ADV01-05_1-298

• 8 •

www.restekcorp.com

RESTEK Advantage

800-356-1688

Analysis time for many HPLC separations can be

drastically reduced by using fast LC. In the example

separations we show here, analysis times of 20 to 40

minutes by conventional HPLC are reduced by 60 to

75%. Further, because columns employed in fast LC

analyses typically are less than 100mm long, analytes

spend less time in the column. Consequently, these

dramatically reduced analysis times are accompa-

nied by improved sensitivity, due to reduced band

spreading.

Fast LC analyses can be performed using either car-

tridge-style fast LC columns or short, conventional

design HPLC columns, typically containing 3µm sili-

ca-based packings. In addition to the performance

improvements attributable to short columns and

small packing particles, gains also can be realized by

using optimized, highly selective stationary phases.

Selective phases improve separation among sample

components with minimal changes in mobile phase

strength. Analysts who reduce mobile phase strength

By Vernon Bartlett, Innovations Team Manager, Terrence S. Reid, HPLC Applications Chemist, and

Rebecca Wittrig, Ph.D., Senior Innovations Chemist

Faster Separations and

Greater Sensitivity

Using Restek HPLC Columns

and Fast LC Cartridge Columns

✔

Save time—significant increases in sample throughput; fast re-equilibration in gradient analyses.

✔

Save money—reduced solvent consumption reduces purchase and disposal costs.

✔

Good screening technique for unknown analytes.

✔

Excellent for LC/MS applications.

Figure 2

— Separate carbamates and

re-equilibrate in 13 minutes, using a

short Ultra Carbamate column.

in attempts to improve selectivity and/or retention

often find k' is increased drastically, and analysis

time is unacceptably prolonged.

There are several precautions to observe before

using fast LC columns and methods. Critical separa-

tions are more sensitive to system volume; evaluate

tubing lengths and system component specifications

with a goal of minimizing internal volume. Also,

highly selective stationary phases can be required for

difficult separations (e.g., structural isomers)—we

recommend discussing your intended application

with our Technical Service group before ordering

columns and attempting a new analysis. Finally, fast

LC is not recommended for normal phase separa-

tions, nor for ion-pairing separations when gradients

are required.

Fast LC Separation of

Digitalis

Derivatives

Figure 1 shows an analysis of

Digitalis

extracts and

derivatives on an experimental 30mm x 4.0mm Ultra

Alkaloids Fast LC cartridge column, using a simple

water:acetonitrile mobile phase gradient. The analy-

sis is completed in 3 minutes, with excellent separa-

tion of the sample components. This is dramatically

reduced analysis time, relative to the cumbersome

analysis on a 30cm C18 column. The fast LC

approach, using this specialized stationary phase, can

be applied to purification and analysis of digoxin-

labeled materials in investigations of biological activi-

ty, and is perfect for high-speed cleaning validations.

Fast LC Separation of Carbamates

Figures 2 and 3 (page 9) illustrate fast LC analyses

of carbamate pesticides by HPLC/UV and LC/MS,

respectively. Total time for the HPLC/UV analysis is

approximately 13 minutes; the LC/MS analysis is

slightly longer, but is less than 20 minutes. Table I

(page 9) summarizes conditions for the LC/MS

analysis. Including re-equilibration time, the conven-

tional analysis on a 250mm x 4.6mm column takes

40 minutes. Designed especially for carbamates

analyses, Ultra Carbamate columns are available in

several dimensions in addition to the 50mm x

4.6mm and 100mm x 4.6mm columns used here.

Fast LC Analyses of Vanillin and Vanilla Extract

Figure 4 (page 9) shows fast LC analyses of

vanillin/ethyl vanillin and vanilla bean extract on a

50mm x 4.0mm Pinnacle™ DB C18 column.

Excellent separations are achieved in less than 5

minutes. Conventional analyses on 150mm x 4.6mm

C8 columns take 25 minutes overall with a mobile

phase gradient, or more than 40 minutes with an

isocratic mobile phase.

In Summary

In diverse applications, fast LC separations enhance

laboratory throughput, reduce solvent waste, and

improve method sensitivity. In some cases, mobile

phase requirements can be simplified, from gradient

elution to isocratic elution, when an optimized sta-

tionary phase is used, dramatically reducing analysis

time.

Column:

Ultra Carbamate

Catalog #:

9177355

Dimensions:

50 x 4.6mm

Particle size:

3µm

Pore size:

100Å

Conditions:

Mobile Phase:

A: 90:10 water:methanol

B: 90:10 methanol:acetonitrile

Time (min):

Flow:

1.5mL/min

Temp.:

27°C

Det.:

UV @ 220nm

LC_0225

Figure 1

—

Digitalis

derivatives separated

in 3 minutes on a fast LC cartridge.

LC_0250

Peak List:

Conc.

Ret. Time (min.)

1. digoxigenin 100µg/mL 0.40

2. gitoxigenin 100µg/mL 0.80

3. digoxin

100µg/mL 1.10

4. gitoxin

~10µg/mL 2.20

5. digitoxin

100µg/mL 2.60

Sample:

Inj.:

10µL

Sample Diluent:

water:acetonitrile (80:20 v/v)

Column:

Ultra Alkaloids Fast LC Cartridge

Catalog #:

custom column

Dimensions:

30 x 4.0mm

Particle Size:

3µm

Pore Size:

100Å

Conditions:

Mobile Phase:

A: water

B: acetonitrile

Time

(min.) %B

0.0

1.5

1.51 35

3.0

3.1

Flow:

2.0mL/min

Temp.:

27°C

Det.:

UV @ 230nm

Peak List:

1. aldicarb sulfone

2. aldicarb sulfoxide

3. oxamyl

4. methomyl

5. 3-hydroxycarbofuran

6. aldicarb

7. propoxur

8. carbofuran

9. carbaryl

10. methiocarb

11. 4-bromo-3,5-dimethylcarbamate

Sample:

Inj.:

5µL

Conc.:

50µg/mL

Solvent:

methanol

Restek standards:

cat.# 32274 and 32273 mixed 50:50