Method Optimization

Gradient Elution Reversed-Phase HPLC

Ion-Pair HPLC
Normal-Phase HPLC
Gradient Elution

• Why choose this separation mode?

• Which HPLC parameters affect a gradient
separation?
• How can I use these parameters to improve my
gradient separation?

Slide 4
Is Gradient or Isocratic Elution the Preferred
Separation Mode for RP-HPLC Assays?

GRADIENT ISOCRATIC
ELUTION ELUTION

Slide 5
Isocratic Elution
Separation occurs using a constant composition
mobile phase

Column: ZORBAX SB-C18

Substituted Anilines 4.6 x 150 mm, 5 µm
3 Mobile Phase: 42% Methanol
58% 25 mM phosphate buffer,
pH 2.5
Flow Rate: 1 mL/min
Temperature: 22°C
Sample: 1. p-anisidine
2. m-toluidine
3. 3-aminobenzonitrile
4 4. p-chloroaniline
5. m-chloraniline
6. o-chloroaniline
5
1
2
6

0 1 2 3 4 5 6 7 8 9
Time (min)

Slide 6
Advantages of Isocratic Elution

• Simple HPLC instrumentation – single pump

• No column re-equilibration necessary
• Easy instrument and method transfer
• Method development better understood

Slide 7
Disadvantages of Isocratic Elution

• Peaks broaden with increasing

retention time
Substituted Anilines
• Sensitivity and LOD decrease with
3 increasing retention time

• Peak broadening limits the number of

compounds in a single
4
chromatogram
5
1
2 • Peak broadening limits the polarity
6
range of compounds eluted in a
0 1 2 3 4 5
Time (min)
6 7 8 9
single chromatogram

Slide 8
Gradient Elution
Separation occurs by continuously increasing
the solvent strength of the mobile phase

Column: ZORBAX 300SB-C8

2.1 x 150 mm, 5 µm
Gradient: 2-62% B in 70 min.
Mobile Phase: A: 100%H2O with 0.1% TFA
B: 0.1% TFA in 80% ACCN,
20% H2O
Flow Rate: 0.2 mL/min
Temperature: 50°C
Sample: 50 pmol of BSA digest in 4M urea

0 10 20 30 40 50 60

Time (min)

Slide 9
Gradient Elution for Reversed-Phase HPLC
Increasing the solvent strength =
Increasing the % organic in the mobile phase
Linear solvent strength gradient = % per min is a constant

90%
90
70% ∆φ = 80%
%ACN 50%
t G = 40 min.
30%
∆φ
10 t G = 2%/min.
0 10 20 30 40 min.
}
}
}
}
∆t 1 = ∆t 2 = ∆t 3 = ∆t 4

For every 20% change in ACN,
t is 10 min.

Slide 10
Why Choose Gradient Elution?

• Case 1:
• To separate samples having components
that vary widely in polarity.

Slide 11
Separation of Herbicides on
ZORBAX SB-C18
Isocratic Elution Gradient Elution
70% aqueous/30% ACN 20 – 60% ACN in 30 min.
1,2
4 Column: ZORBAX SB-C8
4.6 x 150 mm, 5 µm
5
Mobile Phase: A: H2O with 0.1% TFA, 4
5 pH 2
B: Acetonitrile 8
Flow Rate: 1.0 mL/min
Temperature: 35°C
Sample: 1. Tebuthiuron
2. Prometon
3. Prometryne 2

3 4. Atrazine
3
5. Bentazon
6. Propazine 6
1
6 7. Propanil
8 7
8. Metolachlor
7

0 25 50 75 0 5 10 15 20 25 30
Time (min) Time (min)

Slide 12
Why Choose Gradient Elution?

• Case 2:
• To separate low molecular weight
mixtures having a large number of
components

Slide 13
Gradient Elution Analysis of Pesticides
in Drinking Water
Column: ZORBAX SB-C18
3.0 x 250 mm, 5 µm
Gradient: 10% B for 2 min.
10 – 45% B in 70 min
Mobile Phase: A: 2 mM Sodium Acetate
pH 6.5 with 5% ACN
B: Acetonitrile
11 Flow Rate: 0.35 mL/min
10
Temperature: 40°C
Sample: Pesticides
9

7 1. Desisopropylatrazine 15. Chlortoluron

6 2. Metamitron 16. Atrazine
3. Fenuron 17. Monolinuron
5
4. Chloridazon 18. Diuron
4 5. Desethylatrazine 19. Isoproturon
3
6. Metoxuron 20. Metobromuron
7. Carbetamid 21. Metazachlor
2 8. Bromicil 22. Buturon
1 9. Hexazinone 23. Propazine
10. Simazine 24. Dimefuron
0
11. Metribuzin 25. Terbuthylazine
10 20 30 40 50 60 70 80 12. Desethylterbutylazine 26. Linuron
Time (min) 13. Carbutilat 27. Chlorbromuron
14. Methabenzthiazuron 28. Chlorxuron

Slide 14
Why Choose Gradient Elution?

• Case 3:
• To separate high molecular weight
mixtures (i.e., peptides and proteins)

Slide 15
 Larger Molecules are More Sensitive than Small
Molecules to Changes in % Organic
87tg F
100 k* =
Leucine Enkephalin Lysozyme ∆Φ Vm S
s = 11 s = 40

Benzene
10 s = 2.7

k«
∆Φ = change in volume percent of B solvent (%)
S = property of sample compound
1 F = flow rate (mL/min.)
tg = gradient time (min.)
Vm = column void volume (mL)

0.14 0.18 0.22 0.26 0.30 0.34 0.38 0.42

φ

• Lysozyme is 15X more sensitive to changes in organic modifier than

benzene and 4X more sensitive than leucine enkephalin.

Slide 16
 Gradient Separation of Peptides on 300SB

Column: ZORBAX 300SB-C3

4.6 x 150 mm, 5 µm
Gradient: 15-35% B in 19 min.
Mobile Phase: A: 95 : 5 H2O : ACN
with 0.1% TFA
4
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe B: 5 : 95 H2O : ACN
with 0.085% TFA
6
Temperature: 35°C
Detection: UV 215 nm
1
2 Sample: Peptides
3
5

8 1. Leucine Enkephalin
7
Tyr-Gly-Gly-Phe-Leu 2. Angiotensin
3. RNase A
4. Insulin
5. Cytochrome C
6. Lysozyme
7. Myoglobin
8. Carbonic Anhydrase

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0
T im e ( m in )

• Gradient conditions are required for the separation of

Leucine enkephalin (555 daltons) and Lysozyme (14,000 daltons).
Slide 17
Advantages of Gradient Elution

• Complex samples are analyzed in a single HPLC run

• Analysis time is reduced
• All peaks elute with the same bandwidth
• More peaks can be baseline resolved per unit time
• Sensitivity and LOD are unchanged during a gradient
run

Slide 18
Disadvantages of Gradient Elution

• More expensive instrumentation

• Possible precipitation at interfaces, when using multiple
proportioning valves
• Re-equilibration time adds to analysis time
• Instruments vary in their dwell volume (Vd), which can
cause method transfer problems

Slide 19
What Parameters Maximize Gradient
Resolution?

Slide 20
Factors that Maximize Isocratic
Resolution Between Peaks

Increase • Decrease % organic

retention k

• Change the chemistry

Change relative of the mobile or
peak position a
stationary phase
• Change % organic
Reduce peak
• Increase column
width N
length
• Decrease particle size
or flow rate

Slide 21
To Maximize Gradient Resolution
Between Peaks

• INCREASE one or more of the following:

• k* Gradient retention

α selectivity1
•N theoretical plates1

1similar to isocratic elution

Slide 22
Increasing Retention (k*) in Gradient Elution is
Difficult to Visualize

Slide 23
Which of the Following Increases
Gradient Retention?

• A longer gradient time

• A shorter column
• A higher flow rate
• A shorter organic range

Slide 24
All of the Following Increase Gradient Retention
(k*)

• A longer gradient time tG

• A shorter column Vm
• A higher flow rate F
• A shorter organic range
Φ

• Because:
S •
Φ • Vm
• 1/k* ∝Gradient steepness = b = ____________
tG • F

Slide 25
Use of a Longer Gradient Time Increases
Gradient Retention
1,2
1 2
Gradient Time Gradient
3 Time
4 5
10 min. 60 min.
3 Column: ZORBAX SB-C8
4 4.6 x 150 mm, 5 µm
5 7 Gradient: 20 – 60% B
Mobile Phase: A:H2O with 0.1%TFA, pH 2
6,7
B: Acetonitrile
Flow Rate: 1.0 mL/min
Temperature: 35°C
6
Sample: Herbicides

8
8

0 5 10 15 0 25 50
Time (min) Time (min)

• Increased gradient retention improves resolution of several peak pairs – 1,2 and 4,5.

Slide 26
A Shorter Column:
Increases Gradient Retention, Increases Overall Resolution,
Assumes Constant N

1,2

4.6 x 150 mm, 5 mm 4.6 x 75 mm, 3.5 mm

N = 12,000 5 N = 10,000

4
Column: ZORBAX SB-C8
Gradient: 20 – 60% B
3 8 Mobile Phase: A:H2O with 0.1%TFA, pH 2
4 B: Acetonitrile
5
Flow Rate: 1.0 mL/min
7 Temperature: 35°C
2 Sample: Herbicides
3

1 6
7
6

0 5 10 15 0 5 10
Time (min) Time (min)

Slide 27
Gradient Elution

gradient S • ∆Φ • Vm
1/k* ∝ steepness = b =
tG • F

This relationship also says:

If “b” is kept constant from run-to-run

peaks will elute in the same relative pattern.

Slide 28
Two Chromatograms Both Having the Same
Gradient Steepness

Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8. Metolachlor

Column: StableBond SB-C8 Column: Rapid Resolution

4.6 x 150 mm, 5 µm StableBond SB-C8
4.6 x 75 mm, 3.5 µm
4
Gradient 5
5
Time: 30 min. 4 Gradient
Time: 15 min.
Flow Rate: 1.0 mL/min
Flow Rate: 1.0 mL/min
8
8

2 2
Analysis
3 3 Time: 12 min
Analysis
6 1
Time: 24 min 1 6
7
7

0 5 10 15 20 25 0 5 10 15
Time (min) Time (min)

Slide 29
Two Chromatograms Both Having the Same
Gradient Steepness
Sample: 1. Tebuthiuron 2. Prometon 3. Prometryne 4. Atrazine 5. Bentazon 6. Propazine 7. Propanil 8. Metolachlor

Column: StableBond SB-C8 5 Column: StableBond SB-C8

4.6 x 150 mm, 5 µm 4 4.6 x 75 mm, 3.5 µm
Gradient Gradient
Time: 60 min. 1 2 Time: 15 min.
3
Flow Rate: 1.0 mL/min Flow Rate: 2.0 mL/min
4
5 8

Analysis Analysis
7 3
Time: 40 min Time: 7.2 min
1
6
7
6

0 20 40 0 5 10
Time (min) Time (min)

• Multiple gradient parameters can be changed to maintain gradient

steepness and reduce analysis time.
Slide 30
Recommendations for Gradient Elution Method
Development

• Before changing the stationary phase or mobile phase to

generate changes in a, selectivity, explore increasing gradient
retention, k*, by
• Increasing gradient time
• Decreasing column length
• Increasing flow rate
• Shortening organic range
• Before finalizing a gradient method reduce column length (same
N), and adjust the flow rate and gradient time to reduce analysis
time and solvent waster while maintaining resolution.

Slide 31
Break Number 1

Please type your

question into the
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during the presentation.

Slide 32
Reversed-Phase HPLC Doesn’t Always
Do the Job

• Not enough retention of ionized

compounds, i.e., acids and bases
• Too much retention of hydrophobic
compounds
• Peaks overlap no matter what you try

Slide 33
To Resolve Non-Retained Acids and Bases Ion-
Pair Chromatography is Recommended

Slide 34
Ion-Pair Chromatography

C8 or C18

Solute = cation IP-reagent = anion

This diagram demonstrates a mechanism of ion-pair chromatography. The ion-pair

reagent is embedded in the stationary phase and in solution where the analytes
interact with it.

Slide 35
Ion-Pair Chromatography is Preferred
Over Ion-Exchange

• Better column efficiency (N)

• C8 and C18 columns are more stable
and reproducible
• More options to resolve bands

Slide 36
Suggested Experimental Conditions
for Ion-Pair HPLC

• Column: C8 or C18 • Cations – bases

• Mobile Phase: • Buffer: 25 – 50 mM
phosphate, pH 2- 3
• Organic Methanol
preferred • Ion-pair – reagent: 10 – 100
mM hexane sulfonate
• Aqueous Buffered with
appropriate IP- reagent • Anions – acids

• Temperature: Controlled • Buffer: 25 – 50 mM

between 35° and 60°C phosphate, pH 6 – 7
• Ion-pair reagent – 10 – 40
mM tetrabutyl ammonium
phosphate

Slide 37
Comparison Separation of Catecholamines
Reversed-Phase and Ion-Pair HPLC

Reversed Phase HPLC Ion-Pair HPLC

A: 97.5% H2O with 0.1%TFA A: 90% 70 mM sodium phosphate, pH 3
2 B: 2.5% Acetonitrile 1 10 mM hexane sulfonate
B: 10% methanol
2

4
1
3 3
4 5

0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
Time (min) Time (min)

Column: ZORBAX SB-C18, 4.6 x 150 mm, 5 mm Sample: 1. Norepinephrine 2. Vanillyl Mandelic
Acid 3. Tyramine 4. DOPAC 5. Homovanillic acid

Slide 38
Critical Issues with Ion-Pair Chromatography

• Reproducibility
• Temperature control
• Column Equilibration
• IP-reagent in the injection solvent
• IP-reagent concentration in the mobile phase > 10 mM
• Dedicate column to IP-HPLC
• Poor Peak Shape
• 20 mM TEA may be added to the mobile phase to improve
peak shape for basic solutes
• Method may require routine washing with high percent
organic

Slide 39
To Resolve Strongly Retained
Hydrophobic Samples
Normal-Phase HPLC is Recommended

Slide 40
Other Reasons to Choose
Normal-Phase HPLC

• Isomer separation
• Sample injection solvent is non-polar
• Recovery in non-polar solvents is
desirable

Slide 41
Normal-Phase Chromatography

S E E S E E

X Y X Y X Y
b A A A A A

• Polar interactions between solute and stationary phase are most important.
• Retention decreases with increasing polarity of the mobile phase.

Slide 42
Normal-Phase HPLC
Stationary Phase Options

silica > amino > diol > cyano

Strongest Weakest

Cyano and Silica are Preferred

Slide 43
Bonded Silica Preferred Over Silica for
Analytical Work

• Cyano – CN
• Faster equilibration
• Control of water content not needed
• Gradient elution is an option
• Weaker phase
• Silica
• Isomer separation
• Preparative HPLC

Slide 44
Suggested Normal-Phase HPLC Conditions

Bonded-Silica Silica

ZORBAX CN ZORBAX SIL

Column: Packed in normal phase ZORBAX Rx-SIL –
solvents basic solutes

Hexane with Methylene chloride with

Mobile Phase:
1- or 2-propanol 0.05% - 0.5% methanol

Temperature: Ambient - 60°C Ambient - 60°C

Slide 45
Comparison of Silica Types

1 2 ZORBAX SIL 4

3 Column: 4.6 x 150 mm, 5 µm

Mobile Phase: Methylene chloride with
I 0.05% methanol
Flow Rate: 1.0 mL/min
Temperature: ambient
Sample: 1. Toluene
2. Benzanilide
3. Phenol
2 3 ZORBAX Rx-Sil 4 4. Benzyl alcohol
I. Impurity
1

0 2 4 6 8 10 12 14 16
Time (min)

• The low acidity, ZORBAX Rx-Sil improves peak shape of basic compounds.
Slide 46
Critical Issues in Normal-Phase HPLC
• Poor peak shape
• Injection solvent stronger than mobile phase
• Basic samples give better peak shape using high purity silica
• Basic samples may require 20 mM TEA in the mobile phase
• Acidic samples may require 20 mM acetic acid in the mobile
phase
• Strongly retained polar materials can build on the column
and can be removed with water this is slightly acidic
• Reproducibility
• Silica requires addition of water or methanol to maintain
reproducibility
• Use columns packed in normal-phase solvents

Slide 47
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Slide 48