Waters Gen-Pak FAX Columns User Manual

[ Care and Use ManUal ]

Gen-PAk fAX CoLUMns

Contents

I. IntRoDUCtIon

II. InstALLAtIon

III. GeneRAL UsAGe ConsIDeRAtIons

IV. GeneRAL MetHoD DeVeLoPMent GUIDeLInes

V. eXAMPLe UsInG HAe III DIGesteD DnA

VI. tRoUBLsHootInG

Waters Gen-Pak™ FAX columns offer the highest resolution available in
anion- exchange HPLC of nucleicacids. The Gen-Pak FAX column
contains a weak anion exchanger based on DEAE functionalized non-porous
resin. It contains 2.5 µm particles and is well suited for analytical and
micro-preparative applications.

I. IntRoDUCtIon

This manual covers the use of the Waters Gen-Pak FAX column. The

Gen-Pak FAX column is a 4.6 x 100 mm steel column containing

a p o l y m e r - b a s e d h i g h - p e r f o r m a n c e a n i o n - e x c h a n g e p a c k i n g . T h e

c o l u m n i s d e s i g n e d t o p e r f o r m h i g h - r e s o l u t i o n a n a l y s i s a n d

p u r i f i c a t i o n o n v a r i o u s n u c l e i c a c i d s p e c i e s ( u p t o a t l e a s t

5,000 base pairs) such as DNA restriction

reaction (PCR) products, plasmids and synthetic oligonucleotides.

fragments, polymerase chain

Gen-Pak FAX Columns 1

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Tube

Critical distance to be determined by each
application (union, column fitting, etc.)

Compression screw or nut

Ferrule

End must be straight and smooth
to achieve maximum column
efficiency

II. InstALLAtIon

a. Column

Before you attach a new column, attach a union in place of the column and

flush the HPLC system free of previously used solvent.

Note: To achieve optimum performance from each column, use tubing of

0.010 inch i.d. or smaller to connect the

column to the detector.

Procedure to install the column:

1. Remove the end plugs from the column. (Be sure to save the end

plugs for use when the column is removed from the system for

storage.) The column outlet is indicated by an arrow on the label

showing the direction solvent should flow.

2. Finger-tighten the tubing compression screws and then

wrench-tighten them 1/4 to 1/2 turn.

Note: Do not over-tighten; this damages the connection.

3. Make sure that the compression fitting is in good condition and

properly prepared as shown in Figure 1. Because fittings may

vary, it is important to verify that the tubing in your system

bottoms in the column end nuts.

injector to the column and the

3. Slide the compression screw, followed by the ferrule (large end

of the taper first) over the tube. Be certain to bottom the tube in

the fitting seat to assure a leak-free connection.

c. Testing

To validate system and column performance, test each new column

with a standard sample. T his provides a basis for detecting system

component changes and troubleshooting.

Suggested samples include:

• Hae Ill digest of ØX 174 RF DNA

• BstN I digest of pBR322 DNA

III. GeneRAL UsAGe ConsIDeRAtIons

This section presents guidelines that can extend the life of your

column and help you achieve the best possible chromatographic

results.

a. Samples

When preparing and using samples:

• Centrifuge or filter the sample before injection.

Figure 1: Ferrule and Compression Screw Assembly

b. Tubing

Follow the next three steps to cut tubing to connect a new steel

column or to improve the end connections on existing fittings:

1. Using a three-cornered file with a cutting edge, scribe the

circumference of the tubing at the desired break.

2. Grasp the tubing on both sides of the scribe mark with

cloth-covered pliers (to prevent marring the tube surface) and

gently work the tube back and forth until it separates.

Gen-Pak FAX Columns 2

• Do not inject samples containing microparticulates.

b. Solvents

When preparing and using solvents:

• Use pure buffer salts.

• Use of high quality reagents, water, and solvents is critical in

preparing chromatography eluents. Fouling of the Gen-Pak FAX

resin, leading to a loss in retention and / or separation efficiency,

occurs faster on this column chemistry due to the small surface

area of the non-porous resin particles. As such, all prepared HPLC

eluents should be filtered through a solvent compatible 0.45 µm or

0.22 µm depth filter.

• Solvents should be degassed (vacuum filtration, sonication or

helium sparged) prior to use.

• Use of organic solvents other than methanol or acetonitrile at

greater than 10% in water is not recommended.

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Column: Gen-Pak FAX™ (4.6 x 100 mm)
Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
Buffer B: 25 mM Tris/Cl, 1 mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: 30 to 100% B in 30 min., linear
Flow: 0.75 mL/min
Temperature: 30˚C

0.30

0

18

35

Minutes

121 bp

382 bp

1060 bp

929 bp

1857 bp

Column: Gen-Pak FAX (4.6 x 100 mm)
Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
Buffer B: 25 mM Tris/Cl, 1 mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: 30 to 100% B in 30 min., linear
Flow: 0.75 mL/min
Temperature: 30˚C

Absorbance 260 nm

0.30

0

18

35

Minutes

121 bp

382 bp

1060 bp

929 bp

1857 bp

c. Operating Pressure

Do not excee d 4,000 psi op erating pre ssu re or ab out

1 mL/min at 25 °C.

d. pH range

Stay within a pH range of about 1.5 to 12 (do not use concentrated

acids or bases).

e. Flow

Make flow rate changes in a gradual manner (less than 1 mL/min) to

avoid column voiding. Never reverse flow in the column.

f. Cleaning

Clean the column between nucleic acid injections with 3 to 5 mL of

with 3 to 5 mL of 20 - 40% acetic acid.

g. Storage

When you store a column for less than 24 hours, you typically do

not need to follow special storage procedures. However, be sure

that the column never dries out; this can degrade chromatographic

performance.

a. Separating Double Stranded DNA Fragments

DNA fragments are usually isolated using gel electrophoresis.

Although resolution is good, the technique has limited mass capacity,

often gives low yields of extracted fragments, and is time consuming.

The Gen-Pak FAX column is a useful alternative for the

r a p i d p u r i f i c a t i o n a n d a n a l y s i s o f s u c h n u c l e i c a c i d s p e c i e s .

Separations are often accomplished in about 30 minutes. Recoveries

of biologically active material directly from the column are usually

greater than 95%. Direct UV monitoring of the column effluent

provides subnanogram sensitivity without the need for indirect

visualization via ethdium bromide staining or autoradiography.

Depending upon sample complexity, as much as 50 to 100 μg

of DNA can be separated in a single run. Since the separation is

based primarily upon the overall charge of each fragment, smaller

fragments elute prior to larger ones using an ionic strength gradient

as shown in Figure 2.

Figure 2: Separation of 3.0 μg BstN I Digest of pBR322 DNA

For longer term storage, follow the procedure described below:

1. Flush the column with approximately 25 mL of Milli-Q

remove salts.

2. Flush the column with approximately 4 mL of a mixture of 10

percent methanol and 90% Milli-Q water.

3. Disconnect the column.

4. Screw end plugs firmly in place and return the column to its box.

5. Store the column at 4 °C.

Note: Before reusing the column, flush 4 with 25 mL of Milli-Q water

to remove any methanol prior to introducing buffers.

IV. GeneRAL MetHoD DeVeLoPMent GUIDeLInes

This section discusses the use of Waters Gen-Pak FAX columns for

purifying DNA restriction fragments, polymerase chain reaction (PCR)

products, plasmids, and synthetic oligonucleotides.

®

water to

b. Buffers

The preferred chromatographic buffer is 25 mM Tris/Cl. 1mM EDTA,

pH 8.0. The recommended buffers for DNA fragmet separations are:

• Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0

• Buffer B: 25 mM Tris/Cl, mM EDTA, 1.0 M NaCl, pH 8.0

Gen-Pak FAX Columns 3