Waters Sugar-Pak I Column User Manual

[ 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. Prec autions

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. In-Line Filtration

c. Chemical Cleanup of Samples

d. Pre-Injection Cleanup of Proteins and Lipids

e. In-Line Cleanup of Proteins and Lipids

f. Cleanup of Polysacchacrides

g. Removal of Salts and Acids

h. In-Line Cleanup for Samples Not Prone to Hydrolysis

I. IntRoDUC tIon

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 (ID) 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

VIII. oRDeRInG InFoRMAtIon

Sugar-Pak I Columns 1

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COMPRESSION SCREW OR NUT

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 (such as the file included in the

Startup Tool Kit, P/N WAT096146) 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.

FERRULE

TUBE

END MUST BE STRAIGHT
AND SMOOTH TO ACHIEVE
MAXIMUM COLUMN EFFICIENCY

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. The Solvent Clarification Kit is

recommended for solvent filtration (110 V, P/N WAT085113;

240 V, P/N WAT085122) using PES filter membranes (P/N

WAT200538).

 Although the mobile phase will be partially degassed by vacuum

filtration, it is highly desirable that it be thoroughly degassed by

the following procedure:

CRTITICAL DISTANCE TO BE DETERMINED BY

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.

Sugar-Pak I Columns 2

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 C0

) do not redissolve in the mobile

2

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

[ Care and Use ManUal ]

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 C0

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.

) do not

2

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/min

change the flow rate in steps of 0.1 mL/min and wait until the

backpressure stabilizes before the next step change.

2

(0.5 mL/min per minute). It is advisable to

Sugar-Pak I Columns 3