Trends in development of stationary phases

in chromatography

Petr Solich

Charles University

Czech Republic

Separation Techniques, San Francisco, USA - 10 August 2015

CHARLES UNIVERSITY, FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CZECH REPUBLIC

CHARLES UNIVERSITY, FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CZECH REPUBLIC

Faculty of Pharmacy, Charles University, Hradec Kralove

Charles University in Prague - founded 1348- 48 000 students (6.000 foreign) -17 faculties- located in - Prague (14)

- Hradec Králové (2) - Pilsen (1)

Faculty of Pharmacy in Hradec Králové – founded 1969

- 1500 students (100 foreign)- 11 Departments in 2 buildings- 23 research teams

High-performance liquid chromatography

High-pressure pump

Injector – Autosampler

Analytical column (stationary phase)

Detector

Chromatographic software

pump autosampler column detector software

Recent trends in developments of HPLC

sample preparation (automation, sample volume)

increased separation efficiency (columns)

increased selectivity of metods (detectors)

„Green chromatography“ (low consumption of

organic phases)

miniaturization (columns, detectors, instruments)

all to increased the laboratory throughput !

FAST CHROMATOGRAPHY

Recent trends in developments of HPLC

sample preparation (automation, sample volume)

increased separation efficiency (columns)

increased selectivity of metods (detectors)

„Green chromatography“ (low consumption of

organic phases)

miniaturization (columns, detectors, instruments)

all to increased the laboratory throughput !

FAST CHROMATOGRAPHY

Length - 10 - 250 mm

Diameter - 1.0 – 4.6 mm

Particle size - 1 – 10 μμmm

100 mm

Analytical columns – present situation

Length - 10 - 250 mm

Diameter - 1.0 – 4.6 mm

Particle size - 1 – 10 μμmm

100 mm

3.0 mm

Analytical columns – present situation

Length - 10 - 250 mm

Diameter - 1.0 – 4.6 mm

Particle size - 1 – 10 μμmm

100 mm

3.0 mm

3.0 μm

Analytical columns – present situation

1. stationary phase based on silica (normal, reversed)

2. stationary phase for HILIC

(Hydrophilic Interaction Liquid Chromatography)Hydrophilic Interaction Liquid Chromatography)

3. stationary phases based on zirconium oxide

4. polymeric stationary phase

5. monolithic stationary phase

6. hybrid stationary phases (sub-2-micron phase)

7. core-shell stationary phase

Length - 10 - 250 mm

Diameter - 1.0 – 4.6 mm

Particle size - 1 – 10 μμmm

100 mm

3.0 mm

3.0 μm

Analytical columns – present situation

Recent trends in chromatographic separation phases

- smaller particles (2004)- higher operational pressure- low flow rate- low consumption of MP - short time of analysis- large sequences of samples ?

- monolithic rods (2001)- low operational pressure- high flow rate- relatively high consumption of MP- short time of analysis- large sequences of samples

High pressure for separationUHPLC

Low pressure separation(HPLC, SIA, …)

Core-shell technology- porous shell on fused core (2009)- short time of analysis- low consumtion of MP- large sequences of samples ?

Sub-2 m columns

Monolithic columns

Substantial decrease of analysis time !!!

Stationary phases - comparison

B - Monoliths- Porosity 80 %

This special porous character allows relatively high mobile phase flow rates (1–9 ml/min) while keeping the backpressure low..

Monolithic column - made of a highly porous material with two types of pore structure—macropores (size of 2µm) and mesopores (size of 13 nm).

J.Sep.Sci., 2009, 32 (15-16) – special issue devoted to Monoliths

A - Particle based columns- Porosity 65 %

- Comparable speed and effectivity of analysis to sub 2 μm particles in UHPLC, but relatively low back-pressure !!- Shorter diffusion path compared to conventional total porous particles- RP-Amide, Phenyl-Hexyl, HILIC, F5, ES-Cyano- pressure cca 600 psi, pH range 2 – 9

Ascentis, Kinetex, Poroshell, Halo…

Core-shell (Porous shell) particle columns

Solid core

sub-2-microne

porous shell – 20%

w1/2

w1/2

w1/2

3- 5 µm

CORE-SHELL PARTICLES – MASS TRANSFER

Bioanalytical applications

of monolithic columns

Bioanalytical laboratoryCharles University, Teaching Hospital,

Hradec Kralove

Biologically active compounds analysed in Bioanalytical laboratory in hospital

using monolithic columns Retinoic acid in serum Neopterin in urine Retinyl esters = Vitamin A esters in serum Vitamin A, E, D

in human serumin erythrocytesin lipoprotein

fractions

Application:

- in clinical monitoring of patients with cardiovascular problems,

- in monitoring of oncological treatment (side effects, nutritional

status

- in treatment of metabolic diseases (diabetes mellitus,

pancreatitis)

- clinical monitoring of elderly patients (level of antioxidants)

- monitoring of patients with nephorological infections

Bioanalytical applications

of monolithic columns

Determination of retinoic acids (RA)

Determination of retinoic acids (RA)

• Most important RA are 13-cis RA, all-trans RA

• For treatment of hemato-oncological deseases, a formulation Tretinoin is used (contain RA)

• tzv. Retinoidal syndrom – found in many patients (about 25 %)

treated by Tretinoin• Retinoidal syndrom: - high temperatures of body

- acute pulmonary complications- hypotensis- eodems

need for monitoring of RA in patients

treated with formulation Tretinoin

HPLC method for determination of RA, vitamin A (retinol) and vitamin E (tocopherols) in

serumSPE Extraction:• IS (20 µmol.L-1)• 250 µl of serum • Deproteination by ethanol (650 μl, 8 min, 4 ºC). • centrifugation (2 000 ×g, 15 min, 4 ºC)• supernatant placed on SPE colunmn• Elution: by hexan (2 000 μl) • Evaporation to dryiness - 35 ºC• Addition of 250 μl of methanolu

HPLC determination• SP: monolithic column Chromolith Performance RP-18e,

100×4.6mm• MP: ACN : 1% ammonium acetate 95:5 (v/v) 4 min flow rate 1.5 ml.min-

1, 3 min flow rate 3.2 ml.min-1 („flow rate“ gradient !!!)

• injection 20 μl• DAD detection:

of retinol, all-trans- and 13-cis RA 325 nm, alfa-, gama- tokoferol a tocol 295 nm

• Time of analysis - 7 min

Determination of retinoic acids and vitamins A and E in human serum

Krčmová L, Urbánek L, Solichová D, Kašparová M., Vlčková L., Melichar B, Sobotka L., Solich P:

J. Sep. Sci. 32 (15-16), 2009, 2804-2811

4 min - flow rate 1.5 ml.min-1, 3 - min flow rate 3.2 ml.min-1

 

Determination of Vitamin D

(+ vitamins A and E)

Bioanalytical applications

of monolithic columns

  Determination of Vitamin D

Common name for a group of 9,10-secosteroids

Most important – D3 – cholecalciferol – animal origin

created in organism by UV radiation from provitamin D3

D2 –ergocalciferol – plant origin

Both forms have the same activity in organism

Vitamin D is transformed to active form by hydroxylation

Influence on metabolism of phosphorus and calcium in the body

Influence on calcium level in bones

Prevention of osteoporosis

Development of HPLC method for simultaneous determination of vitamines A,

E and D

Vitamin D:• Ergocalciferol (D2)

• Cholecalciferol (D3)

• metabolite calcidiol - 25(OH)D3

• metabolite calcitriol - 1,25(OH)D33

Vitamin E – α-tocopherol

Concentration in serum

19 – 35 μmol/L

Vitamin A - retinol

Concentration in serum

1,05 – 2,27 μmol/L

 

Chromolith RP-18e, 100×4.6mm + 50×4.6 mm

Development of HPLC method for A, E and D

standards

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

-5.0

-2.5

0.0

2.5mAU

264nm,4nm (1.00)

25

(OH

)D3 D

2 D3

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

-2.5

0.0

2.5

5.0mAU

295nm,4nm (1.00)

toko

l

A-t

oko

fero

l

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min

-3

-2

-1

0

mAU325nm,4nm (1.00)

retin

ol

Spiked serum

Sklenářová H., Koblová P., Chocholouš P., Šatinský D., Krčmová L., Kašparová L., Solichová D., Solich P.,

Analytical Letters 44(1-3), 2011, p. 446-456.

Development of HPLC method for A, E and D

Determination of neopterin in urine

Bioanalytical applications

of monolithic columns

reflect the stage of activation of the cellular immune system and pathogenesis and progression of various diseases:

- in viral infections (HIV…)

- in autoimmune or inflammatory diseases

- rejection episodes following allograft

transplantation

- in several malignant diseases

- neopterin concentrations are very closely linked with the progression of these diseases !

Therefore it is of interest for laboratory diagnosis to measure the degree of activation of the human immune system. This is possible in an easy but specific way by the determination of neopterin concentrations.

Neopterin = a diagnostic marker

Chromatogram of urine

Chromolith RP-18 e, 100 x 4.6 mm, flow rate 1,2 ml/min

-5000

0

5000

10000

15000

20000

25000

30000

0 1 2 3 4 5

time (min)

inte

nzi

ta

-10000

0

10000

20000

30000

40000

50000

60000

inte

nzi

ta

290 nm Kys.močová Fluores.Neopterin 230 nm Kynurenin 235 nm Kreatinin

Uric acid

kreatinin

neopterin

kynurenin

Monolithic stationary phases - trends

HR-monolithic columns (introduced 2011)

• Higher separation performance - increased by 50%

(System pressure increased by 60% )

• Performance similar as 2.7 µm core-shell particle columns

• Currently – increasing choice (C18, C8, SILICA, NH2, CN, PHENYL)

• Size - length 5 – 100 mm, diameter 2.0 - 4.6 mm

• Working range of pH 2 – 7.5 (only)

Applications

of sub-2-micro columns

Determination of pharmaceuticals by UHPLC

Particles 5 μm Human hair 60 μm Particles 1.7 μm

Columns with 1,7 μm particles

hybrid sorbent X-Bridge(BEH-Bridged Ethylene Hybrid )- particles 1.7 µm- range pH - 1- 12- pressure up to 1000 bar

working up to 100 MPa (15 000 Psi, 1000 bar)

pressure resistant connections

X-Bridge sorbents

particle size 1.7 μm

special injection system

acquisition rate 80 points/s

special optical cell (500 nl, 10 mm)

Ultra High Performance Liquid Chromatography

(UHPLC)

AU

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

Minutes

0,00 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

met

hylp

arab

en -

0,46

2

prop

ylpa

rabe

n - 0

,754

impu

rity

- 1,0

32

IS/fl

urbi

prof

en -

1,81

1

dicl

ofen

ac -

2,21

3

Minutes

0 2 4 6 8 10 12 14 16 18

mA

U

0

50

100

150

200

250

me

thyl

pa

rab

en

2

,52

pro

pyl

pa

rab

en

4

,66

imp

urit

y-D

PI

7,2

5

IS-f

lurb

ipro

fen

1

1,9

5

dic

lofe

na

c

15

,01

Time of analysis:

16.3 min

Solvent consumption:

11.4 ml

Time of analysis:

2.3 min

Solvent consumption

1.1 ml

Classical C18 column

5 um, 125 x 4.6 mm

X-Bridge column

1.7 um, 50 x 2.1 mm

Nováková L, Solich P.: J.Sep.Sci. 29, 2006, 2433

Speed acceleration 7 x

HPLC

UPLC

0 4 8 12 16 min

0 0,5 1 1,5 2 min

Application of UHPLC – Diclophenac gel

Bioanalytical applications

of sub-2-micro columns

Determination of phenolic compounds by UHPLC

UHPLC separation of 30 phenolic compounds

Acquity BEH C18 (100 x 2.1 mm/ 1.7 μm), gradient 0.1 % formic acid/MeOH/ from 88:12 to 30:70 (v/v), f.rate 0.45 ml/min, UV - 280 nm, 25° C

Gal

lic a

cid

- 1.0

23

GC

- 1

.422

Pro

toca

tech

uic

acid

- 1

.945

Esc

ulin

- 3

.064

EG

C -

3.96

2C

- 4.

198

Chl

orog

enic

aci

d -

5.22

1V

anill

ic a

cid

- 5.

468

Caf

feic

aci

d - 5

.816

Caf

fein

e -

6.04

2

Syr

ingi

c ac

id -

6.9

93E

GC

g -

7.28

5

EC

- 7.

837

GC

g -

8.54

0

Sco

pole

tin -

8.9

93

Fer

ulic

aci

d -

9.60

8E

Cg

- 9.

890

Cg

- 10

.575

o-C

oum

aric

aci

d -

12.0

50

Rut

in -

12.

554

4-hy

drox

ycou

mar

in -

12.8

80

Myr

icet

in -

13.4

83

Que

rcitr

in -

14.0

056-

met

hylc

oum

arin

- 14

.219

Cin

nam

ic a

cid

- 15

.122

Que

rcet

in -

16.2

76

Nar

igen

in -

16.6

97

Lute

olin

- 1

7.32

7

Hes

pere

tin -

17.

792

Kae

mpf

erol

- 19

.043

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

Phenolic acids

Catechines

Coumarins

Flavonoids

Nováková L., Spáčil Z., Seifrtová M., Solich P.: Talanta 80, 2010, 1970-1979

Bioanalytical applications

of core-shell columns

Determination of vitamin A and E

suitable for UHPLC system suitable for HPLC and UHPLC system suitable for HPLC system

!

!

Kučerová B., Krčmová L., Solichová D., Plíšek J., Solich P.: Comparison of new high-resolution monolithic column with core-shell and fully porous columns for THE analysis of retinol and α-tocopherol in human serum and breast milk BY UHPLC, J. Sep. Sci. 23, 2013, p.2223-2230

Comparison of core-shell and HR monolithic columns – for determination of vitamns A and E

Comparison of chromatographic separation

Classical particulate C18150 x 4.6 mm; 5 μm

Monolithic column100 x 4.6 mm

UHPLC – BEH silica100 x 2.1 mm; 1.7 μm

Determination of retinol and alpha-tocopherol in human serum

Where is the future ???

X sub-2 m column monolithic columns

X Very high pressure separation

Two opposite trends

Low pressure separation

Substantial decrease of analysis time !!!

!? Combination of high pressure using monolithic design !?

!? Combination of all features descibed !?

core-shell design X high-temperature LC

Conclusions

Applications of modern technologies (combination of modern HPLC/UHPLC method, new stationary phases with quick and easy sample preparation method) for (bioanalytical) applications allow:

Faster and robust analyses

Large sequences Small volumes of samples Small volumes of solvents No environmental pollution Cheap Increase laboratory throughput

  Acknowledgement

Thank you for your attention