[ 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.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
V.COLUMN REGENERATION PROCEDURE
VI. TEST CONDITIONS
VII. TROUBLESHOOTING
VIII. ORDERING INFORMATION
I. INT RODUCTION
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.
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[ CARE AND USE MANUAL ]
II. P REPARATION FOR OPERATION
a. Column Installation
Remove the end plugs from the steel column with a 5/16-inch openend 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 clothcovered or smooth-faced pliers (to prevent marring the tube surface) and gently work the tube back-and-forth until it separates.
COMPRESSION SCREW OR NUT |
FERRULE |
|
TUBE
END MUST BE STRAIGHT
AND SMOOTH TO ACHIEVE MAXIMUM COLUMN EFFICIENCY
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.
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:
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 C02) 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
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[ 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 C02) 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.
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.
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