Qualification of Analytical Columns Paphomcl 12 128 5r

OMCL Network of the Council of Europe
QUALITY MANAGEMENT DOCUMENT
PA/PH/OMCL (12) 128 5R
QUALIFICATION OF ANALYTICAL COLUMNS
Full document title and Qualification of Analytical Columns

reference
PA/PH/OMCL (12) 128 5R
Document type Recommendation Document
Legislative basis
Date of first adoption
Date of original entry July 2013

into force
Date of entry into force

of revised document
Previous titles/other
references / last valid
version
Custodian The present document was elaborated by the OMCL Network /

Organisation EDQM of the Council of Europe
Concerned Network GEON

PA/PH/OMCL (12) 128 5R 2/20
QUALIFICATION OF ANALYTICAL COLUMNS
1. INTRODUCTION
The present document outlines the general principles for the qualification of analytical
columns used within OMCLs. It has been agreed that analytical columns represent
consumables, but are also an essential part of the chromatographic equipment (Ph. Eur.
2.2.28, 2.2.29).
For this reason, qualification of analytical columns is considered necessary. The purpose
of this document is to give general requirements for the qualification of different types of
analytical columns. Actually, due to the great variety of analytical columns, it is not
possible to provide specific requirements for all of them. The levels of qualification
mentioned in this document refer to the definitions given in OMCL Guideline
PA/PH/OMCL (08) 73 2R.
This document may be used as a guide to OMCLs for planning, performing and
documenting the qualification process upon receipt and usage of analytical columns. It
represents a recommendation document.
2. AIM AND SCOPE OF THE DOCUMENT
This document describes the requirements for the qualification of analytical columns used
in LC and GC. The aim is to provide general criteria for qualification and examples for
testing the most commonly used analytical columns. Other approaches may be used.
3. CONSIDERATIONS FOR LEVEL I QUALIFICATION
Although there is no need for a formal level I procedure, criteria used for the ordering of
columns should comply with the special needs of the intended test procedure.
4. LEVEL II QUALIFICATION
At Level II of the qualification of analytical columns, it is recommended to check the

column for physical damage which may appear during shipping, to verify that the column
received complies with the order and that the supplier has submitted all necessary
documents and information.
The analytical column may be appropriately tested before use by the OMCL according to
a pre-defined procedure. This testing procedure may be performed on receipt or, at the
latest, prior to first use and should be documented in a suitable qualification record (see
Annex 2).
After the qualification, the laboratory should record the results.
The OMCL should implement a system for identification of available columns and should
maintain a list of all columns in the laboratory.
The type of qualification depends on the intended use of the column and can follow
different approaches. Future re-qualifications should follow the same test procedures as
used in level II qualification.
Verification of column performance can be done by one or more of the following
approaches:
a) By testing the column under the same conditions given in the original quality control
chromatogram of the manufacturer. The same parameters have to be checked and the
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results have to be compared with the results of the manufacturer. Alternatively, other test
mixtures may be used.
b) If the column will be used only for specific test methods, it might be appropriate to
perform the qualification with an appropriate system suitability test that is specific for the
test procedure.
c) Alternatively, a common test mixture for columns with similar stationary phases may
be used.
5. LEVEL III QUALIFICATION
The OMCL should ensure that the column is under quality control and column
verification may be performed periodically using the same test compounds. A systematic
record of the results has to be maintained in the laboratory. The qualification may be done
as described in Chapter 4. At least one of the approaches chosen for level II qualification
should be followed in level III qualification.
The first chromatogram obtained on a new column in the laboratory may serve as a
reference and defines column performance at time t = 0 in the OMCL. It should be used
as the base criterion for subsequent verifications. Slight variation may be obtained on
different LC or GC systems due to the quality of the connections, operating environment,
system electronics, etc. These can be reflected in the acceptance criteria used in the
qualification.
A record of the results helps the user to monitor column performance and to know when a
column should be replaced. A column should be replaced whenever its performance falls
outside expectations.
The type and number of samples analysed contributes to column degradation. The
number and type of analyses performed should be traceable for each column. A typical
example for recording could be the following form:
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Qualification parameters:
It is the responsibility of the OMCL to choose the appropriate quality parameters and
corresponding acceptance criteria. These should be appropriate to the intended use of the
column. These parameters could include resolution, symmetry factor, theoretical plates,
etc. Examples are given in chapters 6 and 7.
Frequency of qualification:
The laboratory should define and document the frequency of qualification depending on
the use of the column. For instance, a qualification according to section 4.a (above) could
be performed based on fixed time intervals (e.g. once a year) or, more individually, based
on the frequency of use of a column, e.g. after a defined number of injections.
6. LEVEL IV QUALIFICATION
Level IV qualification corresponds to the system suitability test of the individual test
procedure.
7. GLOSSARY
Terms and definitions:
Height of a theoretical plate (H)
The height of a theoretical plate of a component may by calculated using the following
equation:
L
H= , where
N
L is the length of the column [mm], and

N is the calculated number of theoretical plates of the component.
A good column has a large N and a small H.
Plate per meter

The efficiency of a chromatography column may be expressed for a given component by
the number of theoretical plates per meter.
This parameter is used mainly in GC. It is very useful for comparisons of GC columns.
Table 1 below represents the requirements for the average column efficiency, given in
plates/meter, according to the internal diameter and polarity of the phase.
Table 1. Comparison of average efficiency by phase polarity and internal diameter.

Internal diameter Polarity of the column
Non-polar Intermediate polarity Polar
0.1 mm 10000 - -
0.2 mm 4500 4200 4000
0.32 mm 3200 3000 2500
0.53 mm 1500 1350 1300
Plate number (N) – as specified in Ph. Eur. 2.2.46

The plate number calculated from a non-retained peak is a measure of the column packing
efficiency whereas, in the case of peaks eluted later, mass-transfer processes also
contribute to the plate number. As a general rule, N is higher for retained compounds
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because their band broadening by extra-column volumes (dead volumes) is lower than for
the early eluted peaks. The plate number is a function of the column’s dimensions
(diameter, length and film thickness), the compound and its retention.
Qualification
Qualification is performed to assure that a given system, premises, equipment and column
is able to meet pre-determined acceptance criteria.
Reduced plate height (h)

Information on the number of theoretical plates, or plate height, can cause problems due
to mass transfer, so dimensionless values are much more suitable for absolute
comparisons of a whole range of different types of columns (long, short, thick or thin).
Reduced plate height is calculated as:
H
h= , where
dp
H is the height of a theoretical plate, and

dp is the particle diameter [µm].
Relative retention (r) – as specified in Ph. Eur. 2.2.46.
Resolution (Rs ) – as specified in Ph. Eur. 2.2.46.
Retention time – as specified in Ph. Eur. 2.2.46.
Retention factor (k) – as specified in Ph. Eur. 2.2.46

The larger this value, the greater the amount of a given solute in the stationary phase and,
hence, the greater the retention of the column.
Selectivity (α)
The chromatographic ability of the system to separate two compounds represents its
selectivity.
k2
α= , where
k1
k2 and k1 are retention factors of two compounds with k2 > k1.
Symmetry factor (As) – as specified in Ph. Eur. 2.2.46.
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8. REFERENCES
European Pharmacopoeia chapter 2.2.46.

Column performance test mixture for liquid chromatography - standard reference material
870. NIST.
Spherisorb columns care and use manual, Waters.
HPLC column classification. USP Pharmacopoeia Forum, Vol. 31(2).
Instruction sheet for non-polar test mixture, Supelco.
Instruction sheet for intermediate polarity test mixture, Supelco.
Instruction sheet for polar test mixture, Supelco.
Modern practice of gas chromatography, Fourth edition. 2004. Editors: Robert L. Grob
and Eugene F. Barry. John Wiley & Sons Inc.
High resolution gas chromatography. 1989. Editor: K. J. Hyver. Hewlett Packard Co.
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ANNEX 1:
A. EXAMPLES FOR QUALIFICATION OF ANALYTICAL COLUMNS FOR LC
The chromatographic conditions in the next test examples are for columns with different
stationary phases and dimensions of 250 mm x 4.6 mm. When a column with other
dimensions is tested, acceptance criteria and chromatographic conditions have to be
adapted according to the requirements of Ph. Eur. 2.2.46. Most of the examples given
below have been recommended by column manufacturers.
BACKGROUND INFORMATION ON CHARACTERISTICS OF RP LC-COLUMNS
The characteristics of the stationary phase are fundamentally dependent on their physico-
chemical and chemical properties. Important properties include particle diameter, metal
content, pore size, surface, the type of functional group on the surface, and also the
density of the surface modifier (e.g. carbon load). In addition to these parameters, the
various stationary phases available can also be differentiated based on their
chromatographic properties.
The most important properties of reverse phase models are:
Hydrophobicity – refers to the property of a stationary phase to retain the hydrophobic

compounds as much as possible. The strength of the retention capacity of an individual
phase depends substantially on the surface modification; the longer the hydrocarbon
chain, the greater the hydrophobicity. If the hydrocarbon chains are shorter, the analytes
have less space for adsorbtion, leading to lower hydrophobicity.
Silanol activity – silanol groups have a large influence on the selectivity of an RP-HPLC.
On the one hand, undesired secondary interactions result from the silanol groups,
whereby the basic substances display tailing through interactions with the acidic silanol
groups. On the other hand, such secondary interaction is actually required in some
separations to get the desired selectivity. To minimise silanophilic activity, many
stationary phases are end-capped.
Polar selectivity – refers to the property of a phase to undergo polar interaction with
analytes. RP with alkyl groups shows weak polar selectivity.
QUALIFICATION OF RP-LC C-8 AND C-18 COLUMNS
The following test is appropriate for qualification of this type of column using a mixture
of four compounds (uracil, toluene, phenol and N,N-Diethyl–m-toluamide), with a UV-
Vis detector:
Chromatographic settings:
Mobile phase: acetonitrile:water = 58:42 v/v %
Flow: 1.0 ml/min
Detection: 254 nm
Temperature: room temperature
Injection volume: 5 µl
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Solutions:
Prepare a mixture of four compounds in solvent acetonitrile:water = 58:42 v/v % with the
following concentrations:
- Uracil – 0.005 mg/ml
- Toluene – 4.0 mg/ml
- Phenol – 0.7 mg/ml
- N, N-diethyl-m-toluamide – 0.6 mg/ml
Method:
After the system is equilibrated, inject the prepared mixture at least twice. The retention
time, plate number and symmetry factor have to be recorded for all peaks. Calculate the
retention factor (k), resolution between the adjacent peaks and reduced plate height (h).
Calculate the selectivity for the two compound pairs phenol/toluene and N,N-diethyl-m-
toluamide/toluene.
Criteria for characterisation and limits:

- The elution order is: Uracil, Phenol, N,N-diethyl-m-toluamide and Toluene.
- Uracil – this component is used as an indicator for the hold-up time (tM), which is
required to calculate the retention factor (k).
- Toluenе - the retention of this component is a result of solvophobic interactions.
Calculate the retention factor for this non-polar compound, which provides
information on column strength. It is used to calculate column plate number (N) and
reduced plate height (h).
- N,N-diethyl-m-toluamide – the calculated symmetry factor of this basic compound is
used for the measurement of silanol activity. A more symmetrical peak is indicative of
column deactivation. It is a polar basic analyte that is sensitive to phase loss and
silanol activity. As end-capping is lost, peak shape and efficiency should suffer.
- Phenol – is a weak acid and, together with toluene and N,N-diethyl-m-toluamide, it is
used for determination of the polar selectivity of the stationary phase.
- As = 0.8 to 1.5
- Rs > 1.5 (between adjacent peaks)
- The number of plates N for toluene > 3000
- αphenol/toluene and αN,N-diethyl-m-toluamide/toluene ≥ 1.5
- Reduced peak heights and retention factors are necessary for comparisons between
columns from different manufacturers.
The conditions described above for testing C-8 and C-18 column are also appropriate for
C-4 columns.
QUALIFICATION OF RP-LC CN COLUMNS
Cyano-phases have moderate hydrophobicity compared to alkyl ligands.

The cyano group contains strong dipoles that can interact with other dipoles in solutes and
can separate polar molecules.
The following test is appropriate for qualification of this type of column, using a mixture
of five compounds (uracil, phenol, toluene, 4-Cl-nitrobenzene and naphthalene), with a
UV-Vis detector:
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Mobile phase: methanol:water = 70:30 v/v %
Flow: 1.0 ml/min
Detection: 254 nm
Solutions:
Prepare a mixture of five compounds in solvent methanol:water = 70:30 v/v % with the
- Uracil – 10 µg/ml
- Phenol – 200 µg/ml
- Toluene – 800 µg/ml
- 4-Cl-nitrobenzene – 25 µg/ml
- Naphthalene – 40 µg/ml
Method:
time, plate number, asymmetry and resolution between the adjacent peaks have to be
recorded for all peaks. Calculate the retention factor (k) and reduced plate height (h).

- The elution order is: Uracil, Phenol, Toluene, 4-Cl-nitrobenzene and Naphthalene.
- Toluene and Naphthalene - the retention of these components is a result of
solvophobic interactions. The peaks can be used to calculate the column plate number
(N) and reduced plate height (h).
- Phenol and 4-Cl-nitrobenzene – these polar compounds are used for determination of
polar selectivity.
- As = 0.8 to 1.5
- The number of plates N for Naphthalene > 3000
- Rs ≥ 1.5 (between adjacent peaks)
columns from different manufacturers
QUALIFICATION OF RP-LC PHENYL (phenyl propyl and phenyl hexyl) COLUMNS
The retention characteristics are similar to those of C-8 columns, but with different
selectivity for polycyclic aromatic hydrocarbons.
Phenyl phases are π-basic (electron donating) and are similar in overall retention to alkyl
phases. The alternate selectivity of phenyl phases is often explained by the π-π
interactions available through the phenyl ring. Compounds that appear to exhibit
differential selectivity on the phenyl phase include: small, water soluble molecules and
peptides; π-acceptors; nitroaromatics; polar compounds; dipoles; heterocyclics; etc.
of four compounds (uracil, acetophenone, toluene and naphthalene), with a UV-Vis
detector:
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Mobile phase: acetonitrile:water = 65:35 v/v %
Flow: 1.0 ml/min
Detection: 254 nm
Solutions:
Prepare a mixture of four compounds in solvent acetonitrile:water = 65:35 v/v % with the
- Uracil – 0.01 mg/ml
- Acetophenone – 0.22 mg/ml
- Naphthalene – 9.42 mg/ml
Method:
Calculate the selectivity for the two compound pairs acetophenone/toluene and
toluene/naphtalene.

- The elution order is: Uracil, Acetophenone, Toluene and Naphthalene.
- Naphthalene - the retention of this component is a result of solvophobic interactions.
Calculate the retention factor for this non-polar compound, which provides
information on column strength. It can be used for the calculation of column plate
number (N) and reduced plate height (h).
- The calculated value of α for the compound pair acetophenone/toluene represents
selectivity for specific molecular increments.
- The α value for the compound pair toluene/naphthalene represents selectivity for π-π
interactions.
- As = 0.8 to 1.5
- The number of plates N for Naphthalen > 3000
- αacetophenone/toluene and αtoluene/naphtalen ≥ 1.5
QUALIFICATION OF NP-LC COLUMNS (for Si, NH2, NO2, Diol, CN)
of three compounds (Toluene, Diethyl phthalate and Dimethyl phthalate), with a UV-Vis
detector:
Mobile phase: hexane:ethanol R = 95:5 v/v %
Flow: 1.0 ml/min
Detection: 254 nm
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Solutions:
Prepare a mixture of three compounds in solvent hexane:ethanol R = 95:5 v/v %, with the
- Diethyl phthalate – 1.0 mg/ml
- Dimethyl phthalate – 1.0 mg/ml
Method:
Calculate the selectivity for the compound pair diethyl phthalate/dimethyl phthalate.

- The elution order is: Toluene, Diethyl phthalate and Dimethyl phthalate.
- Toluene – this component is used as an indicator for the hold-up time (tM), which is
- Diethyl phthalate and dimethyl phthalate - the retention of these components is the
result of hydrophylic interactions. Calculate the retention factor for these polar
compounds, which provides information on column strength. Plate number and peak
asymmetry can be assessed using either of the phthalate peaks.
- As = 0.8 to 1.5
- The number of plates N > 3000
- αdiethyl phthalate/dimethyl phthalate ≥ 1.5
QUALIFICATION OF STRONG AND WEAK ION-EXCHANGE COLUMNS (SCX;

WCX; SAX; WAX)
The stationary phase has electric charges on its surface. A resin with SO32- groups is a
strong cation exchanger and a COO- resin is a weak cation exchanger. An anion
exchanger contains NR3+ (strong) or NR2H+ or NH3+ (weak) groups and forms bonds with
negatively-charged anions.
1. Qualification of SCX columns

These types of columns are mainly used in separating basic, water-soluble compounds.
The retention of basic substances is governed by both pH and ionic strength.
of two compounds (Uracil and Cytosine) and a UV-Vis detector:
Mobile phase: 0.15 M di-ammonium hydrogen phosphate buffer pH=6.0
Flow: 1.0 ml/min
Detection: 254 nm
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Solutions:
Prepare a mixture of two compounds in solvent water with the following concentrations:
- Uracil – 7 µg/ml
- Cytosine – 7 µg/ml
Method:
After the system is equilibrated, inject the prepared mixture at least twice. Record the
retention time, symmetry, plate number, resolution and selectivity for the two peaks.
Limits:
- As = 0.8 to 1.5
- Rs > 1.5
- αuracil/cytosine ≥ 1.5
- Reduced peak heights are necessary for comparisons between columns from different
manufacturers.
2. Qualification of SAX columns

This type of column is mainly used for separating anionic analytes.
of two compounds (Uridine and Uridine monophosphate) and a UV-Vis detector:
Mobile phase: 0.05 M sodium di-hydrogen phosphate buffer pH=3.0
Flow: 1.0 ml/min
Detection: 254 nm
Solutions:
Prepare a mixture of two compounds in solvent water, with the following concentrations:
- Uridine – 7 µg/ml
- Uridine monophosphate – 7 µg/ml
Method:
After the system is equilibrated, inject the prepared mixture at least twice. Record the
retention time, symmetry, plate number, resolution and selectivity for the two peaks.
Limits:
- As = 0.8 to 1.5
- Rs > 1.5
- αuridine/uridine monophosphate ≥ 1.5
- Reduced peak heights are necessary for comparisons of columns from different
manufacturers.
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QUALIFICATION OF CHIRAL COLUMNS
It is recommended to test this column type with a test mixture according to the quality
control certificate accompanying the column. After the test is completed, results should
be compared with those in the QC chromatogram of the manufacturer.
SIZE-EXCLUSION COLUMNS
Due to their different pore-sizes, size-exclusion columns differ widely with respect to
their fractionation range. It is therefore not possible to cover all types of size-exclusion
columns with one set of test conditions and specifications. The qualification of a typical
column type is presented here only as an example. A very widely-used column in the
quality control of therapeutic proteins (e.g. immunoglobulin monographs 0338 and 0918
and albumin monograph 0255) is based on hydrophilic silica gel for chromatography R
with a fractionation range for protein of 10000 to 500000.
Mobile phase: dissolve 4.873 g disodium hydrogen phosphate dihydrate R, 1.741 g
sodium dihydrogen phosphate monohydrate R and 11.688 g sodium chloride R in 1 L of
water R.
Column dimensions:
- 10 µm particle size: l = 600mm, i.D. : 7.5 mm
- 5 µm particle size: l = 300mm, i.D. : 7.5 mm
Flow rate: 0.5 mL/min

Detection: 280 nm
Inj. Volume: 20 µL
Solutions:
Dissolve the following compounds in the mobile phase:
- Thyroglobulin (bovine, MW 670,000) 5.0 g/L
- -Globulin (bovine, MW 158,000) 5.0 g/L
- Ovalbumin (chicken, MW 44,000) 5.0 g/L
- Myoglobin (horse, MW 17,000) 2.5 g/L
- Vitamin B12 (MW 1,350) 0.5 g/L
Method:
time, plate number, resolution and symmetry factor should be calculated and used as
acceptance criteria.

- The elution order is thyroglobulin, -Globulin, Ovalbumin, Myoglobin, and Vitamin
B12. There may be additional peaks between thyroglobulin and -Globulin (-
Globulin-dimer peak) and in front of thyroglobulin (aggregates peak).
- Plate number (Vitamin B12): NLT 20,000

- Symmetry factor (Vitamin B12): 0.8 to 1.5
- Resolution (Myoglobin/Ovalbumin): NLT 2.5
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Test mixtures and conditions can be used for several other stationary phases. Limits,
however, have to be individually defined for every column type.
B. EXAMPLES FOR THE QUALIFICATION OF ANALYTICAL COLUMNS

FOR GAS CHROMATOGRAPHY
Background Information:
The separation of the components of the test mixture and its peak shapes serve to monitor
column efficiency and as diagnostic probes for adverse adsorptive effects and the
acid/base properties of a column.
Alcohols and diols are commonly used to detect the presence of active silanol groups in
the capillary column. Ketones and aldehydes show the presence of sites that interact with
Lewis acids. A group of acidic or basic compounds is frequently used to determine how
neutral a column is. Esters and n-paraffins are used to determine column efficiency.
Functional groups of the stationary phases are related to the polarity of the stationary
phases. Polarity is a useful tool because it influences component separation. The
components that co-elute on a non-polar column might separate on a polar column, or
vice versa.
Most of the examples given below have been recommended by column manufacturers.
QUALIFICATION OF NON-POLAR COLUMNS
The following test is proposed for qualification of this type of column, using a mixture of
ten compounds and an FID detector:
Chromatographic settings for narrow-bore columns (0.25 or 0.32 mm ID):

Carrier gas: Helium
Linear gas rate: 20-25 cm/sec
Injection port temperature: 220°C
Oven temperature: 100-135°C
FID temperature: 220°C
Split ratio: 100:1 (5 ng of each component is delivered onto the column)
Chromatographic settings for wide-bore capillary columns (0.53 or 0.75 mm ID):

Dilute the mixture 1:10 in methylenchloride and inject 0.2 µl onto the column in direct
injection mode.
Solutions:
Prepare a mixture of the eight compounds in solvent methylene chloride, with the
- 2-octanone – 0.5 mg/ml
- Decane – 0.5 mg/ml
- 1-octanol – 0.5 mg/ml
- 2,6-dimethylphenole – 0.5 mg/ml
- Undecane – 0.5 mg/ml
- 2,6-dimethylaniline – 0.5 mg/ml
- Dodecane – 0.5 mg/ml
- Tridecane – 0.5 mg/ml
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Method:
After the system is equilibrated, inject the prepared mixture at least twice. The
components in the test mixture provide important information about the column and
system performance. Calculate the retention factor, selectivity for the adjacent peaks and
plates per meter. Calculate the percentage of the peak height of n-alkanes by drawing a
line connecting the peak maxima of these non-adsorbing peaks.
% response = (peak height of active component / height from baseline to curve

connecting hydrocarbon peak apexes) x 100
The % response serves as an internal standard for accurately monitoring column

performance for each active component. This technique includes all types of peak
deformation.

- The elution order is: 2-octanone; N-decane; 1-octanol; 2,6-dimethylphenol; N-
undecane; 2,6-dimethylaniline; N-dodecane and N-tridecane.
- Methane – this is used as an indicator for the hold-up time (tM), which is required
to calculate the retention factor (k) and mean linear gas velocity in the column ( u
= column length in cm/gas hold-up time in cm). Tailing of the peak indicates dead
volume in the system.
- Calculate the retention factor (k) for all peaks.
- 2,6-dimethylaniline - tailing or reduced peak height for this component indicates
that the active sites are acidic.
- 2,6-dimethylphenol – tailing or reduced peak heights for this component indicates
that the active sites are basic.
- α for 2,6-dimethylaniline/2,6-dimethylphenol represents the acid/base ratio.
- 1-octanol - tailing or reduced peak heights indicate silanol or hydrogen-bonding
sites.
- 2-octanol - tailing or reduced peak heights indicates Lewis acid sites.
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- The last eluting hydrocarbons (with k between 5 and 7) are used for the
calculation of the column plate number. Tailing of these peaks indicates dead
volume, which can be caused by: broken columns, ferrule or glass fragments in
columns, leaky connections, incomplete sweeping of the column outlet by the
make-up gas or a damaged phase. A leading peak edge indicates overload, which
can be caused by: too high a sample concentration, too low a split ratio, too low an
oven temperature and too low a carrier gas velocity.
Limits:
- As = 0.8 to 1.5
- The number of plates per meter for the peak with k between 5 and 7: the requirements
are given in Table 1 (see Chapter 7 above) according to the internal diameter and polarity
- α between adjacent peaks ≥ 1.5
- % response for active component ≥ 70%.
QUALIFICATION OF MEDIUM POLAR COLUMNS
nine compounds and an FID detector:
Chromatographic settings for narrow bore column (0.25 or 0.32 mm ID):

Carrier gas: Helium

Dilute the mixture 1:10 with methylenchloride and inject 0.2 µl onto the column in direct
injection mode.
Solutions:
Prepare a mixture of the nine compounds in solvent methylene chloride, with the
- N-decane – 0.5 mg/ml
- N-dodecane – 0.5 mg/ml
- N-tetradecane – 0.5 mg/ml
- N-tridecane – 0.5 mg/ml
- N-undecane – 0.5 mg/ml
- 2.6-dimethylphenole – 0.5 mg/ml
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Method:
After the system is equilibrated, inject the prepared mixture at least twice. Calculate the
retention factor, selectivity for the adjacent peaks and plates per meter. Calculate the
percentage of the peak height of n-alkanes by drawing a line connecting the peak maxima
of these non-adsorbing peaks and calculate the % response for active compounds using
the equation given above in the test for non-polar columns.

- The elution order is: N-decane ; 2-octanone; N-undecane; 1-octanol; N-dodecane;
2,6-dimethylphenol; N-tridecane; 2,6-dimethylaniline; N-tetradecane
- Methane – this component is used as an indicator for the hold-up time (tM), which
is required to calculate the retention factor (k) and mean linear gas velocity in the
column ( u = column length in cm/gas hold-up time in cm). Tailing of the peak
indicates dead volume in the system.
- 2, 6-dimethylphenol – tailing or reduced peak heights for this component indicates
- 1-octanol - tailing or reduced peak heights indicates silanol or hydrogen-bonding
sites. It is a very sensitive probe for deactivation problems.
- 2-octanone - tailing or reduced peak heights indicate Lewis acid sites.
calculation of the column plate number.
Limits:
- As = 0.8 to 1.5
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PA/PH/OMCL (12) 128 5R 18/20
QUALIFICATION OF POLAR COLUMNS
nine compounds and an FID detector:
Chromatographic settings for narrow-bore columns (0.25 or 0.32 mm ID):

Carrier gas: Helium

Dilute the mixture 1:10 with methylene chloride and inject 0.2 µl onto the column in
direct injection mode.
Solutions:
Prepare a mixture of the nine compounds in solvent methylene chloride, with the
- N-eicosane – 0.5 mg/ml
- N-heptadecane – 0.5 mg/ml
- N-hexadecane – 0.5 mg/ml
- N-octadecane – 0.5 mg/ml
- N-pentadecane – 0.5 mg/ml
- 2,6-dimethylphenole – 0.5 mg/ml
Method:
After the system is equilibrated, inject the prepared mixture at least twice. Calculate the
retention factor, selectivity for the adjacent peaks and plates per meter. Calculate the
percentage of the peak height of n-alkanes by drawing a line connecting the peak maxima
of these non-adsorbing peaks and calculate the % response for active compounds using
the equation given above in the test for non-polar columns.
11/07/2013
19/20 PA/PH/OMCL (12) 128 5R

- The elution order is: 2-octanone; N-pentadecane; 1-octanol; N-hexadecane; N-
heptadecane; N-octadecane; 2,6-dimethylaniline; 2,6-dimethylphenol; N-eicosane.
- Methane – this component is used as an indicator for the hold-up time (tM), which
is required to calculate the retention factor (k) and mean linear gas velocity in the
column ( u = column length in cm/gas hold-up time in cm). Tailing of the peak
indicates dead volume in the system.
- 2,6-dimethylphenol – tailing or reduced peak heights for this component indicates
- 1-octanol - tailing or reduced peak heights indicates silanol or hydrogen-bonding
sites. This is a very sensitive probe for deactivation problems.
- 2-octanone - tailing or reduced peak heights indicate Lewis acid sites.
calculation of column efficiency.
Limits:
- As = 0.8 to 1.5
11/07/2013
PA/PH/OMCL (12) 128 5R 20/20
ANNEX 2:
REQUIREMENTS FOR THE CONTENT OF A QUALIFICATION RECORD
Following the qualification, the OMCL should issue a qualification record including the
following information:
- Title and identification of the record
- Page numbers
- Date of qualification
- Reference to the procedure for qualification
- Reference material or reagents used
- Results
- Chromatographic equipment used
- Conclusion (PASS/FAIL)
- Name and signature of the person who performed the qualification
11/07/2013

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OMCL Network of the Council of Europe

QUALITY MANAGEMENT DOCUMENT

PA/PH/OMCL (12) 128 5R

QUALIFICATION OF ANALYTICAL COLUMNS

Full document title and Qualification of Analytical Columns

Document type Recommendation Document

Date of first adoption

Date of original entry July 2013

Date of entry into force

Custodian The present document was elaborated by the OMCL Network /

Concerned Network GEON

QUALIFICATION OF ANALYTICAL COLUMNS

2. AIM AND SCOPE OF THE DOCUMENT

3. CONSIDERATIONS FOR LEVEL I QUALIFICATION

At Level II of the qualification of analytical columns, it is recommended to check the

5. LEVEL III QUALIFICATION

Terms and definitions:

Height of a theoretical plate (H)

L is the length of the column [mm], and

Plate per meter

Table 1. Comparison of average efficiency by phase polarity and internal diameter.

Plate number (N) – as specified in Ph. Eur. 2.2.46

Reduced plate height (h)

H is the height of a theoretical plate, and

Relative retention (r) – as specified in Ph. Eur. 2.2.46.

Resolution (Rs ) – as specified in Ph. Eur. 2.2.46.

Retention time – as specified in Ph. Eur. 2.2.46.

Retention factor (k) – as specified in Ph. Eur. 2.2.46

Symmetry factor (As) – as specified in Ph. Eur. 2.2.46.

European Pharmacopoeia chapter 2.2.46.

A. EXAMPLES FOR QUALIFICATION OF ANALYTICAL COLUMNS FOR LC

BACKGROUND INFORMATION ON CHARACTERISTICS OF RP LC-COLUMNS

The most important properties of reverse phase models are:

Hydrophobicity – refers to the property of a stationary phase to retain the hydrophobic

QUALIFICATION OF RP-LC C-8 AND C-18 COLUMNS

Criteria for characterisation and limits:

QUALIFICATION OF RP-LC CN COLUMNS

Cyano-phases have moderate hydrophobicity compared to alkyl ligands.

Criteria for characterisation and limits:

QUALIFICATION OF RP-LC PHENYL (phenyl propyl and phenyl hexyl) COLUMNS

Criteria for characterisation and limits:

QUALIFICATION OF NP-LC COLUMNS (for Si, NH2, NO2, Diol, CN)

Temperature: room temperature

Criteria for characterisation and limits:

QUALIFICATION OF STRONG AND WEAK ION-EXCHANGE COLUMNS (SCX;

1. Qualification of SCX columns

2. Qualification of SAX columns

QUALIFICATION OF CHIRAL COLUMNS

Flow rate: 0.5 mL/min

Criteria for characterisation and limits:

- Plate number (Vitamin B12): NLT 20,000

B. EXAMPLES FOR THE QUALIFICATION OF ANALYTICAL COLUMNS

QUALIFICATION OF NON-POLAR COLUMNS

Chromatographic settings for narrow-bore columns (0.25 or 0.32 mm ID):

Chromatographic settings for wide-bore capillary columns (0.53 or 0.75 mm ID):

% response = (peak height of active component / height from baseline to curve

The % response serves as an internal standard for accurately monitoring column

Criteria for characterisation and limits:

QUALIFICATION OF MEDIUM POLAR COLUMNS