Basics of Method Development

John Palmer
Agilent Technologies, Inc.
October 16, 2008

Page 1
 The Goal of HPLC Method Development
• Adequate Retention
• Too Little Retention Leads to Poor Resolution
• Too Much Retention Wastes Time and Other Resources
• Suggested Retention 1 > (k) < 10 (actual time depends on Column Size)

• Adequate Resolution
• Minimum, Complete of Adjacent Peaks = 1.5
• Recommend Minimum Acceptable Resolution > 2.0

• Precise
• Accurate
• Reproducible

Page 2
 How Can Resolution Be
Increased in Method Development?

k’ Increase k'
(mobile phase
Starting Point:
chemistry)

N α

Increase Selectivity
Increase Efficiency
(bonded phase,
(physical) mobile phase
chemistry)

Page 3
 Chromatographic Profile
Equations Describing Factors Controlling RS

Retention Factor

(tR-t0)
k=
t0

Selectivity

α = k2/k1

Theoretical Plates-Efficiency

N = 5.54 (tR / W1/2)2

Page 4
 Each Controlling Factor Can Be Combined to
Define and Calculate Resolution

N (α-1) k'
Rs = • •
4 α k'+1
Theoretical Plates Selectivity Retention

Page 5 000990P2.PPT
Resolution as a Function of Selectivity, Column
Efficiency, or Retention
7.00

Selectivity Impacts Resolution Most

6.00
• Change bonded phase
5.00 • Change mobile phase
Resolution

4.00 Increase N
Increase Alpha
Increase k'
3.00

2.00

1.00

Rs = N½/4 • (α-1)/α • k’/(k’+1)

0.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Plates: 5000 10000 15000 20000 25000

Alpha: 1.10 1.35 1.60 1.85 2.1

k’: 2.0 4.5 7.0 9.5 12.0

Page 6
 Chemical Parameters That Influence
HPLC Separations

• Sample Chemistry
• pH, Buffer Choice and Concentration
• Organic Modifier and Concentration
• Column Chemistry

Finding the Correct Interaction of These Parameters

Maximizes the Incredible Separating Power of HPLC

Page 7
Mobile Phase pH and pH Buffers
Why Are These So Important in HPLC?
• pH Effects Ionization
– Silica Surface of Column
– Sample Components of Interest

• Buffers
– Resist Changes in pH and Maintain Retention
– Improve Peak Shape for Ionizable Compounds

• Effects Column Life

– Low pH strips Bonded Phase
– High pH Dissolves Silica

Page 8
pH of Mobile Phase Optimizes Selectivity and Resolution
Changes in Selectivity with pH and Buffer Change
7
pH 2.7 0.1% formic acid/ACN 1. procainamide
1 4 6 2. buspirone
3. pioglitazone
3
4. eletriptan
2 5. dipyridamole
6. diltiazem,
5 7. furosemide

0 2 4 6 8 min

1
pH 4.8 NH4AC/ACN 6
2

7 5 3
4

0 2 4 6 8 min

pH 7 NaPO4/ACN 7 2
6
1 3

4 5
0 2 4 6 8
Conditions: Column: Eclipse Plus C18 4.6 x 100mm, 5um Gradient: 10 – 90% in 10 minutes Detection: UV 254 nm

Page 9
 How Does pH Affect Ionization of Acids?
pH, pKa and Weak Acids

[RCOO-][H+]
RCOOH RCOO- + _
H+ Ka =
[RCOOH]
COOH COO

Ka = 6.4 x 10-5
+ H+ pKa = 4.2

At pH 4.2 – the sample exists as benzoic acid and the benzoate ion in a ratio
of 1:1. Peak shape can be poor
At pH 5.2 – 91% of the sample exists as the benzoate ion. RP retention decreases.
At pH 3.2 – 91% of the sample exists as benzoic acid. RP retention increases.

Page 10
 Effect of pH on Peak Shape at or
Near the Sample pKa
Column: ZORBAX SB-C8 4.6 x 150 mm, 5 mm Mobile Phase: 40% 5 mM KH2PO4: 60% ACN
Flow Rate: 1.0 mL/min. Temperature: RT

CH3CHCOOH

pH 4.4 pH 3.0
CH2CH(CH3)2

Ibuprofen
pKa = 4.4

0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
Time (min) Time (min)

Inconsistent and tailing peaks may occur when operating close to an

analyte pKa and should be avoided.

Page 11
 How Does pH Affect Ionization of Bases?
pH, pKa and Weak Bases

R3NH+ R3N + H+ [R3N][H+]

Ka =
[R3NH+]

∅
+
CH CH
Ka = 1 x 10-9
∅
H pKa = 9
3 3

CHOCH CH N
2 2
CHOCH CH N
2 2 + H+
∅ CH ∅ CH
3 3

At pH 9 – the sample exists as protonated and unprotonated

diphenhydramine in a ratio of 1:1. Peak shape can be poor.
At pH 10 – 91% of the sample exists as unprotonated diphenhydramine.
At pH 8 – 91% of the sample exists as protonated diphenhydramine.

Page 12
 Effect of pH on Peak Shape
Below and Above the Sample pKa
Column: ZORBAX Extend-C18, 4.6 x 150 mm, 5 m m Mobile Phase: See Below Flow
Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm
Sample: 1. Maleate 2. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5.
Chlorpheniramine 6. Triprolidine 7. Diphenhydramine
pH 7 pH 11
30% 20 mM Na2HPO4 30% 20 mM TEA
70% MeOH 70% MeOH
7
4 7
4
2,3
3
1
tR = 8.5 tR = 11.4
5
1
5

6 2
6

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

Improved peak Shape and Retention above pKa

Page 13
 Retention vs. pH for Ionizable Compounds
Effects are Compound Dependent
12

10
Acetylsalicylic acid
Pyridine
pKa 3.5
retention time (min)

8
Codeine
pka 5.2
Procainamide pKa 8.0
6 Amphetamine pKa 9.2
Caffeine pKa 9.9
4 pKa 14

0
pH 2.5 pH 6.5 pH 8 pH 11.5 Mobile Phase:
45% MeOH:
pH 55% 20 mM Phosphate Buffer

Page 14
Common Buffers Used in HPLC
General Purpose Buffers

Potassium dihydrogen phosphate (monobasic) 1.1 – 3.1

Dipotassium hydrogen phosphate (dibasic) 6.2 – 8.2
Tripotassium phosphate (tribasic) 11.3 – 13.3
Sodium dihydrogen citrate 2.1 – 4.1
Disodium hydrogen citrate 3.7 – 5.7
Trisodium citrate 4.4 – 6.4
Sodium acetate 3.8 – 5.8
TRIS [tris(hydroxymethyl)aminomethane] 8.0 – 10.0
Ammonium hydroxide 8.3 – 10.3

Page 15
 SELECTIVITY

• Mobile Phase Interaction

• Bonded Phase Interaction

Page 16
Change In Mobile Phase Organic Alters Selectivity
Acetonitrile vs. Methanol
Column: ZORBAX Eclipse XDB-CN Column Dimensions: 4.6 x 150 mm, 5 μm Mobile Phase: As shown
Flow Rate: 2.0 ml/min Injection Volume: 2.00 μl Column Temperature: 25 °C Detector: UV, 210 nm

Mobile Phase: 35:65 ACN:Water Sample:

1. Estriol (0.00130 μg/μl),
1 2 2. b-Estradiol (0.00130 μg/μl),
3. Ethinyl Estradiol (0.00147 μg/μl),
4. Dienestrol (0.00123 μg/μl),
5. Diethylstilbestrol (0.00128 μg/μl)
4 6. Ethynylestradiol 3-methyl ether
5 (0.00103 μg/μl)
3 7. Ethynodiol Diacetate (0.00139
μg/μl)
6 7

0 2 4 6 8 10 12 14 16 18 min

1
4 Mobile Phase: 47.5:52.5 MeOH:Water
.
2
5
3
6
7

0 2 4 6 8 10 12 14 16 18 min

Page 17
Mobile Phase Optimization on Short RRHT
4.6 x 50 mm, SB-CN Column
1 4 1. Estril
mAU
250 2 3 5 60%B 2. Estradiol
200
Rs 4,5=1.96 3. Ethynyl Estradiol
6
150
4. Dienestrol
100

50
5. Diethylstilbestrol
0 6. Ethynl estradiol methyl ester
0 1 2 3
)
mAU
250
1 4 55%B A: Water
2 3 5 B: 30 % MeOH/70 %MeCN
Rs 4,5=2.59
200

150
1 µl injection, 210 nm
100
6
50

0 1 2 3 4 5
mAU

1 50%B
250

200
2 3 4
150
5 Rs 4,5=3.29
100 6
50

0 1 2 3 4 5 6 7 8 min

¾RRHT columns allow for very fast optimization of % organic in

mobile phase.

Page 18
 Bonded Phase Structure Influences
Method Selectivity

StableBond, pH 1-6 Eclipse XDB, pH 2-9 Bonus-RP, pH 2-8 Extend-C18, pH 2-11.5

1. Uses bulky silanes 1. eXtra Densely Bonded 1. polar alkyl phase 1. unique bidentate structure
2. Non-endcapped dimethylalkylsilanes 2. triple endcapped 2. double endcapped
2. proprietary double-endcapping 3. uses bulky silanes

* R
CH3 R1
O Si
*R1 O Si
C18 C18
O Si PG R
CH3 Si Si
R CH3 R1 O O
OH O Si CH3 CH3
R CH3 Silica Support
Si CH3
CH3
CH3
O Si
O Si R1 R1
CH3
R CH3 O Si PG R
O Si CH3
OH R1
CH3
R CH3 CH3
O Si Si CH3
O Si R1
CH3 CH3

R R1

O Si PG R

R1
At

Page 19
 All C18 Phases Do Not Yield the Same Selectivity
Different Surface Chemistry/Structure-Different Selectivity!

1 2 4 Eclipse Plus C18

3
Mobile phase: (69:31) ACN: water
Flow 1.5 mL/min.
1 2 3 4 5 Temp: 30 °C
Detector: Single Quad ESI
1 2 4 StableBond positive mode scan
3 Columns: RRHT
SB-C18 4.6 x 50 mm 1.8 um

1 2 3 4 5 min Sample:
1. anandamide (AEA)
1 2 4 Eclipse 2. Palmitoylethanolamide (PEA)
3 XDB-C18 3. 2-arachinoylglycerol (2-AG)
4. Oleoylethanolamide (OEA)

1 2 3 4 5

1 4 Extend-C18
2,3

1 2 3 4 5

Page 20
Good Peak Shape Provides Best Foundation
Base Method Development on Optimized Column Packing
1
Mobile Phase: 65% ACN: 35% 25 mM phosphate buffer (pH 7.4)

Eclipse Plus C18, 4.6 x 50mm

1. Nortriptyline Tf = 1.20
2. Dipropylphthalate

1
Competitive C18, 4.6 x 50mm
Tf = 4.86
2

2
Superior peak shape and better selectivity with Eclipse Plus means more resolution, easier
quantitation and better results in your separations.

Page 21
 Different Bonded Phases Within a Column “Family”
Optimize Separation for Speed, Selectivity, Resolution

Eclipse Plus Phenyl Hexyl

0 2 4 6 8 10 min

Elution order reversal between Phenyl-Hexyl and Alkyl chains

Eclipse Plus C8

0 2 4 6 8 10 min

Eclipse Plus C18

0 2 4 6 8 10 min

Mobile Phase 40 % ACN 60 % 25 mM Sodium Phosphate Buffer pH 2.4 Flow Rate: 1.5 ml/min 4.6 x 50mm UV 210 nm
2µl Elution order for Eclipse Plus Phenyl Hexyl: (1) Piroxicam, (2) Sulindac,(3) Tolmetin, (4) Naproxen, (5) Ibuprofen, (6)
Diclofenac, (7) Celebrex (equal portions of approximately 1 mg/ml solutions

Page 22
 Basic Anesthetics on StableBond Columns
Different Bonded Phase Structure Yields Different Selectivity vs. XDB

3
4
SB-C18 1
5
2 Columns: 4.6 x 250 mm
Mobile Phase:
3 0 - 100% B in 18.8 min
1 4
A: 50 mM NaH2PO4,
SB-C8 2
5
pH 2.5 in 95% H2O/5% ACN
B: 50 mM NaH2PO4,
pH 2.5 in 47% H2O/53% ACN
3
1 4 Flow Rate: 1.5 mL/min
5
SB-C3 Temperature: 26°C
2
UV Detection: 254 nm
Sample: 10 μL
1 3 1. Procaine
4 2. Lidocaine
SB-Phenyl 5
3. d-Cinchonine
2
4. Butacaine
5. Tetracaine
1 3 4
SB-CN 2
5
‹ Changes in elution order as the polarity
of the bonded phase changes.
0 5 10 15
Time (min)

Page 23
Many Bonded Phase Options for Method Development
Eclipse Plus Eclipse XDB-Eclipse XDB-
Dimensions Eclipse Plus C18 Eclipse Plus C8 Ph-Hex Eclipse PAH C18 C8 Extend-C18
4.6 x 150 959994-902
4.6 x 100 959964-902 959964-906 959964-912 959964-918 928975-902 928975-906 728975-902
4.6 x 75 959951-902
4.6 x 50 959941-902 959941-906 959941-912 959941-918 927975-902 927975-906 727975-902
4.6 x 30 959931-902 959931-906 959931-912 959931-918 924975-902 924975-906 724975-902
4.6 x 20 926975-902 926975-906 726975-902
3.0 x 150 959994-302
3.0 x 100 959964-302 959964-306 959964-312 928975-302 928975-306 728975-302
3.0 x 50 959941-302 959941-306 959941-312 927975-302 927975-306 727975-302
3.0 x 30 924975-302 924975-306 724975-302
3.0 x 20 926975-302 926975-306 726975-302
2.1 x 150 959794-902
2.1 x 100 959764-902 959764-906 959764-912 959764-918 928700-902 928700-906 728700-902
2.1 x 50 959741-902 959741-906 959741-912 959741-918 927700-902 927700-906 727700-902
2.1 x 30 959731-902 959731-906 959731-912 924700-902 924700-906 724700-902
2.1 x 20 926700-902 926700-906 726700-902
Dimensions SB-C18 SB-C8 SB-Phenyl SB-CN SB-AQ Rx-Sil Bonus-RP
4.6 x 150 829975-902 829975-906 829975-912 829975-905 829975-914
4.6 x 100 828975-902 828975-906 828975-912 828975-905 828975-914 828975-901 828668-901
4.6 x 75 830668-901
4.6 x 50 827975-902 827975-906 827975-912 827975-905 827975-914 827975-901 827668-901
4.6 x 30 824975-902 824975-906 824975-912 824975-905 824975-914
4.6 x 20 826975-902 826975-906
3.0 x 150 829975-302 829975-306 829975-312 829975-305
3.0 x 100 828975-302 828975-306 828975-312 828975-305 828975-314 828975-301 828668-301
3.0 x 50 827975-302 827975-306 827975-312 827975-305 827975-314 827975-301 827668-301
3.0 x 30 824975-302 824975-306 824975-305
3.0 x 20 826975-302 826975-306
2.1 x 150 820700-902 820700-906 820700-912 820700-905
2.1 x 100 828700-902 828700-906 828700-912 828700-905 828700-914 828700-901 828768-901
2.1 x 50 827700-902 827700-906 827700-912 827700-905 827700-914 827700-901 827768-901
2.1 x 30 824700-902 824700-906 824700-912 824700-905 824700-914
2.1 x 20 826700-902 826700-906

Page 24
 Physical Parameters That Influence
HPLC Separations

• Particle Size – Efficiency and Speed

• Column Dimensions – Sensitivity, Sample Load,

Speed

• Flow Rate – Speed, Efficiency

• Temperature – Selectivity, Speed

Page 25
 Van Deemter Curve
HETP vs. Volumetric Flow Rate
Column: ZORBAX Eclipse XDB-C18
H = A + B/u + Cu
0.0030 Dimensions: 4.6 x 50/30mm
Eluent: 85:15 ACN:Water
Flow Rates: 0.05 – 5.0 mL/min
0.0025 Temp: 20°C
Sample: 1.0μL Octanophenone in Eluent
HETP (cm)

0.0020

5.0μm
0.0015 3.5μm
1.8μm
0.0010

0.0005

0.0000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Volumetric Flow Rate (mL/min)

Smaller particle sizes yield flatter curves, minima shift to higher flow rates

Page 26
 ZORBAX Rapid Resolution HT Columns Provides
Efficiency > Longer Columns
Column Resolving Resolving Resolving Analysis
Length Power Power Power Time*
(mm) N(5 µm) N(3.5 µm) N(1.8 µm)
150 12,500 21,000 32,000 -
Efficiency Analysis
100 8,500 14,000 20,000 (N) Time -33%
75 6000 10,500 N.A. -50%
Pressure Peak
Volume -67%
50 4,200 7,000 12,000
Solvent -80%
30 N.A. 4,200 6,500 Usage
15 N.A. 2,100 2,500 -90%

* Reduction in analysis time compared to 150 mm column

• Rapid Resolution HT columns provide high efficiency in longer columns for very
high resolution. N.A. = not available

Page 27
Agilent RRHT Columns Show No Selectivity Differences
with Changes in Particle Sizes Under Same Conditions
1. Caffeine (I.S.)
Column: 4.6 x 50mm Eclipse XDB-C18 Mobile Phase A= pH 4.5 Na Acetate, B=MeOH (60:40)
2. Allobarbital
Flow Rate =1 mL/min Inj. Vol: 2 ul Flowcell: 3uL, 2 mm flow path
3. Phenobarbital
4. Butabarbital
5 µm
t0=0.44 min 5. Hexobarbital
α 3,2=1.20 α 5,4=1.13
α 4,3=1.97
1 2 3 4 5 6 min

3.5 µm t0=0.44 min α 5,4=1.14

α 3,2=1.20
α 4,3=1.97
1 2 3 4 5 6 min

1.8 µm t0=0.46 min α 5,4=1.16

α 3,2=1.22 α 4,3=1.94

1 2 3 4 5 6 min

Page 28
Comparison of Optimal Conditions On Columns with
Different Dimensions – Length, Particle Size, Resolution
1 RRHT SB-CN 4.6 x 50 mm, 1.8um 1. Estril
2. Estradiol
mAU

140
1.5 ml/min 1 µl injection 3. Ethynyl Estradiol
2 4
120
100 3 5 4. Dienestrol
80
6 5. Diethylstilbestrol
60
40 6. Ethynl estradiol methyl ester
20
0
-20

2.5 5
) 1 RRHT SB-CN 4.6 x100 mm, 1.8um A: Water
mAU

4 1.5 ml/min 2 µl injection B: 30 % MeOH/ 70 %MeCN

200 2 40 % B
150 3 5
Detection: UV 210 nm
100
6
50

2.5 5 7.5 10
) 1 SB-CN 4.6 x150 mm, 5um
mAU
4
175
150
2 3 5
1.0 ml/min 3 µl injection
125
100
75 6
50
25
0

2.5 5 7.5 10 12.5 15 17.5 min

RRHT Columns allow rapid resolution optimization during initial method
development. Column lengths compared quickly.

Page 29
Traditional Separation of EPA 610 PAH Mix on
4.6x250mm, 5.0µm Eclipse PAH Column
mAU
1400 Conditions:
Det. 220,4nm No Ref.; Stop time = 26.0min
Flow 2.0 ml/min
Mobile Phase A = Water; B = Acetonitrile
1200 Initial %B = 40
Gradient: Time (Min) %B
0.00 45
17.5 100
1000
24.0 100
25.5 40
27.5 40
Stop Time = 25.0
800
Temp. = 25° C

Rs = 2.2

600

400

200

5 10 15 20 25 min

Page 30
Faster Separation of 16 PAH’s 4.6x100 mm, 3.5µm
Eclipse PAH Column
mAU
Conditions:
Det. 220,4nm No Ref.
1600
Flow 2.0 ml/min
Mobile Phase A = Water; B = Acetonitrile
Gradient: Time (Min) %B
1400 0.00 40
0.90 40
12.00 100
1200 14.5 100
15.5 40
16.5 40
1000 Stop Time = 17.5
Temp. = 25° C
Rs = 2.4
800

600
14 minutes instead of 25 minutes!

400

200

2 4 6 8 10 12 14 min

Page 31
High Resolution and Fast Analysis on Rapid Resolution HT
4.6x50mm, 1.8µm Eclipse PAH Column
mAU
Conditions:
DAD 220,4nm No Ref. DAD Stop Time = 6.0min
2000 Flow 2.00 ml/min
Mobile Phase A = Water; B = Acetonitrile
Gradient: Time (Min) %B
1750 0.00 40
3.5 100
5.2 100
1500 Rs= 2.2 vs. 2.0 for 5.5
6.5
40
40
4.6 x 50mm, 3.5um Stop Time = 7.0
Rs = 2.2 Temp. = 25° C
1250

1000 Higher resolution is achieved in less

time than with the much longer 5u
750 particle size column
500

250

1 2 3 4 min
5

Page 32
 Reduce Particle Size and Maintain Column Length
Increased “N” in Isocratic Separation
DAD1 A, Sig=254,4 Ref =of f (051119A\SIG10003.D)
4.6 x 150, Column:150x4.6
5um mm
30000
mAU 5µm
93 bar 1
2.5
2
N = 7259
Pressure: 93 bar
N: 8213
25000
N∝
1.5 R_S = 1.15
Height: 1.25 mAU 20000 dp

N
S/N: 42.3
1 S/N = 42 15000
Rs = 1.15
0.5 Height = 1.25
t = 14.9 min (1st epimer)
r 10000
0
Noise = 24uAU
Nptp: 2.4 x 10-2 mAU
-0.5 5000
-1 0.2 0.3 0.4 0.5 0.6
0 5 10 15 20 min 1/dp
DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D)
DAD1 A, Sig=254,4 Ref=off (051007D\LC_X000001.D)
Norm. Norm.

2.5 4.6 x 150, 3.5um

Column:150x4.6 mm 3 4.6 xColumn:150
150, 1.8um
x 4.6 mm 1.8µm

2 165 bar
3.5µm 2.5 490 Pressure:
bar 490 bar
Pressure: 165 bar N: 28669
1.5 N = 14862 2
N = 28669
Height:1.78
N: 14862
1.5
1 R_S =Height:1,34
1.37 mAU R_SS/N:
= 43.6
1.80 (+57%)
1
0.5 S/N = S/N:
5050.7 0.5
S/N R=s =44
1.80
R = 1.37 st
tr = 17.2 (1 epimer)
0 Heightt s= 1.34
= 15.3 min (1st epimer)
0 Height = 1.80
r Nptp: 3 x 10-2
-0.5 Noise N=ptp:20uAU
2 x 10-2 mAU -0.5 Noise = 30uAU
-1 -1
0 5 10 15 20 min 10 min

Page 33
 1.8u Particles Reveal More Information and
Improve Detection and Integration
Customer Sample, Translation of Isocratic Impurity Methods, Zoom Critical Time Range (t = 7min)

4 Impurities 7 Impurities 7 Impurities

2 Not Baseline 6 Not Baseline All 7 Baseline
Separated! Separated! Separated!

4.6 x 150, 5μm 4.6 x 150, 3.5μm 4.6 x 150, 1.8μm

93 bar 165 bar 490 bar
N = 7259 N = 14862 N = 28669
R_S = 1.15 R_S = 1.37 R_S = 1.80 (+57%)
S/N = 42 S/N = 50 S/N = 44

Up to 60% higher resolution

without loss in sensitivity

Page 34
Temperature - Higher Temperature as an Aid to
Method Development and Faster Operation
Higher Temperature:
Temperature should always be considered
as a parameter during method development
Provides more rapid mass transfer:
• Improves Efficiency – enhances resolution
• Decreases analysis time – faster separations with no loss in resolution
Decreases Mobile Phase Viscosity
• Lowers backpressure – allows for higher flow rates, faster separations,
greater efficiency and use of sub 2-micron columns
Can change selectivity – optimize resolution

Page 35
Changing Temperature for Selectivity, Resolution
Matches the Expected Results
Salicylic acid 20°C

0 1 2 3 4 min
Coelution
Salicylic acid 30°C
5
4
3
0 1 2

Salicylic acid 40°C

5
4
3
0 1 2

Salicylic acid
60°C

5
4
3
0 1 2
Salicylic acid Column: RRHT SB-C18
4.6 x 50mm, 1.8um 90°C

5
4
3
0 1
2

Page 36
Use Elevated Temperature to Optimize Resolution,
Selectivity
Gradient of Ten Cardiac Drugs on SB-C18 RRHT

50°C
Rs=1.29

0.5 1 1.5 2 2.5 3 3.5 4 min

4.5
60°C
Rs=2.37

0.5 1 1.5 2 2.5 3 3.5 4 4.5 min

70°C
Rs=3.27

0.5 1 1.5 2 2.5 3 3.5 4.5

4 min

Page 37
 Suggested Method Development Path

STEP 1
• Choose C8 or C18 Bonded Phase
• pH If you don’t know, go low (2-3)
• Adjust % ACN or Gradient time for
1.0 <k < 10
Band spacing problems
STEP 2
Change % organic or Gradient Time

Band spacing problems

STEP 3
• Change bonded phase
• CN, Phenyl, other RP(Bonus,Extend)

Band spacing problems

STEP 4
• Change organic modifier
• Adjust % organic for 1< k <10

Page 38
 ISOCRATIC
ELUTION

Page 39
Method Development Example

dipyridamole
propranolol pindolol

disopyramide
β-blocker diltiazem
Anti-arrhythmic
Vasodialator
Ca+ channel blocker
What pH?

Page 40
Fast Scouting Isocratic Runs on Eclipse Plus-C18 RRHT
Cardiac Drugs with Methanol

mAU
Column: ZORBAX RRHT SB-C18, 4.6 x 50 mm, 1.8 mm Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOH
400
Flow Rate: 2.0 mL/min Temperature: 30°C Detection: UV 240 nm
300 Sample: Cardiac Drugs 1.Pindolol 2. Disopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole
200
100
70% MeOH
0
2

mAU
125
100
75
50
25 50% MeOH
0
2

mAU

80
60
40
20
40% MeOH k= 49!!
0
2 4 6 8 10 12 min

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Fast Scouting Isocratic Runs on Eclipse Plus-C18
Cardiac Drugs with Acetonitrile
Column: ZORBAX RRHT EP-C18, 4.6 x 50 mm, 1.8 mm Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: ACN
mAU Flow Rate: 2.0 mL/min Temperature: 30°C Detection: UV 240 nm
Sample: Cardiac Drugs 1.Pindolol 2. Disopyridamide 3.Propranolol 4.Dipyridamole 5. Diltiazem
400
300
200
100 40% ACN
0
2

mAU
125
100
75
50 30% ACN
25
0

mAU

60 20% ACN
40 k= 44!!
20
0
-20
2 4 6 8 10 12 min

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Bonded Phase Selectivity Differences in 30% ACN
mAU
RRHT StableBond-CN
100 4.6 x 50 mm, 1.8 µm
0
1
mAU
RRHT StableBond-Phenyl
100 4.6 x 50 mm, 1.8 µm
0 Dipyridamole
1 2 not excessively
mAU
RRHT StableBond-AQ retained (peak 4)
100 4.6 x 50 mm, 1.8 µm In acetonitrile
0
1 2
mAU
RRHT Eclipse Plus-C18
100
4.6 x 50 mm, 1.8 µm
0
1 2
mAU
RRHT StableBond-C18
100
4.6 x 50 mm, 1.8 µm
0
1 2

Page 43
 GRADIENT
ELUTION

Page 44
 100% B

Changing Gradient Time to

tg= 5 Affect
0% B Retention (k*) and Resolution
100% B
tg F
k* ∝
tg= 10 S Δ%B Vm
0% B
100% B
%B = change in volume fraction of B
solvent
S = constant
tg= 20 F = flow rate (mL/min.)
tg = gradient time (min.)
0% B
Vm = column void volume (mL)
100% B

• S ≈ 4–5 for small molecules

tg= 40
• 10 < S < 1000 for peptides and
0% B
proteins
0 10 20 30 40
Time (min)

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Use of a Longer Gradient Time Increases Gradient
Retention

1,2
Gradient Time Gradient Time
10 min. 1 2
60 min.
3
4 5
Column: ZORBAX SB-C8
4.6 x 150 mm, 5 µm
3 Gradient: 20 – 60% B
4 Mobile Phase: A:H2O
5 7 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.

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A Shorter Column (smaller Vm)
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)

Page 47
Improving Resolution Using Short
Column Length ( Vm ) and Particle Size (N)

300SB-C8, 4.6 x 150 mm, 5µm 300SB-C8, 4.6 x 50 mm, 3.5µm

4 6 4 6
2 5 5
1
10 2 8 10
1 3 7 8 3 7

9 9

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Mobile Phase: A: 95% Water : 5 % ACN, 0.1% TFA Sample: 1. Gly-Tyr 6. Leu-Enk
B: 5% Water : 95% ACN, 0.085% TFA 2. Val-Tyr-Val 7. Angiotensin II
Gradient: 10-60% B in 30 min. 11
3. [Gln ] Amyloid-β- 8. Kinetensin
Flow Rate: 1.0 mL / min. Protein Fragm 1-16 9. RNase
Temperature: Ambient 4. (TYR8) Bradykinin 10. Insulin (Eq.)
5. Met-Enk

Page 48
 Gradient Resolution and Shorter Columns
Reduced Column Volume Improves Gradient Retention
2.1x150mm, 5μm
P/N 883700-922
70 min gradient 120 peaks
0.2 mL/min 60 min.

15 20 25 30 35 40 45 50 55 min

2.1x50mm, 1.8μm 125 peaks

P/N 822700-902 10 min.!
10 min gradient
0.5mL/min
3 4 5 6 7 8 9 10 min

2.1x50mm, 1.8μm
P/N 822700-902 156 peaks!
30 min gradient 25 min
0.5mL/min

5 10 15 20 25 min

Conditions: Mobile Phase A: Water w/ 0.1% TFA, B: ACN w/0.1% TFA, Gradient 2%B to 50%B, Temperature: 50°C
Detection: UV 214 nm Sample: HSA Tryptic Digest

Page 49
 Summary
• Get answers to key chemistry questions about your sample
before you begin method development
• Use buffered mobile phases for ionizable compounds to
get optimum performance
• Use Small Particle columns (short columns with 3.5 u or
1.8u particles) to decrease method development time
• Evaluate methanol AND acetonitrile organic solvents for
the best peak shape and selectivity
• Explore the use of Bonded Phase Selectivity to decrease
analysis time and improve resolution
• Follow Logical Method Development Scheme -Either
Manual or Computer Aided

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