Trends in development of stationary phases in chromatography Petr Solich Charles University Czech...
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Transcript of Trends in development of stationary phases in chromatography Petr Solich Charles University Czech...
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