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 DNAPac PA200   

Document No. 065036 

Page 12 of 25 

 

  

 

5.4   Gradient slope 

Phosphodiester oligonucleotides generally exhibit good peak shape when the gradient slope is ~15 mM/mL (NaCl) 
or ~5 mM/mL (NaClO

4

). Higher values will generally result in shorter run times, but result in poorer resolution. 

Conversely lower values may produce improved resolution, but also require longer run times. 

 

 

5.5   Effect of pH and Solvent on Oligonucleotide Chromatography 

Use of elevated pH offers two advantages over chromatography at neutral pH. First, elevated pH allows control of 
hydrogen bonding interactions. At pH 11 and above, (pH 12.4 is the recommended upper limit for the DNAPac 
columns), Watson-Crick and poly-G hydrogen bonds break. Hence, at high pH chromatographic analysis of 
oligonucleotides with self-complementary sequences results in sharp, well-resolved peaks. Second, for each 
Thymine (T) and Guanine (G) residue, an increase in oligonucleotide charge is generated with rising pH values due 
to ionization of the tautomeric oxygen on these bases. Between pH 9 and 11, oxyanion formation on these bases 
increases retention of oligonucleotides in proportion to the number of T and G residues on the molecule. This offers 
opportunity to control of oligonucleotide selectivity with eluent pH. 

 

 

5.5.1   Effect of pH on Hydrogen Bond Interactions 

In the chart below, the chromatography of a PdG

12-18

 sample at 25°C and pH 8 (bottom trace) reveals the 

absence of identifiable peaks. Under these conditions, poly-G tracts form tetrad ladders with 8 hydrogen bonds 
between each set of bases. Chromatography at 85°C (pH 8, middle trace) dramatically increases the detector 
noise compared to pH8 and 25°C, but still no identifiable peaks are eluted. However, at 25°C and pH 12 each 
of the sample components are clearly eluted, and completely resolved from all the other components. 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

CHART 3 Controlling H-bond interactions in poly-G Tracts:  

Comparison of High pH and High Temperature

 

 

0

8

16

24

0.0

40.0

d(G)

12-18

25 °C, pH8

d(G)

12-18 

A 85 °C, pH8

d(G)

12-18 

25 °C, pH12

mA

26

0

Time (min)

3

2

1

Flow: 1.20 ml/min

0.33M NaClO

4

: 

33.4 %

66.7

75.0

36.4

0

8

16

24

0.0

40.0

d(G)

12-18

25 °C, pH8

d(G)

12-18 

A 85 °C, pH8

d(G)

12-18 

25 °C, pH12

mA

26

0

Time (min)

3

2

1

Flow: 1.20 ml/min

0.33M NaClO

4

: 

33.4 %

66.7

75.0

36.4

Содержание 062998

Страница 1: ...00 Document No 065036 Page 1 of 25 PRODUCT MANUAL DNAPAC PA200 ANALYTICAL COLUMN 4 x 250mm P N 063000 DNAPAC PA200 GUARD COLUMN 4 x 50mm P N 062998 DIONEX Corporation Document No 065036 Revision 01 20 August 2004 ...

Страница 2: ...T SLOPE 12 5 5 EFFECT OF PH AND SOLVENT ON OLIGONUCLEOTIDE CHROMATOGRAPHY 12 5 5 1 Effect of pH on Hydrogen Bond Interactions 12 5 5 2 Effect of pH on Retention 13 5 5 2 Effect of pH on Retention 13 5 5 3 Effect of Solvent on Retention 13 5 5 4 Effect of pH on Selectivity 14 5 6 EFFECT OF TEMPERATURE ON OLIGONUCLEOTIDE RETENTION 15 5 7 EFFECT OF TERMINAL BASE ON SELECTIVITY 16 5 7 1 Selectivity in...

Страница 3: ...TION 24 8 6 UNIDENTIFIED PEAKS APPEAR 24 8 7 DECREASED DETECTION SENSITIVITY 24 8 8 COLUMN PROBLEMS 24 8 9 PEAK EFFICIENCY AND RESOLUTION ARE DECREASING 24 8 10 SYSTEM PROBLEMS 24 8 10 1 High Detection Background Caused by the System 24 8 10 2 No Peaks Poor Peak Area Reproducibility or Unexpectedly Small Peak Area 25 8 10 3 Incorrect or Variable Retention Times 25 8 11 COLUMN CLEANUP 25 8 11 1 Hig...

Страница 4: ...capacity than is possible with conventional non porous materials and good durability This produces a column with oligonucleotide resolution superior to columns using 2 to 3 µm resins Resin Characteristics Particle Size 8 µm Pore Size non porous Cross linking 55 Ion exchange capacity 40 µeq column Latex Characteristics Functional Group quaternary ammonium ion Latex Diameter 130 nm Latex Cross link ...

Страница 5: ...ligonucleotide separations The choice of column depends upon the goal of the separation The DNAPac PA100 consists of a 13 µm substrate particle with 100 nm functionalized MicroBeads This column is available in a variety of formats and should be used when higher capacity is required and if scale up to semi preparative scale separations is anticipated The DNAPac PA200 consists of an 8 µm substrate p...

Страница 6: ...eration Requirements The oligonucleotide analysis systems should be configured with Dionex modules to provide the following attributes a All components of the fluid path are non metallic to eliminate column poisoning b Mobile phase components are kept under helium or nitrogen to minimize out gassing bubble formation in the detector cell On line degassing of eluents may be provided with the eluent ...

Страница 7: ...or H at low pH Temperature Limit 85 C Pressure Limit 4 000 psi Organic Solvent Limit 100 Acetonitrile or methanol if required for cleaning Chaotrope Limit 30 formamide 6 M Urea Note Use of these chaotropes will increase back pressure and reduce column lifetime Typical Eluents High purity water 18 megohm cm sodium chloride sodium perchlorate buffers sodium acetate and sodium hydroxide Detergent Com...

Страница 8: ...ra high purity solvents will usually be of sufficient purity to ensure that your chromatography is not affected by ionic impurities in the solvent At Dionex we have obtained consistent results using High Purity Solvents manufactured by Burdick and Jackson or Optima Solvents by Fischer Scientific When using an ionic eluent with solvent column generated back pressure will depend on the solvent used ...

Страница 9: ...DNAPac PA200 columns packed with both production and test resins are compared This procedure ensures that resins with the highest quality are used and produces consistent column performance Eluent 1 25 mM Tris pH 8 Eluent 2 25 mM Tris pH 8 1 25 M NaCl Flow rate 1 20 mL minute Detection Absorbance 260 nm Injection 25 µL Storage Solution Eluent 2 Gradient Time 1 2 Comments 0 0 68 32 Equilibration so...

Страница 10: ...te eluent The retention time of sulfate is used to measure the capacity of the column Peak efficiency and peak symmetry of sulfate are used to measure the packing quality of the column Retention times and resolution of chloride nitrate and phosphate are used to measure the overall selectivity of the column Eluent 1 1 28mM NaHCO3 1 35 mM Na2CO3 Flow rate 1 00 mL minute Detection Conductivity Suppre...

Страница 11: ...ions Heteropolymer Series Elution is influenced by the base composition especially G T terminal base sequence pH solvent concentration and eluent salt At pH 12 each T or G base contributes a negative charge from tautomeric oxygen atoms as pH shifts from 10 5 12 5 hydrogen bond interactions decrease yielding the expected chromatographic patterns 5 3 Effect of Salt Type on Oligonucleotide Elution 5 ...

Страница 12: ...ses Between pH 9 and 11 oxyanion formation on these bases increases retention of oligonucleotides in proportion to the number of T and G residues on the molecule This offers opportunity to control of oligonucleotide selectivity with eluent pH 5 5 1 Effect of pH on Hydrogen Bond Interactions In the chart below the chromatography of a PdG12 18 sample at 25 C and pH 8 bottom trace reveals the absence...

Страница 13: ...ce retention of oligonucleotides In some cases resolution of closely spaced or co eluting oligonucleotides may be assisted by adding solvent These effects can be seen clearly by comparing Chart 4 with Chart 5 The scales have been aligned to make this comparison easier CHART 5 Effect of Solvent on Retention 0 10 20 pH6 5 pH8 pH9 pH11 pH12 Time min 13 80 14 35 pH10 9 93 6 02 5 42 4 61 Flow 1 20 ml m...

Страница 14: ...he two are only partially resolved However at pH 10 5 and 11 this elution order is reversed due to the relative contributions of T and A to retention at these pH values The base composition of these 23 base oligos is 5 X G4C4A3T7 Y 3 and optimal resolution is observed at pH 10 5 CHART 6 Effect of pH on Selectivity 75 4 12 0 mAU Time Min 10 9 8 7 6 5 4 3 2 WVL 260 nm 1 Flow 1 20 ml min X GA Y TGA p...

Страница 15: ...n Nucleoside monophosphates and very short 2 3 base oligonucleotides may exhibit decreased retention times at elevated temperatures Nucleoside triphosphates and oligonucleotides greater than a few bases long usually exhibit increased retention as the temperature increases The chart below illustrates the influence of increased temperature at constant pH 8 As the chromatographic temperature increase...

Страница 16: ...nd T at either end are pH dependent At pH 9 A contributes more than T but at the higher pH values it contributes less Addition of solvent e g CH3CN tends to reduce retention and minimize hydrophobic interactions This may in some cases improve selectivity and resolution chart not shown CHART 8 Effect of pH on Retention by Terminal Base 15 mM mL NaCl gradient 0 CH3CN 5 X G6C3A5T9 Y3 pH 9 11 5 5 6 5 ...

Страница 17: ...on by Terminal Base 5 mM mL NaClO4 gradient 0 CH3CN 5 X G6C3A5T9 Y3 pH 9 11 7 8 9 10 1 99 mAU Time min 7 6 5 4 3 2 1 Flow 1 20 ml min X C Y A X A Y A X T Y A X G Y A X C Y T X C Y C X C Y G pH 9 pH 10 9 5 10 5 11 5 12 5 1 99 mAU Time min 7 6 5 4 3 2 1 Flow 1 20 ml min X C Y A X A Y A X T Y A X G Y A X C Y T X C Y C X C Y G pH 11 13 14 15 16 1 99 mAU Time min 7 6 5 4 3 2 1 Flow 1 20 ml min X C Y A ...

Страница 18: ... must be resolved eluents maximizing base specific retention would provide the best probability of success Examples of such samples include Identification of all primers in a multiplex PCR amplification cocktail QA QC of multiple primers in amplification based diagnostic kits identification of the different components in n 1 or n 1 impurities when troubleshooting nucleic acid synthesis protocols F...

Страница 19: ...to elute the oligonucleotide and thus the amount of salt eluting with it will be increased d Increasing the pH of the eluent will also generally increase retention of oligonucleotides but in a manner that allows control of oligonucleotide selectivity 6 2 Effect of High Temperature and High pH on Column Lifetime The combination of both high temperature and high pH reduces the useful life of the DNA...

Страница 20: ...adient is effective for resolving the full length oligonucleotide phosphodiesters up to 25 bases long from the n 1 components The same gradient of 5 mM NaClO4 per mL of eluent can also be used to resolve full length from n 1 components between pH 8 and pH 12 using other buffers e g AMPS Na3PO4 etc CHART 11 N N 1 Separation of Phosphorylated Deoxycytosine Oligomers Conditions 22 minute gradient fro...

Страница 21: ...gradient of 15 mM NaCl per mL of eluent can also be used to resolve full length from n 1 components between pH 8 and pH 12 using other buffered eluent systems e g Tris AMPS Na3PO4 etc CHART 12 N N 1 Separation of Deoxythymidine Oligomers Conditions 31 5 minute gradient of 330 to 900 mM NaCl in 20 mM NaOH pH 12 4 Flow rate 1 2 mL minute Injection volume 4 µL Sample 1 A260 mL solution of deoxythymid...

Страница 22: ... an improved anion exchange phase Selectivity control using pH and eluent composition Presented at the Tides 2004 conference in Las Vegas NV April 25 29 2004 Authors J R Thayer Victor Barreto Christopher Pohl Dionex Corporation Sunnyvale CA USA b Control of oligonucleotide resolution on a new strong anion exchange column Optimization using pH and eluent composition Presented at HPLC 2004 in Philad...

Страница 23: ...essure on Column Has Increased The DNAPac PA200 4 x 250 mm operates at 2400 psi at 1 mL min in the absence of solvent If the high backpressure is due to the column first try cleaning the column If the high backpressure persists replace the column bed support Note the color of the original bed support If the inlet side of the used bed support is discolored light brown to grey black metal contaminat...

Страница 24: ...Peak Efficiency and Resolution Are Decreasing Peak deformations may sometimes be caused by sample matrix Always have a spare guard available a Run a standard separation with the Guard column removed from the system If the separation improves with the old Guard removed install a new Guard column It is quite common to replace the Guard column several times during the lifetime of the analytical colum...

Страница 25: ... loss of retention is suspected the column can be cleaned with the following processes 8 11 1 High Salt Wash to Remove Ionic Components This cleanup procedure should be performed when component retention decreases This solution will protonate most organic acids 1 mM HCl pH 3 The NaClO4 will still be largely ionized so it will elute the organic acids The solvent will aid in removing components boun...

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