Which type of HPLC column for which

applications ?
Advantages of Purospher® STAR and Chromolith® HPLC columns in
quality control

Merck KGaA, Darmstadt

Merck Solvents & Chromatography

Page 2
Silica Gel Production Plant - Gernsheim

... the biggest chromatographic

silica gel plant in the world,
1.Generation
LiChroprep
Silica Gel
Page 3
Merck‘s HPLC Stationary Phases

First generation (1970’s): LiChrosorb®

- the first high performance HPLC columns; irregular particles;
1st RP-18 phase

Second generation(1980’s): LiChrospher®, Superspher®

-4 and 5 µm spherical particles with optimised selectivity

Third generation (1990’s): Purospher®

-Metal impurities < 5 ppm. Spherical particles
-Excellent peak symmetry

Fourth generation (2000’s): Purospher® STAR

-3 µm and 5 µm spherical particles
-Extended stability: pH range 1.5 to 10.5

Page 4
LiChrosorb, LiChrospher, Superspher
Reversed phase modified silica gels for RP-Chromatography

Specifications
Sorbents Particle Pore Pore Spec. % C Surface Efficiency (N/m)
size size volume surface coverage
(µm) (Å) (ml/g) area (µmol/m2)
(m2/g)
LiChrosorb® RP-8 5, 10 100 1,0 300 9,5 3,4 55.000, 20.000
LiChrosorb® RP-select B 5, 10 60 0,75 300 11,4 4,21 55.000, 20.000
LiChrosorb® RP-18 5, 10 100 1,0 300 16,2 3,0 55.000, 20.000
LiChrospher® RP-8 5, 10 100 1,25 350 12,5 4,04 55.000, 25.000
LiChrospher® RP-8e 5, 10 100 1,25 350 13,0 4,44 55.000, 25.000
LiChrospher® RP-select B 5, 10 60 0,9 360 11,5 3,55 55.000, 25.000
LiChrospher® RP-18 5, 10 100 1,25 350 21,0 3,61 55.000, 20.000
LiChrospher® RP-18e 5, 10 100 1,25 350 21,6 4,09 55.000, 20.000
Superspher® RP-8 4 60 1,25 350 12,5 4,04 100.000
Superspher® RP-8e 4 60 1,25 350 13,0 4,44 100.000
Superspher® RP-select B 4 60 0,9 360 11,5 3,55 100.000
Superspher® RP-18 4 100 1,25 350 21,0 3,61 100.000
Superspher® RP-18e 4 100 1,25 350 21,6 4,09 100.000

Page 5
LiChrospher® 60 RP-select B (5 µm)

For separations of basic drugs (with acidic buffered eluent)

3 Drugs Betablocker DMD-Test

1 3
1

2 2
2 3
1 4
5

6 4
1

30 min 30 min 30 min

Eluent: Acetonitrile / 0.05 M Eluent: Acetonitrile / 0.02 M KH2PO4 Eluent: Acetonitrile / phosphate buffer pH 2.3
phosphate buffer pH 4.8 pH 4.7 (25/75, v/v) (31.2/86, g/g)
(20/80, g/g) Flow rate: 2 ml/min Flow rate: 1 ml/min
Flow rate: 1ml/min Detection: UV 220 nm Detection: UV 220 nm
Detection: UV 220 nm Sample: 1. Atenolol Sample: 1. Diphenhydramine
Sample: 1. Barbituric acid 2. Metaprolol 2. MPPH
2. Codeine phosphate 3. Bisoprolol 3. Diazepam
3. Atropinium sulfate 4. Propranolol
4. Papaverine
5. Noscapinium chloride
6. Diphenhydramine
Page 6
LiChrospher® RP-select B
Reproducibility

Batch 1

Batch 2

Batch 3

Batch 4
Batch 1
Batch 2 L551205
Batch 3 Batch 5
Batch 4 Batch 6
Batch 5
Batch 6 Batch 7
Batch 7
Batch 8 Batch 8
Batch 9
Batch 10 Batch 9

Batch 10

Page 7
LiChrospher® RP-select B
Reproducibility

Tanaka 1 - Test:

Excellent reproducibility because

1. big masterbatches (10 batches)
2. low variations in specification

L56049418Nr.
601919
L56038218 Nr.
601914
L55120518 Nr.
601895

Page 8
LiChrospher® RP-select B
Reproducibility
Eluent: Methanol/Wasser 30/70
Tanaka 2 - Test: Fluß: 1mL/min
Inj.v.: 5L
Det.: UV254nm
Temp.: 22°C
Probe: 0,01mg/mL Uracil
0,022mg/mL Coffein
0,09mg/mL Phenol gelöst in MeOH/W
30/70

B4186/04 No.601926
L56014218
No.601906
L55057818 No.601874
L55057718
No.601868
No.601857
L55057418 No.601849
L55057318 No.601813
L55057218
No.601804

Page 9
Column hardware

Hibar RT LiChroCART
Ready-to-use Cartridge column
column
Complete column with Cartridge with no
endfittings endfittings (special
cartridge holder - manu-
CART - is necessary)

iD 3, 4 and 4.6 mm iD 2, 3, 4 and 4.6 mm

(same outer diameter) (same outer diameter)

Cost savings

Page 10
Purospher®® RPRP--18 18 endcappedendcap

Chelating Agents

Purospher® STAR RP-18 e

1.

O OH O OH
OH
2.

O OH O OH

Purpurin Quinizarin

0 5 10
Chromatographic conditions:

conventional RP-18 silica

Mobile phase: Methanol / 20 mM Phosphate
buffer
pH 3,0; 75:25 (v/v)
Flow rate: 1.0 ml/min
Detection: UV 254 nm 2.
Temperature: 30 °C
Sample: 1.) Purpurin
2.) Quinizarin 0 5 10

Page 11
Purospher ®

The Difference - Metal Content

Metal content in ppmppm

Sodium Calcium Magnesium Iron

Aluminium

LiChrosorb® 340-400 1300 160-220 20-25 15-20

LiChrospher® 150-250 6-10 4-6 20-40 75-140

Purospher® 1 1 1 3 1
Purospher® STAR

Page 12
Metal impurities of silica gel manufactured
by conventional process

Metal Ion Impurities Silanol Groups

Na + - O OH free
Si Si
OH
- Si
O Si geminal
Fe2+ OH
-
O Si Si O H
OH
OH OH
atrix Si O H vicinal
OH Me OH
M
O
Si O Si Si
O O O O
O O
Si O Si O Si O Si

Page 13
Purospher ®

Manufacture of Silica Gel

Conventional method Purospher® Si

Type A Type B
Si (OC2H5)4
M2O - nSiO2 M=
Na, K, Fe
[-Si (OH)2 - O - [-Si (OC2H5)2 - O -
Si (OH)2- O -]n Si (OC2H5)2 - O
-]n
- Si - O - Si - O - - Si - O - Si - O -
O O O O
- Si - O - Si - O - - Si - O - Si - O -
n n

2(Na, K, Fe) SiO22

Page 14
Purospher ® RP-18 endcapped

Application Procainamides
3

Column: LiChroCART® 125-4

2 Purospher® RP-18 e, 5 µm
Purospher RP-18e Mobile phase: Methanol / 0.02 M Phosphate
buffer pH 7.6 40:60 (v/v)
1 Flow rate: 0.5 ml/min
Detection: UV 254 nm
Temperature: 30 °C
Sample: 1.) Uracil
0 7,5 15
2.) Procainamide
3 3.) N - Acetylprocainamide
1
Conventional RP-18 silica
2

0 7,5 15

Page 15
LiChrospher® RP-18 endcapped uses
conventional surface modification

OH
OH monofunctional
Si
O modification
Si OH CH3 CH2
CH2 CH2 CH2 CH2 CH2
O
Si
X Si CH2 CH2 CH2 CH2 CH2 CH2 C6H13
OH CH3
O
O Si
OH
Free silanol-groups are still
OH CH3 accessible after
CH3 OH
O Si O Si endcapping. Polar
Si
O
CH3 Si
O
CH3
CH3 interactions are still
Endcapping Si CH3
Si OH Si O possible.
OOH O OHCH3
Si O CH3 Si CH3
O
O Si O OH Si
O Si CH3 O Si CH3 CH3
OH O
Si CH3
CH3

Page 16
Purospher® STAR RP-18 endcapped

Polymeric surface modification

O
CH 2 CH 2 CH 2 CH2 CH2 CH 2
Si O Si CH2 CH 2 CH2 CH 2 CH 2 CH 2 C 6H 13

OH O

Si O Si CH3 Purospher® STAR RP-18

OH O endcapped provides a
CH 2 CH 2 CH 2 CH2 CH 2 CH 2 nearly perfect coverage of
Si O Si CH 2 CH2 CH2 CH 2 CH 2 CH 2 C 6H 13 the surface. This prevents
O from polar interactions.
CH 2 CH2 CH 2 CH 2 CH 2 CH 2
Si O Si CH2 CH 2 CH 2 CH 2 CH 2 CH 2 C 6H 13

O
polymeric

Page 17
Purospher® STAR RP-8 endcapped
Separation of caffein & derivatives

Separation of compounds with very similar

structures in highest resolution on Purospher STAR
RP-8 endcapped. Polar substances can be
separated in shorter time on more polar phases.
Chromatographische Bedingungen

Säule: LiChroCART 125-4

Purospher STAR RP-8e (5µm)
Eluent: Methanol / Ammonium-
acetatpuffer pH 3,5 (Gradient)
Fluss: 1,0 mL/min
Detektion: UV 270 nm
Temp.: Raumtemperatur
Injektion: 10 µL
Probe: 1) 1-Methylxanthin
2) 1,3-Dimethylharnsäure
3) Paracetamol
4) Theobromine
5) 1,7-Dimethylharnsäure
6) 1,7-Dimethylxanthin 7)
Theophylline
8) Koffein

Page 18
Purospher® STAR RP-18 endcapped
Separation of Cefazolin

Many antibiotics are chelating compounds.

The separation on conventional HPLC
materials often shows peak tailings.

Cefazolin
Chromatographische Bedingungen

Säule: LiChroCART 125-4

Purospher STAR RP-18e (5µm)
Eluent: Acetonitril / Phosphatpuffer
Gradient
Fluss: 1,2 mL/min
Detektion: UV 254 nm
Temp.: 45 °C
Injektion: 10 µL
Probe: 1) 1H-Tetrazol-1-yl-essigsäure
2) Hydrolysierte Cefazolinsäure
3) unbekannt
4) Cefazolin
5) Cefazolinsäure

Page 19
Purospher® STAR RP-18 endcapped
Highest pH Stability from 1.5 to 10.5

Purospher® STAR has

Longterm stability at pH 10.5
outstanding pH stability.
Various studies have 16,00
shown that Purospher® 14,00 (5)
STAR remains stable 12,00
and reproducible in a pH 10,00
range of 1.5 to 10.5. 8,00 (4)
This provides the 6,00 (3)
required pH stability for 4,00 (2)
99% of common analysis 2,00
and ensures a simple 0,00 (1)
choice in most 0 200 400 600 800 1000 1200 1400
applications. hours

Purospher® STAR RP-18 endcapped, 5 µm, 150-4.6 mm with acetonitrile / water (0,1 % NH3 [25%]; 60:40), 1.0 ml / min at UV
254 nm
Sample: 1. Thiourea, 2. Benzene, 3. Toluene, 4. Propylbenzene, 5. Butylbenzene

Page 20
Merck Chromatography
- Development of Silica Gels

LiChrosorb® irregular

LiChrospher®
Superspher®
Purospher® spherical
Purospher® STAR

Chromolith® monolithic

Page 21
Chromolith® HPLC Columns

Column cladding material is PEEK (poly-ether-ether-

ketone) - stable with all standard HPLC solvents at 200 bar
and 45°C - limitations: 5% DMSO, 5% CH2Cl2, 50% THF

Page 22
 Preparation

Si(OAlk)4
Polymer
H+

Starting Phase Separation Ageing Drying

Sol and Gelation

HPLC
Cladding

Page 23
The Bimodal Pore Structure

SEM of a cross section of a monolithic silica rod

Macropores: 2 m Mesopores: 130 Å

Total porosity 81%
Page 24
Feature of monolithic silica columns

Defined bimodal pore structure

SEM of a cross
section of a
silica monolith

Mesopores: 130 Å
Macropores: 2m

Page 25
Page 26
Chromolith® HPLC columns are robust

Page 27
Chromolith® Specification

• Silica type: High purity

• Particle size: Monolithic shape
• Macropore size: 2 µm
• Mesopore size: 130 Å (13 nm)
• Surface area: 300 m2/g
• Pore volume: 1 ml/g
• Surface modification: RP-18 endcapped
• Carbon content: 18 %
• Surface coverage: 3.6 mmol/m2
• pH range 2 - 7.5
• Total porosity: >81%
Page 28
Purospher STAR®
Specification

Sorbent: High-purity, silica gel

Metal content: Na, Ca, Mg, Al: 1 ppm; Fe: 3 ppm
Particle size: 3 µm and 5 µm
Pore diameter: 120 Å (12 nm)
Specific surface: 330 m2/g
Pore volume: 1.1 ml/g
Surface modification: C-18 endcapped
Carbon load: 17%
Coverage of the surface 3 µmol/m2
Performance 5µm: >85,000; 3µm:>130.000 N/m
pH range: 1.5 - 10.5

Page 29
Column back-pressure
3µm and 5µm columns

Particulate vs. monolithic material

250
3.5 m
200
5 m

150

100

50 Chromolith® Performance

0
0 2 4 6 8 10

Flow rate (mL/min)

Page 30
Column performance
Van Deemter Plot

p/u - curve H/u - curve

198 bar
250
3m 18
200
16 Chromolith 3mm
14
150 12
10
8
100
Chromolith 3mm 6
50 4
3m
2 393 bar
0 0
0,0 0,5 1,0 1,5 2,0 2,5 3,0 0 1 2 3 4 5 6 7 8 9 101112131415

Flow rate (mL/ min) Linear velocity u (mm/s)

Columns: Chromolith® Performance RP-18e, 3mm i.d.

PurospherTM STAR RP 18e, 3m
Page 31
Speed and Quality in Practice
2

1
4 Chromolith® Performance RP-18e
3 5 100 mm x 4.6 mm

5 ß-Blocker

1 mL/min - 17 bar

Mobile Acetonitrile/
0.1% TFA in
3 mL/min - 51 bar water
Phase (20(80; v/v)
Flow rate 1-9 ml/min
Detection 222 nm
Sample 1. Atenolol
2. Pindolol
9 mL/min - 153 bar 3. Metoprolol
4. Celiprolol
5. Bisoprolol
Page 32
Speed and Quality in Practice

2
2
1
4 1
3 4
5 3 5

0.0 1.5
1 mL/min - 17 bar

9 mL/min - 153 bar

3 mL/min - 51 bar

12
9 mL/min - 153 bar
Page 33
Chromolith® HPLC Columns
- unique properties and benefits (1)

1. Columns back-pressure much lower

= faster analysis and higher sample throughput

Page 34
 Chromolith® HPLC

1.51463.0001 Chromolith® Flash RP-18e ( 25 x 4.6 mm)

1.51450.0001 Chromolith® SpeedROD RP-18e ( 50 x 4.6 mm)
1.02129.0001 Chromolith® Performance RP-18e (100 x 4.6 mm)

Page 35
 High throughput analysis with monolithic
silica columns

N. Barbarin, B.D. Mawhinney, R. Black, J. Henion

J. Chromatogr. B, 2003, 783, 73-83

“Determination of methylphenidate (central nervous stimulant)

and its metabolite ritalinic acid in rat plasma with LC/MS”

Chromatographic conditions:

Column: Chromolith® Flash RP-18e, 25 x 4.6mm

flow rate: 3.5ml/ min
analysis time: 15 s

768 protein precipitated samples analysed in 3h 45min !!!!!

Page 36
Chromolith® HPLC Columns
- unique properties and benefits (2)

1. Columns back-pressure much lower

= faster analysis and higher sample throughput

2. Length of column not pressure limited

Page 37
 Chromolith® HPLC Columns
- Higher Separation Efficiency
6
6

1 Chromolith column 100-4.6mm 2 Chromolith columns 100-4.6mm

Back pressure: 30 bar Back pressure: 60 bar
Back pressure of a typical Back pressure of a typical
particulate column (5µm): 150-200 bar particulate column (5µm): 300-400 bar
3
2
5
3
2 5
1 8
1 7
8
7 4
4

0,0 2,0 4,0 min 0,0 3,5 7,0 min

Page 38
Chromolith® HPLC Columns
- unique properties and benefits (2)

1. Columns back-pressure much lower

= faster analysis and higher sample throughput

2. Length of column not pressure limited

= better peak resolution

Page 39
Chromolith® HPLC Products

1.51467.0001
Chromolith Column Coupler

Page 40
Chromolith® HPLC Columns
- unique properties and benefits (3)

1. Columns back-pressure much lower

= faster analysis and higher sample throughput

2. Length of column not pressure limited

= better peak resolution

3. Strong highly porous monolithic silica

= longer column lifetime

Page 41