Gradient conditions

Theory of Gradient Separations: 

Load 

Example: Peptides 

Why do we use gradients? 

Gradient Wash Re-equilibration 

0 3 10 12.5 

Gradient Operation 

Data acquisition ‘Wasted’ cycle time 

Dr. Shulamit Levin 1 

20

Theory of Gradient Separations: 

Example: Peptides 


Load 

Gradient Wash Re-equilibration 

0 3 10 12.5 

20 

Data acquisition ‘Wasted’ cycle time 


The retention (k) of peptides have a steep dependence 

on the % organic in the mobile phase 

Neue 

ln (k) 

6 

4 

2 

0 

0 

Small Molecule 

- The steeper the slope of the line the shallower the 

gradient must be to achieve maximum resolution 

0.2 

Peptide 

0.4 

Protein 

0.6 

Volume Fraction Acetonitrile 

0.8 

1 

© 1998 Waters Corporation 


Why do we use gradients?... 

...because... 

Properties of analytes 

Retention (k) of the solutes has a steep 

dependence on the % organic in the 

mobile phase 

Wide range of differing hydrophobicities 

of the analytes 

Introduction 

Outline 

Theory of Gradient Separations of Peptides 

Optimization of Separations to... 

achieve maximum resolution 

maximize throughput 

maximize reproducibility 

Assay Reproducibility 

Conclusion 

Dr. Shulamit Levin 1-4 


© 1998 Waters Corporation

Column dead 

time 

Calculation of Isocratic 

Mobile Phase 

k-Dependence on Mobile Phase Composition 

in Reversed-Phase HPLC: 

Gradient Retention Times: 

( ) 

t = t + t + G 0 d 1 

b ×S ×ln b×S ×t ×k(Φ ) + 1 

0 0 

System delay 

time 

ln( k ) = ln( k ) - S . Φ 

0 

Gradient slope 

Resolution Equations 

Slope of lnk vs. organic 

-Factors Factors Influencing Resolution for an Isocratic 

Separation RR s 

t N a - 1 k 

~ × × 

w 4 a k +1 

Rs = 

} 

} 

Efficiency Selectivity 

k at gradient starting 

composition 

s = Resolution 

t = Retention time 

w = Peak width 

N = Plate Count 

a = Selectivity Factor 

k = Retention Factor 

-Factors Influencing Resolution for a Gradient Separation 

Rs = 

} 

} 


-Factors are similar, however... 

} 

Retention 

t N 1 

~ × ln a × 

w 4 Bct0 + 1 

} 

Retention 

B = Slope of of ln ln (k) (k) with 

solvent composition (an 

analyte dependent 

property) 

c = Gradient Slope 

t 0 

0= 

= Time of of elution for an an 

unretained peak 



Basic Resolution Equation 

-Factors Factors Influencing Resolution for an 

Isocratic Separation 

t 

Rs = 

w 

N 

~ × 

4 

} 

α − 1 k 

× 

α k +1 

} 

Rs= Resolution Efficiency Selectivity 

N = Plate Count 

t = Retention Time 

w = Average peak width 

α = Selectivity Factor 

k = Retention Factor 

{ 

w1 

} 

t 

{ 

Retention 

Analyte Retention as a Function of Gradient 

Slope 

kk (retention) for each 

analyte changes 

independently as the 

gradient slope changes. 

Thus, the resolution 

between peaks 

changes. 

ln k 

peak 8 

peak 9 

w2 

peak 10 

peak 11 

% Acetonitrile 


Carmody 



What Factors Influence Gradient RP-HPLC 

Separations... 

Part I - Factors influencing efficiency and retention 

Gradient Slope; c 

Column Length; L and N 

Flow Rate; F 

Part II II - Factors influencing selectivity 

Concentration and Type of Modifier 

Temperature 

Chemistry and Pore Size of the Packing Material 

Part III - Factors influencing reproducibility 

Column 

HPLC system 


What Factors Influence Gradient Slope? 

c = %B/minute = 

Two ways to change the slope 

change the percent organic (D ( %) of the mobile 

phase across a specified gradient run time. 

change the gradient run time (t (tg 

g ) while keeping 

the % organic of the mobile phase constant. 

% 

tg 



Rs (Resolution) 

Factors Influencing Resolution in 

Gradient RP-HPLC Separations... 

Part I - Factors influencing efficiency and 

retention 

Gradient Slope; c (%B/min.) - increase in organic 

concentration per unit time 

t N 1 

Rs = ∼ × ln α × 

w 4 Bct0 + 1 

} 

} 

} 

Efficiency Selectivity Retention 

Selectivity 

Retention 

Principle of Gradient Separations 

Neue 

approaching 

isocratic 

conditions 

General slope of the gradient 


0 0.01 0.1 1 10 



0.400 

0.300 

AU 0.200 

0.100 

0.000 

0.300 

0.200 

AU AU 

0.100 

0.000 




Gradient Slope; c (%B/min.) - varied by changing the % organic across across 

a specified gradient run time . . All All other other variables are are kept constant. 

Rs = 

t N 

∼ × lnα 

w 4 

} 


× 

} 

1 

Bct0 + 1 

} 

Retention 

tg = gradient 

run time 

1 

% 

B. . t0 + 1 

Resolution as a Function of Gradient Slope 

30.00 35.00 40.00 

Minutes 

0.62%/min (0-28%) 

30.00 30.00 35.00 35.00 

Minutes Minutes 

40.00 40.00 

Alden 

9 

10 

8 11 

0.66%/min 

9 

10 

8 11 

Rs (btw. 8 and 9) = 2.0 

Rs (btw. 9 and 10) = 5.4 

Rs (btw. 10 and 11) = 6.9 

Rs (btw. 8 and 9) = 2.3 

Rs (btw. 9 and 10) = 6.0 

Rs (btw. 10 and 11) = 6.8 

0.600 

0.500 

0.400 

0.300 

AU 

0.200 

0.100 

tg 

Conditions 

9 

0.71%/min (0-35%) 


-Column: Symmetry300, C 18, 

5 µm, 3.9 x 150 mm 

- Sample: Tryptic digests of 

bovine cytochrome c 

- Injection: 20 µL 

- Mobile Phase: 

Solvent A: 0.1% TFA in water 

Solvent B: 0.1% TFA in acetonitrile 

- Detection: 214 nm 

- Flow rate: 0.75 mL/min. 

- Temperature: 35 ºC 

Rs (btw. 8 and 9) = 1.7 

10 

Rs (btw. 9 and 10) = 4.7 

Rs (btw. 10 and 11) = 6.8 

8 11 

0.000 

30.00 35.00 

Minutes 

40.00 


Resolution as a Function of 

Gradient Slope 

-Slope of the gradient = 0.66%/min 

0.400 

0.300 

AU 0.200 

0.100 

0.000 

0.00 20.00 40.00 

Minutes 

Alden 

8 

9 

10 

11 

Conditions 

-Column: Symmetry300, C 18 , 

5 µm, 3.9 x 150 mm 

- Sample: Tryptic digests of bovine 

cytochrome c 

- Injection: 20µL 



Solvent B: 0.1% TFA in 

acetonitrile 

- Gradient: 0-45 min., 0-30%B 





Analyte Retention as a Function of Gradient 

Slope 

kk (retention) for each 

analyte changes 

independently as as the 

gradient slope changes. 

Thus, the the resolution 

between peaks changes. 

Carmody 

ln k 

peak 8 

peak 9 

peak 10 

peak 11 

% Acetonitrile 



0.2000 

0.1500 

0.1000 

AU AU 

0.1000 0.1000 

AU AU 

0.2000 0.2000 

0.1500 0.1500 

0.0500 0.0500 

0.0000 0.0000 

-0.0500 -0.0500 

0.0500 

0.0000 

-0.0500 -0.0500 




Gradient Slope; c (%B/min.) - - varied by by changing the the 

gradient run time. time. 

All other variables are kept constant. 

t N 

Rs = ∼ × lnα 

w 4 

} 

× 

} 


1 

% 

B. . t0 + 1 

tg 

} 

Retention 

Gradient Modifications: 

Initial ACN% in Peptide Separations 

0.00 0.00 

0-28% in 75 min., 

(0.37%/min) 

20.00 20.00 

40.00 40.00 


6-28% in 60 min., 

9 

(0.37%/min) 

1 

3 

5 

4 

2 

5 

1 4 

3 

2 

6 

7 

8 

60.00 60.00 

80.00 80.00 

0.00 0.00 20.00 20.00 40.00 40.00 60.00 60.00 80.00 80.00 


6 

7 

8 

9 

10 

11 

10 

11 

r.t. = 85 min. 

r.t. = 70 min. 


Gradient SLOPE is 

more important if peaks 

elute during gradient (not 

in initial condition) 

-By changing the initial 

mobile phase conditions, 

but keeping the gradient 

slope the same, the run 

time can effectively be 

shortened without a loss 

in resolution. 


Resolution as a Function of Gradient Duration 

0.1200 

0.1000 

0.0800 

AU 0.0600 

0.0400 

0.0200 

0.0000 

0.00 5.00 10.00 15.00 

Minutes 

0.0800 0.0800 

0.0600 0.0600 

0.0400 0.0400 

AU AU 

0.0200 0.0200 

0.0000 0.0000 

0.00 0.00 10.00 10.00 20.00 


0.0600 

0.0400 

AU 

0.0200 

0.0000 

0.0500 0.0500 

0.0400 0.0400 

0.0300 0.0300 

AU AU 

0.0200 0.0200 

0.0100 0.0100 

0.0000 0.0000 

0.0300 0.0300 

0.0200 0.0200 

AU AU 

0.0100 0.0100 

0.0000 0.0000 

0.00 20.00 40.00 

Minutes 

0.00 0.00 20.00 20.00 40.00 40.00 


60.00 60.00 

78 Peaks 

15 Minutes 

98 Peaks 

25 Minutes 

114 Peaks 

50 Minutes 

137 Peaks 

75 Minutes 

162 Peaks 

150 Minutes 

0.00 0.00 50.00 Minutes Minutes 

100.00 100.00 150.00 150.00 

Conditions 

-Column: Symmetry300, C 18, 5 

µm, 

4.6 x 150 mm 


bovine serum albumin 




Solvent B: 0.1% TFA in 

acetonitrile 





-Longer RT; 

Shallower the slope; 

Increases Rs 

-Rs~ 1/c where c = 

gradient slope. All 

other variables are 

kept constant 

Alden 

Summary of Part I - Gradient Slope 


Gradient Slope is one of the most powerful 

operational parameter you have at your 

disposal 

Resolution increases as gradient slope 

decreases. 

Change in the initial percent organic can 

decrease the run time, maintain the resolution 

of your separation and preserve your elution 

pattern. 

Alden © 1998 Waters Corporation 


Dr. Shulamit Levin 17-20

Volumes in an HPLC System 

A B C 

D 

Proportioning 

Valve 

System Volume 

Waters 2690

Column Volume to to Gradient Volume Relationship (Approach 1) 1) 

-- Gradient volume scaled to to column volume 

50 mm column 

Column volume = 0.5 mL Column volume = 2.5 mL 

5 minute gradient @ 1 mL/min 

gradient volume = tg x f.r. = 5 

Total volume = g.v./c.v. = 10 column vols. 

250 mm column 




Variation in Column Lengths at Equal Ratio of 

Gradient Volumes to Column Volumes 

0.0600 

AU AU0.0400 

0.0400 

0.0200 

0.1500 

0.1000 

AU 

0.0500 

0.1500 

0.1000 

AU 

0.0500 

4.00 5.00 6.00 

Minutes 

10.00 12.00 14.00 16.00 

Minutes 

Alden 

Column Volume = 0.83 mL 

Total Volume = 4.5 c.v. 





20.00 25.00 

Minutes 

5 minutes 

4.6 X 50 mm 


Conditions 

- Column: Symmetry300, C 18 , 5 µm 


serum albumin 





- 0 - 30 %B in the time shown. 


15 minutes 

4.6 X 150 mm, - Detection: 214 nm 


25 minutes 

4.6 X 250 mm, 

- Elution pattern 

stays the same. 

- Resolution inc. as 

the # of plates inc. 

- Run time inc. as 

column length inc. 





Part I I - Factors influencing efficiency and retention 



Rs = 

t 

w 

N 

∼ 

4 

} 

× ln α × 

} 

. % . εt . πr .L/F + 1 

tg 

2 


Retention 

L (column length) is is varied. Gradient 

volume is is scaled in in proportion to to the 

column volume. 

B 

1 

} 

Column Volume to Gradient Volume 

Relationship (Approach 2) 

-- Gradient volume not scaled to column volume 

50 mm column 

Column volume = 0.5 mL Column volume = 2.5 mL 




250 mm column 


Terms are 

constant 


gradient volume = t g x f.r. = 5 






Part I - Factors influencing efficiency and 

retention 



Rs = 

t 

w 

N 

∼ × ln α × 

4 

B 

} 

} 


1 

. % . . εt πr .L/F + 1 

tg 

2 

} 

Retention 


Column Length Effects on Resolution at a Constant 

Gradient Duration (cont'd) 

0.02000 

0.01500 

0.01000 

AU 

0.00500 

0.00000 

-0.00500 

0.00 

20.00 

Minutes 

130 Peaks 

Symmetry300 4.6 X 50 mm, 

C 18, 5 µm 

40.00 

0.1200 

0.1000 

0.0800 

AU 0.0600 

0.0400 

0.0200 

131 Peaks 

Symmetry300 4.6 X 150 

mm, 

C 5 µm 

18, 

0.0000 

0.00 20.00 

Minutes 

40.00 

Conditions 

- Sample: Tryptic digests of bovine serum 

albumin 

- Injection: 20 µL (7 µL for 4.6 X 50 mm) 








-50 mm column has a 

similar resolving power 

as 250 mm column if the 

gradient duration 

remains the same. 


0.2500 0.2500 

0.2000 0.2000 

0.1500 0.1500 

AU AU0.1000 0.1000 

0.0500 0.0500 

1 

0.0000 0.0000 

-0.0500 -0.0500 

0.00 0.00 

0.500 0.500 

0.400 0.400 

0.300 0.300 

AU AU 

0.200 0.200 

0.100 0.100 

0.000 0.000 

0.800 

0.600 

AU 0.400 

0.200 

0.00 0.00 

Column Length Effects on Resolution at a 

Constant Gradient Duration 


Total Volume =40.7 c.v.. 

1 

2 

1 

3 

4 

5,6 

20.00 20.00 


Total Volume =13.5 c.v. 

5,6 

2 4 

3 

20.00 20.00 



0.000 

0.00 20.00 

Minutes 

40.00 

Alden 

2 

3 

4 

7 

5 6 

9 

8 



7 

10 

11 

7 

9 

8 

Symmetry300 4.6 X 50 mm, 

C18, 5 µm 

Conditions 

13 


12 

cytochrome c 







mm, 



10 

C 5 µm 

18, 13 


40.00 40.00 

8 

11 

40.00 40.00 

9 10 

11 

12 


mm, 

C 5 µm 

18, 

13 

12 

-Will observe elution 

pattern changes. 

-Resolution changes 

-Run time remains 

the same. 

Summary Part I - Column Length 

If the gradient volume is scaled proportionally to the 

column volume 

elution pattern does not change 

resolution increases with column length. 


If the gradient volume is not scaled in proportion to the 

column volume 

0.1200 

0.1000 

0.0800 

AU 0.0600 

0.0400 

0.0200 

134 Peaks 


mm, 

C 5 µm 

18, 

elution pattern and resolution changes 

50 mm column exhibits similar resolving power to a 250 

mm column. 

0.0000 

0.00 20.00 

Minutes 

40.00 

Alden © 1998 Waters Corporation 


Dr. Shulamit Levin 29-32

Rs (Resolution) 



Neue 




Flow Rate; FF 



Temperature 



Column 

HPLC system 


Resolution as a Function of Flow Rate at a Constant 

Gradient Duration 

12 

10 

8 

6 

4 

2 

0 

(50 mm Column) 

0.33 

1 mL/min 

Flow Rate (mL/min) 

3.3 





Part I I - Factors influencing efficiency and retention 



Flow Rate; F 

Rs = 

t 

w 

N 

∼ × lnα 

× 

4 

B 

} 

} 


Flow Rate Effects on Resolution 

at a Constant Gradient Duration 

0.0500 

0.0400 

AU 

0.0300 

0.0200 

0.0100 

0.0000 

0.00 

0.0400 

0.0300 

20.00 

Minutes 

40.00 

75 Peaks 

0.3 mL/minute 

100 Peaks 

AU 0.0200 

0.0100 

0.0000 

0.00 20.00 Minutes 40.00 

0.5 mL/minute 

135 Peaks 

0.02500 

0.02000 

0.01500 

AU 0.01000 

0.00500 

0.00000 

-0.00500 

0.00 20.00 Minutes 

1.0 mL/minute 

40.00 

0.02000 

0.01500 

AU AU 

0.01000 

0.00500 

0.00000 

-0.00500 -0.00500 

0.00 0.00 20.00 20.00 Minutes Minutes 40.00 

Alden 

1 

. % . εt . πr .L/F + 1 

tg 

2 

} 

Retention 

F (flow rate) is is varied. All other 

variables are kept constant 

130 Peaks 

2.0 mL/minute 

Conditions 


- Column: Symmetry300, C 18, 

5 µm, 4.6x50mm 


serum albumin 








-Best resolution 

occurred at a flow rate 

of 1.0 mL/min. for this 

peptide under these 

conditions. 

-Elution pattern 

changes. 



Summary of Part I - Flow Rate 

Maximum resolution is achieved at an 

optimal flow rate: 

As flow rate changes N changes 

As flow rate changes the elution pattern 

changes. 

Type of Modifiers 

Solvation 

Ionization 

Ion-pairing 

Volatility (Collection) 









Flow Rate; F 


Concentration and Type of of Modifier 

Temperature 



Column 

HPLC system 

Effects of TFA Concentration on Resolution 

- Typical gradient conditions 

0.300 

0.200 0.200 

AU AU 

0.100 0.100 

0.000 0.000 

Alden 

0.1% TFA in 

solvents A and B 

0.00 0.00 

1 

5,6 

2 4 

3 

9 

7 8 

10 

11 

20.00 Minutes Minutes 40.00 

13 

12 

Conditions 


5 µm, 3.9x150mm, 



cytochrome c 



Solvent A: water 

Solvent B: acetonitrile 







The Power of Different TFA Concentrations in 

Your Mobile Phase 

- 0.05% TFA in 


2 

1 3 

- 0.1% TFA in solvents 

A and B 

- 0.2% TFA in 


2 

1 3 

1 

4 

5 6 

2 4 

3 

5,6 

5,6 

4 

0.00 20.00 40.00 

Minutes 

Alden 

Absorbance (214 nm) 

Alternate Ion Pairing Reagents 

TFA and HFBA (Heptafluorobutyric Acid) 

0.40 

0.02 

0.00 

0.40 

0.20 

0.00 

TFA 

HFBA 

20 40 60 80 

Time (min) 

7 

7 

7 

8 

9 

8 

10 

9 

11 

10 

9 

8 

11 

10 

11 

12 

13 

12 13 

13 

12 


Sample: Rabbit cytochrome c 

tryptic digest, 500 pmol 

Column: Delta-Pak C 18, 5µm, 

300Å, 2.0 x 150 mm 

Eluents: A=water/ 0.1% TFA or 

HFBA 

B=acetonitrile/ 0.1% 

TFA or HFBA 

Gradient: O-60 % B 120 min 

Flow: 0.18 mL/min 

Temp: 35 ºC 



0.200 

0.100 0.100 

0.000 0.000 

The Power of Different TFA 

Concentrations in Your Mobile Phase 

0.300 0.300 

AU AU 

0.3000 

0.2500 

0.2000 

AU 0.1500 

0.1000 

0.0500 

0.0000 

Alden 

0.00 0.00 

0.1% TFA in Solvent A 

0.1% TFA in Solvent B 

1 

1 

2 

3 

20.00 20.00 

2 

3 4 

5,6 

4 


0.05% TFA in Solvent A 

0.1% TFA in Solvent B 

7 

8 

9 

10 

11 

40.00 40.00 

0.00 20.00 40.00 

Minutes 

5 6 

7 

8 

9 

10 

11 

12 

13 

12 13 

Conditions 

- Column: Symmetry300, C 18, 5 µm, 

3.9x150mm 

- Sample: Tryptic Digests of Bovine 

Cytochrome c 


What Factors Influence Gradient 

RP-HPLC Separations... 


Solvent A: water 

Solvent B: acetonitrile 





Part I I - - Factors influencing efficiency and retention 



Flow Rate; FF 

Part II II - - Factors influencing selectivity 


Temperature 


Part III III - - Factors influencing reproducibility 

Column 

HPLC system 




Temperature Effects on Resolution 

Resolution is temperature dependent 

Temperature is a critical parameter to control 

in order to achieve reproducible separations. 





Column Length; L and and N 

Flow Rate; F 

Part II - Factors influencing selectivity 


Temperature 

Pore Size and Chemistry of of the Packing Material 


Column 

HPLC system 




0.300 

0.200 

AU 

0.100 

0.000 

0.300 

0.200 

AU 

0.100 

0.000 

0.300 

0.200 

AU 

0.100 

0.000 

Temperature Effects on Resolution 

Alden 

0.300 0.300 

AU AU 

0.200 0.200 

5, 6 

7 

25.00 30.00 35.00 40.00 

Minutes 

5, 6 

7 

25.00 30.00 35.00 40.00 

Minutes 

5 6 

Rs (5,6) = 0 

Rs (8,9) = 2.39 

Rs (5,6)= 0 

Rs (8,9)= 2.07 

Rs (5,6)= 0.84 

Rs (8,9)= 1.63 

7 

25.00 30.00 35.00 40.00 

Minutes 

8 

8 

9 

9 

8 

9 

10 

10 

10 

11 

11 

11 

30 °C 

35 °C 

40 °C 

Conditions 

- Column: Symmetry300, C 18 , 

5 µm, 3.9x150mm 


cytochrome c 








Pore Size Effects on Resolution 

0.30 

0.20 

AU 

0.10 

0.00 

0.500 0.500 

0.400 0.400 

0.100 0.100 

0.000 0.000 

0.00 0.00 

1 

1 

5 6 

2 4 

3 

-0.10 

0.00 20.00 

Minutes 

40.00 

Carmody 

2 4 

3 

20.00 20.00 

5,6 

7 


8 

7 

Symmetry300, C 18, 5 µm, 

4.6 X 150 mm, 300Å 

10 

13 

9 

Symmetry®, C18, 5µm, 

4.6 X 150 mm, 100Å 

9 

8 

11 

10 

11 

40.00 40.00 

12 

13 

12 

Conditions 



cytochrome c (bovine) 







- Flow Rate: 0.75 mL/min. 


-Different pore 

sizes change 

selectivity. 



Selectivity Differences Between Packings 

Alberta Peptides on Symmetry® Reversed-Phase Columns 

0.10 

AUFS 

AUFS 

0.05 

0.00 

0.10 

0.05 

0.00 

SymmetryShield RP 8, 100Å 

1 

2 

3 

5.00 10.00 15.00 

Symmetry® C 8, 100Å 

1 2 

5.00 10.00 15.00 

Minutes 

3 

4 

4 

5 

5 

Ac-Arg-Gly-X-X-Gly-Gly-Leu-Gly-LysAmide 

-X-X-: 

1: Ala-Gly with free alpha amino group 

2: Gly-Gly 3: Ala-Gly 

4: Val-Gly 5: Val-Val 

Peptide Mapping Validation 

-Robustness Testing 

Choice and quality of enzyme 

Digestion conditions 

HPLC conditions 

Equipment 

System 

Column 

Conditions: 

Columns: 3.9 mm x 150 mm 

Flow Rates: 0.8 mL/min 

Mobile Phase: A. 0.1% TFA aqueous; 

B. acetonitrile with 0.1% TFA 

Gradient: 10% to 40% B in 30 minutes, 

step to 60% B for 5 min 

Sample: 9 µL Alberta Peptides, mix 

with 5 decapeptides 

Detector: 214 nm 

Temperature: 35°C 









Flow Rate; FF 



Temperature 



Column 

HPLC HPLC system system 

Effects of Irreproducible Gradient Delivery 

-- Traditional HPLC System 

Experience resolution 

differences and 

retention time shifts 

0.00 20.00 40.00 

Minutes 

Carmody 




Waters 2690 Separations Module 

Gradient Accuracy & Precision 

0.08 

0.07 

0.06 

0.05 

AU 0.04 

0.03 

0.02 

0.01 

0.00 

10%B 

10.0 20.0 30.0 40.0 50.0 60.0 

Minutes 

Reproducible Gradient Delivery 

-Waters Alliance HPLC System 

-Overlay of 15 Consecutive Injections 

Conditions: 

Column: Symmetry300 C18, 5 µm, 3.9 x 150 nm 

Sample: 20 µL of bovine, cytochrome c (tryptic digest) 

Flow rate: 1 mL/min. 

Temperature: 35 ºC 

Linear gradient: 0 - 28%B in 45 min. 

Mobile phase: A: 0.05% TFA in water 

B: 0.1% TFA in Methanol 

Alden 

1% Step Gradient at 1.0 mL/min 

Accuracy

Complex Gradient Conditions: 

Column: 

Column: 

AccQ 

AccQ 

Tag 

Tag 

Column, 

Column, 

3.9 

3.9 

x 

x 

150 

150 

mm 

mm 

Eluent 

Eluent 

A: 

A: 

AccQ 

AccQ 

Tag 

Tag 

Eluent 

Eluent 

A 

Eluent 

Eluent 

B: 

B: 

Acetonitrile 

Acetonitrile 

Eluent C: C: Water 

Flow 

Flow 

Rate: 

Rate: 

1.0 

1.0 

mL/min. 

mL/min. 

Column 

Column 

Temp.: 

Temp.: 

37 

37 

°C 

°C 

Detection: 

Detection: 

Fluorescence, 

Fluorescence, kk ex = 

ex = 

250 

250 

nm, 

nm, 

kem k 

= 

em= 

395 nm nm 

Sample: 50 50 pmol Hydrolysate Standard 

Sample 

Sample 

Temp.: 

Temp.: 

5 

5 

°C 

°C 

Gradient: 

Gradient: 

Time Flow %A %B %C %D Curve 

0.00 1.00 100.0 0.0 0.0 0.0 * 

0.50 1.00 99.0 1.0 0.0 0.0 11 

18.0 1.00 95.0 5.0 0.0 0.0 6 

19.0 1.00 91.0 9.0 0.0 0.0 6 

29.5 1.00 83.0 17.0 0.0 0.0 6 

33.0 1.00 0.0 60.0 40.0 0.0 11 

36.0 1.00 100.0 0.0 0.0 0.0 11 

65.0 1.00 0.0 60.0 40.0 0.0 11 

100.0 0.00 100.0 0.0 0.0 0.0 6 

"Consideration must be given to 

column-to-column variation attributed to 

production differences from a recommended 

manufacturer. Column-to-column variability 

was recently bemoaned as the 'Achilles heel' in 

the HPLC of protein pharmaceuticals (20). 

Although this problem has been both ignored by 

many and overstated by others, peptide 

mapping does place very high demands on 

column performance. Thus, column-to-column 

variability is of special concern (21,22)." 


Garnick et al. Biologicals (1996) 24 , 255-275 



Why is Column Reproducibility 

Important? 

Validated RP-HPLC assays require HPLC columns to 

be reproducible from column-to-column and 

batch-to-batch. 

Columns which perform reproducibly in terms of 

selectivity and separation characteristics from 

batch-to-batch ensures reliable, reproducible and 

robust assays over the life of of the product. 


Column-to-Column Reproducibility 

Employed 8 different Symmetry300, C 18, , 5 µm 

18 

columns from the same batch (#107) 

Conducted evaluation over a 3-week time period 

Different mobile phase preparations for each run 



Symmetry300 

Batch-to-Batch Reproducibility 

Batch 107 

Batch 106 

Batch 104 

Batch 103 

10 30 50 

Minutes 

Costello 

Conclusions (continued) 

Conditions 


5 µm, 3.9x150mm 


cytochrome c (bovine) 







- Temperature: 35 °C 


For Rugged/Robust gradient method 

development consider... 

System performance 

reproducibility of gradient delivery 

system delay volume 

Column performance 

column-to-column reproducibility 

batch-to-batch reproducibility 

- Instrument: 

Waters Alliance 

HPLC system 




Conclusion 

There are several factors to assess when 

developing/optimizing a gradient RP-HPLC 

method. 

Primary tools for gradient optimization are 

gradient slope, column length and modifier 

type. 

Secondary tools to be used for fine tuning 

your gradient are temperature, modifier 

concentration and flow rate. 

Acknowledgments: 

Bonnie Alden 

Ray Crowley 

Uwe Neue 

Tom Walter 

Ray Fisk 

Dave Costello 

Edouard Bouvier 

...and Waters' Symmetry® Team

Gradient conditions

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