Waters XBridge Peptide BEH C18 130A and 300A Columns User Manual

Download

Page 1

[ CARE AND USE MANUAL ]

XBridge Peptide BEH C18, 130Å and 300Å Columns

CONTENTS I. GETTING STARTED

a. Column Installation b. Column Equilibration c. Initial Column Efficiency Determination

II. COLUMN USE

a. Sample Preparation b. Operating pH Limits c. Solvents d. Pressure e. Temperature

III. SCALING UP/DOWN ISOCRATIC METHODS

IV. TROUBLESHOOTING

V. COLUMN CLEANING, REGENERATING AND STORAGE

a. Cleaning and Regeneration b. Storage

VI. CONNECTING THE COLUMN TO THE HPLC

a. Column Connectors and System Tubing Considerations b. Measuring System Bandspreading Volume c. Measuring Gradient Delay Volume (or Dwell Volume)

Thank you for choosing a Waters XBridge® Peptide BEH C18, 130Å or 300Å Column. The XBridge Peptide BEH C18, 130Å and 300Å packing materials were designed to provide excellent peak shape, high efficiency and excellent stability. The XBridge Peptide BEH C18, 130Å and 300Å packing materials are manufactured in a cGMP, ISO 9002 certified planted using ultra pure reagent. Each batch of XBridge Peptide BEH C18 Column material has been qualified with a peptide separation and the results are held to narrow specification ranges to assure excellent, reproducible performance for peptide separations. Every column is tested and a Performance Test Chromatogram along with a Certification of Acceptance are provided with each column.

VII. ADDITIONAL INFORMATION

a. Use of Narrow-Bore (3.0 mm i.d.) b. Impact of Bandspreading Volume on 2.1 mm i.d. Column Performance c. Non-Optimized vs. Optimized LC-MS/MS System: System Modification Recommendations

Page 2

[ CARE AND USE MANUAL ]

I. GETTING STARTED

Each XBridge Peptide BEH C18, 130Å and 300Å Column comes with a Certificate of Acceptance and a Performance Test Chromatogram. The Certificate of Acceptance is specific to each batch of packing material contained in the Peptide Separation Technology column and includes the batch number, analysis of unbonded particles, analysis of bonded particles, and chromatographic results and conditions. The Performance Test Chromatogram is specific to each individual column and contains the information: batch number, column serial number, USP plate count, USP tailing factor, retention factor, and chromatographic conditions. These data should be stored for future reference.

a. Column Installation

Note: The flow rates given in the procedure below are for a typical

5 µm packing in a 4.6 mm i.d. column. Scale the flow rate up or

down accordingly based upon the column i.d., length, particle size

and backpressure of the Peptide Separation Technology column

being installed. See Scaling Up/Down Isocratic Separations section

for calculating flow rates when changing column i.d and/or length.

See “Connecting the Column to the HPLC” for a more detailed

discussion on HPLC connections.

b. Column Equilibration

XBridge Peptide BEH C18, 130Å and 300Å Columns are shipped in 100% acetonitrile. It is important to ensure mobile phase compatibility before changing to a different mobile phase system. Equilibrate the column with a minimum of 10 column volumes of the mobile phase to be used (refer to Table 1 for a listing of empty column volumes).

To avoid precipitating out mobile phase buffers on your column or in your system, flush the column with five column volumes of a water/organic solvent mixture, using the same or lower solvent content as in the desired buffered mobile phase. (For example, flush the column and HPLC system with 60% methanol in water prior to introducing 60% methanol/40% buffer mobile phase).

c. Initial Column Efficiency Determination

1. Perform an efficiency test on the column before using it in the desired application. Waters recommends using a suitable solute mixture, as found in the “Performance Test Chromatogram,” to analyze the column upon receipt.

2. Determine the number of theoretical plates (N) and use this value for periodic comparisons.

1. Purge the pumping system of any buffer-containing mobile phases and connect the inlet end of the column to the injector outlet. An arrow on the column identification label indicates the correct direction of solvent flow.

2. Flush column with 100% organic mobile phase (methanol or acetonitrile) by setting the pump flow rate to 0.1 mL/min. and increase the flow rate to 1 mL/min over 5 minutes.

3. When the mobile phase is flowing freely from the column outlet, stop the flow and attach the column outlet to the detector. This prevents entry of air into the detection system and gives more rapid baseline equilibration.

4. Gradually increase the flow rate as described in step 2.

5. Once a steady backpressure and baseline have been achieved, proceed to the next section.

Note: If mobile phase additives are present in low concentrations

(e.g., ion-pairing reagents), 100 to 200 column volumes may be

required for complete equilibration. In addition, mobile phases that

contain formate (e.g., ammonium formate, formic acid, etc.) may

also require longer initial column equilibration times.

3. Repeat the test at predetermined intervals to track column performance over time. Slight variations may be obtained on two different HPLC systems due to the quality of the connections, operating environment, system electronics, reagent quality, column condition and operator technique.

XBridge Peptide BEH C18, 130Å and 300Å Columns

Page 3

[ CARE AND USE MANUAL ]

Table 1: Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)

Column internal diameter (mm)

Column Length (mm) 1.0 2.1 4.6 10 19 30

50 0.04 0.17 0.83 3.9 14 35 100 0.08 0.35 1.7 7.8 28 70 150 0.12 0.52 2.5 12 42 106 250 - 0.87 4.2 20 70 176

II. COLUMN USE

To ensure the continued high performance of XBridge Peptide BEH C18, 130Å and 300Å Columns follow these guidelines:

a. Sample Preparation

1. Sample impurities often contribute to column contamination. One option to avoid this is to use Waters Oasis® solid-phase extraction cartridges/columns or Sep-Pak® cartridges of the appropriate chemistry to clean up the sample before analysis.

2. It is preferable to prepare the sample in the operating mobile phase or a mobile phase that is weaker (less organic modifier) than the mobile phase for the best peak shape and sensitivity.

3. If the sample is not dissolved in the mobile phase, ensure that the sample, solvent and mobile phases are miscible in order to avoid sample and/or buffer precipitation.

4. Filter sample with 0.2 µm filters to remove particulates. If the sample is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile, methanol, etc.) ensure that the filter material does not dissolve in the solvent. Contact the filter manufacturer with solvent compatibility questions. Alternatively, centrifugation for 20 minutes at 8,000 rpm, followed by the transfer of the supernatant liquid to an appropriate vial, could be considered.

b. Operating pH Limits

The recommended operating pH range for XBridge Peptide BEH C18, 130Å and 300Å Columns is 1 to 12. A listing of commonly used buffers and additives is given in Table 2. Additionally, the column lifetime will vary depending upon the operating temperature, the type and concentration of buffer used.

XBridge Peptide BEH C18, 130Å and 300Å Columns

Page 4

[ CARE AND USE MANUAL ]

Table 2: Buffer Recommendations for Using XBridge Peptide BEH C18, 130Å and 300Å Columns from pH 1 to 12

Additive/Buffer pK

Buffer

range

TFA 0.3 - Volatile Yes

Acetic Acid 4.76 - Volatile Yes

Formic Acid 3.75 - Volatile Yes

Acetate (NH4CH2COOH)

4.76 3.76 – 5.76 Volatile Yes

Formate (NH4COOH) 3.75 2.75 – 4.75 Volatile Yes

Phosphate 1 2.15 1.15 – 3.15 Non-volatile No

Phosphate 2 7.2 6.20 – 8.20 Non-volatile No

Phosphate 3 12.3 11.3 - 13.3 Non-volatile No

Volatility

(±1 pH unit)

Used for

Mass Spec

Comments

Ion pair additive, can suppress MS signal, used in the 0.02-0.1% range.

Maximum buffering obtained when used with ammonium acetate salt. Used in 0.1-1.0% range.

Maximum buffering obtained when used with ammonium formate salt. Used in 0.1-1.0% range.

Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.

Traditional low pH buffer, good UV transparency.

Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime.

4-Methylmorpholine ~8.4 7.4 – 9.4 Volatile Yes Generally used at 10 mM or less.

Ammonia (NH4OH) 9.2 8.2 – 10.2 Volatile Yes

Keep concentration below 10 mM and temperatures below 30 ˚C.

Used in the 5-10 mM range (for MS work keep source >150 ˚C ). Adjust pH with ammonium

Ammonium Bicarbonate

10.3 (HCO

9.2 (NH

)

8.2 – 11.3 Volatile Yes

hydroxide or acetic acid. Good buffering capacity at pH 10

Note: use ammonium bicarbonate (NH4HCO3), not ammonium carbonate ([NH4]2CO3).

Ammonium (Acetate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range. Ammonium (Formate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.

Borate 9.2 8.2 – 10.2 Non-Volatile No

CAPSO 9.7 8.7 – 10.7 Non-Volatile No

Glycine 2.4, 9.8 8.8 – 10.8 Non-Volatile No

Reduce temperature/concentration and use a guard column to maximize lifetime.

Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.

Zwitterionic buffer, can give longer lifetimes than borate buffer.

1-Methylpiperidine 10.2 9.3 – 11.3 Volatile Yes Used in the 1-10 mM range.

CAPS 10.4 9.5 – 11.5 Non-Volatile No

Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.

Used in the 0.1-1.0% range. Volatile only when

Triethylamine (as acetate salt)

10.7 9.7 – 11.7 Volatile Yes

titrated with acetic acid (not hydrochloric or phosphoric). Used as ion-pair for DNA analysis at pH 7-9

Pyrrolidine 11. 3 10.3 – 12.3 Volatile Yes Mild buffer, gives long lifetime.

XBridge Peptide BEH C18, 130Å and 300Å Columns

Page 5

[ CARE AND USE MANUAL ]

c. Solvents

To maintain maximum column performance, use high quality chromatography grade solvents. Filter all aqueous buffers prior to use. Pall Gelman Laboratory Acrodisc® filters are recommended. Solvents containing suspended particulate materials will generally clog the outside surface of the inlet distribution frit of the column. This will result in higher operating pressure and poor performance.

d. Pressure

XBridge Peptide BEH C18, 130Å and 300Å Columns can tolerate pressures of up to 6,000 psi (400 bar or 40 Mpa) although pressures greater than 4,000 – 5,000 psi should be avoided in order to maximize column and system lifetimes.

e. Temperature

Temperatures between 20 ˚C – 60 ˚C are recommended for operating XBridge Peptide BEH C18, 130Å and 300Å Columns in order to enhance selectivity, lower solvent viscosity and increase mass transfer rates. However, any temperature above ambient will have a negative effect on lifetime which will vary depending on the pH and buffer conditions used.

III. SCALING UP/DOWN ISOCRATIC METHODS

The following formulas will allow scale up or scale down, while maintaining the same linear velocity, and provide new sample loading values:

If column i.d. and length are altered: F2 = F1 (r2/r1) Load2 = Load1 (r2/r1)2 (L2/L1) Injection volume2 = Injection volume1(r2/r1)2 (L2/L1)

Where: r = Radius of the column F = Flow rate L = Length of column 1 = Original, or reference column 2 = New column

Information on column troubleshooting problems may be found in HPLC Columns Theory, Technology and Practice, U.D. Neue, (Wiley-VCH, 1997), the Waters HPLC Troubleshooting Guide (Literature code # 720000181EN) or visit the Waters Corporation website for information on seminars (www.waters.com).

VI. COLUMN CLEANING, REGENERATION AND STORAGE

a. Cleaning and Regeneration

Changes in peak shape, peak splitting, shoulders on the peak, shifts in retention, change in resolution or increasing backpressure may indicate contamination of the column. Flushing with a neat organic solvent, taking care not to precipitate buffers, is usually sufficient to remove the contaminant. If the flushing procedure does not solve the problem, purge the column using the following cleaning and regeneration procedures.

Use the cleaning routine that matches the properties of the samples and/or what you believe is contaminating the column (see Table 3). Flush columns with 20 column volumes each of HPLC-grade solvents (e.g., 80 mL total for 4.6 x 250 mm column) listed in Table 3. Increasing mobile phase temperature to 35-55 ˚C increases cleaning efficiency. If the column performance is poor after cleaning and regeneration, call your local Waters office for additional support.

Table 3: Cleaning and Regeneration Sequence or Options

Polar Samples

1. Water

2. Methanol

3. Isopropanol

Proteinaceous Samples

Option 1: Inject repeated 100 µL aliquots of dimethylsulfoxide (DMSO) using a reduced flow rate delivering 50% Eluent A and 50% Eluent B

Option 2: gradient of 10% to 90% B where: A = 0.1% trifluoroacetic acid (TFA) in water B = 0.1% trifluoroacetic acid (TFA) in acetonitrile (CH3CN)

Option 3: Flush column with 7 M guanidine hydrochloride, or 7 M urea

IV. TROUBLESHOOTING

Changes in retention time, resolution, or backpressure are often due to column contamination. See the Column Cleaning, Regeneration and Storage section of this Care and Use Manual.

Note: To avoid potentially damaging precipitation within your

column (e.g., if your separation eluent contains phosphate buffer),

be certain to flush column with 5 to 10 column volumes of water

BEFORE using suggested organic eluent column wash procedures.

XBridge Peptide BEH C18, 130Å and 300Å Columns

Page 6

[ CARE AND USE MANUAL ]

b. Storage

For periods longer than four days at room temperature, store the column in 100% acetonitrile. Immediately after use with elevated temperatures and/or at pH extremes, store in 100% acetonitrile for the best column lifetime. Do not store columns in highly aqueous (<20% organic) mobile phases, as this may promote bacterial growth. If the mobile phase contained a buffer salt, flush the column with 10 column volumes of HPLC grade water (see Table 1 for common column volumes) and replace with 100% acetonitrile for storage. Failure to perform this intermediate step could result in precipitation of the buffer salt in the column or system when 100% acetonitrile is introduced. Completely seal column to avoid evaporation and drying out of the bed.

Note: If a column has been run with a mobile phase that contains

formate (e.g., ammonium formate, formic acid, etc.) and is then

flushed with 100% acetonitrile, slightly longer equilibration times

may be necessary when the column is re-installed and run again

with a formate-containing mobile phase.

V. CONNECTING THE COLUMN TO THE HPLC

a. Column Connectors and System Tubing Considerations

Tools needed:



3/8 inch wrench



5/16 inch wrench

Handle the column with care. Do not drop or hit the column on a hard surface as it may disturb the bed and affect its performance.

1. Correct connection of 1/16 inch outer diameter stainless steel tubing leading to and from the column is essential for highquality chromatographic results.

2. When using standard stainless steel compression screw fittings, it is important to ensure proper fit of the 1/16 inch outer diameter stainless steel tubing. When tightening or loosening the compression screw, place a 5/16 inch wrench on the compression screw and a 3/8 inch wrench on the hex head of the column endfitting.

Note: If one of the wrenches is placed on the column tube flat

during this process, the endfitting will be loosened and leak.

3. If a leak occurs between the stainless steel compression screw fitting and the column endfitting, a new compression screw fitting, tubing and ferrule must be assembled.

4. An arrow on the column identification label indicates correct direction of solvent flow.

Correct connection of 1/16 inch outer diameter stainless steel tubing leading to and from the column is essential for high-quality chromatographic results. To obtain a void-free connection, the tubing must touch the bottom of the column endfitting. It is important to realize that extra column peak broadening due to voids can destroy an otherwise successful separation. The choice of appropriate column connectors and system tubing is discussed in detail below.

Figure 1. Waters and Parker Ferrule Types.

Waters Ferrule Setting

.130”

Parker Ferrule Setting

.090”

Due to the absence of an industry standard, various column manufacturers have employed different types of chromatographic column connectors. The chromatographic separation can be negatively affected if the style of the column endfittings does not match the existing tubing ferrule settings. This section explains the differences between Waters style and Parker style ferrules and endfittings (Figure 1). Each endfitting style varies in the required length of the tubing protruding from the ferrule. The XBridge Column is equipped with Waters style endfittings that require a

0.130 inch ferrule depth. If a non-Waters style column is presently

being used, it is critical that ferrule depth be reset for optimal performance prior to installing a XBridge column.

In a proper tubing/column connection (Figure 2), the tubing touches the bottom of the column endfitting, with no void between them.

Figure 2. Proper Tubing/Column Connection.

The presence of a void in the flow stream reduces column performance. This can occur if a Parker ferrule is connected to a Waters style endfitting (Figure 3).

Note: A void appears if tubing with a Parker ferrule is connected to

a Waters style column.

XBridge Peptide BEH C18, 130Å and 300Å Columns

Page 7

Diluted/Distorted Sample Band

[ CARE AND USE MANUAL ]

Figure 3. Parker Ferrule in a Waters Style Endfitting.

Void

There is only one way to fix this problem: Cut the end of the tubing with the ferrule, place a new ferrule on the tubing and make a new connection. Before tightening the screw, make sure that the tubing bottoms out in the endfitting of the column.

Conversely, if tubing with a Waters ferrule is connected to a column with Parker style endfitting, the end of the tubing will bottom out before the ferrule reaches its proper sealing position. This will leave a gap and create a leak (Figure 4).

Note: The connection leaks if a Waters ferrule is connected to a

column with a Parker style endfitting.

Figure 4. Waters Ferrule in a Parker Style Endfitting.

SLIPFREE connector features:



Tubing pushed into endfitting, thereby guaranteeing a void-free connection



Connector(s) come(s) installed on tubing



Various tubing i.d’s and lengths available



Fingertight to 10,000 psi – Never needs wrenches



Readjusts to all column endfittings



Compatible with all commercially available endfittings



Unique design separates tube-holding function from sealing function

Table 5: Waters Part Numbers for SLIPFREE Connectors

SLIP FREE Type Tubing Internal Diameter

Tubing Length 0.005” 0.010” 0.020”

Single 6 cm PSL 618000 PSL 618006 P SL 618012 Single 10 cm PSL 618002 PSL 618008 P SL 618014 Single 20 cm PSL 61800 4 PSL 618010 P SL 618016 Double 6 cm P SL 618001 PS L 618007 PSL 618013 Double 10 cm PSL 618003 PSL 61800 9 PSL 618 015 Double 20 cm PSL 618005 P SL 618001 PSL 618017

Gap

There are two ways to fix the problem:

1. Tighten the screw a bit more. The ferrule moves forward, and reaches the sealing surface. Do not overtighten since this may end in breaking the screw.

2. Cut the tubing, replace the ferrule and make a new connection.

Alternatively, replace the conventional compression screw fitting with an all-in-one PEEK™ fitting (Waters Part Number PSL613315) that allows resetting of the ferrule depth. Another approach is to use a SLIPFREE® connector to always ensure the correct fit. The fingertight SLIPFREE connectors automatically adjust to fit all compression screw type fittings without the use of tools (Figure 5).

Figure 5. Single and Double SLIPFREE Connectors.

Band Spreading Minimization

Figure 6 shows the influence of tubing internal diameter on system band spreading and peak shape. As can be seen, the larger tubing diameter causes excessive peak broadening and lower sensitivity.

Figure 6. Effect of Connecting Tubing on System.

0.005 inches

0.020 inches

0.040 inches

XBridge Peptide BEH C18, 130Å and 300Å Columns

Page 8

[ CARE AND USE MANUAL ]

b. Measuring System Bandspreading Volume and

System Variance

This test should be performed on an HPLC system with a single wavelength UV detector (not a Photodiode Array [PDA]).

1. Disconnect column from system and replace with a zero dead volume union.

2. Set flow rate to 1 mL/min.

3. Dilute a test mix in mobile phase to give a detector sensitivity of 0.5–1.0 AUFS (system start up test mix can be used which contains uracil, ethyl and propyl parabens; Waters Part Number WAT034544).

4. Inject 2 to 5 µL of this solution.

5. Measure the peak width at 4.4% of peak height (5-sigma method):

5-sigma Bandspreading (µL) = Peak Width (min) x Flow Rate (mL/min) x (1000 µL/1 mL)

System Variance (µL2) = (5-sigma bandspreading)2/25

1. Replace the column with a zero dead volume union.

2. Prepare mobile phase A (pure solvent, such as methanol) and mobile phase B (mobile phase A with a UV absorbing sample, such as (v/v) 0.1% acetone in methanol).

3. Equilibrate the system with mobile phase A until a stable baseline is achieved.

4. Set the detector wavelength to the absorbance maximum of the probe (265 nm for acetone).

5. Program a 0-100% B linear gradient in 10 min at 2 mL/min (the exact conditions are not critical; just make sure the gradient volume is at least 20 mL) with a hold at 100% B.

6. Determine the dwell time by first locating the time at the midpoint of the formed gradient (t

) (half the vertical

1/2

distance between the initial and final isocratic segments as shown in Figure 8).

Figure 8. Determination of Gradient Delay Volume.

1.0

Figure 7. Determination of System Bandspreading Volume Using 5-Sigma Method.

System Volume

4.4 %h

In a typical HPLC system, the Bandspreading Volume should be no greater than 100 µL ± 30 µL (or Variance of 400 µL2 ± 36 µL2).

In a microbore (2.1 mm i.d.) system, the Bandspreading Volume should be no greater than 20 to 40 µL (or Variance no greater than 16 µL2 to 64 µL2).

c. Measuring Gradient Delay Volume (or Dwell Volume)

For successful gradient-method transfers the gradient delay volumes should be measured using the same method on both HPLC systems. The procedure below describes a method for determining the gradient delay volumes.

0.8

0.6

0.4

0.2

0.0

1/2 Vertical

Distance

1/2

Time

7. Subtract half the gradient time (1/2 tg) (10 min/2 = 5 min in this example) from the gradient midpoint (t

) to obtain the

1/2

dwell time (tD).

8. Convert the dwell time (tD) to the dwell volume (VD) by multiplying by the flow rate (F). Dwell Volume VD = (t

—1/2 tg) x F

1/2

For fast gradient methods, the gradient delay volume (or dwell volume) should be less than 1 mL. If the gradient delay volume is greater than 1 mL, see System Modification Recommendations section on how to reduce system volume.

XBridge Peptide BEH C18, 130Å and 300Å Columns

Page 9

[ CARE AND USE MANUAL ]

VII. ADDITIONAL INFORMATION

a. Use of Narrow-Bore Columns

This section describes how to minimize extra column effects and provides guidelines on maximizing the performance of a narrow-bore column in an HPLC system. A 2.1 mm i.d. column requires modifications to the HPLC system in order to eliminate excessive system bandspreading volume. Without proper system modifications, excessive system bandspreading volume causes peak broadening and has a large impact on peak width as peak volume decreases.

b. Impact of Bandspreading Volume on 2.1 mm i.d.

Column Performance

System with 70 µL bandspreading: 10,000 plates System with 130 µL bandspreading: 8,000 plates (same column)

Note: Flow splitters after the column will introduce additional

band-spreading.

System optimization, especially in a system that contains a flow splitter, can have dramatic effects on sensitivity and resolution. Optimization includes using correct ferrule depths and minimizing tubing inner diameters and lengths. An example is given in Figure 9 where system optimization resulted in a doubling of sensitivity and resolution of the metabolite in an LC-MS/MS system.

Figure 9. Non-Optimized vs. Optimized LC-MS/MS System.

7.00 7.50

Non-optimized LC/MS/MS System Optimized System

8.00

7.00 7.50 8.00

c. Non-Optimized vs. Optimized LC-MS/MS System:

System Modification Recommendations

1. Use a microbore detector flow cell with 2.1 mm i.d. columns.

Note: Detector sensitivity is reduced with the shorter flow cell path

length in order to achieve lower bandspreading volume.

2. Minimize injector sample loop volume.

3. Use 0.009 inch (0.25 mm) tubing for rest of connections in standard systems and 0.005 inch (0.12 mm) tubing for narrowbore (2.1 mm i.d.) systems.

4. Use perfect (pre-cut) connections (with a variable depth inlet if using columns from different suppliers).

5. Detector time constants should be shortened to less than

0.2 seconds.

©2014 Waters Corporation. Waters, The Science of What’s Possible, Oasis, Sep-Pak and XBridge are registered trademarks of Waters Corporation. All other trademarks are property of their respective owners.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com