[ CARE AND USE MANUAL ]

Sugar-Pak I Column

CONTENTS
I. INTRODUCTION
II. PREPARATION FOR OPERATION
a. Column installation
b. Mobile-phase requirements
c. Equilibration
III. CARE AND USE
a. Precautions
b. Storage considerations
(more than 72 hours without use)
c. Starting up after a shutdown
d. Overnight shutdown
(less than 72 hours without use)
e. Starting up after an overnight shutdown
IV. SAMPLE PREPARATION
a. Pre-injection filtration
b. Chemical cleanup of samples
c. Pre-injection cleanup of proteins and lipids
d. In-line cleanup of proteins and lipids
e. Cleanup of polysacchacrides
f. Removal of salts and acids
g. In-line cleanup for samples
not prone to hydrolysis
I. INTRODUCTION
The Waters™ Sugar-Pak™ Column is used for the analysis
of sugar products and process streams in beet, cane, and
starch hydrolysis processing plants; and for incoming quality
inspection of commercial sugar products. Glucose, fructose,
maltose, and maltotriose can be separated from the higher
oligomers found in typical corn syrups. The course of alcoholic
fermentations can be followed by monitoring the reduction of
fermentable sugars and the production of alcohol.
The column can be used to separate many monosaccharides,
polyols, and alcohols. Disaccharides and larger sugars are also
separated, mainly according to molecular weight. Many other
useful separations can also be accomplished (e.g., fruit juices,
non-nutritive sweeteners containing sorbitol or mannitol, cell,
and cell hydrolyzates, etc.) where a variety of monosaccharides
or sugar alcohols require separation and analysis.
The 300 x 6.5 mm (I.D.) Sugar-Pak I Column is packed
with a microparticulate cation-exchange gel in calcium
form. To obtain results comparable to Waters performance
specifications, certain procedures regarding storage,
handling, and operation should be followed carefully,
and as explained in this manual.

V. COLUMN REGENERATION PROCEDURE

VI. TEST CONDITIONS

VII. TROUBLESHOOTING

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[ CARE AND USE MANUAL ]
II. PREPARATION FOR OPERATION
a. Column installation
Remove the end plugs from the steel column with a 5/16-inch
open-end wrench and save them for storage when the column
is removed from the system. The column outlet is indicated
by an arrow on the label (showing the direction solvent
should flow). Tighten the fittings to turn past finger tight.
DO NOT OVERTIGHTEN – this will damage the fitting seat.
A properly prepared and assembled compression fitting in
good condition is all that is required.
If tube cutting is required to connect a new column or
to improve the end connections on your existing fittings,
follow these steps:
1. Use a file with a cutting edge to scribe the circumference
of the tube at the desired break.
2. Grasp the tube on both sides of the scribe mark with
cloth-covered or smooth-faced pliers (to prevent marring
the tube surface) and gently work the tube back-and-forth
until it separates.
Tube Ferrule
End must be
straight and smooth
to achieve maximum
column efficiency
Critical distance to be determined by
Compression screw or nut each application (union, column fitting, etc.)
Figure 1. Ferrule and compression assembly.
3. Inspect the tube at the break for burrs. File the outer edges
at an angle to the tube opening. Do not file flat across the
open tube as this might cause plugging or uneven flow
delivery. Assemble as shown.
4. Slide the compression fitting over the tube, followed
by the ferrule (large end of the taper first).
5. Seat the ferrule by tightly mating the assembly to the
fitting seat in which it will be used. An improperly
positioned ferrule can form unwanted dead volume
which could result in unintentional sample mixing.
Note: Attach a union in place of the column and flush the lines free
of microparticulates before attaching the column.
b. Mobile-phase requirements
This column is packed with a calcium-loaded resin. Hydrogen or other cations
can replace the calcium and cause inversion on the column of sugars such as
sucrose, which are prone to inversion. It is recommended that a small amount
of calcium be used in the mobile phase to maintain the equilibrium and prevent
inversion. The recommended mobile phase is deionized, bacteria-free water
containing approximately 0.0001 M calcium EDTA* (50 mg/L).
* Calcium EDTA has several common names:
– Calcium di-sodium (ethylene dinitrilo) tetra-acetic acid
– Calcium di-sodium ethylene diamine tetraacetate
– Calcium di-sodium edentate
■ Water should be deionized to greater than 2 megohms
resistivity. It is essential that the water used is free of
polyvalent cations, particularly transition and heavy
metals (e.g., iron).
■ Remove bacteria and other particulates from the water
just before use by vacuum filtration.
■ Although the mobile phase will be partially degassed by
vacuum filtration, it is highly desirable that it be thoroughly
degassed by the following procedure:
1. Place the mobile phase in an Erlenmeyer flask on a
stirrer/hot plate.
2. Cover the mouth of the flask with aluminum foil to
minimize evaporation.
3. Bring the mobile phase to its boiling point for a few minutes
just before use, but maintain the temperature between
70–90 °C during use. This practice ensures that gases
(especially CO 2) do not redissolve in the mobile phase.
It also prevents the growth of microorganisms.
4. Keep the mobile-phase reservoir clean and covered and
supply freshly prepared mobile phase every 24 hours.
■ If the system is to be used continually for more than
one day, place the mobile phase in an Erlenmeyer flask
on a stirrer/hot plate. Cover the mouth of the flask with
aluminum foil to minimize evaporation. Bring the mobile
phase to its boiling point for a few minutes just before use,
but maintain the temperature between 70–90 °C during
use. This practice ensures that gases (especially CO 2) do
not redissolve in the mobile phase. It also prevents the
growth of microorganisms.
■ Keep the mobile phase reservoir clean and covered and
supply freshly prepared mobile phase every 24 hours.
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c. Equilibration
To ensure that a new column is fully equilibrated with calcium
prior to analysis, the following procedure is recommended.
1. Install the column with the normal direction of flow
reversed (arrow on label pointing to column inlet tubing).
A backflush valve may also be used.
2. Back flush the column with at least 100 mL of 0.001 M
calcium EDTA solution at 90 °C using a flow rate of
0.5 mL/min. A concentration of 500 mg/L of calcium
EDTA approximates 0.001 M.
3. Reverse the flow direction again (back to the normal
direction) and flush the column with the stable baseline
indicating that the column is ready for use.
III. CARE AND USE
Liquid chromatography columns have a finite life influenced by
their care and use, number of injections, sample and solvent
cleanliness, frequency of solvent changeover, and handling
and storage procedures (among other factors).
If a change is observed in the:
■ retention of a particular compound,
■ resolution between two compounds, or
■ peak shape
take immediate steps to determine the reason for the
changes. Until you have made this determination, you must
not rely upon the results of any separation using the column.
Follow generally accepted procedures for quality control and
methods development when using these columns.
Note: Before running the first analysis on your new column, perform the test
sample separation given in the test conditions section.
Before using your new Sugar-Pak I Column for the first time, it
is recommended that the following procedure be observed:
1. Connect the column (refer to Section II a).
2. Set the flow rate at 0.2 mL/min until the column
temperature reaches 70 °C.
3. Once the column temperature reaches 70 °C, increase
the 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.
a. Precautions
Normal recommended pressure should not exceed: 2000 psi.
Maximum flow rate: 0.6 mL/min (at or above 70 °C).
■ DO NOT use calcium chloride, calcium nitrate, or any other
acidic calcium salt in the mobile phase, as this can corrode
the column and damage the packing.
■ Maximum mobile-phase organic content is 5% V/V. Small
amounts of acetonitrile, ethanol, methanol, and isopropanol
in the sample will not effect column performance.
■ Column temperatures should not exceed 95 °C.
Generally, high temperatures provide greater resolution,
but little improvement occurs between 90 °C and 95 °C.
Temperatures below 70 °C do not provide adequate
resolution of many sugars due to the separation of
anomers. For quantification of ethanol, a column
temperature of 75 °C should be used.
■ Column temperature changes can be rapid without
adverse 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 produce excessive backpressure due to the
higher viscosity of water at lower temperatures.
■ Reverse flow direction through the column (after every
5 liters of mobile phase). This procedure will prolong
column life significantly. Flow rate changes should be
made slowly and not exceed 0.5 mL/min2 (0.5 mL/min
per minute). It is advisable to change the flow rate in
steps of 0.1 mL/min and wait until the backpressure
stabilizes before the next step change.
■ The pressure limit setting on the solvent delivery system
should be set to a few hundred psi above the normal
working pressure to avoid any excessive pressure buildup
while the instrument is unattended.
■ If sugars not prone to inversion are analyzed (e.g.,
hydrolyzates), 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 recommended. Column
regeneration and flow direction changes should be
carried out regularly for optimum column performance.
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b. Storage considerations
(more than 72 hours without use)
■ Turn off the column heater and stop the pump.
■ When the column is cool, remove it from the system and
replace the endcaps originally supplied with the column.
The column should not be allowed to dry out during storage.
The normal mobile phase is suitable as a storage solvent in
the column. Refrigerate the column at 5 °C (not frozen) to
minimize any microbial growth.
■ The end fittings in the system should be joined by a
union after the column is removed. Flush the entire
liquid chromatography system first with water, then
methanol, and store it in methanol.
c. Starting up after a shutdown
Note: Make sure all methanol is flushed from the system with water before
the column is reinstalled.
When starting up a system containing a cold column, turn
on the column heater and start the mobile phase flowing at
no more than 0.2 mL/min. Allow the instrument to stabilize
under these conditions for at least 20 minutes after the
column reaches working temperature before running the
system under usual working conditions.
d. Overnight shutdown
(less than 72 hours without use)
The column may be left in the system with the flow rate
reduced to 0.2 mL/min. Recirculate the solvent by placing
the effluent line in the supply flask. If it is undesirable to
leave the column heater on, shut off the pump and the
column heater and allow them to cool down.
e. Starting up after an overnight shutdown
Turn on the column heater and start the mobile phase
flowing at no more than 0.1 to 0.2 mL/min. Continue at
this flow rate for at least 20 minutes after the column has
reached its working temperature before setting normal
operating conditions for analysis.
IV. SAMPLE PREPARATION
a. Pre-injection filtration
Filter all samples through a 0.45 µm membrane filter just
before injection to remove particulates.
b. Chemical cleanup of samples
Substances such as proteins, lipids, polysaccharides, salts
and acids, which could contaminate your column, should
be removed from samples using the suggested techniques
outlined in sections d through h.
c. Pre-injection cleanup of proteins and lipids
Proteins, or lipids such as fats and oils, can contaminate the
column and cannot be removed by washing with organic
solvents. If samples are contaminated with large amounts of
lipid, the excess can be removed by liquid-liquid extraction
into a solvent such as chloroform.
Residual lipophilic compounds can be removed using a
Sep-Pak™ C18 Cartridge as explained in the following procedure:
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.
2. Wash the methanol from the cartridge with two 5 mL
portions of pure water.
3. Blow excess water from the cartridge with the syringe.
4. Slowly pass the sample through the Sep-Pak C18 Cartridge.
a. Reject the first 2 mL.
b. Collect the next few milliliters for subsequent analysis.
d. In-line cleanup of proteins and lipids
Protect your column from small amounts of lipophilic material
by using the Waters Guard-Pak™ Holder and Guard-Pak C 18
Inserts. As the Guard-Pak Insert becomes contaminated with
lipophilic sample components, it can be simply replaced and
the old one discarded.
e. Cleanup of polysacchacrides
Note: Since C18 -guard columns will adsorb some neutral polysaccharides,
they should not be used for the analysis of such samples.
Neutral polysaccharides will pass through the Sugar-Pak
I Column. For some samples, such as starch hydrolyzates,
the analysis of such polysaccharides is desirable and the
use of guard columns containing resins (as described under
“Removal of Salts and Acids”) is recommended. These resins
remove most residual proteins and lipids, but allow neutral
polysaccharides to pass through.
Sugar-Pak I Column 4
[ CARE AND USE MANUAL ]
f. Removal of salts and acids
Salts and organic acids present in crude samples can
co-elute with sugars and interfere with the analysis.
Typically, salts of strong acids such as sodium chloride,
elute near the void volume. Salts of weak acids, such as
lactate and acetate, elute later.
For some samples of low MW sugars, Waters Sep-Pak
Alumina Type A Cartridges are suitable for removing
salts and acids.
The recommended procedure is as follows:
1. Pass 5 mL of pure water through the Sep-Pak Alumina
Type A Cartridge.
2. Place 10 mL (maximum) of sample in the syringe and
slowly pass the sample through the cartridge.
a. Reject the first 5 mL.
b. Collect the next few milliliters of sample for injection.
Make sure this procedure is suitable for quantitative recovery
for your particular samples.
g. In-line cleanup for samples
not prone to hydrolysis
Use a Waters Guard Column between the column and the
injector to remove salts and acids from the sample. Pack
the guard column with a mixture of strong cation resins (in
the H form) and strong anion resins (in the OH form) with
+ -
a maximum particle size of 200/400 mesh. These resins are
available from a number of sources (e.g., CG120 and CG400
resins made by Rohm and Haas), but have to be converted
into the appropriate ionic forms just before use. In particular,
the anion-exchange resins are unstable in the OH- form
during prolonged storage. Repack the guard column with
fresh resin when necessary.
Note: Do not use a guard column containing the mixed bed resin for sucrose
analysis, as this will cause severe inversion of the sucrose (or other sugars
prone to inversion). Only use the mixed bed resin in the guard column when
100% pure water is used as the mobile phase. Any calcium EDTA in the
mobile phase will saturate the column. Any other type of guard column that
exceeds the bed volume of the Waters Guard Column (about 0.2 mL) is not
recommended for use with Sugar-Pak I Columns. The increased bed volume
will cause excessive band spreading and loss of resolution.
V. COLUMN REGENERATION PROCEDURE
The need to regenerate your Sugar-Pak I Column will vary
according to the relative purity of your samples. The cruder
the samples (particularly those containing heavy or transition
metals or organic acids), the more frequent regeneration will
be required due to inversion of the column.
You can test for inversion by injecting sucrose. If a tail
is produced on the sucrose peak, the column should be
retreated as follows:
■ Make up 500 mL of regeneration solution (500 mg
calcium EDTA/L) 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 regeneration
and returned to normal following regeneration.
When flow is reversed it is important to maintain solvent
temperature in order to ensure proper cleaning. Using cold
solvent will slow the cleaning process.
VI. TEST CONDITIONS
Every column is thoroughly tested and passes strict
manufacturing specifications. It is recommended that
you perform a test sample injection, like the one below,
prior to your first analysis. Record the results along with
all relevant instrument settings. These results can then be
used for comparison throughout the lifetime of the column
to monitor equipment performance, sample variation, and
column condition.
5.80 DP3 Maltoriose
4.22
6.82 DP2 Maltose
9.80 Glucose
Test sample: 42 DE corn syrup
Flow rate: 0.5 mL/min
Temp.: 90 °C
Mobile phase: 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.
Sugar-Pak I Column 5
[ CARE AND USE MANUAL ]

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 in-line
filter or guard column.
If this is not the problem, it is possible that the resin has
partially blocked the outlet frit. Should this occur, shut
down the system. Reverse the direction of flow through the
column and begin pumping the mobile phase at 0.1 mL/min.
Allow 20 minutes after the column has reached its working
temperature before bringing the column up to its working
flow rate.
Refer to column regeneration and other cleanup methods
explained in this manual for actual procedures. Remember
to switch your column to its original flow direction and allow
proper warm-up time before continuing with further analyses.
Caution: The resin in Sugar-Pak I Columns is tightly packed
and elastic. If the column end frits are removed, the packing
may expand out of the column. Thus, opening the end fittings
of the column to inspect or clean the end frits (in an ultrasonic
bath) should be done only as a last resort.

Table 1. Typical column problems and solutions

Problem Cause Solution
Replace the in-line filter, or shut off the pump. When inlet
Excess pressure In-line filters plugged pressure has dropped to zero, disconnect the in-line filter from
buildup with particulates the column and backflush the prefilter ONLY with mobile phase
at 9 mL/min until the particulates are backflushed out.
Column inlet frit plugged Always use an in-line filter or guard column to prevent this. Reverse
with particulates column to try to wash out particulates at normal flow rate.
Exit frit plugged Reverse flow of mobile phase. Check pump and system
Fluctuating
with resin operation before proceeding.
backpressure
Gas in solvent Check degassing procedure.
Faulty pump Inspect pump check valves and carry out “Ramp Test”
operation (consult the operator’s manual for your pump).
Peak “tailing” H+ ions or heavy metals Follow column regeneration procedure using 500 mg calcium
on sucrose (e.g, Fe) on column EDTA per liter as described.
Reverse column flow direction. This may allow the packing
Band broadening Void in column
bed to reform, thus improving peak shape.
“Spurious” peaks, Elution of salts
Check sample cleanup procedures.
not due to sugars and/or acids
Fittings in bad state Tighten fittings properly. DO NOT OVERTIGHTEN.
Leaking mobile phase
of repair Replace worn fittings.
Variations in flow
Variable elution times Check system for faults, leaks, especially pulsations from pump.
rate or temperature

Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
Waters, Sugar-Pak, Guard-Pak, Sep-Pak, and The Science of What’s Possible are trademarks
of Waters Corporation. All other trademarks are the property of their respective owners.
T: 1 508 478 2000
F: 1 508 872 1990
©2020 Waters Corporation. Produced in the U.S.A. January 2020 WAT085454 Rev D IH-PDF www.waters.com