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symmetry columns
i. introduction
Thank you for choosing a Waters Symmetry® c o l u m n . S y m m e t r y
c o l u m n s c o n t i n u e t o s e t t h e s t a n d a r d o f p e r f o r m a n c e f o r
reproducibility. As today’s chemists establish new analytical methods
for the latest pharmaceutical and biopharmaceutical products, the
selection of a reproducible HPLC column is essential. The selected
column needs to provide the same chromatographic results over the
life of the new drug product. The excellent reproducibility of Symmetry, SymmetryShield™ and Symmetry300™ columns is a result
of our commitment to maintaining the tightest specifications in the
HPLC column industry.
Physical C haracteristics
Packing Chemistry Particle Size Particle
Shape
Symmetry C
SymmetryPrep C
SymmetryShield R P18
RP8
Symmetry300 C
C
C
C
18
8
18
8
18
4
3.5, 5 µm
3.5, 5 µm
7 µm
7 µm
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5 µm
3.5, 5 µm
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore
Size
100 Å
100 Å
100 Å
100 Å
100 Å
100 Å
300Å
300Å
Carbon
Load
19%
12 %
19%
12 %
17%
15%
8.5%
2.8%
End-
capped
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
contents
i. introduction
ii. GettinG started
a. Column Installation
b. Column Equilibration
c. Initial Column Efficiency Determination
iii. column use
a. Guard Columns
b. Sample Preparation
c. pH Range
d. Solvents
e. Pressure
f. Temperature
iV. scalinG up/down isocratic methods
V. troubleshootinG
Vi. column cleaninG, reGeneratinG and storaGe
a. Cleaning and Regenerating
b. Storage
Symmetry Columns 1
Vii. connectinG the column to the hplc
a. Column Connectors and System Tubing Considerations
b. Band Spreading Minimization
c. Cartridge Installation
d. Measuring System Bandspreading Volume & System Variance
e. Measuring System Volume
Viii. 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. Waters Small Particle Size (3.5 μm) Columns – Fast Chromatography
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ii. GettinG started
E a c h S y m m e t r y c o l u m n c o m e s w i t h C e r t i f i c a t e o f A n a l y s i s a n d a
Performance Test Chromatogram. The Certificate of Analysis is
specific to each batch of packing material contained in the Symmetry
column and includes the gel 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: gel batch
number, column serial number, USP plate count, USP tailing factor,
capacity 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
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
Symmetry 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. 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
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.
b. Column Equilibration
Symmetry 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
m o b i l e p h a s e . ( F o r e x a m p l e , f l u s h t h e c o l u m n a n d H P L C s y s t e m
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. In addition, mobile phases that
contain formate (e.g., ammonium formate, formic acid, etc.) may also
require longer initial column equilibration times.
c. Initial Column Efficiency Determination
1 . P e r f o r m a n e f f i c i e n c y t e s t o n t h e c o l u m n b e f o r e u s i n g i t .
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
o b t a i n e d o n t w o d i f f e r e n t H P L C s y s t e m s d u e t o t h e q u a l i t y o f
the connections, operating environment, system electronics,
reagent quality, column condition and operator technique.
Table 1. Empty Column Volumes in mL (multiply by 10 for flush
solvent volumes)
Column
Length
20 mm – 0.07 0.1 4 0.33 – – –
30 mm – 0 .1 0.2 0.5 – 8 –
50 mm 0 .1 0.2 0.3 0.8 2.4 14 35
100 mm 0 .1 0.4 0.7 1.7 5 28 70
150 mm 0 .1 0.5 1.0 2.5 7 42 –
250 mm – 0.9 1.8 4 – 70 –
1.0 2 .1 3.0 4.6 7. 8 19 30
Column internal diameter (mm)
Symmetry Columns 2
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iii. column use
To ensure the continued high performance of Symmetry 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 Oasis
solid-phase extraction cartridges/columns or Sep-Pak
cartridges of the appropriate chemistry to clean up the sample
before analysis. Link to www. waters.com/sampleprep
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
i s d i s s o l v e d i n a s o l v e n t t h a t c o n t a i n s a n o r g a n i c m o d i f i e r
(e.g., acetonitrile, methanol, etc.) ensure that the membrane
material does not dissolve in the solvent. Contact the
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.
m e m b r a n e m a n u f a c t u r e r w i t h s o l v e n t c o m p a t i b i l i t y q u e s t i o n s .
Alternatively, centrifugation for 20 minutes at 8,000 rpm,
f o l l o w e d b y t h e t r a n s f e r o f t h e s u p e r n a t a n t l i q u i d t o a n
appropriate vial, could be considered.
c. pH Range
The recommended operating pH range for Symmetry columns is 2 to 8. 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. For example,
the use of phosphate buffer at pH 8 in combination with elevated temperatures will lead to shorter column lifetimes.
Table 2: Buffer Recommendations for Using Symmetry Columns from pH 2 to 8
Additive or Buffer pK
a
Buffer range
(±1 pH unit)
Volatility Used for Mass Spec? Comments
Formic Acid 3.75 Volatile Yes Maximum buffering obtained when used with Ammonium Formate salt.
Used in 0.1-1.0% range.
Acetic Acid 4.76 Volatile Yes Maximum buffering obtained when used with Ammonium Acetate salt.
Used in 0.1-1.0% range.
Ammonium Formate
COOH)
(NH
4
Trifluoroacetic Acid (TFA) 0.3 Volatile Low conc. When used in LC/MS, due to signal suppression, it is generally recommended to
Ammonium Acetate
COOH)
(NH
4CH2
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 guard column to
Symmetry Columns 3
3.75 2.75 – 4.75 Volatile Yes Used in the 1-10 mM range.
Note: sodium or potassium salts are not volatile.
use TFA at concentrations < 0.1%
4.76 3.76 – 5.76 Volatile Yes Used in the 1-10 mM range.
Note: sodium or potassium salts are not volatile.
maximize lifetime.
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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 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
S y m m e t r y c o l u m n s c a n t o l e r a t e p r e s s u r e s o f u p t o 6 , 0 0 0 p s i
( 4 0 0 b a r o r 4 0 M p a ) a l t h o u g h p r e s s u r e s g r e a t e r t h a n
4, 00 0 – 5 ,0 00 p si s ho uld b e avoi de d in or de r t o ma xi miz e
column and system lifetimes.
f. Temperature
Temperatures between 20 ˚C – 45 ˚C are recommended for operating
Symmetry 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.
iV. 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
or
Injection volume
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
2
= Injection volume2 (r2/r1)2(L2/L1)
1
V. 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, click on “Literature Library”, then in the
Information Center Search Box, enter WA20769.
Vi. 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. 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
t h e p r o b l e m , p u r g e t h e c o l u m n u s i n g t h e f o l l o w i n g c l e a n i n g a n d
regeneration procedures. Use the cleaning routine that matches
t h e p r o p e r t i e s o f t h e s a m p l e s a n d / o r w h a t y o u b e l i e v e i s
contaminating the column (see Table 3). Flush columns with 20
c o l u m n v o l u m e s o f H P L C - g r a d e s o l v e n t s ( e . g . , 8 0 m L t o t a l f o r
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 3: Column Sequence or Options
Polar Samples Non-polar Samples Proteinaceous Samples
1. Water 1. Isopropanol (or an
appropriate isopropanol/
water mixture*)
2. Methanol 2. Tetrahydrofuran (THF) Option 2: gradient of
3. Tetrahydrofuran
(THF)
4. Methanol 4. Hexane
5. Water 5. Isopropanol (followed
6. Mobile Phase 6. Mobile Phase
3. Dichloromethane
by an appropriate isopropanol/water mixture*)
* Use low organic solvent content to avoid precipitating buffers.
Option 1: Inject repeated
aliquots of dimethyl
sulfoxide (DMSO)
10% to 90% B where:
A = 0 . 1 % t r i f l u o r o a c e t i c a c i d
( T F A) i n w a te r
B = 0 . 1 % t r i f l u o r o a c e t i c a c i d
(TFA) in acetonitrile (CH
Option 3: Flush column with
7M guanidine hydrochloride,
or 7M urea
CN)
3
Symmetry Columns 4
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0.130 inches
0.0 0 inches
Pa rker Style Fe rrule Settings
Wa ters Fe rrule Settings
G ua r d c ol u mn s n e ed to b e r e pl a c ed at r e gu l ar in te r v al s , a s
determined by sample contamination. When system backpressure
s t e a d i l y i n c r e a s e s a b o v e a s e t p r e s s u r e l i m i t , i t i s u s u a l l y a n
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
For periods longer than four days at room temperature, store the
column in 100% acetonitrile. For elevated temperature applications,
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 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
b u f f e r s a l t i n t h e c o l u m n w h e n 1 0 0 % a c e t o n i t r i l e i s i n t r o d u c e d .
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.
Vii. connectinG the column to the hplc
a . C o l u m n C o n n e c t o r s a n d S y s t e m T u b i n g C o n s i d e r a t i o n s
To o l s ne e d e d :
3 / 8 i n c h w r e n c h
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.
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. Tubing touches the bottom of the 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.
Du e to t he absence of a n industry sta ndard , va rious 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 Symmetry column is equipped with Waters style endfittings
t h a t r e q u i r e a 0 .1 3 0 i n c h f e r r u l e . I f a n o n - W a t e r s s t y l e c o l u m n i s
presently being used, it is critical that ferrule depth be reset for
optimal performance prior to installing a Symmetry column.
Figure 1: Waters and Parker Style Ferrule Types
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,
i t i s i m p o r t a n t t o e n s u r e p r o p e r f i t o f t h e 1 / 16 i n c h o u t e r
diameter stainless steel tubing. When tightening or loosening
Symmetry Columns 5
[ Care and Use ManUal ]
Void
Gap
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 style ferrule is connected to a
Waters endfitting (Figure 3).
Figure 3: Parker Ferrule in a Waters Style Endfitting
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).
Figure 4: Waters Ferrule in 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.
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 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).
Figure 5: Single and Double SLIPFREE Connectors
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
Note: The connection leaks if a Waters ferrule is connected to a column
with a Parker style endfitting.
Symmetry Columns 6
• Unique design separates tube-holding function from sealing
function
Table 4. Waters Part Numbers for SLIPFREE Connectors
SLIPFREE Type and
Tubing Length
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
0.005˝ 0.010˝ 0.020˝
Tubing Internal Diameter
[ Care and Use ManUal ]
Diluted/Distorted Sample Band
0.005 inches
0.020 inches
0.040 inches
0.25 mm (0.009 inch) Tubing
Spacer
C-Clip
Symmetry™ Cartridge Column
Sentry™ Guard Column
End Connector
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. Cartridge Installation
Refer to the installation illustration in Figure 7. Unscrew the end connectors from the old cartridge column, leaving them connected to the inlet
and outlet lines of the instrument.
Figure 7: Installing a Symmetry Cartridge Column with a Sentry Guard Column
Attach the new cartridge column between the connectors so that the direction of the flow arrow on the label is pointing towards the detector.
Finger-tighten all fittings.
Symmetry Columns 7
[ Care and Use ManUal ]
5.69 minutes
- 5.00 minutes
0.69 minutes
50%
Ti me = 5.69 minutes
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0 2 4 6 8 10 12 14 16 18 20 min
Progra mmed time = 5.00 minutes
Sys tem Vo lume:
0.69 min x 1.5 mL/m in = 1.04 mL
Flow Rate = 1.5 mL/m in
AU
System Volume
d. 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 . D i lu t e a t e st m i x i n mo b il e p h a se to gi ve a d et e ct o r
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-sigma Bandspreading (μL) = Peak Width (min) Flow Rate (mL/min x (1000 μL/1 mL)
System Variance (μL2) = (5-sigma bandspreading)2/ 25
Figure 8: Determination of System Bandspreading Volume Using
5-Sigma Method
e. 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 prep column. Compensation for this volume must be
i n c l u d e d i n p l a n n i n g a s c al i n g e x p e r i m e n t t o a v o i d d i s t o r t i n g t h e
chromatography (Figure 9).
Figure 9: Determination of Gradient Delay Volume
1. Remove column.
In a typical HPLC system, the Bandspreading Volume should be
100 μL ± 30 μL (or Variance of 400 μL
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
2
to 64 μL2).
16 μL
Symmetry Columns 8
2
+/- 36 μL2).
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.
[ Care and Use ManUal ]
7.00 7.50
Non-optimized LC/MS/MS System Optimized System
8.00
7.00 7.50 8.00
7.00 7.50 8.00
Viii. 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.
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.
Figure 10: Non-Optimized vs. Optimized LC/MS/MS System
Sys tem w ith 130 µL
bandspreading: 8,000 plates
System with 70 µL 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.
d. Waters Small Particle Size (3.5 μm) Columns –
Fast Chromatography
Waters columns that contain 3.5 μm particles provide faster and
more efficient separations without sacrificing column lifetime. This
section describes five parameters to consider when performing
separations with columns containing 3.5 μm particles.
Note: Columns that contain 3.5 μm particles have smaller outlet frits
to retain packing material. These columns should not be backflushed.
Symmetry Columns 9
[ Care and Use ManUal ]
Sales Offices:
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Belgium 32 2 726 1000
Brazil 55 11 5094 3788
Canada 800 252 4752
China 8621 6495 6999
CIS/Russia +7 495 3367000
Czech Republic 42 02 617 11384
Denmark 45 46 59 8080
Finland +358 9 5659 6288
France (33) 1 30 48 72 00
Germany 49 6196 400600
Hong Kong 852 29 64 1800
Hungary 36 1 350 5086
India and India Subcontinent
91 80 2 837 1900
Ireland 353 1 448 1500
Italy 02 265 0983
Japan (81) 3 3471 7191
Korea (82) 2 820 2700
Mexico 5255 5200 1860
The Netherlands +31 (0)76-50 87 200
Norway 47 63 84 60 50
Poland (48) 22 833 4400
Puerto Rico 787 747 8445
Singapore 65 6273 1221
Spain 34 93 600 93 00
Sweden 46 8 555 11500
Switzerland 41 56 676 7000
Taiwan 886 2 2543 1898
United Kingdom 44 208 238 6100
©2008 Waters Corporation. Waters, The Science of W hat’s
Possible, Symmetry, SymmetryShield, Symmetry300, Oasis
and SepPak are trademarks of Waters Corporation. Acrodisc is
a trademark of Pall Corporation. PEEK is a trademark of Agilent
Technologies. SLIPFREE is a trademark of T hermo Cor poration.
July 2008 WAT047278 Rev 4 KK- PDF
Symmetry Columns 10
Waters Corporation
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Milford, MA 01757 U.S.A.
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