HPLC Workflow Part 2 v3

Classification: Internal
Three Factors Control HPLC and UHPLC Resolution9 Separation

3.0
k α-1 a
Rs = N • •
4 k+1 α 2.5
Resolution (R)
2.0
N = 16 (tR/w)2 or
N = 5.5 (tR/d)2
1.5 N
1.0
k = (tR – t0)/t0 k
0.5
α = k2/k1
0.0
1.00 1.05 1.10 1.15 1.20 1.25 a
All factors are important,
but selectivity is 0 5K 10K 15K 20K 25K N
considered the most 0 5 10 15 20 25 k
powerful term.
1 HPLC workflow - March - 2018 - Dr Matus
9. Yun Mao, PhD Dissertation, University of Minnesota, 2001.
Internal (incl. business partners)
Selectivity Variables in Reversed-Phase * Separation
Continuous variables (solvent): More predictable

• type (organic, water) (modeling software available)
• solvent strength
• pH (especially ionizable solutes) Some analysts may
spend too much time
• additives (type and concentration) here “force feeding”
• temperature C18 columns.
Discontinuous variable (column): Less predictable

• type (phase and substrate) (screening required)
* Excerpted with permission from John Dolan, 2009 Minnesota

Chromatography Forum Spring Symposium; adapted by R. Henry.

Alkyl Bonded Phases (C18 and C8) Separation
Primar
y phase R
• C18 reagents are large and
reagent
can leave some silanols
unreacted (ca. 50%).
Hydrophobic
• C8 reagents are smaller selectivity
and provide better silanol
coverage
“Endcapping” reagent
• At pH >4, silanols can
ionize and add cation-
exchange character.
Si Si
Free silanol
OH O O O
H

Alkyl Amide Bonded Phases (abbreviated: RP-Amide) Separation
• Possible Solute Shielding C9H19

(basic solutes)- alkyl
phases with embedded Hydrophobic
polar groups (EPG) have
H-Bonding
been reported to correlate
much better with logP data
C9H19
than C18 columns due their
higher base deactivation1. OH-Backbinding HN
Amide-Carbonyl: strong C=O
• Possible H-bonding with H-Bonding acceptor
Endcapping-
solutes that are good H- Reagent
bond donors (acids, etc.)
Si NC=O Si Si
Free Silanol groups
OH O OH O O

Phenyl Bonded Phases Separation
• Phenyl is a Lewis base or Primar

y phase
electron donor; π-π
reagent
interaction can occur with
solutes that are deficient in π base
electrons (Lewis acids).
• Due to the rigid nature of
the aromatic ring, solute “Endcapping” reagent
shape can dictate selectivity
(how closely solutes can
approach the ring).
Si Si
Free silanol
OH O O O
H

Pentafluorophenyl (F5) Bonded Phases Separation
• PFP is a Lewis acid or

Primar
electron acceptor; π-π F F y phase
interaction can occur with reagent
solutes that are rich in F F
electrons (Lewis bases). π acid and F
• Due to the rigid aromatic dipole (H-
bond)
ring, solute shape can
dictate selectivity. “Endcapping” reagent
• Dipolar (possible H-bond)

interactions can occur
Si Si
Free silanol
OH O O O
H

Cyano Phases Separation
• Cyano is a strong dipole

that can interact with other
dipoles or induce dipoles on
solutes.
• Cyano phases also have
moderate hydrophobic
character from alkyl ligands.
• Stability of newer Cyano
phases is now comparable
to all other HPLC phases.

Classification by Possible Chemical Interactionsa

Separation
Bonded π-π
Hydrophobic H-Bonding Dipolar Stericb Ionicb
Phase
C18 Very Strong Weak No No No Moderate
C8 Strong Weak No No No Weak
RP- Strong Strong Moderate No Weak Very weak

Amide Acceptor
Phenyl Strong Weak Weak Strong Strong Weak
Acceptor Donor (Rigid)
F5 or Moderate Moderate Strong Strong Strong Moderate
PFP Acceptor Acceptor (Rigid)
Cyano Light to Weak Strong Weak No Moderate
Moderate Acceptor
a. Using Euerby2 variation of Snyder-Dolan-Carr Hydrophobic Subtraction Model3.

b. Steric and Ionic probe data are not very helpful in predicting or interpreting steroid
selectivity results; however, they are always underlying factors with silica bonded phases.
8

Comparison of Selectivity
Hydrophobic Retention Separation
Retention Dispersive Interactions mainly

2
1
4 drive the separation of
3
Pentafluorophenyl hydrophobic molecules.
F5 Retention of butyl benzene and
1 2
4 amyl benzene (4) directly depends
3
on dispersive interaction with the
Phenyl
stationary phase.
1 3
2 Additional selectivity by steric
4 impact (2, 3).
C8
1. Butylbenzene
1 2
2. o-Terphenyl
4 3
3. Triphenylene
RP-Amide 4. Amylbenzene
2
1
4 15x4.6 5um; 65% ACN 35%
3 C18 water
1.5mL/min, 220nm, 5uL

Comparison of Selectivity
Nitro compounds – p-p interactions Separation
NB
TNB 2,4-DNT Nitro groups highly polar and
electron drawing; aromatic
Pentafluorophenyl
compounds are a strong p acid
3NT Pentafluorophenyl, F5
NB 2,4-DNT
TNB  Phase is a strong p acid.
2 & 4NT Phenyl Selectivity is similar to C18, but
with increased retention due to
polar interactions
Phenyl
 p-p interactions change both
C8
selectivity and retention. Di- and
trinitro compounds are retained
most strongly.
RP-Amide
C8, RP-Amide & C18
NB TNB 2,4-DNT  Low hydrophilic retention
HPLC workflow - March - 2018 - Dr Matus C18 15x4.6; 5µm; 50% Methanol 50% water; 10
0 10 20 1.0mL; 210nm; 40C; 10µL

Amide Hydrogen-Bonding Selectivity Example Separation
Phenols and other Natural Phenols (H-bond donors):

Antioxidants 1. p-methoxyphenol
2. phenol
RP-Amide retention enhanced vs C18 3. p-cyanophenol
likely due to amide hydrogen bonding 4. p-fluorophenol
5. p-methylphenol
6. p-nitrophenol
7. p-chlorophenol
6
3
2 5
7 Ascentis Express
1
4 10cm x 4.6mm, 35:65 ACN:H2O, 0.2%
C18 formic acid, 1.8 mL/min, 230nm, 35C, 5µL.
3
2 5
1 4 6 7
RP-Amide
1.0 2.0 3.0

Time (min)

Conclusions Separation
• Selectivity has highest impact on resolution

- Stationary phase impacts selectivity most strongly
- Early screening of stationary phases with highest diversity
 Column phases with optimum retention and selectivity can be identified by fast
screening experiments (before serious mobile phase optimization begins).
• Subsequent method optimization by mobile phase
• Use of dedicated software
• Use all type of our columns for developing selective, high speed separations.

Reference Materials & Certified Reference Materials

When are standards used? Evaluation
• Instrument calibration
• Calibration of a method
• Method validation
• Assessment of precision & trueness (bias)
• Identification
• Internal quality control
• External quality control (proficiency testing)
• Regulatory requirements
NOTE: Some Reference Materials are designed for a specific purpose – for
example USP, EP Primary Standards are for their use in Pharmacopeia
Methods

Regulatory Requirements Evaluation
European Pharmacopoeia 5.12
 „Reference standards are shown to be suitable for their intended purpose; they are not
necessarily suitable for other purposes. If a reference standard is to be used for any purpose
other than that for which it has been established, its suitability for the new use has to be
fully demonstrated. Any value assigned to a reference standard is valid for the intended use
and not necessarily for other uses.“

Regulatory Requirements Evaluation
In pharmaceutical analysis reference standards

 Must be contained in the dossier for marketing authorisation of a drug/pharmaceutical
product
 Must be used in
- Quality control
- Stability testing
- In-process control
- Raw materials control

Qualification – Primary Reference Standard Content Evaluation
 Identity by spectroscopic data from e.g. 1H-NMR, 13C-NMR, MS, UV, IR (check for enantiomers, if
appropriate)
 Content assignment by two independent validated analytical methods such as chromatographic purity
considering residual solvents, water and further impurities
 Residual solvents
 Water content by Karl Fischer titration
 If appropriate elemental analysis or residue on ignition for inorganic impurities
Description of the process of establishment, certification and content of documentation to be filed [1,2]
[1] Veit, M.; Wissel, S. PharmInd 2007, 12, 1475–1480

[2] Veit, M.; Wissel, S. PharmInd 2008, 1, 135–138

Primary Standards from the Authorities Evaluation
European Pharmacopoeia
 EP CRS
 (Certified Reference Substances)
US Pharmacopoeia:
 USP Reference Standards
Now distributed by Sigma-Aldrich/Merck!

Pharmacopoeial Standards through Sigma-Aldrich Evaluation
Just re-selling: Standards are sold with USP/EP labels

For all product information and documentations a link to corresponding
pharmacopoeia is available
Advantages:
 Convenience
 Delivery time
 One-Stop-Shop

Qualification – Secondary Reference Standard Evaluation
A reference material traceable to and qualified against a primary standard, usually

obtained from a National or International Metrological Institute (NIST, BAM, IRMM),
or recognized authority (USP, EP, BP for pharmaceuticals)

Accreditation
CRM Manufacturing Sites Evaluation
In each case, the Scope of the

Accreditation is additionally specified,
as shown in the Merck copy.

Secondary Pharmaceutical Reference Standards Evaluation
 APIs (Active Pharmaceutical Ingredients)

 Impurities
 Excipients
 Melting Point Standards
 Residual Solvents
 Vitamins
21

Certificate of Analysis Evaluation
 Completely ISO Guide 31 compliant

 Downloadable at www.sigmaaldrich.com
 Updated when compendial lots change or new information is added
 Details and results of characterization
- Multi assay values (USP, EP, BP)
 Contains values to be used for compendial purposes

Evaluation

Producing Reference Materials

..... When there is no RM to qualify against? Evaluation
There are countless organics that might be needed

as reference materials, yet not enough
international standards for traceability.
qNMR solves this
Traceability independent of the chemical
identity
qNMR is a relative primary method of value
assignment
From qNMR, the signal intensity is:

Low Uncertainties
• proportional to the number of protons • Expanded uncertainties between 0.1% and 0.5%
• independent of chemical structure • Direct traceability to NIST
(may be impossible by chromatography)
Establishing qNMR at our Buchs, Switzerland facility • Suitable for most organics
has resulted in a complete range of organic CRMs
(TraceCERT )

Tracability
Organic CRMs: Challenges Evaluation
• Number of Organic Compounds is Countless

• Only few international Standard Reference Materials are available
• (short) Traceability?

Tracability
The classical method of content assigment Evaluation
Chromatographic Purity
 Response of target compound and impurities
 Water content?
 Residual solvents?
 Inorganic compounds?
Peak area
determination

Alternative: Quantitative NMR (qNMR)

Relative Primary Method Evaluation
Signal intensity is:

 proportional to the number of protons
 independent of chemical structure
Advantages qNMR
• Traceability across structures
• Low Uncertainties
• Non-destructive
• Structural Information
• Information on Impurities

Tracebility to NIST materials Evaluation
Benzoic acid
NIST Ref. Material (SRM)
Internal
Dimethylsulfone (IS) Standard
P/N 41837
Acenaphthene High-
precision
P/N 05426 weighing
1H-NMR
measurements
Solve
Sample nt

Data processing

Content Assigment by qNMR Evaluation
Internal Standard
Content known
Analyte
 NIST traceable
Acenaphthene Dimethylsulfone
4 protons 6 protons
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
Note:
112.2648
100.0000
29 Measured Sample c
HPLC workflow - March - 2018 - Dr Matus
Acenaphthene >c Dimethylsulfone
–
Quantitative NMR for CRMs
Summary Evaluation
• Lower uncertainty of the assigned value

• Orthogonal to purification techniques (chromatographic)
• qNMR may discover more impurities compared to chromatographic methods
- More reliable assigned content values
• qNMR for content assignment is validated
- Approved by the authorities
• qNMR allows traceability of any organic compound
- Certified Reference Materials available for Chromatography

HPLC Workflow Part 2 v3

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Classification: Internal

Three Factors Control HPLC and UHPLC Resolution9 Separation

Selectivity Variables in Reversed-Phase * Separation

Continuous variables (solvent): More predictable

Discontinuous variable (column): Less predictable

* Excerpted with permission from John Dolan, 2009 Minnesota

2 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Alkyl Bonded Phases (C18 and C8) Separation

3 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Alkyl Amide Bonded Phases (abbreviated: RP-Amide) Separation

• Possible Solute Shielding C9H19

4 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Phenyl Bonded Phases Separation

• Phenyl is a Lewis base or Primar

5 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Pentafluorophenyl (F5) Bonded Phases Separation

• PFP is a Lewis acid or

• Dipolar (possible H-bond)

6 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Cyano Phases Separation

• Cyano is a strong dipole

7 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Classification by Possible Chemical Interactionsa

C8 Strong Weak No No No Weak

RP- Strong Strong Moderate No Weak Very weak

a. Using Euerby2 variation of Snyder-Dolan-Carr Hydrophobic Subtraction Model3.

Internal (incl. business partners)

Retention Dispersive Interactions mainly

Internal (incl. business partners)

0 10 20 1.0mL; 210nm; 40C; 10µL

Amide Hydrogen-Bonding Selectivity Example Separation

Phenols and other Natural Phenols (H-bond donors):

1.0 2.0 3.0

Internal (incl. business partners)

• Selectivity has highest impact on resolution

12 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Reference Materials & Certified Reference Materials

Internal (incl. business partners)

Regulatory Requirements Evaluation

European Pharmacopoeia 5.12

14 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Regulatory Requirements Evaluation

In pharmaceutical analysis reference standards

15 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Qualification – Primary Reference Standard Content Evaluation

[1] Veit, M.; Wissel, S. PharmInd 2007, 12, 1475–1480

16 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Primary Standards from the Authorities Evaluation

Now distributed by Sigma-Aldrich/Merck!

17 HPLC workflow - March - 2018 - Dr Matus

Internal (incl. business partners)

Pharmacopoeial Standards through Sigma-Aldrich Evaluation

Just re-selling: Standards are sold with USP/EP labels