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Agilent ZORBAX Carbohydrate Analysis Manual

1. 4 Maltose 20 mg mL 5 Lactose 20 mg mL 4 6 8 Retention Time min Agilent Zorbax Carbohydrate Analysis Column Datasheet Each column is individually tested in a normal phase mode to ensure a quality packed bed structure The actual QC test and performance of your column is de scribed on the Column Performance Report enclosed with your column Safety Considerations e All points of connection in liquid chromatographic systems are potential sources of leaks Users should be aware of the hazards from such leaks due to the toxicity or flammability of the chosen mobile phases Because of the small particle size dry Zorbax packings are respirable Columns should only be opened in a well ventilated area Operational Guidelines e The direction of flow is marked on the column While generally not harmful to the column reversing flow should be avoided except to attempt removal of inlet pluggage see Column Care section Zorbax Carbohydrate Analysis Columns are shipped containing acetonitrile As the column is to be used with an aqueous mobile phase flush it first with ac etonitrile water 75 25 then with at least 20 column volumes of the chosen mobile phase e Zorbax Carbohydrate Analysis Columns are compat ible with water and all common organic solvents e Use of a guard column is recommended to protect the Zorbax Carbohydrate Anaylsis Column and extend its useful lifetime see Part Numbers e Maxim
2. General Description The Zorbax Carbohydrate Analysis column is an ap plication specific column for the separation of mono and other saccharides The packing is batch tested to ensure the separation of a mixture of sug ars which is representative of the application usage Figure 1 This packing is produced by reacting 3 aminopropylsilane with Zorbax SIL silica par ticles The reaction conditions used to produce Zorbax Carbohydrate were specifically developed to maximize surface coverage with a monolayer bonded phase The uniform spherical Zorbax par ticles are 5 um in diameter and have a controlled pore size to give optimum column efficiency Col umns are packed to a uniform bed density using a proprietary high pressure slurry loading technique Column Characteristics To ensure product quality every batch of packing is tested for its ability to separate a group of mono and disaccharides Typical chromatographic performance for a 4 6 mm ID x 150 mm column is shown in Figure 1 Actual performance of your column may be slightly different Figure 1 Typical Performance of Zorbax Carbohydrate Analysis Column OPERATING CONDITIONS Column Zorbax Carbohydrate Analysis 4 6 mm ID x 150 mm 5 um Mobile Phase 5 25 Acetonitrile Water Flow Rate 1 4 mL min Temperature 30 C Detector HP1100 RID 30 C Sample Volume 3 uL in 50 50 Acetonitrile Water Peak Identity 1 Fructose 3 2 mg mL 2 Glucose 12 mg mL 3 Sucrose 12 mg mL
3. should be filtered before injection into the column Zorbax guard columns and a hardware kit are recom mended for use to extend column life see Part Num bers If solvent flow appears to be restricted high col umn back pressure check first to see that solvent flow is unobstructed up to the column inlet If the column is the source of the high backpressure there may be particulate matter on the inlet frit An initial attempt should be made to clear any inlet debris by back flushing 25 30 mL of mobile phase through the column If this fails to return the column to near its original back pressure the inlet frit should be changed To remove the frit loosen the nut at the col umn inlet taking care not to turn the end fitting it self Then remove the fitting taking care not to dis turb the column bed The frit should drop out when the fitting is tapped sharply on a hard surface Install a new frit and carefully tighten the fitting The columns are shipped in acetonitrile Equilibrate the column with the desired mobile phase e g 75 25 acetonitrile water by volume until a steady baseline is obtained on the detector Many column volumes of mobile phase may be necessary for equilibration to occur after changes in mobile phase composition The number of column volumes required depends on the system in use Since columns have 1 16 terminations a short 1 4 wrench should be used in attaching the column to the HPLC to prevent overtight
4. content that is used the more soluble are the sugars in the mobile phase resulting in decreased retention The typical range for the mobile phase is 60 to 75 acetonitrile Reten tion time of the sugars may decrease with extended column use By increasing the amount of acetonitrile in the mobile phase retention can be adjusted to the appropriate time Temperature Control of temperature is important for maintaining retention time reproducibility and for maintaining re fractometer baseline stability Resolution may be improved when column tempera ture is above room temperature Operation at 30 C or 35 C may give better resolution of a carbohydrate mixture and therefore temperature should be inves tigated for optimizing resolution Small changes in temperature has a noticable effect on baseline stability of the Refractive Index Detector RID Therefore it is important to shield the HPLC column and system from temperature fluctuations Use of a thermostated column compartment and use of a RID with temperature stabilization is highly rec ommended for best performance Flow Rate Adjusting flow rate is appropriate for minimizing analysis time Typical flow rates are 1 to 1 5 mL min The column should not be operated above the back pressure limit 400 bar Column Care The inlet frit on these columns has a nominal poros ity of 2 um Samples that contain particulate matter larger than 2 um may plug the column inlet frit and
5. ening Overtightening the fittings can damage the fitting and necessitate re placement Ordering Information Agilent Carbohydrate Analysis Columns 5 pm Part No 46mm ID x 150 mm 843300 908 4 6 mm ID x 250 mm 840300 908 Guard Column 4 6 mm ID x 12 5 mm 4 Pack 820950 908 Guard Column Hardware Kit 820888 901 Syringe Filters for Aqueous Solutions 100 Pack Regenerated Cellulose 0 45um 13 mm for 1 10 mL sample size 30 mm for 1 50 mL sample size 0061 3365 0061 3364 j Agilent Technologies Printed in USA 7 21 03 Part No 820629 008 C
6. um operating pressure is 400 bar 6000 psi e Maximum operating temperature is 60 C e NOTE Zorbax columns are designed for high stability at low pH e g pH lt 5 However all silica based packings have some solubility in pH gt 6 aqueous mo bile phases Therefore when using silica based columns under conditions of pH gt 6 maximum column lifetime is obtained by operation at low temperatures lt 40 C using low buffer concentrations in the range of 0 01 to 0 02M Column stability at pH gt 6 is also enhanced by avoiding phosphate and carbonate buffers ref H A Claessens M A van Straten and J J Kirkland J Chromatogr A 728 1996 259 Avoid exposure to aldehydes and methyl ketones which may form Schiff s bases with the bonded phase e The column should be filled with 100 acetonitrile for long term storage Sample Preparation The cleaner the sample to be analyzed the longer the column life Care should be taken to remove lip ids and ionic contaminants from the sample before injection onto the column This will prolong column lifetime Routine filtering of samples should be con sidered if there is the possiblity that the samples contain particulate matter see Part Numbers for sy ringe filters Mobile Phase Selection As organic content e g acetonitrile of the mobile phase increases the resolution of the sugars may im prove with an accompanying increase in analysis time The higher the aqueous

Agilent ZORBAX Carbohydrate Analysis Manual

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