ADV01-05_1-298

• 9 •

www.restekcorp.com

Column Performance Test Mixture

✔

Five-component mix for characterizing HPLC column parameters.

✔

Simple, easy, reliable approach to QC evaluations or column classification.

by Vernon Bartlett, HPLC Innovations Manager, and Katia May, Ph.D., R&D Chemist

New Performance Test Mixtures

for Liquid Chromatography

HPLC Operational Qualification Test Mix for UV Detector 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 Check Mix

✔

For simple sugar analysis by HPLC with refractive index detection.

✔

Use with a silica column with amino functionality, as recommended by AOAC International.

✔

Use as a calibration standard for AACC Method 80-04.

✔

Offered dry, for enhanced stability.

The National Institute of Standards and Technology

(NIST) has formulated a mixture of five organic

compounds that is highly effective for characteriz-

ing HPLC columns. The primary column parameters

measured by using the mixture are: efficiency, void

volume, methylene selectivity, retentiveness, and

activity toward chelators and organic bases. Results

obtained by using the mix can be used for column

classification, column selection during method

development, for monitoring column performance

over time, or for quality control during column

manufacturing.

We follow the NIST method for producing this mix,

and we test our material against the NIST 870 stan-

dard. Figure 1 shows the chromatographic profiles

for the Restek reference material 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:

1.5 mL/min.

Temp.:

35°C

Det.:

refractive index @ 35°C

LC_0223

AOAC International recommends an HPLC method

using refractive index (RI) detection and an amino-

based stationary phase for analyses of simple sugars,

i.e., the monosaccharides 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 check mix 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 reference material

in water, lyophilize it, and pack it dry in small

(4mL) vials, for enhanced stability. To perform the

performance test, dissolve the material 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-

thesized materials, 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 linearity measurements, 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 each mixture, a linearity plot, and

coefficient of determination (r

) for the linear plot.

Contains 1mL each of these mixtures.

HPLC OQ Linearity Test Mix Kit

Caffeine at five concentrations in a five ampul kit at 5.0, 25.0,

125.0, 250.0, 500.0 µg/mL in water. Used to determine UV detector

linearity. Sold only as a kit.

Kit

31805

amitriptyline

hydrochloride 2800µg/mL

ethylbenzene

1700

quinizarin

toluene

1400

uracil

In methanol, 1mL/ampul

HPLC Performance Test Mix

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 Column Performance Check Mix

Each

31809

Figure 2

Excellent performance for simple sugars, using a

Pinnacle II

™

Amino HPLC column.

Peak List:

Conc. (µg/mL)

1. uracil

2. toluene

1400

3. ethylbenzene

1700

4. quinizarin

5. amitriptyline

2800

Sample:

HPLC Performance 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 HPLC Column Performance Test Mixture

matches NIST 870 standard.

NIST Mix

Restek

Mix

*Final concentration when reconstituted in 1mL ace-

tonitrile:water (75:25) v/v.

Sample:

Sugar Column

Performance Check Mix

(cat.# 31809)

Inj.: 5µL