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Waters spherisorb columns
contents
i. GettinG started
a. Column Installation
1. Reversed-Phase Columns
2. Normal-Phase Columns
b. Column Equilibration
1. Reversed-Phase Columns
2. Normal-Phase Columns
c. Initial Column Efficiency Determination
ii. column use
a. Guard Columns
b. Sample Preparation
c. pH Range
d. Solvents
e. Pressure
f. Temperature
iii. scalinG up/doWn isocratic methods
vii. additional inFormation
a. Use of Narrow-Bore (≤3.0 mm i.d.) Columns
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
d. Guard Cartridges and Columns Assembly
Thank you for choosing a Waters Spherisorb® column. Spherisorb
analytical columns are durable, high-efficiency chromatographic
columns, featured in thousands of references in chromatographic
literature. The wide range of column lengths and diameters offers
you exceptional flexibility in optimizing methods and reducing
solvent consumption. Follow the guidelines in this manual to obtain
the best performance, reproducibility and durability from your
analytical columns and cartridges.
iv. troubleshootinG
v. column cleaninG, reGeneratinG and storaGe
a. Cleaning and Regenerating
1. Reversed-Phase Columns
2. Normal-Phase Columns
b. Storage
1. Reversed-Phase Columns
2. Normal-Phase Columns
vi. connectinG the column to the hplc
a. Column Connectors and System Tubing Considerations
b. Band Spreading Minimization
c. Measuring System Bandspreading Volume & System Variance
d. Measuring System Volume
Waters Spherisorb Columns 1
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Table 1. Spherisorb Column Physical Characteristics
Chemistry
Silica Spherical 3, 5 and 10 80 220 0.50 n/a n/a
ODS2 (C
ODS1 (C
ODSB (C
C
C
C
Nitrile (CN) Spherical 3, 5 and 10 80 220 0.50 3.1 no
Amino (NH
Phenyl Spherical 3, 5 and 10 80 220 0.50 2.5 no
OD/CN (Mixed Mode) Spherical 5 80 220 0.50 5.0 yes
SAX Spherical 3, 5 and 10 80 220 0.50 4.0 no
SCX Spherical 3, 5 and 10 80 220 0.50 4.0 no
) - Fully End Capped Spherical 3, 5 and 10 80 220 0.50 11.5 yes
18
) - Partially End Capped Spherical 3, 5 and 10 80 220 0.50 6.2 no
18
) - Base De-activated Spherical 5 80 220 0.50 11.5 yes*
18
8
6
1
) Spherical 3, 5 and 10 80 220 0.50 1.9 no
2
Particle
Shape
Spherical 3, 5 and 10 80 220 0.50 5.8 yes
Spherical 3, 5 and 10 80 220 0.50 4.7 yes
Spherical 3, 5 and 10 80 220 0.50 2.2 no
Particle Size
(µm)
Pore Size
(Å)
Surface Area
2
(m
/g)
Pore Volume
(cc/g)
% Carbon
Load
Endcapped
* polar endcapping
i. GettinG started
Each Spherisorb column comes with a Performance Test
Chromatogram. This Performance Test Chromatogram is specific
to each individual column and contains the following information:
gel batch number, column serial number, USP plate count, USP
tailing factor, capacity factor, and chromatographic conditions.
The performance test chromatogram should be stored for future
reference.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical
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
Spherisorb column being installed. See “Scaling Up/Down Isocratic
Separations” 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. Reversed-Phase Columns
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.
2. Normal-Phase Columns
Note: It is assumed that your system has been used for reversed-phase
chromatography. If this is not the case, you can start with step 3.
1. Purge the pumping system of any buffer containing mobile phases.
2. Flush the system thoroughly with acetonitrile.
3. Switch the system over to the mobile phase that you are planning to use
in normal-phase chromatography.
1. Purge the pumping system of any buffer-containing mobile phases and
connect the inlet end of the column to the injector outlet.
2. Flush column with 100% organic mobile phase (methanol or
Waters Spherisorb Columns 2
4. Connect the column and equilibrate it with the mobile phase.
Note: Equilibration with the mobile phase may require a larger amount of
solvent than in reversed-phase chromatography.
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b. Column Equilibration
Spherisorb columns are shipped in test mobile phase. 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 2 for
a listing of empty column volumes).
1. Reversed-Phase Columns
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.)
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.
2. Normal-Phase (Spherisorb Silica, Amino, Cyano) Column
Spherisorb normal-phase (NP) columns are delivered in 96% heptane /
4% isopropyl alcohol. Care should be taken not to pass any mobile
phase through the column that might cause a precipitate. Spherisorb
NP columns are compatible with water and all common organic
solvents, provided that solvent miscibility is accounted for.
Equilibrate normal-phase silica columns in the mobile phase. Very
small quantities of water in the mobile phase can dramatically affect
the activity of normal-phase packings. For good reproducibility,
ensure that the mobile phase always has the same water content.
It is difficult and usually unnecessary to completely eliminate the
water from the mobile phase. Dry mobile phases can take a very long
time to equilibrate the column. A water content of 50 percent of
saturation is recommended for most applications.
To equilibrate your column:
1. Starting at 0.0 mL/min, increase the flow rate in 0.1 mL/min
increments to 1.0 minutes.
2. Purge the column with the mobile phase until you obtain a stable
baseline.
3. Verify that retention times and peak areas for a standard are stable
by comparing 2-3 replicate consecutive injections
Before you perform the first analysis on your new column, perform
an efficiency test to confirm the performance of the column.
Table 2. Empty Column Volumes in mL
(multiply by 10 for flush solvent volumes)
Column
Length
20 mm - 0.07 0.14 0.33 - -
30 mm - 0.1 0.2 0.5 - -
50 mm 0.1 0.2 0.3 0.8 - -
100 mm 0.1 0.4 0.7 1.7 - -
150 mm 0.1 0.5 1.0 2.5 12 24
250 mm - 0.9 1.8 4 20 40
Column Internal Diameter (mm)
1.0 2.1 3.0 4.6 10 20
c. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it. Waters
recommends using a suitable solute mixture, as found in the
“Performance Test Chromatogram”, to analyze the column upon
receipt. However, if the column is used only for a single routine
assay, it may be more convenient to test the column under these
assay conditions. Keep a record of the initial column performance.
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.
Waters Spherisorb Columns 3
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ii. column use
To ensure the continued high performance of Spherisorb columns,
follow these guidelines:
a. Guard Columns
Use a Waters Spherisorb guard column of matching chemistry and
particle size between the injector and main column. It is important to
use a 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 or other
changes in chromatographic performance 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 Oasis
®
columns or Sep-Pak
up the sample before analysis. Link to www.waters.com/sampleprep
cartridges of the appropriate chemistry to clean
solid-phase extraction cartridges/
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. Filter sample with 0.2 μm
membranes to remove particulates. If the sample is dissolved in a
solvent that contains an organic modifier (e.g., acetonitrile, methanol,
etc.) ensure that the membrane material does not dissolve in the
solvent. Contact the membrane 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. pH Range
The recommended operating pH range for Spherisorb columns is 2
to 8. A listing of commonly used buffers and additives is given in
Table 3. Additionally, the column lifetime will vary depending upon
the operating temperature, and the type and concentration of buffer
used. For example, the use of phosphate buffer at pH 8 in combina-
tion with elevated temperatures will lead to shorter column lifetimes.
2. It is preferable to prepare the sample in the operating mobile phase
or a mobile phase that is weaker (less organic modifier in the case
of reversed-phase chromatography, less polar modifier in the case
of normal-phase chromatography or hydrophilic-interaction chro-
matography, less salt in the case of ion exchange) than the mobile
phase for the best peak shape and sensitivity.
Waters Spherisorb Columns 4
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Table 3: Buffer Recommendations for Using Spherisorb Columns from pH 2 to 8
Buffer
Additive or Buffer pK
Formic Acid 3.75 Volatile Yes
Acetic Acid 4.76 Volatile Yes
Ammonmium Formate
(NH
COOH)
4
Trifluoroacetic Acid
(TFA)
Ammonium Acetate
(CH
COONH4)
3
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
3.75 2.75-4.75 Volatile Yes
0.3 Volatile Low Conc.
4.76 3.76-5.76 Volatile Yes
a
Range
(± pH unit)
Volatility
d. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior
®
to use. Pall Gelman Laboratory Acrodisc
(Please refer to the filtration section of the Waters Chromatography
Columns and Supplies Catalog or the Waters web site (www.waters.com)
filters are recommended.
Used for
Mass Spec?
Comments
Maximum buffering obtained when used with ammonium formate salt. Used in
0.1-1.0% range.
Maximum buffering obtained when used with ammonium acetate salt. Used in
0.1-1.0% range.
Used in the 1-10 mM range for LC/MS. Higher concentrations (typically 20 mM)
are recommended for UV applications.
Note: sodium or potassium salts are not volatile.
When used in LC/MS, due at signal suppression, it is generally recommended to
use TFA at coencentrations <0.1%.
Used in the 1-10 mM range for LC/MS. Higher concentrations (typically 20 mM)
are recommended for UV applications.
Note: sodium or potassium salts are not volatile.
Above pH 7, reduce temperature/concentration and use guard column to maxmize
lifetime.
f. Temperature
Temperatures between 20 – 45 ˚C are recommended for operating
Spherisorb columns in order to enhance selectivity, lower
solvent viscosity and increase mass transfer rates. However, any
temperature above ambient may have a negative effect on lifetime
which will vary depending on the pH and buffer conditions used.
for additional product information.) Solvents containing suspended
particulate materials will generally clog the outside surface of
iii. scalinG up/doWn isocratic methods
the inlet distribution frit of the column. This will result in higher
operating pressure and poorer performance. Degas all solvents
thoroughly before use to prevent bubble formation in the pump and
detector. The use of an on-line degassing unit is also recommended.
This is especially important when running low pressure gradients
since bubble formation can occur as a result of aqueous and organic
solvent mixing during the gradient.
e. Pressure
Spherisorb columns can tolerate pressures of up to 6,000 psi (400 bar
or 40 MPa) although long-term, routine operating pressures greater
than 4,000 – 5,000 psi should be avoided in order to maximize column
and system lifetimes.
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:
= F1(r2/r1)2
F
2
or
Injection volume
= Injection volume2 (r2/r1)2(L2/L1)
1
Where: r = radius of the column, in mm
F = flow rate, in mL/min
L = length of column, in mm
1 = original, or reference column
2 = new column
Waters Spherisorb Columns 5
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iv. troubleshootinG
Changes in retention time, resolution, or backpressure are often due
to column contamination. See “Column Cleaning, Regenerating and
Storage”. Information on column troubleshooting problems may be
found in the current Waters Chromatography Columns and Supplies
Catalog. You can also download a copy of the HPLC Troubleshooting
Guide at www.waters.com, and in the Search Box, enter WA20769.
v. column cleaninG, reGeneratinG and storaGe
a. Cleaning and Regenerating
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. Changing the guard column being used
will often restore column performance. If not (or if no guard column
is being used), follow the procedures detailed below. To prevent
potential contamination from affecting detector performance, it is
recommended that any detector(s) be disconnected from the effluent
flow of the column during cleaning. Reversing the direction of the
flow through the column (backflushing) may sometimes improve the
effectiveness of any cleaning procedure.
1. Reversed-Phase Columns
Flushing with a neat organic solvent, taking care not to precipitate
buffers, is usually sufficient to remove most 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 4). Flush
columns with 20-column volumes of HPLC-grade solvents (e.g.,
80 mL total for 4.6 x 250 mm column). Increasing mobile-phase
temperature to 35-55 ˚C increases cleaning efficiency. If the column
performance is poor after regenerating and cleaning, call your local
Waters office for additional support.
Table 4: Column Sequence or Options
Polar Samples Non-polar Samples Proteinaceous Samples
1. Isopropanol (or an
1. Water
2. Methanol 2. Tetrahydrofuran (THF)
3. Tetrahydrofuran 3. Dichloromethane
4. Methanol 4. Hexane
5. Water
6. Mobile Phase 6. Mobile Phase
* Use low organic solvent content to avoid precipitating buffers.
appropriate isopropanol/
water mixture*)
5. Isopropanol (followed by
an appropriate isopropanol/
water mixture*)
Option 1: Inject repeated
aliquots of dimethyl
sulfoxide (DMSO)
Option 2: gradient of
10-90% B where:
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
2. Normal-Phase Columns
To regenerate, pump 20-30 column volumes each of
dichloromethane and isopropanol through the column. Other wash
solvents such as tetrahydrofurane (THF) may also be selected based
on the suspected contamination.
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. Storage
Completely seal the column to avoid evaporation and drying out of
the bed.
1. Reversed-Phase Columns
For periods longer than four days at room temperature, store the
column (with the exception of cyano chemistry columns) in 100%
acetonitrile at room temperature. For elevated temperature applica-
tions, store immediately after use in 100% acetonitrile for the best
column lifetime. Do not store columns in buffered eluents. If the
mobile phase contained a buffer salt, flush the column with 10 col-
umn volumes of HPLC grade water (see Table 2 for common column
volumes) and replace with 100% acetonitrile for storage. Failure to
perform this intermediate step could result in precipitation of the
Waters Spherisorb Columns 6
[ Care and Use ManUal ]
buffer salt in the column when 100% acetonitrile is introduced.
Note: The exception of the storage recommendations above are cyano (CN)
columns used for reversed-phase methods (CN-RP). These columns should never
be stored in 100% intermediate polarity solvents (acetonitrile, methanol, IPA).
Store CN-RP columns in 0.1 M ammonium acetate/acetonitrile 50/50.
2. Normal-Phase Columns
For rapid equilibration upon start-up, store your normal-phase
column in the mobile phase that is commonly used. Completely seal
column to avoid evaporation and drying out of the bed.
vi. connectinG the column to the hplc
a. Column Connectors and System Tubing Considerations
All Spherisorb column and cartridges have Parker style endfittings.
Tools needed for Spherisorb analytical column:
1/2 inch wrench
9/16 inch wrench
Tools needed for Spherisorb analytical cartridge column:
1/4 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.
column endfitting, with no void between them. It is important to realize
that extra column peak broadening can destroy a successful separation.
The choice of appropriate column connectors and system tubing is
discussed in detail below. Due to the absence of an industry standard,
various column manufacturers have employed different types of
chromatographic column connectors. The chromatographic performance
of the 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
Spherisorb column is equipped with Parker style endfittings that require
a 0.090 inch ferrule. If a Waters style column is presently being used,
it is critical that the ferrule depth be reset for optimal performance prior
to installing a Spherisorb column.
0.130 inches
Waters Ferrule Settings Parker Style Ferrule Settings
Figure 1: Waters and Parker Style Ferrule Types
0.090 inches
1. Correct connection of 1/16 inch outer diameter stainless steel tubing
leading to and from the column is essential for high quality chromato-
graphic 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 1/2 inch
wrench on the hex head of the column endfitting.
Note: If one of the wrenches is improperly placed on the inner column hex head (or the
cartridge 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 direc-
tion 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. Tubing touc hes the bottom of the
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 perfor-
mance. This can occur if a Parker style ferrule is connected to a
Waters endfitting (Figure 3).
Waters Spherisorb Columns 7
[ Care and Use ManUal ]
Void
Figure 3: Parker Ferrule in a Waters Style Endfitting
Figure 5: Single and Double SLIPFREE Connectors
Note: A void appears if tubing with a Parker style ferrule is connected to a Waters
style column.
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).
Gap
Figure 4: Waters Ferrule in a Parker Style Endfitting
Note: The connection leaks if a Waters ferrule is connected to a column with a
Parker style endfitting.
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.
SLIPFREE Connectors Features
• Tubing pushed into endfitting, thereby guaranteeing a void-free
connection
• Connector(s) come(s) installed on tubing
• Various tubing i.d. 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
SLIPFREE Type and
Tubing Length
Single 6 cm PSL 618000 PSL 618006 PSL 618012
Single10 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
0.005” 0.010” 0.020”
Tubing Internal Diameter
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. (Note that PEEK fittings are not
recommended for normal-phase applications!) Another approach is to
®
use a Thermo Corporation 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).
Waters Spherisorb Columns 8
[ Care and Use ManUal ]
Diluted/Distorted Sample Band
0.005 inches
0.020 inches
0.040 inches
AU
82
b. 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
c. 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 P/N 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-sig ma Bandspre ading (μL) = Pea k Width (min) x Flow Rate (mL/min) x (1000 μL /1 mL)
System Varianc e (μL2) = (5-sigma bandspreading)2/ 25
System Volume
Figure 8: Determination of System Bandspreading Volume Using
5-Sigma Method
In a typical HPLC system, the bandspreading volume should be 100 μL
2
± 30 μL (or Variance of 400 μL
+/- 36 μL2). In a microbore (2.1 mm
i.d.) system, the bandspreading volume should be no greater than 20
2
to 40 μL (or variance no greater than 16 μL
to 64 μL2).
d. Measuring System Volume
System volume is important in scaling separations because it
creates an isocratic hold at the start of every run. This hold is often
several column volumes on a small scale, but a fraction of the
volume of a preparative column. Compensation for this volume must
be included in planning a scaling experiment to avoid distorting the
chromatography (Figure 9).
Programmed time = 5 min
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
50%
02468101214161
Figure 9: Determination of Gradient Delay Volume
Flow Rate = 1.5 mL/min
5.69 min
-5.00 min
0.69 min
Time = 5.69 minutes
System Volume:
0.69 min x 1.5 mL/min = 1.04 mL
0 Min.
Waters Spherisorb Columns 9
[ Care and Use ManUal ]
1. Remove column.
2. Use acetonitrile as mobile phase A, and acetonitrile with 0.05 mg/mL
uracil as mobile phase B (eliminates non-additive mixing and viscosity
problems).
3. Set UV detector at 254 nm.
4. Use the flow rate in the original method and the intended flow rate on
the target instrument.
5. Collect 100% A baseline for 5 minutes.
6. Program a step change at 5 minutes to 100% B, and collect data for an
additional 5 minutes.
7. Measure absorbance difference between 100% A and 100% B.
8. Measure time at 50% of that absorbance difference.
9. Calculate time difference between start of step and 50% point.
10. Multiply time difference by flow rate.
vii. additional inFormation
a. Use of Narrow-bore (<3.0 mm i.d.) Columns
This section describes how to minimize extra column effects and
provides guidelines on maximizing the performance of a narrowbore
column in an HPLC system. A 3.0 mm i.d. narrow-bore column
usually requires no system modifications. A 2.1 mm i.d. column,
however, 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.
System with 130 L
bandspreading: 8,000 plates
7 8 min7.5 7 8 min7.5
Non-optimized LC/MS/MS SystemOptimized System
Figure 10: Non-Optimized vs. Optimized LC/MS/MS System
System with 70 L bandspreading:
10,000 plates (same column)
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 between pump and injector.
4. 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.
5. Use perfect (pre-cut) connections (with a variable depth inlet if using
columns from different suppliers).
6. Detector time constants should be shortened to less than 0.2 seconds.
b. Impact of Bandspreading Volume on 2.1 mm i.d.
Column Performance
Note: Flow splitters after the column will introduce additional bandspreading.
System optimization, especially in a system that contains a flow
splitter, can have dramatic effects on sensitivity and resolution.
Optimization includes using correct-depth ferrules and minimizing
tubing diameter and lengths. An example is given in Figure 10
where system optimization resulted in a doubling of sensitivity and
resolution of the metabolite in an LC/MS/MS system.
Waters Spherisorb Columns 10
[ Care and Use ManUal ]
d. Guard Cartridges and Columns Assembly
These endfittings and accessories can be used with 4.6 mm, 4.0 mm, or
3.0 mm i.d. cartridges.
Description Qty. Part No.
Removable Column Endfitting 2/pk PSS614100
Frit Assembly (2 µm) 5/pk PSS614103
Frit Assembly (0.5 µm) 5/pk PSS614104
Column Coupler 2/pk PSS614102
Long Tail Endfitting 2/pk PSS614101
Extended Endfitting for use with 10 mm Integral Guard 1/pk PSS614108
Nylon Column Plugs for Storage of Complete Column 1/pk WAT015674
Nylon Column Caps for Storage of
Replacement Cartridge Column
Inline 10 mm Guard Cartridge Holder Kit
for use with above items
2
1
10/pk PSS614113
PSS830008
3
4
Inline Guard Cartridge Holder
10 mm Guard Cartridge (4.6 mm i.d.)
Inline Guard Cartridge Holder
PSS 830008
Extended Endfitting for use with 10 mm Guard Cartridges
10 mm Guard Cartridge (4.6 mm i.d.)
Cartridge
®
Extended Endfitting
for use with 10 mm Guard
PSS 614108
Waters Spherisorb
30 mm Guard Cartridge Coupled to Spherisorb Cartridge
1. 30 mm Stand Alone Guard/Column (endfittings not included)
2. Extended endfitting for use with 10 mm Integral Guard: PSS614108
3. 10 mm Integral Guard Column
4. Column Coupler: PSS614102
Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07, Australia 61 2 9933 1777, Belgium 32 2 726 1000, Brazil 55 11 4134 3788,
Canada 1 800 252 4752, China 86 21 6156 2666, Czech Republic 420 2 617 11384, Denmark 45 46 59 8080, Finland 358 9 5659 6288, France 33 1 30 48 72 00,
Germany 49 6196 400 600, Hong Kong 852 2964 1800, Hungary 36 1 350 5086, India and India Subcontinent 91 80 2837 1900, Ireland 353 1 448 1500, Italy 39 02 265 0983,
Japan 81 3 3471 7191, Korea 82 2 6300 4800, Mexico 52 55 52 00 1860, The Netherlands 31 76 508 7200, Norway 47 6 384 6050, Poland 48 22 101 5900, Puerto Rico 1 787 747 8445,
Russia/CIS 7 495 727 4490/ 290 9737, Singapore 65 6593 7100, Spain 34 93 600 9300, Sweden 46 8 555 115 00, Switzerland 41 56 676 7000, Taiwan 886 2 2501 9928,
United Kingdom 44 208 238 6100, All other countries: Waters Corporation U.S.A. 1 508 478 2000/1 800 252 4752
©2012 Waters Corporation. Waters, Spherisorb, Oasis and SepPak
are registered trademarks of Waters Corporation. The Science of
What’s Possible is a trademark of Waters Corporation. Acrodisc is
a trademark of Pall Corporation. PEEK is a trademark of Agilent
Technologies. SLIPF REE is a trademark of Thermo Corporation.
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
July 2012 WAT094178 Rev B V W-PDF
www.waters.com
Waters Spherisorb Columns 11
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