zorbax hplc column selection guide for biotechnology (5988...

Agilent ZORBAX HPLCColumn Selection Guidefor BiotechnologyResolution that Lasts

The variety and number of bioanalytical HPLC applications continue to grow rapidly. Agilent Technologies’ goal is to offer products that can be used to meet these application needs. This HPLC column guide presents the AgilentTechnologies columns, featuring the extremely durable and reproducible ZORBAX product line, for key bioanalytical separations. In this guide you will find information on columns for reversed-phase HPLC (RP-HPLC), size exclusion chromatography (SEC), ion-exchange chromatography (IEC) and hydrophobicinteraction chromatography (HIC). In addition, this guide provides pertinent information and column choices for some specific bioanalytical applicationsincluding high-resolution separations of amino acids, double stranded DNA and oligonucleotides.

Reversed-phase (RP) HPLC is most often used for the analytical separations ofproteins and peptides due to the high resolution achieved with this technique.Because of the importance of RP-HPLC in complex bioanalytical separations, thisguide opens with a simple strategy for reversed-phase method development ofpeptides and proteins. The use of LC/MS in bioanalytical separations is also dramatically increasing since it can provide detailed molecular information.Following the reversed-phase strategy are a few key tactics for maximizingLC/MS signal intensity.

Review the Table of Contents on the next page to find the Agilent products thatbest meet your bioanalytical chromatographic needs.

Bioanalytical Chromatographyfrom Agilent Technologies

Strategy for Reversed-Phase Peptide/Protein Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Peptide/Protein HPLC Method Development —LC/MS Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Columns for Peptide and Protein Separations

ZORBAX StableBond HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

ZORBAX SB-Aq HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

ZORBAX Poroshell 300SB-C18 HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . 19

ZORBAX Extend-C18 HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

ZORBAX Eclipse XDB HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

ZORBAX Bonus-RP HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

ZORBAX Rx HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Additional Reversed-Phase Columns for BioScience . . . . . . . . . . . . . . . . . . . . 27

ZORBAX GF-250/450 Size Exclusion Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

TSK Gel Filtration Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

ZORBAX SAX and SCX Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

TSK, SynChropak and PL Ion Exchange Columns . . . . . . . . . . . . . . . . . . . . . . . 32

TSK and SynChropak Hydrophobic Interaction Chromatography Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Special Application Columns

ZORBAX Eclipse AAA (Amino Acid Analysis) and AminoQuant Columns . . . 34

ZORBAX Eclipse dsDNA Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

ZORBAX Oligo Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Contents

NEW

2

Protein Separations

Reversed-Phase Columns

Reversed-phase HPLC is a populartechnique for the separation of proteinsand peptides because of its ability toefficiently resolve compounds that maydiffer by as little as one amino acid or asingle modification. Gradient reversed-phase HPLC is the most practicalapproach to separations of peptidesand proteins because the retention ofthese molecules changes dramaticallywith small changes in the % organic ofthe mobile phase and this sensitivityincreases as molecular weight increases. In addition, gradient RP-HPLC provides excellent methoddevelopment flexibility for optimizingthe separations of peptides and proteins. This flexibility is a result ofmany bonded phase column options aswell as the ability to change mobilephases, change gradient separationparameters and temperature. Theretention and resolution of peptides isalso sensitive to the pH of the mobilephase since the overall charge state of these molecules changes with pH.The following method developmentstrategy reviews key steps in themethod development process and considers the effects of low and highmobile phase pH in the separation ofpeptides and proteins.

Protein and Peptide Separations — Start at Low pH

Most reversed-phase protein and peptide separations are performed at low pH with a simple aqueous:organic gradient mobile phase — most often 0.1% TFA in water : 0.09% TFA in acetonitrile. An initial reversed-phasecolumn choice is made following a few basic guidelines summarized here.After the initial separation, a variety of changes can be made to maximize resolution and reduce analysis time. These include changing the gradient conditions, mobile phase, temperature, and bonded phase. ZORBAX 300StableBond and StableBond reversed-phase columns are recommended for low pH protein and peptide separations because of their well-documented stability at low pH.

Peptides and Polypeptides can also be Separated at Mid and High pH

If an optimized low-pH mobile phase does not provide an acceptable separation for peptides and polypeptides, then a mid- or high-pH mobilephase can be used. When using higher pH mobile phases, selectivity isoften very different because the overall charge on a peptide may changesubstantially as the pH increases. Acidic amino acids will become negative-ly charged and some basic amino acids will lose their charge. Therefore,higher pH mobile phases can be used to expand selectivity options withsamples of peptides, polypeptides and small proteins. ZORBAX 300Extend-C18 and Extend-C18 columns are new silica-based columns developed forseparations at high pH. The ZORBAX Eclipse XDB and Bonus-RP columnsare good choices for mid-pH separations of smaller peptides.

The following section on reversed-phase method development offers guidelines on how to choose an HPLC column and optimize the separationfollowing a six-step process.

Strategy for Reversed-Phase HPLCPeptide/Protein Method Development

0 5 10 15 20 25 30 35 40 45

0 2 4 6 8 10 12 14 16 18 20 24 2622

0 1 2 4 5 6 8 973

1 23 4 5 6 7

89 10

40 min.

Time (min)

24 min.

9 min.

3

Choose the initial column and conditionsInitial column parameters - internal diameter, length, particle size, pore size, bonded phase

Start at Low pH

Choosing an initial column for a peptide or protein separation is simple providedthat a few parameters are defined. Some of these parameters will be explored in more depth as we examine method optimization steps.

Choose the column pore size based on sample molecular weightIt is important to select a column with the proper pore size so thathigher molecular weight analytes can efficiently interact with theentire bonded phase. Peak widths can broaden if the pore size is toosmall for the molecules to freely enter and exit the pores (Figure 1).

Choose: 80Å for < 4 - 5 kD300Å for > 4 - 5 kD

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

PW 1

/2

Leu Enke

phalin

M.W. =

556

Angiotensin II

M.W. =

1046

Insulin

B

M.W. =

3,49

6Cyt

C

M.W. =

12,32

7

RNase

M.W. =

13,68

4

Lysozym

e

M.W. =

13,90

0

300SB-C18 (300Å)SB-C18 (80Å)

FIGURE 1Choosing the Best Pore Size for Your Analysis

Protein SeparationsReversed-Phase Columns

Strategy for Reversed-Phase HPLC Peptide/Protein Method Development

1

FIGURE 2Optimize Column Length, Gradient Time and Resolution with Rapid Resolution Columns

Choose the column particle sizeand column length to optimizeefficiency and analysis timeThere are several choices in both particle size and column length. Thecombination of these two parametersdetermine both the efficiency andanalysis time of a separation. A 5 µmparticle size is considered standard,but a smaller 3.5 µm particle size canprovide high efficiency in a shorter column length.

For example, longer columns (250 mm)have been traditionally selected tomaximize resolution when using 5 µmparticles (Figure 2A). However, shorter150 mm, 3.5 µm columns offer reducedanalysis time while maintaining resolu-tion (Figure 2B). For less complex samples and even faster analysis, 50 mm and 30 mm column are frequently selected, especially forLC/MS (Figure 2C).

Choose: 150 mm or shorter, 3.5 µm columns to start

You can optimize the column length and gradient timeto minimize analysis time. When you reduce columnlength, you can reduce particle size to maintain efficiency. The gradient time is reduced proportionalto the decrease in column length, reducing analysistime while maintaining the peak elution pattern. Itmay be possible to choose even shorter columnlengths, further reducing analysis time.

Mobile Phase: A: 95% H2O

5% ACN with 0.1% TFA

B: 5% H2O95% ACN with 0.0.85% TFA

Flow Rate: 1.0 mL/min

Detection: 215 nm

Sample: 1-10 µg protein (10 µL inj.) in 6M Guanidine HCL, pH 7.0

1. Met-enkephalin 6. Insulin (Bov)2. Leu-enkephalin 7. Lysozyme3. Angiotensin II 8. Calmodulin4. Neurotensin 9. Myoglobin5. RNase 10. Carbonic Anhydrase

A. ZORBAX 300SB-C8, 4.6 x 250 mm, 5 µm(Agilent Part No. 880995-902) 10 - 60% B in 50 min

10 - 60% B in 10 min

10 - 60% B in 30 min

C. ZORBAX Rapid Resolution 300SB-C8, 4.6 x 50 mm, 3.5 µm(Agilent Part No. 865973-906)

B. ZORBAX Rapid Resolution 300SB-C8, 4.6 x 150 mm, 3.5 µm(Agilent Part No. 863973-906)

1a

1b

0 10 20 30 40Time (min)

100% B

0% B 100% B

0% B 100% B

0% B 100% B

0% B

tG = 5

tG = 10

tG = 20

tG = 40

Optimize gradient conditions toincrease sample resolution

4



Choose the column internal diameter to optimizesensitivity, sample load and solvent usage

Column Type/i.d. When to Choose

Analytical for standard analytical work4.6 mm i.d.

Solvent Saver to reduce solvent usage by 60% in 3.0 mm i.d. standard analytical work

Narrow Bore when sample limited, for increased 2.1 mm i.d. sensitivity, to save solvent (80% solvent

reduction), or for LC/MS compatibility

Micro Bore for high sensitivity when sample 1 mm i.d limited or for LC/MS compatibility

Capillary for maximum sensitivity with the smallest 0.5, 0.3 mm i.d. samples or for LC/MS compatibility

Semi-Preparative for small scale (milligram) preparative 9.4 mm i.d. separations

Preparative for large scale (hundreds of mgs to gm) 21.2 mm i.d. preparative separations

First, determine the optimum gradientrange (initial and final %B) to elute all components of interest. This willvary from sample to sample. Then several additional parameters maybe changed in order to improve resolution.

• Increase gradient time (Figure 3)

• Decrease column length (Figure 4)

• Increase flow rate

Choose the bonded-phase tooptimize selectivity

• C8 is an excellent starting bondedphase because of its moderatehydrophobicity

• C18 and C8 are generally selected forpeptides but can also be used forproteins

• C3, C4 and CN are generally selectedfor larger, hydrophobic polypeptidesand proteins but can also be used for peptides

Run initial separation conditions foryour sample that provide high effi-ciency and reasonable analysis timeColumn: 300SB-C8, 4.6 x 150 mm

5 or 3.5 µmMobile Phase: A: 95% H2O: 5% ACN

with 0.1% TFAB: 5% H2O: 95% ACN

with 0.085% TFAGradient: 0 - 60% B in 60 minTemperature: 35 - 40°CFlow Rate: 1 mL/min2

FIGURE 3Increasing Gradient Time Increases Resolution

One way to increaseresolution is to increasegradient time (tG) asshown here. This willalso increase the analysis time. Thus,choosing the best compromise betweenresolution and analysistime is important.

Gradient Retention Equation

k* =tG • F

S • Vm • ∆Φ

1c 1d

tG: Gradient TimeVm: Column Volume

F: Flow Rate∆Φ: Organic Range

S: ConstantIncrease k* to increase Rs

Abs(

210n

m)

Time (min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

300SB-C18(Agilent Part No. 883995-902)



300SB-CN(Agilent Part No. 883995-905)

L6

L3 L4

L6

L7 L9

L3L2L1 L4 L6

L7 L5 L8

L2L1 L3

L8

L1

L1

L2

L2

L3 L4

L4

L5

L5

L5

L6

L7

L7

L8

L8

L9

L9

L9

i

i

i

i

i

i

0 2 4 6 8 10 12 14 16

Time (min)

2

4 6

8 101

3

5

7

9

2 4 6

8 101 3

5

7

9

5

FIGURE 5The Advantage of Selectivity Changes Using Different StableBond Phases

Columns: ZORBAX 300SB4.6 X 150 mm, 5 µm

Mobile Phase:Linear Gradient,

0 - 36% B in 30 min.A: 0.1% TFA in H2OB: 0.1% TFA in ACN

Temperature: 40°C

Flow Rate: 1 mL/min

Sample: 2 µg each peptideL1 - LeuGlyLeuL2 - LeuHisLeuL3 - LeuArgLeuL4 - LeuLeuLeu-amideL5 - LeuLeuValTyrL6 - LeuLeuLeuL7 - LeuLeuPhe-amideL8 - LeuLeuPheL9 - LeuLeuValPhe

Columns: ZORBAX 300SB-C8, 3.5 µm

Gradient: 2 - 75% B in 30 min.

Mobile Phase: A: 5% ACN95% H2O 0.1% TFA

B: 95% ACN5% H2O 0.085% TFA

Flow Rate: 1 mL / min.

Injection: 10 µL, 2.6 µg each

Temperature: 35°C

UV: 215 nm

Sample: 1. Gly-Tyr2. Val-Tyr-Val3. [Glu]-β Protein4. [Tyr8 ] Bradykinin5. Met-Enk6. Leu-Enk7. Angiotensin II8. Kinetensin9. RNase

10. Ins (Equine)

4.6 x 150 mm(Agilent Part No. 863973-906)

4.6 x 50 mm(Agilent Part No.865973-906)



This figure shows selectivity changes as a result of changing bonded phase type. The 300SB-CN columnimproves band spacing between peaks L6 and L7, and brings out impurities between L1 and L2 and after L8. In this example the gradient profile and composition are the same on each column.

FIGURE 4Increased Resolution with Shorter Columns

A shorter column can also be used to improve resolution. When a shorter column is used (tG held constant) the column volume is decreased, thereby increasing gradientretention and resolution according to the equation on page 4. This effect is less pronounced if the gradient range is narrow.

Optimize bonded phase

Selectivity and resolution in separa-tions of proteins and peptides can beimproved by choosing a differentbonded phase. Therefore, it is impor-tant for a family of HPLC columns tohave several different selectivitiesavailable, such as those in the300StableBond family. Though SB-C8and SB-C18 bonded phases may bethe most commonly selected for pep-tides, the less polar SB-C3 and SB-CNcolumns can offer dramatically differ-ent selectivity, especially for morepolar peptides. At the same time, theSB-C3 and SB-CN bonded phases aremore commonly selected for largerpeptides and proteins. But, all of thebonded phases can be used to opti-mize sample selectivity and resolution.Figure 5 demonstrates how selectivityvaries with bonded phase for onegroup of peptides.

3

Time (min)0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

80°C

60°C

40°C

*

*

* ?108

9

8

9

10

8

9

10

8

9

10

6

Optimize temperature for maximum efficiency and recovery4



Separations of proteins and peptides can be dramatically influencedby temperature. Higher column temperature often improves efficiencyand, therefore, resolution (Figure 6). In addition, elevated temperaturecan improve sample recovery dramatically (Figure 7). This is especiallytrue for peptides that either aggregate or are very hydrophobic.

FIGURE 6Higher Temperature Can ImproveResolution

FIGURE 7Higher Temperature Can Dramatically Improve Recovery

Column: ZORBAX 300SB-C184.6 X 150, 5 µm(Agilent Part No. 883995-902)

Mobile Phase: A: 0.1% TFA in H2OB: 0.09% TFA in ACN

Gradient: 30 - 90% B in 30 min

Flow Rate: 1 mL/min

Sample: 10 µL crude synthetic peptide in 6M guanidine-HCl (20 µM Tris, pH 8.0)

Columns: ZORBAX 300SB4.6 x 150, 5 µm

Mobile PhaseGradient: 25 - 70% B in 40 min.

A: 0.1% TFA in waterB: 0.09% TFA in

80% ACN/20% water

Flow Rate: 1 mL / min.

Temperature: See Figure

Sample 1. Myoglobin2. Calmodulin3. Carbonic Anhydrase

35°CSeparation of an α-Helical Peptide: Ac-nLGGG(EVSALEK)5-amide

ResolutionImprovement

ResolutionImprovement

60°C



The resolution of these 3 peptides is improved dramatically on both the 300SB-C3 and 300SB-CNcolumns as the temperature is raised from 35 - 60° C.

StableBond columns allow high temperatures to be used — up to 90°C -— to improve sample recovery ofhydrophobic or aggregating peptides. In this case, a temperature of 80°C maximized recovery.

min0 2 4 6 8 10 12 14 16 18

TIC

(150

-150

0 m

/z)

0.1% TFA

20mM NH4OH

LLG LHL

LLL-NH2LLL

LLVF

LLVFLLG

LHL

LLL3.0E7

2.5E7

2.0E7

1.5E7

1.0E7

0.5E7

0

3.0E7

2.5E7

2.0E7

1.5E7

1.0E7

0.5E7

0

LLL-NH2

7



Optimize mobile phase pH forbest selectivity of peptides6A high pH mobile phase is an option for optimizing separations of peptides.Until recently, high pH separations ofpeptides were only carried out on polymer-based columns. Now the ZORBAX Extend-C18 and 300Extend-C18 silica-based columnsare available to take advantage ofselectivity changes in separations ofpeptides and polypeptides that occur athigh pH. With an ammonium hydroxidecontaining mobile phase (pH 10 - 11)the overall charge on a peptide willchange, resulting in a change in selec-tivity in comparison to a TFA-containingmobile phase at low pH (Figure 9). This same high-pH mobile phase is anexcellent option for LC/MS of peptidesbecause of the increased sensitivity itprovides (see page 10). Alternatively,Eclipse XDB or Bonus-RP columns canbe used at mid-range pH (pH 6 - 8).

This simple method development strategy will help optimize reversed-phase separations of proteins and peptides. These steps do not includeall possible optimization choices.Instead, a general outline of the keysteps for successfully optimizing separations of proteins and peptides at low pH with 300StableBond and at high pH with 300Extend-C18 HPLC columns is provided.

FIGURE 9Use Extend-C18 for Different Selectivity at High and Low pH

Column: ZORBAX Extend-C182.1 x 150 mm, 5 µm(Agilent Part No. 773700-902)


Temperature: 25°C


Sample: 4 µL (50 ng each peptide)

Mobile Phase: TFA: A: 0.1% TFA in H2O

B: 0.085% TFA in 80% ACN

NH4OH: A: 20 mM NH4OH in H2OB: 20 mM NH4OH in 80% ACN

Marked changes in the selectivity of peptides can occur using high and low pH mobilephases due to different charge states of acidic and basic amino acids. In addition, astronger ESI/MS signal is seen at high pH with the ammonium hydroxide mobile phase.

A key issue for proper method development iscomplete sample solubilization, leading to bettersample recovery. This table, arranged fromweakest to strongest provides a list of possiblesolubilization solvents that can be used for allpeptides. Highly acidic reagents, such as 99%formic acid and 100% TFA, can be used becauseof the stability of StableBond columns. Dilutebase can be used as a solubilization solvent andmobile phase with the Extend-C18 columnsbecause of its stability at high pH.

Optimize Sample Solubility5 For the best peak shape and recoveryat any pH, it is important to solubilizea sample completely.

Solubilization Solvents for Peptides in Order of Weakest to Strongest• Water / phosphate buffer Weakest• Dilute Acid (TFA, HOAc or HCl)• Neutral pH 6 - 8 M Guanidine —HCl or isothiocynate• 5% HOAc / 6 M Urea• Dilute Acid + aqueous / organic solvents (ACN, MeOH, THF)• Dilute Base (Ammonium Hydroxide)• Neat Organic Solvents — ACN, MeOH, THF• 99% formic acid• HFIP or HFIP/aqueous mixtures• 100% TFA• DMSO or 0.1% - 1% TFA in DMSO• Formamide Strongest

FIGURE 8Solvent Choices to Solubilize Proteins and Peptides

Try Higher pH

(M+H)+ + CF3COO- [ (M+H)+•CF3COO-]"±0"

CF3COO- + RCOOH CF3COOH + RCOO- g g

Strong Ion Pairing with TFA-Anion

Acid Competition (TFA more volatile)

(M+H)+ + CH3COO- [ (M+H)+•CH3COO-]"±0"

Weak Ion Pairing with Acid-Anion

-

-

-

g

Protonated sample molecules can be extracted by the high electric field or ejected from the droplet (ESI).

Neutral acid molecules of sufficient volatility can evaporate, while the droplets are shrinking in size.

FIGURE 10Proposed Mechanism for TFA Signal Suppression and the “TFA-Fix”

The “TFA-fix” using weaker acid substitution is shown here. Strong ion pairing of anions with TFA (top equation)removes ions, and thus reduces MS sensitivity. Swamping out the TFA with a weaker acid (middle equation)removes the more volatile TFA, driving the equilibrium back to the original ions (bottom equation).

Protein Separations


Protein/Peptide HPLC Method Development —LC/MS Considerations

8

LC/MS of proteins and peptides is used to provide information for protein characterization, to accurately identify post-translational modifications of proteins and to determine the molecular weight of synthetic and natural peptides. Most protein and peptide separations are done at low pH with aTFA-containing mobile phase where the TFA functions as an ion-pairreagent and complexes with the basic residues in proteins and peptides. This [protein : TFA] complex is strong enough that ion suppression can occurin LC/MS of proteins and peptides. Several approaches (shown below) can be used to achieve a clearer and stronger MS signal.

To Maximize LC/MS Signal -

Dilute with a different acid post column

TFA can be displaced from the ion-pair complex by exposing theeffluent to a higher concentration of a different acid post column andbefore the MS detector. This isreferred to as the “TFA-Fix”. One acid that works well for this approachis acetic acid. Another acid is a solution of 75% propionic acid : 25% isopropanol. These weaker acids,when used in a higher concentration,will displace the TFA from the protein.In the MS, these weaker acids aremore easily removed during the ionization process, resulting in astronger protein ion signal. In addition, the Agilent MSD is config-ured with an orthogonal flow. Thisimproves the efficiency of sample iontransfer and reduces the amount ofsolvent entering the MSD, therebyimproving spectral clarity.

1

3x10

6

0 10 20 30 40 50 60 min

5% Acetic

0.001% TFA

0.005% TFA

0.01% TFA

MSD1 TIC, MS

9

Time (min)0 5 10 15 20

TIC

(300

- 25

00 m

/z)

0

2,000,000

4,000,000

6,000,000

Aβ (1-28)Insulin

InsBox LysozymeCaD9

CaM

250

2,000,000

4,000,000

6,000,000

Aβ (1-28)Insulin

InsBoxLysozyme

CaD9

CaM

Time (min)0 5 10 15 20 25

FIGURE 11Post-Column Addition of Acetic Acid Increases Signal in TIC of Peptides


Protein/Peptide HPLC Method Development — LC/MS Considerations

Use a lower TFA concentrationIn many cases, you can reduce the TFA concentration in the mobile phasefrom 0.1% to 0.01% or even less and still get good chromatographic results.When you do this, you may improve the MS spectral signal. Figure 12 showsthree total ion chromatograms wherethe concentration of TFA has beenreduced to 0.01%, then 0.005% and finally 0.001%. As the concentration ofTFA is reduced, the strength of the massspectral signal increases; and, for thissample, the chromatographic resolutionand peak shape are excellent with eachmobile phase modification. Samplerecovery also remained high as the TFA concentration decreased on theZORBAX StableBond 300SB-C3 columnselected for this analysis. Other samples and columns may show morechanges in the chromatographic separation as the concentration of TFA is reduced.

2 FIGURE 12LC/MS Sensitivity vs. Mobile Phase ModifierGluC Digest of BSA

Column: ZORBAX 300SB-C32.1 x 150 mm, 5 µm(Agilent Part No. 883750-909)

Mobile Phase: 0-40% B in 20 min

Temperature: 25°C



Sample: 10 µL- 200 ng each component in 6M Urea / 1% HOAc

ESI-MS: Vf Ramped- 60V@ 300 amu; 300V@ 3000 amu; Vcap 4.5 kV, N2- 12L / min, 300°C, 35 psi

Column: ZORBAX 300SB-C82.1 x 150 mm, 5 µm(Agilent Part No. 883750-902)

Temperature: 60°C


Acid Fix: 100% HOAc, 0.1 mL/minNo Addition

Ricker, R.D., Boyes, B.E., Nawrocki,J.P., Pannell, L.K., Stable, StericallyProtected C3 Bonded Phase in LC and LC/MS Applications, 9th ISPPP,Delray Beach, FL, Oct 1999.

Lowering the TFA concentration (bottom chromatograms) improves MS sensitivity. Substituting TFA with a weakeracid in the mobile phase also increases MS sensitivity. The higher concentration of acetic acid — here, 5% (vs. commonly used 1% acetic acid) — improves initial chromatography. The higher acetic acid concentration canbe tolerated by the Agilent 1100/MSD with orthogonal design. When using other MS instruments, where injectionis directly into the MS, a lower concentration of acetic acid should be used.

When the TFA in the mobile phase is replaced post column, with acetic acid, the total ion chromatogram shows a marked increase in signal for a group of peptides. This“TFA-Fix” is very effective for peptide and protein samples analyzed with a TFA-containing mobile phase.

min0 2.5 5 7.5 10 12.50

1.0E6

2.0E6

3.0E6

4.0E6

5.0E6

12.

050

13.

261

AII+AIII

AI

TIC

(200

-150

0 m

/z)

min0 2.5 5 7.5 10 12.50

1.0E7

2.0E7

3.0E7

4.0E7

5.0E7

5.49

9

8.47

7AII

AITIC

(200

-150

0 m

/z)

AIII

9.62

1

m/z500 1000

0

20

40

60

80

100Max: 10889

649

.5

433

.0

227

.1

659

.7

844

.8

250

.9

340

.3

127

7.9

117

9.1

102

5.2

720

.3

132

6.3

398

.0

767

.3

458

.0

146

8.8

137

3.3

+3 +2

m/z500 10000

20

40

60

80

100Max: 367225 6

49.0

433

.0

659

.8

129

6.6 +1

+2

+3

FIGURE 13AImprove Resolution of Peptides at High pH with Extend-C18

Comparison of Angiotensins: Separation with TFA and NH4OH

FIGURE 13BImprove LC/MS Spectral Clarity of Peptides at High pH

Comparison of Angiotensin I Mass Spectra with TFA and NH4OH

Low pHAcidic Conditions A: 0.1% TFA in water

B: 0.085% TFA in 80% ACN

High pHBasic Conditions: A: 10 mM NH4OH in water

B: 10 mM NH4OH in 80% ACN

Signal x10

Column: ZORBAX Extend-C182.1 x 150 mm, 5 µm (Agilent Part No. 773700-902)


Temperature: 35°C

Mobile Phase: As indicated

Gradient: 15-50% B in 15 min.

LC/MS: Pos. Ion ESI- Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12 L/min., 325°C

Sample: Angiotensin I, II, III 2.5 µL sample (50 pmol each)

Angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-LeuAngiotensin II: Asp-Arg-Val-Tyr-Ile-His-Pro-PheAngiotensin III: Arg-Val-Tyr-Ile-His-Pro-Phe

Using a higher pH can improve selectivity and MSclarity. In this example, using ZORBAX Extend-C18,optimized for high pH separations, the threeAngiotensins are only resolved at high pH due to the change in charge on Angiotensin II between low and high pH.

When the Angiotensin peptides are separated athigh pH spectral clarity is dramatically improved.Experiments for this and similar peptide samplesshow up to a 20X improvement in the signal-to-noiseratio of the MS signal at high pH.

Low pHAcidic Conditions A: 0.1% TFA in water

B: 0.085% TFA in 80% ACN

High pHBasic Conditions: A: 10 mM NH4OH in water

B: 10 mM NH4OH in 80% ACN

10



Use a different acid as a mobile phase modifierFigure 12 also demonstrates another option to improve MS signalintensity. Replacing the TFA in the mobile phase with 5% acetic acidproduces excellent chromatography and MS signal intensity for thissample when analyzed using the ZORBAX StableBond 300SB-C3.Lower concentrations of acetic acid may also be used to reduce anypossible chromatographic and spectral interference from impurities inthe acid. It may also be possible to use combinations of acetic acidand other volatile mobile phase ion-pair reagents (i.e. 0.005% HFBA -hexaflurobutyric acid). For this sample, the 5% acetic acid provides alow mobile phase pH, and the Agilent 1100 MSD, with its orthogonalflow design reduces the effect of any impurities in the acetic acid byreducing the amount of acetic acid transferred into the MS.

3

Time (min)0 2 4 6 8 10 12 14 16 18 20

LLG

LHL

LLL

LLL-NH2 LLVF

LLG

LHLLLL

LLL-NH2LLVF

3.0E7

2.5E7

2.0E7

1.5E7

1.0E7

0.5E7

0E7

2.5E7

2.0E7

1.5E7

1.0E7

0.5E7

0E7

Protein SeparationsReversed Phase Columns


11

FIGURE 14RP-HPLC/ESI-MS of Peptides Using NH4OH Yields Both Positive and Negative Ions

Positive Ions

Negative Ions

Column: ZORBAX Extend-C182.1 x 150 mm, 5 µm(Agilent Part No. 773700-902)

Gradient: 5-60% B in 20 min

Mobile Phase: A: 20 mM NH4OH in H2OB: 20 mM NH4OH in 80% ACN

Temperature: 25°C



Use high pH andZORBAX Extend-C18

All of the previous options for improvingMS signal intensity with peptides areattempts to reduce TFA concentrationwhile still working at low pH. Peptidesand polypeptides can also be separat-ed at high pH. An ammonium hydroxidecontaining mobile phase at high pHprovides an alternate selectivity forHPLC separations of peptides, as wellas for improved LC/MS results (i.e.higher S/N, improved spectral clarity).

ZORBAX Extend-C18 and 300Extend-C18columns are efficient, silica-basedcolumns designed for high-pH separa-tions of basic analytes, including peptides. Figure 13A shows one example of a change in selectivity for a peptide at low and high pH on theExtend-C18 column. At high pH allthree Angiotensins are clearly resolved.Figure 13B shows the improvement inspectral clarity for Angiotensin I at highpH. In addition, the Angiotensin +1 ion isvisible and not suppressed as it is withthe TFA-containing mobile phase.

Separations of peptides at higher pHalso allow both positive and negativeions to be detected. Figure 14 shows aseparation of peptides at high pH on theExtend-C18 column where both the pos-itive and negative ions were monitoredin the same run using the Agilent 1100MSD SL. Additional examples of peptide separations at high pH can be found in the Extend-C18 and300Extend-C18 section on page 20.

4

O

R

R

Si

OH

Si

O

Si

R

R

R

O

OH

R

R1

R1

R1

0 10,000 20,000 30,000

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Rela

tive

Rete

ntio

n: T

olue

ne

Column Volumes of Purge

ZORBAX SB-C18 (80Å)ZORBAX SB-C18 (300Å)Commercial C18 Column ACommercial C18 Column BZORBAX Rx C18

FIGURE 1Sterically ProtectedStableBond Bonded Phase

FIGURE 2ZORBAX 300StableBond is Exceptionally Stable at Low pH and High Temperature

Bulky diisobutyl (C18) or diisopropyl (C8, C3, CN,Phenyl, SB-Aq) side-chain groups are used to stabilize both long and short-chain bonded phases.This results in exceptional lifetime at low pH withTFA containing mobile phases.

As an indicator of column breakdown, the retention time of toluene was measuredafter purging the column with a very low pH (pH 0.8 - 0.9) mobile phase at 90°C. The SB-C18 and 300SB-C18 columns are unchanged after the equivalent of 3 working months of use under these conditions.

R = Isopropyl, isobutyl (C18)

R1 = C8, C18, CN, C3, etc.

ZORBAX 300StableBond (300SB)ZORBAX StableBond HPLC Columns

Protein Separations


12

• Exceptional stability and long lifetime at low pH with TFA-containing mobile phases

• Six different bonded phases for selectivity optimization with peptides and proteins

• Excellent reproducibility

• Stable at low pH and high temperature — up to 80 - 90°C

ZORBAX wide-pore StableBond 300SB columns provide long lifetime, high resolution and good peak shape for the separations of peptides and proteins with the most commonly used TFA-containing mobile phases. ZORBAX 300SBcolumns are available in four different bonded phases (C18, C8, C3, and CN) forselectivity and recovery optimization of proteins and polypeptides. Each of thesebonded phases provides excellent reproducibility and long column lifetime. Tofurther increase sample recovery and improve efficiency of difficult proteins,ZORBAX 300SB columns can be used up to 80 - 90°C. For LC/MS separations atlow pH, where 0.1% TFA may not always be suitable, ZORBAX 300SB columnscan also be used effectively with lower concentrations of TFA or with formic acidand acetic acid as mobile phase modifiers.

Purge Solvent:50% methanol/50% water with 1.0% TFA

Temperature: 90°CSolute: Toluene

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

Time (min)

13


ZORBAX 300StableBond (300SB) and StableBond Reversed-Phase HPLC Columns

100

80

60

40

20

00 1000 2000 3000 4000 5000 6000

Column Volumes

% k

Rem

aini

ng

SB-CNSterically Protected Silane

Si-(Me)2PrCNTraditional Silane

FIGURE 3

All StableBond Columns are Exceptionally Stable

Short-chain bonded phases are often not used because they are unstable atlow pH. However, StableBond short-chain phases are also stabilized usingbulky silanes to sterically protect the siloxane bond that attaches the silane tosilica. This reduces hydrolysis at low pH with TFA-containing mobile phases.By contrast, columns using a dimethylsilane are not as stable. This improvesselectivity options for the separations of proteins and peptides. See Figure 5,page 5 for an excellent example using 300SB-CN.

FIGURE 4Extended Column Lifetime of ZORBAX 300SB-C8 — rhGH Tryptic Digest

This figure shows the excellent chromatographic results that can be obtained from the choice of StableBondcolumns. Both long term reproducibility and column lifetime are exceptional. The smallest peaks, as well assome of the incompletely resolved peaks, show reproducible behavior after long column usage. No change isseen in either selectivity or retention.

Columns: StableBond 300SB-C84.6 x 150 mm, 5 µm(Agilent Part No. 883995-906)

Mobile Phase: Linear Gradient, 0 - 60% B in 120 min, pH 2A: 0.1% TFA in WaterB: 0.086% TFA in 80% ACN


Temperature: 40°C

Detection: UV 210 nm

Sample: 50 µg of rhGH Tryptic Digest

After 495 mL

After 13680 mL

25°C

Time (min)

0 5 10 15 20 25 30 35 40

40°C

60°C

80°C

βAP (1-38) βAP ((1-43)* Recovery <10%

Recovery >70%

14


300StableBond (300Å) Reversed-Phase HPLC Columns

FIGURE 5Four Different 300SB Bonded Phases Offer Selectivity Choicesfor the Separation of Large Polypeptides

Columns: StableBond 300SB4.6 x 150 mm, 5 µm

Mobile Phase: Linear Gradient, 25 - 70% B in 40 min.A: 0.1% TFA in WaterB: 0.09% TFA in

80% Acetonitrile/ 20% Water


Temperature: 60°C

Sample: 3 µg each protein1. RNase2. Insulin3. Cytochrome C4. Lysozyme5. Parvalbumin6. CDR7. Myoglobin8. Carbonic Anhydrase9. S-100β

10. S-100α

Conditions: ZORBAX 300SB-C184.6 x 150 mm, 5 µm(Agilent Part No. 883995-902)

Sample: 10 µL injection of 5 mg peptide in 6M Urea/ 5% HOAc

Flow Rate: 1 mL/min.

Mobile Phase: A – 0.1% TFA in water, B – 0.09% TFA in acetonitrile

Gradient: 20–45% B in 35 min.

FIGURE 6High Recovery of Very Hydrophobic βAP (1-43) Using SB-C18 at High Temperature

In this example, the very hydrophobic βAP (1-43)amyloid peptide shows poor recovery at lowtemperature. By contrast, the βAP (1-38) amyloidpeptide, a more hydrophilic peptide, shows goodrecovery at low and high temperatures.Increasing temperature, in this case, dramaticallyimproves recovery of hydrophobic peptides.

12,3

4 56

7 89

10

1 24 5

6,7

89

103

1 24 5

68

9 103

7

Time (min)0 5 10 15 20 25 30 35 40

1 24 5

68

910

37 The 300SB-C18, C8, C3 and CN bonded phases all

provide a different separation of this group ofpolypeptides. This adds an important option forquickly optimizing protein separations. The 300SB-CNcolumn offers unique selectivity for more hydrophilicpolypeptides, whereas C8 and C18 phases betterresolve more hydrophobic polypeptides.






300StableBond (300Å) Reversed-Phase HPLC Columns

15

ZORBAX StableBond 300SB Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits* pH Range* Endcapped Carbon Load

ZORBAX 300SB-C18 300Å 45 m2/g 90°C 1.0 - 8.0 No 2.8%ZORBAX 300SB-C8 300Å 45 m2/g 80°C 1.0 - 8.0 No 1.5%ZORBAX 300SB-C3 300Å 45 m2/g 80°C 1.0 - 8.0 No 1.1%ZORBAX 300SB-CN 300Å 45 m2/g 80°C 1.0 - 8.0 No 1.2%*300StableBond columns are designed for optimal use at low pH (pH 1.8 - 6), but can be used from pH 1 - 8. At pH 6 - 8 highest column stability for all silica-based columnsis obtained by operating at temperatures <40°C and using low buffer concentrations in the range of 0.01 - 0.02M. At mid and high pH, 300Extend is recommended.

ZORBAX StableBond (300Å) Column Ordering Information Column Size Particle 300SB-C18 300SB-C8 300SB-CN 300SB-C3Description (mm) Size (µm) USP L1 USP L7 USP L10Standard Columns and Bulk PackingPreparative 21.2 x 250 7 880995-102 880995-106 880995-109 880995-105Semipreparative 9.4 x 250 5 880995-202 880995-206 880995-209 880995-205Analytical 4.6 x 250 5 880995-902 880995-906 880995-905 880995-909Analytical 4.6 x 150 5 883995-902 883995-906 883995-905 883995-909Analytical 4.6 x 50 5 860950-902 860950-906 860950-905 860950-909Rapid Resolution 4.6 x 150 3.5 863973-902 863973-906 863973-905 863973-909Rapid Resolution 4.6 x 50 3.5 865973-902 865973-906 865973-905 865973-909Solvent Saver Plus 3.0 x 100 3.5 884950-565Narrow Bore 2.1 x 150 5 883750-902 883750-906 883750-905 883750-909Narrow Bore RR* 2.1 x 150 3.5 863750-906Narrow Bore RR 2.1 x 50 3.5 865750-902 865750-906MicroBore RR 1.0 x 150 3.5 863630-902 863630-906MicroBore RR 1.0 x 50 3.5 865630-902 865630-906Bulk Packing, 2 grams 5 820966-306Guard Cartridges, 2/pk 9.4 x 15 7 820675-124 820675-124 820675-124 820675-124Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-921 820950-918 820950-923 820950-924Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-918 821125-918 821125-924 821125-924Guard Hardware Kit 9.4 x 15 840140-901Guard Hardware Kit 4.6 x 12.5 820777-901Guard Hardware Kit 2.1 x 12.5 820777-901

Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8266Capillary 0.5 x 150 5 5064-8264Capillary RR 0.5 x 150 3.5 5064-8268Capillary 0.3 x 250 5 5064-8265Capillary 0.3 x 150 5 5064-8263Capillary RR 0.3 x 150 3.5 5064-8267Guard Cartridges 0.3 x 35 5 5064-8298Guard Cartridges 0.5 x 35 5 5064-8294

*RR: Rapid Resolution 3.5 µm columns.StableBond (80Å) columns are also available for small molecule separations. See page 16 - 17 for descriptions, ordering information and additional information on StableBond columns.

Configurations not shown are available upon request.

16


StableBond (80Å) Reversed-Phase HPLC Columns

ZORBAX StableBond Columns (80Å) Ordering InformationColumn Size Particle SB-C18 SB-C8 SB-CN SB-C3 SB-Phenyl SB-AqDescription (mm) Size (µm) USP L1 USP L7 USP L10 USP L11Standard Columns and Bulk PackingPreparative 21.2 x 250 7 880975-102 880967-106 880975-105 880975-112Semi-Preparative 9.4 x 250 5 880975-202 880967-201 880975-205 880975-209 880975-212Analytical 4.6 x 250 5 880975-902 880975-906 880975-905 880975-909 880975-912 880975-914Analytical 4.6 x 150 5 883975-902 883975-906 883975-905 883975-909 883975-912 883975-914Analytical 4.6 x 50 5 846975-902 846975-906 846975-914Rapid Resolution (RR) 4.6 x 150 3.5 863953-902 863953-906 863953-905 863953-912Rapid Resolution 4.6 x 75 3.5 866953-902 866953-906 866953-905 866953-912Rapid Resolution 4.6 x 50 3.5 835975-902 835975-906 835975-905 835975-912Solvent Saver 3.0 x 250 5 880975-302 880975-306 880975-305 880975-309 880975-312Solvent Saver 3.0 x 150 5 883975-302 883975-306 883975-305 883975-309 883975-312Solvent Saver Plus 3.0 x 150 3.5 863954-302 863954-306 863954-305 863954-312Solvent Saver Plus 3.0 x 100 3.5 861954-306Narrow Bore 2.1 x 150 5 883700-922 883700-906 883700-905 883700-909 883700-912Narrow Bore 2.1 x 50 5 860975-902 860975-906 860975-905 860975-909 860975-912 860975-914Narrow Bore RR* 2.1 x 150 3.5 830990-902 830990-906Narrow Bore RR 2.1 x 50 3.5 871700-902 871700-906MicroBore RR 1.0 x 150 3.5 863600-902 863600-906MicroBore RR 1.0 x 50 3.5 865600-902 865600-906MicroBore RR 1.0 x 30 3.5 861600-902 861600-906Bulk Packing, 2 grams 5 820966-906 820966-905Guard Cartridge, 2/pk 9.4 x 15 7 820675-115 820675-115 820675-124 820675-115Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-920 820950-915 820950-916 820950-922 820950-917 820950-933Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-926 821125-926 821125-924 821125-924 821125-926 821125-933Guard Hardware Kit 9.4 x 15 840140-901 840140-901 840140-901 840140-901 840140-901Guard Hardware Kit 4.6 x 12.5 820777-901 820777-901 820777-901 820777-901 820777-901 820777-901Guard Hardware Kit 2.1 x 12.5 820777-901 820777-901 820777-901 820777-901 820777-901 820777-901

Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8258Capillary 0.5 x 150 5 5064-8256Capillary RR 0.5 x 150 3.5 5064-8262Capillary RR 0.5 x 35 3.5 5064-8260Capillary 0.3 x 250 5 5064-8257Capillary 0.3 x 150 5 5064-8255Capillary RR 0.3 x 150 3.5 5064-8261Guard Cartridges 0.5 x 35 5 5064-8254Guard Cartridges 0.3 x 35 5 5064-8253

*RR: Rapid Resolution 3.5 µm columns.For more information on these and other columns consult the Agilent web site at www.agilent.com.

300Å StableBond columns are also available for protein and peptide separations. See page 15 for descriptions and ordering information


17


StableBond (80Å) Reversed-Phase HPLC Columns

ZORBAX StableBond Columns (80Å) Ordering Information (cont’d)Column Size Particle SB-C18 SB-C8Description (mm) Size (µm) USP L1 USP L7Agilent Cartridge ColumnsAnalytical 4.6 x 250 5 7995218-585 7995208-585Analytical 4.6 x 150 5 7995218-595 7995208-595Rapid Resolution 4.6 x 75 3.5 7995218-344 7995208-344Guard Cartridge, 10/pk 4.0 x 4.0 5 7995118-504 7995118-504Cartridge Holder 5021-1845 5021-1845

High Throughput Cartridge Columns (requires Hardware Kit 820222-901)Rapid Resolution Cartridge 4.6 x 30 3.5 833975-902 833975-906Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 833975-932 833975-936Rapid Resolution Cartridge 4.6 x 15 3.5 831975-902 831975-906Rapid Resolution Cartridge, 3/pk 4.6 x 15 3.5 831975-932 831975-936Rapid Resolution Cartridge 2.1 x 30 3.5 873700-902 873700-906Rapid Resolution Cartridge, 3/pk 2.1 x 30 3.5 873700-932 873700-936Rapid Resolution Cartridge 2.1 x 15 3.5 875700-902 875700-906Rapid Resolution Cartridge, 3/pk 2.1 x 15 3.5 875700-932 875700-936High Throughput Cartridge Column Hardware Kit 820222-901 820222-901

CombiHT High Throughput Preparative Columns (endfittings required)CombiHT 21.2 x 150 5 870150-902 870150-906CombiHT 21.2 x 100 5 870100-902 870100-906CombiHT 21.2 x 50 5 870050-902 870050-906CombiHT End Fittings (required for use) 820400-901 820400-901CombiHT Seal Replacement Kit 820385-901 820385-901

For more information on these and other columns consult the Agilent web site atwww.agilent.com.300Å StableBond columns are also available for protein and peptide separations. See page 15for descriptions and ordering information


ZORBAX StableBond (80Å) Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits* pH Range* Endcapped Carbon Load

ZORBAX SB-C18 80Å 180 m2/g 90°C 1.0 - 8.0 No 10%ZORBAX SB-C8 80Å 180 m2/g 80°C 1.0 - 8.0 No 5.5%ZORBAX SB-C3 80Å 180 m2/g 80°C 1.0 - 8.0 No 4.0%ZORBAX SB-CN 80Å 180 m2/g 80°C 1.0 - 8.0 No 4.0%ZORBAX SB-Phenyl 80Å 180 m2/g 80°C 1.0 - 8.0 No 5.5%ZORBAX SB-Aq 80Å 180 m2/g 80°C 1.0 - 8.0 No N/A*StableBond columns are designed for optimal use at low pH (pH 1.8 - 6), but can be used from pH 1 - 8. At pH 6-8 highest column stability for all silica-based columns isobtained by operating at temperatures <40°C and using low buffer concentrations in the range of 0.01-0.02M. At mid-range pH, Eclipse XDB and Bonus-RP are recommended.

To order or for technical assistance, contact Agilent Technologiesor your local authorized Agilent distributor.

Time (min)0 5 10 15 20

6

23

4

5

1

ZORBAX StableBond Aq (SB-Aq)HPLC Columns

Protein Separations


Column: ZORBAX SB-Aq4.6 x 150 mm, 5 µm (Agilent Part No. 883975-914)

Mobile Phase: 50 mM NaOAc, pH 4.6


Temperature: 35°C


Sample: 1. Cytosine2. Fluorocytosine3. Uracil4. Guanine5. Thymine6. Adenine

18

0.0 0.5 1.0 1.5 2.0Time (min)

1

2

34

FIGURE 1Purines/Pyrimidines

FIGURE 2Separation of Water Soluble Vitamins onthe SB-Aq Column without Ion-Pairing

ZORBAX SB-Aq columns are an idealchoice to increase the retention ofmany difficult lower MW acidic, basicand polar compounds. More retentionmay be obtained than on many conventional ODS HPLC columns.Polar compounds can be analyzedwith excellent reproducibility and peakshape because the SB-Aq columnshave a hydrophilic surface that inhibitsphase collapse, even with 100% aqueous mobile phases. ZORBAXStableBond technology adds exceptional durability and stability at low pH and high temperature — down to pH 1 and up to 80°C.

• Recommended for high resolution analysis of very polar compounds

• Retain polar compounds in 100% aqueous mobile phases

• Obtain reproducible retention without phase collapse

• Provide exceptional durability at low pH

The SB-Aq column is used with a simple mobilephase (no ion-pair reagents) to achieve sufficientretention and high resolution of these B vitamins.

Column: ZORBAX SB-Aq4.6 x 150 mm, 5 µm(Agilent Part No. 883975-914)

Mobile Phase: 95% 0.1% TFA5% MeOH


Temperature: 35°C


Sample:1. Thiamine (B1)2. Nicotinic Acid3. Pyridoxine (B6)4. Niacinamide

ZORBAX SB-Aq Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range EndcappedZORBAX SB-Aq 80Å 180 m2/g 80°C 1.0 - 8.0 No*SB-Aq columns are designed for optimal use at low pH (pH 1.8 - 6), but can be used from pH 1 - 8. At pH 6 - 8 highest column stabilityfor all silica-based columns is obtained by operating at temperatures <40°C and using low buffer concentrations in the range of 0.01 - 0.02M. At mid pH, Bonus-RP is recommended.

For more information on these and other columns consult theAgilent web site at www.agilent.com.


ZORBAX StableBond Aq (SB-Aq) Column Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) NumberAnalytical 4.6 x 250 5 880975-914Analytical 4.6 x 150 5 883975-914Analytical 4.6 x 50 5 846975-914Narrow Bore 2.1 x 50 5 860975-914Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-933Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-933Guard Hardware Kit 820777-901

To order or for technical assistance,contact Agilent Technologies or yourlocal authorized Agilent distributor.

PorousShell

SOLIDCORE

5 µm

19

Protein Separations


ZORBAX Poroshell 300SB-C18HPLC Columns

FIGURE 1Poroshell Particles have a SuperficiallyPorous Surface and a Solid Core

1

0.0 0.5 1.0Time (min)

2

34

5

67

8

FIGURE 2Fast, High Resolution Separation of Peptides and Proteins withPoroshell 300SB-C18 . . . in Seconds

ZORBAX Poroshell 300SB-C18 Column Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) Number

Narrow Bore 2.1 x 75 5 660750-902Available March 2001

• Revolutionary new particle for reversed phase separations of proteins

• High speed and high resolution of proteins and other large molecules

• Ultra-short analysis times with high flow rates

• Outstanding stability at low pH with 300StableBond bonded phase

The Poroshell 300SB-C18 columnsuse a revolutionary new particle forfast, high-resolution reversed-phase separations of proteins, DNA and other large molecules. The porousshell allows rapid mass transfer resulting in very high efficiency andresolution of proteins — at high andlow flow rates. Analysis times can bedramatically reduced with no loss inresolution. In addition, the 300SB-C18bonded phase has superior stabilityat low pH with TFA or other acidicmobile phases.

Spaces between solutes indicates good peak capacity for rapidly separating complex samples.

0.1 0.2 0.3 0.4 0.50

0.10 0.2 0.3 0.4 0.5Time (min)

1,2 34

1

2 3 4

FIGURE 3Very High Resolution at High Flow Rates on Poroshell 300SB-C18 Columns

300SB-C18Totally Porous Particle

Poroshell 300SB-C18(Agilent Part No. 660750-902)

High flow rates can be used on the Poroshell 300SB-C18 column to reduce analysis time with the highestcolumn efficiency. At these high flow rates sharp, efficient peaks are obtained because of the more rapidmass transfer in comparison to the totally porous particle.

The revolutionary new Poroshell 5 µm particle combines a solid core with a 300Å porous surfacelayer bonded with SB-C18. The result is a highly efficient particle for rapid, high-resolution separations of proteins and other large molecules.

Column: 300SB-C182.1 x 75 mm, 5 µm

Mobile Phase:Gradient: 5 - 100% B in 0.7 min.

A: 95% H2O, 5% ACN with 0.1% TFAB: 5% H2O, 95% ACN with 0.07% TFA

Flow: 3.0 mL/min

Temperature: 70°C

Detector: UV 215 nm

Sample: 1µL inj, (200 µg/mL each)1. Neurotensin 3. Lysozyme2. RNaseA 4. Myoglobin

NEW

Column: Poroshell 300SB-C182.1 x 75 mm, 5 µm

Mobile Phase: A: 0.1% TFAB: 0.07% TFA in ACN

Gradient: 5 - 100% B in 1.0 min


Temperature: 70°C

Pressure: 260 bar


Sample:1. Angiotensin II2. Neurotensin3. RNase4. Insulin5. Lysozyme6. Myoglobin7. Carbonic Anhydrase8. Ovalbumin

k Va

lues

0

2

4

6

8

Column Volumes of Mobile Phase

0 10000 20000 30000 40000 50000

1-Chloro-4-nitrobenzene

Trimipramine

20

Protein Separations


ZORBAX 300Extend-C18 andExtend-C18 HPLC Columns

Column: ZORBAX Extend-C18, 4.6 x 150 mm

Mobile Phase: 20% 20 mM NH4OH, pH 10.580% Methanol


Aging at 24°C, tests at 40°C

The unique bidentate chemistry of Extend-C18 gives it long life at high pH. In this example,Extend shows little change after 4 months (40,000 column volumes) of continuous use at pH 10.5.

*At higher pH, successful RP-HPLC of proteins (MW >10 - 15,000) will depend on the conformationalstability achieved in the mobile phase and with the bonded phase.

The combination of the unique bidentate structureand double endcapping permits long life at high pH.

ZORBAX 300Extend-C18 is a wide-pore HPLC column for the high efficiencyseparations of peptides, polypeptides and small proteins* from pH 2 - 11.5. Theunique bidentate bonded phase provides excellent lifetime and reproducibilityat high and low pH. At high pH, the retention and selectivity of peptides canchange dramatically as a result of changes in amino acid charge. Excellentrecoveries of hydrophobic polypeptides have been achieved at high pH. Inaddition, LC/MS sensitivity of peptides can also be improved at high pH using a simple ammonium hydroxide mobile phase. These conditions allow for ions to be detected in both positive and negative ion modes simultaneously. Some other mobile phase modifier options for Extend-C18 columns include triethylamine, glycine, borate and pyrrolidine. Extend-C18 columns may also be used for smaller peptides from pH 2 - 11.5.

• Rugged, high and low pH separationsof peptides, polypeptides and smallproteins (pH 2 - 11.5)

• Different selectivity possible at highand low pH

• High efficiency and good recovery ofhydrophobic peptides at high pH

• Ideal for LC/MS with ammoniumhydroxide mobile phase

FIGURE 2Long Life at High pH with Extend-C18Si

OSi

O

Silica Support

C18C18

C18SiO

O Si C18

C18SiO

O Si C18

CH3

CH3CH3

CH3

FIGURE 1New Bidentate C18-C18 Bonding forExtend-C18 Bonded Phase

ZORBAX Extend-C18 Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Extend-C18 80Å 180 m2/g 60°C* 2.0 - 11.5 Double 12.5%ZORBAX 300Extend-C18 300Å 45 m2/g 60°C* 2.0 - 11.5 Double 4%*Temperature limits are 60°C up to pH 8, 40°C for pH 8 - 11.5.


21

0 5 10 15 20 25Time (min)

Aβ(1-38)Aβ(1-40)

Aβ(1-43)

Aβ(1-42)

Aβ(1-38)

Aβ(1-40)Aβ(1-43)

Aβ(1-42)

Aβ(1-38) Aβ(1-40)Aβ(1-42/43)

FIGURE 4Selectivity Comparisons of Aβ Peptidesat High and Low pH on 300Extend-C18

The 300Extend-C18 shows a different selectivity forthe Aβ(1 - 42) and Aβ(1 - 43) peptides at high andlow pH. In addition, at high pH recovering is markedlyimproved over low pH.

For additional examples of using Extend-C18, see theintroduction section “Strategy for Reversed-PhaseProtein/Peptide Method Development inRelation to pH” and “Protein MethodDevelopment with LC/MS Considerations.”

Low pHTFA Conditions, 80°CA - 0.1% TFA in waterB - 0.085% TFA in 80% ACNGradient: 29-41% B in 30 min.

Low pHTFA Conditions, 25°CA - 0.1% TFA in waterB - 0.085% TFA in 80% ACNGradient: 33 - 45% B in 30 min.

High pHNH4OH Conditions, 25°CA - 20 mM NH4OH in waterB - 20 mM NH4OH in 80% ACNGradient: 26-38% B in 30 min.

Column: ZORBAX 300Extend-C18, 2.1 x 150 mm, 3.5 µm(Agilent Part No. 763750-902)



ZORBAX 300Extend-C18 and Extend-C18 HPLC Columns

Column Volumes of Purge

0 5000 10000 15000 20000 25000 300000.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of T

olue

ne k

Rem

aini

ng

Stablebond SB-C18 (Diisobutyl-C18)

Extend-C18 (Bidentate-C18)

Rx-C18 (Dimethyl-C18)

The 300Extend-C18 column can be used at high and low pH — from pH 2 - 11.5. This chart shows that the300Extend-C18 has the needed stability at low pH for long-term reproducible separations. Therefore, one wide-pore column can be used for selectivity optimization at low and high pH with both TFA and ammoniumhydroxide mobile phases.

Kirkland, J.J., J.B. Adams, M.A. van Straten andH.A. Claessens, “Bidentate Silane StationaryPhases for Reversed-Phase High-PerformanceLiquid Chromatography,” Analytical Chemistry;(1998); 70 (20); 4344-4352.

FIGURE 3300Extend-C18 and Extend-C18 columns are Stable at Low pH

Columns: 4.6 x 150 mm, 5 µmColumn Aging: 50% Methanol : 50% Water + 1% TFA

Flow Rate: 1.5 mL/min, continuousTemperature: 90°C

Column Test: 60% Methanol : 40% WaterFlow Rate: 1.5 mL/minTemperature: RT

Test Solute: Toluene

*RR: Rapid Resolution 3.5 µm columns.


ZORBAX Extend-C18 Column Ordering Information80Å 300Å

Column Size Particle Part PartDescription (mm) Size (µm) Number NumberAnalytical 4.6 x 250 5 770450-902 770995-902Analytical 4.6 x 150 5 773450-902 773995-902Analytical 4.6 x 50 5 746450-902Rapid Resolution 4.6 x 150 3.5 763953-902 763973-902Rapid Resolution 4.6 x 75 3.5 766953-902Rapid Resolution 4.6 x 50 3.5 735953-902 765973-902Narrow Bore 2.1 x 150 5 773700-902Narrow Bore 2.1 x 50 5 760450-902Narrow Bore RR* 2.1 x 150 3.5 763750-902Narrow Bore RR 2.1 x 50 3.5 735700-902 765750-902MicroBore RR 1.0 x 150 3.5 763600-902MicroBore RR 1.0 x 50 3.5 765600-902MicroBore RR 1.0 x 30 3.5 761600-902Bulk Packing, 2 grams 5 820966-402Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-930 820950-932Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-930 821125-932Guard Hardware Kit 820777-901 820777-901

CH3

CH3

CH3

CH3

CH3

CH3

O

SiO

O

O

O

Si

Si

Si

CH3

CH3

CH3

CH3CH3

CH3

Si

The Agilent ZORBAX Eclipse XDB columns — C18, C8 and Phenyl — are designedto provide good peak shape for basic compounds, such as peptides, at mid pH. In the mid-pH region, residual silanols are more active, and tailing interactions are more likely. To overcome these interactions, Eclipse XDB columns are eXtraDensely Bonded and then double endcapped through a proprietary process to coveras many active silanols as possible. Column lifetime at mid pH is enhanced by thedense bonding and the choice of the Rx-SIL support, which resists silica dissolutionin the mid-pH range. As a result, the Eclipse XDB columns can be used over the pH range 2 - 9, with an optimum lifetime from pH 3 - 8. The Eclipse XDB columns areone choice for changing selectivity of peptide separations at mid pH.

Protein Separations


ZORBAX Eclipse XDB HPLC Columns

ZORBAX Eclipse XDB Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Eclipse XDB-C18 80Å 180 m2/g 60°C 2.0 - 9.0 Double 10%ZORBAX Eclipse XDB-C8 80Å 180 m2/g 60°C 2.0 - 9.0 Double 7.6%ZORBAX Eclipse XDB-Phenyl 80Å 180 m2/g 60°C 2.0 - 9.0 Double 7.2%

22

FIGURE 2Eclipse XDB Stability Testing at pH 7.0, 60°C

• Excellent peak shape for bases, acids and neutrals

• Long lifetime at mid pH with extra dense bonding and doubleendcapping

• Extended pH range — pH 2 - 9

0 1 2 3 4 5 6 7 8 9 10 11 120 1 2 3 4 5 6 7 8

1

2 34

2,3

1

Time (min) Time (min)

4

5 5

Column: 4.6 x 150 mm, 5 µm

Mobile Phase: 60% ACN40% 250 mM phosphate buffer, pH 7.0


Temperature: 60°C

Sample: Tricyclic Antidepressants1. Uracil2. Nortriptyline3. Doxepin4. Amitriptyline5. Trimipramine

Eclipse XDB-C8Sol-type silica— Double endcapping

After 1843 Column Volumes

Waters Symmetry C8Sil-type silica— Single endcapping

After 1826 Column Volumes

Double endcapping, dense bonding and the durable sol-type Rx-SIL particles combine to provide long lifetimeat pH 7.0 when compared to single endcapped sil-gel columns. The conditions used for this test — high temperature (60°C) and high salt concentration (250 mM) — accelerate the dissolution of silica, causing premature failure of the sil-gel type column.

FIGURE 1Extra Densely Bonded and DoubleEndcapped Eclipse XDB Bonded Phase

*RR: Rapid Resolution 3.5 µm columns.For more information on these and other columns consult the Agilent web site at www.agilent.com.



0 5 10 15Time (min)

1

2

3

4

5

67

23

FIGURE 3Good Peak Shape for Acids, Bases and Neutrals on ZORBAX Eclipse XDB at Mid-pH

Column: ZORBAX Rapid Resolution Eclipse XDB-C84.6 x 75 mm, 3.5 µm (Agilent Part No. 966967-906)

Mobile Phase: 35% 20 mM NaH2PO4, pH 7.065% MeOH


Temperature: 35°C


Sample:1. Uracil2. Butyl Paraben3. Propranolol4. Naphthalene5. Dipropyl Phthalate6. Acenaphthene7. Amitriptyline

In the mid-pH region, residual silanols can interactmore strongly with analytes, resulting in asymmetricpeaks. The Eclipse XDB column provides good peakshape in this difficult pH range.


ZORBAX Eclipse XDB HPLC Columns

ZORBAX Eclipse XDB (80Å) Column Ordering InformationColumn Size Particle XDB-C18 XDB-C8 XDB-PhenylDescription (mm) Size (µm) USP L1 USP L7 USP L11Standard Columns and Bulk PackingsSemi-Prep 9.4 x 250 5 990967-202 990967-206Analytical 4.6 x 250 5 990967-902 990967-906 990967-912Analytical 4.6 x 150 5 993967-902 993967-906 993967-912Analytical 4.6 x 50 5 946975-902 946975-906Rapid Resolution 4.6 x 150 3.5 963967-902 963967-906 963967-912Rapid Resolution 4.6 x 75 3.5 966967-902 966967-906 966967-912Rapid Resolution 4.6 x 50 3.5 935967-902 935967-906 935967-912Solvent Saver 3.0 x 250 5 990967-302 990967-306 990967-312Solvent Saver 3.0 x 150 5 993967-302 993967-306 993967-312Solvent Saver Plus 3.0 x 150 3.5 963954-302 963954-306 963954-312Narrow Bore 2.1 x 150 5 993700-902 993700-906 993700-912Narrow Bore 2.1 x 50 5 960967-902 960967-906 960967-912Narrow Bore RR* 2.1 x 150 3.5 930990-902 930990-906Narrow Bore RR 2.1 x 50 3.5 971700-902 971700-906MicroBore RR 1.0 x 150 3.5 963600-902 963600-906MicroBore RR 1.0 x 50 3.5 965600-902 965600-906MicroBore RR 1.0 x 30 3.5 961600-902 961600-906Bulk Packing, 2 grams 5 920966-902Guard Cartridges, 4/pk 4.6 x12.5 5 820950-925 820950-926 820950-927Guard Cartridges, 4/pk 2.1 x12.5 5 821125-926 821125-926 821125-926Guard Hardware Kit 820777-901 820777-901 820777-901Agilent Cartridge ColumnsAnalytical 4.6 x 250 5 7995118-585 7995108-585Analytical 4.6 x 150 5 7995118-595 7995108-595Rapid Resolution 4.6 x 75 3.5 7995118-344 7995108-344Solvent Saver 3.0 x 75 3.5 7995230-344Guard Cartridges, 10/pk 4.0 x 4 5 7995118-504 7995118-504Cartridge Holder 5021-1845 5021-1845High Throughput Cartridge Columns (requires Hardware Kit 820222-901)Rapid Resolution Cartridge 4.6 x 30 3.5 933975-902 933975-906Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 933975-932 933975-936Rapid Resolution Cartridge 4.6 x 15 3.5 931975-902 931975-906Rapid Resolution Cartridge, 3/pk 4.6 x 15 3.5 931975-932 931975-936Rapid Resolution Cartridge 2.1 x 30 3.5 973700-902 973700-906Rapid Resolution Cartridge, 3/pk 2.1 x 30 3.5 973700-932 973700-936Rapid Resolution Cartridge 2.1 x 15 3.5 975700-902 975700-906Rapid Resolution Cartridge, 3/pk 2.1 x 15 3.5 975700-932 975700-936Hardware Kit for High Throughput Columns 820222-901 820222-901CombiHT Columns (end fittings required)CombiHT 21.2 x 150 5 970150-902 970150-906CombiHT 21.2 x 100 5 970100-902 970100-906CombiHT 21.2 x 50 5 970050-902 970050-906CombiHT End Fittings (2) (required for use) 820400-901 820400-901Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8286Capillary 0.5 x 150 5 5064-8287Capillary RR 0.5 x 150 3.5 5064-8288Capillary RR 0.5 x 35 3.5 5064-8298Capillary 0.3 x 250 5 5064-8269Capillary 0.3 x 150 5 5064-8291Capillary RR 0.3 x 150 3.5 5064-8271Guard Cartridges 0.5 x 35 5 5064-8296Guard Cartridges 0.3 x 35 5 5064-8297

0 10,000 20,000 30,000 40,000

0.0055

0.0056

0.0045

0.0040

0.0035

0.0030

0.0025

0.0020

Dimethyl-C18/amideBonus-RP

Column Volume of Purge

Tolu

ene

Plat

e He

ight

, cm

0 5000 10,000 15,000 20,000 25,000 30,000 35,000

0.0018

0.0016

0.0014

0.0012

0.0010

Competitive alkyl/amideBonus-RP

Column Volume of Purge

Tolu

ene

Plat

e He

ight

, cm

Protein Separations


ZORBAX Bonus-RP HPLC Columns

24

FIGURE 1ZORBAX Bonus-RP is Stable at Mid and Low pH

• Excellent peak shape for challenging basic compounds at low and mid pH

• Novel bonding technology with embedded polar group and steric protection

• No phase collapse in 100% aqueous mobile phases

• Alternate selectivity to StableBond, Eclipse XDB, Extend-C18 and Rx columnsat low pH

Triple Endcapped

PG = polar amide group

R1 = sterically protecting isopropyl group

R = C14 alkyl chain

Columns: 4.6 x 150 mm

Mobile Phase: 60% 25 mM phosphatebuffer, pH 7.0

40% ACN


Temperature: 23°C

Triple endcapping of Bonus-RP enhances stability at pH 7.Each 10,000 column volumes is approximately one working month.

Bonus-RP is very stable at low pH. Bulky side groups, likethose in StableBond products, stabilize Bonus-RP at low pH.

Sterically protecting side groups provide good lowpH stability and longer column lifetime than similarpolar-alkyl bonded phases.

Mid-pH Stability

Low pH StabilityColumns: 4.6 x 150 mm

Mobile PhaseAging: 50% MeOH

50% 0.1% TFA


Temperature: 60°C

Mobile PhaseTest: 80% MeOH

20% H2O


Temperature: 23°C

The Agilent ZORBAX Bonus-RP column has a polar amide group embedded in a long alkyl chain. This novel bonding reduces interactions between basiccompounds and the silica support, improving peak shape for the most difficult basic compounds. Peak shape and column lifetime are further improved by tripleendcapping. In addition, diisopropyl side groups provide steric protection againstacid hydrolysis for good lifetime at low pH. The Bonus-RP column provides analternate selectivity to C18 and C8 alkyl bonded phases for peptide separations.

O R

Si

Si

O

Si

O R

R 1

CH3

CH3

CH3

CH3

R

CH3

CH3

Si

Si

PG

PG

PG

R 1

R 1

R 1

R 1

R 1

FIGURE 1Unique, Polar Alkyl Bonus-RPBonded Phase


25

0 5 10 15 20 25 30 35

C1

C1

C2

C2

C3

C3

C4

C4

Time (min)40

FIGURE 3Separation of Basic Peptides on Bonus-RP versus Traditional Alkyl Phase

In the mid-pH region, residual silanols caninteract more strongly with basic peptides tocause peak tailing. Bonus-RP eliminates peaktailing of these basic peptides, even withmultiple charges, in comparison to a typicalalkyl C8 bonded phase.

Bonus-RP(Agilent Part No. 883668-901)

Columns: 4.6 x 150 mm, 5 µm

Instrument: HP 1090

Mobile Phase: A: 0.010 M ammonium phosphate, pH 7 / 0.050 M sodium perchlorateB: 0.010 M ammonium phosphate, pH 7 / 0.050 M sodium perchlorate in 50% ACN


Injection: 10 µL

Flow Rate: 1 mL / min

Temperature: 40°C

Detection: 215 nm

Sample: Basic 11-residue peptides with net +1,+2,+3,+4 positive charges at neutral pH

C1: Ac-Gly-Gly-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide

C2: Ac-Lys-Tyr-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide

C3: Ac-Gly-Gly-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide

C4: Ac-Lys-Tyr-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide

Alkyl C8

ZORBAX Bonus-RP Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Bonus-RP 80Å 180 m2/g 60°C 2.0 - 9.0 Triple 9.5%


ZORBAX Bonus-RP HPLC Columns

ZORBAX Bonus-RP Column Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) NumberAnalytical 4.6 x 250 5 880668-901Analytical 4.6 x 150 5 883668-901Rapid Resolution 4.6 x 150 3.5 863668-901Rapid Resolution 4.6 x 75 3.5 866668-901Narrow Bore 2.1 x 150 5 883725-901Narrow Bore 2.1 x 50 5 861971-901Rapid Resolution 2.1 x 50 3.5 861700-901MicroBore RR* 1.0 x 150 3.5 863608-901MicroBore RR 1.0 x 50 3.5 865608-901MicroBore RR 1.0 x 30 3.5 861608-901Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-928Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-928Guard Hardware Kit 820777-901

*RR: Rapid Resolution 3.5 µm columns.For more information on these and other columns consult the Agilent web site at www.agilent.com.Configurations not shown are available upon request.

26

Protein Separations


ZORBAX Rx HPLC Columns

• Rx-C18 is recommended for alternate selectivity at low pH relative to StableBondSB-C18; for higher temperature applications, StableBond is recommended. Rx-C18columns have a higher carbon load than SB-C18 columns (12% vs. 10%).

• Rx-C18 offers high stability and good peak shape for low pH applications.

• Rx-C18 is manufactured using dimethyloctadecylsilane, is non-endcapped andprovides excellent stability up to pH 8.

FIGURE 1Analysis of Diazepam on Rx-C18

ZORBAX Rx Column Ordering InformationColumn Size ParticleDescription (mm) Size (µm) Rx-C18 Rx-C8Standard ColumnsPreparative 21.2 x 250 7 880967-102 880967-106Semi-Preparative 9.4 x 250 5 880967-202 880967-201Analytical 4.6 x 250 5 880967-902 880967-901Analytical 4.6 x 150 5 883967-902 883967-901Rapid Resolution 4.6 x 150 3.5 863967-902 863953-906Rapid Resolution 4.6 x 75 3.5 866967-902 866953-906Solvent Saver 3.0 x 250 5 880967-302 880975-306Solvent Saver 3.0 x 150 5 883967-302 883975-306Narrow Bore 2.1 x 150 5 883700-902 883700-906Bulk Packing, 2 grams 5 820966-906Guard Cartridge packed with Rx/SB-C8, 2/pk 9.4 x 15 7 820675-115 820675-115Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-914 820950-913Guard Cartridge packed with Eclipse XDB-C8, 4/pk 2.1 x 12.5 5 821125-926 821125-926Guard Hardware Kit 9.4 x 15 840140-901 840140-901Guard Hardware Kit 4.6 x 12.5 820777-901 820777-901Guard Hardware Kit 2.1 x 12.5 820777-901 820777-901

NOTE: Rx-C8 is the same product as StableBond SB-C8 For more information on these and other columns consult the Agilent web site at www.agilent.com.


Column: ZORBAX Rx-C183.0 x 250 mm, 5 µm (Agilent Part No. 880967-302)

Mobile Phase: 35% H2O65% MeOH


Sample: 1. Ethylparaben2. Diazepam

An Rx-C18 column is used for this USP analysis ofdiazepam and the internal standard ethylparaben. The Solvent Saver 3.0 mm i.d. Rx-C18 column reducessolvent usage by 60% over what would be used if theanalysis were done on a 4.6 x 250 mm column.

Time (min)

1 2

0.0 2.5 5.0 7.5

ZORBAX Rx Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Rx-C18 80Å 180 m2/g 60°C 2.0 - 9.0 No 12%ZORBAX Rx-C8 80Å 180 m2/g 80°C 1.0 - 8.0 No 5.5%*The Rx-C8 column is designed for optimal use at low pH (pH 1.8 - 6), but can be used from pH 1 - 8. At pH 6 - 8 highest column stability for all silica-based columns isobtained by operating at temperatures <40°C using low buffer concentrations in the range of 0.01 - 0.02M. At mid-pH Eclipse XDB is recommended and at high-pH Extend-C18 is recommended.


SynChropakSynChropak Column SpecificationsBonded Pore Particle Temp pH End MaxPhase Size Size (µm) Limits Range Capped PressureSynChropak C4 300Å 6.5 60°C 2.5 - 6.5 polymeric 335 bar/5000 psiSynChropak C8 300Å 6.5 60°C 2 - 7 no 335 bar/5000 psiSynChropak C18 300Å 6.5 60°C 2 - 7 no 335 bar/5000 psi

VydacVydac Column SpecificationsBonded Pore Particle Temp pH Bonding MaxPhase Size Size Limits Range Type PressureVydac C4(214TP) 300Å 5 µm 60°C 2-6.5 polymeric 335 bar/5000 psiVydac C18(218TP) 300Å 5 µm 60°C 2-6.5 polymeric 335 bar/5000 psi

27

Vydac 300Å Column Ordering InformationSize Particle Pore Stationary Phase(mm) Size (µm) Size C4 (214 TP) C18 (218 TP)

10.0 x 250 5 300Å 79918B3-588 79918O3-5884.6 x 250 5 300Å 79918B3-584 79918O3-5844.6 x 150 5 300Å 79918B3-564 79918O3-5642.1 x 250 5 300Å 79918O3-5821.0 x 250 5 300Å 79918O3-581

SynChropak 300Å Column Ordering InformationSize Particle Pore Stationary Phase(mm) Size (µm) Size C4 C8 C18

4.6 x 100 6.5 300Å 79919B3-554 79919M3-554 79919O3-5544.6 x 250 6.5 300Å 79919B3-584 79919M3-584 79919O3-584

Agilent has available severalother silica-based columns fromdifferent manufacturers for thereversed phase separations ofproteins and peptides. These arewide-pore (300Å) C18, C8 and C4spherical silica-based columnsfrom Vydac and SynChropak.These standard fitting columnsare popular choices for the analysis of proteins and peptides.

Protein Separations


Additional Reversed-Phase Columnsfor BioScience

• Popular 300Å reversed-phasecolumns available through Agilent

• C4, C8 and C18 bonded phasesavailable

• Vydac and SynChropak columnsavailable

• Alternate selectivity to ZORBAXcolumns

28

ZORBAX GF-250 and GF-450 size exclusion columns are ideal for the size separationsof proteins and other biomolecules. The separation range is 4,000 - 900,000 for globular proteins when using a GF-250 and GF-450 column in series. The GF-250/450 size exclusion columns have a hydrophilic diol bonded phase for high recovery ofproteins (typically >90%) and a unique zirconia modification of the silica to extend the pH range from 3 - 8. The GF-250/450 columns are packed with precisely sizedporous silica microspheres with narrow pore size and particle size distributions. The result is a highly efficient, rugged and reproducible size exclusion column forseparations of proteins with flow rates of up to 3 mL/min. These columns are compatible with organic modifiers and denaturants in the mobile phase to eliminateprotein aggregation for proper size determination. Some common applicationsinclude separations of protein monomers, dimers and aggregates, desalting, protein molecular weight estimation and separations of modified proteins.

• High efficiency and reproducibilitywith short analysis times

• Hydrophilic diol bonded phase forgood protein recovery

• Compatible with organic modifiersand denaturants

• Wide usable pH range (pH 3 - 8)

ZORBAX GF-250 and GF-450 Column SpecificationsPore Particle MW Surface pH Flow

Bonded-Phase Size Size (µm) Range Area Range RateGF-250 150Å 4 4,000 - 400,000 140 m2/g 3 - 8 < 3.0 mL/minGF-450 300Å 6 10,000 - 900,000 50 m2/g 3 - 8 < 3.0 mL/min

Protein Separations

Size Exclusion Columns

ZORBAX GF-250/450 Size Exclusion Columns

FIGURE 1Separation of Protein Standards on the ZORBAX GF-250 SEC Column

Column: ZORBAX GF-250, 9.4 x 250 mm(Agilent Part No. 884973-901)

Mobile Phase: 200 mM Sodium Phosphate, pH 7.0

Flow Rate: 2 mL/min

Temperature: Ambient


Sample: BioRad Gel Filtration Standards for Size Exclusion

1. Thyroglobulin 670,000 Da2. Bovine Gamma Globulin 158,000 Da3. Chicken Ovalbumin 44,000 Da4. Equine Myoglobin 17,000 Da5. Vitamin B-12 1,350 Da

Time (min)0 1 2 3 4 5 6 7 8

1

2 3

4

5

The protein standards separated here are acommonly selected set of standards. TheZORBAX GF-250 column shows excellentresolution for this sample. Additional resolution of thyroglobulin can be obtainedby adding the GF-450 column in series.

Column Size Particle Description (mm) Size (µm) Part No.GF-250 21.2 x 250 4 884974-901GF-250 9.4 x 250 4 884973-901GF-250 4.6 x 250 4 884973-701GF-450 21.2 x 250 6 884974-910GF-450 9.4 x 250 6 884973-902GF-250/GF-450 Guard Cartridge, 2/pk 9.4 x 15 4 820675-111GF-250/GF-450 Guard Cartridge, 4/pk 4.6 x 12.5 4 820950-911GF-250, GF-450 Hardware Kit 9.4 x 15 840140-901GF-250, GF-450 Hardware Kit 4.6 x 12.5 820777-901


ZORBAX GF-250 and GF-450 Gel Filtration Columns Ordering Information

2

0 1 2 3 4 5 6 7 8 9 10

1

3

Retention Time (min)

0 5 10Retention Time (min)

1 1

2 2

33

29

High-speed separations are possible on theZORBAX GF-250 and GF-450 columns, as theseseparations show. The GF-450 column wasused for the fast, reproducible separation ofBSA protein monomer and dimer. The GF-250column shows the rapid separation of IgG antibodies. These SEC applications are just acouple of the many types that can be done onthe GF-250/450 columns.

FIGURE 2AHigh-Speed Size Exclusion Separation of Protein Monomersand Dimers on ZORBAX GF-450

TABLE 1High Mass Recovery of Purified Proteinson a ZORBAX GF-250 Column

FIGURE 2BFast Size Exclusion Separation of Antibodies on ZORBAX GF-250

A: BSA and BSA Dimers (duplicate injections)

Inj #1 Inj #2

Column: ZORBAX GF-4509.4 x 250 mm, 6 µm(Agilent Part No. 884973-902)

Flow Rate: 3 mL/min

Eluent: PBS (phosphate buffered saline), pH 7.4


Detector: UV 220 nm

1. BSA dimer2. BSA monomer3. Azide

B: Antibody Separation

Sample:1. IgG Dimer and Aggregate2. Mouse IgG1 (MOPC 31C)3. Salts: Tris, NaPosphate, NaCl

Column: ZORBAX GF-2509.4 x 250 mm, 4 µm(Agilent Part No. 884973-901)

Mobile Phase: 200 mM NaPhosphate Monobasic pH 7.0/0.1% Azide


Detector: UV 225 nm

Sample: 10 mg in 50 mM Sodium Phosphate, pH 7.0

Protein % Recovered

Ovalbumin 100.4%

RNase 99.5%

α-Chymotrypsin 97.3%

β-Amylase 93.8%

Thyroglobulin 92.3%

Cytochrome-C 91.1%

Carbonic Anhydrase 91.5%

Myoglobin 85.6%

Average 93.9%

Protein SeparationsSize Exclusion Columns

ZORBAX GF-250 and GF-450

Mobile Phase: 400 mM Phosphate buffer, pH 7.0

Sample: 5 µg each protein

30

TSK Gel Filtration Column Ordering InformationParticle

Description Size (mm) Size (µm) 2000SW 2000SW-XL 3000SW 3000SW-XL 4000SWPreparative 21.5 x 300 13 79912S2-299Analytical 7.8 x 300 5 79912S2-597 79912S3-597Analytical 7.5 x 600 10 79912S2-107 79912S3-107 79912S4-107 (13 µm)

Analytical 7.5 x 300 10 79912S2-197 79912S3-197 79912S4-197 (13 µm)

Guard Cartridges 7.5 x 75 10 79912S3-147Guard Cartridges 6.0 x 40 5 79912S3-527

GFC Standards Ordering InformationDescription Part No.Polysaccharides 10 x 0.2g of standards in molecular weight range 180 to 850K 1535-4546PEG standards 10 x 0.5g of individual standards in molecular weight range 106 to 20K 1535-4545

Protein Separations

Size Exclusion Columns

TSK Gel Filtration Columns

Silica-based TSK SW and SWXLcolumns are a popular choice for gelfiltration chromatography of proteinsand peptides. TSK SW columns areavailable in 10 and 13 µm particle sizesand the TSK SWXL columns are avail-able in 5 µm particle sizes. These particle sizes allow for high-speed and high-resolution separations from5,000 to 7,000,000 MW. TSK SWcolumns are suitable for the analysisand recovery of proteins while main-taining enzymatic activity. Thesecolumns can be used from pH 2.5 - 7.5.

Properties and Separation Ranges for TSK-GEL SW Type PackingsTSKgel Particle Pore Globular PolytheylenePacking Size (µm) Size Proteins Dextrans Glycols and OxidesG2000SWXL 5 125Å 5,000-150,000 1,000-30,000 500-15,000G2000SW 10, 13 125Å 5,000-100,000 1,000-30,000 500-15,000G3000SWXL 5 250Å 10,000-500,000 2,000-70,000 1,000-35,000G3000SW 10 250Å 10,000-500,000 2,000-70,000 1,000-35,000G4000SW 13 450Å 20,000-7,000,000 4,000-500,000 2,000-250,000Column: two 5 um, 7.8 x 300 mm TSK-GEL SWXL columns in series; two 10 um, 7.5 x 600 mm TSK-GEL SWcolumns in seriesElution: proteins 0.3 M NaCl in 0.1 M (0.05 M for SWXL columns) phosphate buffer, pH 7; dextrans and PEOs: dis-tilled water

TSK Gel Filtration Column Specifications Pore Flow pH Particle Max

Type Size Max Range Configuration Size (µm) Pressure2000SW 125Å 1.2 mL/min 2.5 - 7.5 21.5 x 300 mm 13 10 bar

7.5 x 600 mm 10 40 bar7.5 x 300 mm 10 20 bar

2000SWXL 125Å 1.2 mL/min 2.5 - 7.5 7.8 x 300 mm 5 70 bar3000SW 250Å 1.2 mL/min 2.5 - 7.5 21.5 x 300 mm 13 15 bar

7.5 x 600 mm 10 50 bar7.5 x 300 mm 10 25 bar

3000SWXL 250Å 1.2 mL/min 2.5 - 7.5 7.8 x 300 mm 5 70 bar4000SW 450Å 1.2 mL/min 2.5 - 7.5 7.5 x 600 mm 13 30 bar

7.5 x 300 mm 13 15 bar

• TSK SW and SWXL columns are a popular choice for SEC of peptides and proteins

• High resolution with 5 µm particle size

• Analytical and preparative sizes available

• Compatible with denaturants such as urea and SDS


31

ZORBAX SAX and SCX Column SpecificationsBonded Phase Pore Size Surface Area pH Range Functionality Max PressureZORBAX Bio Series SCX 300Å 50 m2/g 2.5 - 8.5 Sulfonic acid 350 barZORBAX Bio Series SAX 300Å 50 m2/g 2.5 - 8.5 Quaternary amine 350 barZORBAX 300SCX 300Å 50 m2/g 2 - 7 Sulfonic acid 350 barZORBAX SAX 70Å 300 m2/g 2 - 7 Quaternary amine 350 bar

ZORBAX SAX and SCX Column Ordering InformationParticle Bio Series Bio Series

Description Size (mm) Size (µm) SAX (70Å) SAX (300Å) SCX (300Å) SCX (300Å)Semi-Preparative 9.4 x 250 5 880952-203 880952-204Analytical 4.6 x 250 5 880952-703 880952-704Analytical 4.6 x 150 5 883952-703 883952-704Analytical 6.2 x 80 6 820944-903 820944-904Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-903 820950-904Reusable Cartridge Hardware 820777-901 820777-901

Protein Separations

Ion Exchange Columns

ZORBAX SAX and SCX Columns

FIGURE 1Separation of Anti-PTN Monoclonal IgG fromMouse Ascites Fluid on ZORBAX Bio Series SAX

Column: ZORBAX Bio-SAX6.2 x 80 mm, 6 µm

Mobile Phase: Gradient: 0 to 100% B in 20 min. A = 0.02 M BIS-TRIS, pH 7.0,B = A+1.0 M NaCl

Flow Rate: 2 mL/min



Sample: 20 µL mouse ascites fluid1. IgG2. Albumin

0 5 10 15Time (min)

1

2

• ZORBAX Bio Series SAX and SCX columns are stable from pH 2.5 - 8.5.

• They use unique zirconium oxide stabilized silica.

• ZORBAX 300SCX and SAX are stable from pH 2.5 - 7.

• All provide high efficiency, rapid separations.

ZORBAX has two different types ofstrong ion-exchange columns. TheZORBAX BioSeries SAX and SCXcolumns have a unique zirconiumoxide stabilized wide-pore (300Å) silica. These columns are good for ionexchange separations of biomoleculesfrom pH 2.5 - 8.5 and can be used atflow rates up to 6 mL/min for rapid separations. The ZORBAX 300SCX column also has the same 300Å poresize for separations of biomoleculesbut may have different selectivity, as the silica is not modified with zirconia. The SAX (70Å) is good for the ion-exchange separations of smaller molecules.

32

Anion Exchange Column Ordering InformationStationary Phase

Size Particle(mm) Size (µm) pH Range TSK DEAE-5PW TSK DEAE NPR PL 1000SAX SynChropak WAX SynChropak SAX

4.6 x 35 2.5 2 - 12 79912DE-3244.6 x 100 6.5 2 - 8 79919DE-754 79919QA-7544.6 x 250 6.5 2 - 8 79919DE-784 79919QA-7845.0 x 50 8 1 - 13 79931QA-8357.5 x 75 10 2 - 12 79912DE-147

21.5 x 150 10 2 - 12 79912DE-169

Cation Exchange Column Ordering InformationStationary Phase

Size Particle(mm) Size (µm) pH Range TSK SP-5PW TSK SP-NPR SynChropak WCX SynChropak SCX

4.6 x 35 2.5 2–12 79912SP-3244.6 x 100 6.5 2–8 79919CM-754 79919SP-7544.6 x 250 6.5 2–8 79919CM-784 79919SP-7847.5 x 75 10 2–12 79912SP-147

Protein Separations

Ion Exchange Columns

TSK, SynChropak and PL Ion Exchange Columns

• TSK and PL porous and TSK nonporous resins (NPR) available

• TSK nonporous resins provide high speed, recovery and sensitivity

• Silica-based SynChropak anion and cation exchangers

• Compatible with organic solvents, denaturants and surfactantsAgilent provides a selection of silica-and resin-based ion-exchangecolumns from TSK, PL andSynChropak. These are useful for theseparations of proteins and peptidesas well as separations of nucleotidesand oligonucleotides. Both salt andpH gradients can be used for sampleelution on these ion-exchangecolumns. The SynChropak 300Åcolumns are recommended for pro-teins and peptides of MW <200,000and are compatible with non-ionicdetergents such as urea or Triton.Larger proteins can be analyzedusing the 1000Å TSK and PL columns.

Specifications of TSK, PL and SynChropak Ion Exchange ColumnsPore Particle Flow pH Max Size Functionality Size (µm) Max Range Pressure

TSK DEAE-5PW1 1000Å diethylamine 10 1.2 mL/min 2 - 12 20 barTSK DEAE-NPR2 - diethylamine 2.5 1.6 mL/min 2 - 12 200 barTSK SP-5PW1 1000Å sulfopropyl 10 1.2 mL/min 2 - 12 20 barTSK SP-NPR2 - sulfopropyl 2.5 1.6 mL/min 2 - 12 200 barPL 1000 SAX1 1000Å polyethyleneimine 8 4 mL/min 1 - 13 55 barSynChropak WAX3 300Å polyethyleneimine 6 4 mL/min 2 - 8 300 barSynChropak SAX3 300Å quaternary amine 6 4 mL/min 2 - 8 300 barSynChropak WCX3 300Å carboxymethyl 6 4 mL/min 2 - 8 300 barSynChropak SCX3 300Å sulfonic acid 6 4 mL/min 2 - 8 300 bar1porous resin2nonporous resin3silica based


33

Hydrophobic Interaction Column Ordering InformationStationary Phase

Size Particle(mm) Size (µm) pH Range TSK Phenyl-5PW TSK Ether-5PW SynChropak H-Propyl

4.6 x 100 6.5 2–8 79919P3-7547.5 x 75 10 2–12 79912PH-147 79912ET-147

21.5 x 15.0 10 2–12 79912PH-169

Hydrophobic Interaction Chromatography (HIC) Column SpecificationsColumn Particle Flow Rate pH Max Type Size (µm) Max Range PressureTSK Phenyl-5PW 10 1.2 mL/min 2 - 12* 20 barTSK Ether-5PW 10 1.2 mL/min 2 - 12* 20 barSynChropak Phenyl 6 >1 mL/min 3 - 8*** polymer based column** silica based column

Accessories Ordering InformationItem Part No.Pre-column hardware kit for self-packing guard columns 79916KT-101Phenyl guard column kit 79912PH-707

Protein Separations

Hydrophobic Interaction Chromatography Columns

TSK and SynChropak HydrophobicInteraction Chromatography Columns

• The most popular TSK Phenyl phaseavailable for HIC

• TSK Ether phase is less hydrophobicfor different retention and shorteranalysis time.

• TSK columns are compatible withaqueous solvents, some organic andcan be base washed (pH 2 - 12).

• SynChropak Propyl is an all purposeHIC phase with mild hydrophobicity.

• SynChropak Propyl is compatible withaqueous solvents from pH 3 - 8 andmany organic solvents.

Hydrophobic interaction chromatography (HIC) is a powerful technique forthe purification of proteins. HIC is based on the hydrophobic interaction ofproteins with the stationary phase, and is used when it is critical to maintainthe enzymatic activity and tertiary structure of a protein. In HIC, a high saltconcentration is used to bind the proteins to a weakly hydrophobic stationaryphase, and a decreasing salt gradient is used to elute the proteins. Therefore,HIC is ideal after salt precipitation or ion exchange. Very small amounts oforganic solvents (or none) can be used on the TSK and SynChropak columns;proteins are not denatured and sample recovery is very high. HIC is a com-plementary technique to ion exchange or size exclusion chromatography, andthe columns available here can be used to purify 5 - 20 mg of crude protein.

34

Amino Acid Separations

Special Applications Columns

ZORBAX Eclipse AAA (Amino Acid Analysis) Columns

FIGURE 1High Sensitivity, High Resolution Analysis of Amino Acids Using Different DetectionModes and the ZORBAX Eclipse AAA Protocol

0 5 10 15 20Time (min)

mAU

0

5

10

mAU

0

5

10

0

500

1000

1250mAU

21

3

4

56

7

8

9

10 11 12 13

14

15

1617

18

19

2021

21

2223

24

21

3

4

5

6

7

8

9

10 11 1214

15

1617

18

19

20

21

22 23

24

Column: ZORBAX Eclipse AAA4.6 x 150 mm, 3.5 µm(Agilent Part No. 963400-902)

HPLC Agilent 1100 HPLC binary system

Detection: A) Primary amino acids, DAD 338 nm. B) Secondary amino acids, DAD 262 nm. C) Fluorescence

• High resolution and rapid analysis of 24 amino acids

• Use tested for Amino Acid Analysis

• Uses well known OPA and FMOC derivatization chemistry

• Easily automated using a detailed on-line derivatization protocol available for use with Agilent 1100 Autosampler

The new ZORBAX Eclipse AAA highefficiency HPLC column rapidly separates amino acids following anupdated and improved protocol for usewith the Agilent 1100 HPLC. Totalanalysis from injection to injection can be achieved in as little as 14 min.(10 min. analysis time) on the short, 7.5 cm length column and 24 min. (16 min. analysis time) on the 15 cmcolumn length. Exceptional sensitivity(5 – 50 pmol with DAD, FLD) and reliability are achieved using both OPAand FMOC derivatization chemistries inone fully automated procedure usingthe Agilent 1100 HPLC instrument.

ZORBAX Eclipse AAA Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) NumberAnalytical routine sensitivity 4.6 x 150 5 993400-902Analytical high sensitivity, high-resolution work using the FLD 4.6 x 150 3.5 963400-902Analytical routine sensitivity, high-throughput 4.6 x 75 3.5 966400-902Solvent Saver high sensitivity, high-resolution 3.0 x 150 3.5 961400-302Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-931Guard Hardware Kit 820777-901


Injection: 125 pmoles each amino acid

Mobile Phase: (A) 40 mM Na2HPO4, pH 7.8, (B) ACN: MeOH: water (45:45:10, v/v)

Sample:1. Aspartate 9. Citrulline 17. Tryptophan2. Glutamate 10. Arginine 18. Phenylalanine3. Asparagine 11. Alanine 19. Isoleucine4. Serine 12. Tyrosine 20. Leucine5. Glutamine 13. Cystine 21. Lysine6. Histidine 14. Valine 22. Hydroxyproline7. Glycine 15. Methionine 23. Sarcosine8. Threonine 16. Norvaline 24. Proline

Time (min) Ex/Em (nm)0 340/450

8.5* 266/305*specific time will vary underactual use conditions

A.

B.

C.

Improved resolution between peak #21 (lysine)and peak #22 (hydroxyproline) makes wavelengthswitching easy when using the FLD while per-forming a high resolution amino acid analysis.

For a detailed description of the Eclipse AAAprotocol using the Agilent 1100 HPLC, requestpublication number 5980-1193E, or download itfrom the Agilent website at www.agilent.com.


35

Eclipse AAA and AminoQuant Additional Ordering InformationDescription Part No.

Reagents for Eclipse AAA and AminoQuant Amino Acid AnalysisOPA reagent, 10 mg/ml each in 0.4 M borate buffer o-phthalaldehyde (OPA) and 3-mercaptopropionic acid, 6 ampoules 5061-3335

FMOC reagent, 2.5 mg/ml in acetonitrile, 9-fluorenylmethylchloroformate, 1 ml 5061-3337

Borate buffer, 100 ml 5061-3339DTDPA (Dithiodipropionic) reagent for analysis of cysteine, 5 g 5062-2479

Reagents for AminoQuant Amino Acid AnalysisAminoQuant checkout sample kit: Includes 1 ampoule each of Amino acid standards 1 nmol/ml, 100 pmol/ml, 10 pmol/ml, OPA reagent, FMOC reagent 5061-3353

Amino Acid Standards*Each amino acid standard contains the following amino acids:Glycine L-tyrosine L-serine L-glutamic acid L-arginine L-lysineL-cystine L-leucine L-alanine L-proline L-threonine L-aspartic acidL-histidine L-methionine L-phenylalanine L-isoleucine L-valineAmino acid standard (1 nmol/ml), 10 1-ml ampoules 5061-3330Amino acid standard (250 pmol/ml), 10 1-ml ampoules 5061-3331Amino acid standard (100 pmol/ml), 10 1-ml ampoules 5061-3332Amino acid standard (25 pmol/ml), 10 1-ml ampoules 5061-3333Amino acid standard (10 pmol/ml), 10 1-ml ampoules 5061-3334Amino acids supplement kit: Includes 1 g each of norvaline, sarcosine, asparagine, glutamine, tryptophan, and 4-hydroxyproline 5062-2478

AminoQuant Amino Acid Separation Kit for use with 1100* The AminoQuant Amino Acid separation kit was originally developed for the HP 1090 and can also be used in the 1100 HPLC

AA kit includes: AA column, guard cartridges, guard cartridge holder, connecting capillary, AA standards and reagents, technical note, method disk 5063-6588

Columns for AminoQuant Amino Acid SeparationsColumn for AminoQuant amino acid analysis, 200 x 2.1 mm 79916AA-572Guard columns, Hypersil ODS, 20 x 2.1 mm, 3/pk 79916KT-110Guard cartridge holder 79900CH-010Connecting capillary 35 mm, 0.12 mm id, 2/pk 79841-87609*Buy limited quantities because of shelf-life considerations.

Time (min)0 4 6 8 10 122

2

1 34

5 6

7

8

9

1011

12

13

14 15

1617

19

18

32

4 56

7

89

10

11

12

13

14 15 1617

18

19

1

FIGURE 2Analysis of Free Amino Acids in California Cabernet Sauvignons by ZORBAX Eclipse AAA Protocol

Pedroncelli ‘97

Monthaven ‘98

Column: ZORBAX Eclipse AAA4.6 x 75 mm, 3.5 µm(Agilent Part No. 966400-902)

HPLC Agilent 1100 HPLC binary system

Detection: DAD 338 nm

Mobile Phase: (A) 40 mM Na2HPO4, pH 7.8, (B) ACN: MeOH: water (45:45:10, v/v)

Sample: See Figure 1 for peak identities

Amino AcidsSpecial Applications Columns and Kits

ZORBAX Eclipse AAA (Amino Acid Analysis) Columns

36

DNA Separations


ZORBAX Eclipse dsDNA Column

Wild Type Variant wt/variant wt/wt variant/varianthomoduplexesheteroduplexes

Time (min)0 1 2 3 4 5 6 7

FIGURE 2Mutation Detection by Denaturing HPLC (DHPLC)

FIGURE 1High Resolution Separation on a ZORBAX Eclipse dsDNA Analysis Column

0 10 20 30 40 50 60 70 80 min

mAU302520151050

67 72 89 110 118 147 190 194

234 242271

281310

353404

489501

603872

10781353

Column: Eclipse dsDNA, 2.1 x 150 mm, 3.5 µm

Mobile Phase: Gradient: 45 – 75% B in 90 minutesA: 0.1 M TEAA*, 0.1 mM EDTA, pH 7.0B: A + 25% ACN


Temperature: 50°C

*TEAA = triethylammonium acetate


Sample: 200 ng restriction digest standards

Instrument: Agilent 1100, 1 µL flow cell, binary pump

The Eclipse dsDNA Analysis column can be used for denaturing HPLC — DHPLC — andother specialized techniques. In this example a DHPLC analysis of the Y chromosomeSTS, DYS271 shows ideal separation of the homo- and heteroduplexes formed.

• For DHPLC and dsDNA fragment sizingapplications

• High sensitivity and resolution fordsDNA fragments

• Separate from 20 - 1500 base pairs

• 20-50X the capacity of non-porouscolumns

The ZORBAX Eclipse dsDNA analysiscolumn provides high-resolution, automated quantitative separations of dsDNA fragments by HPLC. Up to 1 bp resolution can be achieved depending on fragment length and gradient analysis time. For high sensitivity, load up to 10 µg and detectless than 1 ng/band using the 2.1 mm i.d.columns. Or, inject up to 40 µg on the 4.6 mm i.d. columns for higher capacityquantitative separations. This column isuseful for applications such as PCR fragment separations, restriction digest separations, DHPLC, RFLP and RAPD.The HPLC separation of dsDNA on theEclipse dsDNA Analysis column canreplace conventional gel electro-phoresis and provide easy samplerecovery of DNA in a simple, automated quantitative analysis.

ZORBAX Eclipse dsDNA Ordering InformationColumn Dimension Particle PartDescription (mm) Size (µm) NumberAnalytical 2.1 x 75 3.5 945750-902Analytical 2.1 x 150 3.5 993850-902Analytical 3.0 x 75 3.5 945750-302Preparative 4.6 x 75 3.5 992750-902Guard Cartridge, 4/pk 2.1 x 12.5 3.5 821125-929Guard Cartridge, 4/pk 4.6 x 12.5 3.5 820950-929Guard Hardware Kit 820777-901

DHPLC analysis of the Y chromosome STS, DYS271 shows ideal separation

Oefner, P.J., Underhill, P.A. (1995)Comparative DNA Sequencing byDenaturing High-Performance LiquidChromatography (DHPLC), Am. J. Hum.Genet., 57 [Suppl.] A266 (Abstract)

37

Oligomer Separations


ZORBAX Oligo Columns24

.03 27

.28

30.4

6

33.5

636

.59

39.4

742

.21

Time (min)0 5 10 15 20 25 30 35 40 45

FIGURE 1Separation of Standard p(DT) 12–18 onZORBAX Oligo Column

Column: ZORBAX Oligo6.2 x 80 mm, 5 µm

(Agilent Part No. 820940-901)

Mobile Phase:Gradient: 35 - 80% B in 90 min.

A: 20:80 ACN: 20 mM Sodium Phosphate pH 7.0B: 20:80 ACN: 20 mM Sodium Phosphate

pH 7.0 + 0.8 M NaCl

Flow: 1.0 mL/min


Detector: UV 260 nm

Sample Volume: 50 µL, 5 units/mL

Time (min)0 5 10 15 20 25 30 35 40 45

FIGURE 2Semi-Preparative Separation of 24 and 25-mer Oligonucleotides

Column: ZORBAX Oligo6.2 x 80 mm, 5 µm(Agilent Part No. 820940-901)

Mobile Phase: Gradient: 35% B to 60% B in 90 min.A = 20% Acetonitrile: 80% 0.02 M Sodium PhosphateB = A + 1.0 M NaCl


Detector: UV 260 nm

Sample: 1.0 mL


ZORBAX Bio Series Oligo Column SpecificationsBonded Phase Pore Size Surface Area pH Range Bonded Functionality ZORBAX Oligo 150Å 140 m2/g 2.5 – 8.5 Proprietary diol

ZORBAX Oligo Column Ordering InformationSize Particle Part(mm) Size (µm) Number

6.2 x 80 5 820940-901 9.4 x 250 5 884973-903

21.2 x 250 5 884974-915 Configurations not shown are available upon request.

• Separate oligonucleotides up to 60 - 80 mer lengths

• High-resolution gradient analysis with mixed mode ion-exchange andreversed-phase column

• Isolate oligonucleotides with >95% purity

• Denaturants can be added to the mobile phase

The ZORBAX Oligo column is designed for the high-resolution gradient separationand purification of oligonucleotides. Oligonucleotides from 3 - 48 bases have beenpurified using this column, while it is possible to separate oligonucleotides up to the 60 - 80 mer range. The Oligo column uses a mixed mode ion-exchange andreversed-phase bonded phase developed for oligonucleotides in order to effectivelyisolate them with high purity. Simple gradient mobile phases are used for oligomerseparations with the possible addition of denaturants, such as urea, to prevent self-hybridization. The Oligo column will provide high resolution for samples from less than 20 micrograms up to 300 micrograms in a volume of up to 1 mL.

This preparative separation of a 24 and 25-mer oligonucleotide shows high resolution witha 1 mL injection volume. The 6.2 x 80 mm column can be used for analytical ( > 20 µg) andsemi preparative (up to 300 µg) separations.

®Copyright 2001 Agilent Technologies, Inc.Printed in the USA March 1, 20015988-2092EN

For the latest information on the complete line of Agilent Technologiescolumns and supplies for analyticalinstruments, see our online catalog at www.agilent.com/chem on theWorld Wide Web, or contact yourlocal Agilent sales office. For all other areas contact Agilent or your localauthorized distributor.

Information, descriptions and specifications in this publication aresubject to change without notice.

An Online Resource for Applications and Products

Visit our web site at www.agilent.com/chem toaccess HPLC applications and to browse the latestinformation for all the columns and consumablesyou need for your day-to-day analytical instrumentoperations.

Many applications can be accessed on the website by simply clicking on Literature Library underthe Publications heading. Search by general application category to find applications of interestto you. Some of the applications available showchromatograms of amino acids, chiral compounds,pharmaceuticals, pesticides and herbicides, aswell as proteins and peptides. More applicationsare being added everyday!

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