Chromatography Reverse Phase

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Reverse-Phase Chromatography

RP-HPLC for Peptides

Reverse-phase high performance liquid chromatography (RP-HPLC) is an extremely useful tool for analytical biochemists. However, unlike small molecule HPLC, separations of proteins and peptides are nearly always performed under gradient conditions. There are other differences that one needs to be aware of in order to develop RP-HPLC separations of proteins and peptides as efficiently as possible. The general guidelines given in this short article may help reduce your method development time.

RP-HPLC of complex peptide or protein mixtures remains a general method of choice because of the resolution it provides. Unlike small organic molecules whose chromatographic behavior is described by a finite partitioning equilibrium between the stationary phase and the mobile phase, proteins and peptides typically do not exhibit such an effect. Instead, they exhibit an adsorption phenomenon in which the polypeptide is adsorbed onto the stationary phase and elutes only when the solvent strength of the mobile phase is sufficient to compete with the hydrophobic forces keeping it there. For this reason, elution of peptides or proteins from reverse-phase supports is by gradients of increasing solvent strength. When run under isocratic conditions, peaks for proteins and peptides are typically much broader than their small molecule counterparts.

Getting Started

Column: A good starting point for separating peptide or polypeptide mixtures is to start with a C18-bonded silica column designed for these applications. The Discovery® BIO Wide Pore C18 is an ideal choice. Begin with 5 μm particles packed into 15 cm L x 2.1 mm I.D. columns with a mobile phase flow rate of 0.2 mL/min. The 2.1 mm I.D. or “narrowbore” column configuration is a good balance between sensitivity (with 4.8-times the sensitivity of a 4.6 mm I.D. column) and analysis time. If the dwell volume* of your HPLC system is greater than 500 μL, a 4.6 mm I.D. column run at 1.0 mL/min is likely to be a better dimension to begin with.

Mobile phase: Choice of mobile phase will in part be dictated by the means of detection. If detection is by mass spectrometry (MS), then the options are more limited and also generally don’t provide optimal chromatography. Commonly used MS-compatible ionic mobile phase additives are acetate, formate, and carbonate and their corresponding ammonium salts. The organic component is most often acetonitrile (CH3CN).

If detection is by traditional UV absorption, then mobile phases can be selected that provide for superior chromatography, which is largely conferred by including ion pairing reagents like TFA (trifluoroacetic acid) and HFBA (heptafluorobutyric acid). Typical concentrations are 0.05–0.1% (v/v). A good starting point is to prepare mobile phase A to be 0.1% TFA

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Reverse-Phase Chrom

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in water and mobile phase B to be 0.1% TFA in a mixture of 50:50 water:acetonitrile. Alternatively, having some acetonitrile in both A and B minimizes the refractive index changes that occur upon mixing and can provide more stable baselines.

An initial run should begin with a gradient of 10 to 100% B in 45 minutes. An example of this starting gradient for peptides derived from a protein digest is shown in Figure A. While this separation does not provide significant resolution of component peptides, there are means that can be employed to improve the resolution for virtually all parts of the chromatogram.

The effect of these changes is shown in Figure B where the gradient profile was changed from 10 to 70% B in 60 minutes. The shallower gradient provided some improvements in resolution in the first half of the gradient. However, just as significant is the difference in selectivity in various groups of peaks. This is a fundamental effect due to the differential responses of the sample components to changes in the gradient slope. Also note that there is still wasted space at the end of the chromatogram that can be reduced.

To see if a lower initial concentration of acetonitrile improves resolution of early eluting peaks, a third run is needed. This run has the same slope as previous runs, but in order to

maximize the resolution of early-eluting peaks, the gradient starts at 100% aqueous. Under these conditions, the bonded phase is better able to distinguish between small differences in the hydrophobicity of the peptides. Although this may come at a cost of longer run time and may result in some wasted space in the initial part of the chromatogram, the improved resolution afforded may offset these consequences. The chromatogram in Figure C shows that this change had the desired effect. Peaks eluting before 25 minutes were better resolved than in Figure B.

Another option to improve resolution is to increase the run time. Although this reduces through-put, it may in some cases be the best way to obtain the required resolution. In Figure D the run time is increased to 130 minutes: 0 to 65% B in 130 minutes (0.25% CH3CN/minute) as compared to Figure B where the slope is 0.5% CH3CN/minute.

Figure A. Initial Chromatographic Run of Complex Peptide Sample

Column: Discovery BIO Wide Pore C18, 15 cm x 2.1 mm I.D., 5 μm (568202-U) Mobile Phase: (A): 0.1% TFA (v/v) in water (B): 50:50, (0.1% TFA, v/v in water): (0.1% TFA, v/v in CH3CN) Flow Rate: 0.208 mL/min Temp.: 35 °C Det.: 215 nm Inj.: 10 μL Sample: Carboxymethylated b-Lactoglobulin B tryptic digest Gradient: Min %A %B 0 90 10 45 0 100

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One last option, but not illustrated here, is column length. Increased column length may provide the desired improvements in resolution. The separations in this article were all run on 15 cm columns, but very often 25 cm columns are used.

In conclusion, the gradient profile (rate of change in % CH3CN and the starting and ending % CH3CN) has enormous power to increase resolution and decrease analysis time. By combining gradient methodology with a highly-efficient RP-HPLC material, like Discovery® BIO Wide Pore C18, one has all the tools needed to maximize the separation of peptides and protein digests.

Reference:

Geng, X. and Regnier, F.E. 1984. Retention Model for Proteins in Reversed-Phase Liquid Chromatography. J. Chrom 296, 15.

*Dwell volume is the volume from and including the mixer to the column inlet. Low pressure mixing systems generally have larger dwell volumes than high pressure mixing systems.

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Conditions for Figures B-D Column: Discovery BIO Wide Pore C18, 15 cm x 2.1 mm I.D., 5 μm (568202-U) Mobile Phase: (A): 0.1% TFA in water (B): 50:50, (0.1% TFA in water): (0.1% TFA in CH3CN) Flow Rate: 0.208 mL/min Temp.: 35°C Det.: 215 nm Inj.: 10 μL Sample: Carboxymethylated -Lactoglobulin B tryptic digest

Figure B. Change Gradient Profile Gradient: Min %A %B 0 90 10 60 30 70

Figure C. Enhanced Resolution Early in the Gradient

Gradient: Min %A %B 0 100 0 65 35 65

Figure D. Further Refine Gradient Slope Gradient: Min %A %B 0 100 0 130 35 65

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Reverse-Phase Chrom

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Effect of Stationary Phase on Selectivity of Reversed-Phase HPLC Separations of PolypeptidesRP-HPLC separations of peptides and polypeptides is influenced by the chemistry of the bonded phase. The objective of most peptide separations is to obtain as much information about the sample as possible, especially when working with peptide maps. Therefore, it is to the researcher’s advantage to run the sample on different stationary phases, like a C18, C8, and C5.

What is Meant by Selectivity?

The resolution equation:

RS = (1/4) N1/2 {(a-1)/a} {k/(1+k)}

tells us that retention (k), efficiency (N), and selectivity (a) each play a role in a chromato-graphic separation. There are few improvements that can be made to column efficiency if one is working with small particles and modern packing materials. Retention also gives limited options because of the need to keep analysis times as short as possible. However, selectivity wields great power to increase resolution. Selectivity can be thought of as peak spacing. The further the peaks are spaced from one another, the better the selectivity. Selectivity, or separation factor, between peaks 1 and 2 is measured by the equation:

a = k2/k1

where k = (tR–t0)/t0

Of course, improvements in selectivity beyond allowing for complete baseline resolution of all sample components is of no additional benefit. Application Note 166 (T302166) showed that improvements in selectivity (and thus resolution) for a complex peptide sample can be achieved by altering the gradient slope and start conditions for the run. These are the most common strategies for optimizing selectivity with polypeptide samples, but there are other tools as well. In this short article, we will discuss the effect of stationary phase chemistry on the selectivity of peptide separations.

How Does the Stationary Phase Effect Selectivity?

Unlike the partitioning mechanism exhibited by small molecules, retention of polypeptide analytes on a reversed-phase matrix is by differential adsorption to the stationary phase, primarily due to differences in their hydrophobicity. More hydrophobic peptides are retained longer by the bonded phase, and vice versa. By reducing the alkyl chain length of the bonded phase, not only is the hydrophobicity reduced, but also the total surface area that is in contact with the peptide analytes. For small molecule separations, a C18 and C8 will usually give the same selectivity, although different retention. However, because of different


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retention mechanisms for peptide and polypeptide separations, the differences in selectivity between a C18, C8, and C5 can be dramatic. The same sample run on C18, C8, and C5 phases will yield different information about the sample, an important consideration for peptide mapping.

There are other factors that affect adsorption to the matrix as well, even when comparing only linear aliphatic alkyl bonded phases. These other factors involve polar or H-bonding interactions with the silica surface itself, or the indirect effects of the silica surface chemistry on the conformation of the bonded phase. Thus, not only differences in the hydrophobicity of the bonded phase can influence selectivity, but also secondary effects impacted by the bonding chemistry and surface silanols: bonding density, extent of endcapping of silanols, and type of bonding (mono, di, or trifunctional).

An example of selectivity differences conferred by bonded phase chemistry is shown in Figures A and B. Here, a proteolytic digest of apohemoglobin is chromatographed on the three Discovery® BIO Wide Pore reversed-phases C18, C8, and C5. The chromatograms displayed only represent a portion of the entire run to better illustrate the subtle, but significant, differences in selectivity conferred by each phase. Each of the phases displays better selectivity in different parts of the chromatogram. If the goal is purification of a specific peptide, then this has particular utility. If the goal is the best overall resolution of the entire sample, then a decision process should be applied which evaluates the performance of each phase with its optimized method.

In conclusion, different bonded phase chemistries give subtle yet significant differences in selectivity toward peptides and polypeptides. Running the sample on each of the three Discovery BIO Wide Pore reversed-phase chemistries will yield different, useful information about the sample.


Reverse-Phase Chrom

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Figure A. Proteolytic Digest Discovery BIO Wide Pore C18, C8, and C5 (0-16 minute window)

Mobile Phase: (A): 95:5, (0.1% TFA (v/v) in water): (0.1% TFA (v/v) in CH3CN) (B): 50:50, (0.1% TFA (v/v) in water): (0.1% TFA (v/v) in CH3CN) Column: Discovery BIO Wide Pore, 15 cm x 4.6 mm I.D., 5 μm particles Flow Rate: 1.0 mL/min Temp.: 30 °C Det.: UV, 215 nm Inj.: 50 μL Sample: tryptic digest of carboxymethylated apohemoglobin Gradient: Min %A %B 0 100 0 65 0 10

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Figure B. Proteolytic Digest Discovery BIO Wide Pore C18, C8, and C5 (28-38 minute window)

Mobile Phase: (A): 95:5, (0.1% TFA (v/v) in water): (0.1% TFA (v/v) in CH3CN) (B): 50:50, (0.1% TFA (v/v) in water): (0.1% TFA (v/v) in CH3CN) Column: Discovery BIO Wide Pore, 15 cm x 4.6 mm, 5 μm Flow Rate: 1.0 mL/min Temp.: 30 °C Det.: UV, 215 nm Inj.: 50 μL Sample: tryptic digest of carboxymethylated apohemoglobin Gradient: Min %A %B 0 100 0 65 0 100

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Discovery BIO Wide Pore C18







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Products for Reverse-Phase ChromatographyDiscovery® BIO Wide Pore HPLC columns and capillaries provide sensitive, stable, reproducible separations of proteins and peptides. The different phase chemistries and separation modes provide unique selectivity options. Separations are scalable from analytical to preparative.

Please visit sigma.com/discovery for complete information on our range of high-specification HPLC columns and visit sigma.com/proteomics_literature to request a copy of the full Discovery BIO catalogue.

Particle Length I.D. Type Size (µm) (cm) (mm) Cat. No.

Reverse-Phase ColumnsDiscovery BIO Wide Pore C5

Capillary 3.0 5.0 0.2 65609-U 3.0 5.0 0.3 65531-U 3.0 5.0 0.5 65520-U 3.0 10.0 0.2 65611-U 3.0 10.0 0.3 65532-U 3.0 10.0 0.5 65521-U 5.0 5.0 0.2 65612-U 5.0 10.0 0.2 65613-U 5.0 15.0 0.2 65614-U 5.0 15.0 0.3 65533-U 5.0 15.0 0.5 65522-U

Microbore 3.0 5.0 1.0 65511-U 3.0 10.0 1.0 65512-U 5.0 15.0 1.0 65513-U

Narrowbore 3.0 5.0 2.1 567226-U 3.0 10.0 2.1 567227-U 3.0 15.0 2.1 567228-U 5.0 5.0 2.1 568400-U 5.0 10.0 2.1 568401-U 5.0 15.0 2.1 568402-U 5.0 25.0 2.1 568403-U

Guards Pkg of 2 3.0 2.0 2.1 567278-U Kit 3.0 2.0 2.1 567279-U Pkg of 2 5.0 2.0 2.1 568470-U Kit 5.0 2.0 2.1 568471-U


Standard Analytical 3.0 5.0 4.6 567229-U 3.0 10.0 4.6 567230-U 3.0 15.0 4.6 567231-U 5.0 5.0 4.0 568410-U 5.0 5.0 4.6 568420-U 5.0 10.0 4.0 568411-U 5.0 10.0 4.6 568421-U 5.0 15.0 4.0 568412-U 5.0 15.0 4.6 568422-U 5.0 25.0 4.0 568413-U 5.0 25.0 4.6 568423-U 10.0 25.0 4.6 567232-U


Semi-preparative 5.0 25.0 10.0 568430-U 10.0 5.0 10.0 567233-U 10.0 15.0 10.0 567234-U 10.0 25.0 10.0 567235-U

Guards Pkg of 1 10.0 1.0 10.0 567286-U

Preparative 10.0 5.0 21.2 567236-U 10.0 15.0 21.2 567237-U 10.0 25.0 21.2 567238-U


Reverse-Phase Chrom

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Discovery® BIO Wide Pore C8






Guards

Pkg of 1 10.0 1.0 10.0 567284-U

Preparative 10.0 5.0 21.2 567223-U 10.0 15.0 21.2 567224-U 10.0 25.0 21.2 567225-U



Capillary 3.0 5.0 0.2 65603-U 3.0 5.0 0.3 65526-U 3.0 5.0 0.5 65517-U 3.0 10.0 0.2 65604-U 3.0 10.0 0.3 65527-U 3.0 10.0 0.5 65518-U 5.0 5.0 0.2 65606-U 5.0 10.0 0.2 65607-U 5.0 15.0 0.2 65608-U 5.0 15.0 0.3 65529-U 5.0 15.0 0.5 65519-U

Microbore 3.0 5.0 1.0 65504-U 3.0 10.0 1.0 65506-U 5.0 15.0 1.0 65508-U 5.0 25.0 1.0 65509-U




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Discovery® BIO Wide Pore C18 (Cont.)




Guards Pkg of 1 10.0 1.0 10.0 567282-U

Preparative 10.0 5.0 21.2 567210-U 10.0 15.0 21.2 567211-U 10.0 25.0 21.2 567212-U

Description Cat. No.

Reverse Phase Media

C18 packing pkg of 10 g 58419

HYPERPREP C18 SILICA GEL, 30 μm, 100 g 13326

Polyamide for column chromatography, 6 02395

Silica gel 90 C18-Reversed phase for column chromatography, fully endcapped 60757



Silica gel 100 C18-Reversed phase for column chromatography, not endcapped 60758

Silica gel 100 C8-Reversed phase for column chromatography 60759


Hydrophobic Interaction Chromatography (HIC) ColumnsTSK-GEL®

Butyl-5PW, 3.5 cm × 4.6 mm I.D., 2.5 μm 814947

Ether-5PW, 7.5 cm × 7.5 mm I.D., 10 μm 808641

Phenyl-5PW, 7.5 cm × 7.5 mm I.D., 10 μm 807573

Guard Column Kit: Phenyl-5PW 1 cm × 6.0 mm I.D., 20 μm. Kit contains one cartridge, a stand-alone holder, 5 mL packing, 5 cm of 1/16 in. tubing, and 2 nuts and ferrules. 807652

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Chromatography Reverse Phase