• 9 •
Column Performance TestMixture
✔
Five-component mix for characterizingHPLC column parameters.
✔
Simple, easy, reliable approach toQC evaluations or column classification.
by Vernon Bartlett, HPLC InnovationsManager, and KatiaMay, Ph.D., R&DChemist
NewPerformance TestMixtures
for Liquid Chromatography
HPLCOperational Qualification TestMix for UVDetector Linearity
✔
Simple test of a detector's ability to produce a linear response to varied concentrations.
✔
Concentrations suitable for clear indication of linear range.
Sugar Column Performance Qualification CheckMix
✔
For simple sugar analysis by HPLCwith refractive index detection.
✔
Usewith a silica columnwith amino functionality, as recommended by AOAC International.
✔
Use as a calibration standard for AACCMethod 80-04.
✔
Offered dry, for enhanced stability.
The National Institute of Standards and Technology
(NIST) has formulated amixture of five organic
compounds that is highly effective for characteriz-
ing HPLC columns. The primary column parameters
measured by using themixture are: efficiency, void
volume, methylene selectivity, retentiveness, and
activity toward chelators and organic bases. Results
obtained by using themix can be used for column
classification, column selection duringmethod
development, formonitoring column performance
over time, or for quality control during column
manufacturing.
We follow the NISTmethod for producing thismix,
andwe test ourmaterial against the NIST 870 stan-
dard. Figure 1 shows the chromatographic profiles
for the Restek referencematerial and the NIST 870
standard are nearly identical.
Peak List:
Conc.
(mg/mL)
1. fructose
2.0
2. glucose
2.1
3. sucrose
4.0
4.maltose
4.5
5. lactose
4.4
Column:
Pinnacle II
™
Amino
Catalog #:
9217365
Dimensions:
150 x 4.6mm
Particle size:
3µm
Pore size:
110Å
Conditions:
Mobile phase:
water:acetonitrile (25:75, v/v)
Flow:
1.5mL/min.
Temp.:
35°C
Det.:
refractive index@35°C
LC_0223
AOAC International recommends anHPLCmethod
using refractive index (RI) detection and an amino-
based stationary phase for analyses of simple sugars,
i.e., themonosaccharides fructose and glucose and
the disaccharides sucrose, maltose, and lactose.
The performance qualification test (PQ), part of
laboratory instrument validation, determines the
precision of the HPLC system. Usually the PQ test
results show the retention time variations. Our new
performance checkmix for PQ of HPLC / RI con-
sists of five simple sugars in varied concentrations.
Because sugar solutions are subject to bacteriologi-
cal degradation, we prepare the referencematerial
inwater, lyophilize it, and pack it dry in small
(4mL) vials, for enhanced stability. To perform the
performance test, dissolve thematerial in 1mL ace-
tonitrile/water (75:25, v/v).
Figure 2 shows an excellent resolution of the sim-
ple sugars, achieved by using a Pinnacle II
™
Amino
HPLC column (cat.# 9217365)—a propylamino
stationary phase bonded to 3µm silica. Because we
manufacture this new column from in-house syn-
thesizedmaterials, we have strict control of col-
umn-to-column reproducibility.
Regulatory documentation for installation qualifica-
tion (IQ), operation qualification (OQ), and per-
formance qualification (PQ) verifies the system's
performance for regulatory agencies. An important
part of OQ for HPLC instruments is detector lineari-
ty. Analyzed compounds can vary in concentration,
so it is important that the detector produce linear
responses to concentration variations. Response
should be proportional to the concentration of the
analyte at constant injection volume.
For this purpose, Restek chemists developed a new
analytical standards kit consisting of 1mL each of five
aqueous solutions of caffeine at concentrations of 5,
25, 125, 250 and 500 µg/mL. These solutions are
convenient for linearitymeasurements, and can be
used to generate simple plots of response versus
concentration. Further, the correlation coefficient
between sample concentration and response is easily
calculated. Our Certificate of Analysis includes caf-
feine concentration (5–500µg/mL), calculated vari-
ance in preparing eachmixture, a linearity plot, and
coefficient of determination (r
2
) for the linear plot.
Contains 1mL each of thesemixtures.
HPLCOQ Linearity TestMix Kit
Caffeine at five concentrations in a five ampul kit at 5.0, 25.0,
125.0, 250.0, 500.0 µg/mL inwater. Used to determine UV detector
linearity. Sold only as a kit.
Kit
31805
amitriptyline
hydrochloride 2800µg/mL
ethylbenzene
1700
quinizarin
94
toluene
1400
uracil
28
Inmethanol, 1mL/ampul
HPLCPerformance TestMix
Each
5-pk.
10-pk.
31699
31699-510
—
w/data pack
31699-500
31699-520
31799
glucose
2.0µg/mL*
fructose
2.1
lactose
4.4
maltose
4.5
sucrose
4.0
Dry components in 4mL screw-cap vial.
Sugar ColumnPerformanceCheckMix
Each
31809
Figure 2
Excellent performance for simple sugars, using a
Pinnacle II
™
AminoHPLCcolumn.
0
1
2
3
4
5
6
1
2
3
4
5
Peak List:
Conc. (µg/mL)
1. uracil
28
2. toluene
1400
3. ethylbenzene
1700
4. quinizarin
94
5. amitriptyline
2800
Sample:
HPLCPerformance Text Mix (cat.# 31699)
Inj.:
5.0µL
Sample diluent:
methanol
Column:
Pinnacle II
™
C18
Catalog #:
9214565
Dimensions:
150 x 4.6mm
Particle size:
5µm
Pore size:
110Å
Conditions:
Mobile phase:
A: 5mM potassium
phosphate, pH 7.0
B: MeOH
20%A: 80%B
Flow:
2.0mL/min.
Temp.:
23°C
Det.:
UV@ 254nm
LC_0242
LC_0244
Figure 1
Restek HPLCColumnPerformance Test Mixture
matches NIST 870 standard.
NISTMix
Restek
Mix
*Final concentration when reconstituted in 1mL ace-
tonitrile:water (75:25) v/v.
Sample:
Sugar Column
Performance CheckMix
(cat.# 31809)
Inj.: 5µL
