Reversed Phase Mobile phase

Chromatographic Process
B+A

Mobile phase

Reversed Phase: Mobile Phase and Stationary Phase

B Dr. Shulamit Levin, Medtechnica Shula_Levin@medtechnica.co.il www.forumsci.co.il/HPLC A

Stationary Phase

Distribution: K = Cs/Cm
0.34 0.32 0.30 0.28 0.26 0.24 0.22 0.20 0.18 0.16 AU 0.14 0.12 0.10 0.08 0.06 0.04 0.02

Chromatogram

Stable RT
Area A = 300,000 Area B = 100,000

Peak Shape

A-93.448

Baseline Noise

B-94.776

Elution through the Column-movie

0.00 -0.02 90.50 91.00 91.50 92.00 92.50 93.00 93.50 94.00 94.50 95.00 95.50 96.00

Time

Minutes

Chromatographic Process: Reversed Phase

Mobile phase: Water, Buffers MeOH, Acetonitril, IPA B+A
Hydrophobic Stationary Phase

Reversed Phase Elution Order

0.30 OH 0.25

OH
0.20

A
AU 0.15

B Distribution: K = C s/C m

H3C

B (More

H3 C Hydrophobic)
0.10

Methylparabene
0.05

Propylparabene
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

A
0.00

Migration through the Column

Chromatogram

Minutes

Reversed Phase Mobile phase

MOBILE PHASE MOBILE PHASE

* * * * * *

TYPE OF MODIFIER (MeOH, ACN) MOBILE PHASE COMPOSITION (% modifier) pH TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration) ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)

* * * * * *

TYPE OF MODIFIER (MeOH, ACN)

MOBILE PHASE COMPOSITION (% modifier) pH TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration) ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)

OPTIMIZATION: CHOICE OF SOLVENTS

Elution Strength

20% MeOH

20% ACN

20% EtOH

20% THF

main solvent: H2O

Reversed Phase Mobile phase

MOBILE PHASE
SOLVENT STRENGTH Analyte Retention as a Function of % Modifier
* * * * * * TYPE OF MODIFIER (MeOH, ACN)

MOBILE PHASE COMPOSITION (% modifier)

pH TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration) ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)

k (retention) for each analyte changes independently as % Modifier changes.

lnk

Thus, the resolution between peaks changes.

peak 8 peak 9

peak 10 peak 11

% Acetonitrile

OPTIMIZATION: % SOLVENTS

k vs Modifier and pH
k vs % Organic
K'

k vs pH k6
5 4
5N-2,4,6T 4A-6C-1,3B HCT TMT

20% MODIFIER 3 40% MODIFIER

5N-2,4,6T 4A-6C-1,3B HCT TMT

2 1

3 2 1

60% MODIFIER 0 16 80% MODIFIER 20 18 22 % Acetonitrile 24

2 3

4 5

pH of 50 mM NaH2PO4 Buffer

Reversed Phase Mobile phase

MOBILE PHASE
0. 036 0. 027

pH as a Selectivity Tool For Impurities

1
U A 0. 018

pH 9 Ammonium Bicarbonate

0. 009

* * * * * *

TYPE OF MODIFIER (MeOH, ACN) MOBILE PHASE COMPOSITION (% modifier)

U A

0. 000

0. 50

1.00

1. 50

2.00

2. 50 Minut es

3. 00

3.50

4. 00

4.50

5.00

0.036

0.027

pH 7 Sodium Phosphate

0.018

pH
TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration)

0.009

0.000

0.50

1.00

1. 50

2.00 Minut es

2. 50

3. 00

3.50

4. 00

4.50

5.00

0. 048

0. 032

pH 5 Ammonium Acetate

0. 016

0. 000

-0. 016

0. 50

1. 00

1. 50

2. 00

2. 50 Minut es

3. 00

3.50

4. 00

4. 50

5.00

0. 048

0. 036

pH 2 Sodium Phosphate

ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)

0. 024 U A

0. 012

0. 000

0. 50

1.00

1. 50

2. 00

2.50 Minut es

3. 00

3.50

4. 00

4.50

5.00

Ionization of Acids and Bases

Dissociation of Molecule

Ionization of Acids and Bases

Dissociation of Molecule

Acid HA
(Un-ionized) 50% 100% 0% @ pKa Low pH High pH

Base H
+

+ A
50% 0% 100%

(Ionized)

B
(Un-ionized) 50% 0% 100% @ pKa Low pH High pH

BH

+ OH

(Ionized) 50% 100% 0%

K
a

Acids( Un-IonizedpKa =4.8(HA))

]

k HA 1 k A[ HK a [] H

Un-IonizedB

Neutral