peptide and protein analysis by capillary hplc ... hplc – optimization of chromatographic and...
TRANSCRIPT
Peptide and protein analysis by capillary HPLC – Optimization of chromatographic and instrument parameters
Application
Abstract
The analysis of biologically active peptides, peptides from digests and
proteins is influenced by many parameters – typical chromatographic
parameters, for example, column and gradient, but also instrumental
parameters, such as pump, injector or detector setup. The Agilent 1100
capillary system is designed such that it can be treated and set up like a
standard LC instrument. Having optimized all instrumental and chro-
matographic parameters, the relative standard deviation for retention
times for peptides and proteins can be expected to be smaller than 0.5
%. This also allows to use retention times in long shallow gradients for
confirmation of peak identity, complementary to MS detection.
Angelika Gratzfeld-Huesgen
This application hasbeen verified using anAgilent 1200 SeriesLC system, and
showed comparable oreven better performance.
Application AreaPeptide and protein analysis
The samples used were purchasedfrom Agilent, Sigma and Michrom:- Sigma HPLC Peptide standard
H-2016- Sigma HPLC Protein Standard
H-2899- Sigma Lysozyme L-6876- Sigma Cytochrom C-7752- Sigma Myoglobin M-1882- Sigma Ribonuclease R-5503- Sigma Insulin I-5500- Sigma Met-Lys-Bradykinin B-5014- Sigma Leu-Enkephalin E-8757- Sigma Met-Enkephalin M-6638- Sigma Angiotensin II A-9525 - Agilent Angiotensin I G
1982-85000- Michrom tryptic digest of
Apotransferrin 910/000/12/26
The amino acid sequence of theused peptide and the formula andmolecular weight ranges of peptideand proteins are listed in table 1.
Equipment
For the experiments the Agilent1100 capillary LC system used con-sisted of:
• Micro vacuum degasser with aninternal volume of 1 ml for fastsolvent changeover
• High-pressure capillary pump forstable flow rates in the low µlrange
• Micro well-plate autosampler forprecise injections in the nl- andµl-ranges with sample coolingdevice to avoid sample decom-position
• Thermostatted column compart-ment for stable retention times atany temperature between 10 °Cdegrees below ambient and 80 ºC.
• Diode array detector with a 500 nlcell for sensitive analysis of smallpeak elution volumes
Introduction
Capillary reversed phase HPLChas become an indispensable toolfor analysis of peptides, proteinsand digests. Columns with smallinternal diameter provide improvedsensitivity when sample volumeand/or sample concentration is very low. In 1999 Agilent Technologies introduced the Agilent 1100 Series capillary LCsystem, which fulfills theserequirements, offering excellentperformance and robustness.
The Agilent 1100 Series capillarypump is based on the Agilent 1100Series high-pressure binary pump.Flow rates down to 1 µl/min aredelivered using an electronic flowcontrol (EFC) device, which con-sists of an electromagnetic pro-portioning valve and an electronicflow sensor. The column flow isactively measured and kept stableeven at changing pressures.4
In this Application Note wedemonstrate how chromatographicand instrument parameters can beoptimized for best performancewhen analyzing peptides, proteinsand digests. The influence of thefollowing parameters is discussed:
Chromatographic parameters• column• mobile phase• gradient• column temperature• different pHInstrument parameters• injector• detector• pump • column compartment
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Amino acid sequence Formula and molecular weight ranges
Met-Enkephalin: Try-Gly-Gly-Phe-MetLeu-Enkephalin: Try-Gly-Gly-Phe-LeuLeu-Enkephalin-Arg: Tyr-Gly-Gly-Phe-Leu-ArgAngiotensin 1: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-LeuAngiotensin 2: Asp-Arg-Val-Tyr-Ile-His-Pro-PheMet-Lys-Bradikinin: Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Gly-Tyr and Val-Tyr-Val: FW 239- 381Met-Enkephalin, Leu-Enkephanlin and Leu-Enkephalin-Arg: FW 557-713Met-Lys-Bradikinin, Angiotensin 1and 2: FW 1046-1320Apo-myoglobin: MW ~ 17800Cytochrom c and Ribonuclease a: MW ~12173- 13700Holo-transferrin: MW ~76000 - 81000
The following capillary columnswere used:1. Zorbax 150 x 0.3 mm 300Å
Extend C-18, 3.5 µm, (Agilent part number 5065-4464). Thesecolumns incorporates bidentate
silane, combined with a double-endcapping process that pro-tects the silica from dissolutionat high pH.
2. Zorbax 150 x 0.3 mm 80Å XDB C-18, 5 µm (Agilent part number
Table 1Amino acid sequence and formula and molecular weight ranges of peptides and proteins
5064-8291). The densely-bonded,double endcapped silica surfaceminimizes any silanol interactions.
3. Zorbax 150 x 0.3 mm 80Å SBC18, 5 µm (Agilent part number5064-8255). SB columns aremade using bulky unique silanesthat sterically protect the silox-ane bond. Stable bond materialis not endcapped.
4. Zorbax 150 x 0.3 mm 300Å SBC18, 3,5 µm (Agilent part number 5064-8267).
5. Zorbax 150 x 0.3 mm 300 SB C-8,3.5 µm (Agilent part number5065-4460).
6. Zorbax 150 x 0.3 mm 80ÅEclipse XDB C-18, 5 µm, (Agilent part number 5064-8291).The densely-bonded, double-endcapped silica surface minimizesany silanol interaction and pro-tects the silica support from dissolution.
7. Hypersil 120Å ODS C-18 150 x0.3 mm, 5 µm (Agilent part number 5064-8290). A very polarpacking, adds a monolayer ofoctadecyl silane (C-18) to theHypersil support. Columns areendcapped to reduced silanolinteraction.
For more column informationrefer to literature reference 1,which is a reference guide forcolumns and supplies1. This guideis updated on a yearly base andcontains detailed informationabout different column properties.
Results and discussion
Influence of different column materialsReversed phase columns are frequently used for the separation
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of peptides, proteins and trypticdigests, since reversed phase provides optimum resolution. It has been demonstrated thatreversed-phase HPLC providesseparation of polypeptides of near-ly identical sequences.2 The sepa-ration mechanism of peptides andproteins on reversed phase col-umn is based on small differencesin the “hydrophobic foot” of thepolypeptide, based on differencesin amino acid sequences and differences in confirmation.2
Whereas the separation mechanismof small peptide on reversed phaseis assumed to be a mixture of
adsorption and partition, the sepa-ration of big proteins is based onadsorption only.3 The elution ofthese big molecules dependsmainly on the organic phase con-centration. If for one of these mol-ecules, which is adsorbed at thesurface of the column material,the critical percentage of organicphase is reached the moleculeleaves the surface and is trans-ferred to the detector without further interference with the stationary phase.
To demonstrate the influence ofdifferent column material on the
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120 Å Hypersil ODS C-18, 5 μm
300 Å SB C-8, 3.5 μm
80 Å Eclipse XDB C-18, 5 μm
300 Å SB C-18,5 μm
80 Å SB C-18, 5 μm
300 Å SBC-18, 3.5 μm
300 Å Extend C-18, 3.5 μm
1 = Gly-Tyr, 5 ng/100 nl 4 = Leu Enkephalin, 20 ng/100 nl2 = Val-Tyr-Val, 20 ng/100 nl 5 = Angiotensin II, 20 ng/100 nl3 = Met Enkephalin, 20 ng/100 nl
Figure 1 Analysis of a peptide standard on different capillary columns of the same length and internal diameter.
Chromatographic conditions
Column flow: 5.5 µl/minPrimary flow: low solvent consumption: 200-500 µl/min
(Optimum pump settings will be explained later.)Mobile phases: water + 0.05 % TFA, pH = 2.2 = A, acetronitrile + 0.045 % TFA = BGradient 0.5 % B/min: at 0 min = 1 % B, at 60 min = 31 %B, at 70 min = 50 % B,
at 75 min = 85 % B, at 80 min = 85 % B, at 81 min = 1 % B, at 110 min = 1 % BDAD UV detector: 206/10 nm, ref 450/80 nmInjection volume: 0.1 µl, automatic delay volume reduction was activated(will be explainedlater)Column temperature: 30 °C
separation of polypeptides sevendifferent capillary columns were tested. All tested capillarycolumns are available from Agilent Technologies. The followingdifferent column material was tested:
• Different C-18 materials, silicamaterial end-capped vs. non end-capped, polar vs. non-polar
• Different pore size, 80Å vs. 300Å• Different polarity of reversed
bonded phases, C-18 vs C-8
Figure 1 shows an overlay of chro-matograms of a peptide standard,analyzed on seven differentreversed phase columns. TheHypersil ODS material, which is apolar packing, gives long retentiontimes and tailing peak shape. Thelast peak, which is Angiotensin IIwith a formular weight of 1046.2already shows a significant tailing.All other tested columns with lesspolar material provide better peakshapes. The Zorbax 300Å Extendmaterial and all other 300Å showshorter retention times and goodpeak shape. All tested 300Å materials are able to separate theinjected compounds under theconditions used. The 80Å materialsalso show good peak shape andshort retention times but are notable to completely separate compounds 4 and 5. The 300Å C-8 material is also able to separate all peptides and together with the300Å Extend C-18 material offersthe shortest retention times.In figure 2 another peptide standardwas analyzed using the samecolumns and the same chromato-graphic parameters as in figure 1.The polar ODS Hypersil columnalso shows long retention timesand peak tailing. The 300Å C-8 and
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120 Å Hypersil ODS C-18, 5 μm
300 Å SB C-8, 3.5 μm
80 Å Eclipse XDB C-18, 5 μm
300 Å SB C-18, 5 μm
80 Å SB C-18, 5 μm
300 Å SBC-18, 3.5 μm
300 Å Extend C-18, 3.5 μm
10 ng/100 nl each1 = Leu Enkephalin Arg 4 = Angiotensin II2 = Met Enkephalin 5 = Angiotensin I3 = Met LysBradikinin
Figure 2 Analysis of another peptide standard using the same columns and chromatographic conditions as infigure 1.
80 Å Eclipse XDB C-18, 5 µm
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80 Å SB C-18, 5 µm
300 Å Extend C-18, 3.5 µm
300 Å SB C-18, 5 µm
300 Å SB C-8, 3.5 µm
120 Å Hypersil ODS C-18, 5 µm
10 ng/100 nl each1 = Ribonuclease a 3 = Holo-transferrin2 = Cytochrome c 4 = Apomyoglobin
Figure 3 Analysis of proteins on six different capillary columns with a length of 150 mm and an internal diameter of 0.3 mm.
Chromatographic conditions
Column flow: 5.5 µl/minPrimary flow: low solvent consumptionMobile phases: water + 0.05 % TFA = A, acetonitrile + 0.045 % TFA = BGradient: At 0 min = 20 % B, at 60 min = 85 % B, at 61 min = 90 % B, at 65 min = 90 % B,
at 66 min = 20 % B, at 80 min = 20 % BDAD UV detector: 206/10 nm, ref 450/80 nmInjection volume: 100 nl, automatic delay volume reduction activatedColumn temperature: 30°C
the 300Å Extend C-18 offer theshortest retention times. Here onlythe 80Å C-18 columns are able toseparate all compounds to baseline.Obviously it is not possible to recommend just one column forthe separation of peptides with MW < 2000. Further experiments arestill necessary to find the optimum column for a specific application.In the beginning of method devel-opment it makes sense to test threeor four columns of the samelength, using the same chromato-graphic conditions, and to comparethe separation efficiency on:
• Non-polar packing materials withpore size of 300 vs. 80Å
• C-18 vs C-8 bonded phase material or others bonded phaseslike phenyl or C-4
This should help to find the optimum column for a specific application.
The next experiments show theseparation of proteins with molec-ular weights > 10000. Again, pre-diction based on molecular weightis not possible. Holo-tranaferrin,which is by far the biggest mole-cule is not the last one in the chro-matogram, as may be expected.Figure 3 shows the chromatogramsof a protein standard, analyzed onsix different columns. It is quiteobvious that the polar Hypersil col-umn is not suited for this type ofapplication. Very good peak shapecould be achieved on the 300Extend C-18 column. This columnis specifically designed forreversed-phase separation of pep-tides, proteins and the analysis ofpeptide fragments from enzymaticdigests.
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For protein analysis it is importantthat the column used has “seen”proteins before. This means thatseveral injections of a protein stan-dard should be done onto a newcolumn. If after several injectionsthe chromatograms all look thesame and retention time precisionis at least below 0.5 % RSD, calibra-tion and sample runs can be started.
Figure 4 is an example of how different chromatograms appear on a new column and after severalprotein runs. Not all columns showsuch a significant difference.Columns, which are speciallydesigned for protein analysis, show less difference for the firstand later runs. Having selected thecolumn, other parameters suchmobile phase, gradient, concentra-tion of modifier detection wave-length, pH and column tempera-ture have to be optimized. For all
further experiments we will usethe column 150 x 0.3 mm 300ÅExtend C-18, 3.5 µm.
Influence of mobile phase fluctuations on retention timesFor all chromatograms abovewater and acetonitrile with trifluo-racetic acid (TFA) as modifierwere used. TFA is the preferredion-pair reagent for best peakshape. Another advantage is thatTFA absorbs in the UV rangebelow 200 nm and thereforeshows minimal interference withdetection of peptides above 200 nm.One disadvantage of TFA is that itcauses baseline drift due to spec-tral absorption differences withchanging acetonitrile percentage.To reduce this drift as much aspossible the TFA concentration inorganic phase, here acetonitrile,should be 15 % lower than in thewater phase.
First injection of proteinsonto a new column
Fifth injection of proteinsonto a new column
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60150 x 0.3 mm 80 Å Eclipse XDB C-18, 5 µm
1 = Ribonuclease a2 = Cytochrome c3 = Holo-transferrin4 = Apomyoglobin
Figure 4 Change of chromatogram from the first protein injection to the fifth injection of proteins, chromatographic conditions as figure 3, column 150 x 0.3 mm 80Å Eclipse XDB C-18, 5 µm.
Unfortunately, TFA is not the bestchoice for mass spectrometers,because it suppresses the ion generation. Ion suppression canbe partially overcome by loweringthe TFA concentration in themobile phase. For some columnslowering the TFA concentrationmight lead to broader and tailingpeaks, however the 300Extend C-18 column does not have such aproblem, as shown in figure 5. In this case lowering the TFA concentration resulted in the separation of the first two peaks,which were not separated usingthe higher TFA concentration.
In figure 6 an example is given onhow lower TFA concentrationinfluences the separation of pep-tides from a myoglobin digest. Noadditional peak tailing can beobserved and separation efficien-cy is also not negatively effected.Nevertheless, in MS applications ithas become good practice to useformic acid (FA) as modifierinstead of TFA. Formic acid is alsovolatile and does not suppress iongeneration like TFA.
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Gradient: 0.5 % B/min
10 ng/100 nl each1 = Met Enkephalin 4 = Angiotensin II2 = Leu EnkephalinArg 5 = Angiotensin I3= MetLysBradikinin
Figure 5Analysis of peptides at different TFA concentrations, all other conditions are the same as in figure 1.
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10203040506070
0.05 % TFA
0.01 % TFAColumn: 150 x 0.3 mm 300 Å
Extend C-18, 5.5 µm Gradient: 0.5 % B/min
Figure 6Analysis of myoglobin digest using different TFA concentrations. All other conditions are the same as in figure 1, except that the injection volume was 3 µl, which represents an injection of 3 pmol.
Figures 7, 8 and 9 show the influ-ence of formic acid on peak shapeand elution series of a peptidestandard, a digest and a proteinstandard. Analyzing peptides andproteins with formic acid insteadof TFA may lead to broader peaksand different elution times for theinjected compounds. In our exam-ple the elution order has changed.Met-Lys-Bradikinin has moved tolower retention time and nowelutes between Leu-Enkephalin-Arg and Met-Enkephalin. This isdue to the fact that Bradikinin has2 Arg in its sequence which bothform ion pairs with TFA. Thisresults in longer retention timesfor Bradikinin, if TFA is used asmobile phase modifier.
Using formic acid as mobile phasemodifier for the analysis of pep-tides shows no or only minor disadvantages for peak shape. For big proteins as seen in figure 7formic acid is not ideal.
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0100200300400500 Mobile phase:
Water/ACN with TFA
Mobile phase: Water/ACN with 0.1 % formic acid
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10 ng/100 nl each
1 = Met Enkephalin2 = Leu EnkephalinArg3 = MetLysBradikinin4 = Angiotensin II5 = Angiotensin I
Figure 7Analysis of a peptide standard using different mobile phases, all other conditions are the same as in figure 1.
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Mobile phase: Water/ACN with TFA
Mobile phase: Water/ACN with 0.1 % formic acid
Figure 8Analysis of a peptides from a Myoglobin digest standard using different mobile phases. All other conditions are the same as in figure 1, except that the injection volume was 3 µl, which represents an injection of 3 pmol.
Influence of gradient settingsAs already mentioned smallchanges for chromatographicparameters can influence peptideor protein separation significantly.This is definitely true if gradientsettings are changed. In figure 10two different gradients wereapplied to the same sample. Theshallower gradient gives at least apartial separation of the first twopeaks. Using shallow gradient alsoprolongs retention times signifi-cantly. For specific peptide sepa-rations it is of advantage to testmore rapid gradients first. Thiscan help to save cycle times, labortime and costs.
Using shallow gradients for pep-tide maps offers a much betterseparation performance for theanalysis of peptides from a Apotransferrin digest (figure 11).Shallow gradients for peptidemaps are recommended, if opti-mum separation for identificationshould be obtained.
Typically, shallow gradients pro-vide better resolution for peakclusters. In the example above the peak cluster at approximately20 and 40 minutes show betterresolution with 0.25 % organicphase increase per minute.
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Mobile phase: Water/ACN withTFA
Mobile phase: Water/ACN with 0.1 % formic acid
Time [min]0 5 10 15 20 25 30 35 40 45
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1 = Ribonuclease a2 = Cytochrome c3 = Holo-transferrin4 = Apomyoglobin
Figure 9Analysis of a peptides from a protein standard using different mobile phases, all other conditionsare the same as in figure 3.
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Gradient: 0.5 % B/min
Gradient: 0.25 % B/min
10 ng/100 nl each1 = Met Enkephalin 4 = Angiotensin II2 = Leu Enkephalin Arg 5 = Angiotensin I3 = Met LysBradikinin
Figure 10Analysis of peptide standard using different gradients. All other conditions are the same as infigure 1 except the gradient. The gradient was changed to reach 31 % B in 120 min to provide an increase for the gradient of 0.25 %/min.
Influence of column temperatureDifferent column temperaturescan also be an important tool toimprove separation efficiency. Infigure 12 the influence of the col-umn temperature on the separa-tion of a peptide standard isshown. In this example increasingthe column temperature providesbetter separation of the first twopeaks. A further effect is that therun time could be shortened by 10minutes.
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0.25 % B/min
150 x 0.3 mm 300 Å Extend C-18, 3.5 µm
3 pmol 0.5 % B/min
Figure 11 Analysis of Apotransferrin digest using different gradients. All other conditions are the same asin figure 1 except the gradient. The gradient was changed to reach 31 % B in 120 min to providean increase for the gradient of 0.25 %/min. Injection volume was 3 µl.
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Column temperature: 30º C
Column temperature: 60º C
10 ng/100 nl each
1 = Met Enkephalin2 = Leu Enkephalin Arg3 = Met LysBradikinin4 = Angiotensin II5 = Angiotensin I
Figure 12Analysis of peptide standard at different column temperatures, all other conditions are the sameas in figure 1.
Influence of different pHThe separation of peptide is alsostrongly influenced by the pH, dueto protonation and deprotonationprocesses of basic or acidic sidechains. Figure 13 shows howstrong the influence on the elutionof peptides can be. A peptide stan-dard was analyzed using 0.001 mNaH2PO4 with pH 7 and 0.05 %TFA modifier with pH 2.2 asmobile phases. The elution seriesis completely different for Met-Enkephalin, Leu-Enkephalin andAngiotensin II, if the pH changesso drastically. The smaller pep-tides are less effected in this case.It is also obvious that with thehigher pH the retention times shiftto shorter value.
Detector settings for the Agilent 1100Series diode array detectorFor combination with low flowrates the 1100 Series diode arraydetector (DAD) is equipped with a500-nl cell, which provides lowestpeak dispersion in combinationwith capillary columns with idsdown to 300 mm. The 1100 SeriesDAD offers the possibility toacquire several UV signal simulta-neously. Figure 14 shows how dif-ferent UV signals can provideadditional information.
In addition to the absorptionaround 206 nm cytochrome has amaximum at 400 nm. Proteins con-taining Trypthophan or/and Tyro-sine also show some absorption at280 nm. In combination with forexample MS data, this can help toidentify peptides or proteins moreeasily.
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1 = Gly-Tyr, 5 ng/100 nl2 = Val-Tyr-Val, 20 ng/100 nl3 = Met Enkephalin, 20 ng/100 nl4 = Leu Enkephalin, 20 ng/100 nl5 = Angiotensin II, 20 ng/100 nl
pH = 70.001 m NaH2PO40.5 % B/min
pH = 2.20.05 % TFA0.5 % B/min
Figure 13Analysis of a peptide standard at different pH, all other conditions are the same as in figure 1.
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UV spectrum Cytochrome c
Absorbance[mAU]
UV spectrum ofRibonuclease a
1 = Ribonuclease a2 = Cytochrome c3 = Holo-transferrin4 = Apomyoglobin
Sig: 400/20 nm
Sig: 280/20 nm
Sig: 206/10 nm
Figure 14Analysis of a protein standard at different wavelength, all other conditions are the same as infigure 3.
Injector settings for the Agilent 1100capillary instrumentIn its standard version the Agilent1100 Series micro-well plate sam-pler is equipped such to allowinjection volumes from 0.01 up to8 µl. Changing the loop capillaryextends the injection volume to 40 µl. Typically this gives suffi-cient flexibility in the analysis ofpeptides, proteins and digests. The area precision for differentinjection volumes is given intable 2. For more informationrefer to literature reference 4.
The lower the flow rates the moreimportant the system delay vol-ume gets. For a capillary systemsmall delay volumes betweenpoint of injection and columnhead and column end and detectorcell are of utmost importance forlow peak dispersion. Also, thedetector cell should have a lowvolume. The delay volumes for the 1100 Series capillary systemare 0.08 µl before and after thecolumn. The internal volume ofthe detector is 500 nl. For lowestdelay volume it is recommendedto activate the “automatic delayvolume reduction” function in the setup screen for the microwell-plate sampler. This allows to bypass the autosampler delayvolume after the sample hasreached the top of the column.This saves 300 µl delay volume ofthe autosampler and is the mainstep to reduce the system delayvolume. The delay time is about 50 % lower.
Pump settingsThe column flow is set according tothe internal diameter of the column.The primary flow rate, whichdetermines the flow rate deliveredby the pump up to the electromag-netic proportioning valve, can beselected within three ranges.
1. Low solvent consumption, 200 to 500 µl/min
2. Medium solvent consumption,500 to 800 µl/min
3. High solvent consumption, 800 to 1300 µl/min for rapid gradient changes
In the electromagnetic proportion-ing valve the flow is split into thecolumn flow and the “waste”flow. The range selected for the primaryflow depends on the application.For typical peptide and proteinanalysis the first range is selected.If speed of analysis is an importantissue range 2 or 3 can be selected.
Other important pump settings,which influence pump perfor-mance, are compressibility andthe calibration settings for theflow sensor. Because the flowmeasuring process is based onheat capacities, each solvent mixneeds its individual calibrationcurve, which is available from theChemStation or control modulefor most common solvents andsolvents mixtures. Also, for thecompressibility parameter theChemStation proposes values forthe most common solvents andsolvent mixtures.
Having entered all settings accord-ing to the used solvents, the preci-sion of retention times is < 0.3 %relative standard deviation (figure 15).
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Table 2Precision of areas at different injection volumes
Injection volume of Met-Enkephalin (ml) RSD retention times in % RSD areas in %(concentration = ~1ng/µl) (n=6 each) (n=6 each)
0.03 0.14 7.590.1 0.19 2.690.5 0.06 1.91
Recommendations for the chromatographic parameters are somewhat more complex.• A good start is to select a capil-
lary column which is speciallydesigned for the separation ofpeptides and proteins, such asthe Agilent 300 Extend C-18 capillary column which is available in different lengths.
• The next step is to decide whichmobile phase is appropriate forthe detector. If a mass spectrom-eter is used a good choice is touse 0.1 % formic acid as modifier.If a UV detector is used, Trifluoro-acetic acid should be used asmodifier.
• If the peptides of a digest gradi-ent are be analyzed a shallowgradient is recommended. Forthe separation of known pep-tides or proteins it is of advan-tage to select a gradient whichprovides sufficient separation incycle times as short as possible.
• If the separation cannot beachieved with the first set ofparameters it is recommendedto change the gradient first, fol-lowed by changing the columntemperature, mobile phase con-centration and/or pH.
• If a separation can still not beachieved a different stationaryphase with other pore size and/or differently bonded phaseshould be selected.
Column compartment set upIt is recommended to connect theflow connection capillary from the injection valve directly to thecapillary column and not to themobile phase pre-heating device.This saves run and cycle time andthe compounds do not have contactwith metal surface of this device.The capillary column should beinstalled with column holder to provide good contact with heatzone.
Conclusion
The analysis of peptides, peptidesfrom digests and proteins is influ-enced by many parameters. Theseinclude typical chromatographicparameters, such as column andgradient, but also instrumentalparameters, for example, set up ofpump, injector or detector. In prin-ciple there is no significant differ-ence to standard, narrow-bore ormicro-bore HPLC, except that thedimensions are different.
The Agilent 1100 Series capillaryLC system is designed such that itcan nearly be treated and set uplike a standard LC instrument.Only a few settings should beselected with care and the recom-mendations given by the helpfunction should be followed.
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1
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Peak RSD RT RSD Area 1 0.26 0.702 0.06 0.70 3 0.06 0.90 4 0.04 1.30
Figure 15Precision of retention times for peptide of a myoglobin digest. All other conditions are the same as in figure 1 except the injection volume with 3 µl.
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Recommended start conditions for reversed phase separation
Parameter Peptides with MW<2000 DA Peptides/proteins >2000 DA
Column selection 150 x 0.3 mm Zorbax 80 Å SB-18 150 x 0.3 mm Zorbax 300 Å or/and Extend C-18150 x 0.3 mm Zorbax 300 Å Extend C-18
Flow rate 5.5 µl/min 5.5 µl/min
Mobile phase For UV detectors: water and For UV detectors: water and acetonitrile with 0.05 %TFA as acetonitrile with TFA asmodifier modifierFor MS detectors: water and For MS detectors: water and acetonitrile with 0.1 % formic acetonitrile with formic acid acid as modifier as modifier
orFor MS detectors: water and acetonitrile with 0.01% TFA as modifier
Gradient 1 to 31% ACN with 0.5 %/min 20 to 85 % ACN with ACN increase ~1.1%/min ACN increase
Table 4 Recommended start conditions
Having optimized all instrumentaland chromatographic parametersthe relative standard deviation forretention times can be expectedtypically < 0.5 % for a runtimerange from 0 to 100 minutes. The area precision for a digestwith an injected concentration of3 pmol and an injection volume of 3 µl is typically < 2 % RSD.Tables 3, 4 and 5 should help tofind optimum separation condi-tions quickly and conveniently.The optimization procedure is limited to reversed phase applications.
Selection of separation mechanism
Peptide with MW<2000 DA Peptides/proteins Membrane proteinswith MW>2000DA
Reversed phase separation Reversed phase separation, Ion exchange separationlimit is ~30 – 36 k DA oror Size exclusion with detergentsIon exchangeorSize exclusion
Table 3 Recommendation for selecting the best column material
Recommended optimization process of start conditions
Parameter Known peptides and proteins Peptide maps with unknown compounds
Gradient If the start conditions do not More shallow gradients with provide baseline separation, 0.25 %/min ACN increase are use shallow gradients with recommended.0.25 %/min ACN increase.
Oven temperature Start temperature is typically Typically 40 ºC are used40 ºC. For improving separation temperature can be raised to 60 ºC
Modifier concentration Increase and decrease of Typically TFA concentration modifier concentration can be ≤ 0.1 % is used for UV detectionused to optimize separation.
If an MS is used as detector 0.1 % formic acid is a recommended concentration
pH The pH can be varied from Acidic conditions are typically acidic to basic conditions as used, such as pH=2.2 with far as the column can tolerate 0.05 % TFAthe change
Table 5 Recommended optimization process
References
1.Chromatography and Spectros-copy Supplies, Reference GuideAgilent publication number 5988-4785EN, 2002.
2.J.Rivier, R.McClintock,”Reversed-phase high performance liquidchromatography of insulins fromdifferent species”, J.Chrom.268,
pp 112-119, 1983.
3.X.Geng, F.E.Regnier, “Retentionmodel for proteins in reversed-phase liquid chromatography”,J.Chrom. 296, pp15-30, 1984.
4.“Performance Characteristics ofthe 1100 Series capillary HPLCsystem using diode-array UV andmass spectrometers as detectionsystem”, Agilent Technical Note,
publication number 5988-7511EN,
2002.
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© 2003 - 2007 Agilent Technologies
Published May 1, 2007Publication number 5988-8628EN
www.agilent.com/chem/1200
Angelika Gratzfeld-Huesgen
is Application Chemist at
Agilent Technologies, Waldbronn,
Germany.