reversed phase chroma

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Reversed PhaseChromatography

Principles and Methods

18-1134-16

Edition AA

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Reversed PhaseChromatography


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Contents1. Introduction .............................................................................................. 5

Theory of r eversed phase chrom atography ............................................... 6The matr ix ................................................................................................ 9The ligands ............................................................................................. 11Resolution in r eversed phase chromatography ........................................ 13Resolution .............................................................................................. 13Capacity factor ....................................................................................... 14Efficiency ........................... ........................... ........................... ............... 15Selectivity........... .......................... ........................... ........................... ..... 17Binding capacity .....................................................................................18

Critical parameters in reversed phase chromatography ........................... 19Column length ...................................................................................19Flow rate............................................................................................19Temperatur e ....................................................................................... 20Mobile phase .....................................................................................2 0Organic solvent..................................................................................20Ion suppression .................................................................................. 21Ion pairing agents .............................................................................. 22Gradient elution ................................................................................. 23

Mode of use ............................................................................................24Desalting............................................................................................24High resolution separations ............................................................... 25Large scale p reparative purification ................................................... 25Stages in a purification scheme .......................................................... 26Captu re .............................................................................................. 26Intermediate stages.............................................................................27Polishing ........................... ........................... ........................... ........... 27

2. Product Guide ......................................................................................... 29SOURCE RPC ........................ ........................... .......................... ........ 30

Product description ............................................................................ 30High chemical stability ......................................................................32Excellent flow/pressure characteristics................................................34High capacity.....................................................................................35Availability ........................ ........................... ........................... .......... 36

RPC C2/C18 ......................................................................................... 37

Product description ............................................................................ 37Chemical and physical stability .......................................................... 38Flow/pressure characteristics ......................... .......................... ........... 38Capacity ............................................................................................38Availability ........................ ........................... ........................... .......... 39

Sephasil Protein/Sephasil Peptide ......................................................... 39Product description ............................................................................ 39Chemical and physical stability .......................................................... 40Flow/pressure characteristics ......................... .......................... ........... 40Availability ........................ ........................... ........................... .......... 40


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3. Methods ................................................................................................. 41Choice of separat ion medium ................................................................. 41

Unique requirements of the application .............................................. 41Resolution..........................................................................................41Scale o f the purification .....................................................................4 2Mobile phase conditions ....................................................................4 2Throughput and scaleability ..............................................................42Molecular weight of the sample components ...................................... 42Hydrophobicity of the sample components ........................................ 43Class of sample compo nents ..............................................................43

Choice of mobile phase ........................................................................... 44The organ ic solvent ........................... ........................... ...................... 44pH ..................................................................................................... 46Ion pairing agents .............................................................................. 47

Sample preparat ion ................................................................................. 49Mobile phase preparat ion ....................................................................... 50

Storage of mobile phase .....................................................................5 0Solvent disposal ................................................................................. 50

Detection ................................................................................................ 51Ghosting ............................................................................................51Mobile phase balancing .....................................................................5 1

Column conditioning .............................................................................. 52Elution conditions ...................................................................................5 3Column re-equilibration ......................................................................... 55Column cleaning.....................................................................................55Column storage ...................................................................................... 56

4. Applications............................................................................................ 57Designing a biochemical purification ......................................................57Na tura lly occurring peptides and proteins ..............................................5 8

Purification of platelet-derived growth factor (PDGF) ........................ 59Trace enrichment ............................................................................... 59Purification o f cholecystokinin-58 (CCK-58) from pig intestine ......... 60

Recombinant peptides and proteins ........................................................62Process purification of inclusion bodies..............................................63Purification of recombinant hu man epidermal growth factor ............. 63

Chemically synthesised peptides..............................................................65Purification of a phosphorylated PDGF -receptor derived peptide....65Structura l characterisation of a 165 kDa protein ............................... 66

Protein fragments from enzyme digests ................................... ................ 66Protein characterisation at the micro-scale ......................................... 66Protein identification by LC-MS.........................................................69

Chemically synthesised oligonucleotides ........................... ...................... 705. Fault finding chart .................................................................................. 726. References ............................................................................................... 817. Ordering information .............................................................................84


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5

Chapter 1

Introduction

Adsorption chromatography depends on the chemical interactions between solutemolecules and specifically designed ligands chemically grafted to achromatography matrix. Over the years, many different types of ligands havebeen immobilised to chromatography supports for biomolecule purification,exploiting a variety of biochemical properties ranging from electronic charge tobiological affinity. An important addition to the range of adsorpt ion techniques forpreparative chromatography of biomolecules has been reversed phasechromatography in which the binding of mobile phase solute to an immobilisedn-alkyl hydrocarbon or aromatic ligand occurs via hydrophobic interaction.

Reversed phase chromatography has found both analytical and preparativeapplications in the area of biochemical separat ion and purification. M oleculesthat possess some degree of hydrophobic character, such as proteins, peptides andnucleic acids, can be separated by reversed phase chromatography with excellentrecovery and resolution. In addition, the use of ion pairing modifiers in themobile phase allows reversed phase chromatography of charged solutes such asfully deprotected oligonucleotides and hydrophilic peptides. Preparative reversedphase chromatography has found applications ranging from micropurification ofprotein fragments for sequencing (1) to p rocess scale purification of recombinantprotein products (2).

This handbook is intended to serve as an introduction to the principles andapplications of reversed phase chromatography of biomolecules and as a practicalguide to the reversed phase chromatographic media available from AmershamPharmacia Biotech. Among the topics included are an introductory chapter onthe mechanism of reversed phase chromatography followed by chapters onproduct descriptions, applications, media handling techniques and orderinginformation. The scope of the information contained in this handbook will belimited to preparative reversed phase chromatography dealing specifically with

the purification of proteins, peptides and nucleic acids.


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Theory of reversed phase chromatographyThe separation mechanism in reversed phase chromatography depends on thehydrophobic binding interaction between the solute molecule in the mobile phaseand the immobilised hydrophobic ligand, i.e. the stationary phase. The actual

nature of the hydrophobic binding interaction itself is a matter of heated debate (3)but the conventional wisdom assumes the binding interaction to be the result of afavourable entropy effect. The initial mobile phase binding conditions used inreversed phase chromatography are primarily aqueous which indicates a highdegree of organised water structure surrounding both the solute molecule and theimmobilised ligand. As solute binds to the immobilised hydrophobic ligand, thehydrophobic area exposed to the solvent is minimised. Therefore, the degree oforganised water structure is diminished with a corresponding favourable increasein system entropy. In this way, it is advantageous from an energy point of viewfor the hydrophobic moieties, i.e. solute and ligand, to associate (4).

Fig. 1. Interaction of a solute with a typical reversed phase medium. Water adjacent tohydrophobic regions is postulated to be more highly ordered than the bulk water. Part of thisstructured water is displaced when the hydrophobic regions interact leading to an increase in theoverall entropy of the system.

Reversed phase chromatography is an adsorpt ive process by experimental design,which relies on a partitioning mechanism to effect separation. The solute

molecules partition (i.e. an equilibrium is established) between the mobile phaseand the stationary phase. The distribution of the solute between the two phasesdepends on the binding properties of the medium, the hydrophobicity of thesolute and the composition of the mobile phase. Initially, experimental conditionsare designed to favour adsorption of the solute from the mobile phase to thestationary phase. Subsequently, the mobile phase composition is modified tofavour desorption of the solute from the stationary phase back into the mobilephase. In this case, adsorption is considered the extreme equilibrium state wherethe distribut ion of solute molecules is essentially 100% in the stationary phase.Conversely, desorpt ion is an extreme equilibrium state where the solute is

essentially 100% distributed in the mobile phase.

Protein Protein Protein+

a b c

MatrixStructured water


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Fig. 2. Principle of reversed phase chromatography with gradient elution.

Reversed phase chromatography of biomolecules generally uses gradient elutioninstead of isocrat ic elution. While biomolecules strongly adsorb to the surface ofa reversed phase matrix under aqueous conditions, they desorb from the matrixwithin a very narrow window of organic modifier concentration. Along withthese high molecular weight biomolecules with their unique adsorptionproperties, the typical biological sample usually contains a broad mixture ofbiomolecules with a correspondingly diverse range of adsorption affinities. Theonly practical method for reversed phase separation of complex biologicalsamples, therefore, is gradient elution (5).

In summary, separations in reversed phase chromatography depend on thereversible adsorption/desorption of solute molecules with varying degrees ofhydrophobicity to a hydrophobic stationary phase. The majority of reversedphase separation experiments are performed in several fundamental steps asillustrated in Figure 2 .

The first step in the chromatographic process is to equilibrate the column packedwith the reversed phase medium under suitable initial mobile phase conditions ofpH, ionic strength and polarity (mobile phase hydrophobicity). The polarity of

the mobile phase is controlled by adding organic modifiers such as acetonitrile.Ion-pairing agents, such as tr ifluoroacetic acid, may also be appropr iate. Thepolarity of the initial mobile phase (usually referred to as mobile phase A) mustbe low enough to dissolve the partially hydrophobic solute yet high enough toensure binding of the solute to the reversed phase chromatographic matrix.

In the second step, the sample containing the solutes to be separated is applied.Ideally, the sample is dissolved in the same mobile phase used to equilibrate thechromatographic bed. The sample is applied to the column at a flow rate whereoptimum binding will occur. Once the sample is applied, the chromatographic

bed is washed further with mobile phase A in order to remove any unbound andunwanted solute molecules.

1Starting

conditions

2Adsorptionof sample

substances

3Start of

desorption

4End of

desorption

5Regeneration


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Bound solutes are next desorbed from the reversed phase medium by adjusting thepolarity of the mobile phase so that the bound solute molecules will sequentiallydesorb and elute from the column. In reversed phase chromatography th is usuallyinvolves decreasing the polarity of the mobile phase by increasing the percentageof organic modifier in the mobile phase. This is accomplished by maintaining a

high concentrat ion of organic modifier in the final mobile phase (mobile phase B).Generally, the pH of the initial and final mobile phase solutions remains the same.The gradual decrease in mobile phase polarity (increasing mobile phasehydrophobicity) is achieved by an increasing linear gradient from 100% initialmobile phase A containing little or no organic modifier to 100% (or less) mobilephase B containing a higher concentration of organic modifier. The bound solutesdesorb from the reversed phase medium according to their individualhydrophobicities.

The four th step in the process involves removing substances not previously

desorbed. This is generally accomplished by changing mobile phase B to near100% organic modifier in order to ensure complete removal of all boundsubstances prior to re-using the column.

The fifth step is re-equilibration of the chromatographic medium from 100%mobile phase B back to the initial mobile phase conditions.

Separat ion in reversed phase chromatography is due to the different bindingproperties of the solutes present in the sample as a result of the differences in theirhydrophobic properties. The degree of solute molecule binding to the reversed

phase medium can be controlled by manipulating the hydrophobic properties ofthe initial mobile phase. Although the hydrophobicity of a solute molecule isdifficult to quantitate, the separation of solutes that vary only slightly in theirhydrophobic properties is readily achieved. Because of its excellent resolvingpower, reversed phase chromatography is an indispensable technique for the highperformance separation of complex biomolecules.

Typically, a reversed phase separation is initially achieved using a broad rangegradient from 100% mobile phase A to 100% mobile phase B. The amount oforganic modifier in both the initial and final mobile phases can also vary greatly.

However, routine percentages of organic modifier are 5% or less in mobile phaseA and 95% or more in mobile phase B.

The technique of reversed phase chromatography allows great flexibility inseparation conditions so that the researcher can choose to bind the solute ofinterest, allowing the contaminants to pass unretarded through the column, or tobind the contaminants, allowing the desired solute to pass freely. Generally, it ismore appropriate to bind the solute of interest because the desorbed solute elutesfrom the chromatographic medium in a concentrated state. Additionally, sincebinding under the initial mobile phase conditions is complete, the starting

concentrat ion of desired solute in the sample solution is not critical allowingdilute samples to be applied to the column.


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The specific conditions under which solutes bind to the reversed phase mediumwill be discussed in the appropriate sections in greater detail.

Ionic binding may sometimes occur due to ionically charged impuritiesimmobilised on the reversed phase chromatographic medium. The combinat ion

of hydrophobic and ionic binding effects is referred to as mixed-mode retentionbehaviour. Ionic interactions can be minimised by judiciously selecting mobilephase conditions and by choosing reversed phase media which are commerciallyproduced with high batch-to-batch reproducibility and stringent quality controlmethods.

The matrixCritical parameters that describe reversed phase media are the chemical

composition of the base matrix, particle size of the bead, the type of immobilisedligand, the ligand density on the surface of the bead, the capping chemistry used(if any) and the pore size of the bead.

A reversed phase chromatography medium consists of hydrophobic ligandschemically grafted to a porous, insoluble beaded matrix. The matrix must beboth chemically and mechanically stable. The base matrix for the commerciallyavailable reversed phase media is generally composed of silica or a syntheticorganic polymer such as polystyrene. Figure 3 shows a silica surface withhydrophobic ligands.

Fig. 3. Some typical structures on the surface of a silica-based reversed phase medium.The hydrophobic octadecyl group is one of th e most common ligands.

SiOH

Si

Si

O

SiOSi(CH2)17CH3

CH3

CH3

CH3

SiOSiCH2CH3

CH3

Residual silanol group

Ether; source of silanols

Octadecyl group

C2 capping group


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Silica was the first polymer used as the base matrix for reversed phasechromatography media. Reversed phase media were originally developed for thepurification of small organic molecules and then later for the pur ification of lowmolecular weight, chemically synthesised peptides. Silica is produced as porousbeads which are chemically stable at low pH and in the organic solvents typically

used for reversed phase chromatography. The combination of porosity andphysical stability is important since it allows media to be prepared which haveuseful loading capacities and high efficiencies. It is worth not ing that, a lthoughthe selectivity of silica-based media is largely controlled by the properties of theligand and the mobile phase composition, different processes for producingsilica-based matrices will also give media with different patterns of separation.The chemistry of the silica gel allows simple derivatisation with ligands of variouscarbon chain lengths. The carbon content, and the surface density anddistribution of the immobilised ligands can be controlled during the synthesis.

The pr imary disadvantage of silica as a base matr ix for reversed phase media is itschemical instability in aqueous solutions at h igh pH. The silica gel matrix canactually dissolve at h igh pH, and most silica gels are not recommended forprolonged exposure above pH 7.5.

Synthetic organic polymers, e.g. beaded polystyrene, are also available as reversedphase media. Polystyrene has traditionally found uses as a solid support inpeptide synthesis and as a base matrix for cation exchange media used forseparation of amino acids in automated analysers. The greatest advantage ofpolystyrene media is their excellent chemical stability, particularly under strongly

acidic or basic conditions. Unlike silica gels, polystyrene is stable at all pH valuesin the range of 1 to 12. Reversed phase separat ions using polystyrene-basedmedia can be performed above pH 7.5 and, therefore, greater retention selectivitycan be achieved as there is more control over the degree of solute ionisation.

CH2CHCH2CHCH2CHCH2CHCH2CH





Fig. 4. Partial structure of a polystyrene-based reversed phase medium.


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Fig. 5. Reverse phase media with wide pores allow the most efficient transfer of large solutemolecules between the mobile and the stationary phases.

The surface of the polystyrene bead is itself strongly hydrophobic and, therefore,when left underivatised unlike silica gels that have hydrophobic ligands grafted toa hydrophilic surface.

The porosity of the reversed phase beads is a crucial factor in determining theavailable capacity for solute binding by the medium. Note that this is not thecapacity factor (k ) but the actual binding capacity of the medium itself. Mediawith pore sizes of approximately 100 are used predominately for small organicmolecules and peptides. Media with pore sizes of 300 or greater can be used inthe purification of recombinant peptides and proteins that can withstand the

stringent conditions of reversed phase chromatography.

The ligandsThe selectivity of the reversed phase medium is predominantly a function of thetype of ligand grafted to the surface of the medium. Generally speaking, linearhydrocarbon chains (n-alkyl groups) are the most popular ligands used inreversed phase applications. Some typical hydrocarbon ligands are shown inFigure 6.

Fig. 6. Typical n-alkyl hydrocarbon ligands. (A) Two-carbon capping group, (B) Octyl ligand,(C) Octadecyl ligand.

Restricted mass transfer More efficient mass transfer

50100Narrow pore

300Wide pore

OSiCH2CH3

CH3

CH3

OSiCH2CH2CH2CH2CH2CH2CH2CH3

CH3

CH3

OSiCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2C

CH3

CH3

(A)

(B)

(C)


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efficiency and selectivity). Analytical and small scale preparative applications areusually performed with 3 and 5 m beads while larger scale preparat iveapplications (pilot and process scale) are usually performed with par ticle sizes of15 m and greater. Micropreparative and small scale preparative work can beaccomplished using particle sizes of 3 m since the limited capacity of the smallcolumns packed with these media is not a severe problem when only smallquantities of material are purified.

Resolution in reversed phase chromatographyResolution

Adequate resolution and recovery of purified biological material is the ultimategoal for reversed phase preparat ive chromatography. Resolution, Rs, is generallydefined as the distance between the centres of two eluting peaks as measured byretention time or volume divided by the average width of the respective peaks(Fig. 8). For example, an Rs value of 1.0 indicates 98% purity has been achieved(assuming 98% peak recovery). Baseline resolution between two well formed

peaks indicates 100% purity and requires an Rs value greater than 1.5 (Fig. 9).

Calculating Rs is the simplest method for quantitating the actual separationachieved between two solute molecules. This simple relationship can be expandedto demonstrate the connection between resolution and three fundamentalparameters of a chromatographic separation. The parameters have been derivedfrom chromatographic models based on isocratic elution but are still appropriatewhen used to describe their effects on resolution when discussing gradient elution(consider a continuous linear gradient elution to be a series of small isocraticelution steps). The parameters that contribute to peak resolution are column

selectivity, column efficiency and the column retention factor.

Fig. 8. Determination of the resolutionbetween two peaks.

v2

v1

w2w1

v2v1

(w2 + w1)/2Rs=


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Fig. 9. Separation results with different resolution.

98%B

A B

98%A

98%B Rs=1

A B

100%A

100%B Rs=1.5

Resolution Rs is a function of selectivity , efficiency (number of theoreticalplates N) and the average retention factor, k, for peaks 1 and 2.

Capacity factor

The capacity factor, k, is related to the retention t ime and is a reflection of theproportion of time a particular solute resides in the stationary phase as opposedto the mobile phase. Long retention times result in large values of k . Thecapacity factor is not the same as the available binding capacity which refers tothe mass of the solute that a specified amount of medium is capable of bindingunder defined conditions. The capacity factor, k , can be calculated for every peakdefined in a chromatogram, using the following equations.

Capacity factor=k=

TR

TO

VR

VO

TO

VO

where TR

and VR

are the retention time and retention volume, respectively, of thesolute, and T

oand V

othe retention t ime and retention volume, respectively, of an

unretarded solute.

moles of solute in stationary phasemoles of solute in mobile phase

k=

Rs=1 (-1)

4 ( N )

k

1 + k


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In the resolution equation previously described, the k value is the average of thecapacity factors of the two peaks being resolved. Unlike non-adsorptivechromatographic methods (e.g. gel filtra tion), reversed phase chromatographycan have very high capacity factors. This is because the experimental conditionscan be chosen to result in peak retention t imes greatly in excess of the totalcolumn volume.

Efficiency (N)

The efficiency of a packed column is expressed by the number of theoreticalplates, N. N is a dimensionless number and reflects the kinetics of thechromatographic retention mechanism. Efficiency depends primarily on thephysical properties of the chromatographic medium together with thechromatography column and system dimensions. The efficiency can be altered bychanging the par ticle size, the column length, or the flow rate. The expressionnumber of theoretical plates is an archaic term carried over from thetheoretical comparison of a chromatography column to a distillation apparatus.The greater the number of theoretical plates a column has, the greater itsefficiency and correspondingly, the higher the resolution which can be achieved.

The column efficiency (N) can be determined empirically using the equationbelow based upon the zone broadening that occurs when a solute molecule iseluted from the column (Fig. 11).

The number of theoretical plates, N , is given by

N = 5.54 (V1/W

1/2)2

where V1

is the retention volume of the peak and W1/2

is the peak width (volume)at half peak height.

Fig. 10. Hypothetical chromatogram.VO=void volume, VC=total volume,V1, V2, and V3 are the elution(retention) volumes of peaks 1, 2 and 3,respectively.

1 2 3

vc

v0v1

v2v3


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The number of theoretical plates is sometimes reported as plates per metre ofcolumn length (N /L).

The height equivalent to a theoretical plate, H , is given by

H = L/N

where L is the column length and N is the number of theoretical plates.Any parameter change tha t increases N will also increase Rs. The relationshipbetween the two is defined by the square root of N. For example, an increase inN from 100 to 625 will improve the resolution by a factor of 2.5, rather than6.25. The main contr ibution to column efficiency (N) is part icle size and theefficacy of the column packing procedure.It should be noted that the smaller the par ticle size, the more difficult it is to packan efficient column. This is the reason why reversed phase media with part iclesizes less than 10 m are commercially available only in pre-packed formats.

v1

w1/2

Peakheight

Volume

Abs

Fig. 11. Measurements for determiningcolumn efficiency. V1 is the retentionvolume of the peak and W1/2 is the peakwidth (volume) at ha lf peak height.


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Selectivity

Selectivity () is equivalent to the relative retention of the solute peaks and, unlikeefficiency, depends strongly on the chemical propert ies of the chromatographymedium.

The selectivity, , for two peaks is given by

= k2 /k

1 = V

2- V

0/V

1 V

0= V

2/V

1

where V1

and V2

are the retention volumes, and k2 /k

1 are the capacity factors,

for peaks 1 and 2 respectively, and V0

is the void volume of the column.

Selectivity is affected by the surface chemistry of the reversed phase medium, thenature and composition of the mobile phase, and the gradient shape.

Fig. 12. Selectivity comparison between different silica based media at pH 2.0 and pH 6.5.A mixture of closely related angiotensin peptides was used as sample. (Work by AmershamPharmacia Biotech AB, Uppsala, Sweden.)

pH 2

a) d)c)b)

e) h)f) g)

pH 2 pH 2 pH 2

pH 6.5 pH 6.5 pH 6.5 pH 6.5

1

2+3

4

5+6

7+8

0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min

12

34

5+67+8

12

34

5+67+8

1

24

3

6

7+8

5

0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min

0.0 5.0 10.0 15.0 20.0 25.0 min

1

2

3+4

6

5

87

0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min

1

234

65

8

7

1 2

64 3 5 8

7

1

2

34

5+6

7+8

Sephasil Protein C4 Sephasil Peptide C8 Sephasil Peptide C18 RPC C2/C18

1. Val4-lle7-AT III (RVYVHPI)2. Ile7-AT III (RVYIHPI)3. Val4-AT III (RVYVHPF)4. Sar1-Leu-AT II (Sar-RVYIHPL)

(Sar=sarcosine, N-methylglycine)5. AT III (RVYIHPF)6. AT II (DRVYIHPF)7. des-Asp1-AT I (RVYIHPLFHL)8. AT I (DRVYIHPFHL)

Columns: a) and e) Sephasil Protein C4 5 m 4.6/100b) and f) Sephasil Peptide C8 5 m 4.6/100c) and g) Sephasil Peptide C18 5 m 4.6/100d) and h) RPC C2/C18 ST 4.6/100

Eluent A (pH 2): 0.065% TFA in distilled waterEluent B (pH 2): 0.05% TFA, 75% acetonitrileEluent A (pH 6.5): 10 mM phosphateEluent B (pH 6.5): 10 mM phosphate, 75% acetonitrileFlow: 1 ml/minSystem: KTApurifierGradient: 595% B in 20 column volumes


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Fig. 13. The effect of selectivity and efficiency on resolution.

Both high column efficiency and good selectivity are important to overallresolution. H owever, changing the selectivity in a chromatographic experiment iseasier than changing the efficiency. Selectivity can be changed by changing easilymodified conditions like mobile phase composition or gradient shape.

Binding capacity

The available binding capacity of a reversed phase medium is a quantitat ivemeasure of its ability to adsorb solute molecules under static conditions. Thedynamic binding capacity is a measure of the available binding capacity at aspecific flow rate. Both values are extremely important for preparat ive work.

The amount of solute which will bind to a medium is proportional to theconcentration of immobilised ligand on the medium and also depends on the typeof solute molecule being adsorbed to the medium. The available and dynamicbinding capacities depend on the specific chemical and physical properties of thesolute molecule, the properties of the reversed phase medium (porosity, etc.) andthe experimental conditions during binding.

The porosity of the bead is an important factor which influences bindingcapacity. The entire hydrophobic surface of macroporous media is available forbinding solute. Large solute molecules (i.e. high molecular weight) may beexcluded from media of smaller pore size and only a small fraction of the wholehydrophobic surface will be used. When maximum binding capacity is required, amedium with pores large enough to allow all the molecules of interest to enterfreely must be used.

Highefficiency

Lowefficiency

Highefficiency

Lowefficiency

Good selectivity Poor selectivity


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Critical parameters in reversed phasechromatography

Column length

The resolution of high molecular weight biomolecules in reversed phaseseparations is less sensitive to column length than is the resolution of smallorganic molecules. Proteins, large peptides and nucleic acids may be purifiedeffectively on short columns and increasing column length does not improveresolution significantly. The resolution of small peptides (including some peptidedigests) may sometimes be improved by increasing column length. For example,the number of peaks detected when a tryptic digest of carboxamidomethylatedtransferrin was fractionated by RPC increased from 87 on a 5 cm long column to115 on a 15 cm long column and 121 on a 25 cm long column (6).

The partition coefficients of high molecular weight solutes are very sensitive tosmall changes in mobile phase composition and hence large molecules desorb in avery narrow range of organic modifier concentration. The retention behaviour oflarge molecules may be considered to be governed by an on/off mechanism (i.e. alarge change in partition coefficient) which is insensitive to column length. Whensmall changes in organic modifier concentration result in small changes in thepartition coefficient, longer column lengths increase resolution.

The use of gradient elution further reduces the significance of column length forthe resolution of large biomolecules by reversed phase chromatography. Gradientsare required since most biological samples are complex mixtures of moleculesthat vary greatly in their adsorption to the reversed phase medium. Due to thisvariety of adsorption affinities, the mobile phase must have a broad range ofeluting power to ensure elution of all the bound solute molecules. Under theseconditions, especially with moderate to steep gradient slopes, column length isnot a critical factor with regard to resolution.

Flow rate

Flow rate is expected to be an important factor for resolution of small molecules,including small peptides and protein digests, in reversed phase separat ions.

However, reversed phase chromatography of larger b iomolecules, such as proteinsand recombinantly produced peptides, appears to be insensitive to flow rate. Infact, low flow rates, typically used with long columns, may actually decreaseresolution due to increased longitudinal diffusion of the solute molecules as theytraverse the length of the column.

The flow ra te used dur ing the loading of the sample solution is especiallysignificant in large scale preparative reversed phase chromatography, althoughnot critical during analytical experiments. Dynamic binding capacity will varydepending on the flow rate used during sample loading. When scaling up apurification, the dynamic binding capacity should be determined in order to


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assess the optimum flow rate for loading the sample. Dynamic binding capacity isa property of the gel that reflects the kinetics of the solute binding process. Theefficiency of this step can have enormous consequences for the results of a largescale preparative purification.

Temperature

Temperature can have a profound effect on reversed phase chromatography,especially for low molecular weight solutes such as short peptides andoligonucleotides. The viscosity of the mobile phase used in reversed phasechromatography decreases with increasing column temperature. Since masstransport of solute between the mobile phase and the stationary phase is adiffusion-controlled process, decreasing solvent viscosity generally leads to moreefficient mass transfer and, therefore, higher resolution. Increasing thetemperature of a reversed phase column is par ticularly effective for low molecular

weight solutes since they are suitably stable at the elevated temperatures.

Mobile phase

In many cases, the colloquial term used for the mobile phases in reversed phasechromatography is buffer . However, there is little buffering capacity in themobile phase solutions since they usually contain strong acids at low pH withlarge concentrations of organic solvents. Adequate buffering capacity should bemaintained when working closer to physiological conditions.

O rganic solvent

The organic solvent (modifier) is added to lower the polarity of the aqueousmobile phase. The lower the polarity of the mobile phase, the greater its elutingstrength in reversed phase chromatography. Although a large variety of organicsolvents can be used in reversed phase chromatography, in practice only a few areroutinely employed. The two most widely used organic modifiers are acetonitrileand methanol, although acetonitrile is the more popular choice. Isopropanol (2-propanol) can be employed because of its strong eluting propert ies, but is limitedby its high viscosity which results in lower column efficiencies and higher back-pressures. Both acetonitrile and methanol are less viscous than isopropanol.

All three solvents are essentially UV transparent. This is a crucial property forreversed phase chromatography since column elution is typically monitored usingUV detectors. Acetonitrile is used almost exclusively when separating peptides.Most peptides only absorb at low wavelengths in the ultra-violet spectrum(typically less than 225 nm) and acetonitrile provides much lower backgroundabsorbance than other common solvents at low wavelengths.


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The retention, or capacity factor (k ), for a given solute is a function of the mobilephase polarity. The elution order can be affected by changing the type of organicmodifier or by the addition of ion pairing agents. Changes in elution o rder aremost pronounced for proteins that are denatured in organic solvents. Denaturationof the protein can result in a change in its hydrophobicity.

Ion suppression

The retention of peptides and proteins in reversed phase chromatography can bemodified by mobile phase pH since these par ticular solutes contain ionisablegroups. The degree of ionisation will depend on the pH of the mobile phase.The stability of silica-based reversed phase media d ictates that the operating pHof the mobile phase should be below pH 7.5. The amino groups contained inpeptides and proteins are charged below pH 7.5. The carboxylic acid groups,however, are neutralised as the pH is decreased. The mobile phase used inreversed phase chromatography is generally prepared with strong acids such as

trifluoroacetic acid (TFA) or ortho-phosphoric acid. These acids maintain a lowpH environment and suppress the ionisation of the acidic groups in the solutemolecules. Varying the concentration of strong acid components in the mobilephase can change the ionisation of the solutes and, therefore, their retentionbehaviour.

The major benefit of ion suppression in reversed phase chromatography is theelimination of mixed mode retention effects due to ionisable silanol groupsremaining on the silica gel surface. The effect of mixed mode retention isincreased retention times with significant peak broadening.

(A) Reversed phase chromatography

(B) Mixed-mode

Fig. 14. Typical effects of mixed-mo deretention. Peaks are broader and skewed,and retention time increases.


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Mixed mode retention results from an ion exchange interaction between negativelycharged silanol groups exposed on the surface of the silica and the positivelycharged amino groups on the solute molecules. Silanol groups on the surface ofsilica-based media can ar ise from two primary sources. The first is due toinadequate end-capping procedures during the manufacture of the gel. It is critical

to choose a manufacturer that produces a gel with reproducibly low mixed moderetention effects, since these artefact can affect resolution.

The other source of surface silanol groups is column ageing. The silica gel surfaceis cont inually eroded dur ing the life of the column, resulting in exposed silanolgroups and progressive deterioration in column performance. Prolonged exposureto aqueous solutions can accelerate column ageing.

The low pH environment (usually less than pH 3.0) of typical reversed phasemobile phases suppresses the ionisation of these surface silanol groups so that the

mixed mode retention effect is diminished.

Ion suppression is not necessary when dealing with reversed phase media basedon polystyrene or other synthetic organic polymers. Polystyrene media are stablebetween pH 1-12 and do not exhibit the mixed mode retention effects that silicagels do with mobile phases at high pH .

Ion pairing agents

The retention t imes of solutes such as proteins, peptides and oligonucleotides canbe modified by adding ion pairing agents to the mobile phase. Ion pairing agents

bind to the solute by ionic interactions, which results in the modification of thesolute hydrophobicity. Examples of ion pairing agents are shown in chapter 3.

Fig. 15. Ion pair format ion with (A)anionic or (B) cationic ion pairing agents.

++ +

++

+ +

+ +

+ +

+ +

+

+

+

Negatively charged ionpairing agent withhydrophobic surface

Positively charged ionpairing agent withhydrophobic surface

Positively charged peptide

Negatively charged oligonucliotide


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Both anionic and cationic ion pairing agents are used depending on the ioniccharacter of the solute molecule and the pH of the mobile phase. For example, atypical ion pairing agent for peptides at pH less than 3.5 is trifluoroacetic acid.The ion pairing agent used with oligonucleotides, which contain a negativecharge at neutral to high pH , is typically triethylamine.

In some cases the addition of ion pairing agents to the mobile phase is anabsolute requirement for binding of the solute to the reversed phase medium. Forexample, retention of deprotected synthetic oligonucleotides, i.e. without thetrityl protecting group attached, requires triethylamine in the mobile phase. Thesame is true for hydrophilic peptides where binding is negligible in the absence ofa suitable ion pairing agent such as tr ifluoroacetic acid.

The concentrat ion of ion pa iring agents in the mobile phases is generally in therange 0.1 - 0.3% . Potential problems include possible absorbance of UV light by

the ion pairing agent and changes in extinction coefficient with concentrat ion oforganic modifier. This can result in either ascending or descending baselinesduring gradient elution.

Gradient elution

Gradient elution is the method of choice when performing preparative reversedphase chromatography of biomolecules. The typical gradients for preparativereversed phase chromatography of proteins and peptides are linear and binary, i.e.involving two mobile phases. Convex and concave gradients are used

occasionally for analytical purposes par ticularly when dealing with multi-component samples requiring extra resolution either at the beginning or at theend of the gradient.

The concentrat ion of organic solvent is lower in the initial mobile phase (mobilephase A) than it is in the final mobile phase (mobile phase B). The gradient then,regardless of the absolute change in percent organic modifier, always proceedsfrom a condition of high polarity (high aqueous content, low concentration oforganic modifier) to low polarity (lower aqueous content, higher concentration oforganic modifier).

Gradient shape (combinations of linear gradient and isocratic conditions),gradient slope and gradient volume are all important considerations in reversedphase chromatography. Typically, when first performing a reversed phaseseparation of a complex sample, a broad gradient is used for initial screening inorder to determine the optimum gradient shape.

After the initial screening is completed, the gradient shape may adjusted tooptimise the separation of the desired components. This is usually accomplishedby decreasing the gradient slope where the desired component elutes and

increasing it before and after. The choice of gradient slope will depend on how


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closely the contaminants elute to the target molecule. Generally, decreasinggradient slope increases resolution. H owever, peak volume and retention timeincrease with decreasing gradient slope. Shallow gradients with short columns aregenerally optimal for high molecular weight biomolecules.

Gradient slopes are generally reported as change in percent B per unit time (% B/min.) or per unit volume (%B/ml). When programming a chromatography systemin time mode, it is important to remember that changes in flow rate will affectgradient slope and, therefore, resolution.

Resolution is also affected by the to tal gradient volume (gradient time x flowrate). Although the optimum value must be determined empirically, a good ruleof thumb is to begin with a gradient volume that is approximately ten to twentytimes the column volume. The slope can then be increased or decreased in orderto optimise resolution.

Mode of use

Desalting

Desalting is a routine laboratory procedure in which low molecular weightcontaminants are separated from the desired higher molecular weightbiomolecules. The procedure is sometimes simply referred to as buffer exchange.Non-chromatographic techniques for buffer exchange include ultra-filtration anddialysis.

Desalting is used in the laboratory primar ily for sample prepara tion, e.g.

desalting fractions obtained by other methods such as ion exchangechromatography. Size exclusion chromatography (gel filtration) with SephadexG-25 is commonly used for desalting proteins and nucleic acids, and SephadexG-10 is used for desalting small peptides. Size exclusion chromatography is avaluable method for desalting due to its simplicity and gentleness, although itsuffers from the unwanted side effect of sample dilution.

Proteins, peptides and oligonucleotide samples can be conveniently desalted usingreversed phase chromatography. When desalting samples using reversed phasetechniques, the samples can be recovered and reconstituted into small volumes

thereby avoiding the sample dilution effects of gel filtration.

The sample is passed through a small reversed phase column where it binds andconcentrates on the reversed phase medium. Unlike gel filtra tion, reversed phaseis an adsorption technique and sample volume is not limited. Reversed phasechromatography columns can concentrate large volumes of dilute samples at thesame time as desalting them.

After the entire sample has been processed, the bound solute is eluted using asmall volume of low polarity mobile phase, typically acetonitrile. If the solvent is

volatile, as acetonitrile is, it can then be removed by evaporat ion and the sampleresidue re-suspended in the desired volume of new buffer.


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Fig. 16. Desalting by (A) gel filtration and (B) reversed phase chromatography. The largemolecules elute first in gel filtration; the salt elutes without changing the eluent. The salt elutesfirst in reversed phase chromatography; a less-polar eluent is needed to elute proteins and othermolecules which are retained on the column.

High resolution separations

Reversed phase chromatography is most typically used as a high resolutiontechnique, where its inherent robustness is especially advantageous. However,certa in applications push the resolving power o f the reversed phase technique toits limit. These tend to be in the intermediate stages of preparative applications orwhen isolating structurally similar components from a complex mixture.Examples include isolation of specific peptides from enzymatic digests or

pur ification of oligonucleotides from a complicated mixture of oligonucleotidecontaminants. In these cases, a great many peaks must be resolved from eachother and recovered in sufficient amounts for further analysis. Reversed phasemedia of very small particle size, typically 3 and 5 m beads, are usually requiredtogether with painstaking attention given to details such as column temperature,gradient slope and mobile phase composition. When dealing with smaller solutes,such as shor t oligonucleotides, digested protein fragments and short peptides, theoptimisation of other factors such as flow rate and column length may also benecessary in order to maximise resolution.

Large scale preparative purificationThe large scale purification of biomolecules such as synthetic oligonucleotidesand peptides, and recombinant peptides and proteins by reversed phasechromatography requires both high resolution separation together with theability to scale up the purification. In these cases, the purification is optimisedusing a small particle reversed phase medium and then scaled up accordinglyusing a medium with similar selectivity but with a larger par ticle size. Thetechniques of scale up used with reversed phase chromatography are similar tothose used with other chromatographic techniques such as ion exchange. Specificexamples of preparative, large scale reversed phase purification of biomolecules

are shown in chapter 4.

vo

(A) Gel filtration

Protein Salt

vc vo vc

Salt

(B) Reversed phasechromatography

Protein etc

Non-polareluent


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Fig. 17. Stages in a purification scheme.

Stages in a purification schemeOnce the source for a biomolecule has been determined, whether microbial,chemical, natural or other, and the starting material has been produced insufficiently large quantities, the desired substance must then be purified fromcontaminants present in the crude sample. There are essentially three functionalstages in the purification of a biomolecule from a crude preparation or extract.These are referred to as Capture, Intermediate Purification and Polishing.The suitability of any separa tion technique, including reversed phasechromatography, at any stage of purification will always depend on the specificsample, the specific separation problem at hand and the intended use of the

purified material.

Capture

Capture is the first step in the purification procedure. At this stage, the samplevolume is usually at its largest and the sample may contain part iculates or viscousmaterials. The purpose of the capture step is to isolate, concentrate and stabilisethe ta rget molecule from the crude preparation rapidly, and with good recovery.The captu re step is not expected to be highly resolving but is required to isolatethe molecule of interest from contaminating substances that are dissimilar to thedesired molecule. The capture step may be considered as a group separation

rather than as a high resolution purification. Consequently, time, capacity andrecovery are more important than resolution in a successful capture step.Reversed phase chromatography is a suitable method for the capture of syntheticpeptides and synthetic oligonucleotides. However, it is usually less suitable forcapture of peptides and proteins from biological sources. This is because of thepresence of lipids and other highly hydrophobic solutes which bind strongly,reduce the dynamic capacity for the molecule of interest, and can be difficult toremove from the column. Additionally, the small particle size of most reversedphase media requires particulates to be removed from the sample to prevent thecolumn from clogging. Ion exchange chromatography and hydrophobic

interaction chromatography using bead diameters greater than 90 m are bettersuited for capture in these instances.

Purification stage

Capture

Intermediatepurification

Polishing

Start material

Demands

Throughputtolerate crude feedcleaning in place

Resolutionreproducibility

cleaning in place

Pure product


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Intermediate stages

In the intermediate purification phase the focus is to separate the target moleculefrom most of the bulk impurities such as other proteins, peptides, nucleic acids,

endotoxins and viruses. An ability to resolve similar components is of increasedimportance since contaminants at th is stage are often similar to the targetmolecule in terms of functional or structural propert ies. The critical requirementsare recovery and resolution. Reversed phase chromatography is a suitabletechnique for th is stage of the purification because of the high resolution that canbe achieved.

Polishing

Polishing is the final step in the preparation of a pure product. The polishing stepis used to remove trace contaminants and impurities. The purified biomolecule

should be in a form suitable for its intended use. Contaminants at the polishingstage are often very similar to the ta rget molecule. Typical contaminants mayinclude conformers and structural variants of the target molecule. Structuralvariants can include dimers, oligomers, aggregates, oxidised amino acids,protease-clipped molecules, desamidated amino acids etc. Other micro-heterogeneities may also occur. In process related applications, polishing alsoremoves final traces of leachables, endotoxins, viruses etc.

The goals of the polishing stage might be product purity of 100% in less thantwo steps with a recovery of greater than 99% . Polishing can be performed using

size exclusion, especially when dimers and aggregates must be removed. However,when dealing with slight structural variants and micro-heterogeneities, reversedphase chromatography with its excellent resolving power is the method of choice.

Apply sample Elute peptides,proteins etc

Elute unboundsugars, salts etc

Fig. 18. Steps during capture.


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Chapter 2

Product GuideReversed phase media from Amersham Pharmacia Biotech provide a broad rangeof selectivity for different applications for use at analytical, laboratory andproduction scale.Table 1 below reviews briefly the main characteristics of the media together withtheir application suitability.

Table 1

Media

SOURCE 5RPC

SOURCE 15RPC

SOURCE 30RPC

RPC C2/C18

Sephasil Protein C4Sephasil Peptide C8Sephasil Peptide C18

Sephasil Protein C4Sephasil Peptide C8Sephasil Peptide C18

Sephasil C8

Sephasil C18

Medium

Polystyrene/

divinyl-benzene

Polystyrene/divinyl-benzene

Polystyrene/divinyl-benzene

Silica

Silica

Silica

Silica

Particle size(approx)

5 m mono-

sized

15 m mono-sized

30 m mono-sized

3 m

5 m

12 m

5 m

Applications

High resolution analysis andsmall scale purification.

Alternative selectivity to silica,especially for separationsperformed at high pH. Ideal forrecombinant and syntheticpeptides and oligonucleotides.

Preparative purification ofproteins, peptides andoligonucleotides. Alternativeselectivity to silica, especiallyfor separations performed athigh pH. Excellent pressure/

flow characteristics.Large scale purification ofproteins, peptides andoligonucleotides. Alternativeselectivity to silica, especiallyfor separations performed athigh pH. Excellent pressure/flow characteristics.

High efficiency media, forpeptide mapping, analysis and

micropurification.High resolution analysis andpurification. Suitable forrecombinant and syntheticpeptides.

Preparative purification ofpeptides, proteins andoligonucleotides.

SMART system pre-packed

columns. Micropurification andanalysis.


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SOURCE RPCProduct Description

SOURCE RPC media are designed for analytical and preparative chromatographyof synthetic peptides, oligonucleotides and proteins. SOURCE RPC is based on

rigid, monosized, polystyrene/divinyl benzene beads (Fig. 19) that give rapid,reproducible, high capacity separations with excellent resolution at high flowrates.

SOURCE RPC is a useful alternative to RPC matrices based on silica, especiallyfor separations which must be performed at high pH or when different selectivityor h igher capacity are required.

The pore size distribution, batch-to-batch reproducibility (Fig. 20) and excellentscalability (Fig 21) of SOURCE RPC ensure outstanding chromatographicproperties at any scale of operation.

5 m

Fig. 19. Scanning electronmicrograph of SOURCE 15RPC.

Note the uniform size distribution.


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Retention time (min)13

12

11

10

9

8IIe7 Angio III VaI4 Angio III Angio III Angio I

Batch 1Batch 2

Batch 3

Sample: (Ile7) angiotensin III (0.5 mg/ml)(Val4) angiotensin III (0.5 mg/ml)Angiotensin III (0.5 mg/ml)Angiotensin I (0.5 mg/ml)25 l applied.

Column: RESOURCE RPC, 1 ml (i.d. 6.4, length 30 mm)Eluent A: 0.1% TFA in water

Eluent B: 0.1% TFA, 60% acetonitrile in waterGradient: 15-65% B in 20 minFlow: 1 ml/min

Fig. 20. Reproducibility ofthree production ba tches ofSOURCE 15RPC. (Workby Amersham PharmaciaBiotech AB, Lillestrm,Norway.)

Fig. 21. Excellentscalability ofSOURCE 30RPC.

Column: SOURCE 30RPC, 10 mm i.d. x 300 mm column (24 ml)200 mm i.d. x 300 mm column (10 l)

Sample: Mixture of Angiotensin II, Ribonuclease A and InsulinSample load: 0.064 mg/ml medium, total loadSolution A: 0.1% TFA/0.05 M NaClSolution B: 0.1% TFA/60% n-propanolFlow: 150 cm/hGradient: 2070% B, 5 column volumes (cv)

HR 10/30

column

FineLINE 200L

column

1 2

2.0

1.0

00 3 4 5 6 7 8 9

(cv)

A280 nm

2.0

1.0

00 1 2 3 4 5 6 7 8 9

(cv)

A280 nm

FineLINE 200Lcolumn


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Characteristics of SOURCE RPC are shown in Table 2.

SOURCE 5RPC SOURCE 15RPC SOURCE 30RPC

Base matrix and Polystyrene/divinyl Polystyrene/divinyl Polystyrene/divinylstationary phase benzene benzene benzene

Particle size 5 m 15 m 30 m

Particle size monosized monosized monosizeddistribution

Typical separation 100 -480 200 - 900 100 - 1 000flow velocity (cm/hr)

pH stability 1 - 12 1 - 12 1 - 12(operational)

pH stability 1 - 14 1 - 14 1 - 14(cleaning range)

Dynamic binding ~ 80 mg ~ 10 mg BSA/ml ~ 14 mg BSA/mlcapacity (per ml bacitracin/ml ~ 50 mg insulin/ml ~ 72 mg insulin/mlmedium at 300 cm/h)

Table 2.

High Chemical Stability

SOURCE RPC has an operating range between pH 1 - 12 allowing a free choicefor running conditions. Since peptide solubility is frequently pH dependent,

successful separations of some peptides may require conditions at high pH .SOURCE RPC therefore offers much greater pH stability and flexibility thansilica based reversed phase matrices.

Figure 22 demonstrates the chemical stability of SOURCE 30RPC, showing theseparation of angiotensins before and after incubation of the medium for oneweek at 40C in 1M HCl and, similarly, in 1M N aOH.

The extremely high pH tolerance (1 - 14) gives full flexibility for frequentcleaning procedures improving both media lifetime and overall economy at every

scale of application. Figure 23 shows results from a SOURCE 5RPC 4.6/150column on which a thousand runs were performed, including four cleaning-in-place steps with 1 M NaOH and 1.0 M HCl, during a 21 days cycle.The resolution and retention times remained unchanged.


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A214 nm

0.10

0.05

0.00

0.0 5.0 10.0 15.0 20.0(min)

= before incubation= after incubation with 1 M HCl

Column: SOURCE 30RPC, 5 mm i.d. x 50 mm column (1 ml)Sample: Mixture of (Iie7) Angiotensin III, (Val4) Angiotensin III,

Angiotensin III and Angiotensin IISample load: 0.13 mg/ml media of each peptideSolution A: 0.1% TFASolution B: 0.1% TFA/60% AcetonitrileFlow: 300 cm/h

Gradient: 1565% B, 20 column volumes (cv)

Fig. 22. Separation o f model protein mixture on SOURCE 30RPC before and after incubation for oneweek at 40 C in 1 M HCl, and similarly, in 1 M N aOH.

Fig. 23a and 23b. Separation of peptides on SOURCE 5RPC ST 4.6/150 . Figure a shows the firstinjection of the p eptide mixture and Figure b the 1000 th injection. CIP with 1 M H Cl and 1 M N aOHwas performed after 275, 400, 600 and 800 runs.

Column: SOURCE 5RPC ST 4.6/150System: KTAexplorer 10S systemSample mixture: 1. (lle7) Angiotensin III

2. (Val4)Angiotensin III3. Angiotensin III4. Angiotensin I

Sampleconcentration: 0.1 mg/ml of each peptideSample volume: 20 lEluent A: 20 mM Boric acid/NaOH, pH 10.0Eluent B: AcetonitrileFlow: 1.0 ml/minGradient: 535% B over 15 minutes (6 CV)

0.10

0.05

0.00

0.0 5.0 10.0 15.0 20.0(min)

A214 nm = before incubation= after incubation with 1 M NaOH

0

100

200

10.08.0 12.0 14.0 min

A214 nm

a)1

2

3 4

0

100

200

8.0 10.0 12.0 14.0 min

b)

12

3

4

A214 nm


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SOURCE RPC media and FineLINE, HR, RESOURCE RPC and ST columns areresistant to all solvents commonly used in reversed phase chromatography, such as0.1% TFA in water and 0.1% TFA in acetonitr ile. SOURCE RPC is resistant toother organic solvents such as methanol, isopropanol, ethanol, acetic acid, andtetrahydrofuran. Due to the inert aromatic/hydrocarbon structure of the polystyr-

ene/divinylbenzene matrix, SOURCE RPC is stable to more disruptive chemicalreagents, such as 6 M guanidine hydrochloride and 0.1% SDS.

Excellent Flow/ Pressure Characteristics

The uniform bead size and spherical shape of SOURCE RPC beads give stabledensely packed beds with excellent flow properties unlike media with a widerange of part icle sizes. The low operat ing back-pressure generated by SOURCERPC allows higher flow rates to be used during separations and cleaningprocedures while still giving excellent resolution, as demonstrated in Figure 24

which shows the performance maintained by SOURCE 30RPC even at high flowrates.

1.0

0.5

0

0 50 100(min)

Flow of 150 cm/hA280 nm

0 10 20

A280 nmFlow of 600 cm/h

(min)

1.0

0.5

0

Column: SOURCE 30RPC, 10 mm i.d. x 100 mm column (8 ml)Sample: Mixture of Ribonuclease A, Insulin and AlbuminSample load: 1 mg/ml medium, total loadSolution A: 0.1% TFASolution B: 0.1% TFA/60% AcetonitrileFlow: 150 and 600 cm/hGradient: 2080% B, 20 column volumes (cv)

Fig. 24. The influence of increasing flow velocity on resolution.

Actual pressure values generated during a run will depend upon the solvent usedand the operating temperature.

Figure 25 shows pressure versus flow curves with several solvents forRESOURCE RPC columns, packed with SOURCE 15RPC, and FineLINEcolumns, packed with SOURCE 30RPC.


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High Capacity

The controlled uniform pore size distribution in SOURCE RPC is responsible forthe high capacities obtained for peptides, proteins and oligonucleotides.The dynamic binding capacity of SOURCE 15RPC is illustrated in Figure 26while Figure 27 shows an example of the performance maintained by SOURCE30RPC even with h igh sample loads.

Fig. 26. Dynamic binding capacity of SOURCE 15RPC. (Work by Amersham Pharmacia BiotechAB, Lillestrm, Norway.)

Fig. 25a. Pressure:flow curves for (a) RESOURCE RPC, 1 ml and (b) RESOURCE RPC, 3 ml withvarious organic solvents and water. (Work by Amersham Pharmacia Biotech AB, Lillestrm,Norway.)

Fig. 25b . Pressure/flow characteristics of SOURCE 30RPC in various organic solvents and water atroom temperature. The pressure/flow velocity data were determined in a FineLINE column witha) 15 cm and b) 30 cm bed height.

180 cm/h

360 cm/h

1800 cm/h

900 cm/h

Insulin

00

10

20

30

40

50

60

0.0 10.0 20.0 Volume (ml)

%

Pressure (MPa)

3.0

2.0

1.0

3.0

0 180 360 540 720 900 1080 1260 1440

Linear flow (cm/h)

a

Pressure (MPa)

3.0

2.0

1.0

3.0

0 180 360 540 720 900 1080 1260 1440

Linear flow (cm/h)

b

Isopropanol

Ethanol

Water

Acetonitrile

Isopropanol

Ethanol

Water

Acetonitrile

10

8

6

4

2

0

2-Propanol

EthanolAcetonitrile

Water

Pressure(bar)

0 200 400 600 800 1000 1200

Flow velocity (cm/h)

a) 15 cm bed height

10

8

6

4

2

0

0 200 400 600

2-Propanol

Ethanol

Acetonitrile

Water

Flow velocity (cm/h)

Pressure(bar)

b) 30 cm bed height


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Availability

SOURCE 15RPC and SOURCE 30RPC are available in 10 ml, 200 ml, 500 ml,1 litre and 5 litre pack sizes and should be packed in HR or FineLINE columnsaccording to the scale of the separat ion.

SOURCE 15RPC is also supplied pre-packed in PEEK or stainless steel STcolumns: RESOURCE RPC 1 ml is recommended for rapid screening experimentswhere as RESOURCE RPC 3 ml and SOURCE 15RPC 4.6/100 are better suited forapplications where higher resolution is necessary.

SOURCE 5RPC is supplied in stainless steel 4.6/150 columns and isrecommended for small scale or analytical separat ions which require the higherresolution which can be achieved using the smaller 5 m bead size.All pre-packed columns are fully compatible with KTAdesign and other highperformance liquid chromatography systems. Ordering information is shown inSection 7.

Table 3. Pre-packed SOURCE RPC columns

Column Dimensions Recom- Efficiency Maximum Maximum(i.d. x bed mended N/m flow operatingheight) mm flow (ml/min) pressure

(ml/min) (MPa, bar,psi)

RESOURCE 1 ml 6.4/30 1.0 - 5.0 > 12 000 10 3, 30, 435RESOURCE 3 ml 6.4/ 100 1.0 - 5.0 > 12 000 10 3, 30, 435SOURCE 15RPC ST 4.6/100 4.6/100 0.5 - 2.5 > 20 000 5.0 4, 40, 580

SOURCE 5RPC ST4.6/150 4.6/100 1.0 > 60 000 1.5 40, 400, 5800

1.0

0.5

0

0 50 100(min)

Sample load of 1 mgA280 nm

A280 nm

2.0

1.0

0

0 50 100(min)

Sample load of 10 mg

a)

b)Column: SOURCE 30RPC,

10 mm i.d. x 100 mm column (8 ml)Sample: Mixture of Ribonuclease A,

Insulin and AlbuminSample load: 1 and 10 mg/ml media, total loadSolution A: 0.1% TFASolution B: 0.1% TFA/60% Acetonitrile

Flow: 150 cm/hGradient: 2080% B, 20 column volumes (cv)

Fig 27. The influence of increasing sample load on resolution.


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RPC C2/C18 is supplied in 2 pre-packed column formats. RPC C2/C18 PC 3.2/3can be used at relatively high flow rates in many micro-preparative applications.The longer bed height of RPC C2/C18 SC 2.1/10 produces extremely highresolution of complex mixtures where long, shallow elution gradients are oftenused. Characteristics of these columns are shown in Table 4.

RPC C2/C18 RPC C2/C18 SCPC 3.2/3 2.1/10 (stainless(glass column) steel column)

Dimensions i.d. x bed height (mm) 3.2 x 30 2.1 x 100

Efficiency (N/m) >90 000 >100 000

Operational pressure limit 15, 150, 2250 25, 250,3625(MPa, bar,psi)

Pore size 120 120

Particle size 3 m 3 m

Recommended flow (ml/min) 0.01 - 1.2 0.01 - 0.25

Practical loading capacity 0.2 - 500 0.01 - 500(g protein/peptide/column)

Table 4.

Chemical and Physical Stability

RPC C2/C18 can be used with aqueous and organic solvents miscible in water inthe pH range 2 - 8. As with all silica based media extended exposure to pH

extremes should be avoided as the matrix begins to degrade at pH values greaterthan 7 - 8 and less than 2 - 3. SOURCE 5RPC media should be chosen wheneverseparation conditions demand pH conditions to be above pH 8.0.

Additives such as guanidine hydrochloride, urea, formic acid (< 60% ) anddetergents may be used with RPC C2/C18.Both columns may be operated overthe temperature range of 4 - 40 C up to the maximum pressures shown inTable 4.

Flow/Pressure Characteristics

Higher flow rates than those specified in Table 4 are possible with low viscositysolvents, but the integrity of the packing may be compromised if the pressurelimit is exceeded.

Capacity

The maximum capacity of peptides for the RPC C2/C18 PC 3.2/3 column isapproximately 1 - 3 mg and for RPC C2/C18 SC 2.1/10 approximately 1 - 2 mg.However, to minimise the risk of losing unbound material in the flow-throughfractions or losing resolution, lower and more practical loading ranges arerecommended. The detection limit for one peak may be below 1 ng under

optimal conditions.


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Availability

Both columns are designed specifically for use with SMART System. They can beconnected to KTApurifier or other high performance chromatography systemsvia a Precision Column Holder (Code No. 17-1455-01). Ordering information isshown in Section 7 .

Sephasil Protein/Sephasil PeptideProduct Description

Sephasil are porous silica-based media giving excellent resolution and offeralternative selectivities compared to SOURCE RPC media. Carefully controlledproduction conditions ensure batch-to-batch reproducibility for consistentperformance in both analytical and process scale applications.

Sephasil Protein and Sephasil Peptide are available with three differentselectivities C4, C8 and C18. Sephasil Protein C4 is based on a wide-pore 300silica that makes it par ticularly suitable for proteins, whereas Sephasil Peptide isbased on a 100 silica which is more suitable for smaller biomolecules. Sephasil5 m media are recommended for high resolution analysis and purification andSephasil 12 m media for preparative purification of peptides, proteins oroligonucleotides. Characteristics of Sephasil pre-packed columns are shown inTable 5.

Column Pore Specific Maximum Efficiency Recom-(bonded phase particle size size pore operating (N/m) mendeddimensions i.d. mm/bed () volume pressure* flow

height mm) (ml/g) (MPa, bar, psi) (ml/min)Sephasil Protein C4 5 m ST 4.6/100 300 0.6 25, 250,3625 >70 000 0.5 - 2.0Sephasil Peptide C8 5 m ST 4.6/100 100 0.7 Sephasil Peptide C18 5 m ST 4.6/100 100 0.7

Sephasil Protein C4 5 m ST 4.6/250 300 0.6 25, 250,3625 >70 000 Sephasil Peptide C8 5 m ST 4.6/250 100 0.7 Sephasil Peptide C18 5 m ST 4.6/250 100 0.7

Sephasil Protein C4 12 m ST 4.6/250 300 0.6 25, 250,3625 >40 000 0.5 - 2.0Sephasil Peptide C8 12 m ST 4.6/250 100 0.7

Sephasil Peptide C18 12 m ST 4.6/250 100 0.7 Sephasil Protein C4 12 m ST 10/250 300 0.6 25, 250,3625 >40 000 2 - 8Sephasil Peptide C8 12 m ST 10/250 100 0.7 Sephasil Peptide C18 12 m ST 10/250 100 0.7

Sephasil Protein C4 12 m ST 20/250 300 0.6 25, 250,3625 >40 000 5 - 20Sephasil Peptide C8 12 m ST 20/250 100 0.7 Sephasil Peptide C18 12 m ST 20/250 100 0.7

Sephasil Protein C4 12 m ST 50/250 300 0.6 14, 140, 2000 >40 000 20 - 60Sephasil Peptide C8 12 m ST 50/250 100 0.7 Sephasil Peptide C18 12 m ST 50/250 100 0.7

*refers to pressure above which bed compression may begin

Table 5.


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Chapter 3

3. Methods

The following sections will discuss the practical steps involved in planning andimplementing a reversed phase separation. Critical aspects of selecting theappropriate stationary phase, mobile phase and gradient conditions are discussed.Practical considerations of sample preparation, solvent handling, and potentialpitfalls which may be encountered will also be presented.

Choice of separation mediumThe proper choice of reversed phase medium is critical for the success of aparticular application. This choice should be based on the following criteria:

1) The unique requirements of the application, including scale and mobilephase conditions.

2) The molecular weight, or size of the sample components.3) The hydrophobicities of the sample components.4) The class of sample components.

Unique requirements of the application

ResolutionIt is essential to choose a medium which will yield the required resolution for agiven purification. Applications involving fractionation of multi-componentsamples, such as peptide mapping, require extremely high resolution. Preparat ivereversed phase applications, such as the purification of synthetic peptides, aremore concerned with throughput, and resolution is routinely traded off againstboth speed and capacity.

Resolution in reversed phase chromatography depends on both the selectivity andthe efficiency of the column. Selectivity for a specific separation depends on the

nature of the immobilised hydrophobic ligand, the chemistry used to derivatisethe silica matrix and chromatographic conditions used, such as the compositionof the mobile phase.

The maximum efficency of a reversed phase column depends on the process usedto pack the column and the particle size of the medium. The smallest par ticles,e.g. RPC C2/C18, give the highest efficiency followed by the larger 5 m media,such as SOURCE 5RPC, Sephasil Protein 5 m and Sephasil Peptide 5 m. Lowerefficiency is seen with larger 12 m, 15m and 30 m particles such as SephasilProtein 12 m, Sephasil Peptide 12 m, SOURCE 15RPC and SOURCE 30 RPC.


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Scale of the purification

For micro-purification narrow bore columns packed with 3 or 5 m par ticlesshould be used e.g. RPC C2/C18 or SMART columns Sephasil C8 and SephasilC18. The ligands C2/C18, C8 and C18 will offer significantly differentselectivities from which to choose.

For typical laboratory scale purification 5 m media can be selected e.g.SOURCE 5RPC, Sephasil Protein 5 m and Sephasil Peptide 5 m. It may bemore suitable to use 12 m media for part icularly crude samples or SOURCE15RPC media, if a high pH or alternat ive selectivity to silica is required for asuccessful separation.

Media with larger part icle sizes should always be chosen for pilot and processscale purification, using their equivalent smaller particle size media for methodscout ing prior to scale up e.g. RESOURCE RPC pre-packed columns exhibit

similar selectivity to SOURCE 30RPC.

Mobile phase conditions

Choice of reversed phase matrix may also be influenced by the composition of themobile phase. If the stability or solubility of the sample components dictates theuse of a specific solvent system, then the stability of the base matrix in thatsolvent needs to be considered. For example, when the reversed phasechromatography will involve mobile phases with pH above 7.5, a polystyrene-based matrix, such as SOURCE RPC, should be used.

Throughput and scaleability

The amount of sample material that can be processed within a defined time (i.e.throughput) is determined by such properties as capacity, flow characteristics andthe size of the column the medium is packed into. SOURCE 15RPC, SOURCE30RPC, Sephasil Protein 12 m and Sephasil Peptide 12 m have been optimisedfor throughput in large scale preparat ive chromatography with easy scale upunder high performance conditions.

Molecular weight of the sample components

The accessibility of the sample components to the immobilised hydrophobicligands will determine the available capacity of the reversed phase medium for aspecific biomolecule. Accessibility depends greatly on the pore size and the porevolume of the bead. Reversed phase media with large pores will typically givebetter capacity and resolution with larger biomolecules than media with smallpores. Generally, pore sizes of 300 or larger are recommended for theseparation of proteins. Pore sizes less than 300 are recommended for theseparation of peptides and oligonucleotides. The pore sizes and capacities for thereversed phase media supplied by Amersham Pharmacia Biotech are given inChapter 2.


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Hydrophobicity of the sample components

Unlike other chromatographic techniques, such as ion exchange and size exclusionchromatography, it is difficult, if not impossible, to predict the retention ofbiomolecules in reversed phase chromatography. Attempts to predict retention onthe basis of hydrophobicity factors derived from studies of standard peptides have

been the subject of several studies. One which provides an a lgorithm which hasbeen shown to mimic quite closely the retention behaviour of protein samplessubjected to proteolytic digestion has been described by Sakamoto (7). The generalbenefit from such studies has been an improved understanding of the bindingprocess. The important parameters affecting the retention of a peptide appear to bea combinat ion of the amino acid sequence of the peptide together with anysecondary structure, such as a-helices and b-pleated sheets, that the peptide maypossess. The situation for proteins is further complicated by their tertiary structure.

The selection of a reversed phase medium must, therefore, be made empirically,

depending on the nature of the biomolecule components in the sample. Someprediction of resolution can be made based on the hydrophobicity of the samplerelative to the immobilised ligand, i.e. the more hydrophobic the sample, the lesshydrophobic the immobilised ligand should be. Consequently, a medium with C8ligands, is generally recommended for preparative purification of more hydro-phobic biomolecules than those purified by the corresponding C18 medium.

Choice of reversed phase matrix may also be influenced by the composition of themobile phase. When the reversed phase chromatography will involve mobilephases with pH above 7.5, a polystyrene-based matrix, such as SOURCE RPC,should be used. When the stability of the sample components dictates the use of a

specific solvent system, then the stability of the base matrix in tha t solvent needsto be considered.

Class of sample components

Reversed phase chromatography is used for purification of many classes ofbiomolecules.

Whilst the conditions of reversed phase chromatography usually have no harmfuleffects on the chemical integrity of oligonucleotides and peptides, the question ofthe stability and biological activity of proteins must be considered carefully.Polypeptide interactions with a hydrophobic surface and organic solventsgenerally leads to some loss of tertiary structure. The loss in tertiary structuremay then give rise to different conformational states for a given biomolecule (8)and each of these states may interact differently with the reversed phase medium.

The widespread use of reversed phase chromatography for the large-scalepur ification of recombinant and synthetic proteins and peptides, such as insulin,growth hormone, growth factors and many others, indicates, however, thatproblems caused by denaturation can often be overcome. Loss of structure and,consequently, loss of activity can be minimised by proper treatment. The kinetics


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be reversed by transferring the protein to conditions under which its nativestructure is favoured. N ote that complex enzymes and multi-component proteinsare more likely to lose activity than small peptides or highly stabilised and cross-linked proteins. However, when a protein or peptide is pur ified for primarystructure determination, denaturation is not a problem unless precipitation occurs

and reversed phase chromatography has found widespread use in preparing purebiomolecules for subsequent sequencing.

Choice of mobile phaseThe mobile phase in reversed phase chromatography of biomolecules generallycontains a buffer component, an organic modifier and, often, an ion pairingagent added to the mobile phase to affect selectivity.

All solvents, buffering salts, ion pairing agents, as well as the water used to

prepare the mobile phases, must be of high chemical pur ity and should be free ofany metal ions. Solvents, salts and water should be labelled as H PLC grade toensure sufficient pur ity. Chemical purity is important in preparat ive reversedphase chromatography, since any contaminants in the mobile phase may affectthe chromatography by producing unwanted extra peaks, ghost peaks, and maycontaminate the purified biomolecule.

The organic solvent

The organic solvent is added to the mobile phase to lower its polarity. Twosolvents with different polarity, such as an aqueous low pH solution and

acetonitrile, may be mixed together resulting in a solvent with polarityintermediate between those of the original components. The lower the polarity ofthe solvent mixture, the higher its eluting power in reversed phasechromatography. There is a large number of water miscible organic solvents thatcan be used in reversed phase chromatography but few of them are used inpractice. The properties of some typical organic solvents used in reversed phasechromatography are shown in Table 6.

Table 6. Solvents used in reversed phase chromatography.

Solvent Boiling UV cut-off* Viscosity Comments

point (C) (nm) (cP at 20 C)

Acetonitrile 82 190 0.36 More powerfuldenaturant thanalcohols. Toxic.

Ethanol 78 210 1.20Methanol 65 205 0.601-propanol 98 210 2.26 Viscous(n-propanol)2-propanol 82 210 2.30 Viscous(iso-propanol)Water 100


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Acetonitrile and methanol are the most commonly used since both have lowviscosity (even when mixed in aqueous solution) and are UV transparent.2-Propanol has the advantage of lower polarity and therefore higher elutingstrength. It is UV transparent and is excellent for cleaning the reversed phasecolumn. H owever, use of 2-propanol as the organic modifier results in high

viscosity mobile phases. High viscosity mobile phases are undesirable since theyresult in poor mass transport of solute between the mobile and stationary phasesand high back-pressure even at moderate to low flow rates. Figure 29 shows thechange in pressure during gradient formation due to the viscosity effects ofvarious solvents.

Fig. 29. Variation of pressure during elution with a gradient from 100 % water to 100% organicsolvent at constan t flow. Note that the pressure increases by ca. 50% from the starting value (purewater) during elution with methanol. The increase in the case of acetonitrile is much smaller,ca. 10%. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.)

100

80

60

40

20

02 4 6 8 10 12 14 16

100

80

60

40

20

02 4 6 8 10 12 14 16

Time (min)

Time (min)

Column: C18 silica, 10 mm (i.d. 4.6 mm, length 250 mm).Eluent A: 100% waterEluent B: (A) 100% acetonitrile

(B) 100% methanolGradient: 0-100% B in 10 min

100% B for 2 min100-0% B in 3 min0% B for 2.5 min

Flow: 1.75 ml/min

acetonitrile

methanol


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Only high quality organic solvents should be used in reversed phasechromatography. H PLC grade solvents are desirable since small par ticulates thatcan clog reversed phase columns have been removed. They provide adequatechemical pur ity with good transparency to low wavelength UV light. Organicsolvent in the eluting buffer is generally removed from the recovered biomolecule

by evaporat ion and residual organic contaminan ts, such as acrylic acid, can bedamaging to the biological integrity of the recovered sample. UV transparency isespecially important since many reversed phase separations are monitored below220 nm for optimum detection sensitivity (e.g. peptides, pro teins lackingsignificant amounts of Trp or Tyr, etc.). Th

reversed phase chroma

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