efficient hplc method development using fluorescence ... · from: turro, n.j., modern molecular...

PittCon 2005

Tanya JenkinsSenior Applications Chemist

Jeannine JordanProduct Manager – Detection Systems

Worldwide MarketingWaters Corporation34 Maple StreetMilford, Massachusetts01757 USA

Efficient HPLC Method Development Using Fluorescence Spectral Data

©2005 Waters Corporation

Jenkins, Jordan

Overview

• Why Choose Fluorescence Detection?

• Fundamentals of Fluorescence Detection

• Design of Fluorescence Detectors

• HPLC Considerations for Fluorescence

• Challenges of Fluorescence Detection Method Development

• Advantages of Using Fluorescence Spectral Data


Jenkins, Jordan

Why Fluorescence Detection?

• Fluorescence detectors (FLDs) are probably the most sensitive among modern HPLC detectors.

• Typical sensitivity is 10-1000 times greater than that of UV/VIS.– Sensitivity typically in pg/fg range however even a single analyte

molecule can be detected in the flow cell.

• Fluorescence detectors are very specific and selective when compared to other optical detectors.

• Technique is simple and non-destructive


Jenkins, Jordan


SENSITIVITY

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Fluorescence S/N = 1258

AbsorbanceS/N = 18

70x Better Sensitivity for Anthracene


Jenkins, Jordan

Why Fluorescence Detection?E

U

0.00

500.00

1000.00

1500.00

2000.00

2500.00

3000.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

230ex/350em280ex/450em230ex/310em

EU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

SELECTIVITYPDA @ 240 nm

Expanded

∗∗

∗

∗∗ ∗

∗

∗

Licorice Extract


Jenkins, Jordan


• When other detection techniques, such as UV/Vis, are too insensitive or not selective.– Environmental

PolyAromatic HydrocarbonsPhenols, Carbamates

– Food and BeverageAflatoxinsMycotoxinsVitamins (B2, B6)Dyes

– Biotech and PharmaceuticalsDrugs and their metabolitesDerivatized amino acids (AccQ-Tag) or (OPA)

• When no UV/VIS Chromophores exist.– Label with Fluorescence tags


Jenkins, Jordan

Fundamentals of Fluorescence

A fluorescent compound absorbs light (UV or Vis) and its molecules reach an excited state. The phenomenon of light emission during this process of returning to the ground state is called fluorescence.

ExcitedState

GroundState

S1

S0

Excitation

Vibration Energy

Emission(1)

(2)

(3)

(1) Molecules enter an excited state after absorbing UV or visible light. Molecules reach an unstable state of high energy.

(2) Electrons lose excess energy as vibration energy and reach the lowest level of excited singlet state.

(3) Fluorescence occurs when electrons lose energy and reach the ground state


Jenkins, Jordan


• The most intense fluorescence is found in compounds containing aromatic functional groups with low energy π→π* transition levels.

• Fluorescence can be observed in aliphatic and alicyclic carbonyl structures and highly conjugated double-bond structures.

• Most unsubstituted aromatic hydrocarbons fluoresce in solution with the quantum efficiency increasing with the number of rings, however simple heterocyclics do not fluoresce

• Substitution of the rings causes shifts in absorption maxima and efficiency.

• Fluorescence is favored in rigid structures.


Jenkins, Jordan


O

O

O

NH

CH3

CH3

CH3

O

O

O

O O

OCH3

Aflatoxins

Application Areas

Carbamate Pesticides

OH

CH3

CH3CH3CH3 CH3

VitaminsPoly-aromatic Hydrocarbons

O

NH2

OH

CH3

CH3

Amino acidsNH

NH

NH

O

O

OH

CH3

CH3

+ ACQ =


Jenkins, Jordan

Detector Design Considerations

• Sensitivity– Fluorescence is used over UV/Vis because of high sensitivity,

therefore the detector must optimize its light intensity.

• Noise Performance– The second half of sensitivity is minimizing noise. Optics that

reduce stray light and minimize scatter will improve noise performance.

• Low Dispersion– Low dispersion is important for the integrity of the separation and

preserving the concentration band.


Jenkins, Jordan

Detector Design ConsiderationsAxially Illuminated Flow Cell Design

• What is it?– Excitation energy enters the rectangular flow cell along its long axis

allowing the excitation energy to be reflected back along the axis of the cell.

• What’s the advantage?– Provides best S/N specification on the market with lowest noise and

minimal RI effects– Flow cell axial walls consist of a geometrically matched lens and

curved mirror. This gives the light a second opportunity to be absorbed, or exit through the lens, minimizing stray light.

λEm

λEx


Jenkins, Jordan

Detector Design ConsiderationsLow Dispersion Flow Cell Design

• What is it?– The 2475 excitation and emission optics are at right angles, and

also in opposite planes, to minimize stray light

• What’s the advantage?– A long, thin rectangle disperses liquids less, has less stray light, less

volume and more path length than the traditional cubic flow cell.– Increase in response, peak areas and heights because path length is

longer than conventional cuvette-shaped cells.

2475 Flow Cell Design Conventional Flow Cell DesignλEm

λEx

λEm

λEx


Jenkins, Jordan

Detector Design ConsiderationsUse of High Energy Xenon Source

Conventional Xenon Source

Area of Least Intense Emission

Intensity

Image

2475 Xenon Source Lamp

Area of Most Intense Emission

Image

Intensity

• What is it?– The 2475 Excitation optics uses a curved mirror to focus the most intense

part of Xenon emission on to the small flow cell entrance.

• What’s the advantage?– Decrease in noise, especially at longer Ex wavelengths– Improved wavelength accuracy because of smaller bandwidth– FLR signal receives the highest quality of excitation light entering the flow

cell.


Jenkins, Jordan

Detector Design ConsiderationsMore Light into the Flow Cell

Relative Lamp Output, Continuous Arc Xenon lamp versus Xenon Flash Lamp

0

50

100

150

200

250

300

350

400

450

200 300 400 500 600 700Wavelength, nm.

Phot

ocur

rent

, mic

roam

ps

Xenon Flash Lamp

Continuous Arc Xenon Lamp


Jenkins, Jordan

Detector Design ConsiderationsMirrors Not Lenses

• What’s the advantage?– Stray light is reduced– Lenses tend to absorb more light than

mirrors and are less efficient– mirror based optics optimize signal energy

throughput– Ex and Em exit can use the same long path

length of the rectangular flow cell design

Traditional Optics

• less optical complexity

• optics are at 90º in 2 different planes.

Xe LampFlow cell

Flow cell

Mirror Ex Optics -Top View

Grating

Grating

Mirror Em Optics –Side View

PMT


Jenkins, Jordan

Detector Design ConsiderationsNormalized Emission Units

• What is it?– Auto-Optimize Gain

uses the Raman peak of water and will determine the recommended Gain setting for an analysis

• What’s the advantage?– Normalized EUs eliminate peak dependence on gain settings.– Factors that normally influence FLR measurements, such as lamp or

optics degradation, can be compensated for.– Superior bench-to-bench reproducibility.


Jenkins, Jordan

Detector Design ConsiderationsNormalized Emission Units

• Comparison for naphthalene– Excitation : 219 nm– Emission : 329 nm

• % RSD with normalization : 18 %

• % RSD w/o normalization : 60 %

Naphtalene, emission units (normalised mode)

0

2000000

4000000

6000000

8000000

10000000

12000000

Beta 10 Beta 15 S/N J01475003N

Are

a co

unt

Naphtalene, energy units

1000000150000020000002500000300000035000004000000

Are

a co

unt

0500000

Beta 10 Beta 15 S/N J01475003N


Jenkins, Jordan

HPLC Considerations for Fluorescence Detection

• Physical characteristics that effect sample fluorescence– Quenching effects of solvent (e.g., MeOH vs. ACN).– The presence of buffers or ion-pairing reagents in solvent.– The concentration of the solvent in sample fraction.– The pH of the solvent.– The temperature of solvent.– The presence of dissolved oxygen in solvent.– The concentration of sample being separated.– The co-elution of other compounds with the sample.– The quality and reproducibility of the HPLC solvent and sample

delivery system.

Note: All spectra, including UV/VIS, are also influenced by many of the factors listed above. However, these effects are significantly magnified when utilizing fluorescence detection techniques.


Jenkins, Jordan


Comparison of Response Degassed and Undegassed Solvents

1- Benzo(b)flouranthene- 400 ppb2- Benzo(k)fluoranthene- 200 ppb3- Benzo(a)pyrene- 200 ppb

Column- Waters PAH Column @ 27º CEluent A: WaterEluent B: AcetonitrileGradient: 60% B to 100% B using curve 9 in 12 minutesHold 11 minutesFlow Rate 1.2 ml/minInjection: 20ul

Degassed

Not Degassed


Jenkins, Jordan


Effect of Concentration on Pyrene Excitation Maxima

Emis

sion

λ

Excitation λ

~ 10–3 M

~ 10 –5 MIn n-heptane

From: Turro, N.J., Modern Molecular PhotochemistryMenlo Park CA, Benjamin/Cummings Publishing, 1978


Jenkins, Jordan

Method Development Challenges for Fluorescence Detection

• Each compound has a unique excitation and emission wavelength

• To get a fluorescence signal you must determine the excitation and emission maxima for each component in the sample and use these exact wavelengths to maximize sensitivity

• Method development can be very tedious if there are many compounds in your sample

• One option is to use a bench top spectrophotometer, but what if you don’t have pure standards?


Jenkins, Jordan

Method Development Challenges for Fluorescence Detection

Naphthalene: Excitation = 245 nm, Emission = 325 nm

Ener

gy

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

*Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

PAH 610 Standard – 16 Components, 15 Fluoresce


Jenkins, Jordan

• What’s the advantage?– 3D data allows for faster methods

development – Enhanced peak identification

through the use of spectral libraries

• What is it?– Allows the user to collect 3-

dimensional emission or excitation spectral data on the fly

– Software designed to process and analyze the data gives the user results faster

3D Capabilities Reduce Detection Method Development Time


Jenkins, Jordan


Polyaromatic Hydrocarbons Structures


Jenkins, Jordan


Polyaromatic Hydrocarbons Method

Column: PAH Column 4.6 x 250mm, 5µm Mobile Phase A: WaterMobile Phase B: AcetonitrileFlow Rate: 1.2 mL/min Gradient: 60%-100% B over 12min curve 9, hold 11minInjection Volume: 20.0 µLSample Diluent: 50/50 Water/ACN (0.1-0.001ng/µL)Needle Wash: 5:1:1 = ACN:Water:IPASeal Wash: 95/5 Water/ACNTemperature: 27˚CFLD Detection: as describedSampling rate: 2 pts/sec Time Constant: 2.0PMT Gain: 1Instrument: Alliance 2695/2475 FLD


Jenkins, Jordan


Injection #1 - Scan to Determine Optimal Emission λ


Jenkins, Jordan


Injection #2 - Scan to Determine Optimal Excitation λ


Jenkins, Jordan


Automatically Build a 2D Method


Jenkins, Jordan


Injection #3 - Confirm Optimized 2D Method

Ene

rgy

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

800.00

900.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Nap

htha

lene

- 5.

751

Ace

naph

thal

ene

- 8.2

34Fl

uore

ne -

8.84

1

Phe

nant

hren

e - 1

0.39

2

Ant

hrac

ene

- 12.

006

Fluo

rant

hene

- 13

.226

Pyr

ene

- 13.

935

Ben

z(a)

anth

race

ne -

15.4

20C

hrys

ene

- 15.

806

Ben

zo(b

)fluo

rant

hene

- 16

.922

Ben

zo(k

)fluo

rant

hene

- 17

.783

Ben

zo(g

hi)p

eryl

ene

- 18.

696

Dib

enz(

ah)a

nthr

acen

e - 2

0.22

0

Ben

zo(a

)pyr

ene

- 21.

332

Inde

no(1

23-c

d)py

rene

- 22

.559


Jenkins, Jordan


Why Create a 2D Method from Spectral Data?

SENSITIVITY

• Base sampling rate of detector is a set value

• With lower sampling rates, points are averaged to provide the required sampling rate

• Averaging of data points in 2D Mode at lower data rates results in better baseline noise


Jenkins, Jordan

Noise Comparison between 2D and 3D Mode

Ene

rgy

-0.10

0.00

0.10

0.20

0.30

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Ene

rgy

-0.10

0.00

0.10

0.20

0.30

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

2D Mode1 pt/s


3D Mode1 pt/s


Jenkins, Jordan

Library Matching Capabilities

Another Benefit of 3D Spectral Information

• Spectra of samples/standards can be collected and stored in a library for comparison.

• Spectral contrast theory measures the differences between the spectra in the library and the spectrum collected for the unknown peak.

• The probability that the two spectra match depend on the degree of difference between the spectra as compared to spectral differences due to non-ideal behavior such as noise, linearity, and solvent effects


Jenkins, Jordan

Library Matching CapabilitiesSpectral Contrast Theory

• Each compound has a unique spectrum that is represented by a vector in space.

• Spectral Contrast Angle is the angle between vectors, “the differences”.– A value of zero degrees the vectors overlay and suggest that the

two spectra are equivalent.

– A value of 90 degrees demonstrates maximum differences in the two spectra


Jenkins, Jordan


Noise AngleNoise Angle is the is the gray area, the area of gray area, the area of uncertaintyuncertainty

The vector length is The vector length is proportional to proportional to absorbanceabsorbance

Spectrum A

Spectrum B

Noise

Absorbance

θ

Detection limits for impurities and ability to identify very small peaks are Detection limits for impurities and ability to identify very small peaks are directly link to the noise.directly link to the noise.A good detector must provide A good detector must provide at the same timeat the same time both high sensitivity both high sensitivity and high resolutionand high resolution


Jenkins, Jordan


Spectral Contrast 53 Degrees

200.00200.00 240.00240.00 280.00280.00 320.00320.00

Wavelength (nm)Wavelength (nm)

Ethyl-PABAEthyl-PABAEthylparabenEthylparaben

Abs

orba

nce

A

bsor

banc

e

53 degrees 53 degrees is a large is a large spectral spectral differencedifference


Jenkins, Jordan


Spectral Contrast 10 Degrees

230.00230.00 250.00250.00 270.00270.00 290.00290.00 310.00310.00


TheophyllineTheophyllineDyphyllineDyphylline

Abs

orba

nce

Abso

rban

ce

Similar spectra Similar spectra for structurally for structurally related related compounds compounds


Jenkins, Jordan


Spectral Contrast 0.5 Degrees

200.00200.00 240.00240.00 280.00280.00 320.00320.00


MethylparabenMethylparabenEthylparabenEthylparaben

Abso

rban

ceAb

sorb

ance

Very similar Very similar spectra, CH2 spectra, CH2 differencedifference

Spectral Spectral Contrast can Contrast can differentiate differentiate these spectrathese spectra


Jenkins, Jordan

Fluoroquinolone MethodColumn: Atlantis Column 4.6 x 150mm, 3µm Mobile Phase A: 0.2% HFPA in WaterMobile Phase B: AcetonitrileMobile Phase C: MethanolFlow Rate: 1.1 mL/min Isocratic: 75:22:3 A:B:C Injection Volume: 10.0 µLSample Diluent: 0.1ng/µL in WaterNeedle Wash: 5:1:1 = ACN:Water:IPASeal Wash: 95/5 Water/ACNTemperature: 30˚CFLD Detection: ex280nm, em400-500nm,

extracted 460nmSampling rate: 1 pts/sec Time Constant: 1.0PMT Gain: 100Instrument: Alliance 2695/2475 FLD

NN N

COOH

F

CH3

NH N

N

O

COOH

FN

N

CH3 O

N

CH3

O

COOHF

NH

N

F

N

CH3

O

COOHF

CH3

O

NHN N

CH3

COOHF

Norfloxacin

Lomefloxacin

Ofloxacin

Ciprofloxacin

Enrofloxacin



Jenkins, Jordan


Separation of 5 FluorquinolonesE

U

0.002.004.006.008.00

10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.0032.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

9.90

110

.294

11.1

51

13.6

19

15.4

26ex280nm, em400-500nm, extracted 460nm


Jenkins, Jordan


Emission Data for all 5 Fluoroquinolones


Jenkins, Jordan


Sample: Enrofloxacin


Jenkins, Jordan


Library Matching adds Increased Confidence in Peak Identification

EU

0.00

5.00

10.00

15.00

20.00

25.00

30.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Nor

floxa

cin

- 9.9

01O

floxa

cin

- 10.

294

Cip

roflo

xaci

n - 1

1.15

1

Lom

eflo

xaci

n - 1

3.61

9

Enr

oflo

xaci

n - 1

5.42

6


Jenkins, Jordan


Vitamin AnalysisMobile Phase A: IsopropanolMobile Phase B: HexaneFlow Rate: 2.0 mL/min Dial-a-Mix: 1% A, 99% BInjection Volume: 5.0 µLSample Diluent: HexaneNeedle Wash: IPASeal Wash: IPATemperature: 25˚CFLD Detection: ex. 295, em. 325Sampling rate: 1 pts/sec Time Constant: 2.0PMT Gain: 1Instrument: Alliance 2695/2475 3D FLD

Vitamin A

Vitamin E


Jenkins, Jordan


Vitamin Separation

Ex λ 295nm; Em λ 305-405nm, extracted 325


Jenkins, Jordan


Overlay of Emission Spectra for Vitamin A and E


Jenkins, Jordan


Overlay of Vitamin E Emission Spectra with 2 Closest Library Matches


Jenkins, Jordan


Overlay of Excitation Spectra for Vitamin A and E


Jenkins, Jordan


Overlay of Vitamin A Emission Spectra with 2 Closest Library Matches


Jenkins, Jordan

Peak Purity with Fluorescence Spectral Data

• Peak purity algorithms compares the spectrum collected at the apex of a peak to the other spectra collected across the peak.

• For a peak purity test to be successful, the spectra of the co-eluters needs to be notably different.

• Fluorescence detectors have large bandwidths to increase sensitivity which decreases the spectral definition.


Jenkins, Jordan


Good optical resolution gives good quality spectral information

230.00 250.00 270.00nm

Benzenespectra maxima spaced 2.5nm

Less resolution at 3.6 nm vs. 1.2 nm

UV maxima shifted


Jenkins, Jordan


Larger spectral differencesθ

B not detectable

More of B relative to A

θ

θ

A

B

A

A

A

BB

B

Detection of coelution B when analyzing for A

Peak Purity should not be used with Fluorescence Data


Jenkins, Jordan

Summary

• Fluorescence is used for detection when increased sensitivity or selectivity is needed

• Method development for fluorescent methods can be much more difficult because the excitation and emission maxima must be determined

• 3D scanning capabilities can help to drastically reduce detection method development time by allowing for excitation and emission spectra to be collected to determine optimal wavelengths

• Library matching is a powerful tool which helps with compound identification


Jenkins, Jordan

The Fluorescence Method Development Solution

Waters 2475 Multi λ Fluorescence Detector

Performance by Designfor

Efficient Method Development


Jenkins, Jordan

Appendix 1 – Separation Conditions for Anthracene

Chromatographic Conditions:Column: Atlantis 4.6 x 150mm, 3µm Mobile Phase A: WaterMobile Phase B: AcetonitrileFlow Rate: 1.0 mL/min Isocratic: 30/70 Water/ACN Injection Volume: 2.0 µLSample Diluent: 50/50 Water/ACN (100ng/µL)Needle Wash: 5:1:1 = ACN:Water:IPASeal Wash: 95/5 Water/ACNTemperature: 30˚CPDA Detection: 210-400nm, extracted 249nmTime Constant: 0.5FLD Detection: ex249, em402Sampling rate: 5 pts/sec Time Constant: 0.5PMT Gain: 10Instrument: Alliance 2695/2475 3D FLD/2996 PDA


Jenkins, Jordan

Appendix 2 – Separation Conditions for Licorice Extract

Chromatographic Conditions:Column: Atlantis 4.6 x 150mm, 3µm Mobile Phase A: WaterMobile Phase B: AcetonitrileFlow Rate: 1.0 mL/min Gradient: 10%-98% B over 20min, hold 10minInjection Volume: 10.0 µLNeedle Wash: 5:1:1 = ACN:Water:IPASeal Wash: 95/5 Water/ACNTemperature: 40˚CPDA Detection: 200-300nmTime Constant: 1.0FLD Detection: ex230nm, em300-400nmSampling rate: 1 pts/sec Time Constant: 0.5PMT Gain: 1Instrument: Alliance 2695/2475 3D FLD/2996 PDA

efficient hplc method development using fluorescence ... · from: turro, n.j., modern molecular...

Documents