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WAters IC-PAk C M/d CoLuMn
I. IntroduCtIon
This manual describes procedures for using the Waters IC-Pak™ C M/D
Ion Chromatography separation of monovalent and divalent cations.
Please take a few moments to read this manual carefully. Follow its
recommendations to obtain reproducible chromatography and column
stability.
a. Waters IC-Pak C M/D Column Description
Waters IC-Pak C M/D column is designed for ion chromatography
applications. Use the M/D column to simultaneously analyze
monovalent and divalent cations isocratically. The column separates
and allows quantitation of Li
+
, Na+, NH
+
, +*, Mg
4
+
’ Ca+, Sr
2
+
, and Ba
2
+
2
cations at ppb levels.
b. Specifications
Column IC-Pak C M/D
Dimensions 3.9 x 150 mm
Particle size 5 µm
Capacity 1.5 ± 0.2 meq/g
Packing material Silica base coated with polybutadi-
ene/maleic acid copolymer
Shipped in 20/80 (v/v} acetonitrile/water
Contents
I. IntroduCtIon
a. Waters IC-Pak C M/D Column Description
b. Specifications
II. InstALLInG tHe CoLuMn
a. Preparing the Ion Exchange System
b. Installing the Column
c. Equilibrating the Column
III. PrePArInG MoBILe PHAse And sAMPLes
a. Preparing the Mobile Phase
b. Preparing the Sample Preparing
IV. usInG tHe CoLuMn
a. Chromatography Guidelines
b. Efficiency Testing
V. CAre & MAIntenAnCe
a. Storing the Column
b. Troubleshooting
VI. orderInG InForMAtIon
Waters IC-Pak C M/D Column 1
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Compression Screw or Nut
Ferrule
0.009-inch ID Tubing
(0.23 mm)
II. InstALLInG tHe CoLuMn
a. Preparing the Ion-Exchange System
To prepare the system, you need two 3/8-inch wrenches and a zero
dead-volume union. Before installing the column in the flow path:
1. Directly connect the injector to the detector by replacing the old column
with a zero dead-volume union.
2. Flush the lines and the detector of microparticulates and previous sol-
vents. Use fresh eluent at a flow rate of 1.0 mL/min. Flush the injector
loop if applicable.
3. Remove the union.
4. Install the column between the injector and detector (see Section II. b).
b. Installing the Column
Remove the end plugs from the column and save them for use when
you store the column. To install the column:
3. Slide the compression fitting, followed by the ferrule (large end of the
taper first) over the tube.
Note: Properly bottom the tubing in the fitting seat. Otherwise, dead
volume could result in sample bond spreading.
Figure 1: Ferrule and Compression Screw Assembly
c. Equilibrating the Column
1. Precondition new columns by flushing with 100% acetonitrile for 30
minutes at 0.5 mL/min.
1. Thread the inlet and outlet fittings into the column until finger-tight.
An arrow on the label indicates the direction of flow.
2. Use the wrenches to tighten the fittings 1 /4 to 1 /2 turn. Do not
overtighten. Overtightening damages the connection.
3. Prepare a new tubing-ferrule connection (Figure 1) when connecting a
new column or when removing a damaged compression screw or worn
ferrule.
Note: The tubing distance beyond the ferrule may differ for each
column type. Resize the tubing to the correct distance by replacing
the ferrule.
Preparing a New Connection
To prepare a new tubing-ferrule connection:
1. Scribe the circumference of the tubing at the desired break using either
a file with a cutting edge or a tube cutter.
2. Grasp the tubing on both sides of the scribe mark with cloth-covered
pliers (to prevent marring the tube surface), and gently work the tube
back and forth until it separates.
Note: Ensure that tubing end is straight, open, and free of burrs.
2. Flush the column with mobile phase for 5 minutes at 0.5 mL/min,
and them for 30 minutes at 1.0 mL/min before performing the first
separation.
III. PrePArInG MoBILe PHAse And sAMPLes
a. Preparing the Mobile Phase
Chemical requirements
The IC-Pak C M/D uses 0.1 mM EDTA/3.0 mM HNO3 as a mobile
phase. Store mobile phase at room temperature for up to one month.
• ACSReagentgradeorequivalentqualityfree-acidEDTA
• Qualitynitricacid
• Quality18megohmwater
• Plasticwaretoprepareandstoreallmobilephases,standards,and
samples
Waters IC-Pak C M/D Column 2
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Filtration
Before filtering the mobile phase, flush the filters with 100 mL of mobile
phase. Discard the filtered mobile phase. Filter and degas mobile phases
®
with a compatible Millex
of the column.
Use an in-line precolumn filter to remove particulates in the mobile phase
and contaminants which the 0.45 µm filter does not remove.
0.45 µ filter for optimum long-term performance
Glass Containers
Sodium leaching from glass viols causes artifacts when analyzing cations.
Use plastic containers for all solutions.
Organic Mobile Phases
Samples containing organic amines may exhibit hydrophobic interaction
between the mobile phase and packing. You may use a water-miscible
organic mobile phase, such as acetonitrile, as a modifier to reduce this.
Do not use methanol; over time it causes a significant decrease in cation
8
retention time. Pretreat the sample with a C1
remove hydrophobic compounds.
Sep-Pak® cartridge to
Changeover to Mobile Phases Containing Salts
Perform changeover between on organic solvent and water containing
salts gradually to avoid salt precipitation. Use 18 megohm water as the
intermediate solvent. Do not exceed salt concentrations of 0.1 M.
Preparing the Mobile Phase
b. Preparing the Sample Preparing
Sample cleanup with Sep-Pak cartridges prevents alteration of the column
chemistry by strongly adsorbing or precipitating sample components. Pass
the first few drops of sample to waste.
Filter prepared samples with a microporous filter to prevent excessive
pressure buildup due to particulate matter. Rinse the filter with 10 mL of
18 megohm water. Pass the first few drops of Sample through the filter
to waste.
IV. usInG tHe CoLuMn
a. Chromatography Guidelines
The life of a column is directly related to its care and use. Contamination
from samples and mobile phases and improper handling and storage
reduce column life.
Note: Before running the first analysis on the new column, perform the test
sample separation given in Section IV. b, Efficiency Testing.
Physical Limits
Maximum Pressure 27 MPa (27 atm or 4000 psi)
Flow Rote 1 m min
pH Range 2 to 7
Temperature:
Normal operation
Limits
25 °C
≤ 50 °C (do not freeze column)
1. Put800mLofMilli-Q™waterintoaone-litervolumetricflask.
2. Add 0.0292 g EDTA, free acid (reagent grade). Stir or place in ultra-
sonic bath for 15 minutes.
3. Add 189 µL Nitric Acid (Ultrex).
4. DilutethesolutiontooneliterwithMilli-Qwaterandmixthoroughly.
5. Filter any undissolved EDTA from the solution and degas with one
of the following Millipore membranes (refer to Section ??? for part
numbers):
Aqueous Replacement Filters (0.45 µm, 47 mm)
®
Durapore
Waters IC-Pak C M/D Column 3
Filters (0.22 µm, 47 mm)
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General considerations
• Filterallaqueousbuffers.Donotuseturbidorcloudybuffers.
• Donotreusealiquotstakenfromthesamplecontainer;disposeof
them.
• Protectthecolumnfromvibration,mechanicalshock,andrapid
changes in pressure, flow rate or mobile phase composition. Any
thermal, physical or chemical shock (such as changing mobile phases
rapidly) can cause a loss of efficiency.
• Whenusingwater,treatitwithawatersystemcapableofdelivering
18 megohm water. Neither deionized water nor HPLC-grade bottled
water are acceptable because they may contain organic compounds
which alter column selectivity.
• Highlyconcentratedsamples(greaterthan10ppmperion)can
overload the packing material, resulting in poor peak shape. Dilute
the sample before injection. W hen analyzing an unknown, start with a
1:100 dilution.
• DO NOT inject concentrated samples directly into the mobile phase.
Direct injection may cause precipitation of the salts in the sample.
Dissolve (or dilute) samples in an appropriate volume of the mobile
phase first. If you must use other solvents, watch for precipitation upon
injection into the eluent. Always filter samples before use.
Preparing Mobile Phase
The IC-Pak C M/D column uses 0.1 mM EDTA/3.0 mM HNO3 as a
mobile phase. See phase Section III. a, Preparing the Mobile Phase,
for procedures for mobile phase preparation.
Guidelines for the Calibration Standard
Standard concentrates are available from most major chemical
suppliers. Use reagent-grade or analytical standard-grade
solutions. Several anionic species can cause the precipitation of
alkali and alkaline earth metals. Consult solubility tables to avoid
these species. Avoid hygroscopic salts. Select the highest purity
salt available. Certain atomic absorption standards are made
from ammonium salts and must not be used to prepare calibration
standard.
Preparing Individual 1000 ppm Stock Standards
1. Weigh the specified amount of salt (refer to Table 1).
2. Add the salt to a plastic 1 liter volumetric flask.
3. Fill the flask to the mark with 18 megohm water.
4. Store the stock solutions in clean plasticware at room temperature for
up to 6 months.
Table 1: Salt Weight for Stock Solution Preparation
b. Efficiency Testing
Perform on efficiency test before attempting the first analysis.
Run the test sample using the calibration standards detailed in the
following pages. Record the retention time and the settings used
There are four parts to performing the efficiency test:
1. Preparing the mobile phase
2. Preparing the stock calibration standards
3. Running the calibration (working) standard
4. Calculating column efficiency
If you experience problems during normal operation, repeat the
conditions for the initial efficiency test and compare the results.
Differences may indicate a problem with the column.
Waters IC-Pak C M/D Column 4
Cation Compound Weight (g)
+
(Lithium) UOH•H20 6.0476
Li
+
(Sodium) NaCI 2.5421
No
+
(Ammonium) NH4CI 2.9640
NH
4
+
(Potassium) KCI 1.9067
K
+
(Magnesium) Mg(NO3)2•6H20 10.5466
Mg
2
+
(Calcium) Ca(N03)2•4H20 5.8919
Ca
2
+
(Strontium) Sr(NO3)2•4H20 3.2377
Sr
2
+
(Barium) BaCl2•2H20 1.7786
Ba
2
The following equation is an example of how these weights were
determined:
+
/L x 74.553 g KCI/39.100 g K+ x 1 L = 1.9067 g KCI
1 g K
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T
R
Inject
W
1/2
1/2h
h
Column eciency (plates)
Time to peak apex (minutes)
Width of peak at 1/2 peak height (minutes)
N = 5.54 x
2
Time (minutes)
0510 15 20 25
15.0 µS
1
2
3
4
5
6
7
8
1. Lithium
2. Sodium
3. Ammonium
4. Potassium
0.25 ppm
1.0 ppm
1.0 ppm
3.0 ppm
5. Magnesium
6. Calcium
7. Strontium
8. Barium
2.0 ppm
3.0 ppm
5.0 ppm
5.0 ppm
To prepare 1 liter of working standard:
1. Measure the appropriate quantity of stock standard (refer to Table 2).
2. Add the stock standard to a plastic 1000 ml volumetric flask.
3. Fill the flask to the mark with 18 megohm water. Table 2 shows
the concentrations for the monovalent and divalent cation working
standards.
4. Store working standards in clean plasticware at room temperature for
up to one month.
Table 2: Working Standard Solutions and Final Concentrations
Cation Use This Volume of
Stock Standard
+
Li
(Lithium) 0.250 ml 0.25
+
(Sodium) 1.000 ml 1.00
Na
+
NH4
Ammonium) 1.000 ml 1.00
+
(Potassium) 3.000 ml 3.00
K
+
agnesium) 2.000 ml 2.00
Mg
2
+
(Calcium 3.000 ml 3.00
Ca
2
+
(Strontium 5.000 ml 5.00
Sr
2
+
(Barium) 5.000 ml 5.00
Ba
To Yield This Concentration of
Working Standard (ppm)
Calculating Efficiency
Use the potassium peak (#4) to measure your column efficiency
with the half-height method equation in Figure 3.
Figure 3: Half-Height Method Test Calculations
V. CAre & MAIntenAnCe
Determining Column Efficiency
Use a flow rate of 1.0 ml/min. The peak for potassium should yield
>2,000 plates by the half-height method on a low dispersion Waters
ion chromatograph.
Figure 2 shows the conditions and resulting chromatogram for
monovalent and divalent cation standards.
Figure 2: IC-Pak C M/D Standard Cation Chromatogram
Waters IC-Pak C M/D Column 5
a. Storing the Column
For short-term storage, leave the mobile phase in the column with
the column connected to the system.
If the column will not be used for more than 72 hours, store it in
20/80 acetonitrile/water (v/v).
When storing columns:
DO NOT store the column in methanol or methanol-containing
Conditions
Column IC-Pak C M/D
Eluent 0.1 mM EDTA/
3 mM HNO3
Detector Waters 431
Flow Rate 1.0 mL/min
Injection
Volume
Conductivity
100 µL
solutions. Over time, methanol causes significant decreases in
cation retention times. To prevent growth of bacteria, fill with 20/80
acetonitrile/water (v/v), replace the end plugs, and return the column
to its box.
DO NOT allow the column to dry out. Allowing the column packing to
dry out can result in poor chromatographic performance.
Store at 15 °C to 35 °C. Freezing during storage degrades
performance.
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b. Troubleshooting
Table 3: Column Problems and Solutions
Symptom Conditions Corrective Action
Excess pressure
buildup
Filters plugged with
particulates. Check for
Replace filter.
injector and pump seal
shedding.
Clogged tubing Unclog or replace tubing.
Failing injector Repair the injector.
Sample precipitates
on column
Sample not soluble in
eluent
Backflush column with
mobile phase for 30 minutes at 0.1 mL/min.
Use a compatible mobile
phase.
Loss of resolution,
broad peaks, low
plate counts
Insufficient equilibration Continue equilibration
Incorrect diameter stainless steel tubing
Install 0.0009-inch stainless steel tubing.
Contaminated column Flush column with 100%
acetonitrile at 0.5 mL/min
for 30 minutes.
VI. orderInG InForMAtIon
Sales Offices:
Austria and European Export
(Central South Eastern Europe,
CIS and Middle East) 431 877 18 07
Australia 2 9933 1777
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 39 02 274 211
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 62 889 2030
Taiwan 886 2 2543 1898
United Kingdom 44 208 238 6100
To order these parts, contact the nearest subsidiary (see back cover).
Table 4: Spare Parts and Accessories
Part Number
IC-Pak C M/D Column, 3.9 x 150 mm WAT036570
Replacement frit for 3.9 mm column WAT015931
IC-Pak C M/D Guard-Pak™ Inserts, 10/pkg WAT098801
© 2007 Waters Corporation. Waters, The Science of W hat’s Possible, IC-Pak, Sep-Pak, and Guard-Pak are trademarks of Waters
Corporation. Millex, Milli-Q, and Durapore are trademarks of
Millipore Corporation.
November 2007 WAT036549 Rev 4 VW-P DF
Waters IC-Pak C M/D Column 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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