General Principles of HPLC Method Developmentfortunesci.com/download document/siminar2_56/General Principles of... · 5 Basis of Chromatographic Separation Separation of compounds

Philip J. Koerner, Ph.D.

ThailandSeptember 2013

General Principles General Principles of HPLC Method of HPLC Method

DevelopmentDevelopment

Part 1. General Chromatographic Theory

Part 2. The Stationary Phase:

An Overview of HPLC Media

Part 3. Role of the Mobile Phase in Selectivity

3

The Liquid Chromatographic Process

4Mikhail Tsvet, 1872-1919

The Beginning of Liquid

Chromatography

Empty Column

Adsorbent particles added

Sample is loaded onto the top of

the column

Solvent is added to the

top of the column

Separation occurs as the bands

move down the column

Column Chromatography:

5

Basis of

Chromatographic Separation

Separation of compounds by HPLC depends on the interaction of

analyte molecules with the stationary phase and the mobile phase.

Mobile Phase Stationary Phase

6

The Liquid

Chromatographic Process

Mobile Phase

Stationary Phase

Analytes

7

Mobile Phase

Stationary Phase

Analytes

The Liquid


8

Non-Polar Stationary Phase (e.g. C18)

O H

H

OH

H

O

HH

N

N

N

N

N

O H

H

O

HH

Polar/AqueousMobile Phase

Benzo(a)pyrene Ethyl sulfate

The Liquid


9

In any separation, almost never get a pure, single mode of separation. In RP, performance will be dictated by mixture of:

1. Hydrophobic interactions

2. Polar interactions

3. Ionic interactions

Method Development = modulating stationary phase and mobile phase conditions to optimize these interactions and achieve a specific separation goal.

OH

N

CH3

CH3

CH3

CH3

Tapentadol

Mechanisms of Interaction

In RP Chromatography

10

Hydrophobic Interactions

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO HCH 3

OH O H OH O H O H O H OH O H O H O H

O O O- O H

O O OO

S iS i S i

S iS iCH 3

C H 3

CH 3C H 3

CH 3C H 3

CH 3 C H 3

C H 3C H 3 C H 3 CH 3

C H 3

C H 3

C H 3O H

OH

N

CH3

CH3

CH3CH3

Hydrophobic• Weak, transient interactions between a non-polar stationary phase and the molecules

• Hydrophobic & van DerWaals interactions

• Retention will be predicted by

Log P values

11

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO HCH 3


O O O- O H

O O OO

S iS i S i

S iS iCH 3

C H 3

CH 3C H 3

CH 3C H 3

CH 3 C H 3

C H 3C H 3 C H 3 CH 3

C H 3

C H 3

C H 3O H

OH

N

CH3

CH3

CH3CH3

Polar

• Interactions between polar functions groups of analyteand residual silanols or polar groups on media

• Hydrogen bonding, dipole-dipole interactions

• Relatively weak and transient

Polar Interactions

12

Ion-exchange Interactions

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO HCH 3


O O O- O H

O O OO

S iS i S i

S iS iCH 3

C H 3

CH 3C H 3

CH 3C H 3

CH 3 C H 3

C H 3C H 3 C H 3 CH 3

C H 3

C H 3

C H 3O H

OH

NH+

CH3

CH3

CH3CH3

Ion-Exchange

• Interactions between ionizable functional groups on analyte and counter-charged moiety on stationary phase

• Ion-exchange

• Strong, slow interactions

13

Chromatographic Measurements

14

Chromatographic

Measurements

k’ Asym N α

15

Void Volume

Analytes which do not interact with the adsorbent elute from the column in a volume equal to the void volume in the column. The void volume of a column is the amount of mobile phase in the column between the adsorbent particles and in the pores of the porous adsorbent particles.

Mobile phase occupies the space

between the particles or the interstitial volume.

Mobile phase fills the pores of the

porous adsorbent particles.

16

Void Volume

A compound which does not interact with the adsorbent at all elutes at what is termed the void volume or the solvent front. The time that it takes for non-retained components to elute is the void time or t0.

• void volume

• solvent front

• t0

17

The retention factor of the eluting compound is its elution volume (time) relative to the elution volume (time) of an unretained compound. The k’value for a given analytes will be determined by its relative affinity for the stationary phase and mobile phase.

t0

Retention factor (k’)=

tR – t0

t0

Retention Factor (k’)

18

Retention Factor (k’)

For any given analyte, the k’ value will be most readily modified by changing the % of strong mobile phase (e.g. methanol or ACN).

Example: The Separation of Steroids:

Column used: Phenomenex Luna C18(2) 150 x 4.6 mm

19

Retention Factor (k’):

Effect of Changing % ACN

20

Peak Asymmetry (Asym)

The asymmetry value (Asym) for a peak is a measure of the divergence of

the peak from a perfect Gaussian peak shape. Peaks with asymmetry values > 1.0 are said to be “tailing”, those with asymmetry values < 1.0 are said to be “fronting”.

Asymmetrical peaks can be attributed to a number of factors:(1) Strong secondary interactions (e.g. ionic interactions between basic compounds and residual silanols)(2) Sample overloading(3) Sample solvent incompatibility(4) Poor packing

Peak Tailing due to

Secondary Interactions

Classical peak tailing in reversed-phase methods is most commonly caused by strong ionic interactions between basic analytes and residual silanolson the surface of the silica.

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO

S iO HCH 3

OH O H OH O H O H O H OHO H O H O H

O O O- O H

O O OO

S iS i S i

S i

S iCH 3

C H 3

CH 3C H 3

CH 3C H 3

CH 3 C H 3

C H 3C H 3 C H 3

CH 3

C H 3

C H 3

C H 3O H

O H

NH+

CH 3

C H 3

C H 3CH 3

Ion-Exchange

21

2222

Example: The Separation of Tricyclic Antidepressants:

Column used: Kinetex 2.6µ XB-C18 50 x 2.1 mm

Brand H 2.7µ C18 50 x 2.1mm

Peak Tailing due to


Amitriptyline (pKa 9.4) Nortriptyline (pKa 9.7) Protriptyline (pKa 8.2)

23

Peak Tailing due to


XIC of +MRM (8 pairs): 264.2/191.1 Da ID: Protrip from Sample 28 (Halo C18-50x2, 2.7 um, MeOH:0.1% ... Max. 1.1e5 cps.

3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min

1

2 3

45

6

7 8

XIC of +MRM (8 pairs): 264.2/191.1 Da ID: Protrip from Sample 28 (Halo C18-50x2, 2.7 um, MeOH:0.1% ... Max. 1.1e5 cps.

3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min

4

6

Mobile phase: A = 0.1% Formic acid in water, B = 100% Methanol,Gradient: 15 to 60% B in 5 min, hold for 1 minFlow rate: 400 µL/min1. Amoxapine2. Imipramine3. Desipramine4. Protriptyline*5. Amitriptyline6. Nortriptyline*7. Clomipramine8. Norclomipramine

XIC of +MRM (8 pairs): 278.2/191.2 Da ID: Amitrip from Sample 7 (TCA-Kin-XB-C18, 50x2, 2.6, MeOH 0.... Max. 1.2e5 cps.

3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min

2 3

1

4

5

6

78

1

5

XIC of +MRM (8 pairs): 264.2/191.1 Da ID: Protrip from Sample 13 (Kinetex XB-C18-50x2, 2.6 um, MeO... Max. 1.2e5 cps.

3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0Time, min

4

6

Kinetex 2.6µ XB-C18 50x2.1mmBrand H 2.7µ C18 50x2.1mm

Peak tailing

24

Strongly basic analytes are very sensitive to sample loading, and will display peak tailing effects as a function of increasing loading (µg on column):

min12 12.5 13 13.5 14 14.5 15 15.5 16 16.5

mAU0

20

40

60

80

100

DAD1 C, Sig=254,4 Ref=300,100 (DJ040611\DJGSK 2011-04-06 12-28-48\GSK000010.D)

14.157

12 µg on column; USP Tailing = 1.87

min12 12.5 13 13.5 14 14.5 15 15.5 16 16.5

mAU0

10

20

30

40

50


14.169

5 µg on column;

USP Tailing = 1.34

min12 12.5 13 13.5 14 14.5 15 15.5 16 16.5

mAU0

5

10

15

20

25

30


14.208

2.5 µg on column;USP Tailing = 1.17

Peak Tailing due to

Sample Overloading

pKa 9.7

25

min1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7

mAU0

250

500

750

1000

1250

1500

1750

2000

2250

DAD1 A, Sig=240,10 Ref=off (MT042211\DJGSK 2011-04-22 09-12-57\MUPIROCIN000001.D)

1.745

1.905

2.145

2.495

Fronting due to overload

Peak Fronting due to

Sample Overloading

Neutral and acidic compounds will typical show peak fronting when the column is overloaded.

Detector saturation!

26

Peak fronting can also be due to sample solvent effects:(1) Sample insolubility

(2) Sample solvent is too strong:

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

0.24

1.791.36

0.970.51 1.71 1.85 3.972.15 3.213.36

Sample in 100% Methanol

Peak Fronting due to

Sample Solvent Effects

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

1.06

1.77

Sample in 50% Methanol

Morphine

Hydromorphone

Norhydrocodone

Breakthrough!

Fronting

27

Selectivity (αααα)

Selectivity is a measure of the difference in the interactions of two compounds with the stationary phase. Selectivity is a function of both the stationary phase and the mobile phase.

α = k2/k1

2828

The choice of stationary phase will often have a dramatic effect on the selectivity of analytes.

OH

H H

OCH

3

H

Estrone

OH

H H

OHCH

3

H

Estradiol

Selectivity (α)

m i n1 2 3 4 5

m A U

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5 C18 Column1+2

α = 1.0

Phenyl Column 1

2

m i n1 2 3 4 5

m A U

0

2 0

4 0

6 0

8 0

1 0 0

α = 2.3

29

Selectivity (αααα)

But mobile phase is also a very powerful tool in modulating selectivity.

35% Methanol

20% Acetonitrile

Column: Gemini 5 µm C6-Phenyl 150 x 4.6mm

Mobile phase: 20mM KH2PO4, pH 2.5; % organic as noted

Flow rate: 1.0 mL/minDetection: UV at 220nm

1. Saccharin2. p-Hydroxybenzoic Acid3. Sorbic Acid4. Dehydroacetic Acid5. Methylparaben

30

Column Efficiency (N)

W

W1/2

tR

Peak Width (W)

The amount of band (peak) broadening or dispersion that occurs in the column is measured by calculating the column efficiency (N) expressed as the number of theoretical plates in the column:

• Columns that cause a lot of peak broadening have few theoretical plates.

• Columns that produce very narrow peaks (little peak broadening) have a large number of theoretical plates.

31

Column Efficiency (N) is a

Function of Particle Size

Efficiency of a well-packed column will be a function of several factors, one of

which will be particle size. As particle size decreases, efficiency will increase. In addition, back-pressure also increases as particle size decreases.

10 µm

50,000 P/m

5 µm

100,000 P/m

3 µm

150,000 P/m

sub-2 µm

300,000 P/m

Efficiency

Back-Pressure

Core-Shell

300,000 P/m

Columns packed with coreColumns packed with core--shell particles will deliver shell particles will deliver

significantly higher efficiency (N) than columns packed with significantly higher efficiency (N) than columns packed with

fullyfully--porous particles of the same diameter.*porous particles of the same diameter.*

6

w1/2

tRInjected

Sample Band

Fully Porous Particles

* Gritti et al., Journal of Chromatography A, 1217 (2010) 1589

The Core-Shell Advantage

Fully Porous Particles




Kinetex™ Core-Shell Particles

5 6

w1/2

tR



Core-Shell Particles

34

Fully Porous 3 µm C18; 150 x 4.6 mm

N = 166,500 p/m

Kinetex™ 2.6 µm C18; 150 x 4.6 mm

N = 295,340 p/m

78% Increase in Efficiency!






Comparison

*Farcas et al., HPLC 2012 Conference




0

50

100

150

200

250

300

350

400

450

5 3.5/3.6 2.5/2.6 1.7 1.3

Particle Diameter (µm)

Eff

icie

ncy(

p/m

)

Fully Porous Core-Shell


Efficiency vs. Diameter

36

The van Deemter Equation

The three principle factors that cause band broadening and a decrease in column efficiency are described by the van Deemter equation:

e

*e

A · dp + B/µ + C · de2 · µH =

Simplified version:

Particle size

Linear velocity (flow rate)Mobile phase viscosity

Particle sizeLinear velocity (flow rate)Mass Transfer

A · dp + B/u + C · de2 · uH =

Eddy Diffusion

Multi-path Effect

6

w1/2

tR


A-term

A · dp + B/u + C · de2 · uH =

Longitudinal diffusion

6

w1/2

tR


B-term

39

Dispersion due to resistance to mass transfer

A

B

A · dp + B/µ + C · de2 · µH =

Mass Transfer (Kinetics)


Fully Porous C-term

Dispersion due to resistance to mass transfer

A

B

A · dp + B/µ + C · de2 · µH =

Mass Transfer (Kinetics)


Core-Shell C-term

A · dp + B/µ + C · de2 · µH =

0 5 10 15 200

5

10

15

4.6 x 100 mm Kinetex-C18

Re

du

ce

d p

late

he

igh

t h

Reduced velocity νννν

Eddy dispersion


Solid-liquid mass transfer

*Gritti and Guiochon, 2012. LC-GC 30(7) 586-595.

0 5 10 15 200

5

10

15

4.6 x 100 mm Luna-C18

(2)

Eddy dispersion


Solid-liquid mass transfer

Re

du

ce

d p

late

he

igh

t h

Reduced velocity νννν


h vs. v Comparison

42

For a given particle size, column efficiency will be directly proportional to the length of the column. However, pressure and elution time will also be directly proportional to the column length.

Effect of Column Length

on Efficiency

25cm 25,000 Plates40 min250 Bar




43

Balancing Column Length

and Particle Size

Column

Length

(mm)

Efficiency dp

5µµµµm

250 25,000

150 15,000

100 10,000

50 5,000

44

Column

Length

(mm)

Efficiency dp

5µµµµm

Efficiency dp

3µµµµm

250 25,000 37,500

150 15,000 22,500

100 10,000 15,000

50 5,000 7,500


and Particle Size

45

Column

Length

(mm)

Efficiency dp

5µµµµm

Efficiency dp

3µµµµm

Efficiency

sub-2µµµµm /

Core-shell

250 25,000 37,500

150 15,000 22,500 45,000

100 10,000 15,000 30,000

50 5,000 7,500 15,000


and Particle Size

46

Column

Length

(mm)

Efficiency dp

5µµµµm

Efficiency dp

3µµµµm

Efficiency

sub-2µµµµm /

Core-shell

%Reduction in

Analysis Time

250 25,000 37,500

150 15,000 22,500 45,000 33

100 10,000 15,000 30,000 60

50 5,000 7,500 15,000 80

Use shorter columns packed with smaller particles

to reduce analysis time while maintaining/improving

efficiency!!


and Particle Size

47

Effect of Flow Rate on Column Efficiency (100x4.6mm)

Effect of Flow Rate on

Efficiency

0

5000

10000

15000

20000

25000

30000

35000

0 0.5 1 1.5 2 2.5 3 3.5

Flow rate (ml/min)

N (

Pla

tes/c

olu

mn

)

Core-Shell 2.6

Luna 3u

Luna 5u

5µ ~1 mL/min

3µ ~1.5 mL/min

Core-Shell ~2 mL/min

48

Quick Review

The chromatographic measurements that we have discussed so far will all play a significant role in method development.

1. Retention factor (k’) – retention of analyte relative to void t0

• Controlled by modulating % strong mobile phase

2. Peak asymmetry – peak shape (fronting, tailing, symmetrical)• Result of secondary interactions (e.g. Ionic in RP mode)• Sensitive to sample loading & sample solvent effects

3. Selectivity (αααα) – difference in the k’ of two analytes• Will be determined by mobile phase composition and nature of

stationary phase

4. Efficiency (N) – function of peak width and retention• Determined by particle size, column length, flow rate• Column packing will affect efficiency (vendor)

49

Any Questions?Any Questions?

50

Resolution: The Goal of Chromatography

51

Resolution: The Goal of

Liquid Chromatography

The goal and the purpose of liquid chromatography is to resolve the individual components of a sample from each other so that they may be

identified and/or quantitated.

52

Resolution is a measure of how well two peaks are separated from each other.

It is calculated as the difference in retention time of two eluting peaks divided by the average width of the two peaks at the baseline.

R (resolution) = RTB - RTA / .5 (widthA + widthB)



53

(1) Retention time for the two peaks will be a function of retention factor (k’).

(2) The selectivity (αααα) will also affect the retention time values for the two peaks.

(3) Peak width will be a function of column efficiency (N) and asymmetry (Asym).

k’

α

N, Asym



54

Selectivity

Retention factorEfficiency

The equation below allows us to calculate the relative importance of each of

these three factors in overall resolution:

It is important to note that you (the analyst) have control over each of those factors through your choice of HPLC column and running conditions:

1. Efficiency (N)→ Particle size/morphology and column length

2. Selectivity (αααα) → Stationary phase and mobile phase3. Retention factor (k’) → Stationary phase and mobile phase



55

Resolution: The Relative

Effectiveness of k’, αααα, and N

Ineffective after k’ ~10

Constant increase in resolution

Most important determinant of resolution!!

56

The Impact of Efficiency

on ResolutionModulating column efficiency is a very effective way to optimize resolution. There is a strong, linear correlation between N and Rs, but it is not a 1:1 ratio. Column efficiency is a flexible tool because we can easily modify it via changes in particle size and column length.

Column

Length

(mm) Efficiency 5µµµµm Efficiency 3µµµµm

Efficiency

sub-2µµµµm /

Core-shell

250 25,000 37,500

150 15,000 22,500 45,000

100 10,000 15,000 30,000

50 5,000 7,500 15,000

More efficiency/resolution

Lo

ng

er

Ru

nT

ime

More Back-Pressure

Mo

re P

res

su

re

57


on Resolution

0

0.5

1

1.5

2

2.5

0 10000 20000 30000 40000

Rel

ativ

e R

esol

utio

n

Column Efficiency (Plates)

Doubling column

efficiency increases

Rs by a factor of 1.4x


on Resolution

min0 0.5 1 1.5 2 2.5 3 3.5

mAU

0

20

40

60

80

100

5 µm

80,000 P/m

min0 0.5 1 1.5 2 2.5 3 3.5

mAU

0

20

40

60

80

100

120

140

3 µm

150,000 P/m

58

59

Optimizing Efficiency for

Maximum Resolution

1. For method development, start with an intermediate column length, packed with the smallest particle size that system pressure limitations will allow.

• Conventional HPLC → 3µm 150x4.6mm or core-shell 100x4.6mm

• UPLC → sub-2µm or core-shell particle• Work at optimal flow rate for that particle size

2. Fine-tune for maximum productivity:

• Excessive resolution → shorter column, increase flow rate• Insufficient resolution → longer column; modify flow rate to compensate

for pressure

60

The Impact of Retention

Factor on Resolution

Retention factor is the most important, yet limited, factor in determining resolution. It is crucial to have a reasonable k’ value because analytes must be retained in order to separate them. The drawback is that at high k’ values, passive diffusion causes extensive band broadening and loss of performance.

61

Optimizing Retention Factor

for Maximum Resolution

1. Adjust k’ value to be between 2 and 10

• In RP, adjust % of organic (acetonitrile or methanol)• Altering nature of stationary phase/media can modulate k’ as well

2. At k’ < 2, have sub-optimal resolution• May also have interference from solvent, non-retained components

3. At k’ values > 10, band broadening due to diffusion limits resolution gain• In RP, complex mixtures of polar and non-polar components will require

gradient for optimal performance/run time balance• Polar stationary phases can the “total elution window” of complex

mixtures in isocratic mode

62

The Impact of Selectivity

on Resolution

Small changes in selectivity can have a dramatic effect on retention. This is one of the reason why the same stationary phases from different manufacturers can sometimes give very different results, and also why changes to mobile phase composition can alter the results so strongly.

63

Method Development Exercise 1:

Optimization to Reduce Analysis Time and

Increase Productivity

64

Column: C8 250 x 4.6mm 5µm

Mobile phase: 70 / 30 0.1M Ammonium acetate / THF

Flow rate: 1.0 mL/min

Components: 1-6 = Impurities A - G

7. Mupirocin

Mupirocin Impurity Profile

Mupirocin

min0 2 4 6 8 10 12 14 16

mAU0

25

50

75

100

125

150

175

DAD1 A, Sig=240,10 Ref=off (Z:\1\DATA\MT050211\DJGSK 2011-05-02 15-34-44\MUPIROCIN000001.D)

Mupirocin: Original Method

1

2 3

5

6

7

~16 min

Column: Luna 5µ C8(2) 250 x 4.6mm

Mobile phase: 70/30 0.1M Ammonium acetate pH 5.7/THF

Flow rate: 1.0 mL/min

4

Rs 3/4 = 0.63; k’ = 2.3

65

66

Step 1. Adjust k’ for better

Resolution

Step 1. Reduce % organic to increase k’:• Increases Rs

• Increases run time

67

Step 2. Optimize Efficiency

and Length

Column

Length

(mm)

Efficiency dp

5µµµµm

Efficiency dp

3µµµµm

Efficiency

sub-2µµµµm /

Core-shell

%Reduction in

Analysis Time

250 25,000 37,500

150 15,000 22,500 45,000 33

100 10,000 15,000 30,000 60

50 5,000 7,500 15,000 80

Step 2. Switch to 150x4.6mm 3 µm media:• Reduces analysis time• Maintains efficiency

0

5000

10000

15000

20000

25000

30000

35000

0 0.5 1 1.5 2 2.5 3 3.5

Flow rate (ml/min)

N (

Pla

tes/c

olu

mn

)

Core-Shell 2.6

Luna 3u

Luna 5u

68

Step 3. Optimize the Flow

Rate

Step 3. Increase flow rate to 1.5 mL/min:• Optimizes efficiency for 3 µm

min0 2 4 6 8 10 12 14 16 18

mAU

0

20

40

60

80

VWD1 A, Wavelength=240 nm (JL050211\MUPI0003.D)

Mupirocin: Intermediate

Method

1

23

4

5

6

7

Column: Luna 3µ C8(2) 150 x 4.6mm

Mobile phase: 80/20 0.1M Ammonium acetate pH 5.7/THFFlow rate: 1.5 mL/min

Rs increased from 0.63 to 1.6

Run time increased from 16 to 20 minutes

20 min

k’ = 9; Rs 3/4 = 1.6

69

70

Step 4. Switch to Core-Shell Media

Column

Length

(mm)

Efficiency dp

5µµµµm

Efficiency dp

3µµµµm

Efficiency

sub-2µµµµm /

Core-shell

%Reduction in

Analysis Time

250 25,000 37,500

150 15,000 22,500 45,000 33

100 10,000 15,000 30,000 60

50 5,000 7,500 15,000 80

Step 4. Switch to 100x4.6mm Core-Shell media:• Reduce analysis time• Increase efficiency

min0 1 2 3 4 5 6 7 8 9

mAU

0

50

100

150

200

250


Mupirocin: Final Optimized Method

Column: Kinetex 2.6µ C8 100 x 4.6mm

Mobile phase: 80/20 0.1M Ammonium acetate pH 5.7:THFFlow rate: 1.5 mL/min (2mL/min if pressure allows)

Rs increased from 1.6 to 2.3Run time decreased from 20 to 8 minutes

8 min

1

5

6

7

23

4

Rs 3/4 = 2.3

71

Mupirocin: Final Optimized Method

min0 1 2 3 4 5 6 7 8 9

mAU

0

50

100

150

200

250


8 min1

5

6

7

23

4

Rs 3/4 = 2.3

Final Optimized Method:

m in0 2 4 6 8 1 0 12 14 16

m AU0

25

50

75

1 00

1 25

1 50

1 75

D AD 1 A , S ig= 240,1 0 R ef=o ff ( Z:\1 \DAT A\MT 050 211\ DJ G SK 201 1-0 5-0 2 15 -34 -44 \MU PIRO C IN00 0001 .D)

1

2 3

5

6

7

16 min

4

Rs 3/4 = 0.63

Initial Method:

72

73


74


Part 2. The Stationary Phase:

An Overview of HPLC Media


75

Tetraethoxysilane Silica Sol-Gel

Polymerization

Alkaline

75

99.9% of reactive surface area is internal

Fully Porous Silica

76

Advantages:• Ability to derivatize with numerous bonded phases

• High mechanical strength• Excellent efficiency• Highly amenable to modulation of material characteristics (pore size, surface area, etc.)

Disadvantages:• Dissolution of silica at pH > ~7.5 (may extend with bonded phase)• Hydrolysis of bonded phase at pH <1.5

76

Fully Porous Silica

7777

Conventional Silica Particle Organosilica Hybrid Particle

Dissolution at pH > 7.5 Stable to pH ~12

SiloxaneBridge

Ethane linkage

Organosilica Hybrid Particle

78

Advantages:• Extended pH range from 1-12• Performance and strength of

conventional silica particle• Unique selectivity

Disadvantages:• Fewer stationary phases available compared to conventional silica (e.g.

cyano, amino)

78

Organosilica Hybrid Particle

79

1.9 µm Solid Core

0.35 µm Porous Shell

2.6 µm Core-Shell

Particle

79

Core-Shell Particle

80

Advantages:

• 3x the efficiency of 5 µm fully-porous media & 2x the efficiency of

3 µm media• Pressures compatible with conventional HPLC systems*

Disadvantages:

• Pressure is still higher than 3 µm media

• More sensitive to system extra-column volumes• More sensitive to overload in some cases

80

Core-Shell Particle

81

Alkyl bonded phases (C18, C8, C4):

Phenyl phases (Phenyl, PFP):

F F

F

FF

Polar-embedded phases:

Fusion

Polar-endcapped phases:

Hydro

RP Stationary Phase Classes

82

We use the methylene selectivity test to determine the ability of stationary phase to separate molecules based upon differences in their hydrophobic character. In general, very hydrophobic bonded phases (e.g. C18) will display

higher levels of methylene selectivity than less hydrophobic phases.

m i n2 4 6 8 1 0

m A U

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

m i n2 4 6 8 1 0

m A U

0

1 0 0

2 0 0

3 0 0

4 0 0

C18

Ph

en

yl

Methylene Selectivity

83

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

0.180

0.200

Luna C18(2)

Synergi Hydro-RP

Jupiter C18

Synergi Max-RP

Luna C8(2)

Luna C5 Luna Phe-Hex

Jupiter C4

Synergi Polar-RP

Prodigy Phenyl

Luna Cyano

Luna Amino

Slo

pe

of l

og

k v

s. #

-C

H2-

un

its

C18 > C8 > C5 ≥ Phenyl > CN > Amino


84

Columns: 5µm C18 150x4.6mm

5µm C8 150x4.6mm

5µm Phenyl 150x4.6mm

Mobile phase: 65:35 Acetonitrile:Water

Flow rate: 1 mL/min

Components: Two steroids:

1. Testosterone

2. Methyltestosterone

O

H H

CH3

H

CH3

OH

CH3

Methyltestosterone

O

H H

CH3 H

CH3

OH

Testosterone


Columns: C18

Phenyl

Dimensions: 150 x 4.6 mm

Mobile phase: 75:25 Methanol:water

Flow rate: 1 mL/min

Components: 1. Estrone

2. EstradiolOH

H H

OHCH

3

H

Estradiol

OH

H H

OCH

3

H

Estrone

85

Phenyl Selectivity

86

m in1 2 3 4 5

m A U

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5

m in1 2 3 4 5

m A U

0

2 0

4 0

6 0

8 0

1 0 0

C18

Phenyl

1+2

1

2

OH

H H

OHCH

3

H

Estradiol

OH

H H

OCH

3

H

EstroneC18

Phenyl

Phenyl Selectivity

87

Nucleic Acid Bases:

Day 2

min2 4 6 8 10 12 14

0

Day 1

2 4 6 8 10 12

0

Phase Collapse!

Luna C18(2)

Day 1

0 2 4 6 8 10 12

0

Day 6

2 4 6 8 10 12 14

0

Polar-Endcapped C18

Aqueous Stability of

Embedded Phases

88

LC/MS/MS Analysis of ETG & ETS in Urine:

XIC of -MRM (6 pairs): 221.200/75.000 Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR600... Max. 9020.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.549 96 144 191 239 287 334 382 429 477 525 572 620

Time, min

0.00

1000.00

2000.00

3000.00

4000.00

5000.00

6000.00

7000.00

8000.00

9000.00

1.00e4

1.10e4

Intensity, cps

ETG

ETS

XIC of -MRM (6 pairs): 221.200/75.000 Da ID: ETG-1 from Sample 6 (P-2_Hyro-RP_100x4.6_4u_FR600... Max. 9020.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.549 96 144 191 239 287 334 382 429 477 525 572 620

Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

Intensity, cps

ETG

ETS

Polar-Endcapped 2.5 µm C18 100x3.0mm

10mM Ammonium formate

1. Ethyl glucuronide2. Ethyl sulfate

ETG

ETS

Aqueous Stability of

Embedded Phases


Part 2. Overview of HPLC Media


Solvents for RP

Chromatography

Mobile phase selection is much more challenging that stationary phase selection because the options are limitless. However, in practical method development, we can dramatically narrow down the options to focus on those conditions which will give us the highest likelihood of success.

Typical RP Solvents:

Weak Solvent: Water/Buffer

Strong Solvent: Acetonitrile (64) Methanol (34)Composite mixtures (1)THF (1)

Frequency of use

The elution strength of a given solvent is determined by its hydrophobicity (e.g. heptane would be stronger than hexane because it is more hydrophobic). The

selectivity of a solvent, however, is determined by its polar characteristics

(e.g. heptane and hexane would have the same solvent selectivity).

Acetonitrile has a dipole

moment but is only a very weak proton acceptor in hydrogen

bonding.

N CH3δ+δ−

Tetrahyrofuran is able to

accept a proton in hydrogen bonding but cannot donate a

proton.

O

Methanol is a strong proton

donor and a strong proton

acceptor in hydrogen bonding.

CH3 OH

Solvent Selectivity

Optimum Separation of 4 Steroids in Different Solvents:

Solvent Selectivity

1. Start at high % acetonitrile and work backwards until k’ is 2-10 (if possible)

80% ACN

k’ = 0

25% ACN

k’ ~ 6

21% ACN

k’ ~ 11

40% ACN

k’ ~ 0.8

Solvent Screening for

Isocratic Methods

2. Repeat with alternative solvent:

Solvent Screening for

Isocratic Methods

= Typical for LC/MS

= Typical for LC/UV

Buffer Selection for

RP-HPLC

O

Si

O

Si

O

Si

OH

OH

OH

O

O

O

Si

Si

Si

pH <3.5

O

Si

O

Si

O

Si

O

OH

O

O

O

O

Si

Si

Si

pH >3.5

Any silica-based RP material will have some residual silanols left after bonding and end-capping. These Si-OH groups can be deprotonated at

values above pH ~3.5. The deprotonated silanols are more likely to engage in ion-exchange with basic analytes, leading to peak tailing.

• Silanols protonated• Less ion-exchange

• Less peak tailing

• Silanols deprotonated• Increased ion-exchange

• Increased peak tailing

Effect of pH on

Base Silica

The primary mechanism of retention in RP chromatography is hydrophobic interaction. Ionizing compounds will cause them to behave as more polar

species, and reduce their hydrophobic interaction with the stationary phase, leading to decreased retention.

The ionization state of a molecule will be determined by the pH of the mobile phase. Therefore, mobile phase pH will dictate retention behavior of

analytes with ionizable functional groups.

• More hydrophobic

• More strongly retained

• Less hydrophobic

• Less strongly retained

• More hydrophobic

• More strongly retained

• Less hydrophobic

• Less strongly retained

Effect of pH on Analyte

IonizationR

ete

nti

on

Facto

r (k

’)

Acidic Compounds:

Rete

nti

on

Facto

r (k

’)

Basic Compounds:


Ionization

H+

Alkaline

Acidic

Aqueous Mobile PhaseAlkyl Stationary Phase


Ionization

H+

Alkaline

Acidic

Aqueous Mobile PhaseAlkyl Stationary Phase

H+


Ionization

B

NN

A

B

NN

AA

NN

B

Amitriptyline (pKa 9.4) = (B)ase Toluene = (N)eutral Naproxen (pKa 4.5) = (A)cid


Retention

The gradient slope is analogous to solvent strength in isocratic elution.

Isocratic Solvent Strength:

Increasing the solvent strength reduces analysis time but also

reduces resolution.

Decreasing the solvent strength increases resolution at the cost

of increased analysis time.

Solvent strength sometimes

affects selectivity

Gradient Slope:

Increasing the gradient slope reduces analysis time but also

reduces resolution.

Decreasing the gradient slope increases the resolution at the cost of increased analysis time.

Gradient slope sometimes affects selectivity

The goal of gradient elution is to optimize resolution while minimizing analysis time.

Gradient Analysis

The use of temperature in HPLC method development presents a challenge because it can have unpredictable effects on selectivity.

The use of elevated temperatures will:

1. Reduce mobile phase viscosity and back-pressure. This can allow you to operate at higher flow rates, or to use longer columns/smaller particle sizes.

2. Reduce elution time.

3. Improve method reproducibility (as opposed to operating at room temperature).

However, it is impossible to determine if the use of elevated temperatures will help or hinder a specific separation. For complex separations, improvements

in one portion of the chromatogram are almost always accompanied by decreases in another part of the same chromatogram.

Temperature in

HPLC Methods

104

End of Section II


End of the HPLC Method Development Portion

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