Fast peptide separations at elevated temperatures by ... · 16th International Symposium on Column Liquid Chromatography - June, 1992 Poster #140 Fast Peptide Separations at Elevated

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Poster Presentation

16th International Symposium on Column LiquidChromatography - June, 1992

Poster #140

Fast Peptide Separations at ElevatedTemperatures by Microbore HPLC

P. Young, H. Richardson, T. Wheat and G. VellaMillipore Corporation, Waters Chromatography Division

34 Maple Street, Milford, MA. 01757 U.S.A.

MILLIPORE Waters Chromatography Division34 Maple StreetMilford, MA 01757508 478-2000

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:tABSTRACT '

Conventional gradient HPLC peptide separations are

performed on silica-based reversed phase columns having

alkyl bonded phases. Peptides are retained by the

hydrophobic packing material in the presence of aqueous

trifluoroacetic acid (TFA) and eluted with a gradient of

increasing organic solvent, usually acetonitrile. The

separation is normally performed at ambient or slightly

elevated temperature (~35°C). Peptide separations can be

optimized by adjustments in flow rate, column diameter and

length, particle size, gradient slope and temperature.

Separations with shallow gradients on long, narrow columns

packed with small particles provide maximum resolution, but

may require reduced flow rates and extended run times.

Separations on large-bore columns packed with large

particles can provide rapid separations through increased flow

rates, but often result in reduced resolution. When sample

amount is limiting, narrow bore (i.d.= 2 ram) and microbore

(i.d.=l ram) columns packed with small particles provide

increased sensitivity since peptides are eluted in a higher

concentration. At ambient temperature, separations on narrow

bore columns are performed at reduced flow rates (<1

ml/min). Operating at elevated temperatures will decrease

solvent viscosity and increase the fluidity of the alkyl bonded

phase (C18). This can result in altered selectivity and

decreased run times. Such alterations can lead to optimized

high-speed peptide separations on narrow bore columns.

.J

_'" MATERIALS AND METHODS

q

Tryptic Digestion

Cytochrome c (Sigma Chemical Co) from bovine heart

mitochondria (lmg/500_l) were suspended in 0.1M ammonium

bicarbonate (Sigma) buffer, pH 8.0. TPCK-treated Trypsin

(Worthington Biochemical Corp.) was dissolvedat a concentration

of 0.2mg/lml buffer. Trypsin solution (500111)was added to the

cytochrome c suspensions and incubated for 24h at 37°C.

Followingincubation,trypsin was deactivated by heating at 100°C

for 5 rain. Aliquots of 100 !_1were frozen (-20°C). Prior to HPLC,

digestswere diluted 1:10 with aqueousTFA,

HPLC System

Samples were chromatographed on a 625 LC System (Waters

Division of Millipore) equipped with a column heater and

autosampler (Waters model 715). Samples were analyzed by

photodiode array detection (Waters model 991) in a wavelength

range of 190-425 nm with 1.3 nm resolution. Peptide mixtureswere

separated on Waters columns (2 mm x 150 mm) packed with C4,

C8, or C18 Delta-Pak_ reversedphase supports. All particleswere

51_mwith 300A pore sizes.

i.

,.,, .,,' Effectof Temperature on Peptide Mapping: C4 Reversed Phase Supportb,

°-_I_5_:"-'.... ,... , ..., ..., . , ,................... Sample: Tryptic Digest

• __ Eluants: A=water/0/1B°vineCyt°chr°mecO__ (400 pmol)Column: C4 (2mm x 150 mm)n

-0 ; % TFA• :J35°C ........ i...................... _iiiiiiiiii ..."_..__ ...... B=acetonitrile/0.1%TF

Flow: 180 Id/minGradient: 0-60% B/120 min

(46 column volumes)

-0.01 ; ...........!..............................._..............................0.2

g_ t T

_t

< -0.01 _ .... ...... • .*-___0.22i65oC............................ . .................. ..

o.22|zs°c ...... _-I

0 _T-" _ • I ' '20

40 60 80

_ High-SpeedSeparationonNarrow-Bore(:4 FlowRate:180_l/min.Columnat 75°C Gradient:.0.5%B/rain.

0.4 columnvol./min.Time:80 min.

EluantsA = Water/0.1% TFAB = Acetonitrile/0.1% TFA

.b 8'O

Flow Rate: 360 _l/min.Gradient:.1% B/rain.

"E" 0.8 columnvol./min." Time:40 min.

o

J_D

2b .bI

FlowRate:720 IJI/min.Gradient:.2% B/min. I

1.6 columnvol./min.Time:20 min.

I

t I

Time (min.I Ib 20

_-_JuuLu, ,u_l=perature on repuge Mapping: (Jl_ Heversed Phase Support,(,,_ D

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0.22 ........... l.., ! i ....... i •, : ..... i , ,25oC ...... _......................................................... : ................. Sample: Tryptic Digest

_L Bovine Cytochrome c(400 pmol)Column: C18 (2mm x 150 mm)Eluants: A=water/0/1 % TFA-o.o1=,_,-,,,-,,__.. _ ............:............. - - ................................. ; ............................. B=acetonitrile/0.1% TFA0.2,_ "............... : ............................ ................................ ; .......................

35°6 _ ,_ Flow: 180 I.LI/min

Gradient: 0-60% B/120 rnin

L (46 column volumes)-o.o, ...... _....0.22 ..................................... : .............................. i..

q

2 0.22 ....... " ! _ "8 55°C

-0.01__ ____;_ _ _ - . - - -

0.22 6500 .... , ...................... :............................ : .......................... ,,,.._,

0.22 .... ".....................75oC ...................... : ......

-o.o,,:--,----,_-C---,.,;.. "-_- . ,.,( .... --1" I " " ! .... I .... I20 40 60 80

, High-SpeedSeparation•; onNarrow-BoreCls Column FLowRate:180IJI/min.Gradient:0.5% B/min.

at 75 ° C 0.4 columnvot./min.Time:80 min.

EluantsA = Water/0.1% TFAB= Acetonitrile/0.1% TFA

FlowRate:360 IJI/min.Gradient:.1% B/rain.

0.8 column vol./min.E Time:40 min.C

@UCoe,i

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2'0 4b

FlowRate:720 pl/min.Gradient:.2% B/min.

1.6 columnvol./min.Time: 20 rain.

Time(min.) lb 2'0

_ ";" ,. Selectivity Differences Between Reversed Phase Supports at 75°CSample: Tnjptic Digest

.... _ ....... L ..... l ............ _ , , i ................ i , Bovine Cytochrome c0 22 ..... :................................ (400 pmol)

C4 11 13 Eluants: A=water/0/l%TFA12 B=acetonitrile/0.1% TFA

9 Flow: 180 ld/min6 Gradient: 0-60% B/120 min

5 8 (46 column volumes)

......... _.............................. o ......

--. 0.22E i .................... • .... i

'- C8 11 " 12 139I"-"

65O

,-" 2 4 8 10_ 7o 3

-0.01 ........ _ .............. -: ...... " " - .. : ............................. _ ............0.22 ....... :....................................................... '

'i ............................. i .........

C18 113

9. 126

2 451 _ 7 8_ 1_

o(_I f ........../_...............---- ..............................' ' " ' I ' " ''"r'--l'---'_-----'- " --'----I-" '-' ' '"-'--I " " '"" " I " " " • i ' ' '-" I '"--"--_--"-'--

0 20 40 60 80-lime (rain)

Q

Amino acid sequence for tryptic peptides derived from bovine

cytochrome c.

Peptide Bovine

number Sequence

1 (1-5) GDVEK

2 (74-79) YIPGTK

3 (73-79) KYIPGTK

4 (9-13) IFYQK

5 (39- 53) KTGQAPGFSYTDAN K

6 (40-53) TGQAPGFSY-I'DANK

7 (92-97) EDLIAY

8 (80-86) MIFAGIK

9 (28-38) TGPNLHGLFGR

10 (92-99) EDLIAYLK

11 (14-22) CAQCHTVEK (+Heme)

12 (56-73) GITWGEETLMEYLENPKK

13 (56-72) GITWGEETLMEYLENPK

1

0

Resultsfrom SequencingPeptide4 (IFYQK)

It is possiblethat exposureto hightemperature(75°C) may causedeaminationof glutamine (Q) to glutamicacid (E). To test the effectofhightemperatureon the recoveryof glutamine,peptide4 was isolatedfrom separationsrunat 35°C and 750Cand analyzedon aProSequencer_ (Milliporemodel6625) automatedpeptide sequencer.

PTH Standard

I , _ M "=, I ! J

2.69"_.47 , '

T=35°C S,OI

7,88-1.38.269_ sub _._, A _ ........

• .."-e._. V-_,_,

T=75°C

I

., -_3C.... -_ --- ...... - - -..... ------------ ----- " _- -_"--- --

These data indicatethat isolationof peptides havingamide-basedresidues are not adversely affected by purification at 75°C.

f :.i ¸

-X

Effectof Temperatureand,,:, GradientSlope:Separationof

Insulinon Narrow BoreCleColumnTemp:26°CFlow:0.2ml/min.Gradient:.1% B/rain.Time:60 rain.

EluantsA - Water/0.1% TFAB= Acetonitrile/0.1% TFA

JLI I

30 60

Temp: 75°CFlow:0.2mi/min.Gradient:.1% B/min.

_- Time:60 min.c

@

0

.,D.<

Temp:75°CFlaw: 1.2ml/min.Gradient:.6% B/min.Time: 10 min.

I

5 ]bIt Time (min.)

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I

CONCLUSIONS

• Fast peptide separations can be performed atelevated flow rates and temperatures on silica-

' based reversed phase supports.

• High-speed HPLC methodologies can be used toprepare peptides for sequencing and amino acidanalysis. These techniques can be useful torapidly monitor the progress of proteolyticdigestions, preparative processes, chemicalmodifications, and other on-going protocols.

• For columns operated at the same flow rate,increasing the operating temperature enhancedresolution. At 75°C, increasing the flow rate andgradient slope resulted in equivalentchromatograms in less time.

. For each chain length studied (C4,C8, or C18),increasing column temperature resulted inincreased resolution and altered selectivity.

4

139 Use of Short Microcolumn for Fast Analysis of Proteins and Peptides. RICHARD LUDWIG, Supelco,Inc., Supelco Park, Bellefonte, PA 16823-0048 USA.

The mass sensitivity and low solvent consumption of small diameter columns have been clearly demonstrated(1). The use of short 1.0mm diameter columns packed with wide pore materials for the rapid analysis of proteinand peptide microsamples will be demonstrated. These columns give high sensitivity/recovery with limitedsample sizes. In addition the low flow rates employed facilitate coupling with mass spectrometers. Whencoupled to MS/MS systems both molecular weight and sequence information can be obtained for peptides (2).(1)Small Bore Liquid Chromatography Columns: Their Properties and Uses, Vol. 72 Chemical Analysis Series,

R.P.W. Scott (Ed.) John Wiley & Sons, New York (1984).(2)E.C. Huang, T. Wachs, J.J. Conboy, and J.D. Henion, Anal. Chem., 62, 713A (1990).

:_L 140 Fast Peptide Separations at Elevated Temperatures by Mierobore HPLC. PATRICIA YOUNG',HAROLD RICHARDSON, THOMAS WHEAT, and GEORGE VELLA, Millipore Corporation, 34 Maple St.,Milford, MA 01757 USA.

Conventional gradient HPLC peptide separations are performed on silica-based reversed phase columns havingalkyl bonded phases. Peptides are retained by the hydrophobic packing material in the presence of aqueoustrifluoroacetic acid (TFA) and eluted with a gradient of increasing organic solvent, usually acetonitrile. Theseparation is normally performed at ambient or slightly elevated temperature (- 35 °C). Peptide separations canbe optimized by adjustments in flow rate, column diameter and length, particle size, gradient slope andtemperature. Separations with shallow gradients on long, narrow columns packed with small particles providemaximum resolution, but may require reduced flow rates and extended run times. Separations on large-borecolumns packed with large particles can provide rapid separations through increased flow rates, but often resultin reduced resolution. When sample amount is limiting, narrow bore (i.d. = 2 mm) and microbore (i.d. = Imm) columns packed with small particles provide increased sensitivity since peptides are eluted in a higherconcentration. At ambient temperature, separations on narrow bore columns are performed at reduced flowrates (< 1 ml/min). Operating at elevated temperatures decreases solvent viscosity and increases the fluidityof the alkyl bonded phase (C18). This can result in altered selectivity and decreased run times. Such alterationscan lead to optimized high-speed peptide separations on narrow bore columns.

141 Analysis and Process Control of Polymers by HPLC and GPC. STEPHAN ROSE*, MARIO MELZI,NICO VONK and WILL VERSTRAETEN, Chrompack Int. B.V., P.O. Box 8033,4330 EA Middelburg, THENETHERLANDS.

This paper describes two methods for polymer analysis which can be used for process control as well as for

quality control: 1. Low temperature PET analysis. 2. Analysis of isocyanate polymers. !Low temperature PET analysis. Polyethylene Terephtalates (PET) are used more and more as packagingmaterial for food and beverages. For this reason the product is subjected to various residual monomer anddegradation studies The GPC method presented allows molecular weight (MW) distribution analyses of PETat ambient temperatures and can be used as a MW- screening technique. Data analysis of the low MW fractiongives information about residual monomers, additives and degradation products. Analysis of the overall MWdistribution allows monitoring of polymer degradation.Thus the method described gives the complete information of both monomer content as well as molecular sizedistribution in one single analysis.Analysis of isocyan_[e polymers, The analysis of isocyanates is not only important for process control but alsofor environmental analysis, because of the toxicological problems related to the monomers and oligomers.The polymerization process of isocyanates like MDI and TDI can be monitored by low molecular weight GPC,as well as by RPLC. In this paper we compare both techniques. The RPLC method uses a specially modifiedODS column and can be used for rapid process control of oligomeric samples as well as for environmentalmonitoring. Both methods are compared on speed, resolution, MW data and application range.

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