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VIRIDIs sFC CoLUMns
Contents
I. IntRoDUCtIon
II. GettInG stARteD
a. Safety Considerations
b. Column Installation
c. Column Equilibration
d. Initial Column Efficiency Determination
III. CoLUMn Use
a. Sample Preparation
b. Solvents
c. Pressure
IV. sCALInG UP/DoWn IsoCRAtIC MetHoDs
V. ConneCtInG tHe CoLUMn to tHe sFC sYsteM
I. IntRoDUCtIon
Thank you for choosing a Viridis® SFC column. The Viridis SFC packing
materials are designed to provide excellent stability, minimal column
bleed, and high mass loading. The Viridis SFC packing materials are
manufactured in an ISO 9000 certified plant using ultra-pure reagents.
Each batch of Viridis SFC material is tested and the results are held to
narrow specification ranges to assure excellent, reproducible performance.
Every column is individually tested and a Performance Chromatogram is
provided with each column along with the Certificate of Batch Analysis.
Table 1. Physical Characteristics
Viridis
SFC
Chemistry
2-Ethylpyridine
Silica Spherical 5 10 0 340 0.90 n/a n/a
Particle
Shape
Spherical 5 10 0 340 0.90 8 No
Particle
Size
(µm)
Pore
Size
(Å)
Surface
Area
(m2/g)
Pore
Volume
(cc /g)
Carbon
Load
(%)
End-
capped
a. Column Connectors and System Tubing Considerations
b. Band Spreading Minimization
VI. tRoUBLesHootInG
VII. CoLUMn CLeAnInG, ReGeneRAtInG AnD stoRAGe
a. Cleaning and Regeneration
b. Storage
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II. GettInG stARteD
Each Viridis SFC column comes with a Certificate of Analysis and a
Performance Test Chromatogram. The Certificate of Analysis is specific
to each batch of packing material and includes the gel batch number,
analysis of unbonded particle and analysis of bonded particles. The
Performance Test Chromatogram is specific to each individual column
and contains the gel batch number, column serial number, USP plate
count, USP tailing factor, capacity factor, and chromatographic condi-
tions under normal-phase LC conditions. These data should be stored
for future reference.
a. Safety Considerations
An SFC column while being used is, by design, under pressure. The
major safety concern for even a small leak with just CO
is frostbite.
2
Pay attention to any frosting on the column or system connections.
This indicates a leak, usually with temperatures far below 0 °C. Any
small leak with solvent introduced could produce a situation where
the LEL (lower exposure limit) is reached.
b. Column Installation
Note: Scale the flow rate up or down accordingly based upon the i.d.,
length, particle size, and backpressure of the Viridis SFC column being
installed. See “Scaling Up/Down Isocratic Methods” for calculating
flow rates when changing column i.d. and/or length. See “Connecting
the Column to the SFC system” for a more detailed discussion on SFC
connections.
c. Column Equilibration
Viridis SFC columns are shipped dry. Equilibrate the column with a mini-
mum of 10 column volumes of the mobile phase prior to use. (Refer to
Table 2 for a listing of empty column volumes.)
d. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it. Waters
recommends using a suitable solute mixture to analyze the column
upon receipt.
2. Determine the number of theoretical plates (N) and use this value
as a benchmark for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained on
two different SFC systems due to the quality of the connections,
operating environment, system electronics, reagent quality and
column condition.
Table 2. Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Column
Dimension
50 mm 0.8 4 14 35 98
100 mm 1.7 8 28 70 196
150 mm 2.5 12 42 106 294
250 mm 4 20 70 176 490
4.6 mm 10 mm 19 mm 30 mm 50 mm
Column Internal Diameter (mm)
1. Make sure your co-solvent pump is primed and has an adequate
solvent/modifier supply before performing injections.
2. Connect both the inlet and outlet of the column to the SFC system.
3. If the column is still filled with a solvent, use a low flow rate and
back pressure setting (100 bar) to start pumping CO
and modifier
2
through the column.
4. If you see frosting on the column at the inlet or outlet, tighten the
compression screw on that side. If you see frosting on the column,
turn off the CO
and vent the system. Allow the column to depres-
2
surize fully before disconnecting the inlet or outlet to troubleshoot
the leaking issue.
III. CoLUMn Use
To ensure the continued high performance of Viridis SFC columns,
follow these guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination.
®
Use Oasis
Pak
sample before analysis. Link to www.waters.com/sampleprep.
2. Prepare the sample in the SFC mobile-phase modifier (typically,
methanol) for the best peak shape and sensitivity.
2Viridis SFC Columns
solid-phase extraction cartridges/columns or Sep-
®
cartridges of the appropriate chemistry to clean up the
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3. If the sample is not dissolved in the mobile-phase modifier,
ensure that the sample, solvent and mobile phases are miscible
in order to avoid sample precipitation. Filter sample with 0.2 μm
membranes to remove particulates. If the sample is dissolved in
a solvent, ensure that the membrane material does not dissolve
in the solvent. Contact the membrane manufacturer with solvent
®
compatibility questions. Pall Gelman Laboratory Acrodisc
filters are recommended. (Refer to the Waters Chromatography
Columns and Supplies Catalog for additional information.)
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. Solvents
To maintain maximum column performance, use high quality chro-
matography grade solvents. Solvents containing suspended particulate
materials will generally clog the outside surface of the inlet distri-
bution frit of the column. This will result in higher operating pres-
sure, reduced column lifetime, and compromised performance.
c. Pressure
Table 3 may be helpful in scaling-up the flow rate to maintain sepa-
rations in larger column internal diameters.
Table 3. Flow Rate Scaling Chart
Column Diameter (mm)
4.6 10 19 30 50
2.5 11. 8 4 2.7 106.3 295.4
3 14 .2 51.2 127. 6 354.4
3.5 16 .5 59.7 148. 9 413. 5
4 18 .9 68.2 170.1 472.6
4.5 21.3 76.8 191.4 5 31.7
5 23.6 85.3 212.7 590.7
6 28.4 102.4 255.2 708.9
Flow rate (g/min)
7 3 3.1 119.4 297.7 8 27.0
8 37. 8 13 6.5 340.3 945.2
9 42.5 15 3.5 382.8 10 63.3
10 47.3 170.6 425.3 1181. 5
V. ConneCtInG tHe CoLUMn to tHe sFC sYsteM
Viridis SFC 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.
IV. sCALInG UP/DoWn IsoCRAtIC MetHoDs
The following formulas are used for scaling separations.
If column i.d. and length are altered:
F
= F1(r2/r1)
2
or
Injection volume
Where: r = Radius of the column (mm)
F = Flow rate (g/min)
L = Length of column (mm)
1 = Original, or reference column
2 = New column
2
= Injection volume2 (r2/r1)2 (L2/L1)
1
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 fit-
tings, it is important to ensure proper fit of the 1/16 inch
outer diameter stainless steel tubing. When tightening or loos-
ening 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.
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0.090 inches
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 tub-
ing 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. Extra column peak broadening
can destroy a successful separation. The choice of appropriate column
connectors and system tubing is discussed in detail below.
Figure 1: Waters and Parker Ferrule Types
0.130 inches
Waters Ferrule Settings
Parker Style Ferrule Settings
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 Viridis
SFC column is equipped with Waters style endfittings that require a
0.130 inch ferrule. 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 Viridis SFC column.
In a proper tubing/column connection (Figure 2), the tubing touches
the bottom of the column endfitting, with no void between them.
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 end-
fitting (Figure 3).
Figure 3: Parker Ferrule in a Waters Style Endfitting
Void
Note: A void appears if tubing with a Parker ferrule is connected to a
Waters style column.
To fix a void, 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
Gap
Note: The connection leaks if a Waters ferrule is connected to a column
with a Parker style endfitting.
There are two ways to fix a leak:
Figure 2: Proper Tubing/Column Connection
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.
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b. Band-Spreading Minimization
Figure 5 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 5: Effect of Connecting Tubing on System
0.005 inches
0.020 inches
0.040 inches
Diluted/Distorted Sample Band
VI. tRoUBLesHootInG
VII. CoLUMn CLeAnInG, ReGeneRAtInG 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 additives, is usually suffi-
cient to remove the contaminant. If the column performance is poor
after regenerating and cleaning, call your local Waters office for
additional support.
b. Storage
1. Ensure all additives are removed from the column by flushing
with an appropriate organic solvent. Allow the column to sta-
bilize at room temperature before pumping the desires single
solvent through the column for storage.
2. Do not store columns that might be under pressure or can
thermodynamically become pressurized.
1. One of the most common problems with regards to columns is
incorrect or non-priming of the co-solvent/modifier pump. If
no peaks are observed after an injection or unusually long
retention times occur, check the priming of the co-solvent/
modifier pump first.
2. If you see frosting on the column at the inlet or outlet, tighten
the compression screw on that side. If tightening doesn’t work,
depressurize the system and the column then replace the fitting
that is not sealing correctly.
3. If you see frosting on the column, turn off the CO
system. Allow the column to depressurize fully before discon-
necting the inlet or outlet. Please contact your Waters repre-
sentative for additional support.
4. Excessive flow rates could damage the internal frits and accel-
erate column failure. Try to keep the pressure drop (∆P) below
100 bar across the column.
and vent the
2
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Sales Offices
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(Central South Eastern Europe, CIS
and Middle East) 43 1 877 18 07
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91 80 2837 1900
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United Kingdom 44 208 238 6100
All other countries:
Waters Corporation U.S.A.
1 508 478 2000
1 800 252 4752
www.waters.com
©2011 Waters Corporation. Waters, The Science of W hat’s
Possible, Viridis, Oasis and SepPak are trademarks of Waters
Corporation. Acrodisc is a trademark of Pall Corporation.
May 2011 720003441EN Rev B KK-PDF
Viridis SFC Columns 6
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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