Waters Gen-Pak FAX Columns User Manual

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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

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The inclusion of EDTA in the buffers is recommended to protect the

DNA from nucleases during and after the chromatography1. Filter and

vacuum degas the buffers before use.

Useful chromatography can be obtained with sodium phosphate

buffer, but the phosphate coprecipitates with the DNA in the common

ethanol-precipitation procedures.

c. Equilibration and flow

Rigorous control of column equilibration is essential for predictable
and reproducible chromatography. Before injection, equlibrate the

column with 10 column volumes of buffer at initial chromatographic

conditions. The column can be used at flow rates from below 0.5 to

1 mL/min, but 0.75 mL/min is recommended for general use.

d. Temperature Effects

The conformation and intermolecular hydrogen bonding of DNA

fragments, and therefore the Gen-Pak FAX separation of those

fragments, are affected by temperature. At higher temperatures, the

structure of DNA is more open or relaxed so more ionic groups can

interact with the column. In general, higher ionic strength is required

as temperature is elevated.

Since conformation is dependent on base sequence, variations in

temperature over the range of 30 to 60 °C can be used to optimize a

particular separation. Where elevated temperatures are not required,

the column should still be operated at a controlled temperature,

typically 5 °C above ambient, to ensure reproducibility.

e. Detection

The separation of subnanogram quantities of DNA can be monitored

at 260 nm, provided that the buffers are pure and free of UV

a b s o r b i n g c o n t a m i n a n t s . A s s e s s b l a n k g r a d i e n t s o n a p r o p e r l y

equilibrated column prior to performing actual sample

chromatography.

f. Sample Size

D e p e n d i n g u p o n t h e s i z e d i f f e r e n c e s a m o n g t h e f r a g m e n t s t o b e

isolated, as much as 50 to 100 μg of DNA can be applied to the

column. Injection volume is essentially unlimited as long as the ionic
strength of the sample is at least 0.1 M less than that required to
elute the fragment of interest. For injection volumes above 100 μL,

the sample should be at pH 8.

Excellent recoveries, in most cases more than 95 percent, of

biologically active material have been obtained purifying samples

containing fragments of 5000 base pairs and less.

g. Sample Treatments

DNA fragments generated by restriction enzyme digestion or

from the polymerase chain reaction may be extracted with phenol/

chloroform prior to chromatorgraphy. However, the reaction may be

injected directly since most proteins are completely unretained under

conditions used for the separation (i.e., 0.30 M NaCl).

In either case perform these steps before injection:

1. Remove particulate material from samples by centrifugation or

filtration through a 0.45 µm filter.

2. Heat treated samples at 37 °C for 10 min prior to injection to

disrupt any reannealed termini of the DNA fragments.

h. Gradients

Typical DNA fragments elute from the Gen-Pak FAX column between

0.4 and 0.75 M NaCl at 30°C. Equilibrate the column to 0.1 M below
the ionic strength required to elute the smallest fragment of interest.
Useful gradients are usually 5 to 10 mM/min at 0.75 mL/min.

i. Column Washing

Upon completion of each separation, wash the column with a small

volume of 20 - 40% acetic acid to maintain its chromatographic

characteristics as well as to eliminate sample carry-over from injec­tion to injection.

Procedure

After the last fragment elutes, perform the following procedure:

1. Flush the column with 5 mL of buffer B.

2. Pump or inject 3 to 5 mL of 20 - 40% acetic acid onto the

column. This may be performed with the column at 60 °C.

3. Flush the column with about 5 mL of buffer B.

4. Reequilibrate the column for the next sample injection.

Note: Washing the column with sodium hydroxide does not perform as

well as the above acetic acid procedure and is not recommended.

Gen-Pak FAX Columns 4

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Absorbance at 260 nm

Minutes

0

10 35

1

2

3

4

5-7

8-11

0.2

Column: Gen-PakôFAX (4.6 mm x 100 mm)
Buffer A: 25 mM Tris/Cl, 1mM 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

V. eXAMPLe UsInG HAe III PLAsMID DIGesteD DnA

This section shows an example of how to develop a method using

Gen-Pak FAX columns. The example in this chapter can serve as a a

model for adapting this technique to the requirements of a particular

sample.

The example describes the evolution of the separation of a restriction

enzyme digest, the Hae III digest of ØX 174 RF DNA. This DNA sample
contains 11 fragments ranging in size from 72 to 1353 base pairs.

Table 1: Number of Base Pairs in Each Fragment

Fragment Base Pairs

1 72

2 118

3 194

4 234
5 271

6 281
7 310

8 603
9 872
10 1078

11 135 3

a. Initial Gradient

Chromatography is initally performed using a relatively steep

ionic strength gradient. As Figure 3 shows, these conditions do not

completely resolve the 234, 271, 281, and 310 (peaks 4-7) nor the
603, 872, 1078, and 1353 (peaks 8-11) base pair fragments.

Figure 3: Initial Separation uf 2 μg of Hae III Digest of ØX 174 RF
DNA at 30°C using a Steep Salt Gradient

b. Gradient Optimization

Resolution can often be improved by systematic adjustment of

gradIent conditions. In the current example, examination of the inital

chromatorgram indicates a more shallow gradient is required.

c. Calculating NaCl Concentration

It is possible to calculate the approximate NaCl concentration

required to elute the first (72bp) and last ( 1353 bp) restriction

fragments from the data obtained in the inital chromatography.

First, determine the system delay volume from the point of gradient

formation to the detector cell.

d. Calculating System Delay Volume

To calculate the system delay volume from point of gradient

formation to detector cell:

1. Remove the column from the system, insert a union, and set the

detector to 260 nm.

2. Flush the pump and solvent lines with eluent A ( Milli-Q water)

3. Start the flow of eluent B (0.01% acetone in Milli-Q water)

4. Monitor the detector output until it rises to the new, higher value

of about 0.032 absorbance units if a cm detector cell is used.

The system delay volume corresponds to the volume that has flowed

from the start of eluent B to the midpoint of the absorbance rise plus

the excluded column volume of 0.6 mL.

e. Using a More Shallow Gradient

The system delay volume for this instrument was 7.5 mL,
corresponding to 10 minutes at 0.75 mL/minute.

Under inital conditions, elution occurs as follows:

Gen-Pak FAX Columns 5

• The 72 base-pair fragment elutes at 22 minutes, reflecting

the NaCl concentration at 12 minutes in the gradient table of

0.56 M NaCl

• The 1353 base-pair fragment elutes at 0.65 M NaCl

Figure 4 shows the results of changing to a more shallow NaCl

gradient, from 0.54 to 0.67 NaCl, over the same 30 minute time

interval. As this figure shows, the change significantly imrproved the

resolution.

[ Care and Use ManUal ]

Absorbance at 260 nm

0.2

0

10

1

2

3

4

5-7

8

9

10

11

35

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, 1mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: Load at 30% B to 54% B in 0.01 min.

Then 54 to 67% in 30 min., linear.
Flow:
Temperature:

Minutes

0.75 mL/min

30˚C

Absorbance at 260 nm

1

2

3

4

5-7

8

9

10

11

0

0.2

Column: Gen-Pak

ô

FAX (4.6 x 100 mm)

Buffer A: 25 mM Tris/Cl, 1 mM EDTA, pH 8.0
Buffer B: 25 mM Tris/Cl, 1mM EDTA, 1.0 M NaCl, pH 8.0
Gradient: Load at 30% B to 54% B in 0.1 min.

Then 64 to 77% in 30 min., linear.
Flow: 0.75 mL/min
Temperature:

Minutes

60˚C

35

10

Figure 4: Separation of 2 μg of Hae III Digest of ØX 174 RF DNA at
30 ˚C using a Shallow Salt Gradient

f. Temperature Optimization

Ad ju st ment s of gra di ent s lo pe m ay no t pro du ce a de qu ate

r e s o l u t i o n o f D N A f r a g m e n t s i n e v e r y c a s e . H o w e v e r , c h a n g e s i n t h e

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

relative retention of the fragments. As Figure 5 shows,

chromatography of the Hae III digest of ØX 174 at 60 °C yields
slightly less resolution of the 72 and 118 base-pair fragments.

Since it is not possible to predict the best separation temperature for

a particular sample, it is often useful to compare separations at two

temperatures, such as 30 °C and 60 °C. Although increased NaCl

concentration is required to elute DNA fragments from the column at

elevated temperatures, chromatography at such temperatures does

not shorten the column life nor affect the biological activity of the

collected samples.

Figure 5: Separation of 2 μg of Hae III Digest of ØX 174 RF DNA at
60 °C using a Shallow Gradient

VI. tRoUBLsHootInG

Liquid chromatography columns have a finite life that is directly

related to the care and use they recieve. Column life is influenced

by the number of injections, cleanliness of sample and solvent,
frequency of solvent changeover, and procedures for handling and

storage.

If you observe a change in the following areas, take immediate steps

to determine the reason for the changes.

• Retention of a particular compound

• Resolution between two compounds

• Peak shape

Until the cause of the change is determined, you should question the

validity of the results of any separation using the column

a. Pressure Buildup/Loss of Resolution

With continued use, it is possible that excessive pressure buildup

(more than 4,000 psi at 1.0 mL/min at 25°C) or loss of resolution

may be experienced. These may both be caused by the buildup of

particulates from samples or eluents on the inlet filter. Filtering

samples and eluents greatly extends inlet filter and column life.

b. Possible Corrective Actions

If pressure buildup or loss of resolution occurs, any of the following

corrective actions, listed in order preference, may help:

• Perform a 20 - 40% acetic acid wash as described in Column

Washing Section. The length of the wash can be extended to

10 to 15 minutes.

• R e p l a c e t h e i n l e t f i l t e r w i t h a n e w o n e ( W a t e r s P a r t N u m b e r
WA T015 715)

Gen-Pak FAX Columns 6

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©2008 Waters Corporation. Waters, The Science of W hat’s
Possible, and Gen-Pak are trademarks of Waters Corporation.
Milli-Q is a trademark of Millipore Corporation.

October 2009 WAT015493 Rev 6 VW-P DF

Gen-Pak FAX Columns 7

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