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[ Care and Use ManUal ]

XBridge

™

Columns 5

III. sCalInG up/down IsoCratIC methods

The following formulas will allow scale up or scale down, while maintaining

the same linear velocity, and provide new sample loading values:

If column i.d. and length are altered:

= F

)

Load

= Load

)

Injection volume

= Injection volume

)

Where: r = Radius of the column

F = Flow rate

L = Length of column

1 = Original, or reference column

2 = New column

IV. Troubleshooting

Changes in retention time, resolution, or backpressure are often due to column

contamination. See the Column Cleaning, Regeneration and Storage section of

this Care and Use Manual. Information on column troubleshooting problems

may be found in HPLC Columns Theory, Technology and Practice, U.D. Neue,

(Wiley-VCH, 1997), the Waters HPLC Troubleshooting Guide (Literature code

# 720000181EN) or visit the Waters Corporation website for information on

seminars (www.waters.com).

V. Column CleanInG, reGeneratIon and storaGe

a. Cleaning and Regeneration

Changes in peak shape, peak splitting, shoulders on the peak, shifts in reten-

tion, change in resolution or increasing backpressure may indicate contamina-

tion of the column. Flushing with a neat organic solvent, taking care not to

precipitate buffers, is usually sufficient to remove the contaminant. If the

flushing procedure does not solve the problem, purge the column using the

following cleaning and regeneration procedures.

Use the cleaning routine that matches the properties of the samples and/or

what you believe is contaminating the column (see Table 4 ). Flush columns

with 20 column volumes each of HPLC-grade solvents (e.g., 80 mL total for

4.6 x 250 mm column) listed in Table 4. Increasing mobile phase temperature to

35-55 ˚C increases cleaning efficiency. If the column performance is poor after

cleaning and regeneration, call your local Waters office for additional support.

Flush XBridge HILIC columns with 50:50 acetonitrile:water to remove polar

contaminants. If this flushing procedure does not solve the problem, purge the

column with 5:95 acetonitrile:water.

To clean polar contaminants from XBridge Amide columns, run a 25 minute

gradient from 0-100% water. Please note that as aqueous concentration

increases, backpressure will rapidly increase as well. Reduce flow rate when

operating at greater than 60% aqueous. Repeat if necessary.

Table 4: Cleaning and Regeneration Sequence or Options

* Use low organic solvent content to avoid precipitating buffers.

b. Storage

For periods longer than four days at room temperature, store the reversed-

phase XBridge columns and XBridge Amide columns in 100% acetonitrile. Im-

mediately after use with elevated temperatures and/or at pH extremes, store in

100% acetonitrile for the best column lifetime. Do not store columns in highly

aqueous (<20% organic) mobile phases, as this may promote bacterial growth.

If the mobile phase contained a buffer salt, flush the column with 10 column

vol umes of HPLC grade water (see Table 1 for common column volumes) and

replace with 100% acetonitrile for storage. Failure to perform this intermedi-

ate step could result in precipitation of the buffer salt in the column or system

when 100% acetonitrile is introduced. Run a gradient to 100% ACN in order

to flush all aqueous solvent from an XBridge Amide column prior to storage

in 100% ACN. Completely seal column to avoid evaporation and drying out

of the bed. For periods longer than four days, store XBridge HILIC columns in

95:5 acetonitrile:water. Do not store in buffered solvent. If the mobile phase

contained a buffered salt, flush the column with 10 column volumes of 95:5

acetonitrile:water (see Table 1 for common column volumes).

Polar Samples Non-polar Samples Proteinaceous Samples

1. water

1. isopropanol (or anappropriate

isopropanol/water mixture*)

Option 1: Inject repeated

aliquots of dimethyl sulfoxide

(DMSO)

2. methanol 2. tetrahydrofuran (THF) Option 2: gradient of 10% to 90%

B where:

A = 0.1% trifluoroacetic acid

(TFA) in water

B = 0.1% trifluoroacetic acid

(TFA) in acetonitrile (CH

CN)

3. tetrahydrofuran (THF) 3. dichloromethane

4. methanol 4. hexane

5. water

5. isopropanol

(followed by an appropriate

isopropanol/water mixture*)

Option 3: Flush column with 7M

guanidine hydrochloride, or 7M urea

6. mobile phase 6. mobile phase

1 2 3 4 5 6 7 8 9 10 11 12

[ Care and Use ManUal ] 1

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Waters XBridge Columns Manuel d'utilisateur Page 5

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