ADV01-05_1-298

• 12 •

www.restekcorp.com

800-356-1688

by Terrence Reid, HPLC Applications Team Chemist

Enhanced Retention of Polar

Analytes by HPLC

Using the New

Ultra Aqueous C18 Column

✔

Excellent peak shape for basic analytes.

✔

Compatible with 100% aqueous to 100% organic mobile phases.

✔

Compatible with MS detection.

The newest addition to our selection of HPLC

columns, the Ultra Aqueous C18 column, is

designed to enhance the retention of polar com-

pounds by reversed phase HPLC. The Ultra Aqueous

C18 stationary phase is a true C18 chain (USP L1),

but it is immobilized on the silica surface through a

new

unique chemistry that creates polar groups on the

silica surface, between the C18 chains (Figure 1).

This secondary polar character has several benefits.

First, polar analytes that are insufficiently retained

on a conventional C18 column interact with the

polar groups in an Ultra Aqueous C18 column, pro-

ducing enhanced retention. Second, the polar

groups aid the retention of polar compounds by

keeping the stationary phase completely wetted,

even in 100% aqueous mobile phases. In theory,

eliminating organic solvent from the mobile phase

should maximize retention in reversed phase HPLC,

and this is true for Ultra Aqueous C18 columns. In

contrast, many conventional C18 columns lose abil-

ity to retain analytes in highly aqueous mobile phas-

es because the C18 chains self-associate or fold

down on the silica (Figure 1), a phenomenon

sometimes referred to as chain folding. Third, the

polar groups on the Ultra Aqueous C18 stationary

phase shield analytes from active silanol sites on

the silica surface, ensuring excellent peak shape for

basic analytes (Figure 2).

Although they were designed to be used with highly

aqueous mobile phases, Ultra Aqueous C18

columns also are completely compatible with highly

organic mobile phases. The ability to cover the full

range of mobile phase composition, from 100%

aqueous to 100% organic, is useful for developing

gradient methods for analyzing samples containing

Figure 1

An Ultra Aqueous C18 column is compatible

with mobile phases from 100% organic

content to 100% aqueous.

Ultra Aqueous

C18 phase is

stable in highly

organic or

highly aqueous

mobile phase

Conventional

C18 chains

collapse and

lose capacity

for retention in

highly aqueous

mobile phases

C18

chains

polar

groups

both highly polar and nonpolar analytes. For exam-

ple, Figure 3 shows water-soluble vitamins are elut-

ed from an Ultra Aqueous C18 column with excel-

lent resolution and as sharp, symmetric peaks.

Ultra Aqueous C18 columns also are compatible

with MS detection-the minimal noise generated by

an Ultra Aqueous C18 column is comparable to

background in a blank analysis with no column in

line (Figure 4). LC/MS is the analytical approach

for a steadily increasing variety of analytes, and the

versatile Ultra Aqueous C18 column is an obvious

column choice.

If you are analyzing samples containing polar ana-

lytes, or mixtures of polar and nonpolar analytes,

and are contending with unsatisfactory resolution,

insufficient retention of polar compounds, poorly

shaped peaks, and/or complicated mobile phases,

an Ultra Aqueous C18 column can be the solution

to your problems.

Peak

Conc. (mg/mL)

1. thiamin (B1)

250

2. ascorbic acid (C)

1000

3. unknown

n/a

4. nicotinic acid (B3)

1000

5. unknown

n/a

6. pantothenic acid (B5)

1000

7. folic acid (B9)

500

8. riboflavin (B2)

250

9. methyl paraben

0.2

Sample:

Analytes in water; initial dilutions of

B1 and B2 basified with ammonium

hydroxide

Column:

Ultra Aqueous C18 (cat.# 9178575 )

Dimensions:

250 x 4.6mm

Particle size:

5µm

Pore size:

100Å

Mobile Phase:

A: 25mM potassium phosphate, pH 2.00:

methanol (95:5, v/v)

B: methanol:25mM potassium phosphate,

pH 3.5 (60:40, v/v)

Time (min.)

0-6

Hold 0

6.01

Step to 25

6.01-11

25-100

11-16

Hold 100

Flow:

1.0mL/min.

Temp.:

27°C

Det.:

UV @ 254nm

LC_0141

Figure 2

Silanol-shielding polar groups on the Ultra Aqueous C18 stationary phase ensure symmetric

peaks for basic analytes, compared to other C18 phases.

Column:

Ultra Aqueous C18 (cat.# 9178565)

Dimensions:

150 x 4.6mm;

Particle Size:

5µm

Pore Size:

100Å

10 min.

Column:

Base-deactivated C18

Dimensions:

150 x 4.6mm;

Particle Size:

5µm

Pore Size:

100Å

10 min.

1. uracil

5.0µg/mL

2. pyridine

0.1µL/mL

3. phenol

1.86mg/mL

LC_0143

Sample:

5µL

Mobile Phase:

20mM potassium

phosphate pH 7.0:

acetonitrile (80:20)

Flow:

1.0mL/min.

Temp:

25°C

Det.:

UV @ 254nm

Figure 3

Excellent resolution and peak shapes for

water-soluble vitamins eluted from an Ultra

Aqueous C18 column.

5 6

0 2 4 6 8 10 12 14 min.