[ Care and Use Manual ]

Dextro-pak cartridge

I. INTRODUC T ION

Contents I. introduction

The Waters Dextro-Pak™ cartridge that you have purchased is an 8 mm x 100 mm cartridge packed with silica particles bonded

II. column conditioning

with octadecyl saline. The chemical and physical stability of this

III. solvent preparation

reversed-phase packing material provides rapid, reliable sugar analysis and minimal reaction with samples. The Dextro-Pak system

IV. sample filtering

operates at ambient temperature an uses a pure water mobile phase. It is particularly suited to the separation of: •

Carbohydrate oligomers, such as starch hydrolysates

•

Relatively simple sugar mixtures

•

Sugar derivatives such as methyl glycosides and ethanol

V. Test conditions Vi. column efficiency vII. cartridge life

Please take a few moments to read this manual carefully. Use these

VIII. operating tips

procedures to ensure that you obtain quality results and take full advantage of the features that your Waters cartridge offers.

IX. care & trobleshooting procedures x. warranty xi. ordering information

Dextro-Pak Cartridge

1

[ Care and Use Manual ]

Figure 1. Different glucose syrups separated on a Dextro-Pak cartridge Column: Dextro-Pak, 10 µm, 8 mm x 100 mm Mobile phase: Water Flow rate: 1mL/min Detector: M410, R1, X18

High Maltose-Enzyme Digest High DP-Enzyme Digest 62DE-Acid Digest 42DE-Acid Digest

In sugar analyses, the smallest sugars elute first and usually, as retention time increases, the molecular weight of the sample increases. The table below shows typical retention times of some carbohydrates on the Dextro-Pak cartridge using water at 1 mL/min as the mobile phase. These retention times are to be used only as a guide an are not absolute values. Flow rate, cartridge age and cartridge condition may affect the retention time of the analysis. Table 1. Typical Retention Times Compound

Retention Time (min)

Compound

Retention Time (min)

Adonitol

2.87

Maltotriose

4.40/4.75 (doublet)

Arabinose

3.00

Mannitol

2.87

Cellobiose

3.70

Mannose

2.87

2-Deoxyglucose

3.42

Melibiose

3.12

Ethanol

6.83

Methanol

3.90

Ethylene Glycol

3.50

Panose

4.95

Fructose

2.92

Raffi nose

7.00

Fucose

4.07

Rham nose

3.27

Galactose

2.77

Ribose

3.07

Glucose

2.82

Sorbitol

2.85

Glucose-6-phosphate

2.10

Sorbose

2.82

Glycerol

3.15

Stachyose

6.25

lactu lose

3.82

Sucrose

4.37

Lactose

3.00/3.22 (doublet)*

Xylitol

2.87

Lycose

2.97

Xylose

2.82 (doublet)*

Maltose

3.45

Xylulose

3.12

* Some sugars such as lactose, xylose and glucose oligomers above a DP of 2, produce two partly resolved peaks which are attributable to partial separation of anomers. Dextro-Pak Cartridge

2

[ Care and Use Manual ]

Installation

III. SOLVENT PREPARAT ION

Note: Follow storage, handling and operation procedures carefully 1.

to obtain results comparable to Waters performance specifications.

The recommended mobile phase for all analyses is 100% particulate-free water.

Use the Dextro-Pak cartridge with a Waters 8x10 Holder (P/N WAT082887).

2. Use vacuum filtration through a membrane of 0.45 µm porosity.

Read each of the two sections on solvent preparation and sample

Waters Solvent Clarification Kit, P/N WAT085122, is recom-

filtering carefully. Follow the procedures as explained before

mended. Suitable filters are Triton-Free Aqueous Replacement

performing any separations.

Filters, P/N WAT200538. 3. Remove most dissolved gases using a vacuum filtration of 635

Perform and permanently record the test procedures on your new

mm (25-inch). Some gases will redissolve from the air and

column to serve as a performance standard.

some growth of microorganisms will occur over a period of time,

Cheetham, N.W.H., Sirimanne, P. and Day, W.R., (1981). Highperformance Liquid Chromatographic Separation of Carbohydrate Oligomers. Journal of Chromatography, 207, 439-444.

1

even in purified water. Avoid long term problems by heating the mobile phase in a 1 or 2 liter Erlenmeyer flask on a stirrer/ hotplate. Heat the water to a boil that cannot be stirred down, then switch off the stirrer and reduce the heat to approximately

II. COLUMN CONDIT IONING

70 °C. Cover the neck of the flask with a piece of aluminum foil to help minimize evaporation.

Unlike steel columns (which are packed in solvent), radially

4. Add fresh mobile phase prepared as above when needed to

compressed cartridges are supplied dry. Before use, the cartridge

allow the system to run continuously.

must be wetted with a solvent such as methanol or acetonitrile. To

Detector stability will not be affected because the low flow rate

provide a suitable surface for adsorption of the sample: 1.

Place a new cartridge into the 8x10 Holder.

2.

Prepare solvents as described in the following section before

enables the mobile phase to cool down in the solvent supply line before it reaches the pump. Caution: The use of salts and solvent as additives in the mobile phase

using them to condition your column. 3.

will impair cartridge performance and is not recommended.

Pump about 50 mL of methanol or acetonitrile through the cartridge at a flow rate of 1 mL/min.

4.

Flush the system thoroughly with water. The cartridge is now ready for use.

If the cartridge is kept wet, this wetting operation need only be carried out the first time a cartridge is used.

Dextro-Pak Cartridge

3

[ Care and Use Manual ]

IV. SAMPLE FILTERING

4. Slowly pass the sample through the Sep-Pak C18 cartridge, reject the first 2 mL, and collect the next few milliliters for analysis.

1.

Filter all samples through a 0.45 µm membrane filter just

b. Removal of Salts and Acids

before injection to remove particulates. Suitable replacement filters are provided in the Waters Aqueous Sample

Do not expose silica bonded phase packing to a pH of greater than 8

Clarification Kit, P/N WAT026865. Suitable disposable filters

or less than 2. If samples are strongly acidic or alkaline, neutralize

with a larger surface area are also available.

prior to injection. Many organic acids and salts elute at or near the void volume. Some

2. Use an (in-line Precolumn Filter (P/N WAT084560) immedi-

larger organic acids may be retained long enough to interfere with

ately before the column to protect it from any particulate matter

the analysis of the sugars. Remove these materials by using ion

in the sample or mobile phase.

exchange resins.

3. Sugar samples often contain polysaccharides or other high

•

molecular weight materials, such as proteins, which will adsorb

Pass the sample containing the ionizable compounds through a small open column (such as a Pasteur pipette) filled with resin.

to the cartridge packing and become difficult or impossible to •

remove. Use guard columns packed with C18 for all samples,

Alternately, add a small amount of resin to the sample and shake.

except very pure sugar solutions, to avoid contamination of the cartridge packing material.

When using ion-exchange resins, keep in mind that the resins are supplied in a wetted condition and that this water may dilute the

4. Place the insert inside the inlet of the Dextro-Pak cartridge.

sample to a significant degree.

5. The Guard-PakTM inserts are supplied dry and must be wetted before use. To wet, place a few inserts under methanol in a

Some sugars may be adsorbed by the resin and must be washed

Buchner flask and draw a vacuum of 635 mm (25-inch) for a

out for full recovery. Certain resins may contain a strongly acidic

few minutes, breaking the vacuum a couple of times, to ensure

functionality causing inversion of sugars, such as sucrose that are

that all air is sucked from the inserts. The inserts can be stored

prone to hydrolysis.

in methanol in a small jar until needed. When the insert in use

A mixed bed, ion-exchange resin that has been used successfully for

becomes plugged, simple remove it and replace it with a fresh,

sample cleanup is Amberlite MB-i®. Amberlite MB-i® is available

wetted insert and restart the flow. Flush the methanol from the

from Sigma Chemical Company, St. Louis, MO (Catalogue Number A

system for about 15 minutes.

7393).

a. Removal of Lipids and Polymeric Materials V. TEST CONDIT IONS

Use the following procedure to extract lipids and polymeric material from your sample:

Waters cartridges are thoroughly tested for adherence to our

1. Fill a 10 mL syringe with 5 mL methanol and slowly flush

specifications. However slight variations in your results may occur

the methanol through a Waters SepPak® C18 cartridge (P/N

depending on the equipment used. Therefore before attempting

WAT020515) to activate the packing material.

the first analysis, test sample makeup, equipment settings and

2. Wash the methanol from the cartridge with two 5 mL portions of

condition, perform the test sample run given here for your new

pure water.

cartridge and record the results (retention time and the settings

3. Blow excess water from the cartridge with the syringe.

used). Use these results for comparison throughout the life of your cartridge.

Dextro-Pak Cartridge

4

[ Care and Use Manual ]

a. Cartridge Test Procedure

V I. COLUMN EFFICIENC Y

1. Install cartridge and condition it according to the startup Waters measures cartridge efficiency by using the 5 sigma plate

procedure.

count method. This is determined by measuring the peak width

2. Make up a standard solution using:

at 4.4% of the peak height. Unlike half-peak height and tangent

25 mg glucose

methods used by other manufacturers, this stringent method takes

45 mg maltose

into consideration peak asymmetry.

80 mg maltotriose

N = 25

160 mg ethanol

2

Confirm the efficiency of the Dextro-Pak cartridge by performing the

3. Weigh out 160 mg of ethanol in a capped sample vial, then add

test below.

4 mL water.

1. Inject a 2% w/V solution of sucrose at a flow rate of 1 ml/min

4. Weight out the sugars and dissolve in the water/ethanol.

( ) VR W

and a chart speed of 10 cm/min.

Note: Accuracy for this test need only be ± 5% unless this mixture

2. Record the plate count efficiency of new cartridges before they

is being used as a quantitation standard. Use this mixture the

are used.

same day as prepared to avoid microbial degradation.

3. Track subsequent loss of efficiency by repeating this test.

5. Use water as the mobile phase at 1 mL/min, Model 410 Refractive Index Detector at 64x, sensitivity 20. Inject 10

Note: Lack of required efficiency is not always dependent on the condi-

to 40 µL.

tion of the cartridge. It also depends on the condition of the liquid chromatographic system.

6. If you do not obtain a chromatogram similar to that shown in the

Contact our Waters representative for details on how to test or

figure below repeat steps 1 through 3.

band broadening caused by the LC system. Retain the results as a performance test.

V III. CARTRIDGE LIFE Liquid chromatography cartridges have a finite life influenced by such factors as their care and use, number of injections, sample an solvent cleanliness, frequency of solvent changeover and handling and storage procedures. If a change is observed in the retention of a particular compound, resolution between two compounds, or peak shape, tae immediate steps to determine the reason for the change. Until you have made this determination, you must not rely upon the results of any separation using the cartridge.

K = Dextro-Pak Cartridge

Important Note: Before running the first analysis on your new

Vn - V 0

cartridge, perform the test sample separation given in the test

V0

conditions section. 5

[ Care and Use Manual ]

IX. OPERAT ING T IPS

X. CARE & TROUBLESHOOT ING PROC EDURES

•

a. Column Storage

The usual flow rate is 1 mL/min. Slower flow rates will produce slightly improved separations. Faster flow rates are limited

If the Dextro-Pak cartridge is to be stored for a significant period (up

only by the permissable upper limit of the 8x10 Holder. •

to 12 hours), lace the effluent line in the mobile phase supply vessel

Dedicate your cartridge to sugar analysis. Using the cartridge

and recirculate the mobile phase at a reduced flow rate o 0.2 ml/

for other reversed-phase analyses may adversely affect its abil-

min.

ity to properly separate sugars. •

b. Long Term Storage

Avoid strong solvents to prolong useful cartridge life. The water

•

mobile phase will hydrolyze the bonded phase very slowly.

of time (more than 12 hours), a 20:80 acetonitrile: water

Solvents such as 100% methanol or acetonitrile may wash off

mixture will inhibit microbial growth during cartridge storage.

some of the hydrolyzed bonded phase, with a consequent partial

If stored in this manner; the Dextro-Pak cartridge is ready for

loss of retention. Rely on guard columns to protect the cartridge

immediate use once the solvent is flushed from the system.

from adsorbed compounds and stray particulate matter. •

If it is necessary to store the cartridge for an extended length

•

If it is necessary to wash the cartridge, use a mobile phase of

If the cartridge is removed from the 8x10 Holder, it must be sealed with the caps originally supplied with the cartridge.

20% acetonitrile/80% water. Because this is a weak solvent,

•

the hydrolyzed bonded phase remains in place and continues to

If the cartridge dries out during storage, re-wet the cartridge using the startup procedure. To avoid drying of the cartridge,

provide the required sample retention.

store in a stoppered test tube filled with 20:80 acetonitrile: water. This will also ensure that no microorganism growth will occur inside the packing.

VR INJ ECT W N = Column efficency ( plates) V R = Volume to pea k a pex (mL) W = Volume at 4.40% of pea k heig ht (mL)

Dextro-Pak Cartridge

6

[ Care and Use Manual ]

Table Troubleshooting Procedures Problem

Cause

Solution

Excess pressure buildup

In-line filters lugged 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 cartridge and backflush the prefilter only with mobile phase at 9 ml/min until the particulates are backflushed out.

Excess pressure buildup

Guard column clogged with particles or adsorbed materials

Replace spent guard column with fresh packing.

Fluctuating backpressure

Cartridge inlet frit plugged with particulates.

Always use an in-line filter or guard column to prevent this. Reverse cartridge to try to wash out particulates at normal flow rate.

Gas in mobile phase

Check degassing procedure.

“Spurious” peaks, not due to sugars

Faulty pump operation Elution of salts and/or acids

Keep mobile phase heated to 70 °C. Inspect pump check valves and carry out “Ramp Test” (consult the operator’s manual for your pump). Check sample cleanup procedures.

Leaking mobile phase

Fittings in bad state

Tighten fittings properly.

Variable elution times of repair

Variations in flow rate

DO NOT OVERTIGHTEN. Replace worn fittings,o-rings, or seals. Check system for faults, leaks, especially pulsations from pump.

Band broadening or loss of resolution

Guard column fouled

Replace spent guard column with fresh packing.

Analytical cartridge fouled or old

Try washing with 20% acetonitrile, 80% water. If cartridge does not recover, use a new cartridge.

XI. WARRANTY

XII. ORDERING INFORMAT ION

Waters warrants its high-performance liquid chromatography

Item

Part Number

cartridges in accordance with the following terms and conditions:

Compression screws and ferrules (5 pk)

WAT025604

Waters will repack or replace (at our discretion) without cost, any

Guard-Pak~” Inserts Resolve”’ C1e (10 pk)

WAT085824

cartridge that fails to perform satisfactorily if notified within 60 days from receipt of the cartridge. Any cartridge returned must have a prior Return Authorization Number assigned by the Waters Customer Service Department. Approval is subject to the following exclusions: •

Physical damage to the cartridge because of misuse or abuse.

•

Chemical damage to the packing material because of use with incompatible solvents or buffers, or at an incorrect pH. •

Physical damage to the packing material because of operation at incorrect temperatures or pressures.

•

High internal pressures which have occurred in the cartridge due to the improper solvent or sample filtration practices causing particulate buildup or precipitation in the cartridge or end fittings due to high internal pressures which have occurred because of improper solvent or sample filtration practices.

Dextro-Pak Cartridge

7

[ Care and Use Manual ]

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

©007 Waters Corporation. Waters, The Science of What’s Possible, Dextro-Pak, Guard-Pak, and Sep-Pak are trademarks of Waters Corporation. Amberlite and Triton are registered trademarks of Rohm and Haas Company.

November 2007 WAT010261TP Rev 2 VW-PDF

Dextro-Pak Cartridge

8

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com