What’s Wrong With My Chromatography - Agilent Technologies

What's Wrong With My Chromatography

Rita Steed LC Columns Application Engineer January 13, 2015

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Outline of Presentation

I. Common Problems, Symptoms, and Solutions

- Baseline Shifts/Noise - Peak Shapes - Pressure - Retention Shifts

II. Preventative Maintenance/Good Practices

- In-line Devices - Sample Preparation - Operational Limitations

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Common Problems

Noisy Baselines

Possible Causes

? Dirty flow cell ? Lamp failing ? Pulses from pump (if periodic) ? Temperature effects on detector ? Air bubbles passing through detector

Time (min.)

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Non-spec Lamp

Chromatographic Results with Second Source Lamp at 214 nm

Lamp from Agilent Technologies

Peak 1 Peak 2 Peak 3

S/N = 150 S/N = 400 S/N = 300

Lamp from Second Source

Peak 1 Peak 2 Peak 3

S/N = 15 S/N = 50 S/N = 50

Drifting Baseline

Possible Causes

? Gradient elution

o Mobile phase component

? Temperature unstable (RID) ? Contamination in mobile phase ? Mobile phase not in equilibrium with

column ? Contaminant bleed in system

o Hardware chemical compatibility

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Effect of TFA on Baseline

A: 0.1% TFA in H2O B: 0.1% TFA in ACN

Temperature: 35?C

Sample: 1. Phenacetin 2. Tolmetin 3. Ketoprofen 4. Fenoprofen 5. Ibuprofen 6. Phenylbutazone 7. Mefenamic acid 8. Flufenamic acid

Eclipse XDB-C8 4.6 x 150 mm, 5 m

Gradient: 5 - 100% B in 30 3 min

Flow Rate: 2.0 mL/min

6 8 7

Wavelength ?215nm

?254nm less impact

2

5

1

4

Adjust TFA concentration in Solvent B to level baseline

0

5

10

15

20

Time (min)

Know the UV Cutoff of your mobile phase components

January 13, 2015Month ##, 200X A.WennerGroup/Presentation Title

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Peak Shape

Ghost Peaks ? Appear When No Sample is Injected

60

20% - 100% MeOH

15

No Sample Injected

30

15

0

3

7

Problem ? Dirty mobile phase ? Sample carryover

? May imply poor recovery

? Pe15ak from17an early run (isocratic)

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Solvent Contamination

___ Solvent Source 1 ___ Solvent Source 2 ___ Solvent Source 3 ___ Solvent Source 4, Lot 1 ___ Solvent Source 4, Lot 2

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