Waters XSelect CSH130 C18 3.5 μm and 5 μm Columns User Manual

[ CARE AND USE MANUAL ]

XSELECT CSH130 C18, 3.5 µm AND 5 µm COLUMNS

CONTENTS

I. GETTING STARTED

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

II. COLUMN USE

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

III. SCALING UP/DOWN ISOCRATIC METHODS

IV. TROUBLESHOOTING

V. COLUMN CLEANING, REGENERATING AND STORAGE

a. Cleaning and Regeneration
b. Storage

Thank you for choosing a Waters XSelect™ CSH™ HPLC column. XSelect
CSH130 C18, 3.5 µm and 5 µm Columns feature Waters Charged Surface
Hybrid (CSH) Technology which provides excellent peak shape, high
efficiency and loading capacity for basic compounds when using acidic,
low ionic strength mobile phases. This same particle technology is used
in the ACQUITY UPLC CSH130 C18, 1.7 µm and 2.5 µm XP family of
HPLC columns, thus enabling seamless transferability between HPLC and
UPLC® system platforms. The XSelect CSH130 C18 packing materials
were designed and are manufactured in a cGMP, ISO 9001 certified
manufacturing facility using ultra pure reagents. Each batch of XSelect
CSH130 C18 material is tested chromatographically with acidic, basic
and neutral analytes as part of qualification for use in peptide mapping.
The results are held to narrow specification ranges to assure excellent,
reproducible performance. XSelect CSH130 C18 batches are also QC tested
with a gradient separation of a tryptic digest of cytochrome c using 0.1%
formic acid containing eluents. Finally, every shipped column is individually
tested for packed bed efficiency and a Performance Chromatogram and
Certificate of Batch Analysis is available upon request.

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)

VII. ADDITIONAL INFORMATION

a. Use of Narrow-Bore (2.1 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

XSelect CSH130 C18 Columns 1

[ CARE AND USE MANUAL ]

I. GETTING STARTED

Each XSelect CSH130 C18 Columns 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.

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.

b. Column Equilibration

Peptide Separation Technology 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.

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.

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.

XSelect CSH130 C18 Columns 2

[ 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

50 0.04 0.17 0.83 3.9 14 35 98

100 0.08 0.35 1.7 7.8 28 70 196

150 0.12 0.52 2.5 12 42 106 294

II. COLUMN USE

To ensure the continued high performance of XSelect CSH130 C18 Columns,

follow these guidelines:

a. Guard Columns

Use a Waters guard column of matching chemistry and particle size between

the injector and main column. It is important to use a high performance

matching guard column to protect the main column while not compromising

or changing the analytical resolution.

Guard columns need to be replaced at regular intervals as determined by

sample contamination. When system backpressure steadily increases above

a set pressure limit, it is usually an indication that the guard column

should be replaced. A sudden appearance of split peaks is also indicative

of a need to replace the guard column.

b. 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.

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.

c. Operating pH Limits

The recommended operating pH range for XSelect CSH130 C18, 3.5 µm

and 5 µm columns are 1 to 11. 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.

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.

XSelect CSH130 C18 Columns 3

[ CARE AND USE MANUAL ]

Table 2: Buffer Recommendations for Using XSelect CSH130 C18 Columns from pH 1 to 11

Additive/Buffer pKa Buffer range Volatility

TFA 0.3 Volatile Yes Ion pair additive, can suppress MS signal, used in the 0.02-0.1% range.

Acetic Acid 4.76 Volatile Yes Maximum buffering obtained when used with ammonium acetate salt. Used in 0.1-1.0% range.

Formic Acid 3.75 Volatile Yes Maximum buffering obtained when used with ammonium formate salt. Used in 0.1-1.0% range.

Acetate (NH4CH2COOH) 4.76 3.76 – 5.76 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.

Formate (NH4COOH) 3.75 2.75 – 4.75 Volatile Yes Used in the 1-10 mM range. Note that sodium or potassium salts are not volatile.

Phosphate 1 2.15 1.15 – 3.15 Non-volatile No Traditional low pH buffer, good UV transparency.

Phosphate 2 7.2 6.20 – 8.20 Non-volatile No Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime.

Phosphate 3 12.3 11.3 - 13.3 Non-volatile No 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.

Ammonium Bicarbonate 10.3 (HCO

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

CAPSO 9.7 8.7 – 10.7 Non-Volatile No Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.

Glycine 2.4, 9.8 8.8 – 10.8 Non-Volatile No 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.

Triethylamine

(as acetate salt)

-

)

8.2 – 11.3 Volatile Yes Used in the 5-10 mM range (for MS work keep source >150 ˚C ). Adjust pH with

3

+

9.2 (NH

)

4

10.7 9.7 – 11.7 Volatile Yes Used in the 0.1-1.0% range. Volatile only when titrated with acetic acid (not hydrochloric or phosphoric).

(±1 pH unit)

Used for

Mass Spec

Comments

ammonium hydroxide or acetic acid. Good buffering capacity at pH 10

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

Used as ion-pair for DNA analysis at pH 7-9

d. 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.

e. Pressure

XSelect CSH130 C18 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.

f. Temperature

Temperatures up to 80 ˚C are recommended for operating XSelect CSH130

C18 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.

XSelect CSH130 C18 Columns 4

[ CARE AND USE MANUAL ]

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:

F

= F1 (r2/r1)2

2

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

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. 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.

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 Proteinaceous Samples

1. water Option 1: Inject repeated 100 µL aliquots of dimethylsulfoxide

2. methanol Option 2: gradient of 10% to 90% B where:

3. isopropanol

(DMSO) using a reduced flow rate delivering 50% Eluent A
and 50% Eluent B

A = 0.1% trifluoroacetic acid (TFA) in water
B = 0.1% trifluoroacetic acid (TFA) in acetonitrile (CH

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

CN)

3

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.

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 con-

tained 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.

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

XSelect CSH130 C18 Columns 5

[ CARE AND USE MANUAL ]

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 high-

quality 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 compres-

sion 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.

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.

Figure 3: Parker Ferrule in a Waters Style Endfitting

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 lead-

ing 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” .090”

XSelect CSH130 C18 Columns 6

Parker Ferrule Setting

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.

Void

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.

[ CARE AND USE MANUAL ]

Figure 4: Waters Ferrule in a Parker Style Endfitting

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 Keystone, Inc.

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

Table 5: Waters Part Numbers for SLIPFREE Connectors

SLIPFREE Type Tubing Internal Diameter

Tubing Length 0.005” 0.010” 0.020”

Single 6 cm PSL 618000 PSL 618006 PSL 618012

Single 10 cm PSL 618002 PSL 618008 PSL 618014

Single 20 cm PSL 618004 PSL 618010 PSL 618016

Double 6 cm PSL 618001 PSL 618007 PSL 618013

Double 10 cm PSL 618003 PSL 618009 PSL 618015

Double 20 cm PSL 618005 PSL 618001 PSL 618017

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

SLIPFREE Connector Features:

• Tubing pushed into endfitting, thereby guaranteeing

a void-free connection

• Connector(s) come(s) installed on tubing

• Various tubing IDs 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

XSelect CSH130 C18 Columns 7

Diluted/Distorted Sample Band

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.

[ CARE AND USE MANUAL ]

1.0

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

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

System Volume

5

4.4 %h

In

a typical HPLC system, the Bandspreading Volume should be no greater than

100 µL ± 30 µL (or Variance of 400 µL ± 36 µL).

In a microbore (2.1mm i.d.) system, the Bandspreading Volume should be no

greater than 20 to 40 µL (or Variance no greater than 16 µL to 64 µL).

c. Measuring Gradient Delay Volume (or Dwell Volume)

Figure 8: Determination of Gradient Delay Volume

0.8

0.6

Au

0.4

0.2

0.0

1/2 Vertical

Distance

Time

t

1/2

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 (t1/2) (half the vertical distance

between the initial and final isocratic segments as shown in

Figure 8).

7. Subtract half the gradient time (1/2 tg) (10 min/2 = 5 min in

this example) from the gradient midpoint (t1/2) to obtain the

dwell time (tD).

8. Convert the dwell time (tD) to the dwell volume (VD) by

multiplying by the flow rate (F).

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.

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).

XSelect CSH130 C18 Columns 8

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.

[ 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.

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.

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

XSelect CSH130 C18 Columns 9

[ CARE AND USE MANUAL ]

Figure 10: Representative Test Chromatography and Conditions for Separation of a Cytochrome c Tryptic Digest

1.20

1.10

1.00

0.90

0.80

0.70

0.60

0.50

0.40

0.30

0.20

0.10

0.00

2 345678910 11 12 Min.

Peak Identification

T1 N-AcGDVEK
T13-T14 KYIPGTK
T14 YIPGTK
T4 IFVQK
T9-T10 KTGQAPGFSYTDANK
T10 TGQAPGFSYTDANK
T8 TGPNLHGLFGR
T15 MIFAGIK
T19C EDLIAY
T19 EDLIAYLK
T12-T13 GITWGEETLMEYLENPKK
T12 GITWGEETLMEYLENPK
T5 CAQCHTVEK (heme attached)

Retention Time (min) Result

T19C 6.69

Retention Time Difference (min) Result

T4 and T14 0.312
T10 and T9-T10 0.780
T19 and T19C 0.461
T12 and T12-T13 0.290

Chromatographic Conditions 

Column: 2.1 mm x 50 mm

Sample: Tryptic digest of bovine cytochrome c (P/N 186006371)

Reconstituted w/ 200µL of MP A, 5.0µL injection

Flow rate: 0.2 mL/min

Temperature: 40°C

Mobile Phase A: 0.1% formic acid in 100% waterMPA

Mobile Phase B: 0.085% formic acid in 75% acetonitrileMPB

Gradient: 1 to 21%B in 5 min; 21 to 31.4% B in 2.5 min; 31.4 to

95% B in 2.5 min; 95% B for 1.5min; 1%B for 2.5 min

Detection: UV 214 nm

XSelect CSH130 C18 Columns 10

[ CARE AND USE MANUAL ]

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©2013 Waters Corporation. Waters, The Science of W hat’s Pos­sible, Oasis, Sep-Pak, CSH, and XSelect, are trademarks of Waters
Corporation. Acrodisc is a trademark of Pall Life Sciences.
SLI PFR EE is a trademark of Keystone, Inc.

January 2013 720004585EN Rev A IH-PDF

XSelect CSH130 C18 Columns 11

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T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com