[ Care and Use Manual ]
SUGAR-PAK I COLUMN
Contents
I. INT RODUC T ION
I. INTRODUCTION
The Waters Sugar-Pak™ Column is used for the analysis of sugar
products and process streams in beet, cane, and starch hydrolysis
II. PREPARATION FOR OPERATION
processing plants; and for incoming quality inspection of commercial
a. Column Installation
sugar products. Glucose, fructose, maltose, and maltotriose can be
b. Mobile-Phase Requirements
separated from the higher oligomers found in typical corn syrups. The
c. Equilibration
course of alcoholic fermentations can be followed by monitoring the
reduction of fermentable sugars and the production of alcohol.
III. CARE AND USE
The column can be used to separate many monosaccharides,
a. Precautions
polyols, and alcohols. Disaccharides and larger sugars are also
b. Storage Considerations (more than 72 hours without use)
separated, mainly according to molecular weight. Many other
c. Starting Up After a Shutdown
useful separations can also be accomplished (e.g., fruit juices,
d. Overnight Shutdown (less than 72 hours without use)
non-nutritive sweeteners containing sorbitol or mannitol, cell, and
e. Starting Up After an Overnight Shutdown
cell hydrolyzates, etc.) where a variety of monosaccharides or sugar
IV. SAMPLE PREPARATION
alcohols require separation and analysis.
a. Pre-Injection Filtration
The 300 x 6.5 mm (ID) Sugar-Pak I Column is packed with a
b. In-Line Filtration
microparticulate cation-exchange gel in calcium form. To obtain
c. Chemical Cleanup of Samples
results comparable to Waters performance specifications, certain
d. Pre-Injection Cleanup of Proteins and Lipids
procedures regarding storage, handling, and operation should be
e. In-Line Cleanup of Proteins and Lipids
followed carefully, and as explained in this manual.
f. Cleanup of Polysacchacrides
g. Removal of Salts and Acids
h. In-Line Cleanup for Samples Not Prone to Hydrolysis
V. COLUMN REGENERATION PROCEDURE
VI. TEST CONDITIONS
VII. TROUBLESHOOTING
VIII. ORDERING INFORMATION
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[ Care and Use Manual ]
II. P RE PA RAT ION FOR O P ERAT ION
b. Mobile-Phase Requirements
a. Column Installation
This column is packed with a calcium-loaded resin. Hydrogen or
other cations can replace the calcium and cause inversion on the
Remove the end plugs from the steel column with a 5/16-inch open-
column of sugars such as sucrose, which are prone to inversion.
end wrench and save them for storage when the column is removed
It is recommended that a small amount of calcium be used in the
from the system. The column outlet is indicated by an arrow on the
mobile phase to maintain the equilibrium and prevent inversion.
label (showing the direction solvent should flow). Tighten the fittings
The recommended mobile phase is deionized, bacteria-free water
to turn past finger tight. DO NOT OVERTIGHTEN – this will damage the
containing approximately 0.0001 M calcium EDTA* (50 mg/L).
fitting seat. A properly prepared and assembled compression fitting in
* Calcium EDTA has several common names:
good condition is all that is required.
- Calcium di-sodium (ethylene dinitrilo) tetra-acetic acid
If tube cutting is required to connect a new column or to improve the
- Calcium di-sodium ethylene diamine tetraacetate
end connections on your existing fittings, follow these steps:
- Calcium di-sodium edentate
1. Use a file with a cutting edge (such as the file included in the  Water should be deionized to greater than 2 megohms resistivity.
Startup Tool Kit, P/N WAT096146) to scribe the circumference of It is essential that the water used is free of polyvalent cations,
the tube at the desired break.
particularly transition and heavy metals (e.g., iron).
2. Grasp the tube on both sides of the scribe mark with cloth-

Remove bacteria and other particulates from the water just
covered or smooth-faced pliers (to prevent marring the tube before use by vacuum filtration. The Solvent Clarification Kit is
surface) and gently work the tube back-and-forth until it recommended for solvent filtration (110 V, P/N WAT085113;
separates.
240 V, P/N WAT085122) using PES filter membranes (P/N
WAT200538).
COMPRESSION SCREW OR NUT
FERRULE
 Although the mobile phase will be partially degassed by vacuum
TUBE
filtration, it is highly desirable that it be thoroughly degassed by
END MUST BE STRAIGHT
AND SMOOTH TO ACHIEVE
MAXIMUM COLUMN EFFICIENCY
the following procedure:
CRTITICAL DISTANCE TO BE DETERMINED BY
EACH APPLICATION (UNION, COLUMN FITTING, ETC.)
1. Place the mobile phase in an Erlenmeyer flask on a
stirrer/hot plate.
Figure 1. Ferrule and compression assembly.
2. Cover the mouth of the flask with aluminum foil to minimize 3. Inspect the tube at the break for burrs. File the outer edges at an evaporation.
angle to the tube opening. Do not file flat across the open tube as this might cause plugging or uneven flow delivery. Assemble 3. Bring the mobile phase to its boiling point for a few
as shown.
minutes just before use, but maintain the temperature between 70-90 °C during use. This practice ensures that 4. Slide the compression fitting over the tube, followed by the gases (especially C02) do not redissolve in the mobile ferrule (large end of the taper first).
phase. It also prevents the growth of microorganisms.
5. Seat the ferrule by tightly mating the assembly to the fitting seat 4. Keep the mobile-phase reservoir clean and covered and supply in which it will be used. An improperly positioned ferrule can freshly prepared mobile phase every 24 hours.
form unwanted dead volume which could result in unintentional sample mixing.

If the system is to be used continually for more than one
day, place the mobile phase in an Erlenmeyer flask on a
Note: Attach a union in place of the column and flush the lines stirrer/hot plate. Cover the mouth of the flask with aluminum
free of microparticulates before attaching the column.
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[ Care and Use Manual ]
foil to minimize evaporation. Bring the mobile phase to
Note: Before running the first analysis on your new column, perform
its boiling point for a few minutes just before use, but
the test sample separation given in the test conditions section.
maintain the temperature between 70-90 °C during use.
Before using your new Sugar-Pak I column for the first time, it is
This practice ensures that gases (especially C02) do not
recommended that the following procedure be observed:
redissolve in the mobile phase. It also prevents the growth of
1. Connect the column (refer to Section II a)
microorganisms.
2. Set the flow rate at 0.2 mL/min until the column temperature  Keep the mobile phase reservoir clean and covered and supply
reaches 70 °C.
freshly prepared mobile phase every 24 hours.
3. Once the column temperature reaches 70 °C, increase the c. Equilibration
flow rate to 0.6 mL/min in steps of 0.1 mL/min. Wait for the backpressure to stabilize between each 0.1 mL/min increase.
To ensure that a new column is fully equilibrated with calcium prior to
analysis, the following procedure is recommended.
a. Precautions
1. Install the column with the normal direction of flow reversed Normal recommended pressure should not exceed: 2000 psi.
(arrow on label pointing to column inlet tubing). A backflush Maximum flow rate: 0.6 mL/min (at or above 70 °C).
valve may also be used.
 DO NOT use calcium chloride, calcium nitrate, or any other acidic
2. Back flush the column with at least 100 mL of 0.001 M calcium
calcium salt in the mobile phase, as this can corrode the column
EDTA solution at 90 °C using a flow rate of 0.5 mL/min. A and damage the packing.
concentration of 500 mg/L of calcium EDTA approximates  Maximum mobile-phase organic content is 5% V/V. Small amounts
0.001 M.
of acetonitrile, ethanol, methanol, and isopropanol in the sample
3. Reverse the flow direction again (back to the normal direction) will not effect column performance.
and flush the column with the stable baseline indicating that the  Column temperatures should not exceed 95 °C. Generally, high
column is ready for use.
temperatures provide greater resolution, but little improvement
occurs between 90 °C and 95 °C. Temperatures below 70 °C do not
III. CA RE AND USE
provide adequate resolution of many sugars due to the separation
Liquid chromatography columns have a finite life influenced by their
of anomers. For quantification of ethanol, a column temperature of
care and use, number of injections, sample and solvent cleanliness,
75 °C should be used.
frequency of solvent changeover, and handling and storage procedures
 Column temperature changes can be rapid without adverse
(among other factors). If a change is observed in the:
effects. If the column temperature is below 60 °C, a maximum
flow rate of 0.2 mL/min should be used. Higher flow rates may
 retention of a particular compound,
produce excessive backpressure due to the higher viscosity of
 resolution between two compounds, or
water at lower temperatures.
 peak shape
 Reverse flow direction through the column (after every 5 liters
take immediate steps to determine the reason for the changes. Until you
of mobile phase). This procedure will prolong column life
have made this determination, you must not rely upon the results of any
significantly. Flow rate changes should be made slowly and not
separation using the column. Follow generally accepted procedures for
exceed 0.5 mL/min2 (0.5 mL/min per minute). It is advisable to
quality control and methods development when using these columns.
change the flow rate in steps of 0.1 mL/min and wait until the
backpressure stabilizes before the next step change.
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[ Care and Use Manual ]
e. Starting Up After an Overnight Shutdown
 The pressure limit setting on the solvent delivery system should
be set to a few hundred psi above the normal working pressure
Turn on the column heater and start the mobile phase flowing at no
to avoid any excessive pressure buildup while the instrument is
more than 0.1 to 0.2 mL/min. Continue at this flow rate for at least
unattended.
20 minutes after the column has reached its working temperature
 If sugars not prone to inversion are analyzed (e.g., hydrolyzates),
before setting normal operating conditions for analysis.
it is not essential to use calcium EDTA in the mobile phase. Pure
water can be used but it should be filtered and degassed as
IV. Sample Preparation
recommended. Column regeneration and flow direction changes
a. Pre-Injection Filtration
should be carried out regularly for optimum column performance.
b. Storage Considerations (more than 72 hours without use)
Filter all samples through a 0.45 µm membrane filter just before
injection to remove particulates. Suitable replacement filters are
 Turn off the column heater and stop the pump.
provided in the Waters Aqueous Sample Clarification Kit (P/N
 When the column is cool, remove it from the system and replace the
WAT026865). Suitable disposable filters with a larger surface area
endcaps originally supplied with the column. The column should not
are also available (HV Filter Unit, P/N WAT085996).
be allowed to dry out during storage. The normal mobile phase is
b. In-Line Filtration
suitable as a storage solvent in the column. Refrigerate the column
at 5 °C (not frozen) to minimize any microbial growth.
If a Guard-Pak™ Pre-Column module is being used, disposable
pre-column area filter inserts (P/N WAT032472) for use instead of
 The end fittings in the system should be joined by a union after the
Guard-Pak Pre-Column inserts. If guard columns are not being used,
column is removed. Flush the entire liquid chromatography system
it is advisable to use an in-line pre-column filter (P/N WAT084560)
first with water, then methanol, and store it in methanol.
immediately before the column to protect it from any particulate
c. Starting Up After a Shutdown
matter in the samples or mobile phase.
Note: Make sure all methanol is flushed from the system with water
c. Chemical Cleanup of Samples
before the column is reinstalled.
Substances such as proteins, lipids, polysaccharides, salts and acids,
When starting up a system containing a cold column, turn on the
which could contaminate your column, should be removed from samples
column heater and start the mobile phase flowing at no more than
using the suggested techniques outlined in sections d through h.
0.2 mL/min. Allow the instrument to stabilize under these conditions
d. Pre-Injection Cleanup of Proteins and Lipids
for at least 20 minutes after the column reaches working temperature
before running the system under usual working conditions.
Proteins, or lipids such as fats and oils, can contaminate the column
and cannot be removed by washing with organic solvents. If samples
d. Overnight Shutdown (less than 72 hours without use)
are contaminated with large amounts of lipid, the excess can be
The column may be left in the system with the flow rate reduced to
removed by liquid-liquid extraction into a solvent such as chloroform.
0.2 mL/min. Recirculate the solvent by placing the effluent line in the
Residual lipophilic compounds can be removed using a Sep-Pak® C18
supply flask. If it is undesirable to leave the column heater on, shut off
Cartridge (P/N WAT051910) as explained in the following procedure:
the pump and the column heater and allow them to cool down.
1. Fill a 10 mL syringe with 5 mL of methanol and slowly flush the methanol through the cartridge to condition the
Sep-Pak C18 packing.
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[ Care and Use Manual ]
a. Reject the first 5 mL.
2. Wash the methanol from the cartridge with two 5 mL portions of pure water.
b. Collect the next few milliliters of sample for injection.
3. Blow excess water from the cartridge with the syringe.
Make sure this procedure is suitable for quantitative recovery for your
4. Slowly pass the sample through the Sep-Pak C18 Cartridge.
particular samples.
a. Reject the first 2 mL.
h. In-Line Cleanup for Samples Not Prone to Hydrolysis
b. Collect the next few milliliters for subsequent analysis.
Use a Waters Guard Column (P/N WAT084550) between the column
and the injector to remove salts and acids from the sample. Pack the
e. In-Line Cleanup of Proteins and Lipids
guard column with a mixture of strong cation resins (in the H+ form)
Protect your column from small amounts of lipophilic material by
and strong anion resins (in the OH- form) with a maximum particle
using the Waters Guard-Pak Holder (P/N WAT080040) and Guard-
size of 200/400 mesh. These resins are available from a number of
Pak C18 Inserts (P/N WAT085824). As the Guard-Pak Insert becomes
sources (e.g., CG120 and CG400 resins made by Rohm and Haas), but
contaminated with lipophilic sample components, it can be simply
have to be converted into the appropriate ionic forms just before use.
replaced and the old one discarded.
In particular, the anion-exchange resins are unstable in the OH- form
during prolonged storage. Repack the guard column with fresh resin
f. Cleanup of Polysacchacrides
when necessary.
Note: Since C18-guard columns will adsorb some neutral polysaccharides,
Note: Do not use a guard column containing the mixed bed resin for
they should not be used for the analysis of such samples.
sucrose analysis, as this will cause severe inversion of the sucrose (or
Neutral polysaccharides will pass through the Sugar-Pak I Column.
other sugars prone to inversion). Only use the mixed bed resin in the
For some samples, such as starch hydrolyzates, the analysis of such
guard column when 100% pure water is used as the mobile phase.
polysaccharides is desirable and the use of guard columns containing
Any calcium EDTA in the mobile phase will saturate the column. Any
resins (as described under “Removal of Salts and Acids”) is
other type of guard column that exceeds the bed volume of the Waters
recommended. These resins remove most residual proteins and lipids,
guard column (about 0.2 mL) is not recommended for use with Sugar-
but allow neutral polysaccharides to pass through.
Pak I Columns. The increased bed volume will cause excessive band
spreading and loss of resolution.
g. Removal of Salts and Acids
Salts and organic acids present in crude samples can co-elute with
V. COLUMN REGENE RAT ION P ROC EDU RE
sugars and interfere with the analysis. Typically, salts of strong acids
such as sodium chloride, elute near the void volume. Salts of weak
The need to regenerate your Sugar-Pak I Column will vary according
acids, such as lactate and acetate, elute later.
to the relative purity of your samples. The cruder the samples
(particularly those containing heavy or transition metals or organic
For some samples of low MW sugars, Waters Sep-Pak Alumina
acids), the more frequent regeneration will be required due to
Type A Cartridges (P/N WAT051800) are suitable for removing
inversion of the column.
salts and acids.
You can test for inversion by injecting sucrose. If a tail is produced on
The recommended procedure is as follows:
the sucrose peak, the column should be retreated as follows:
1. Pass 5 mL of pure water through the Sep-Pak Alumina Type A 
Make up 500 mL of regeneration solution (500 mg calcium EDTA/L)
Cartridge.
and allow it to run through the column overnight at 0.5 mL/min at
90 °C. Make sure the direction of flow is reversed before starting
2. Place 10 mL (maximum) of sample in the syringe and slowly regeneration and returned to normal following regeneration.
pass the sample through the cartridge.
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[ Care and Use Manual ]
When flow is reversed it is important to maintain solvent temperature
the mobile phase at 0.1 mL/min. Allow 20 minutes after the column
in order to ensure proper cleaning. Using cold solvent will slow the
has reached its working temperature before bringing the column up to
cleaning process.
its working flow rate.
Refer to column regeneration and other cleanup methods explained in
V I. TEST CONDIT IONS
this manual for actual procedures. Remember to switch your column
to its original flow direction and allow proper warm-up time before
Every column is thoroughly tested and passes strict manufacturing
continuing with further analyses.
specifications. It is recommended that you perform a test sample
injection, like the one below, prior to your first analysis. Record the
Caution: The resin in Sugar-Pak I Columns is tightly packed and elastic.
results along with all relevant instrument settings. These results
If the column end frits are removed, the packing may expand out of the
can then be used for comparison throughout the lifetime of the
column. Thus, opening the end fittings of the column to inspect or clean
column to monitor equipment performance, sample variation, and
the end frits (in an ultrasonic bath) should be done only as a last resort.
column condition.
Table 1. Typical Column Problems and Solutions
4.22
5.80 DP3 Maltoriose
6.82 DP2 Maltose
9.80 Glucose
Problem
Cause
Solution
Excess
In-line filters
pressure buildup plugged with
particulates
Replace the in-line filter, or shut off the
pump. When inlet pressure has dropped to
zero, disconnect the in-line filter from the
column and backflush the prefilter ONLY
with mobile phase at 9 ml/min until the
particulates are backflushed out.
Fluctuating
backpressure
Column inlet
frit plugged with
particulates
Always use an in-line filter or guard
column to prevent this. Reverse column
to try to wash out particulates at normal
flow rate.
Exit frit plugged
with resin
Reverse flow of mobile phase. Check pump
and system operation before proceeding.
Gas in solvent
Check degassing procedure.
Faulty pump
operation
Inspect pump check valves and carry
out “Ramp Test” (consult the operator's
manual for your pump).
Peak “tailing”
on sucrose
H+ ions or heavy
metals (e.g, Fe)
on column
Follow column regeneration procedure
using 500 mg calcium EDTA per liter as
described.
Band
broadening
Void in column
Reverse column flow direction. This may
allow the packing bed to reform, thus
improving peak shape.
“Spurious”
peaks, not
due to sugars
Elution of salts
and/or acids
Check sample cleanup procedures.
in-line filter or guard column.
Leaking mobile
phase
Fittings in bad
state of repair
Tighten fittings properly. DO NOT
OVERTIGHTEN. Replace worn fittings.
If this is not the problem, it is possible that the resin has partially
Variable
elution times
Variations in
flow rate or
temperature
Check system for faults, leaks,
especially pulsations from pump.
Test Sample:
Flow Rate:
Temp.:
Mobile Phase:
42 DE Corn Syrup
0.5 mL/min
90 ˚C
Deionized water
Figure 2: A typical chromatogram of sugar standards analyzed with a Sugar-Pak I Column.
Note the high resolution of glucose, maltose and maltotriose. Glucose should elute in less
than 10 minutes. Maximum backpressure should be less than 2000 psi.
VII. TROUBLESHOOTING
The Sugar-Pak I Column is tightly packed with a resin in a swollen
form. If good sample preparation is practiced, most problems that
arise will relate to the resin packing. The resin packing is delicate and
can be forced into the end frits of the column by pump pulsations due
to faulty check valves or by sudden or excessive pressure buildup.
Should pressure buildup occur, check for trapped particulates in the
blocked the outlet frit. Should this occur, shut down the system.
Reverse the direction of flow through the column and begin pumping
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[ Care and Use Manual ]
V III. O RDE RING INFORMAT ION
Description
Part Number
Compression Screws and Ferrules, 5/pk
WAT025604
Guard-Pak Holder , 1/pk
WAT088141
Guard-Pak Inserts, 10/pk
WAT085824
Inline Pre-Column Filter Kit
WAT084560
LC/GC Certified Vials, 12 x 32 mm, 100/pk
186000272C
Solvent Clarification Kit, 110V
WAT085113
Solvent Clarification Kit, 220V
WAT085112
47mm PES Filters, 0.45 μm, 100/pk
WAT200538
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 22 833 4400, 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 is a registered trademark
of Waters Corporation. Sugar-Pak, Guard-Pak, Sep-Pak, and
T he Science of W hat’s Possible are trademarks of Waters
Corporation. All other trademarks are the property of their
respective owners.
May 2012 WAT085454 Rev C VW-PDF
Sugar-Pak I Columns
7
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com