Gradient Considerations in LC Operation in Gradients of Solvent Strength in LC

Layout

Gradient Application link

•Introduction % Organic

Final Hold

•LC System Considerations

Gradient Time

•Gradient Parameters Affecting Resolution

Inject Hold-up time

Re-equilibration Total Cycle Time 1998 Waters Corporation

Why do we use gradients?... Gradient Separations: Example: Peptides

...because... Properties of analytes

Why do we use gradients?

Retention (k) of the solutes has a steep dependence on the % organic in the mobile phase Wide range of differing hydrophobicities of the analytes

1

Gradient Considerations in LC Why do we use gradients?

Analyte Retention as a Function of Gradient Slope

The retention (k) of peptides have a steep dependence on the % organic in the mobile phase - The steeper the slope of the line the shallower the gradient must be to achieve maximum resolution 50 40

Peptide

Protein

30 Small Molecule

Slope=B

3.2

k (retention) for each analyte changes independently as the gradient slope changes.

3

Thus, the resolution between ln k peaks changes.

20 ln (k)10

Slope=B

3.1

peak 8 peak 9 peak 10 peak 11

2.9 2.8

0 -10

2.7

-20

2.6

-30 Neue

Volume Fraction Acetonitrile

% Organic

•LC System Considerations

0.67

0.71

System Considerations When Working with Gradients

Layout

•Introduction

0.62

% Acetonitrile

Carmody

Final Hold Gradient Time Inject

•Gradient Parameters Affecting Resolution

Hold-up time

Re-equilibration Total Cycle Time

2

Gradient Considerations in LC Different Configurations Create Different Time Delays

Volumes of Pumping Systems High Pressure Mixer: Multi-Pump Smaller System Volume = Dwell volume Pump 1

Mixer

Injector

Column

Detector

Pump 2

Low Pressure Gradient: Single Pump Larger System Volume = Dwell volume Solvent delivery

B

Mixing Valve Low – More Pipe Volume – More Time Delay

Mixing Valve High – Less Pipe Volume – Less Time Delay

A

C D

Injector

Delay Volume is Significant in Narrow BoreLow Flow Applications Theoretical Gradient No column , 1.00 mL/min 3.9x150 mm, 1.00 mL/min 2.1x150 mm, 0.29 mL/min

System Volume Column Volume A

D

C

Solvent delivery

Injector

Column

Detector

Proportioning Valve

Delay Volume and Extra-Column Band Broadening Volume

B

Column

%B Detector

Proportioning Valve

Extra Column Volume

3

Delay Volume 0

5

Re-equilibration Time 10

15 Minutes

20

25

30

Gradient Considerations in LC Application

Linear Gradient Separation 1 mL/min, 200 and 50 µL/min

System Considerations When Working with Gradients

0.12

1 mL/min 0.10

% Organic

0.08 AU 0.06

200 µ L/min

0.04

50 µ L/min 0.02 0.00

Final Hold Gradient Time Inject Adjust Hold-up time

Re-equilibration Total Cycle Time

10.0

20.0

30.0 Minutes

40.0

50.0

60.0

Delay Volume is Significant in Narrow BoreLow Flow Applications

Re-Equilibration Time For good system/column equilibration

Theoretical Gradient No column , 1.00 mL/min 3.9x150mm, 1.00 mL/min 2.1x150mm, 0.29 mL/min

%B

Delay Volume 0

4

5

where: tr is the re-equilibration time in minutes, VT is the total system volume, Vc is the column volume in mL F is the flowrate in mL/min. column volume = 0.7(π πr2L)

Re-equilibration Time 10

15 Minutes

20

25

tr = (3VT + 5Vc)/F

For example, Alliance system’s volume = 650 µL

30

Gradient Considerations in LC Basic Resolution Equation

Layout

-Factors Influencing Resolution for an Isocratic Separation t w

~

Selectivity

Resolution Equations -Factors Influencing Resolution for an Isocratic Separation Rs =

t

~

w

α-1 α

}

} Efficiency

Selectivity

k k +1

}

N 4

Retention

-Factors Influencing Resolution for a Gradient Separation Rs =

t w

~

ln α Selectivity

1 Bct0 + 1

}

Efficiency

}

}

N 4

Retention

-Factors are similar, however...

5

Rs = Resolution t = Retention time w = Peak width N = Plate Count α = Selectivity Factor k = Retention Factor

w1

w2

Analyte Retention as a Function of Gradient Slope

B = Slope of ln (k) with solvent composition (an analyte dependent property) c = Gradient Slope

t0 = Time of elution for an unretained peak

Retention

t {

•Gradient Parameters Affecting Resolution

Rs = Resolution Efficiency N = Plate Count t = Retention Time w = Average peak width α = Selectivity Factor k = Retention Factor

}

•LC System Considerations

k k +1

α-1 α

}

}

N 4

{

Rs =

•Introduction

3.2

k (retention) for each analyte changes independently as the gradient slope changes. Thus, the resolution between ln k peaks changes.

Slope=B

3.1 3

peak 8 peak 9 peak 10 peak 11

2.9 2.8 2.7 2.6

Carmody

0.62

0.67

% Acetonitrile

0.71

Gradient Considerations in LC What Factors Influence Gradient RP-HPLC Separations...

Part I - Factors influencing efficiency and retention Gradient Slope; c

Factors Influencing Resolution in Gradient RP-HPLC Separations... Part I - Factors influencing efficiency and retention

Column Length; L and N Flow Rate; F

Gradient Slope; c (%B/min.) - increase in organic concentration per unit time

Part II - Factors influencing selectivity Concentration and Type of Modifier

Rs =

t

~

w

Part III - Factors influencing reproducibility

Efficiency

ln α Selectivity

1 Bct0 + 1

}

}

N 4

}

Temperature Chemistry and Pore Size of the Packing Material

Retention

Column HPLC system

Principle of Gradient Separations What Factors Influence Gradient Slope? % tg

Two ways to change the slope change the percent organic (∆ ∆ %) of the mobile phase across a specified gradient run time.

Rs (Resolution)

c = %B/minute =

change the gradient run time (tg) while keeping the ∆% organic of the mobile phase constant.

0 0.01 Neue

6

approaching isocratic conditions

0.1

1

General slope of the gradient

10

Gradient Considerations in LC Resolution as a Function of Gradient Slope

What Factors Influence Gradient RP-HPLC Separations...

-Slope of the gradient = 0.66%/min

Part I - Factors influencing efficiency and retention Gradient Slope; c (%B/min.) - varied by changing the % organic across a specified gradient run time. All other variables are kept constant.

Conditions

10

AU

-Column: Symmetry300™, C18, 5 m, 3.9 x 150 mm

0.400

Rs =

t

~

w

N 4

1

ln a

9

Bct0 + 1

Selectivity

8

Retention

tg = gradient run time

1 % tg

B.

- Injection: 20µ µL

0.200

}

}

} Efficiency

- Sample: Tryptic digests of bovine cytochrome c

0.300

0.100

- Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile

0.000

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

0.00

.t

0+1

20.00

11

- Flow rate: 0.75 mL/min.

40.00

Minutes

- Temperature: 35 C - Detection: 214 nm

Alden

1998 Waters Corporation

Resolution as a Function of Gradient Slope Analyte Retention as a Function of Gradient Slope Conditions

0.66%/min 0.400 0.300

-Column: Symmetry300™, C18, 5 m, 3.9 x 150 mm - Sample: Tryptic digests of bovine cytochrome c - Injection: 20 L - 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 C

Rs (btw. 8 and 9) = 2.0 Rs (btw. 9 and 10) = 5.4 Rs (btw. 10 and 11) = 6.9

10

9

0.200 AU

11

8

0.100 0.000 30.00

35.00

40.00 Minutes

0.71%/min

0.62%/min 10

9

0.200 AU 0.100

8

11

0.600

Rs (btw. 8 and 9) = 1.7 Rs (btw. 9 and 10) = 4.7 Rs (btw. 10 and 11) = 6.8

0.500 0.400 0.300 AU 0.200

9 8

30.00

35.00

Alden

Minutes

40.00

0.000 30.00

Slope=B

3.1 3

peak 8 peak 9 peak 10 peak 11

2.9 2.8 2.7

11

2.6

0.100

0.000

7

Thus, the resolution between ln k peaks changes.

10

Rs (btw. 8 and 9) = 2.3 Rs (btw. 9 and 10) = 6.0 Rs (btw. 10 and 11) = 6.8

0.300

3.2

k (retention) for each analyte changes independently as the gradient slope changes.

35.00 Minutes

40.00

Carmody

0.62

0.67

% Acetonitrile

0.71

Gradient Considerations in LC Resolution as a Function of Gradient Duration

What Factors Influence Gradient RP-HPLC Separations...

78 Peaks 15 Minutes

0.1200 0.1000 0.0800 AU 0.0600 0.0400 0.0200 0.0000 0.00

5.00

10.00 Minutes

-Column: Symmetry300™, C18, 5 m, 4.6 x 150 mm - Sample: Tryptic digests of bovine serum albumin - Injection: 20 L - Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - Gradient: 0-45 min., 0-30%B - Detection: 214 nm - Flow rate: 0.75 mL/min. - Temperature: 35 C

15.00

98 Peaks 25 Minutes

0.0800 0.0600

Part I - Factors influencing efficiency and retention

Conditions

AU 0.0400 0.0200

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

0.0000 0.00

time. All other variables are kept constant.

10.00

20.00

Minutes

114 Peaks 50 Minutes

0.0600 0.0400 AU 0.0200 0.0000

Rs =

t

~

w

0.00

N 4

ln α

}

}

Selectivity

B.

% tg

. t0 + 1

0.0400 0.0300 AU

20.00

40.00

Minutes

-Longer RT; Shallower the slope; Increases Rs

137 Peaks 75 Minutes

0.0500

0.0200 0.0100

-Rs ~ 1/c where c = gradient slope. All other variables are kept constant

0.0000

}

Efficiency

1

0.00

Retention

40.00 Minutes

20.00

60.00

162 Peaks 150 Minutes

0.0300 0.0200 AU 0.0100 0.0000 0.00

Gradient Modifications: Initial ACN% in Peptide Separations 0-28% in 75 min., (0.37%/min)

0.2000

0.1500

9

1

4

3 0.0500

5

Gradient SLOPE is more important if peaks elute during gradient (not in initial condition)

7 6

2

0.0000

r.t. = 85 min.

0.0500-0.0500

0.00

20.00

40.00 Minutes Minutes

6-28% in 60 min., (0.37%/min)

0.2000

60.00

80.00 80.00

11

9 10

0.1500

8 0.1000

1 3

AU 0.0500

4

5

7 6

2

0.0000

r.t. = 70 min.

0.0500 0.0500--

0.00

Alden

8

20.00

40.00 Minutes

Minutes

100.00

150.00

Alden

1998 Waters Corporation

Summary of Part I - Gradient Slope

11 10

8 0.1000 AU

50.00

60.00

80.00

-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.

• 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.

Gradient Considerations in LC 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 - Factors influencing selectivity

Part I - Factors influencing efficiency and retention

Rs =

t w

~

N 4

ln α

} Selectivity

Approach 2: do not scale the gradient volume in proportion to the column volume (such as keep the gradient run time constant while changing the column length).

9

0

+1

1 B.

% tg

. ε . πr2 .L/F + 1 t

Column Volume to Gradient Volume Relationship (Approach 1)

-Gradient volume scaled to column volume 5 minute gradient = 1 c.v./min Total volume = 10 column volumes

50 mm column

Approach 1: scale gradient volume in proportion to the column volume such as change the gradient run time with the column length).

.t

Retention

Column HPLC system

2 Approaches:

% tg

B.

}

Efficiency

1

Part III - Factors influencing reproducibility

The Number of Column Volumes per Minute Impacts Resolution



Gradient Slope; c – Column Length; L and N

}

Concentration and Type of Modifier Temperature Chemistry and Pore Size of the Packing Material

What Factors Influence Gradient RP-HPLC Separations...

250 mm column

Column volume = 0.5 mL

Column volume = 2.5 mL

5 minute gradient @ 1 mL/min

25 minute gradient @ 1 mL/min

gradient volume = tg x f.r. = 5 Total volume = g.v./c.v. = 10 column vols.

gradient volume = tg x f.r. = 25 Total volume = g.v./c.v. = 10 column vols.

Gradient Considerations in LC Variation in Column Lengths at Equal Ratio of Gradient Volumes to Column Volumes

What Factors Influence Gradient RP-HPLC Separations...

Column Volume = 0.83 mL Total Volume = 4.5 c.v.

Part I - Factors influencing efficiency and retention

5 minutes 4.6 X 50 mm

0.0600

Gradient Slope; c Column Length; L and N

AU0.0400 0.0200 4.00

5.00

6.00 Minutes

Rs =

t w

~

N

0.1500

1 B.

% tg

. ε . πr2 .L/F + 1 t

Selectivity

AU 0.0500

10.00

Retention

L (column length) is varied. Gradient volume is scaled in proportion to the column volume.

0.1500

Terms are constant

15 minutes 4.6 X 150 mm,

0.1000

}

Efficiency

}

}

4

ln α

Column Volume = 2.5 mL Total Volume = 4.5 c.v.

Conditions - Column: Symmetry300™, C18, 5 m - Sample: Tryptic digests of bovine serum albumin - Injection: 20 L - Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - 0 - 30 %B in the time shown. - Flow rate: 0.75 mL/min. - Detection: 214 nm - Temperature: 35 C

12.00

14.00 Minutes

- Elution pattern stays

16.00

Column Volume = 4.2 mL Total Volume = 4.5 c.v.

the same. 25 minutes 4.6 X 250 mm,

0.1000

- Resolution inc. as the # of plates inc.

AU 0.0500

- Run time inc. as column 20.00

length inc.

25.00

Minutes Alden

Column Volume to Gradient Volume Relationship (Approach 2) -Gradient volume not scaled to column volume 5 minute gradient = 1 c.v./min Total volume = 10 column volumes 50 mm column 250 mm column

5 minute gradient @ 1 mL/min

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.

gradient volume = tg x f.r. = 5 Total volume = g.v./c.v. = 2 column vols.

Column volume = 0.5 mL

1998 Waters Corporation

What Factors Influence Gradient RP-HPLC Separations...

Part I - Factors influencing efficiency and retention Gradient Slope; c Column Length; L and N

Rs =

t w

~

10

Selectivity

1 B.

% tg

. ε . πr2 .L/F + 1 t

}

Efficiency

ln α

}

}

N 4

Retention

Gradient Considerations in LC Column Length Effects on Resolution at a Constant Gradient Duration Column Volume = 0.83 mL Total Volume =40.7 c.v..

Symmetry300™ 4.6 X 50 mm,C18, 5 µm 10

13

5,6

0.2500 0.2000

2

0.1500 AU0.1000

1

8

4 3

0.0500

12

9 11

7

0.0000 0.0500 0.0500-20.00

0.00

40.00

Minutes

Column Volume = 2.5 mL Total Volume =13.5 c.v.

Symmetry300™ 4.6 X 150 mm C18, 5µm 5,6

0.500

13

10

0.300 AU 0.200

4

2

8

3

1

0.100

11

7

-Will observe elution pattern changes.

0.000

Column Length Effects on Resolution at a Constant Gradient Duration (cont'd) Conditions 130 Peaks Symmetry300™ 4.6 X 50 mm, C18, 5 µm 0.02000 0.01500 0.01000 AU 0.00500 0.00000 0.00500 0.00500-0.00

20.00

40.00 Minutes

131 Peaks µm Symmetry300™ 4.6 X 150 mm, C18, 5µ

0.1200 0.1000 0.0800 AU0.0600 0.0400 0.0200 0.0000 0.00

20.00

Minutes

40.00

40.00

20.00

0.00

- Sample: Tryptic digests of bovine cytochrome c - Injection: 20 L - Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - Gradient: 0-45 min., 030%B - Flow rate: 0.75 mL/min. - Detection: 214 nm - Temperature: 35 C

12

9

0.400

Conditions

Minutes

0.800

Column Volume = 4.2 mL Total Volume = 8.0 c.v.

Symmetry300™ 4.6 X 250 mm, C18, 5µ µm 9 10

0.600

56

AU0.400

4

2 1

0.200

8

12

13

11

-Resolution changes -Run time remains the same.

7

3

0

Alden

0.000 0.00

20.00

Minutes

0.00

20.00

Minutes

-50 mm column has a similar resolving power as 250 mm column if the gradient duration remains the same.

40.00 1998 Waters Corporation

40.00

Summary Part I - Column Length

What Factors Influence Gradient RP-HPLC Separations...

If the gradient volume is scaled proportionally to the column volume

Part I - Factors influencing efficiency and retention

Elution pattern does not change Resolution increases with column length.

If the gradient volume is not scaled in proportion to the column volume Elution pattern and resolution changes 50 mm column exhibits similar resolving power to a 250 mm column.

11

134 Peaks µm Symmetry300™ 4.6 X 250 mm, C18, 5µ

0.1200 0.1000 0.0800 AU0.0600 0.0400 0.0200 0.0000 0.0

- Sample: Tryptic digests of bovine serum albumin - Injection: 20 L (7 L for 4.6 X 50 mm) - Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - Gradient: 0-45 min., 0-30%B - Temperature: 35 C - Flow rate: 0.75 mL/min. - Detection: 214 nm

Gradient Slope; c Column Length; L and N Flow Rate; F Part 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

Alden

Gradient Considerations in LC What Factors Influence Gradient RP-HPLC Separations...

Resolution as a Function of Flow Rate at a Constant Gradient Duration

Part I - Factors influencing efficiency and retention Gradient Slope; c Column Length; L and N Flow Rate; F

12

Rs (Resolution)

10

F (flow rate) is varied. All other variables are kept constant

Rs =

t w

~

N 4

1

ln α

Efficiency Selectivity

% tg

. εt . πr2 .L/F + 1

}

}

}

B.

Retention

8

6

1 mL/min

4

2

0

0.33

3.3

Flow Rate (mL/min) Neue 1998 Waters Corporation

Flow Rate Effects on Resolution at a Constant Gradient Duration

Conditions

75 Peaks AU

0.0500 0.0400 0.0300 0.0200 0.0100 0.0000 0.00

20.00

Minutes

40.00

0.3 mL/minute

100 Peaks AU

0.0400 0.0300 0.0200 0.0100 0.0000

0.5 mL/minute 0.00

AU

0.02500 0.02000 0.01500 0.01000 0.00500 0.00000 0.00500 0.00500-0.00

20.00

40.00

135 Peaks

Minutes

20.00

Minutes

40.00

1.0 mL/minute

130 Peaks 0.02000

- Column: Symmetry300™, C18, 5µ µm, 4.6x50mm - Sample: Tryptic digests of bovine serum albumin - Injection: 20µ µL - Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - Gradient: 0-45 min., 0-30%B - Temperature: 35 C - Detection: 214 nm

-Best resolution occurred at a flow rate of 1.0 mL/min. for this peptide under these conditions.

0.01500

AU

0.01000 0.00500 0.00000 0.00500-0.00500 0.00

12

2.0 mL/minute 20.00

Minutes

40.00

-Elution pattern changes. 1998 Waters Corporation

Alden

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.

Gradient Considerations in LC What Factors Influence Gradient RP-HPLC Separations...

Type of Modifiers

Part I - Factors influencing efficiency and retention Gradient Slope; c Column Length; L and N Flow Rate; F

– Solvation

Part II - Factors influencing selectivity – Ionization

Concentration and Type of Modifier Temperature Chemistry and Pore Size of the Packing Material

– Ion-pairing

Part III - Factors influencing reproducibility

– Volatility

Column HPLC system

The Power of Different TFA Concentrations in Your Mobile Phase

Effects of TFA Concentration on Resolution - Typical gradient conditions Conditions - Column: Symmetry300™, C18, 5 µm, 3.9x150mm,

0.1% TFA in solvents A and B

- Injection: 20µ µL

0.200

13 12

9 5,6 2

0.100

1 0.000

4

8 7

3

11

- Mobile Phase: Solvent A: water Solvent B: acetonitrile

- Gradient: 0-45 min., 0-30%B - Flow rate: 0.75 mL/min.

0.00

1

Minutes

1

1

- Temperature: 35 C - Detection: 214 nm

13

12 11

8

7

10 13 9

5,6

2

4

7

3

12

8

11

10

- 0.2% TFA in solvents A and B

0.00 Alden

4

3

- 0.1% TFA in solvents A and B

40.00

20.00

13 9

56

2

0.300

AU

10

- 0.05% TFA in solvents A and B

- Sample: Tryptic digests of bovine cytochrome c

10

(Collection)

13

5,6 2

3

4

7

20.00

9 8

12 11

40.00

Minutes Alden

Gradient Considerations in LC The Power of Different TFA Concentrations in Your Mobile Phase

Alternate Ion Pairing Reagents TFA and HFBA (Heptafluorobutyric Acid)

Conditions 10

0.1% TFA in Solvent A 0.1% TFA in Solvent B

- Column: Symmetry300™, C18, 5µ µm, 3.9x150mm

0.200

9

12

5,6

AU

2

0.100

1

4

8 7

3

11

20.00

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

0.05% TFA in Solvent A 0.1% TFA in Solvent B

0.2500

- Sample: Tryptic Digests of Bovine Cytochrome c

- Mobile Phase: Solvent A: water Solvent B: acetonitrile

40.00

Minutes

0.3000

10

- Flow rate: 0.75 mL/min.

0.2000

12 13 AU 0.1500

1

0.0500

9

56

2

0.1000

TFA

7

3 4

8

Column: Delta-Pak™ C18, 5 µm, 300 , 2.0 x 150 mm

0.02

Eluents: A=water/ 0.1% TFA or HFBA B=acetonitrile/ 0.1% TFA or HFBA

- Injection: 20µ µL

0.000

0.00

11

Gradient: O-60 % B 120 min

0.00

0.40

Temp: 35 C

0.20

0.00 20

20.00

Flow: 0.18 mL/min

HFBA

- Temperature: 35 C - Detection: 214 nm

0.0000 0.00

Sample: Rabbit cytochrome c tryptic digest, 500 pmol

0.40

13

Absorbance (214 nm)

0.300

40.00

40

60

80

Time (min)

Minutes

Alden

What Factors Influence Gradient RP-HPLC Separations...

Temperature Effects on Resolution

Part I - Factors influencing efficiency and retention Gradient Slope; c Column Length; L and N Flow Rate; F

Part 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

14

Resolution is temperature dependent Temperature is a critical parameter to control in order to achieve reproducible separations.

Gradient Considerations in LC What Factors Influence Gradient RP-HPLC Separations...

Temperature Effects on Resolution Conditions

10

Rs (5,6) = 0 Rs (8,9) = 2.39

0.300

30 C

- Column: Symmetry300™, C18, 5µ µm, 3.9x150mm

9

0.200

5, 6 AU

Part I - Factors influencing efficiency and retention 

11

8

7

0.100

- Sample: Tryptic digests of bovine cytochrome c

0.000 25.00

30.00

35.00

Gradient Slope; c – Column Length; L and N –

40.00

Minutes

- Injection: 20µ µL

0.200

10

Rs (5,6) = 0 Rs (8,9) = 2.07

0.300

35 C

AU

8

7

0.100

Flow Rate; F–

- Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile

9

5, 6

11

0.000

Part II - Factors influencing selectivity

- Gradient: 0-45 min., 0-30%B 25.00

30.00

35.00

40.00

Minutes

Rs (5,6) = 0.84 Rs (8,9) = 1.63

0.300

0.200

- Flow rate: 0.75 mL/min.

10

40 C - Detection: 214 nm

Part III - Factors influencing reproducibility 

9

5 6

AU

8

7

0.100

Column– HPLC system –

11

0.000 25.00

Alden

30.00

35.00



Concentration and Type of Modifier – Temperature– Pore Size and Chemistry of the Packing Material

40.00

Minutes

Pore Size Effects on Resolution

Selectivity Differences Between Packings

Conditions

Symmetry , C18, 5µm, 4.6 X 150 mm, 100 10

0.30

4

23

AU 0.10

13 12

9

56

0.20

8

1

11

7

0.00

0.10 0.10-0.00

20.00

40.00 Minutes

Symmetry300™, C18, 5µm, 4.6 X 150 mm, 300

- Sample: Tryptic digests of cytochrome c (bovine) - Injection: 20µ µL - Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - Gradient: 0-50 min., 030%B - Temperature: 35 C - Flow Rate: 0.75 mL/min. - Detection: 214 nm

10 13

5,6

0.500

12

9

0.400 0.300 AU 0.200

4

2 1

0.100

8

3

7

0.10

5 4

AUFS 0.05

1

2

3

0.00 5.00

10.00

5 4

AUFS 0.05

1 2

Carmody

20.00

Minutes

40.00

Gradient: Sample:

3

Detector: Temperature:

0.00

0.00

Conditions: Columns: Flow Rates: Mobile Phase:

15.00

Symmetry C8, 100A

0.10

-Different pore sizes change selectivity.

Ac-Arg-Gly-X-X-Gly-Gly-Leu-Gly-LysAmide -X-X-: 1: Ala-Gly with free alpha amino group 3: Ala-Gly 2: Gly-Gly 5: Val-Val 4: Val-Gly

SymmetryShield™ RP8, 100A

11

0.000

15

Alberta Peptides on Symmetry Reversed-Phase Columns

5.00

10.00

15.00 Minutes

3.9 mm x 150 mm 0.8 mL/min A. 0.1% TFA aqueous; B. acetonitrile with 0.1% TFA 10% to 40% B in 30 minutes, step to 60% B for 5 min 9 L Alberta Peptides, mix with 5 decapeptides 214 nm 35 C

Gradient Considerations in LC What Factors Influence Gradient RP-HPLC Separations...

Peptide Mapping Validation -Robustness Testing

Part I - Factors influencing efficiency and retention Gradient Slope; c Column Length; L and N

Choice and quality of enzyme Digestion conditions HPLC conditions Equipment

Flow Rate; F

Part II - Factors influencing selectivity Concentration and Type of Modifier Temperature Chemistry and Pore Size of the Packing Material

System Column

Part III - Factors influencing reproducibility Column HPLC system

Separations Module

Effects of Irreproducible Gradient Delivery Traditional HPLC System

-

Gradient Accuracy & Precision

Experience resolution differences and retention time shifts

10%B

0.08 0.07

1% Step Gradient at 1.0 mL/min Accuracy