Small Particle Columns for Faster High Performance … Dorothy J. Phillips, Mark Caparella, Zoubair El Fallah, and Uwe D. Neue Table of Contents Small Particle Columns.....1-7 Sample

1

Dorothy J. Phillips, Mark Caparella, Zoubair El Fallah, and Uwe D. Neue

Table of Contents

Small Particle Columns .........................1-7

Sample Dilution IncreasesSensitivity and Resolution....................8-11

Electrophoretic Mobilities..................12-19

Literature Corner..............................20-21

The Choice is Yours ..............................21

What’s New........................................22

How to Reach us ..................................22

Application Notes...............................23-26

EPA Method 604: PhenolsRapid Analysis on Symmetry®

3.5µm Column

Editor: Uwe Neue

ISSN # 1084-0540

AbstractThe customer need for fast and efficient

separations on complex samples such

as pharmaceuticals and biologicals led

Waters to develop fast, efficient, and

reproducible Symmetry® 3.5 µm

columns. Separations using 3.5 µm

columns are 30 to 50 % faster at equal

plate-count compared to 5 µm columns.

For many analytical methods, switching

to Symmetry® 3.5 µm columns can save

time and reduce costs.

Separation methodologies using

Symmetry® 5 µm columns are easily

modified to accommodate Symmetry®

3.5 µm columns because efficiency,

resolution and sensitivity remain the

same. It is shown that Symmetry®

3.5 µm columns have column lifetimes

comparable to Symmetry® 5 µm

columns and are more stable than other

3 and 3.5 µm columns.

IntroductionHigh-performance liquid chromatogra-

phy (HPLC) columns packed with 3 µm

packing materials were first commercial

ised in the early 1980s (1, 2, 3). Until

recently, however, HPLC system com-

patibility and column lifetime issues

have precluded the widespread use

of the 3 µm columns. Relative to the

traditional 5 µm analytical packing

materials, the 3 µm required a shorter

column to compensate for increased

backpressure. The smaller column

volume of the shorter columns resulted

in an increase of the influence of

extra column effects, which negated

the separation efficiency improvements

expected from the smaller particle size.

Volume VIIssue 2, 1996

Scanning Electron Micrographs Symmetry® Silica 5 µm

Small Particle Columns for Faster HighPerformance Liquid Chromatography

2

The extra column effects were a direct

result of the HPLC system’s incompati-

bility with small column volumes.

Columns made with 3 µm packing

materials also had shorter lifetimes due

to plugging of the frits. The smaller pore

diameter frits are more prone to plug-

ging by particulates that are either pre-

sent in the sample or arise from abrasion

of system seals.

This paper compares the separation

efficiency of the Symmetry® 3.5 µm

column (4.6 x 100 mm) with the

Symmetry® 5 µm column

(3.9 x 150 mm and 4.6 x 150 mm).

Chromatographic data on several phar-

maceutical compounds show the speed

advantage of the Symmetry® 3.5 µm

column for fast chromatography. Column

lifetimes for Symmetry® columns are

compared with Merck Supersher® 100

RP-18 and Zorbax® SB C18 column.

ExperimentalSulphanilamide, sulphadiazine,

sulphathiazole, sulphamerazine,

sulphamethazine, succinylsulphathia-

zole, beclomethasone, paclitaxel

(taxol), and tamoxifen were from

Sigma Chemical Corporation

(St. Louis, MO). Testosterone propionate,

testosterone benzoate and testosterone

acetate were from U.S. Pharmacopeia

Reference Standards (Rockville, MD).

Butalbital and butabarbital were from

Alltech (Deerfield, IL). Rabbit serum was

from Equitech-Bio (Ingram, TX). Buffers

and mobile phases were made with

reagent grade chemicals and HPLC

grade solvents. Mobile phases were

filtered and degassed.

Symmetry® C18 and C8 columns

(3.9 x 150 mm – 5µm; 4.6 x 150 mm

– 5µm; 4.6 x 100 mm – 3.5 µm)

and Symmetry Sentry™ guard columns

were from Waters (Milford, MA).

Merck Superspher 100 RP-18

(4 mm x 125 mm) was purchased from

EM Separations (Gibbstown, NJ)

Zorbax SB C18 (4.6 x 75 mm, 3.5 µm)

was obtained from MacMod (Chadds

Ford, PA).

HPLC SystemsThe HPLC system used in this study con-

sisted of either a Waters 625 or a 616

LC System, 717plus Autosampler and

the 996 Photodiode Array Detector.

Stability studies with biological samples

were done on the Waters

Figure 1: Plate Count N versus Flow Rate for 5 µm and 3.5 µm Columns

600E Multisolvent Delivery System,

715 Ultra WISP Sample Processor and

490 Programmable Multiwavelength

detector. Control and results manage-

ment were provided by the Millennium®

2010 Chromatography Manager.

The components of the HPLC system for

column lifetime studies with the standard

sulpha drug mixture consisted of a

Waters Model 712 WISP,™590

Programmable Solvent Delivery Module,

and 441 Fixed Wavelength UV/Visible

detector. The Waters 845

Chromatography Data and Control

Station with Expert-Ease™ v.3.0 was

used for system control and data

acquisition.

Stability StudiesStability studies with sulpha drugs and

rabbit serum were done with precolumn

filters between the pumps and the injec-

tors. The temperature was controlled at

25 °C on each HPLC system using a

EuroMark™ Spark Holland Mistral®

Column Thermostat.

The column stability study using 10 µL

of the sulpha drug mixture (10 to

39 mg/mL) was injected on a

Symmetry® C8 Sentry guard column

(3.9 x 20 mm, 5 µm) in line with a

Symmetry® C8 column (4.6 x 100 mm,

3.5 µm). The mobile phase was water/

methanol/ glacial acetic acid 79:20:1

and was recycled during the course

of the experiment. The flow rate was

1.5 mL/min and the detection wave-

length was 254 nm.

Rabbit serum was deproteinated by

mixing two parts acetonitrile to one part

serum. The sample was then centrifuged

at 1500 g for 5 minutes and spiked

with 5 µg/mL of butalbital and

5 µg/mL of butabarbital. The mobile

phase was 0.1 M potassium phos-

phate, pH 6.9/ acetonitrile/ water,

20:30:50 v/v. In this study, the mobile

phase was not recycled. The injection

volume was 15 µL and the mobile

phase flow rate was 1.4 mL/min

(1.25 mL/min for the Superspher

column). The detection wavelength

was 214 nm.

Results and DiscussionEfficiency TheoryThe objective of the Symmetry® 3.5 µm

column is to offer fast separations with-

out sacrificing chromatographic resolu-

tion. Resolution is a function of: the

selectivity, α, which is a measure of the

relative retention of two components in

a mixture, the capacity factor, k, which

is a measure of how well the analyte is

retained and the efficiency, N,

expressed in theoretical plates. Equation

1 shows the dependence of resolution

on α, k and N.

Rs = 1/4 N 1/2 (α-1) [k/(k+1)]

(Equation 1)

The same efficiency can be obtained in

different ways, one can either use long

columns with larger particles or short

N

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0 1 2 3 4 5

F [mL/min]

150 mm 3.5 µm

150 mm 5 µm

100 mm 3.5 µm

75 mm 3.5 µm

3

columns packed with smaller particles.

The key to faster separations is to use

shorter columns packed with smaller

particles. In this manner, the increase in

efficiency due to the smaller particle

size compensates for the loss in plate

count due to a shorter column length.

Figure 1 shows a plot of the theoretical

plate count, N vs. flow rate for 5 µm

and 3.5 µm Symmetry® columns and

illustrates the following points:

• The 3.5 µm,150 mm column gives

a higher efficiency than the 5 µm

column of the same length, but is

limited in flow rate due to the high

column backpressure.

• The short 75 mm column packed

with 3.5 µm particles yields very fast

separations with still a good plate-

count.

• The 100 mm, 3.5 µm columns and

the 150 mm, 5 µm columns give

similar efficiencies at a given flow

rate, which translates into a faster

analysis time due to the decreased

column length.

In order for the theoretical results to hold

true experimentally, the columns need to

be packed to the same quality, for

example, to the same reduced plate

height (h). Figure 2 shows the plot of

reduced plate height versus linear veloc-

ity for the Symmetry® C18 5 µm column

and the Symmetry® C8 3.5 µm column.

The curves overlap, showing that the

columns are packed equally well. Also,

the reduced plate height of 2.4

obtained at the minimum is excellent.

It should be noted that the minimum of

the HETP occurs at different flow rates

depending on the mobile phase viscosi-

ty. For example, the minimum is found at

1.8 mL/min for 50:50 acetonitrile /

water, whereas for 50:50 methanol /

water, a more viscous mobile phase, the

minimum occurs at 0.9 mL min.

Efficiency and SpeedFigure 3 illustrates the similarity in

efficiency between 5 µm, 150 mm

columns and 3.5 µm, 100 mm columns.

Chromatograms for beclomethasone

and its internal standard testosterone

propionate using two Symmetry® C18

columns: a. 3.9 x 150 mm, 5 µm and

b. 4.6 x 100 mm, 3.5 µm are shown.

Both columns have a resolution of 18

which meets the U.S. Pharmacopeia

Figure 3: Beclomethasone USP Method: Faster Analysis with Symmetry® 3.5 µm Column

assay (4) specification (Rs ≥ 5). Both

columns have similar plate counts of

approximately 8000 and similar back-

pressures under the chromatographic

conditions chosen. The analysis time for

the 3.5 µm column (b) is about 50%

lower compared to the 5 µm column

(a). The reduction in analysis time clear-

ly illustrates the point of fast chromatog-

raphy without a sacrifice of separation

efficiency. Many analytical methods

could become more cost effective by

merely changing particle size from

5 µm to 3.5 µm.

1

2

1

0 5 10

0 5Minutes

Minutes

10

2

For your free copy of the Symmetry®

Applications Notebook, please checkbox 3 on the Business Reply Cardand return today.

1000

1

2

3

4

5

6

7

8

20

linear velocity, u, mm/sec

30 40 50

h 5µm C18

3.5µm C8

Curve-Fit

Figure 2: Reduced Plate Height

Column: Symmetry ® C18

a. 5 µm 3.9 x 150 mmb. 3.5 µm 4.6 x 100 mm

Mobile phase:Acetonitrile/Water (65:35)Flow Rate: a. 1.0 mL/min.

b. 1.4 mL/min

Detection: UV at 254 nmInjection volume: 15 µLSample: 0.1 mg/mL in methanol of each

1.beclomethasone, dipropionate and 2. testosterone propionate (internal standard, I.S.)

4

Figure 4: Tamoxifen: Comparison of Symmetry® 5 µm versus 3.5 µm Columns

Figure 5: Comparison of Taxol Impurities Profile on Symmetry® 3.5 µm and 5 µm Columns

b: Symmetry C18 3.5 µm (4.6 x 100mm)

a: Symmetry C18 5 µm (3.9 x 150mm)

1

2

1

2

0.004

0.002

0.000

0.004

0.002

0.000

10.00 20.00

10.00 20.00

Minutes

Minutes

AU

AU

a: 5 µm 3.9 x 150 mm

b: 5 µm 3.9 x 150 mm

0 5 10

0

.01

.01

0

15 20

0 5 10 15 20

AU

AU

Minutes

Minutes

4 6

9

4 69

Performance equivalence between

3.5 µm and 5 µm packing is further

illustrated in Figures 4 and 5 where

impurity profiles of pharmaceutical com-

pounds are shown. Figure 4 and Figure

5 show the assays of tamoxifen and

taxol, respectively, on a Symmetry® C18

(3.9 x 150 mm, 3.5 µm) column and

a Symmetry® C18 (4.6 x 100, 5 µm)

column. In Figure 4, both columns

resolve the impurity peaks. However,

the 3.5 µm column separation is

markedly faster. Also, a calculation of

the plate counts for the impurity peaks

actually shows a slight improvement in

efficiency for the shorter 3.5 µm

column.

Essentially the same observations were

made with the taxol assay (Figure 5).

The shorter 3.5 µm column gave a

much shorter analysis time combined

with a slight improvement in plate count

and resolution. A plate count of 8900

was measured for the taxol peak on the

100 mm, 3.5 µm column, while a plate

count of 7100 was obtained on the

150 mm, 5 µm column. This is an

added benefit, since this increased

resolution provides an additional buffer

against column deterioration.

The 3.5 µm columns can be operated

at even faster flow rates to further

reduce analysis times. Figure 6 shows

the separation of testosterone at 1.4

mL/min and 2.8 mL/min. The separa-

tion efficiency at 2.8 mL/min is still

6800 plates (compared to 7500 plates

at the lower flow rate) and the run time

is reduced by half. This is possible due

to the use of acetonitrile as the organic

modifier in the mobile phase.

Acetonitrile-water mixtures have a much

lower viscosity than methanol-water

mixtures, resulting in a much reduced

backpressure. For still faster analysis or

to accommodate highly aqueous mobile

phases within normal pressure limits,

the 3.5 µm packing is also available in

75 mm columns.

Mobile Phase: 50 mM potassium phosphate, pH 3/acetonitrile 60:40

Flow Rates a. 0.7 mL/minb. 1.05 mL/min

Detector: UV at 240 nmSample: 4 µL of 5 mg/mL

Columns: Symmetry® C8

Mobile Phase: 20 mM ammonium acetate, pH 5/acetonitrile/methanol 50:40:10

Flow Rates a. 1.0 mL/minb. 1.4 mL/min

Detector: UV at 230 nmSample: taxol at 100 µg/mLInjection Volume: 25 µl injected

5

Figure 6: Faster Testosterones Analysis on Symmetry® 3.5 µm ColumnColumn LifetimeFor column lifetime studies, a Symmetry®

C8 column (4.6 x 100 mm, 3.5 µm)

was used with Sentry guard columns

containing 5 µm C8 packing material.

The guard column was replaced every

time the backpressure increased or reso-

lution decreased. The sample was a

mixture of sulpha drug standards. The

results are shown in Figures 7 and 8.

In Figure 7 the plate count of one of the

analytes is plotted against the number of

injections. In Figure 8 the system back-

pressure is plotted. As one can see,

neither a significant decline in efficiency

nor a significant increase in backpres-

sure was observed over10,000 injec-

tions. This demonstrates that excellent

column life can be achieved with

Symmetry® 3.5 µm columns, when they

are protected by Sentry guard columns.

This result parallels the results of a

previous study using Symmetry® 5 µm

packing.

In another lifetime study, deproteinated

rabbit serum spiked with butalbital and

buta was injected 1000 times on a

Symmetry® C18 3.5 µm column, a 3 µm

Merck Superspher 100 RP-18 column

and a 3.5 µm Zorbax SB C18 column.

Figure 9 is a comparison between

theoretical plates versus number of

injections for the Symmetry® and the

Supersher column. The Superspher col-

umn had a 25 % loss in efficiency

compared to a ≤ 5 % decrease for the

Symmetry® column. As shown in Figure

10, the Zorbax column did not reach

1000 injections due to a sharp increase

in backpressure ( < 3500 psi).

Figure 7: Efficiency of Succinyl Sulphathiazole Versus Number of Injections

1

2

3

1

2

3

0 2 4 6 8Minutes

0 2 4 6 8Minutes

a.

b.

11000

9000

7000

5000

20000 4000 6000 8000 10000

Number of Injections

UPS

Pla

tes

Column: Symmetry® C18 3.5 µm (4.6 x 100mm)Mobile Phase: Acetonitrile/Water (70:30)Flow Rates a. 2.8 mL/min

b. 1.4 mL/minDetector: UV at 254 nmInjection volume: 10µLSample: 0.1 mg/mL in methanol of each

1. testosterone acetate2. testosterone propionate3. testosterone benzoate

For more Information on

Symmetry® 3.5 µm columns, please check box 4 on the Business Reply Card and return today.Symmetry® 5 µm columns, please check box 5 on the Business Reply Card and return today.SymmetryPrep™ 7 µm columns, please check box 6 on the Business Reply Card and return today.

6

Compared to other 3 µm columns,

Symmetry® has a larger mean particle

size (3.5 µm). Also, the particle size dis-

tribution about the mean is very narrow

(90 % / 10 % volume ratio of 1.5). The

increase in particle size and the narrow

distribution is the likely cause of the

increase in the lifetime of the Symmetry®

3.5 µm columns.

ConclusionsThe Symmetry® 3.5 µm columns demon-

strate 30 to 50 % faster analysis times

over Symmetry® 5 µm columns.

Separation methodologies using

Symmetry® 5 µm columns can be

easily translated into methods using

Symmetry® 3.5 µm columns since the

efficiency, resolution and sensitivity

remain the same. Also, unlike other

3 µm columns, Waters Symmetry®

3.5 µm columns are as stable as their

5 µm counterparts.

References:

1. L. R. Snyder, J. L. Glajch and J. J. Kirkland, PracticalHPLC Method Development”, John Wiley & Sons, 1988,pp. 46-50.

2. R. W. Stout, J. J. DeStefano and L. R. Snyder, “Larger-Diameter Small-Particle Columns for Fast High-PerformanceLiquid Chromatographic Separations with ConventionalEquipment”, J. Chromatog. 261 (1983)189-212.

3. L. R.. Snyder and P. E. Antle, “Selecting Columns forReversed-Phase HPLC Part II: Column and PackingConfiguration”, LC Mag. 3 (1985) 98.

4. U.S. Pharmacopeia, Volume 23, 1995, page 167

Figure 9: Butalbital Efficiency Versus Number of Injections

Figure 10: Backpressure Versus Number of Injections

2600

2400

2200

2000

18000 200 400 600 800 1000


UPS

Pla

tes

Symmetry

Superspher

3600

3200

2800

2400

2000

16000 200 400 600 800 1000


UPS

Pla

tes

Symmetry

ZORBAX

Figure 8: Backpressure Versus Number of Injection: Sulpha Drugs

3700

3200

2700

220020000 4000 6000 8000 10000

Number of Injectionsps

i


Millennium® ChromatographyManager, please check box 7on the Business Reply Cardand return today.

7

Dimension 1.0 mm x 150 mm 2.1 mm x 50 mm 4.6 mm x 50 mm 4.6 mm x 75 mm 4.6 mm x 100 mm 4.6 mm x 150 mmC8 WAT248059 WAT200650 WAT200625 WAT066224 WAT066220 WAT200632

C18 WAT248072 WAT200624 WAT200620 WAT066200 WAT066204 WAT200630

Dimension 2.1 mm x 150 mm 3.0 mm x 150 mm 3.9 mm x 150 mm 4.6 mm x 150 mm 4.6 mm x 250 mmC8 WAT056955 WAT054230 WAT046970 WAT045995 WAT054270

C18 WAT056975 WAT054200 WAT046980 WAT045905 WAT054275

Dimension 3.9 mm x 150 mm 4.6 mm x 150 mm 4.6 mm x 250 mmC8 WAT054440 WAT054442 WAT054444

C18 WAT054452 WAT054454 WAT054456

Dimension 3.0mm x 150 mm 3.9 mm x 150 mm 4.6 mm x 150 mm 4.6 mm x 250 mmC8 WAT054434 WAT046955 WAT054435 WAT054438

C18 WAT054446 WAT047210 WAT054448 WAT054450

Dimension 3.9 mm x 50 mm 3.9 mm x 150 mm 4.6 mm x 150 mm 4.6 mm x 250 mmC8 WAT054240 WAT054235 WAT054255 WAT054245

C18 WAT054220 WAT054205 WAT054210 WAT054215

Ordering Information

Symmetry® Analytical Steel Columns

Symmetry® Steel Cartridge Columns (All cartridge columns require reusable endfittings.)

Symmetry® C8 5 µm 2/pkg 3.9 mm x 20 mm WAT054250

Symmetry® C18 5 µm 2/pkg 3.9 mm x 20 mm WAT054225

Integrated Guard Holder

(for Waters steel cartridge columns** only) WAT046905

Universal Guard Holder (for any HPLC column) WAT046910

Sentry™ Guard Columns* and Guard Holders

Symmetry® Validation Kit (three steel columns or steel cartridges from 3 different batches of packing materials)

Particle Size Quantity Dimensions Part No.

End Connector Kit (includes 2 sets of reusable endfittings, 2 C-Clips and Kalrez o-rings) WAT037525

Part No.

* Sentry guard columns can be used with either the steel cartridge columns but require the appropriate guard holder.

** For Waters 3.9 mm x 50 mm cartridge column, you must use the Universal Guard Holder.

For more Information onSymmetry® columns, pleaserefer to box numbers 4, 5,6, on the Business ReplyCard. To receive informationsimply check the appropriatebox number.

Symmetry 5 µm Columns

Symmetry 5 µm Cartridge Columns

Symmetry 5 µm Columns

Symmetry 5 µm Cartridge Columns

Symmetry 3.5 µm Columns

Dimension 4.6 mm x 75 mm 4.6 mm x 100 mmC8 WAT066260 WAT066265

C18 WAT066210 WAT066215

Symmetry 3.5 µm Cartridge Columns

8

Introduction and OverviewMarginal or insufficient sensitivity is

not uncommon in chromatographic

analysis. It may be encountered in the

analysis of the metabolic fate of a drug

or in the investigation of environmental

pollutants. In this paper we look at a

specific set of circumstances where low

sensitivity can be improved through a

simple operation.

The principle that we employed is the

use of the analytical column as an

enrichment column. Sample enrichment

permits a significant increase in the

injection volume which results in a pro-

portional increase in sensitivity. A very

specific example is used to demonstrate

the principle. But the general principle

is useful in many other circumstances

as well.

In this example, the analytical method

is an isocratic reversed-phase method.

The sample is subjected to a solid

phase extraction step prior to chromato-

graphic analysis for sample cleanup

and enrichment. A typical instance is

a metabolite assay from a biological

matrix like serum or urine. In this case,

the solid-phase extraction step fre-

quently is a reversed-phase method as

well, using a Waters Sep-Pak® cartridge

with the same or similar chemistry as

the analytical column. In a commonly

followed procedure, the reversed-phase

Sep-Pak cartridge is first conditioned

and the sample is then loaded onto the

cartridge. Then the adsorbed sample is

washed with a polar eluent, typically

water or buffer containing a small

amount of organic solvent.

Subsequently, the sample is eluted from

the solid-phase extraction cartridge with

an organic solvent such as methanol.

The methanol extract is then evaporated

to dryness, and the sample is redis-

solved in mobile phase for HPLC analy-

sis. This sequence of steps usually results

in an enrichment of the sample as well

as a partial sample clean-up.

Sample Dilution Increases Sensitivity and ResolutionUwe D. Neue and Ed Serowik

A significant disadvantage of this proce-

dure is the fact that a fairly broad spec-

trum of hydrophobicity lies between the

sample washing step and the elution

step. Sample constituents with a larger

hydrophobicity than the metabolites are

eluted from the Sep-Pak cartridge

together with the analytes. They are

retained on the analytical column more

strongly than the analytes of interest.

Consequently, they will appear either

late in the analytical chromatogram,

resulting in excessively long analysis

times, or they may even interfere with

subsequent analyses. The latter case

would necessitate a washing step after

each chromatogram, which together

with the accompanying reequilibration

of the column significantly increases

the run time for an assay and reduces

productivity.

A better approach is an elution proce-

dure, in which a solvent is used that is

just strong enough to elute the metabo-

lites efficiently, but leaves compounds

of higher hydrophobicity on the Sep-Pak

cartridge. If the bonded-phase used for

sample preparation is the same as or

similar to the bonded phase in the ana-

lytical column, a good elution solvent

would be the chromatographic mobile

phase with the amount of organic modi-

fier increased by about 20 %. Therefore,

if the mobile phase for the analytical

column is 30 % methanol / 70 % buffer,

the elution solvent for the Sep-Pak

cartridge should be 50 % methanol /

50 % buffer.

However, using mixtures of organic sol-

vent and water or buffer as the elution

solvent has one major disadvantage: it

is very difficult and time-consuming to

evaporate aqueous mixtures to dryness

for reconstitution of the sample. If on

the other hand, the sample is injected

without this step, there is, a limitation

amount that can be injected onto the

column without loss of resolution. To

solve this dilemma, the method of

simply diluting the sample with water or

buffer, and injecting a larger sample

volume was employed. To use a dilution

step to increase sensitivity is counterintu-

itive, but the increased sample volume

that can be injected overcompensates

for the dilution.

ExperimentalWe used ethyl- and propylparaben at

a concentration of 0.2 µg/mL as our

model system. The column used was a

Symmetry® C18 5 µm 3.9 mm x

150 mm column. The mobile phase

consisted of 35 % acetonitrile and 65 %

water, resulting in an elution time of

about 5 minutes for ethylparaben and

about 10 minutes for propylparaben.

The sample was dissolved in 55 % ace-

tonitrile, 45 % water to mimic the elution

conditions from a C18 Sep-Pak cartridge

needed for a quick and complete elu-

tion of the analytes. For the enrichment

experiments, the sample was diluted

with water in ratios of 1:1, 2:1, 4:1,

5:1, 6:1, 7:1, 11:1, and 15:1. The

amount injected was varied in propor-

tion to the dilution such that a constant

mass of sample was injected.

DiscussionIn order to maximise sensitivity under

fixed mobile phase conditions, we

would like to inject the largest mass of

sample onto the column that is possible.

Ideally, the entire sample should be

injected (although there may be cases

where one chooses to retain a portion

of the sample for repeat analysis). The

factor that limits how much can be

injected is the loss of resolution due to

volume overload. Sometimes, we have

enough sample available that this limit is

encountered using a column with a stan-

dard diameter. However, in most cases

the sample amount is limited, and a col-

umn with a smaller diameter should be

considered.

An increase in injection volume leads to

a broadening of the peak and to a loss

in resolution. Let us first consider the

9

case in which the sample is dissolved in

mobile phase. Table 1 shows the injec-

tion volume as a function of the retention

factor k for different column dimensions

that result in either a 5 % or 10 % loss

in resolution.

If we are willing to accept about 10 %

loss in resolution between peaks, we

can inject about 50 % of the peak-vol-

ume on the column. If we are willing to

accept only a 5 % loss in resolution, we

are still able to inject about 33 % of the

peak-volume on the column. Therefore,

typical tolerable injection volumes for a

3.9 mm x 150 mm, 5 µm column

would be between 40 and 160 µL,

depending on the retention factor of the

sample and the resolution requirement.

The situation actually gets worse in the

case when the sample is dissolved in a

solvent that has a higher elution strength

than the mobile phase. In Figure 1 we

plot the % resolution versus injection vol-

ume for the paraben sample dissolved

in 55 % acetonitrile with a mobile

phase of 35 % acetonitrile for a

3.9 mm x 150 mm Symmetry® C18

column. Already at an injection volume

of only 25 µL, the resolution has

dropped by about 15 %. For a 50 µL

injection, the resolution is decreased

by 35 %.

However, the situation changes a great

deal if the sample is injected in a sol-

vent that has a higher water content

than the mobile phase. We diluted the

sample 5:1 with water, resulting in a

water content of 90 %. At this dilution,

we were able to inject 1200 µL of the

sample without a loss in resolution

(Figure 2). The mass of sample injected

was the same as in the 200 µL of undi-

luted sample, which had resulted in a

fourfold loss of resolution. Also, due to

the fact that the peak-volume was small-

er than in the former case, the height of

the signal was larger by nearly a factor

of 4. As can be seen in Figure 2, the

peaks obtained at the larger injection

volume are delayed compared to the

peaks at the small injection volume. This

is due to the injection volume itself. The

1.2 mL of sample is loaded onto the top

of the column first, and peak migration

will not start until the loading step is

complete.

k’ 2.1 3 3.9 4.6 2.1 3 3.9 4.62 12 24 40 56 18 36 62 863 16 32 54 76 24 48 82 1145 24 48 82 114 36 72 122 1707 32 64 108 152 48 96 164 228

10 44 88 150 208 66 132 224 312

Table 1: Maximum Injection Volume [µL] for a 5 % or 10 % Loss in Resolution for 5 µm, 150 mm Columns

5 % 10 %Column I. D. [mm] Column I. D. [mm]

Figure 1: Relative Resolution as a Function of the Injection Volume

The experience of thousands of other scientists atyour fingertips. For more information about Solid Phase Extraction Applications Guide andBibliography, please check box 8 on the Business Reply Card.

1

0.8

0.6

0.4

0.2

00 50

Injection Volume [µL]

100 150 200

Rela

tive

Reso

lutio

n

This method is based on the interplay

between the dilution of the sample by

water and the enrichment of the sample

on the surface of the packing due to the

increased water content of the sample

solvent. The enrichment factor increases

exponentially with the water content of

the sample. For small molecules the

enrichment increases by a factor of

roughly 2.5 to 3 per 10% increase in

water content. Therefore, as long as a

2:1 dilution results in a change in water

content that exceeds 10 %, a gain in

the amount injectable onto the column

for a tolerable loss in resolution is

realised. Since this is only a rule of

thumb, experimentation is required to

determine the actual optimal dilution.

10

Figure 2: Overlay of a 200 µL Injection of the Original Sample and a 1200 µL Injection afterDilution

Figure 3: Resolution versus Injected Amount for Different Dilutions

Figure 3 shows the results of some dilu-

tion experiments. Resolution is plotted

against the amount of sample injected.

In order to inject a constant amount of

sample, the injection volume was

increased in proportion to the dilution.

For example, while a 40 ng injection

corresponded to 200 µL of undiluted

sample, it corresponded to 1600 µL of

1:7 diluted sample. One can see that

for the 40 ng injection, a dilution of

about 1:5 to 1:6 gave optimal results,

while for the 20 ng sample any dilution

between 1:2 and 1:7 gave equivalent

results.

There is an optimal dilution factor that

maximises the amount of sample that

can be injected without peak distortion.

It can be modeled mathematically, if the

relationship of the retention factor with

solvent composition is known. However,

one can assume some typical values

and use this model as a guide. In our

model, we assumed that the sample has

been eluted from the Sep-Pak cartridge

using an eluent that contains 20 % more

organic solvent than the mobile phase.

We then calculated the optimal dilution

factor as a function of the mobile phase

composition. The enrichment factor is

the ratio of the sample volume that can

be injected after dilution compared to

the sample volume that can be injected

if the sample is dissolved in mobile

phase. Table 2 shows the results of the

calculation for different mobile phase

compositions. The lower values are

more typical when methanol is used as

organic modifier. The higher values are

more typical for acetonitrile. It is clear

that the dilution method works best for

mobile phases containing large amounts

of organic solvents.

00

20

40

60

80

100

%

4 6 8 10Minutes

14

12

10

8

6

4

2

00 5 10 15 20

Amount [ng]

25 30 35 40

No Dilution1:1 Dilution1:2 Dilution1:5 Dilution1:6 Dilution1:7 Dilution

Reso

lutio

n

Waters Applications Notes

1.5 AU

02

1

2 3

4

5

6

7

8

9

10

11

12

46

810 12 14 16 18 20

Abs

orba

nce

230

nm

Minutes

Rapid Polyolefin Additive Separation Using Nova-Pak ® C18

Objective:The objective of this application note is to

demonstrate the utility of reversed-phase

HPLC for large scale purifica-tions of DMT

protected synthetic oligonucleotides.Details:Synthetic oligonucleotides are success-fully

used in a wide variety of applica-tions

ranging from use as hybridization probes to

utilization as primers for DNA sequencing

and the polymerase chain reaction* (PCR).

Most recently, studies have shown the

effectiveness of using standard (i.e. phosphodiester, P=0) or phosphole back-

bone modified (i.e. phosphoro-thioated,

P=S) oligonucleotides to selectively inhibit

the production of targeted proteins within

cells. As such, “antisense and related

technology investigations” require milligram

to multigram amounts of purified synthetic

DNA. Reversed-phase HPLC provides a

rapid and cost effective technique for the

isolation of either phosphodiester or phosphorothioated full length

oligonucleotide products for these applications.

Column: Bondapak® HC18 HA (8 x 100mm

Radial-Pak™ cartridge)Eluent A: 100mM ammonium acetate, pH

7.2/methanol (95:5, v/v)

Eluent B: Methanal/Milli-Q® water (95:5, v/v)

Sample: Phosphorothioated 21 mer (DMT

protected)Injection: 12 mLs, containing 624 O.D.260nm

units (21 mgs) of oligonucleotide

in 70% A and 30% B, injected into

Milligram quantities of DMT protected synthetic oligonucleotides can be rapidly purified from undesired synthesis reaction by-products using Bondapak

HC18 HA reversed-phase material in an 8 x 100mm Radial-Pak 0

10

20

1

2

3

4

56

78

9

10

1112

13

14

15

16

17 18

30

Minutes

140

1 = AMQ2 = Asp3 = Ser4 = Glu5 = Gly6 = His

7 = NH38 = Arg9 = Thr10 = Ala11 = Pro12 = Tyr

13 = Val14 = Met15 = Lys16 = IIe17 = Leu18 = Phe

Fluo

resc

ence

Res

pons

em

V

Analysis of Hydrolyzed Bovine Serum Albumin Using Waters

AccQ•Tag™ Method for Amino Acid Analysis

Objective:The objective of this application note is to





























HC18 HA reversed-phase material in an 8 x 100mm Radial-Pak cartridge. This same packing is available in a variety of column dimension for gram scale

purifications as required for antisense therapeutic investigations.

Moffat, A.S. 1988 (Nov/Dec). “Researchers Pursue

Anti-Sense Technology In Quest for Novel Drugs and Agriproducts”,

Genetic Engineering News. Vol 8. Pages 1 and 7.

Klauser. A. 1990 . “Antisense Start-Ups Surveyed”,

Bio/Technology. Vol 8. Pages 303 - 304.

Detritylated failuresand protecting groups

DMT blocked failures

Abs

orba

nce,

300

nm

DMT blocked full length product

2.4

0

05

1015

2025

3035

40

Minutes

HPLC Purification of Phosphorothioated DNA for

Antisense Therapeutic InvestigationsObjective:

The objective of this application note is to





























HC18 HA reversed-phase material in an 8 x 100mm Radial-Pak cartridge. This same packing is available in a variety of column dimension for gram scale

purifications as required for antisense therapeutic investigations.

Moffat, A.S. 1988 (Nov/Dec). “Researchers Pursue

Anti-Sense Technology In Quest for Novel Drugs and Agriproducts”,

Genetic Engineering News. Vol 8. Pages 1 and 7.

Klauser. A. 1990 . “Antisense Start-Ups Surveyed”,

Bio/Technology. Vol 8. Pages 303 - 304.

W a t e r s

A P P L I C A T I O N SN O T E S

For your free Application Note File Folder,please check box 9 on the Business ReplyCard and return today.

Column: Symmetry® C8 3.9 x 150 mmMobile Phase: 35% Acetonitrile

65% H2OFlow: 1.0 ml/min

11

Table 2: Optimal Dilution Factor and Resulting Enrichment Factors as a Function of theMobile Phase Composition

20 2 - 3 1 - 3.530 3 - 4.5 3.5 - 2040 4 - 6 10 - 10050 5 - 7.5 35 - 50060 6 - 9 100 - 250070 7 - 10 300 - 1000080 8 - 12 800 - 50000

% Organic Dilution Factor d Enrichment Factor e

As an example: the sample of interest

has a k’ of 5 on a 3.9 mm x 150 mm

column in 40% methanol / 60% buffer.

It was eluted from the Sep-Pak car-

tridge with 60% methanol / 40% buffer

and diluted by a factor of 4 based on

the data in table 2. The sample volume

that would result in a 10 % loss in

resolution in Table 1 is 122 µL, if the

sample were dissolved in mobile

phase. The enrichment factor from the

last table is at least 10, so we can

inject at least about 1200 µL on a

3.9 mm x 150 mm column. If the elu-

tion volume from the Sep-Pak cartridge

was 500 µL and we diluted the sample

to 2500 µL, then this procedure means

that we can inject about 50 % of the

total amount of sample available onto

the column. That is quite reasonable.

In the experiment shown in Figure 3,

the optimal dilution factor was found to

be about 5, and the enrichment factor

about 40, in good agreement with the

estimation in Table 2. This means the

method is quite robust.

THE BENEFITS OF SAMPLE FILTRATION

Filtration offers two main benefits for chromatography: 1 it helps prevent instrument downtime, and

2 it helps maintain accuracy and precision of chromatography results.

Some of the ways in which sample filtration helps prevent downtime are:• extending the life of the HPLC (analytical) column

• preventing plugging of critical valves and small bore tubing• preventing wear and abrasion of internal components

such as gaskets by particulate

Ways that sample filtration can help maintain accuracy (a true value) and precision (reproducibility of values) of chromatographic results are:

• eliminating background peaks caused by particulate in the detector that could interfere with the analysis

• eliminating dissolved gases in the mobile phase that may cause “noise” in the detector and interfere with the analysis

At the higher enrichment factors, the

desired injection volumes exceed the

volume of the injector loop in a typical

injector. However, one can compensate

for this by choosing a smaller diameter

column.

ConclusionA method for sample enrichment has

been outlined that is simple in practice

but complex in concept. We have pro-

vided some guidance on how the tech-

nique should be used. Also we have

demonstrated in a model system that the

technique works in practice.

For more Information onGelman Sciences Acrodisc Filtersfrom Waters, please check box12 on the Business Reply Cardand return today.

12

A Resolution Equation for EKC Based onElectrophoretic Mobilities

AbstractA resolution equation for EKC was

developed starting from the resolution

equation for electrophoresis. The equa-

tion was used to predict the influence of

the migration window and partitioning

on resolution in EKC. It is theoretically

shown that the migration window can

have a dramatic effect on resolution in

EKC. Using a novel chiral surfactant,

the influence of the migration window

and partitioning on the separation of

benzoin enantiomers was experimental-

ly determined. The results obtained

agreed with predictions based on the

equation. The ability to obtain very high

resolution values by migration window

manipulation was demonstrated for the

separation of N-methylpseudoephedrine

enantiomers (α=1.3). Specifically, the

resolution was 2.4 under conditions of

robust EOF, but increased to 11 using

conditions of low EOF.

IntroductionElectrokinetic chromatography (EKC),

invented by Terabe, is a subset of capil-

lary electrophoresis (CE) (1). In EKC,

analytes partition between the bulk

aqueous CE phase and an additive.

The most powerful technique of EKC

is the use of micelles (MEKC). Resolution

of two analytes is achieved in EKC by

one or both of the following mecha-

nisms:

1) differences in their mobilities in the

bulk aqueous phase (capillary zone

electrophoresis), and/or

2) differences in their partitioning

between the bulk aqueous phase

and the additive, with the further

requirement that the mobility of the

analyte-additive complex is different

from the mobility of the analyte in

the bulk aqueous phase.

The second mechanism results in a

migration window in EKC. For instance,

MEKC is usually performed with sodium

dodecyl sulphate (SDS) micelles. SDS

micelles are anionic and have an

Jeffrey R. Mazzeo, Michael E. Swartz and Edward R. Groverreprinted with permission from Anal. Chem., Vol. 67 (1995), pp. 2966-2972

electrophoretic mobility towards the

anode. Uncoated fused silica capillaries

are typically used in MEKC, and a bulk

electroosmotic flow toward the cathode

is produced at pH > 2.0. Above pH

6.0, the electroosmotic velocity is usual-

ly faster than the electrophoretic velocity

of the SDS micelles, causing the micelles

to have a net movement toward the

cathode. This situation leads to a migra-

tion window, which for neutral analytes,

is defined by the electroosmotic flow

marker (no partitioning) and the micelle

marker (complete partitioning). All neu-

tral analytes must migrate between

these two boundaries.

The existence of a migration window

leads to an additional term in the resolu-

tion equation for MEKC compared to

the standard resolution equation for

chromatography. As developed by

Terabe (1), the resolution equation for

neutral analytes in MEKC is:

[1]

where N is the theoretical plate count,

α is the selectivity term, k1 and k2 are

the capacity factors for the two ana-

lytes, t0 is the electroosmotic flow time,

and tmc is the micelle marker time.

Capacity factors of neutral compounds

are calculated using the following

equation (1):

[2]

where tr is the observed migration time

of the analyte.

The resolution and capacity factor

equations for MEKC were derived for

neutral analytes under conditions where

the micelles and the bulk aqueous

phase move toward the same electrode

(1). Charged analytes which do not

interact with the micelles will not migrate

at the electroosmotic flow time. Several

researchers have proposed alternative

equations for calculating capacity fac-

tors and resolution of charged analytes

(2-3). We proposed that taq be substi-

tuted for t0 in the resolution and capaci-

ty factor equations, taq being defined as

the time in the aqueous phase (3).

k = t r − t 0

t 0 1− t rtmc

Figure 1: Theoretical Plot of Resolution versus Electroosmotic Mobility Using Equation 10 (See Theory Section)

10

9

8

7

6

5

4

3

2

1

0

0.00

005

0.00

015

0.00

025

0.00

035

0.00

045

0.00

055

0.00

02

0.00

03

0.00

04

0.00

05

0.00

06

0.00

01

EOF (cm2/Vs)

Reso

lutio

n

Rs = N1

2

4

a − 1

a

k2

k2+1

1− t 0

tmc

1+ t 0

tmc

k1

13

The taq value of a charged analyte will

be a function of the electroosmotic

mobility, its own electrophoretic mobility,

and its interaction, if any, with non-

micellised surfactant. The value of taq

can be determined by measuring the

analyte’s electrophoretic mobility in the

MEKC buffer without micelles, adding

it to the electroosmotic mobility in the

MEKC buffer with micelles, and convert-

ing the resulting mobility to a migration

time. Analyte interaction with free sur-

factant molecules is assumed to be

negligible.

It is possible for the micelles and the

aqueous phase to have net mobilities

toward opposite electrodes. This situa-

tion occurs when the electrophoretic

velocity of the anionic micelles toward

the anode is greater than the electroos-

motic velocity toward the cathode (4).

In this case, an unpartitioned neutral

analyte will migrate toward the cath-

ode, while a completely partitioned

analyte will migrate toward the anode

(4). Depending on its partitioning value,

a neutral analyte can migrate toward

the cathode with a migration time from

taq to infinity, or toward the anode with

a migration time from tmc to infinity. The

resolution and capacity factor equations

are written with migration times, so the

practice has been to use negative val-

ues for the migration times when migra-

tion is toward the anode (4).

Consequently, negative resolution values

can also be obtained. A negative reso-

lution value indicates that the more high-

ly retained analyte migrates faster (5).

The migration window or elution range

is defined as t0/tmc, so the case where

the net micelle movement is toward the

anode has been referred to as a nega-

tive migration window.

We are interested in determining the

influence of the migration window and

partitioning on resolution in EKC, espe-

cially with negative migration windows.

Therefore, we have derived a resolution

equation which is applicable to all forms

of EKC. This paper shows the derivation

of this equation. Using the equation,

the influence of the migration window

and partitioning on resolution were

predicted. The predictions were then

verified experimentally.

TheoryThe resolution equation for two analytes

in electrophoresis is defined as (6):

[3]

where N is the average theoretical

plate count, and µapp is the apparent

mobility. Assuming that diffusion is the

only cause of band broadening, the

average plate count for two analytes in

CE is given by (7):

[4]

where V is the applied voltage, l is the

capillary length from injection to detec-

tion, D is the diffusion coefficient, and Lis the total capillary length.

For two analytes partitioning between

the aqueous phase and some additive

(micelles, cyclodextrins, etc.), apparent

mobilities can be calculated:

µapp,1 = x1(µadditive) + (1-x1)(µfs,1) [5]

µapp,2 = x2(µadditive) + (1-x2)(µfs,2) [6]

where x is the fraction of time the

analyte associates with the additive,

µadditive is the mobility of the analyte-

additive complex, and µfs is the mobility

of the analyte in the aqueous phase.

The mobilities of the analyte-additive

complexes for the two analytes are

assumed to be the same in order to

simplify the resulting equations.

The apparent mobilites are determined

from the electrophoretic and electroos-

motic mobilities:

µadditive = µadditive,ep + µos [7]

µfs = µfs,ep + µos [8]

0.0004

µ1

µ2

0.0003

0.0002

0.0001

0.00

005

0.00

01

0.00

015

0.00

02

0.00

025

0.00

03

0.00

035

0.00

04

0.00

045

0.00

05

0.00

06

0.00

0550

0

–0.0001

–0.0002

–0.0003

EOF (cm2/Vs)

mob

ility

(cm

2 /Vs

)

Figure 2: A. Plot of Analyte Mobilites versus Electroosmotic Mobility Using Equations 5,6,7, and 8

EOF (cm2/Vs)

350000

300000

250000

200000

150000

100000

50000

0

0.00

004

0.00

008

0.00

012

0.00

016

0.00

020.

0002

40.

0002

80.

0003

20.

0003

60.

0004

0.00

044

0.00

048

0.00

052

0.00

056

0.00

060

Ave

rage

Pla

te C

ount

Figure 2: B. Plot of Average Theoretical Plates versus Electroosmotic Mobility Using Equations 4

N =

1

2µapp,1 + µapp,2( )Vl

2DL

Rs = N1

2

4

µapp , 1 − µapp , 2

1

2µapp , 1 + µapp , 2( )

14

Substituting equations 4-8 into equation 3 and canceling terms leads to the

following equation:

The main assumptions of equation 9 are:

1) diffusion is the sole cause of band broadening,

2) the analytes have the same diffusion coefficient, and

3) the mobilities of the analytes when interacting with the additive are the same.

If the free solution electrophoretic mobilities of the two analytes are the same, i.e.,

they are enantiomers, equation 9 simplifies to:

Notice that the denominator of the second term in brackets in equations 9 and 10

can equal 0. Therefore, the resolution can go to infinity. A resolution of infinity means

that either one analyte is not moving (net mobility of zero) and the other is, or the

two analytes have net movements toward opposite electrodes. An example of the

second case has been demonstrated for the separation of some peptides with SDS

micelles at low pH (8).

We have defined x to be the fraction of time the analyte spends associated with the

additive. The x values are related to the more commonly used capacity factor, k, by

the following equation (9):

Thermodynamic alpha values, which are defined as k2/k1, are related to x values

with the following equation:

α =χ2 1− χ1( )χ1 1− χ2( )

χ = k

k +1

The influence of EOF (and therefore the

migration window) on resolution can be

calculated using equation 9 assuming

constant values for x, electrophoretic

mobility of the additive, and free solu-

tion electrophoretic mobility of the two

analytes. As an example, assume neu-

tral analytes (µfs = 0), negatively

charged micelles with µadditive,ep =

-4.4 x 10-4 cm2/Vs, x1 = 0.50 and x2

= 0.51 (α=1.04), V = 15,000 volts,

L= 60 cm, l = 52.5 cm and D =

8 x 10-6 cm2/Vs (typical value for an

analyte with MW < 500).

Since the analytes have the same elec-

trophoretic mobility, equation 10 can

be used. Resolution of the two analytes

was plotted as a function of electroos-

motic mobility, Figure 1. A symmetrical

curve was generated, centered about a

point at which the resolution is infinity.

This point occurs at an electroosmotic

mobility of +2.222 x 10-4 cm2/Vs for

these assumed values.

Using equations 5, 6, 7, and 8, the

apparent mobilities of each analyte can

be calculated as a function of electroos-

motic mobility. Using equation 4, the

plate count can be calculated. These

results are plotted in Figure 2. As the

electroosmotic mobility approaches

+2.222 x 10-4

cm2/Vs, the average

apparent mobility decreases. Since the

plate count is directly proportional to the

average apparent mobility, the plates

also decrease.

For the given x values (0.50 and 0.51),

at electroosmotic mobilities less than

+2.222 x 10-4 cm2/Vs, the net mobili-

ties of the analytes are toward the

anode, while at values greater than

+2.222 x 10-4 cm2/Vs the net mobili-

ties are toward the cathode.

Furthermore, below electroosmotic

mobilities of +2.222 x 10-4 cm2/Vs,

the more highly retained analyte (x2)

has the higher mobility, while above

+2.222 x 10-4 cm2/Vs, the more high-

ly retained analyte has the lower mobili-

ty. Therefore, an inversion of migration

order is expected. Electroosmotic mobil-

ities near +2.222 x 10-4 cm2/Vs lead

to very low analyte mobilities and long

analysis times. Clearly, a tradeoff exists;

higher resolution can be obtained at the

expense of longer analysis time.

0.05 0.1

0.15 0.2

0.25 0.3

0.35 0.4

0.45 0.5

0.55 0.6

0.65 0.7

0.75 0.8

0.9

0.95 1

0.85

0

X1

7

6

5

4

3

2

1

0

Reso

lutio

n

d dc cb

b

a

a

e

e

Figure 3: Plot of Resolution versus Partitioning (x Value) for Several Electroosmotic Mobilities

a) µos=+6.0 x 10-4 cm2/Vs

b) µos=+4.4 x 10-4 cm2/Vs

c) µos=+4.0 x 10-4 cm2/Vs

d) µos=+2.2 x 10-4 cm2/Vs

e) µos=0

[10]

[11]

[12]

Rs = Vl

16LD

1

2 x1 − x2( ) µadditive,ep − µ fs,ep( )x1 + x2( ) µadditive,ep − µ fs,ep( ) + 2µos + 2µ fs,ep

1

2

Rs = Vl

16LD

1

2 x1 µadditive,ep − µ fs,1,ep( ) − x2 µadditive,ep − µ fs,2,ep( ) + µ fs,1,ep − µ fs,2,ep

x1 µadditive,ep − µ fs,1,ep( ) + x2 µadditive,ep − µ fs,2,ep( ) + 2µos + µ fs,1,ep + µ fs,2,ep

1

2

[9]

15

The influence of x on resolution for sev-

eral migration windows can also be cal-

culated using equation 12 in conjunc-

tion with equation 10. Note that when

varying x, both the average apparent

mobility and the difference in apparent

mobility change (equation 10). Assume

neutral analytes (µfs = 0), negatively

charged micelles with µadditive,ep = -4.4 x

10-4 cm2/Vs, α=1.04, V = 15,000

volts, L= 60 cm, l = 52.5 cm and D =

8 x 10-6 cm2/Vs. The influence of x on

resolution for several migration windows

is shown in Figure 3.

With a finite migration window (i.e.,

the micelles move in the same direction

as the electroosmotic flow but at a slow-

er rate), an optimum value for x is seen

and the resolution goes to zero when

x = 1 (Figure 3a, µos = 6.0 x 10-4

cm2/Vs, µmc = 1.6 x 10-4

cm2/Vs).

With an infinite migration window (the

micelles do not move), resolution is high-

er at all x values and the optimum xvalue is higher (Figure 3b, µos = 4.4 x

10-4 cm2/Vs, µmc,app = 0 x 10-4

cm2/Vs). This shift in the optimum xvalue is in agreement with the Foley

equation, which shows that the optimum

capacity factor in MEKC depends on

the migration window (10).

With a negative migration window

where the micelles move slowly toward

the anode, the optimum x value increas-

es compared to an infinite migration

window, and infinite resolution can be

obtained (Figure 3c, µos = 4.0 x 10-4

cm2/Vs, µmc = -0.4 x 10-4 cm2/Vs).

Resolution is higher at x values from x =

0 to 0.97 (the infinite resolution point)

for the negative migration window (3c)

compared to the infinite migration win-

dow (3b). However, at x values > 0.97,

better resolution is obtained with an infi-

nite migration window.

With a symmetrical, negative migration

window (the micelles move toward the

anode with the same velocity that the

bulk aqueous phase moves toward the

cathod), the optimum x value is lower

than with a negative migration window

where the micelles move slowly toward

the anode (Figure 3d, µos = 2.2 x 10-4

cm2/Vs, µmc = -2.2 x 10-4 cm2/Vs).

Note the similarities of this curve to the

one in Figure 1, where the migration

window was varied while x was

constant.

Finally, with an infinite migration win-

dow where the micelle moves toward

the anode but the bulk aqueous phase

does not move, the optimum x value

shifts lower compared to a symmetrical,

negative migration window (Figure 3e,

µos = 0 x 10-4 cm2/Vs, µmc = -4.4 x 10-4

cm2/Vs). Also note that the curve is the

same shape as with an infinite migration

window where the micelle does not

move (3b), but is reversed.

To determine if equation 10 correctly

predicts the influence of electroosmotic

flow and x on resolution, the plots in

Figure 1 and Figure 3d will be verified

experimentally.

ExperimentalSeparations were performed on a

Waters Quanta® 4000E system

(Milford, MA). AccuSep™ uncoated

fused silica capillaries (50 µm x 60 cm,

52.5 cm injection to detection) were

rinsed with 0.5 M NaOH initially,

and with 0.1 M NaOH at the begin-

ning and end of the day. A coated

capillary with minimal electroosmotic

flow (eCAP™ neutral, 50 µm x 45 cm,

35 cm injection to detection) was

obtained from Beckman (Fullerton, CA).

Injections were performed hydrostatical-

ly (10 cm height) for times of 2-10 sec-

onds. Between injections, the capillaries

were purged with run buffer for 5 min-

utes. Electropherograms were recorded

and analysed using the Millennium®

Figure 4: Surfactant Structures

Chromatography Manager (Waters).

The micelle marker was sulconazole,

while the EOF marker was methanol.

Sodium phosphate (monobasic and

dibasic), sodium acetate, sodium

hydroxide, phosphoric acid, benzoin

(racemate and (+)-enantiomer), and

N-methylpseudoephedrine (individual

enantiomers) were obtained from

Sigma (St. Louis, MO).

The synthesis of the novel chiral surfac-

tant (S)-2-[(1-oxododecyl)amino]-(3S)-

methyl-1-sulphooxypentane will be

described in a future publication. The

synthesis of (S)-N-dodecoxycarbonylva-

line has been described (3). Structures

of the two surfactants are shown in

Figure 4.

ResultsTo demonstrate the influence of the

migration window on resolution, the

dependence of EOF on pH was exploit-

ed (4). A phosphate-acetate buffer (25

mM each, sodium salts) was employed

over the pH range 3-6, with the elec-

troosmotic mobility ranging from 5.3 x10-4 cm2/Vs (pH 6) to 0.5 x 10-4

cm2/Vs (pH 3). The enantiomers of the

neutral (µfs = 0) analyte, benzoin, were

separated (3:1 ratio of (+):(-)). A novel

chiral surfactant, (S)-2-[(1-oxododecyl)

amino]-(3S)-methyl-1-sulphooxypentane

(Figure 4a, patent pending), was

employed. Its electrophoretic mobility

(µphase,ep = -4.4 x 10-4 cm2/Vs) was

constant over the pH range studied.

O N

O

O

H

OH

H

O N

O

O

O

O

SH H

OH

H

4A (S)-2-[(1-OXODODECYL)AMINO]-(3S)-METHYL-1-SULFOOXYPENTANE

4B (S)-N-DODECOXYCARBONYLVALINE

16

It was initially assumed that the x values

of the benzoin enantiomers would not

change over the pH range of 3-6. The

observed differences in x over the pH

range are attributed to joule heating as

no attempt was made to perform the

experiments at constant ionic strength.

Table 1 lists the values of Tm(+), Tm(-), Tos,

Tmc, resolution, plate counts, x, and

alpha at the different pH-values. From

the apparent mobilities of the analyte,

micellar phase, and electroosmotic

flow, the x value of the analyte was

determined using the following

equation:

[13]This equation is simply a rearrangement

of equation 5. Alphas values were cal-

culated using equation 12.

At pH ≤ 4.5, injection was performed

at the cathode, while at pH ≥ 5.2,

injection was performed at the anode.

Some of the separations are shown in

Figure 5. The separations at pH 5.2

(Figure 5b) and pH 4.5 (Figure 5c)

show that the migration order of the

enantiomers was reversed. Experiments

were also performed at pH 4.8 and

5.0. However, injecting from both the

anode and cathode led to no peaks up

to 90 minutes. These pH-values led to

electroosmotic mobilities close to the

point where resolution approaches infin-

ity; unfortunately analysis time

approaches infinity as well.

Figure 6 is a graph of experimental

resolution versus electroosmotic mobility.

The experimental results show the same

symmetrical profile as seen in Figure 1.

Since the x values and alpha values

remained relatively constant with elec-

troosmotic mobility, the change in reso-

lution was due to changes in the migra-

tion window.

The ability of equation 10 to quantita-

tively predict changes in resolution with

electroosmotic flow was tested for this

data set. The diffusion coefficient of

the enantiomers was calculated with

equation 10 using the experimental

x = µapp − µfs

µphase − µfs

Figure 5: Separation of Benzoin Enantiomers at pHs 5.5 (a), 5.2 (b), 4.5 (c) and 4.0 (d).

8

7

6

5

4

3

2

14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19Minutes

mA

U

Buffer: 25 mM (S)-2-[(1-oxododecyl)amino]-(3S)-methyl-1sulphooxypentane25 mM sodium acetate/25 mM monosodium phosphate50 mm x 60 cm uncoated capillary

Voltage: + or - 15 kV2 second hydrostatic injection

Sample: 3:1 ratio of (+):(-) benzoin, 0.4 mg/mL in buffer

-2.5

-2.0

-1.50

-1.0

-0.5

0.0

0.5

1.0

33 3433.5 3534.5 3635.5 3736.5 3837.5Minutes

mA

U

-5.4

-5.2

-5.0-4.8

-4.4

-4.2

-4.0

-3.8-3.6

-4.6

41 41.5 42 42.5 43 43.5 44 45 45.5 4644.5Minutes

mA

U

-3

-2

-1

0

1

21 21.5 22 22.5 23 23.5 24 24.5 25 25.5Minutes

mA

U

(a)

(b)

(c)

(d)

17

resolution at pH 6.0 as well as the x

values, electroosmotic mobility, additive

electrophoretic mobility, capillary

lengths and voltage. The diffusion

coefficient was calculated to be

6.45 x 10-6 cm2/s. Then, using this

value for the diffusion coefficient and

assuming constant x values, resolution

was calculated as a function of elec-

troosmotic mobility at each pH. Table 2

gives the calculated and experimental

resolution at each pH, as well as the %

difference. Generally, the predicted

value using equation 10 is within 10%

of the actual resolution value. The one

exception is at pH 4.5, where the dif-

ference was 21%. This large difference

is attributed to the fact that the x values,

which were assumed to be the same as

at pH 6.0, were significantly different

at pH 4.5 (see Table 1). If the x values

measured at pH 4.5 are used, the pre-

dicted resolution is 4.19, only a 3.7%

difference from the experimental resolu-

tion. Another possible source of error

could be improperly measured values

for the electroosmotic flow and micelle

marker time.

Trends in resolution similar to those in

Figure 6 can also be generated with

a negative, symmetrical migration win-

dow by changing the analytes’ x values

(Figure 3d). In an equilibrium distribution

process, such as MEKC, an analyte’s xvalue, or partitioning, can be altered

thermodynamically (by changing the

nature of one of the phases) or through

phase ratio (by changing the concentra-

tion of one of the phases in relation to

the other). The advantage of changing

the partitioning through phase ratio is

that selectivity (α) does not change. To

demonstrate this concept, the separation

of benzoin enantiomers was performed

at pH 5.0 with surfactant concentrations

of 60, 30, 15 and 10 mM. At pH

5.0, µos = +2.1 x 10-4 cm2/Vs and

µmc = -2.3 x 10-4 cm2/Vs, which is

essentially a symmetrical, infinite migra-

tion window. The separations at the four

surfactant concentrations are shown in

Figure 7. Note the similarity to those

shown in Figure 5 and the fact that a

reversal of migration order was

obtained. The general trend in resolu-

tion with x (surfactant concentration) is

similar to the theoretical curve (Figure

3d). Quantitative predictions of resolu-

tion as a function of x values using

equation 10 are problematic since the

electroosmotic flow changes with the

change in surfactant concentration.

The alpha value for benzoin using (S)-2-

[(1-oxododecyl)amino]-(3S)-methyl-1-

sulphooxypentane is 1.04, which was

sufficient for baseline resolution under all

the conditions investigated. In many

cases, lower selectivities are found. By

optimising the migration window and xvalues for a given system using equation

9 or 10, solute pairs with alpha values

less than 1.04 can be baseline

resolved. Very high resolution values can

be realised by optimising the migration

window and/or x values when the

alpha value is higher. To demonstrate

this point, separation of the enantiomers

of N-methylpseudoephedrine was

Table 1: Migration Times, Resolution, Plate Counts, Partitioning (x), and Alpha at Each pH

6.0 13.193 13.397 6.590 38.890 2.15 263,000 0.60 0.61 1.04

5.5 16.187 16.540 7.393 116.990 2.64 230,000 0.58 0.59 1.04

5.2 35.037 36.827 9.890 (40.698) 4.17 107,000 0.59 0.60 1.04

4.5 (44.820) (42.323) 21.875 (9.210) 4.35 82,000 0.52 0.53 1.04

4.0 (24.450) (23.630) 43.750 (9.722) 2.80 155,000 0.55 0.56 1.04

3.0 (19.183) (18.707) 69.750 (9.000) 2.32 210,000 0.55 0.56 1.04

All migration times are in minutes; values in parentheses indicate migration towardthe anode.

performed with (S)-N-dodecoxycar-

bonylvaline (Figure 4b, µmc,ep = -4.5 x

10-4 cm2/Vs) under high and low EOF

conditions. The alpha value for this pair

of enantiomers is 1.3 with (S)-N-dodec-

oxycarbonylvaline (3). Separations

of the enantiomers were performed in

50 mM Na2HPO4 adjusted to pH 8.0,

where N-methylpseudoephedrine is

positively charged (µfs,ep= +2.1 x 10-4

cm2/Vs). Separations were performed in

an uncoated capillary with robust EOF

(µos = +5.5 x 10-4 cm2/Vs, µmc = +1.0

x 10-4 cm2/Vs) and in a coated capil-

lary with low EOF (µos = +0.5 x 10-4

cm2/Vs, µmc = -4.0 x 10-4 cm2/Vs). In

the uncoated capillary, a finite migration

window was obtained, while with the

coated capillary a negative migration

window was obtained.

Figure 6: Plot of Resolution versus Electroosmotic Mobility (Data in Table 1)

3.6

EOF (10-4 cm2/Vs)

4.75.31.5

2

2.5

3.5

4

4.5

3

0.50.81.5

Rs

Conditions: Used For Data Generated in Table 1:Buffer: 25mM (S)-2-[(1-oxododecyl)amino]-(3S)-methyl-1-sulphooxypentane

25mM sodium acetate/25 mM monosodium phosphate 50 µm x 60 cm uncoated capillary; + or - 15 kV; 2 second hydrostatic injection

Sample: 3:1 ratio of (+):(-) benzoin, 0.4 mg/mL in buffer

pH Tm(+) Tm(-) Tos Tmc Rs Navg. x1 x2 α

18

Figure 7: Separation of Benzoin Enantiomers at 60 (a), 30 (b), 15 (c), and 10 (d) mMSurfactant. Buffer pH 5.0.

-1.5

-1

-1.5

1.5

-.5

0

1

21 21.5 22 22.5 23 23.5 24 24.520 20.5

Minutesm

AU

0

.5

1

1.5

50 51 52 53 54 55 56 57 58

Minutes

mA

U

-5.0

-4.5

-4.0

-3.5

31.5 32 32.5 33 33.5 34 34.5 35Minutes

mA

U

Figure 8 shows the two separations,

with the resolution increasing from 2.4

(uncoated capillary) to 11.0 (coated

capillary). The same buffer was used

for both separations, the only difference

being the capillary employed (and

hence the magnitude of electroosmotic

flow).

ConclusionsA resolution equation for EKC based on

electrophoretic mobilities is a useful tool

in predicting the influence of the migra-

tion window and partitioning on resolu-

tion. Several points can be made about

the simulated resolution vs. migration

window and partitioning curves pre-

pared using equation 10. First, the

migration window is a powerful para-

meter to optimise resolution in EKC.

Infinite resolution is possible. Second,

for compounds which spend more time

with the additive than in bulk solution,

i.e., x values > 0.50, an infinite migra-

tion window where the additive does

not move is preferable to an infinite

migration window where the additive

does move. For compounds with x val-

ues < 0.50, the latter case is preferred.

This situation is due to the fact that the

lower the average apparent mobility

of the analytes, the better the resolution.

When the additive does not move,

analytes which spend more time with

the additive will have lower average

apparent mobility. When the bulk solu-

tion does not move, analytes which

spend more time in it (and therefore less

time with the additive) will have the

lower average apparent mobility.

Finally, through a combination of EOF

and/or x value manipulation, reversals

in migration order can be obtained in

EKC.

The system employed here to verify the

predictions of equation 10 is an ideal

one. The analytes are enantiomers

and neutral over a wide pH range; the

surfactant is fully ionised over a wide

pH range. However, the separation of

enantiomers is an important practical

problem, and the thought process

demonstrated in this paper makes the

optimisation of such a separation by

chiral MEKC both rational and efficient.

-11

-10

-9

-8

-7

50 52 54 56 58 60

Minutes

mA

U

(a)

(b)

(c)

(d)

Table 2: Comparison of Experimental Resolution and Resolution Calculated using Equation 10

5.5 2.64 2.43 8.05.2 4.17 3.81 8.64.5 4.35 3.42 21.44.0 2.80 2.58 8.03.0 2.32 2.39 3.0

pH Experimental Rs Calculated Rs %Difference

19

Figure 8: Separation of N-Methylpseudoephedrine Enantiomers using Uncoated (top) andCoated Capillary (bottom)

Ordering Information

References1. Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985,57, 834-841.

2. Khaledi, M.G.; Smith, S.C.; Strasters, J.K. Anal.Chem. 1991, 63, 1820-1830.

3. Mazzeo, J.R.; Grover, E.R.; Swartz, M.E.; Petersen,J.S. J. Chromatogr. 1994, 680, 125-135.

4. Otsuka, K.; Terabe, S. J. Microcolum Sep., 1989, 1,150-154.

5. Zhang, C.X.; Sun, Z.P.; Ling, D.K. J. Chromatogr.1993, 655, 309-316.

6. Giddings, J.C. Sep. Sci., 1969, 4, 181-189.

7. Jorgenson, J. W.; Lukas, K. D.; Anal. Chem., 1981,53, (1298-1302)

8. Beijersten, I.; Westerlund, D. Anal. Chem. 1993, 65,3484-3488.

9. Karger, B.L.; Snyder, L.R.; Horvath, C. An Introductionto Separation Science; John Wiley & Sons, Toronto,1973; Chapter 1.

10.Foley, J.P. Anal. Chem. 1990, 62, 1302-1308.

-1.6

-1.4

-1.2

-1.0

-0.8-0.6

-0.4

-0.2

-0.0

.20

.40

20 40Minutes

mA

U

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

20 40Minutes

mA

U

Conditions: 10 mM (S)-N-dodecoxycarbonylvaline50 mM Na2HPO4pH 8.0214 nm detection10 second injection2:1 ratio of (+):(-)-N-methylpseudoephedrine inbuffer, 0.4 mg/mL

Uncoated Capillary:50µm X 45 cm, 35 cm effective+8 kV

Coated Capillary:50µm X 45 cm, 35 cm effective-8 kV

Waters EnantioSelect Chiral Test Kit WAT066270

Waters EnantioSelect (S)-Val-1 (25) WAT066274

Waters EnantioSelect (R)-Val-1 (25) WAT066272

Description Part No.

For more InformationPlease check box 10 onthe Business Reply Cardand return today.

20

Literature Corner:

“Analysis of Cisapride in Neonatal Plasma Using High-Performance LiquidChromatography with a Base-Stable Column and Fluorescence Detection”

Y. Preechagoon and B. G. Charles, J.Chromatogr. B 670 (1995), 139

“Development of a High Performance LiquidChromatographic Method for the Analysis ofStaurosporine”

L. R. Gurley, K. O. Umbarger, J. M. Kim, E.M. Bradbury and B. E. Lehnert, J.Chromatogr. B 670 (1995), 125

The authors describe a method for measuring the level of staurosporine which is a protein-

kinase inhibitor in blood. The assay required that the acetone extract be diluted 1:1 in order

for the staurosporine to bind to the µBondapak C18 Radial-Pak cartridge (8 mm x 100 mm).

Direct injection of the plasma sample was not possible because the protein and drug would

co-elute using a linear elution gradient of acetonitrile with 0.2% TFA. The UV detection

allowed a range from 0.1 to 2 µg staurosporine. This method was also used to determine

the pharmacokinetics of the drug in vivo.

Dorothy J. Phillips

Simple, rapid and robust methods are most suitable for drug analyses. For the analysis of

drugs in plasma a high degree of sensitivity and selectivity are also required. A simple, selec-

tive, sensitive and precise high-performance liquid chromatography method was developed

for the basic drug cisapride. The plasma extracts were analysed on a Waters Symmetry® C8

column (3.9 mm x 150 mm) with a simple mobile phase of acetonitrile and phosphate buffer.

No mobile phase additives were required for sharp, symmetrical peaks. The linear range for

cisapride was 5 to 250 ng/mL. The specificity of the assay was confirmed by showing that

cisapride did not co-elute with its metabolite nor with twelve other drugs used in neonatal

pharmacotherapeutics. The short analysis time of 10 minutes also made the assay suitable

for clinical assays. This is one of the first reported clinical applications in neonatal medicine

of the Symmetry® column, which is a recently developed column from Waters that is ideally

suited for this type of analysis.

Dorothy J. Phillips

“High-Performance Liquid ChromatographyMethod for Potency Determination ofCephalexin in Commercial Preparations andfor Stability Studies”

M. Hsu, Y. Lin and H. Chung, J. Chromatogr.A 692 (1995), 67

The U. S. Pharmacopeia method for cephalexin (an antibiotic) requires the use of both an ion-

pairing reagent and a silanol blocking agent . Also the method uses an uncommon internal

standard. The British Pharmacopeia method for the analysis of cephalexin oralpreparation is

an iodometric titration. The US Code of Federal Regulations describe both an iodometric titra-

tion and a microbiological method. The authors of this paper have developed a method which

has a simple mobile phase (methanol/1.25% glacial acetic acid) with the readily available

drug acetaminophen as the internal standard. The µBondapak C18 column (3.9 mm x 300 mm)

gave a short analysis time of 15 minutes with a cephalexin concentration range of 0.2 to

1.75 mg/mL. It was shown that excipients from commercial formulations do not interfere with

the assay. The assay was also used for product degradation studies and to determine peak

purity with a photodiode array detector. The results from the HPLC method were compared

to those of the microbiological assay and showed that the chromatographic method is a

suitable substitute. In addition, the HPLC assay is more specific than either the titration or the

microbiological assay.

Dorothy J. Phillips

“Stereospecific High-Performance LiquidChromatographic Assay of Iomefloxacin inHuman Plasma”

R. T. Foster, R. A. Carr, F. M. Pasutto, J. A.Longstreth, Journal of Pharmaceutical andBiomedical Analysis 13 (1995), 1243

Several methods have been reported in the literature for the quantitation of iomefloxacin, a

chiral quinolone carboxylic acid antibiotic. The literature methods are not able to discern the

quantities of the individual enantiomers of this drug in biological fluids. The method reported

in this paper describes an HPLC assay for quantitation of the enantiomers of iomefloxacin.

The drug is extracted from plasma at pH 7 in the presence of the internal standard, racemic

acebutolol. A two-step precolumn derivatization with unichiral (S)-(+)-naphthylethyl isocyanate

and achiral ethyl chloroformate is then carried out. The enatiomers are separated as diastere-

omers on Waters Nova-Pak® silica packing in the 8 mm x 100 mm Radial-Pak™column

segment.

Dorothy J. Phillips

“Simultaneous Determination ofPreservatives, Sweeteners and Antioxidantsin Food by Paired-Ion LiquidChromatography”

B. H. Chen and S. C. Fu, Chromatographia41 (1995), 43

The authors developed a paired-ion HPLC method for the simultaneous determination of

sweeteners (dulcin, saccharin-NA and acesulphame-K), preservatives (acetic, sorbic, salicylic,

benzoic, succinic, methyl-p-hydroxybenzoic, ethyl-p-hydroxybenzoic, n-propyl-p-hydroxyben-

zoic and i-propyl-p-hydroxybenzoic acids) and antioxidants (3-t-butyl-4-hydroxy-anisole and

t-butyl-hydroquinone). Detailed sample preparation procedures are given for soy sauce, dried

roast beef and sugared fruit using solid-phase extraction with Waters Sep-Pak C18 cartridges.

Uwe D. Neue

Pharmaceutical Applications:

fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl



Food Applications:

21

Waters™ Columns now available from Phase Separations

“Structure of Hydroxycinnamic AcidDerivatives Established by High-Performance Liquid Chromatography withPhotodiode-Array Detection”

L. Bengoechea, T. Hernández, C. Quesada, B. Bartolomé, I. Estrella and C. Gce Liquid Chromatography with Photodiode-Array

Hydroxycinnamic acid derivatives are widely distributed throughout the plant kingdom and

play an important role in the secondary metabolism in plants. In this paper, hydroxycinnamic

acid derivatives are separated by reversed-phase HPLC using a Nova-Pak C18 column and

categorised using a Waters Model 991 Photodiode Array Detector. p-Coumaric acid deriva-

tives are distinguishable from caffeic and ferulic acid derivatives, but it is not possible to distin-

guish between the latter two groups due to overlap of the ranges of spectral characteristics.

Uwe D. Neue

Food Applications:

fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl

The Choice is Yours


Spherisorb Columns, pleasecheck box 21 on the BusinessReply Card and return today.

We credit the tremendous success of Symmetry® columns to the fact that their development is a

direct result of a dialogue with our customers. So in our search for a better way to sell and

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What we’ve found is that you are happy with our products and support, but would like to pur-

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Our search for a better way to sell and deliver Waters™ columns led us to Phase Separations, a

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Spherisorb® Columns now available from Waters

Spherisorb®, one of the world’s widely used HPLC columns was first developed by Phase

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HPLC, Phase Sep and Waters are taking steps to bring a higher level of quality and consistency

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22

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EPA Method 604: Phenols

System:

The HPLC system consisted of the Waters 626 LC System,717plus Autosampler, and the 996 Photodiode Array Detector.Control and results management were provided by a Millennium® 2010Chromatography Manager.

Waters Corporation 34 Maple Street, Milford, Massachusetts 01757-3696 U.S.A. 508 478-2000

Details:

Phenols are important industrial wastewatercontamimants which must be monitored forcompliance with discharge regulations. Phenols, particularly nitrophenols, often display inconsis-tent response when analyzed by GC, evenwhen time consuming derivatization steps areemployed.

EPA Method 604 phenols are separated in20 minutes using a Symmetry® C8 5 µmcolumn.

1

2

3

45

6

7 8

9

10

11

0 5 10 15

Minutes

Column: Symmetry® C8 3.9 mm x 150 mmMobile Phase: A: 1% Acetic Acid in Water

B: 1% Acetic Acid in AcetonitrileGradient: (T,%B)(0,30), (20,100)Flow Rate: 1.2 mL/minDetection: UV @ 280 nm (0.08 AUFS)Injection Volume: 40 µLConcentration: 1.0 µg/mL Each Component

1: Phenol

2: 4-Nitrophenol

3: 2-Chlorophenol

4: 2-Nitrophenol

5: 2,4-Dinitrophenol

6: 2,4-Dimethylphenol

7: 4-Chloro-3-Methylphenol

8: 2,4-Dichlorophenol

9: 4,6-Dinitro-2-Methylphenol

10: 2,4,6,-Trichlorophenol

11: Pentachlorophenol

Millennium® 2010 Chromatography Manager

Advanced computer technology simplifies accessing, interpreting, and reporting your results.

The productivity of today’s chromatography is centered around

the ability to find, generate, summarise, and report results in a

way that meets your needs and complies with any regulatory

requirements. The Millennium relational database lets you create

your own view filters to customise the way you sort your informa-

tion, such as “lotCode”.

• Using the database, you can track a method history giving

you audit trail capability that ensures total security of your

raw data and results. In addition, you can specify “Access

Type” which allows you to “lock” your methods for even

greater security.

• The database keeps track of your instruments, triggering an

alarm when scheduled maintenance is required. The

Millennium relational database lets you view the injection

information on any calibration point at any particular point

in time on a processing sequence. This gives you a

detailed account of the processing method for that injec

tion, ensuring an accurate audit trail for regulatory

compliance.

• The Millennium Chromatography Manager can provide

acquisition and control for as many as four chromato-

graphic systems, including three HP5890 GC.

• The Millennium Report Publisher has the capability to give

you exactly what you want.

Waters 996 Photodiode Array Detector

Complete Spectral Data-Complete Confidence in Peak Purity and Identity.

With Waters 996 Photodiode Array Detector, you can be

confident that if there is an impurity in your sample, you will

find it; that your compound is really what you report it to be

and not something else. Every spectral detail of your sample

is rapidly, easily, and accurately detected. Every peak is

analysed with mathematical precision so you can be sure to

asses the purity of peaks and confirm their identity. And you

will never have to trade off resolution against sensitivity.

• The 996 PDA Detector lets you look at your results in a

variety of ways - even while a separation is under way.

• Absorbency measurement over the entire 190-800 nm

wavelength range.

• The MaxPlot feature of the Millennium PDA Software

automatically detects UV/Vis absorbing compounds

anywhere within the 190-800 nm range. It measures

their absorbence maxima, and plots a peak for each

compound at its absorbence maximum, creating a

composite chromatogram of all of the compounds it

has detected.

Waters 717plus Autosampler

Waters 717plus Autosampler is a fully programmable

autosampler for High-Pressure Liquid Chromatography.

Waters 626 LC System

Innovative LC technology.

The Waters 626 LC System combines advanced polymeric

technology and low dispersion system volume into a liquid

chromatograph that provides the highest level of performance

available in a non-metallic system. The 626 LC System is

optimised for ion exchange, gel filtration, affinity, hydropho-

bic interaction and reversed-phase chemistries used in micro-

bore, analytical or micro preparative methods relevant to the

chemist. The 626 LC System has maximum system operating

pressure of up to 4000 psi.

For more Information onFor more information on these products please check box 17-21 on the Business Reply Card.

Rapid Analysis on Symmetry® 3.5 µm Column

2.00 4.00 6.00Minutes

0.00

0.02

0.04

AU

AU

2.00 4.00 6.00Minutes

0.00

0.02

0.04

12

3 4 5

6

1

2

34 5

6

a.)

b.)

Objective:

To demonstrate that 3.5 µm packing allow short-

er analysis times without sacrificing efficiency or

sensitivity.

Details:

Efficiency is directly related to column length.

Therefore, reducing column length results in a

decrease in efficiency. Decreasing the particle

size from 5 µm to 3.5 µm gives an increase in

efficiency which compensates for this loss when

using shorter columns. Hence, faster analysis

can be obtained with the shorter 3.5 µm column

without a decrease in efficiency. Maintaining the

efficiency on the similar volume columns also

results in no change in sensitivity.

System:

The HPLC system consisted of the Waters

626 LC System, 717plus Autosampler, and the

996 Photodiode Array Detector. Control and

results management were provided by a

Millennium® 2010 Chromatography Manager.

Waters Corporation 34 Maple Street, Milford, Massachusetts 01757-3696 U.S.A. 508 478-2000

Columns: a. Symmetry® C18 4.6 x 100 mm 3.5 µm

b. Symmetry® C18 3.9 x 150 mm 5 µm

Mobile Phase: Water/Methanol/Glacial acetic acid

(79:20:1)

Flow Rate: a. 1.5 mL/min. b. 1.0 mL/min

Detection: UV at 254 nm

Injection volume: 3 µL of mixture with 6 sulphur drugs

Sample

1.Sulphanilamide 9.8 µg/mL

2.Sulphadiazine 24.5 µg/mL

3.Sulphathiazole 24.5 µg/mL

4.Sulphamerazine 29.4 µg/mL

5.Sulphamethazine 39.2 µg/mL

6.Succinylsulphathiazole 39.2 µg/mL

Results:Peak 6

Column USP Plates TailingVolumes Factorsa.) 1.66 mL 6280 1.1b.) 1.79mL 5960 1.1

Waters 996 Photodiode Array Detector

Complete Spectral Data-Complete Confidence in Peak Purity and Identity.

With Waters 996 Photodiode Array Detector, you can be

confident that if there is an impurity in your sample, you will

find it; that your compound is really what you report it to be

and not something else. Every spectral detail of your sample

is rapidly, easily, and accurately detected. Every peak is

analysed with mathematical precision so you can be sure to

asses the purity of peaks and confirm their identity. And you

will never have to trade off resolution against sensitivity.

• The 996 PDA Detector lets you look at your results in a

variety of ways - even while a separation is under way.

• Absorbency measurement over the entire 190-800 nm

wavelength range.

• The MaxPlot feature of the Millennium PDA Software

automatically detects UV/Vis absorbing compounds

anywhere within the 190-800 nm range. It measures

their absorbence maxima, and plots a peak for each

compound at its absorbence maximum, creating a

composite chromatogram of all of the compounds it

has detected.

Waters 626 LC System

Innovative LC technology.

The Waters 626 LC System combines advanced polymeric

technology and low dispersion system volume into a liquid

chromatograph that provides the highest level of performance

available in a non-metallic system. The 626 LC System is

optimised for ion exchange, gel filtration, affinity, hydropho-

bic interaction and reversed-phase chemistries used in micro-

bore, analytical or micro preparative methods relevant to the

chemist The 626 LC System has maximum system operating

pressure of up to 4000 psi.


For more information on these products please check box 17-21 on the Business Reply Card.

Millennium® 2010 Chromatography Manager

Advanced computer technology simplifies accessing, interpreting, and reporting your results.

The productivity of today’s chromatography is centered around

the ability to find, generate, summarise, and report results in a

way that meets your needs and complies with any regulatory

requirements. The Millennium relational database lets you create

your own view filters to customise the way you sort your informa-

tion, such as “lotCode”.

• Using the database, you can track a method history giving

you audit trail capability that ensures total security of your

raw data and results. In addition, you can specify “Access

Type” which allows you to “lock” your methods for even

greater security.

• The database keeps track of your instruments, triggering an

alarm when scheduled maintenance is required. The

Millennium relational database lets you view the injection

information on any calibration point at any particular point

in time on a processing sequence. This gives you a

detailed account of the processing method for that injec

tion, ensuring an accurate audit trail for regulatory

compliance.

• The Millennium Chromatography Manager can provide

acquisition and control for as many as four chromato-

graphic systems, including three HP5890 GC.

• The Millennium Report Publisher has the capability to give

you exactly what you want.

Waters 717plus Autosampler

Waters 717plus Autosampler is a fully programmable

autosampler for High-Pressure Liquid Chromatography.

Documents

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