectrometry analysis.

Figure 1Fractionation strategies with the Agilent 1100 Series micro fraction collection system

Micro Fraction Collection

• Time based • Peak based

Separation of sample compounds with differentchromatographic LC methods and column types.

• Analysis of fractions• Further processing and analysis• Next separation dimension and analysis

Equipment

An Agilent 1100 Series micro frac-tion collection system 1 consistingof the following modules was used(figure 3):

• Agilent 1100 Series capillary pump with 20-µL or 100-µL flow sensor for capillary columns with 0.3–1.0 mm i.d. and micro vacuum degasser

• Agilent 1100 Series thermostattedmicro well-plate autosampler

• Agilent 1100 Series diode-array detector with 500-nL cell or 80-nLcell

• Agilent 1100 Series thermostattedcolumn compartment

• Agilent 1100 Series thermostattedmicro fraction collector

• Agilent ChemStation A10.02

3

LC/MSD Trap XCT

Micro fractioncollector/spotter

ESI withmicro sprayer

Column effluentfrom capillary LC

MALDI TOF

Biological screening

Scintillation screening ofradioactively labeled metabolites

Splitter

Figure 2Parallel analysis with ESI MS and other downstream analytical methods of the collected microfractions

Figure 3Components of the Agilent 1100 Series micro fraction collection system

Solvent tray

Degasser

Capillarypump

Micro-WPS

Cooler

TCC + micro 6 port valve

DAD

Micro fraction collector

Cooler

Results and discussion

The Agilent 1100 Series micro fraction collector

The liquid contact control, a uniquecollection principle which isillustrated in figure 4, has beendeveloped to reliably collect smallvolumes in the lower µL range. TheAgilent 1100 Series micro fractioncollector ensures that the dropletforming at the capillary tip is incontact with the bottom of thewell at the beginning and in con-stant contact with the solvent dur-ing the remaining fractioncollection process. At the start offraction collection, the dropletbeing formed is delivered to thebottom of the well. As the well isbeing filled the capillary tip movescontinuously upwards, keeping inconstant contact with the solventsurface. At the end of fractioncollection, the capillary tip movessharply upwards, delivering thedroplet currently being formedinto the well. As the speed at whichthe well is filled is not only depen-dent on flow rate but also on thegeometry of the well, both factorsare taken into consideration dur-ing the collection process. Theparameters of the liquid contactcontrol mode, which can be con-trolled by the ChemStation soft-ware, are the distance between thecapillary and the well bottomrespective to the surface of theliquid as well as the capillaryextraction speed. This methodproduces accurately collectedfractions free from air bubbles.Cross contamination between frac-tions is also prevented using thisapproach. It is also possible to setthe collection time for each frac-tion to a minimum of 3 seconds in

order to collect very small fractionvolumes using a time-based methodand a given flow rate.

Any component in the flow pathwhich could contribute to addi-tional delay volume, peak disper-sion5 or could be considered as asource of cross contamination iseliminated due to the low flowrates and liquid volumes. In par-ticular a diverter valve, whichswitches the flow path betweenwaste and fraction collection, iffor example, a peak is detected,can be considered such an item.Therefore, there is no divertervalve in the micro fraction collec-

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tor. The micro fraction collectorhas a complete flow-throughdesign without any critical parts in the flow path between thedetector and capillary tip. Thedelay volume caused by the capil-lary itself can be calculatedapproximately subject to the innerdiameter of the capillary, which isused according to the flow rate(table 1). A delay calibration isrecommended for a more precisedetermination of the delay vol-ume, which also includes the cap-illary, the connection to the detec-tor cell and the detector responsetime. To avoid possible cross cont-amination of the collected fraction

Capillary i.d. Flow rate Delay volume[µm] [µL] [nL]

25 < 4 250

50 4 - 30 1000

100 30 -100 4000

Table 1Delay volume and corresponding flow rate of the capillaries used in the Agilent 1100 Series microfraction collector

Figure 4Liquid contact control is the basis for collecting micro fractions without cross contamination andair bubbles

1 2 3

4 5 6

5

01 02 03 04 05 06 07 08 09 10 11 12 13

min0 5 10 15 20 25 30 35 40

mAU

0

250

500

750

1000

1250

1500

1750

2000

100 mM NaCl

500 mM NaCl

Figure 5Time based fraction collection of a complex cell lysate from yeast

with waste compounds, thedroplet formed at the capillary tipis continuously directed to thewaste container by means of aspecial flap septum, which is incontact with the capillary when itis at the waste position.

The fractions can be collected intoa variety of standard vials or well-plates of different geometry andbrands with the Agilent 1100Series micro fraction collector. Inaddition, Eppendorf cups (2.0 mL,1.5 mL , 0.5 mL) can be used ascollection devices. A wide varietyof collection vessels are alreadypreconfigured and can be chosenvia the ChemStation software.Alternatively, user-defined collec-tion devices may be added. Toensure that the liquid is accuratelydelivered into the well, the exact

position and height of the capil-lary is automatically measured bya sensor pad during initialisationof the micro fraction collector. Inaddition, it is possible to calibratethe capillary position in the diag-nosis mode of the ChemStationsoftware especially for 384 well-plates.

The eluting compounds can befractionated in a time-based or ina peak-based manner with themicro fraction collector. Forinstance, a complex cell lysatefrom yeast (Saccharomyces cere-

visiae) was fractionated using atime-based method from an SCXchromatography in the firstdimension of an offline two-dimensional LC/MS proteomeanalysis (figure 5). The fractionswere collected every three

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1-P1-A

-01

min10 15 20 25 30

mAU

200

400

600

800

1000

0

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-02

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-15

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-16

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-17

Figure 6Peak based separation of a crude plant extract from Rheum palmatum with the Agilent 1100 Series micro fraction collection system

product extracts often only thesingle compounds are of interestfor an initial biological screeningaccomplished with a small amountof the material. In this case, it ispossible to fractionate the mixturewith the micro fraction collectorusing a peak-based method (figures 6 and 7). Figure 6 showsthe chromatogram with the peak-based collected fractions from acomplex crude extract from theplant Rheum palmatum. Thesefractions were subsequentlyreanalysed, which proves the goodseparation of the compounds. Thisholds especially true for the peakseluting between 16 and 17 minutes(figure 7). In this case, fractions

with only a few micro liters weredeposited precisely on the bottomof a conical shaped 384-well-plate.

Often it is necessary to obtainmore material for screening tests.In this event the instrument allowspooling of fractions from multipleruns into the same wells of a givenwell-plate. In order to avoid anyloss of column effluent from veryvaluable samples during peak-based collection, the ChemStationsoftware “recovery on the track”tool was designed for the microfraction collector. By using thisfeature, the complete columneffluent in between fractions canbe collected in the well-plate for

minutes at a flow rate of 5 µL/min.The tryptic peptides contained inthese fractions were separated inthe second dimension by nano RPchromatography and subsequentlyanalyzed by MS/MS. After a data-base search with the acquiredMS/MS spectra and comparison ofthe obtained protein hits with oth-er methods, it was determinedthat the offline 2D LC/MS methodusing a micro fraction collectionsystem for the first dimension sep-aration is superior to other online2D LC/MS methods for proteomicsapplications2.

In complex compound mixtures asobtained, for example, from natural

min10 12 14 16 18 20 22 24 26

mAU

0

100

200

300

400

500

600

700

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Figure 7 Reanalysis of collected fractions and separation of peaks eluting between 16 and 17 minutes

Column: ZORBAX SB C18, 0.3 x 150 mm, 5 µm

Capillary pumpSolvent: A = water + 0.1 % formic acid, B =

AcN + 0.1 % formic acid.Gradient: 0 min 5 % B, 3 min 5 % b, 33 min

65 % B, 36 min 85 % B, 45 min 85 % B.

Stop time: 45 minPost time: 15 min

ColumnFlow rate: 10 µL/min

Micro well-plate autosamplerInjection: 1 µL for purification and 10 µL for

reanalysis (fractions were diluted to 10 µL with water).

Injector valve: Bypass at 3 min, needle wash

for 10 sec, cleaning procedure for lowest carry-over at 40 min

Diode-arraydetector: 80 nL flow cell, 254 nm +/- 8, ref.

360 nm.Fraction collector: Peak based between 8 min and

33 min, threshold = 50, up and down slope = 5

reanalysis and a new fraction iscollected only when a peak isdetected and matches the userdefined peak-triggered fractioncollection parameter. After thecompound is fractionated, themicro fraction collector starts tocollect the column effluent againin a new well. It is certainlypossible to equip the micro fractioncollector with a cooler, whichprevents evaporation and decom-position of valuable samples. Thisthermostat is able to regulate thefractions between 4º C and 40º C.

The Agilent 1100 Series capillary pump

For robust and reliable fractioncollection in the lower micro literrange it is crucial that the pumpdelivers the flow with higheststability, thus ensuring retentiontime stability of the separatedcompounds. The Agilent 1100Series capillary pump is equippedwith a special Electronic FlowControl (EFC) which delivers avery stable and robust liquid flowin the micro liter flow rate rangeinstead of a non-regulated passivesplitter. The EFC divides the pri-mary flow rate into the micro litercolumn flow and a waste flow. Themicro liter column flow is moni-tored with a special flow sensor tocontrol the active splitting ratio.The calibration curves for all com-mon HPLC solvents and their mix-tures are included in the ChemSta-tion software for this sensor. Thesensor signal drives the split ratioat the electromagnetic proportion-ing valve (EMPV) (figure 8a). Theflow sensor6 (figure 8b) consists ofa stainless steel capillary, two tem-perature sensors and a heateraround the capillary. If there is noflow through the capillary the tem-perature profile around the heateris symmetric. If there is a flowthough the capillary the tempera-ture profile shifts downstream.The shift in the temperature pro-file represents a temperature dif-ference caused by the heat trans-port of the flowing fluid. The heat

transport is proportional to theflow rate. Therefore, the sensormeasures the flow of the fluid anddelivers a feedback to the EMPV(figure 8c). This ensures an out-standing flow stability independentfrom system backpressure fluctua-tions. There are two flow sensorsavailable, a 20-µL sensor and a 100-µL sensor for the capillary pump.

The composition accuracy and pre-cision as well as the mixing noisewas calculated with the resultsobtained from three runs with atracer gradient from 0 to 10 % inincrements of 1 %. For every pumpthis is the most critical perfor-mance range. The injection valvewas switched into the bypass modeafter one minute to exclude thewell-plate sampler volume from thesystem delay volume. The primaryflow rate was set to low solventconsumption and the column flowrate to 4 µL/min.

The main features of the capillarypump include:• Accuracy of step height has an

average deviation of 0.036 -0.066 %.• The step height precision typically

shows a standard deviation of 0.024 - 0.041 %.

• Mixing noise was typically < 0.027 % with the autosampler set to the mainpass position, 420-µL mixer installed and low solvent consumption.

8

9

Figure 8The electronic flow control of the Agilent 1100 Series capillary pump used in the Agilent 1100Series micro fraction collection system.

FilterDamper

Electronicflow control (EFC)

To samplingunit andcolumn

To waste

Mixing point

SensorEMPV

These results demonstrate theextreme robustness of the capil-lary pump flow rates, whichensures the reliable and strongflow for fraction collection, andretention time stability. The mea-surements are described in detailin a separate Application Noteabout the Agilent 1100 Series cap-illary LC system7.

Agilent 1100 Series micro well-plate autosampler

The application field of the Agilent 1100 Series micro fractioncollection system covers a broadrange of accessible sample vol-umes. Therefore, it is essentialthat the autosampler included inthe micro fraction collection sys-tem is able to extract very lowsample as well as larger samplevolumes with high precision. TheAgilent 1100 Series micro well-plate autosampler offers two dif-ferent sample loops, an 8-µL and a40-µL loop. The injection volumesare set in 10-nL steps beginning at20 nL. The recommended mini-mum injection volume is 200 nL.The injection precision was deter-mined by 1-µL injections of a100 fmol BSA digest and measure-ment of detected peak areas ofselected ions with ion trap MS.Under these conditions the RSD ofthe peak area was between 2.58 %and 3.45 %8. Due to the high valueand limited volume of some sam-ples it is also very important thatthe autosampler is able to extractas much sample volume as possi-ble from a given volume out of thesample vial. In this case, with avial-optimized method, the Agilent1100 Series micro well-plateautosampler is able to extract

HeaterThermocouples

T1 T2

Valve

Core Plate

Seat

Decoupling pin

Magnetic system

Column flow

Primary flow

Waste flow

c

a

b

ing procedure only < 0.01 % carryover of azithromycin couldbe detected compared to 0.95 %without this procedure6.

Summary of the main features:• Two different loop sizes – 200 nL

minimum injection volume in 10-nL steps

• RSD of peak area is 2.58 % • Four full 2-µL injections out of

10 µL• Lowest carryover <0.01 % with

special cleaning function

nearly the complete sample vol-ume out of the vial. This can beaccomplished using the “bottomsensing” feature of the autosam-pler. To inject low volume samples300-µL wide open conicalpolypropylene vials (Agilent partnumber 5182-0549) and 100-µLpolypropylene inserts (Agilentpart number 9301-0978) arerecommended. The Agilent 1100Series micro well-plate autosampleris able to extract four full 2-µLinjections out of 10-µL samplevolume from these vial inserts7 .

Carryover of compounds betweendifferent samples is always acritical issue for the autosampler.To determine the carryover for asmall pharmaceutical molecule aswell as for a large peptide severalexperiments were carried out7,8.Azithromycine, a macrolide antibi-otic compound, which is very criti-cal for carryover because it sticksto the valve in the autosampler,was tested to develop a specialcleaning method to avoid carry-over from the autosampler in gen-eral. This procedure consists of acapillary wash prior to the injectionto clean the capillary exterior.

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After the elution of all samplecompounds the autosampler valveis switched two or three timesbetween the bypass and mainpathposition to clean the valve groovesin the high organic solvent stream.Figure 9 shows the resultsobtained using this cleaningmethod are shown in the extract-ed ion chromatograms. After theinjection of 2-µg azithromycinewater was injected to evaluate thecarryover. Using the special clean-

Azithrmycine

O

OCH3

CH3

H3C

HO

HO

H3C

H3C

H3CN

CH3CH3

CH3

CH3

CH3 CH3

CH3

CH3

O

O O

HO

OH

HOO

O

N

Intensity

2

4

6

8

x108

Base peak chromatogram

0

2

4

x106

5 10 15 20 25 30 35Time [min]

0

Sample injection 2 µg azithromycine

0.01 % carryover in blind solvent injectionwith 10 seconds needle wash and injector program

Figure 9Analysis of azithromycine and evaluation of carryover using a special cleaning procedure

The Agilent 1100 Series diode-array detector with 500-nL and 80-nL flow cell

An Agilent 1100 Series diode-arraydetector equipped with an easilyexchangeable 500-nL or 80-nL flowcell is integrated into the Agilent1100 Series micro fraction collectionsystem for peak detection and fortriggering the micro fraction collec-tor in the peak-based fractioncollection mode. Both flow cellshave an optimized geometry andflow path for minimized dispersion,high sensitivity and high resolution.Due to the low internal cell volumein the 80-nL flow cell, the peakwidth, the resolution and thechromatographic plates areimproved compared to the 500-nLcell. More details about both flowcells are outlined in table 2 anddescribed in a separate Applica-tion Note9.

Conclusion

The Agilent 1100 Series microfraction collection system is a toolfor fractionation of samples in thelower micro liter range with lowestdelay volume and peak dispersion.The fractions can be collectedeither in a high precision time orpeak based manner using the“liquid contact control” mode,which precisely deposits smallamounts of liquid with a minimumof 2 µL. To protect valuable samplesfrom decomposition it is possibleto equip the micro fraction collec-tor with an additional cooler.Therefore, the Agilent 1100 Seriesmicro fraction collection systemcan be used in proteomics,metabolomics and natural productpurification. The Agilent 1100Series micro fraction collectionsystem includes the Agilent 1100Series capillary pump, whichdelivers high precision micro literflow rates, the Agilent 1100 Seriesmicro well-plate autosampler, whichis capable of injecting samplevolumes in a broad range suitableto a large field of applicationsrequiring highest precision andlowest carry over and the Agilent1100 Series diode-array detectorwith the 500-nL or the 80-nL flowcell for highest sensitivity andresolution.

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Flow cell Path length Operating Sensitivity of Noise of Recommended Recommendedvolume [µL] [mm] range [bar] standard cell standard cell flow rate [µL] column [mm i.d.]

500 10 0 - 50 1/3 3 x * 50 - 200 1 - 2

80 6 0 - 50 1/10 10 x** 0 - 50 0.3 - 0.5* +/- 30µAU = 60µAU according to ASTM methode at 254 nm ** +/-100µAU = 200 µAU according to ASTM methode at 254 nm

Table 2Performance of the 500-nL and 80-nL flow cells

6.Boer HJ. “Mass flow controlledevaporation system.” 5 (C5):

961-966, J. Physique IV 1995.

7. “Performance characteristics ofthe Agilent 1100 Series capillaryLC system using diode-array UVand MS for detection.” Agilent

Application Note, Publication

Number 5988-7511EN, 2002.

8. Performance characteristics of theAgilent 1100 Series Nanoflow LCsystem for MS. Agilent Application

Note. Publication Number

5988-9010EN, 2004.

9.“Performance of the 80-nL UVflow cell”, Agilent Application

Note, Publication number

5989-0642EN, 2004.

The information in this publication is subject tochange without notice.

Copyright © 2004 Agilent TechnologiesAll Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited, except as allowed under the copyright laws.

Published May 1, 2004Publication number 5989-0954EN

www.agilent.com/chem/1100

Edgar Nägele is Application

Chemist and Martin Vollmer

is R&D Biochemist,

both at Agilent Technologies,

Waldbronn, Germany

References

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number 5989-0718EN, 2004.

2. “Improved protein identification:Off-line multidimensional LC/MSas an effective tool for proteomicsresearch”, Agilent Application

Note, publication number 5988-

9913EN, 2003.

3. Keil O., LeRiche T., Deppe H.,Volmer D.A. Rapid Commun.

Mass Spectrom. 16: 814-820,

2002.

4. Hillisch A., Hilgenfeld R. (Eds.)“Modern Methods of DrugDiscovery.” Birkhäuser Verlag

Basel, Boston, Berlin, 2002.

5.“Innovative fraction collectionwith the Agilent 1100 Series purifi-cation platform”, Agilent Applica-

tion Note, Publication number

5988-9250EN, 2003.