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Strength in Technology…Strength in Technology…

Ken Butchart, Sandra Hanson. • Fortis Technologies Ltd, 45 Coalbrookdale Road, Cheshire, CH64 3UG, UK

Evaluation of HPLC Stationary Phases for Environmental Analysis

IntroductionIn environmental monitoring there are many compounds which have similarstructure and this can make the analysis of pollutants difficult to achieve in asimple cost effective way.Chromatography provides the option to gain resolution and sensitivity of manyenvironmental compounds in a short time, the use of UV detection or morecommonly Mass Spectrometer detection provides accurate and rapidconfirmation.

Pesticides, Polyaromatic Hydrocarbons (PAHs), PhenoxyHerbicides, Aldehydesand Explosives all present a challenge in the diversity of structures, requiredmobile phases and stationary phase choice. This means that a wide range ofchromatographic techniques must be utilised in order to gain the resolutionrequired.

Here we present data on a variety of applications and highlight whereimprovements in current methodology have been made.

Improving Transition IdentificationBy selecting the correct stationary phase for the analysis of both polar and non-polar compounds, resolution can be improved due to the sharp peak shapes andimproved retention..

FortisTM, is a trademarks of Fortis Technologies Ltd

Fortis Technologies Ltd recognises the trademarks of all other manufacturers

Atlantis ® is a registered trademark of Waters Corporation

Shim-pack® is a registered trademark of Shimadzu

All columns are original manufacturers packed columns

ConclusionBy selection of correct stationary phase coupled with UHPLC technology we areable to improve compound identification in several environmental applicationswhilst also significantly reducing analysis time.

Application of UHPLC to Reduce Analysis Times

Mobile phase A = aq 10 mM ammonium acetate

Mobile phase B = methanol

FIGURE 1. Analysis of 135 Pesticide Transitions – Apple Matrix

2 4 6 8 10 12 14 16 18 20 22 24 26Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e5

4.4e5

4.6e54.8e5

3.32

7.74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

3.16

7.696.064.822.85

10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0Time, min

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

13.20

13.5811.79

10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0Time, min

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

13.23

13.52

Thaibendazole 0.9% Column carryover FortisTM C18, 150x2.1mm, 3um

Thaibendazole 1.9% Column carryover Atlantis® d C18, 150x2.1mm, 3um

FIGURE 4. PAH Analysis

NaphthaleneAcenaphthyleneAcenaphthaleneFluorenePhenanthreneAnthraceneFluoranthenePyreneBenz[a]anthraceneChryseneBenzo[b]fluorantheneBenzo[k]fluorantheneBenzo[a]pyreneDibeno[a,b]anthraceneBenzo[ghi]peryleneIndeno[1,2,3,cd]pyrene

Shimadzu LC-20AA: WaterB: ACNTime %B0 7020 7027 10030 7040 70

1.0ml/min40oC254nm

Shimpack VP-ODS 250x4.6mm 5um Fortis C18 50x2.1mm, 1.7um

35 min 3 min

FIGURE 3. Increased speed of Aldehyde Analysis

Conventional AnalysisShim-pack VP-ODS 250x4.6mm, 5um

Fast AnalysisFortis C18 50x2.1mm, 1.7um

FormaldehydeAcetaldehydeAcetoneAcroleinPropionaldehydeCrotonaldehydeButyraldehydeBenzaldehydeIsovaleraldehydeValeraldehydeo-Tolualdehydem,p-TolualdehydeHexaaldehyde2,5-Dimethylbenzaldehyde

Figure 1. Shows how the analysis of 105 pesticide compounds in 135 transitionsis achievable, the Fortis column providing greater no. of identifications whilst alsohaving less sample column carryover.

As well as improving compound identification selection of the correct UHPLCcolumn technology allows the analyst to reduce run times whilst improving peakresolution. Figure 3 shows the analysis time of 14 aldehydes successfullyreduced from 20 to 4 minutes.

Failure to meet maximum exposure limits imposed by many Food agencies canprove a barrier to trade, therefore accurate measurement of pesticide levels inagricultural products is important. Figure 2 shows that selection of a sub 2umparticle size can increase number of resolved peaks even when using a shortercolumn length – thereby generating less column backpressure and wear on thesystem.

Fortis C1830x2.1mm 1.7um

FIGURE 2. KFDA 83 59 Shim-pack® VP-ODS 150x2.1mm 2.2um

Fortis C1850x2.1mm 1.7um

A: Water/THF/MeOH 7/2/1

B: Water/ACN 4/6

Time %B

0 50

0.5 50

4 90

5 50

Flow: 0.4ml/min

Temp: 50oC

UV: 360nm

Phenoxy AcidsExplosives

Column: 1.7um Fortis Cyano, 50x2.1mm

Mobile Phase: 80/15/5 H2O/MeOH/ACN

Wavelength: 254nm

Flow rate: 0.2ml/min

Temperature: 50C

Column: 1.7um Fortis Cyano, 50x2.1mm

Mobile Phase: 80/20 H2O + 0.2% Acetic acid/ACN

Wavelength: 280nm

Flow rate: 0.2ml/min

Temperature: 25C

2.5mins14mins

FIGURE 5. Making Use of Alternate Selectivity

0.67

71.

2037

444

1.47

00.

7433

024

1.77

01.

34

2.00

01.

580

3.01

31.

0221

8298

3.21

71.

2433

6504

4.62

8

0.85

1936

71

5.24

2

5.85

80.

9126

0620

6.21

01.

0927

2054

6.75

3

7.53

50.

9425

0183

12.9

800.

00

0.00