poster hplc 2010 - generic approach to hplc analysis...0 1 2 3 4 5 6 7 8 9 10 retentiontime...
TRANSCRIPT
0 1 2 3 4 5 6 7 8 9 10
Retention Time (minutes)
Water Atlantis dC18
SiliCycle SiliaChrom AQ C18
N
HO
O OF
NNH
F
N
OF
O N
OH
O
NN
H3C CH3
Marbofloxacin
Sarafloxacin
SiliaChrom® Generic approach for HPLC analysis: The modernsilica-based HPLC columns; SiliaChrom Series.
Charles Lévesque*, Vincent Bédard and François Béland
SiliCycle® Inc., 2500 Parc-Technologique Blvd,Quebec City (Quebec), G1P 4S6, Canada
Introduction
HPLC method development for the analysis of mixtures is a task that usually requires much expertise and is also extremely time-consuming. In spite of advances in chromatographic theory, HPLC method development is still based mainly on “trial and error”. Consequently, many attempts have been made to use computer programs to facilitate this process. In general, these are stand-alone programs, such as ChromSword® (Merck KGaA), DryLab® (Molnár-Institute) and others. But these approaches are very limited due to the fact that they require the use of an universal HPLC column.
Based on the United States Pharmacopia (USP) website, there are about 380 different HPLC C18 columns on the market and this number increases each year. It’s very challenging to find the best HPLC column to suit specific applications. The chromatographer needs a lot of experience to elaborate a quick and efficient HPLC method. To address this, SiliCycle has developped several C18 chemistries covering a very wide range of HPLC applications in combination with a generic approach in order to determine the best SiliaChrom column.
For further informationPlease contact [email protected]. More information on this poster and related projects can be obtained at www.silicycle.com/silica-application-library. You may as well get this poster in PDF version at www.silicycle.com/poster
Worldwide HeadquartersSiliCycle Inc., 2500, Parc-Technologique Blvd, Quebec City (Quebec) G1P 4S6 CANADA,
Phone: 1 418.874.0054, Toll Free: 1 877.745.4292 (North America only)
Fax: 1 418.874.0355, Website: www.SiliCycle.com, E-mail: [email protected]
The ion H3O+ doesn’t have access to the surface to break the O-Si bond (Steric Effect)
SiO
O
Si
OSi
CC
O
O
Si
OH
OO
Si
C
H3CH3C CH3
CH3
CH3
H3C
C CH3
CH3H3C
CCH3H3C
R
R
CH3
CCH3
CH3
CH3
CH3
SiliaChrom SB C18 surface is treated with an organic form of silicon to increase the number of silanol groups on the surface. After this step, the surface is bonded with a silane containing two fonctions. One of them has a protecting group that shields the area and protects the surface from an acid attack from the mobile phase.
HPLC Column for Low pH (0.5-7.5)SiliaChrom SB C18 or C8
Si
Si Si
Si
OHO
HO
HO
HO
HO
HOOH
OH
OHOH
OHOH
OH
OHO
O O
O O
OO
O
OO O
OO
OO
O
O
OO
O O
OO
O
OO
OO
OO
O
OO
O
OO
O
O
O
O
OO
O
O
O
Si
Si
Si
Si
Si
Si
SiSi
SiSi
Si
Si
Si
Si
Si
Si
Si
Si
SiSi
Si
Si
Si
Si
Si
Si
Si
Si
SiSi
SiSi
Si
SiSi
Si
Si
Si
Si
HPLC Column for High pH (1.0-12.0)SiliaChrom XT C18 FidelityThe key is to have a hybrid surface to reduce the solubility of silica at high pH. In fact, SiliaChrom XT C18 and SiliaChrom XT C18 Fidelity are silica coated with a monomeric ethyltriethoxysilane/tetraethoxysilane prepolymer, followed by a special thermic treatment to get a rigid surface that is less soluble than silica itself at high pH.
R = C18
Stability Study of SiliaChrom SB C18 and SiliaChrom XT C18 Fidelity
R
C
C
C
C
R
R
R
R
R
R R
R
R
R
R
R
R
R
R
R
R
R
RR
Si Si
SiOH
OH
OH
HO
HO
OH
O
O
O O
OO
OO
O
O
O O
O
O
OO
O
O
O
OO
O
O
O
O
O
OO
O
O
O
O
OSi
Si
Si
SiSi
Si
Si
Si
Si
Si Si
Si
Si
Si
Si
Si
Si
Si
Si
Si
Si
Si
Si Si
Si
Si
Si
Si
Si
SiSi
Si
Si
Si
SiO
O
SiO
OSi
O
O
O
Si
Si
Si
CH3
OO
H3C
O
CH3
OSi
O
Si
Si
Si
H3C
CH3
CH3
H3C
CH3
CH3
CH3
CH3
(CH2)17CH3
0
0 hour
Conditions
Column: SiliaChrom SB C18, 5µmColumn Size: 4.6 x 150 mmSiliCycle P/N: H101805H-N150Mobile Phase: 2% TFS in ACN/Water (60/40)Solution pH = 1.00Temperature: 23 ºCFlow Rate: 1.00 mL/minDetector: UV at 270 nm
Conditions
Column: SiliaChrom XT C18 Fidelity, 5µmColumn Size: 4.6 x 150 mmSiliCycle P/N: HF171805H-N150Mobile Phase: 0.2% TEA in ACN/Water (55/45)Solution pH = 11.50Temperature: 23 ºCFlow Rate: 1.00 mL/minDetector: UV at 270 nm
SiliaChrom SB C18Time
(hour)RT
(min)TF
(USP)N
(USP)0 5.91 1.01 14 014
24 5.89 1.02 14 085
48 5.77 1.02 14 023
72 5.83 1.02 14 076
96 5.85 1.01 14 087
120 5.84 1.02 14 050
MeanRSD (%)
5.850.84
1.020.51
14 0560.23
SiliaChrom XT C18 FidelityTime
(hour)RT
(min)TF
(USP)N
(USP)0 13.35 1.01 13 623
24 13.29 1.01 13 648
48 13.27 1.01 13 689
72 13.25 1.00 13 604
96 13.24 1.00 13 649
120 13.28 1.00 13 582
MeanRSD (%)
13.280.29
1.010.54
13 6330.28
0 1 2 3 4 5 6 7 8
0
120 hours
0 hour
N
NH
H
O
O
OH
CH3
CH2CH3
Retention Time (minutes)
0 2 4 6 8 10 12 14 16
N
NH
H
O
O
OH
CH3
CH2CH3
Retention Time (minutes)
SiliaChrom AQ C18: Purity of 99.999%SiliaChrom dt C18: Purity of 99.9999% (no metal content)
FeaturesRapid equilibrationUnique reversed-phase selectivity (universal HPLC column)Exceptional stability from pH 1.5 to 9.0Reduce need for mobile-phase modifiersCompatible with 100% aqueous mobile phase (buffer)Long column lifetime
Surface area: 380 m2/gCarbon load: 18 %Endcapped: Partially endcapped (unique feature)USP L-code: L1
Universal HPLC Column - SiliaChrom AQ C18 (or C8) and SiliaChrom dt C18
SiO
O
Si
OSi
CH3
O
O
Si
O
O
OH
OHCH3
CH3
SiHO
OH
OSi R
CH3
CH3
O
R = C18 or C8
SiliaChrom AQ (or dt) adsorbent employs an optimum ratio of C18 (or C8), short TMS chains and some free silanol groups. This new technology shows good peak shapes for any type of molecule (acid, neutral and basic).
Optimum Ratio of TMS-Silanol-C18 (or C8)
SiliaChrom AQ Applications
0 1 2 3 4 5 6 7 8 9
Retention Time (minutes)
OHHO
HONH2
OHHO
NH2
OH
H2N
OHO
OH
H2N
OHO
OH
OH
HO
HONH
CH3
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5
Retention Time (minutes)
N
NN
N
H3C
CH3
O
CH3
O
NH
HO
Cl
SNH2
O
O
O
N
HN
NH
O
O CH3S
H3C
O
OH
O
O
O
OH
O
O
O
O
O CH3
O
CH3
1
2
3
4
5
6
1
2
3
4
5
6
Catecholamines with SiliaChrom AQ C18 - In Acidic Water as Mobile Phase
Rapid HPLC with SiliaChrom AQ C18 - Test Mix from Pharmaceutical Company
Conditions
Column: SiliaChrom AQ C18, 5 µmColumn Size: 4.6 x 150 mmSiliCycle P/N: H151805E-N150Mobile Phase: 1 % Acetic Acid in WaterTemperature: 23 ºCFlow Rate: 1.00 mL/minDetector: UV at 265 nmInjection Volume: 5 µL
Conditions
Column: SiliaChrom AQ C18, 5 µmColumn Size: 3.0 x 30 mmSiliCycle P/N: H151805E-H030Mobile Phase: MPA: 0.1 % TFA in ACN/Water (5/95) MPB: 0.1 % TFA in ACN/Water (95/5)Linear Gradient: MPA to MPB, 2.25 min.Temperature: 23 ºCFlow Rate: 1.75 mL/minDetector: UV at 254 nmInjection Volume: 5 µL
Peak Shape Evaluation for Acidic and Basic Fluoroquinolones
Conditions
Column: SiliaChrom AQ C18, 5 µm Waters Atlantis dC18, 5 µmColumn Size: 4.6 x 150 mmSiliCycle P/N: H151805E-N150Mobile Phase: 2.5 mM potassium phosphate monobasic (adjust to pH 2.50 with H3PO4)/MeOH (68/32)Temperature: 23 ºCFlow Rate: 1.00 mL/minDetector: UV at 275 nmInjection Volume: 10 µL
Evaluation of Resolution and Peak Shape
0 2 4 6 8 10 12 14 16 18 20
Retention Time (minutes)
Phenomenex Luna C18 (2)(230 nm)
SiliaChrom AQ C18(230 nm)
Phenomenex Luna C18 (2) (210 nm)
SiliaChrom AQ C18 (210 nm)1
1
1
1
2
2
2
2
3
3
4
4
4
4
5
5
6
6
7
7
8
8
1
OHO
OOH
2
H
HO
HN CH3
O
3
CH3
OH
NHH3C 4
Cl
N
NH3C
CH3
5
OH
O
6OH
OOH
7
OH OH
ClCl8
H3C
CH3
CH3
OH
O
Conditions
Column: SiliaChrom AQ C18, 5 µm Phenomenex Luna C18 (2), 5 µmColumn Size: 4.6 x 150 mmSiliCycle P/N: H151805E-N150Mobile Phase: MPA: 5 mM potassium phosphate monobasic (adjust to pH 2.50 with H3PO4)/ACN (90/10)MPB: 5 mM potassium phosphate monobasic (adjust to pH 2.50 with H3PO4)/ACN (10/90)Linear Gradient: MPA to MPB, 20 min.Temperature: 23 ºCFlow Rate: 1.00 mL/minDetector: UV at 210 and 230 nmInjection Volume: 20 µL
Different Hydrophobicity (%C) SiliaChrom XDB & XDB1 C18 (or C8) and SiliaChrom XDB2 C18
SiO
Si O
OSi
(CH2)7CH3H3C
CH3
Si(CH3)3C8
SiO
Si O
O
Si(CH3)3
Si(CH2)17CH3
H3C
CH3
C18Carbon LoadingSiliaChrom XDB C8 (8%) and SiliaChrom C18 (15%)SiliaChrom XDB1 C8 (14%) and SiliaChrom C18 (22%)SiliaChrom XDB2 C18 (18%)
ConclusionsGeneric Approach with SiliaChrom Series
GenericConditions(Acidic, Basicor Neutral)
Retention &Resolution Issues
Polarity Type HPLC Solutions
SiliaChrom AQ or dt (MeOH/H2O) or (ACN/H2O), pH=1.5-9.0
Good retention but not enough
resolution
Not enoughretention
Acidic
Basic
Neutral
SiliaChrom SB pH=0.5-7.5
SiliaChrom XT pH=1.0-12.0
SiliaChrom XDB, XDB1 or XDB2
Carbon Loading=8-22%
SiliaChrom SB or XT
(low and/or high pH)
It is possible, with SiliaChrom HPLC products, to consider a generic approach for HPLC method development by using just a few HPLC phases. Using only these three phases, we are able to obtain over 90% succes in separating most compounds from different matrices. In this poster, we have shown phases that are effective in low and high pH conditions and a new polar phase that works for practically any type of analyte (acidic, neutral and basic, limited to pH between 1.5-9). We have demonstrated that it is possible to have high efficiency at low cost by using SiliaChrom generic approach.
B
B
C
C
DA
B
A
C D
or
R = C18 or C8
A Generic Approach to determine the best HPLC column to use
Asymmetry (USP)
SiliaChrom AQ C18 Waters Atlantis dC18 1.12 1.29 1.09 1.14
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The authors would like to thank Ms. Stéphanie Parent for her contribution to this poster.
SiliaChrom SB C18 SiliaChrom SB C18after 120 hours
SiliaChrom XT C18 SiliaChrom XT C18after 120 hours
120 hours
Stability studies were performed on both low and high pH conditions using SiliaChrom SB C18 and XT C18 Fidelity HPLC columns respectively. Each HPLC column was used under extreme conditions and even after 5 days of continuous injections, the number of theoretical plates (N), the tailing factor (TF) and the retention times (RT) are comparable. Both sorbents kept their chemical and structural properties as proven by similar chromatograms and scanning electron microscope pictures (SEM) before and after 120 hours of use.