fluorescence detection in liquid chromatography (agilent technologies)

7/27/2019 Fluorescence Detection in Liquid Chromatography (Agilent Technologies)

1/54

Fluorescence detection in

liquid chromatography

A new approach tolower limits of detection andeasy spectral analysis


2/54

2


3/54

3

A primer

A new approach to

lower limits of detectionand easy spectral analysis

Applications offluorescence detectionin liquid chromatography

Rainer Schuster andHelmut Schulenberg-Schell


4/54

4

Copyright 2000 Agilent Technologies

All rights reserved. Reproduction, adaption,

or translation without proir written permission

is prohibited, except as allowed under the

copyright laws.

Printed in Germany 01/00

Publication number

5968-9346E


5/54

5

Acknowledgment The authors would like to thank our Agilent Technologiescolleagues Thomas Drr, Angelika Gratzfeld-Huesgen and

Ludwig Huber for fruitful discussions and review of the

manuscript. Udo Huber and Angelika Gratzfeld-Huesgen

contributed to the applications.


6/54

6

Preface High performance liquid chromatography (HPLC) is themethod of choice for separation and quantitation of polar

and nonvolatile compounds. Retention time is the main tool

for identification of analytes. The complexity of real world

analytical problems requires the confirmation of peak

identity through additional qualitative information. This can

be provided from several detector types: diode array, mass

spectrometric or fluorescence detectors.

In 1982 HPs chemical analysis group (now part of Agilent

Technologies) introduced the first commercially available

diode array detector (DAD) for HPLC. This detector addeda third dimensionwavelengthto the chromatogram in

addition to retention time and signal intensity. The diode

array detector records UV/Visible absorption spectra in

milliseconds while compounds are eluting from a column.

A single chromatographic run provides quantitative results

from the signal intensity and qualitative spectral information

for peak confirmation. The improvements in detector

design and the lower price have made it a sensitive and

cost-effective tool that is about to replace single wavelength

UV-detectors, even those performing routine analyses.

Since the mid 1990s, mass spectrometric detectors for liquid

chromatography (LC-MSD) have been available to provide

analysts with both molecular weight and structural informa-

tion for peak confirmation. The ruggedness and easy use of

the instrumentation make this technique available for the

chromatographer. From research and development, the

technique will make its way into the cost-sensitive routine

QA/QC laboratories.

The fluorescence detector (FLD) is one of the most sensitive

detectors in liquid chromatography. Both excitation and

emission fluorescence spectra help to characterize indi-

vidual compounds. While excitation spectra are identical

to UV/Visible absorption spectra, emission spectra can giveadditional information. Until recently, however, FLDs have

been built to provide single-wavelength information.


7/54

7

Fluorescence spectra had to be acquired under stop-flow

conditions and data analysis was time-consuming and

cumbersome compared to data analysis of UV/Visible

spectra.

A completely new approach to fluorescence detector design

has overcome these drawbacks and even improved the

sensitivity. The Agilent 1100 Series fluorescence detector

acquires spectra online simultaneously with the detector

signal. During method development, one or two chromato-

graphic runs can be sufficient to optimize wavelength

settings for a series of analytes. This replaces tedious stop-flow experiments for each individual compound. Fluores-

cence spectral data are analyzed with the same easy-to-use

software tools as diode array spectra. In addition, up to four

wavelengths can be recorded simultaneously to replace

timetable-based wavelength switching. This ensures

maximum sensitivity and selectivity without sacrificing the

reliability of the analytical method in routine analysis of

real-world samples.

This primer introduces the new fluorescence detector

technology and describes new strategies for rapid method

development. A selection of applications demonstrates the

wealth of information available with this new approach to

fluorescence detection.


8/54

8

Tableofco

ntent

Chapter 1Fluorescence detector technology ..................................................................................... 9How the fluorescence detector works .......................... .............. .............. .............. .............. ........ 11

Data handling ................................................................................................................................... 12

How to measure limits of detection .............................................................................................. 14

Chapter 2Strategies for rapid method development ....................................................................... 15Step 1: Check the HPLC system for impurities............. .............. .............. .............. .............. ....... 17

Step 2: Optimize limits of detection and selectivity ................................................................... 18

Procedure I Take a fluorescence scan............... .............. ............... ............... .............. ........... 19

Procedure II Take two HPLC runs with FLD ......................................................................... 21Procedure III Make a single run with the Agilent 1100 Series DAD/FLD combination ... 23

Step 3: Set up routine methods ...................................................................................................... 25

Multi wavelength detection ........................................................................................................ 25

Fluorescence spectral libraries for peak confirmation.............. .............. ............. .............. .... 26

Chapter 3The applications ................................................................................................................... 29Environmental:

Polynuclear aromatic hydrocarbons ......................................................................................... 30

Carbamates ................................................................................................................................... 33

Glyphosate .................................................................................................................................... 37

Food:

Mycotoxins ................................................................................................................................... 40

Aflatoxins B1/B2 and G1/G2 ............... .............. .............. .............. .............. .............. ............... ... 40

Ochratoxin A ................................................................................................................................ 42

Vitamins B2 and B6............. ............... .............. ............... .............. .............. ............... .............. .... 43

Pharmaceutical:

Quinidine....................................................................................................................................... 46

Warfarin......................................................................................................................................... 48

Amino acids ............ .............. .............. .............. .............. .............. .............. .............. ................ .... 50

References........................................................................................................................................ 52

Index ................................................................................................................................................. 53


9/54

9

Chapter 1

Fluorescencedetector

technology


10/54

10

Fluorescenc

edetectortechnology

Fluorescence detectors offer high selectivity combined

with superior limits of detection (LOD) compared to UV

detectors. Only about 10 percent of organic molecules

have fluorophore structures, which enable the molecules

to absorb light over a range of wavelengths. This takes

electrons in the molecule to an excitation level as with any

other molecule containing a chromophore. The fluorescent

molecule has the ability to emit the absorbed energy at

longer wavelengths (ref. 1).

If the fluorescent light intensities are recorded while the

excitation wavelength is changed and the emission wave-

length is fixed, an excitation spectrum can be obtained.Or the excitation wavelength may be fixed and the emission

wavelength changed. This procedure provides an emission

spectrum of the analyte. Compounds without this native

fluorescence may be derivatized to attach a fluorescent

marker molecule in a pre- or post-column reaction (ref. 2).

Fluorescence detectors offer limits of detection down to the

ppt level. The signal intensities are very low compared to UV

absorption and they are measured ideally versus a very low

background noise level. This is inherently more sensitive

than comparing two relatively large signals from a blank and

a sample as done in UV absorption spectroscopy. However,

the sensitivity of fluorescence detection is dependent on

both the fluorophore properties and the detector design and

settings. The response of a fluorophor is characterized by

molar absorptivity and quantum yield at the applied experi-

mental conditions. The sensitivity of the fluorescence

detector depends on several factors: source intensity,

efficiency of the optical system, bandwidth and so forth.


11/54

11

How the fluorescencedetector works

Figure 1Optical design of the Agilent 1100 Seriesfluorescence detector

Fluorescenc

edetectortechn

ology

Xenon

flash lamp

Excitation

monochromator

Emission

monochromator

Sample

Photo-

multiplier

Lens

Mirror

Photodiode

Lens

Previous fluorescence detectors were equipped with motor-

driven gratings for programmable excitation and emission

wavelengths. The detector layout enabled positioning for a

single wavelength at a time and required stop-flow condi-

tions for acquisition of spectra. Thus, acquiring fluores-

cence spectra was time-consuming and optimizing excita-

tion and emission wavelengths was not possible without

disturbing chromatographic separations.

The new optical design of the Agilent 1100 Series fluores-

cence detector is illustrated in figure 1.A Xenon flash lamp

is used to offer the highest light intensities for excitation inthe UV range. The flash lamp ignites only for microseconds

to provide light energy. Each flash causes fluorescence in

the flow cell and generates an individual data point for the

chromatogram. Since the lamp is not powered on during

most of the detector operating time, it offers a lifetime of

several thousand hours. No warmup time is needed to get a

stable baseline. A holographic grating is used as a mono-

chromator to disperse the polychromatic light of the Xenon

lamp. The desired wavelength is then focused on the flow

cell for optimum excitation. To minimize stray light from

the excitation side of the detector, the optics are configured

such that the emitted light is recorded at a 90 degree angle

to the incident light beam. Another holographic grating is

used as the emission monochromator. Both monochroma-

tors have optimized light throughput in the visible range.

A photomultiplier tube is the optimum choice to measure

the low light intensity of the emitted fluorescence light.

Since flash lamps have inherent fluctuations with respect to

flash-to-flash intensity, a reference system based on a

photodiode measures the intensity of the excitation and

triggers a compensation of the detector signal.

Since the vast majority of emission maxima are above

280 nm, a cut-off filter (not shown) prevents stray light

below this wavelength to enter the light path to the emis-sion monochromator. The fixed cut-off filter and bandwidth

(20 nm) avoid the hardware checks and documentation


12/54

12

Data handling

Fluorescenc

edetectortechn

ology

work that is involved with an instrument that has exchange-

able filters and slits.

The excitation and emission monochromators can switch

between signal and spectral mode. In signal mode they are

moved to specific positions that encode for the desired

wavelengths, as with a traditional detector. This mode offers

the lowest limits of detection since all data points are

generated at a single excitation and emission wavelength.

The spectral mode is used to obtain multi-signal or spectral

information. The ignition of the flash lamp is synchronized

with the rotation of the gratings, either the excitation or

emission monochromator. The motor technology for the

gratings is a long-life design as commonly used in high-

speed PC disk drive hardware. Whenever the grating has

reached the correct position during a revolution, the Xenon

lamp is ignited to send a flash. The flash duration is below

two microseconds while the revolution of the grating takes

less than 14 milliseconds. Because of the rotating mono-

chromators, the loss in sensitivity in the spectral mode is

much lower compared to conventional dual-wavelength

detection with UV detectors.

In addition to its use as a detector for liquid chromatogra-

phy, the Agilent 1100 Series FLD offers capabilities for off-

line measurements in a refillable cuvette to obtain excita-

tion and emission spectra in a single task for a pure com-

pound. The result of this fluorescence scan can be viewed in

a three-dimensional plot showing excitation wavelength,

emission wavelength and fluorescence intensity on the axis.

The sequence of data handling is shown schematically in

figure 2. The example describes the collection of excitation

spectra and a signal with Ex 250 nm/Em 350 nm. Alternating

flashes are used either for chromatographic signals or tocontribute to spectra acquisition. About every 14 millisec-


13/54

13

Figure 2Schematic representation of data flowin spectral modeOne signal at Ex 250 nm/Em 350 nm isrecorded and excitation spectra are takenstarting at Ex 230 nm.

Spectrum

Chromatogram

Wavelength

Time

Flash# Time Ex Em Intensity

[msec] [nm] [nm]

1* 0 250 350 5

2 14 230 350 4

3 28 250 350 8

4 42 240 350 15

5 56 250 350 15

6 70 250 350 30

7 84 250 350 30

8 98 260 350 25

*flash duration = 1 microsecondFluorescenc

edetectortechn

ology

With chromatographic data in three dimensions (emission

or excitation wavelengths, retention time and intensity),

the analysis can be displayed either in a three-dimensional

plot or in a two-dimensional isofluorescence plot. Differ-

ent colors signify different fluorescence intensities.

Spectra can be evaluated against spectra from customized

spectral libraries to determine compound identity or to

control peak purity within a peak. While the quality of

fluorescence spectra has proven to be useful for several

applications, it has to be emphasized that UV/Vis spectraobtained with a diode array detector are of superior quality,

especially for trace analysis.

onds, a data point is obtained. The spectrum starts at

230 nm. Just as with a DAD system, the fluorescence spectra

and signals can be watched online and both types of data

are stored on the PC hard disk. Additional signals can be

extracted from the spectral data set during post-run data

analysis. Spectral data points are corrected automatically

for intensity changes over time based on the signal data.


14/54

14

Fluorescenc

edetectortechnology

Figure 3Raman signal-to-noise (S/N)measurementSignal and ASTM noise at 397 nm (water),excitation at 350 nm, PMT=12, responsetime 8 s

How to measure limitsof detection

Fluorescence spectrophotometers can be qualified by the

signal-to-noise ratio (S/N) for the Raman band for water or

as a limit of detection (LOD) for a specific fluorescent

compound.(ref.3)

The Raman band is a result of Raman light scattering and

not due to fluorescence. It simulates the phenomenon of

fluorescence as it involves an initial light signal at a specific

wavelength that causes a signal to occur at a longer wave-

length. The specification based on Raman stray light is given

as the ratio of signal to noise with the excitation wavelength

at 350 nm and emission at 397 nm. The short-term noise is

measured at 397 nm (figure 3) according to the procedures

described in ASTM method 1657/94. For both values, the

dark value at 450 nm (where no stray light appears) is taken

as a reference point for the scale. Raman values greater than

400 are typical for fluorescence detectors in HPLC.

If anthracene is used to measure detector specifications,

limits of detection as low as 10 femtogram anthracene are

possible.

LU

38

36

34

32

30

28

26

24

0 2.5 5 7.5 10 12.5 [min]2017.515

Signal and ASTM noise at 350/397 nm

Dark value at 350/450 nm

Raman S/N =Signal (397 nm) dark value (450 nm)

ASTM noise (397 nm) dark value (450 nm)


15/54

15

Chapter 2

Strategies forrapid method

development


16/54

16

Methoddevelop

ment

Fluorescence detectors are used in liquid chromatography

when superior limits of detection and selectivity are re-

quired. Thorough method development, including spectra

acquisition, is fundamental to achieve good results. This

chapter describes three different steps that can be taken

with the Agilent 1100 Series fluorescence detector. Table 1

gives an overview of how to benefit from the operation

modes during these steps.

Table 1Steps for thorough method development

Step 1: Step 2: Step 3:Check system Optimize limits of Set up routine

detection and methodsselectivity

Fluorescence Find impurities Determinescan (for example, in simultaneously the

solvents and excitation and emissionreagents) spectra of a pure

compound

Signal mode Perform wavelength Use for lowestswitching limits of detection

Spectral mode/ Determine Ex/Em Collect onlinemulti-wave- spectra for all spectra, perform

length separated compounds library search,detection in a single run determine peak

purityActivate up to four Replacewavelengths wavelengthsimultaneously switching


17/54

17

Step 1:Check the HPLC systemfor impurities

A critical issue in trace level fluorescence detection is to

have an HPLC system free of fluorescent contamination.

Most contaminants derive from impure solvents. Taking a

fluorescence scan is a convenient way to check the quality

of the solvent in a few minutes. This can be done, for

example, by filling the FLD cuvette directly with the

solvent for an offline measurement even before the start of

a chromatographic run. The result can be displayed as an

isofluorescence plot or a three-dimensional plot. Different

colors reflect different intensities.

Figure 4 shows a sample of slightly impure water which wasplanned for use as mobile phase. The area where fluores-

cence of the contaminated water sample can be seen is

between the stray light areas: the first- and second-order

Raleigh stray light and Raman stray light. Since excitation

and emission wavelength are the same for Raleigh stray

light, the area of first-order Raleigh stray light is visible in

the left upper area of the diagram. The Raman bands of

water are seen below the first-order Raleigh stray light.

Since the cut-off filter cuts off light below 280 nm, the

second-order Raleigh stray light starts above 560 nm.

Stray light acts in the same way as impurities in that it

simulates background noise. In both cases, a higher noise

level and therefore a higher limit of detection are obtained.This indicates that high sensitivity measurements should be

done away from wavelength settings that have a high stray

light background.

Figure 4Isofluorescence plot of a mobile phaseA pure water sample was put into theflow cell. Spectra were recorded at 5 nmstep sizes.

Impurity 1. order Raman 2. order

450 nm

220 nm

Emission

Excitation

600 nm

Methoddevelop

ment


18/54

18

Methoddevelop

ment

Wavelength [nm]

250 300 350 400 450 500 550

Norm.

0

5

10

15

20

25

30

35

40

Excitation Emission

600

Figure 5Excitation and emission spectra ofquinidineExcitation spectrum with emission at440 nm, emission spectrum with excitation

at 250 nm of 1 g/ml quinidine.Detector settings: step size 5 nm, PMT 12,Response time 4 s.

Step 2:Optimize limits ofdetection andselectivity

To achieve optimum limits of detection and selectivity,

analysts must find out about the fluorescent properties of

the compounds of interest. Excitation and emission wave-

lengths can be selected for optimum limits of detection and

best selectivity. In general, fluorescence spectra obtained

with different instruments may show significant differences

depending on the hardware and software used. (ref. 4)

The traditional approach is to extract an appropriate

excitation wavelength from the UV spectrum that is similar

to the fluorescence excitation spectrum (see figure 5) and

to record the emission spectrum. Then with an optimumemission wavelength determined, the excitation spectrum

is acquired.

These tasks have to be repeated for each compound using

either a fluorescence spectrophotometer or stop-flow

conditions in HPLC. Usually each compound requires a

separate run. As a result, a set of excitation and emission

spectrum is obtained (figure 5) for each compound. Since

this is a tedious procedure, it is applicable only when there

is a limited number of compounds of interest.


19/54

19

Methoddevelop

ment

The Agilent 1100 Series HPLC offers three different

ways to obtain complete information on a compounds

fluorescence:

Procedure I - Take a fluorescence scan offline for a single

compound as described above for the mobile phase. This is

done preferably with a manual FLD cuvette when pure

compounds are available.

Procedure II - Use two HPLC runs with the Agilent 1100

Series FLD to separate the compound mix under known

conditions and acquire emission and excitation spectraseparately.

Procedure III - Use an Agilent 1100 Series FLD/DAD

combination and acquire UV/Visible spectra (equivalent to

excitation spectra) with the DAD and emission spectra with

the FLDboth in a single run.

Procedure I Take a fluorescence scan

Because fluorescence spectra traditionally have not been

easily available with previous HPLC fluorescence detectors,

standard fluorescence spectrophotometers have been usedin the past to acquire spectral information for unknown

compounds. Unfortunately this approach limits optimiza-

tion, as there are differences expected in optical design

between an HPLC detector and a dedicated fluorescence

spectrophotometer, or even between detectors. These

differences can lead to variations for the optimum excita-

tion and emission wavelengths.

The Agilent 1100 Series fluorescence detector offers a

fluorescence scan that delivers all spectral information

previously obtained with a standard fluorescence spectro-


20/54

20

Methoddevelop

ment

Figure 6Characterization of a pure compoundfrom a fluorescence scanAll excitation and emission spectra ofQuinidine (1 g/ml) are shown in onegraphic. Fluorescence intensity is plottedvs. excitation and emission wavelengths.Figure 5 gives detector settings.

straylight1. order 350 nm Ex 315 nm Ex 250 nm Ex

Ex-axis Em-axis

photometer, independent of the HPLC fluorescence detec-

tor. Figure 6 shows the complete information for quinidine

as obtained with the Agilent 1100 Series FLD and a manual

cuvette in a single offline measurement. The optima for

excitation and emission wavelengths can be extracted as

coordinates of the maxima in the three dimensional plot.

One of the three maxima in the center of the plot can be

chosen to define the excitation wavelength. The selection

depends on the additional compounds that are going to be

analyzed in the chromatographic run and the background

noise that may be different upon excitation at 250 nm,

315 nm or 350 nm. The maximum of emission is observedat 440 nm.

440 Em


21/54

21

Procedure II Take two HPLC runs with theFLD

The conditions for the separation of organic compounds

such as polyaromatic nuclear hydrocarbons (PNAs) are well

described in various standard methods, including commonly

used EPA and DIN methods. Achieving the best detection

levels requires checking for the optimum excitation and

emission wavelengths for all compounds. Yet taking fluores-

cence scans individually makes this a tedious process. A

better approach is to acquire spectra online for all com-

pounds during a run. This speeds up method development

tremendously. Two runs are sufficient for optimization.

During thefirst run, one wavelength is chosen in the low

UV range for the excitation wavelength and a spectral range

for the emission wavelength. Most fluorophores show strong

absorption at these wavelengths. Excitation is sufficient for

collecting emission spectra.

Figure 7 contains all emission spectra obtained in a single

run from a mix of 15 PNAs. This set of spectra is used to set

up a timetable for optimum emission wavelengths for all

compounds.

The individual compound spectra in the isofluorescence

plot show that at least three emission wavelengths are

needed to detect all 15 PNAs properly:

0 min: 350 nm for naphthalene to phenanthrene

8.2 min: 420 nm for anthracene to benzo(g,h,I)perylene

19.0 min: 500 nm for indeno(1,2,3-cd)pyrene

In the second run, three setpoints for emission wavelengths

are entered into the time-program and excitation spectra

are recorded, as shown in figure 8. The area of high intensity

(red) is caused by stray light when emission spectra overlapwith the excitation wavelength. This can be avoided by

fitting the spectral range automatically. Excitation at

260 nm is most appropriate for all PNAs.

Methoddevelop

ment


22/54

22

Methoddevelop

ment

Conditions for figure 7 and 8Column Vydac, 2.1 200 nm,

PNA, 5 mMobile phase A = water; B = acetonitrileGradient 3 min, 60 %B;

14 min, 90 %B;22 min, 100 %B

Flow rate 0.4 ml/minColumntemperature 18 CInjectionvolume 5 lFLD settings PMT 12,

response time 4 s,

step size 5 nm

Figure 8Optimization of the time-program for theexcitation wavelength

Figure 7Optimization of the time-program for theemission wavelengthThis shows the isofluorescence plot ofemission spectra for 15 PNAs (5 g/ml)with a fixed excitation wavelength (260 nm).

LU

10

20

30

40

50

60

220 nm

400 nm

350 nm 420 nm 500 nm Emissionswitching

1

2

34

5

67

8

9

10

11

12

13

14

1 Naphthalene

2 Acenaphthene

3 Fluorene

4 Phenanthrene

5 Anthracene6 Fluoranthene

7 Pyrene

8 Benz(a)anthracene

9 Chrysene

10 Benzo(b)fluoranthene

11 Benzo(k)fluoranthene

12 Benz(a)pyrene13 Benzo(g,h,i)perylene

14 Indeno(1,2,3-cd)pyrene

Excitationspectra

0 2.5 7.5 10 12.5 15 17.5 20 22.5Time [min]

5

LU

10

20

30

40

50

60

300 nm

600 nm

1

2

34

5

67

8

9

10

11

12

13

14

1 Naphthalene

2 Acenaphthene

3 Fluorene

4 Phenanthrene

5 Anthracene

6 Fluoranthene

7 Pyrene

8 Benz(a)anthracene

9 Chrysene



12 Benz(a)pyrene

13 Benzo(g,h,i)perylene


Em-spectra

fixed Ex

0 2.5 5 7.5 10 12.5 15 17.5 20 22.5Time [min]


23/54

23

Table 2Timetable for the analysis of 15 poly-nuclear aromatic hydrocarbonsThis timetable gives the conditions foroptimum detection based on the results

of two chromatographic runs.

Time Excitation Emission[min] wavelength wavelength

[nm] [nm]

0 260 3508.2 260 420

19.0 260 500

Methoddevelop

ment

The obtained data are combined to setup the time-table for

for best limit of detection and selectivity. The optimized

switching events for this example are summarized in

table 2.

Procedure III Make a single run with theAgilent 1100 Series DAD/FLD combination

For most organic compounds, UV-spectra from diode array

detectors are nearly identical to fluorescence excitation

spectra. Spectral differences are caused by specific detectorcharacteristics such as spectral resolution or light sources.

In practice, combining a diode array detector with a fluores-

cence detector in series gives the full data set needed to

achieve the optimum fluorescence excitation and emission

wavelengths for a series of compounds in a single run. With

the UV/Visible/excitation spectra available from the diode

array detector, the fluorescence detector is set to acquire

emission spectra with a fixed excitation wavelength in the

low UV range.

The example is taken from the quality control of carbam-

ates. Samples are analyzed for the impurities 2,3-diamino-

phenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP).

Reference samples of DAP and AHP were analyzed with

diode array and fluorescence detection. Figure 9 shows the

spectra obtained from both detectors for DAP. The excita-

tion spectrum of DAP is very similar to the UV absorption

spectrum from the diode array detector. Figure 10 shows

the successful application of the method to a carbamate

sample and a pure mixture of DAP and AHP for reference.

The column was overloaded with the non-fluorescent

carbamate (2-benzimidazole carbamic acid methylester/

MBC) to see the known impurities, AHP and DAP.


24/54

24

Methoddevelop

ment

Wavelength [nm]

200 250 300 350 400 450 500 550

0

5

10

15

20

25

30

35

Excitation Emission

430 nm

265 nm

DAD-spectra

UV

Norm.

Time [min]0 2 4 6 8 10 12

LU

0

0.2

0.4

0.6

0.8

430/540 nm

Standard

Unknown2-amino-3-OH-phenazine

2,3-diaminophenazine

MBC

265/540 nm

Figure 9UV-spectrum and fluorescence spectra for 2,3-diaminophenazine (DAP)This is an impurity of carbamates. The excitation spectrum in a second runshows the similiarity of UV-spectra and fluorescence excitation spectra. Anexcitation wavelength at 265 nm was used for taking the emission spectrum andan emission wavelength at 540 nm was used for taking the excitation spectrum.

ConditionsColumn Zorbax SB, 50 2 mm,


10 min, 15 %B;Flow rate 0.4 ml/minColumntemperature 35 CInjectionvolume 5 lFLD settings PMT 12,

response time 4 s,step size 5 nm, Ex 265 nm

and 430 nm, Em 540 nm

Figure 10Qualitative analysis of MBC(2-benzimidazole carbamic acidmethylester) and impuritiesThe two upper traces are obtained usingtwo different excitation wavelengths.The lower trace is a pure standard of theknown impurities.


25/54

25

Methoddevelop

ment

Step 3:Set up routine methods

In routine analysis, sample matrices can have a significant

influence on retention times. For reliable results, sample

preparation must be thorough to avoid interferences or

HPLC methods must be rugged enough. With difficult

matrices, simultaneous multi-wavelength detection offers

more reliability than timetable-controlled wavelength

switching. The Agilent 1100 Series FLD can, in addition,

acquire fluorescence spectra while it records the detector

signals for quantitative analysis. Therefore qualitative data

are available for peak confirmation and purity checks in

routine analysis.

Multi wavelength detection

Time-programmed wavelength switching traditionally is

used to achieve low limits of detection and high selectivity

in routine quantitative analysis. Such switching is difficult

if compounds elute closely and require a change in excita-

tion or emission wavelength. Peaks can be distorted and

quantitation made impossible if wavelength switching

occurs during the elution of a compound. Very often this

happens with complex matrices, influencing the retention

of compounds.

In spectral mode, the Agilent 1100 Series FLD can acquire

up to four different signals simultaneously. All of them can

be used for quantitative analysis. Apart from complex

matrices, this is advantageous whenwatching for impurities

at additional wavelengths. It is also advantageous for

reaching low limits of detection or increasing selectivity

through optimum wavelength settings at any time. The

number of data points acquired per signal is reduced and

thus limits of detection may be higher, depending on the

detector settings compared to the signal mode.

PNA analysis, for example, can be performed with simulta-

neous multi wavelength detection instead of wavelength-

switching. With four different wavelengths for emission, all

15 PNAs can be monitored (figure 11).


26/54

26

ConditionsColumn Vydac, 2.1 250 mm,


14.5 min, 90 %B;22.5 min, 95 %B


response time 4 s

1 excitation WL at 260 nm

4 emission WL at 350, 420,

440 and 500 nm

Ex=260, Em=350

Ex=260, Em=420

Ex=260, Em=440

Ex=260, Em=500

Ex=275, Em=350, TTReferencechromatogramwith switching events

0 5 10 15 20 25

LU

0

20

40

60

80

100

120

140

160

180

Time [min]

12

3

5

6

7

8

9

10

11

12

13 14154

1 Naphthalene

2 Acenaphthene

3 Fluorene

4 Phenanthrene

5 Anthracene

6 Fluoranthene

7 Pyrene

8 Benz(a)anthracene

9 Chrysene



12 Benz(a)pyrene

13 Dibenzo(a,h)anthracene

14 Benzo(g,h,i)perylene


Figure 11Simultaneous multi wavelengthdetection for PNA-analysisThe upper trace was received withtraditional wavelength switching.

Fluorescence spectral libraries for peakconfirmation

Previously, only diode array detectors and mass spectromet-

ric detectors could deliver spectral information on-line to

confirm peak identity as assigned by retention time.

Now, fluorescence detectors provide an additional tool for

automated peak confirmation and purity control. No

additional run is necessary after the quantitative analysis.

During method development, fluorescence excitation and

emission spectra are collected from reference standards and

entered into a libraryat the choice of the method devel-

oper. All spectral data from unknown samples can then becompared automatically with library data. Table 3 illustrates

Methoddevelop

ment

Ex/Em = 260/420 nm

Ex/Em = 270/440 nm

Ex/Em = 260/420 nm

Ex/Em = 290/430 nm

Ex/Em = 250/550 nm


27/54

27

this principle using a PNA analysis. The match factor given

in the report for each peak indicates the degree of similarity

between the reference spectrum and the spectra from a

peak. A match factor of 1,000 means identical spectra.

In addition, the purity of a peak can be investigated by

comparing spectra obtained within a single peak. When

a peak is calculated to be within the user-defined purity

limits, the purity factor is the mean purity value of all

spectra that are within the purity limits.

The reliability of the purity and the match factor dependson the quality of spectra recorded. Because of the lower

number of data points available with the fluorescence

detector in general, the match factors and purity data

obtained show stronger deviations compared to data from

the diode array detector, even if the compounds are identi-

cal.

Table 3Peak confirmation using a fluorescencespectral libraryThis shows an automated library searchbased on the emission spectra from a

PNA reference sample.

Meas. Library CalTblRetTime Sig Amount Purity Library Name

[min] [min] [min] [ng] Factor # Match

4.859 4.800 5.178 1 1.47986e-1 1 993 Naphthalene@em

6.764 7.000 7.162 1 2.16156e-1 1 998 Acenaphthene@em

7.137 7.100 7.544 1 1.14864e-1 1 995 Fluorene@em

8.005 8.000 8.453 1 2.56635e-1 1 969 Phenanthrene@em

8.841 8.800 9.328 1 1.76064e-1 1 993 Anthracene@em

9.838 10.000 10.353 1 2.15360e-1 1 997 Fluoranthene@em

10.439 10.400 10.988 1 8.00754e-2 1 1000 Pyrene@em

12.826 12.800 13.469 1 1.40764e-1 1 998 Benz(a)anthracene@em

13.340 13.300 14.022 1 1.14082e-1 1 999 Chrysene@em

15.274 15.200 16.052 1 6.90434e-1 1 999 Benzo(b)fluoranthene@em

16.187 16.200 17.052 1 5.61791e-1 1 998 Benzo(k)fluoranthene@em

16.865 16.900 17.804 1 5.58070e-1 1 999 Benz(a)pyrene@em

18.586 18.600 19.645 1 5.17430e-1 1 999 Dibenz(a,h)anthracene@em

19.200 19.100 20.329 1 6.03334e-1 1 995 Benzo(g,h,i )perylene@em

20.106 2 0.000 21.291 1 9.13648e-2 1 991 Indeno(1,2,3-cd)pyrene@em

Methoddevelop

ment


28/54

28

Methoddevelop

ment


29/54

29

Chapter 3

The applicationsEnvironmental

Polynuclear aromatic hydrocarbons Carbamates

Glyphosate

Food

Aflatoxins B1/B2 and G1/G2

Ochratoxine A

Vitamins B2 and B6

Pharmaceutical

Quinidine

Warfarin

Amino acids


30/54

30

Environmentalapplica

tions

Polynuclear aromatic hydrocarbons (PNA) are formed

during pyrolysis or incomplete combustion in industrial or

private heaters, automobile exhaust fumes and tobacco

smoke. These compounds are in many oil products such as

diesel fuel, gasoline and bitumen. PNAs have become a

ubiquitous class of compounds found in all environmental

matrices, including air, soil and water.

Many PNAs have been found to be carcinogenic or mu-

tagenic. Because their structures differ, some are more

carcinogenic than others. Benzo(e)pyrene, for example, is a

weak carcinogen, while isomeric benzo(a)pyrene is a strongcarcinogen. For this reason, maximum concentration limits

have been set for each individual PNA in air, soil and

especially in water samples.

Most environmental regulations in the U.S., many countries

in Asia, and eastern Europe require PNA analysis according

to U.S. Environmental Protection Agency (EPA) methods.

These include analysis of 16 individual PNAs. In western

Europe, most official methods describe the analysis of the

same set of compounds as the EPA methodsexcept the

European methods do not include acenaphthylene, a com-

pound which does not show any fluorescence. A rapid

method for analyzing only six PNAs using a fast isocratic

HPLC method is described in the German standard method

DIN 38 407 F8.

Experiments and results

With the rapid HPLC method, a PNA analysis is achieved in

less then 10 min on a 25 cm microbore column (shown in

figure 12). The lowest limits of detection for five of the six

compounds is found at Ex 360 nm/Em 429 nm. For the last

compound, the optimum is found at Ex 360/Em 490 nm and

the compromise (Ex 360/Em 465 nm) gives medium limits

Polynuclear aromatichydrocarbons


31/54

31

ConditionsIsocratic methodColumn Vydac, 2.1 250 nm,

PNA, 5 mMobile phase Water/acetonitrile = 10/90


response time 4 s,step size 5 nm,Ex 360 nm, Em 465 nm

LU1

24

5

31 Fluoranthene



4 Benz(a)pyrene

5 Benzo(ghi)perylene


2 4 6 8 10 12 14 16 18

2

4

6

8

10

Position-mode

75ul ( 2ppb) = 150 pg

LOD - 26ppt

LOD - 7.6ppt

LOD - 3.3ppt

LOD - 4.9ppt

6

Time [min]

of detection for all. The detection limit for five of the

PNAs is down to 100 fg and 1 pg absolute for indeno(1,2,3-

cd)pyrene (Ex 360 nm/Em 490 nm), the last eluting com-

pound. Increasing the injection volume achieves even

better detection limitsdown to low ppt levels.

Figure 12Determination of PNAs according to DIN 38407 F8 down to a low ppt-levelA 2 ppb reference standard was analyzed at Ex 360 nm/Em 465 nm, with aninjection volume of 75 l. The limits of detection (LOD) are given at the S/N ratio=2.

Most PNA standards contain the antioxidant trichlorophenol,

which coelutes with chrysene. The antioxidant is so far

observed only in the DAD analysis because of the UV

absorption spectrum. With simultaneous multi wavelength

detection, both chrysene at Ex 260/Em 420 nm and

trichlorophenol at Ex 260/Em 310 nm can be quantitated

selectively, as shown in figure 13. This is useful information

when checking the stability of PNA standards.


tions


32/54

32


tions

ConditionsGradient methodColumn Vydac, 2.1 250 nm,

PNA, 5 mMobile phase A = water, B = acetonitrileGradient 3 min, 50 % B,

14 min, 90 % B,22 min, 100 % B,


response time 4 s,step size 5 nm, Ex=260 nm,Em=310 and 420 nm

Figure 13Control of antioxidants in PNA reference standards with the Agilent 1100 Seriesfluorescence detectorWith multi wavelength detection, the antioxidant trichlorophenol and thePNAs could be analyzed in a single run, each at a specific wavelength.

Time [min]

LU

14 16 18 20 22

5

10

15

20

25

30

Chrysene Ex=260, Em=420

Trichlorophenol Ex=260, Em=310

Conclusions

Fluorescence detection is the most sensitive HPLC

detector for PNA analysis. Detection limits are in the

low ppt range.

Simultaneous multi wavelength detection can replace

time-programmed wavelength switching.

Spectral mode fluorescence detection makes PNA

methods more reliable, as it can provide information on

additives and impurities not seen in single-wavelength

detection.


33/54

33

Carbamates Pesticides are regarded as essential to protect the qualityof food during production, storage and distribution. The

persistence of these chemicals requires monitoring of all

major pesticides in crops as well as the environment.

Carbamates are mainly used as insecticides on fruits and

vegetables.

According to EPA method 531.1, carbamates like the

herbicide glyphosate need a postcolumn derivatization step.

To meet the specified limits of detection, a fluorophor is

attached to compounds separated on a dedicated column.

Ortho-Phthalaldehyde is the reagent used most frequently.


Carbamates are separated on dedicated columns. After

hydrolysis of the compounds in the effluent, the derived

methylamines react with an o-Phthalaldehyde/Thiofluor

solution to the corresponding isoindole.

Figure 14Excitation (Ex=450 nm) and emission spectra (Em=330 nm) of the OPA-derivativeof a carbamate

ConditionsColumn Pickering Carbamate

column, 150 4.6 nm,Mobile phase A = water, B = methanolGradient 0 min, 1 5 % B,

29 min, 100 % B,Flow rate 1.0 ml/minColumntemperature 42 CInjectionvolume 1 lFLD settings PMT 12,

response time 4 s,step size 5 nm,

Post-column conditions in thePickering system:OPA reagent for derivatization, reactor forhydrolysis at 100 C, 23 sec dwell time,Derivatization: Ambient, 100 l, 4 s dwell

timeWavelength [nm]

Norm.

250 300 350 400 450 500

0

0.5

1.0

1.5

2.0

2.5

3.0330 nm

450 nm

Excitation Emission


tions


34/54

34


tions

From the collected fluorescence spectra the lowest limit of

detection is achievable when the detector is adjusted to

Ex 230 nm/Em 450 nm or Ex 330 nm/Em 450 nm (figure 14).

The individual carbamate residues do not shift the excita-

tion and emission maxima significantly. The latter choice

is slightly different from the literature where Ex 330 nm/

Em 465 nm is proposed. (ref. 5) Figure 15 compares the two

wavelength settings found with the Agilent 1100 Series FLD.

Watching the ratio of the two signals (Ex 230 nm/Em 450 nm

and Ex 330 nm/Em 450 nm) allows control of peak purity.

In a post-column reaction, the reagent is constantly flowing

through the detector. To measure selectively the fluores-

cence of the derivatized carbamates, it is important to

characterize the fluorescent properties of the reagent,

impurities and the derivatized compounds completely.

The fluorescence of the OPA-reagent in the mobile phase

water/methanol is visible in a three-dimensional plot from afluorescence scan (shown in figure 16). Excitation at 330 nm

and emission at 450 nm are the best choices to achieve the

Figure 15Analysis of carbamates at two differentexcitation wavelengths, 230 and 330 nm

ConditionsSee figure 14.

12

4

53

1 Aldicarb sulfonate

2 Aldicarb sulfoxide

3 Oxamyl

4 Methomyl

5 3-OH-Carbofuran

6 Aldicarb

6

78

9

10

7 Propoxur

8 Carbofuran

9 1-Naphthol

10 Carbaryl

11 Methiocarb

12 BDMC

0 5 10 15 20 25 30 35 40

5

10

15

20

25

30

35

40

230/450 nm

330/450 nm

2.5 ng

LU

Time [min]

1211

45


35/54

35


Figure 16Characterization of the mobile phase,including OPA reagent for carbamatesThe wavelength pair (Ex 330 nm/Em 450 nm)for quantitation of carbamates is separa-ted from the reagents fluorescence.Chromatographic conditions wereestablished and the fluorescence scanwas taken under stop-flow conditions.

Food samples are complex matrices that need some sample

preparation steps. In contrast, water can be clean enough to

be injected directly. With high injection volumes, detection

limits down to 100 ppt (S/N > 2) can be achieved (figure 17).

Figure 17

Analysis of carbamate standard (250 ppt)in waterWith an injection volume of 800 to 900 l, adetection limit of 100 ppt can be achieved(S/N > 2).


tions

lowest limits of detection when monitoring selectively

derivatized compounds independent of the background from

the reagent.

LU

Time [min]

10 15 20 25 30 35

14

15

16

17

18

19

330/450 nm

900 l

800 l

1

24

5

3

1 Aldicarb sulfonate2 Oxamyl3 Methomyl4 3-OH-Carbofuran5 Aldicarb

6 7 8 9

10

6 Propoxur7 Carbofuran8 1-Naphthol9 Carbaryl10 Methiocarb

390

330

220280 450 480


36/54

36


tions

Conclusions

Optimized wavelength settings allow superior limits

of detection. Changes in chromatographic conditions may

change optimum wavelengths.

Simultaneous multi wavelength detection enables

chromatographers to control peak purity by watching

the ratio of the two signals.

Information on reagent properties and possible fluores-

cent impurities ensures reliable quantitative results.


37/54

37

Glyphosate


tions

One of the most often used non-selective and post-emer-

gence herbicides today is glyphosate (commonly found

under the retail names of Roundup and Basta). Because

of an increasing number of plants that are genetically

engineered to resist glyphosate, the quantities applied may

increase. Monitoring of glyphosate in soil, food and water is

therefore becoming mandatory.

The method of choice for analyzing glyphosate and its meta-

bolite aminomethyl phosphonic acid (AMPA) is postcolumn

derivatization based on a two-step mechanismoxidation

with hypochlorit and reaction with o-phthalaldehyde. (ref.6)Several standard methods are currently being optimized,

including DIN 38 407 F22 in Germany.


Similar to the carbamate analysis, the optimum wavelengths

for derivatizing glyphosate and AMPA are 230 nm excitation

and 450 nm emission, as shown in figure 18.

Because of the very high fluorescence background from the

hypochlorit solution and OPA, the excitation wavelength

340 nm results in a signal-to noise ratio two times higherthan the excitation wavelength 230 nm (see figure 19),

although Ex 230 nm gives a much higher intensity.

The limit of detection can be lowered to 5 pg (S/N>2).

Depending on the injection volume, this is equivalent to

0.5 g/l (500 ppt) or even lower (see figure 20).


38/54

38


tions

Conditions for figures 18, 19 and 20Column Pickering Glyphosate

column, 150 4.6 nm,Mobile phase A, 5 mM KH

2PO

4, pH 2.0,

B, 100 % 5 mM potassiumhydroxide

Gradient 015 min, 100 % A,1517 min, 100 % B,1725 min, 100 % A,

Flow rate 0.4 ml/minColumntemperature 55 CFLD settings PMT 12,


Post-column conditions in thePickering system:0.3 ml/min Pickerings hypochloritereagent for oxidation, 0.3 ml/min ofPickerings OPA reagent for derivatization,reactor for oxidation at 36 C, 500 l,43 s dwell timeDerivatization: Ambient, 100 l,4 s dwell time

Wavelength [nm]

Norm.

200 250 300 350 400 450

0

25

50

75

100

125

150

175

200

Excitation Emission340 nm

450 nm230 nm

Time [min]

Norm.

0 2.5 5 7.5 10 12.5 15 17.5

200

400

600

800

1000

230/450 nm

340/450 nm

Figure 18Fluorescence spectra of a glyphosate derivative10 ng/ml glyphosate dissolved in water).

Figure 19Analysis of glyphosate and AMPA atdifferent wavelengthsExcitation at 340 nm offers a lower noisecompared to Ex 230 nm.


39/54

39


tions

Time [min]

LUGlyphosate

AMPA

0 2.5 5 7.5 10 12.5 15 17.5 20

43.5

43.6

43.7

43.8

43.9

44.0

44.1

44.2

340/450 nmFigure 20Trace level analysis of glyphosate andAMPAInjection of 500 ppt glyphosate andAMPA with a10-l injection volume(5 pg absolute)

Conclusions

Excitation of glyphosate can be achieved in the UV

or visible range.

Excitation at the 340 nm offers better selectivity because

excitation at 230 nm gives high background noise andresults in higher limits of detection.

Glyphosate and AMPA can be detected down to the

ppt level.


40/54

40

Foodapplica

tions

Aflatoxins and ochratoxin A belong to a large family of

compounds produced by funghi. These mycotoxins are

highly toxic compounds to protect themselves against other

organisms. All kinds of plant tissue can be growth media for

funghi, and therefore all types of food can be contaminated

with mycotoxins. Storage conditions define the extent of

fungal growth.

Aflatoxins are known to cause degradation of fruits and

vegetables. Ochratoxine is the prominent mycotoxin found

in cereals, flour and figs. Because of the carcinogenic,

teratogenic and mutagenic character of mycotoxins, foodsamples require careful control down to trace levels.

A suitable clean-up procedure and optimized fluorescence

or mass spectrometric detection are fundamental in achiev-

ing the required limits of detection in the low parts per

billion (ppb) range.


Two FLD runs produce optimum excitation and emission

wavelengths, as described in chapter 2. The fluorescence

spectra shown in figure 21 illustrate the result. Both the

B- and G-type aflatoxins show similar spectra. The optimum

excitation wavelength for both is 365 nm. The optimum

emission wavelengths are different, 455 nm for the G-type

and 445 nm for the B-type. These wavelengths deviate from

the literature (ref. 7), which may be due to differences in

experimental conditions such as the pH, the eluent compo-

sition or the instrumentation used.

With the optimized conditions listed below, the limit of

detection is down in the low nanogram range, as shown in

figure 22. This means that a sample of only a few grams can

be sufficient to detect at the low ppb level.

Mycotoxins

Aflatoxins B1/B2 andG1/G2


41/54

41

Foodapplications

Figure 21Fluorescence spectra for aflatoxins G2and B2Reference standards were dissolved inmethanol (step size 5 nm).

Time [min]

LU

0 2 4 6 8 10 12 14 16 18

2

4

6

8

10

12

365/445 nm

365/460 nm

0.3 ng G2

1 ng G1

0.3 ng B2

1 ng B1

ConditionsColumn Hypersil ODS 100*2.1 mm,

3 mMobile phase H

2O/MeOH/CH

3CN =

63/26/11Flow rate 0.3 ml/minColumntemperature 25 CInjectionvolume 5 lFLD settings PMT 12,


Figure 22Analysis of aflatoxins at two differentwavelengths

Wavelength [nm]

Norm.

200 250 300 350 400 450 500

0

2

4

6

8

Excitation Emission

365 nmAflatoxin B29

445 nm

Norm. 365 nmAflatoxin G2 455 nm

200 250 300 350 400 450 500

-1

0

1

2

3

4

5

Excitation Emission

Time [min]


42/54

42

Foodapplica

tions

Ochratoxin A is found in degrading plant materials as a

product of aspergillus or penicillium funghi. Through the

food chain, this compound may become enriched in animal

tissue and act as a cancerogenic. It can also be a substance

that is directly toxic.


The chromatogram in figure 23 shows a 125 pg of ochra-

toxine A at two different excitation wavelengths, 230 nm

and 333 nm, with lower limits of detection at 230 nm but

better selectivity over the matrix background at 333 nm.

Ochratoxin A

ConditionsColumn Zorbax SB C18, 150 2 mm,

3.5 mMobile phase Water/acetonitrile = 50/50Flow rate 0.4 ml/minColumntemperature 40 CInjectionvolume 5 lFLD settings PMT 12,

response time 4 s,step size 5 nm

Figure 23Simultaneous multi wavelength detectionfor ochratoxine analysis125 pg ochrotoxine was injected. Themost appropriate wavelength can bechosen for quantitation depending onmatrix conditions. Conclusions

Optimization of wavelength settings is mandatory if any

change in experimental setup or chromatographic

conditions occurs.

The ability to detect simultaneously at multiple wave-

lengths helps to obtain the best limits of detection and

selectivity for the different species of aflatoxins.

Ochratoxine A may be excited in the UV or visible range

depending on the need for a higher signal or a more stable

baseline.

Time [min]

LU

0 2 4 6 8 10 12 14

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

333/460 nm

230/460 nm

Ochratoxin A


43/54

43

Foodapplica

tions

Vitamins B2 and B6 Vitamins are essential to avoid human and animal malnutri-tion. Improper storage may rapidly damage the natural

vitamin content of food products or supplemental pharma-

ceutical formulations.

Vitamins are classified as either water-soluble or fat-soluble

compounds. Each is analyzed by different methods. Among

the water-soluble vitamins, only B2 (riboflavin and phospho-

rylated riboflavins) and B6 (pyridoxamine, pyridoxal and,

pyridoxine) show fluorescence. All compounds are sepa-

rated in a single run. With previous fluorescence detectors

they were detected using time-programmed wavelengthswitching. (ref.8)


The optimal wavelengths for B6 vitamins are Ex 270/Em

400 nm and for vitamin B2 Ex 270/Em 530nm, as shown in

figure 24. The detection limit for B2 is down to 20 pg; the

detection limit for B6 is 200 pg (S/N > 2).

Figure 24Excitation and emission spectra ofvitamins B2 and B6Spectra were extracted from two FLD runsfor excitation and emission spectra(1 g/ml dissolved in water, step size 5 nm).

Norm.

Wavelength [nm]

200 250 300 350 400 450 500 550

05

1015202530

35

Excitation Emission

450 nm 530 nm

362 nm262 nm

Norm.

Wavelength [nm]

200 250 300 350 400 450 500 550

05

101520

2530

35

Excitation Emission

400 nm280 nm


44/54

44

Foodapplica

tions

ConditionsColumn Zorbax SB 50 2.0 mm,

5 mMobile phase A = 0.005 mM KH

2PO

4,

pH 2.5 - H2SO

4,

B = acetonitrileGradient 025 % B in 10 minFlow rate 0.5 ml/minColumntemperature 35 CInjectionvolume 5 lFLD settings Response time 4 s

During metabolism as well as during sample preparation

from complex matrices, vitamins are subject to various

transformations. The goal is therefore to monitor precursors

and modified species as well as the main compounds in a

single run. Figure 25 demonstrates this for vitamins B2 and

B6. The small impurities close to Riboflavine (seen in figure

26) can be identified through excitation spectra as belonging

to the vitamin B2 complex. Reference standards help to

identify them as monophosphates and diphosphates.

Time [min]

0 2 4 6 8

LU

0

20

40

60

80

270/530 nm

270/400 nm

10

Riboflavine

Pryridoxamine

Pryridoxal

Pryridoxine

Figure 25

Analysis of vitamins B2 and B6Multi wavelength detection is used todetect both vitamins selectively andsensitive.


45/54

45

Foodapplica

tions

Time[min]

0 2 4 6 8 10 12 14

LU

-10

0

10

20

30

40

270/530 nm

Wavelength [nm]250 300 350 400 450

Norm.

0

10

20

30

40

50

60

70

80

1 Pyridoxamine

2 Pyridoxal

3 Pyridoxine

4 Riboflavine-diphosphate

5 Riboflavine-5'monophosphate

6 Riboflavine

12 3

4

56


Figure 26Confirmation of byproducts of vitamin B2FLD excitation spectra (the red line is5monophosphat, the blue line is riboflavin)show the similarity of spectra from thephosphorylated and unphosphorylatedriboflavin (20 g/ml dissolved in mobilephase A).

Conclusions

The fluorescent vitamins B2 and B6 can be analyzed

selectively at the pg level.

Because of the difference in excitation and emission

maxima, simultaneous multi wavelength detection is

essential to detect both vitamins and byproducts at trace

levels.

Online spectra ensure confirmation of minor compounds

throughout the complete HPLC run.


46/54

46

Pharm

aceuticalapplica

tions

Quinidine occurs as two stereoisomers and has been used in

anti-malaria and antiarrhythmic drugs since the beginning of

the twentieth century. Before that, American Indians used

plant material containing quinidine for its antipyretic activity.

This compound has a complex chemical structure containing

a fluorophor and groups (tert. amine) that can be protonated

(illustrated in figure 27). An HPLC separation with well

shaped peaks requires a buffered eluent as described in the

literature. (ref. 9) With a new generation of more stable

column materials (such as Zorbax Stablebond), this com-

pound has to be analyzed under acidic conditions. As muchas retention times change with the type of stationary phase,

a different pH of the mobile phase changes UV absorption

and fluorescence behavior.


As pH decreases, quinidine shows a bathochrome shift:

The emission wavelength changes from 380 nm to 450 nm

as the pH shifts from pH 7 to pH 2.5 (shown in figure 28).

This is important in maintaining optimum limits of detection.

If slight changes in pH occur, these can contribute to

non-reproducible separation and quantitation results.Recording spectra can help to view these changes. Conse-

quently, spectral data can be used in routine work to check

separation conditionsa prerequisite for reproducing

quantitative results.

Quinidine

Figure 27Chemical structure of quinidineThe arrows indicate the functionalgroups that can be protonated.

N

N

HHO

CH3O


47/54

47

Pharm

aceuticalapplications

Conditions

Method 1 pH 7Column Purosphere 125 4 mm,

5 mMobile phase H

2O pH 7 (H

2SO

4)/

acetonitrileFlow rate 0.6 ml/minColumntemperature 40 CInjectionvolume 1 lFLD settings PMT 12,

response time 4 s,

step size 5 nm

Method 2 pH 2.5Column BDS 100 2.1 mm,

3 mMobile phase H

2O pH 2.5 (H

2SO

4)/

acetonitrileFlow rate 0.3 ml/minColumntemperature 40 CInjectionvolume 1 lFLD settings PMT 12,


Norm.

Wavelength [nm]

350 400 450 500 550 600

0

5

10

15

20

25

30

Emission

pH 2.5pH 7

450 nm380 nm35

Emission

Figure 28Influence of pH on emission spectra for quinidine

Conclusions

Fluorescence spectra are an excellent indicator of

pH changes in the eluent.

Quantitation of acidic or basic type substances requires

strict control of pH not only to control retention times

but for reliable quantitation as well.


48/54

48

Pharm

aceuticalapplica

tions

Warfarin Warfarin is an anticoagulant drug that is used in post-surgerytreatment. The chemical structure derives from coumarine

and has phenolic character.

In the literature, warfarin is analyzed mainly at neutral condi-

tions (phosphate buffer pH 7.5) with fluorescence measured

at Ex 290 nm/Em 390 nm or Ex 310 nm/Em 370 nm. (ref.10)


Warfarin can be analyzed on a Zorbax Stablebond columnat pH 2.5. Under these conditions, excitation and emission

maxima shifted to 272 nm and 355 nm, respectively (shown

in figure 29).

The resulting comparison between the analysis based on

literature and actual optimized fluorescence wavelengths

is shown in figure 30. Actual optimized fluorescence wave-

lengths show about three times better limits of detection.

Compared to UV detection, the Agilent 1100 Series FLD has

a limit of detection about 20 times lower.

Wavelength [nm]

200 250 300 350 400 450 500

Norm.

0

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Excitation Emission

272 nm 355 nmConditionsColumn Zorbax SB C18, 50 2.1 mm,

3 mMobile phase 0.005 M KH

2PO

4,

pH 3/acetonitrileGradient 20 % B to 80 % B in 10 minWash 80 % B to 20 % B in 2 minColumntemperature 25 CInjectionvolume 1 lFLD settings PMT 12,


Figure 29

Fluorescence spectra for warfarin underacidic conditions (pH=3)10 g/ml dissolved in phosphate buffer.


49/54

49

Pharm

aceuticalapplications

Time [min]

LU

0 1 2 3 4 5 6 7 8 9

0.5

1.0

1.5

2.0

2.5

3.0

3.5

272/355 nm

290/390 nm

accord. Lit.

10 ng Warfarin

Figure 30Analysis of warfarinResponse under literature conditions(ref. 10) and actual optimized fluorescencewavelengths.


Conclusions

New types of columns that produce different retention

behavior require a rework of chromatographic conditions.

This may induce significant shifts in fluorescence spectra,

which in turn can influence the limits of detection with

the fluorescence detector.

In routine analysis, fluorescence spectra can be taken

automatically and reviewed to check for accidental

changes in chromatographic conditions.


50/54

50

Amino acids are essential components for biological

systems and have been found already during early stages of

life on earth. For human nutrition a set of essential amino

acids is needed that cannot be formed in the body but must

be taken up from the daily food. As building blocks of the

proteins they form the vast majority of all enzymes in

biochemical transformations. A wide range of decease and

drug research is targeted on protein biochemistry to under-

stand the role of amino acids, peptides and proteins.

After acidic or enzymatic hydrolysis amino acid composi-

tion can be determined using reversed phase columns which

will give typically a better resolution than ion chromatogra-

phy. For sensitive detection amino acids are derivatized

in a two-step pre-column derivatization. A post-column

derivatisation is suitable for higher selectivity as required

for complex biological samples. Fluorescence detection is

used for concentrations below 100 pmol/l. UV-detection

is a choice for higher concentrations up to the nanogram

range. A standard HPLC system can be used for this straight-

forward and cost-effective approach to amino acid analysis.

Experimental and resultsPre-column derivatisation of primary amino acids is

achieved with ortho-phthalaldehyde (OPA) and 9-fluorenyl-

methylchloroformate (FMOC) is used for secondary amino

acids. Seventeen different amino acids that are all found in

protein hydrolysates were analyzed as shown in figure 31.

A wavelength switching program is used to detect Proline.

The limits of detection for the amino acids are listed in

table 4 based on a signal-to-noise > 2. This is close to 100

times more sensitive than UV-detection at 338nm. Retention

time precision is typically below 0.2 % and peak area

precision is typically below 5 %.

Pharm

aceuticalapplica

tions

Amino Acids

Compound LOD for FLD FLDFLD

(pmol) (fmol)

Asp 19 0.139 0.924

Glu 18 0.155 0.576Ser 21 0.156 1.015His 29 0.155 1.778Gly 21 0.118 1.124Thr 21 0.113 0.739Ala 20 0.120 0.767Arg 17 0.094 0.905Tyr 19 0.062 1.614Cys-SS-Cys not measuredVal 17 0.058 0.919Met 16 0.045 1.236Phe 17 0.048 1.079Ile 16 0.050 0.759Leu 18 0.040 0.952Lys 57 0.060 5.107Pro 22 0.044 4.379

Table 4LODs for fluorescenceand UV detection


51/54

51

Pharm

aceuticalapplica

tions

Conclusion

Reversed phase chromatography combined with pre-

column derivatization is an ideal tool for automated cost-

effective amino acid analysis on a standard HPLC setup.

With the use of OPA and FMOC and fluorescence

detection it is possible to push the limits of detection

signifantly below 100 femtomoles for most amino acids.

This technique is worked out to offer precise results for

protein hydrolysates in a routine laboratory.

Time [min]0 2 4 6 8 10 12 14 16 18

LU

0

25

50

75

100

125

175Asp

Glu

Ser

His

Gly

Thr A

la

Arg

Try

C

ys-S

S-Cys

Val

Met

Phe

Ile

Leu

Lys

Pro

150

10 pmol standardConditionsColumn 200 2.1 mm AA column

and guard columnMobile phase A = 20 mMol NaAc + 0.018%

TEA adjusted to pH 7.2 with1-2 % acetic acid, B = 20 %of 100 mMol NaAc adjustedto pH 7.2 with 1-2 % aceticacid + 40 % ACN and 40 %MeOH

Flow rate 0.45 ml/minGradient start with: 100 %A, at 17 min

60 %B, at 18 min, 100 %B,at 18.1 min flow 0.45, at18.5 min flow 0.8, at 23.9 minflow 0.8, at 24 min 100%Band flow 0.45, at 25 min 0%B

Oven temp. 40 CPost time 5 minInjector program1 Draw 5.0 l from vial 10borate buffer2 Draw 1.0 l from vial 11OPA reagent3 Draw 0.0 l from vial 12water4 Draw 1.0 l from sample5 Draw 0.0 l from vial 12water6 Mix 8 l in air, max speed, six times7 Draw 1.0 l from vial 14FMOC8 Draw 0.0 l from vial 12water9 Mix 9 l in air, max speed, 3 times10 InjectFLD settings Excitation 340 nm

Emission 450 nmPTM gain 12

at 14.5 minExcitation =266 nmEmission = 305 nmPTM Gain 11

Figure 31Analysis of 10 pmol/l amino acids with fluorescence detection


52/54

52

Refere

nces

1. Lakowicz, J.R., Principles of Fluorescence Spectros-

copy,Plenum Press, New York, 1983.

2. Schuster, R., A comparison of pre- and post-column

derivatization for the analysis of glyphosate,

Agilent Technologies Application Note 5091-3621E, 1991.

3. Froehlich, P.,Internatl Laboratory, No. 10, 42-44,1989.

4. Brownrigg, J.T. and Sullivan, M. J.,Spectroscopy, Vol.1,

No.2, 1989.

5. Pickering Laboratories,Product brochure, Publ. No.

B-CA 4, 1992.

6. Pickering Laboratories,Application Note, Publ. No.

B-CA 5, 1993.

7. Official Methods of Analysis, Food Compositions;

Additives, Natural Contaminants, 15th edition: AOAC:

Arlington, VA, Vol. 2.; AOAC Official method 980.20,

aflatoxins in cotton seed products, 1990.

8. Mc Calley, D.V., J. Chrom., 357, 221, 1986.

9. Chu, Y-Q. and Wainer, I.W.,Pharm. Res. 5, 680, 1988.

10. Gratzfeld-Huesgen, A. and Schuster, R., HPLC for Food

Analysis,Agilent Technologies Application Primer,

5968-9345E, 2000.

11. Gratzfeld-Huesgen, A., Sensitive and Reliable Amino

Acids Analysis in Protein Hydrolysates using the Agilent

1100 Series HPLC,Agilent Technologies Technical Note

5968-5658E, 1999.


53/54

53

Index

Aaflatoxins, 4041

B1/B2, 4041G1/G2 4041

amino acids, 50AMPA, 3739antioxidant, 31anthracene, 14, 22, 26applications, 29ASTM, 142-amino-3-hydroxyphenazine (AHP),23,24

B

bandwidth, 10112-benzimidazole carbamic acidmethylester (MBC), 23, 24

Ccarbamates, 3336cut-off filter, 11, 17cuvette, 12, 17, 19, 20chromophore, 10

Ddata handling, 1213data analysis, 13derivatization, 33detector

diode array, 6, 13, 23, 26, 31mass spectrometric, 6, 26, 40

2,3-diamino-phenazine (DAP), 23, 24DIN method, 30, 37

EEPA methods, 30, 33emission

monochromator, 1112spectra, 6, 1014, 1627, 33, 43, 46, 47wavelength, 1014, 1625, 40

excitationmonochromator, 1112spectra, 6, 1014, 1626, 33, 43wavelength, 1014, 1625, 40,

Ffluorescence spectra, 13, 19, 25, 34, 38,40, 41, 48, 49

fluorescence scan, 12, 17, 21, 34fluorophore, 21, 33, 46

Ggerman standard method DIN, 30, 37glyphosate, 3739grating, 1112

Iimpurities, 17, 23, 25isofluorescence plot, 13, 17, 22

Llamp, 11

Xenon, 1112light sources, 23limit of detection (LOD), 12, 1618, 23, 25,

30, 31, 33, 34, 35, 36, 37, 39, 40, 42, 43, 46, 48,

Mmass spectrometric, 6, 26, 40match factor, 27method development, 16272-benzimidazole carbamic acidmethylester (MBC), 23, 24mobile phase, 17, 34, 35, 46monochromator, 1112multi wavelength detection, 16, 25, 3132,36, 42, 4445,mycotoxins, 40

Oo-phthalaldehyde (OPA), 3335

ochratoxin A, 42

Ppeak, 2527

purity, 13, 27, 34identity, 26

pesticides, 33photo-multiplier, 11photodiode, 11Pickering, 38polynuclear aromatic hydrocarbons(PNA), 21, 25, 3032precolumn derivatization,postcolumn derivatization, 33, 37purity factor, 27pyridoxal, 43, 44pyridoxamine, 43, 44pyridoxine, 43, 44

Qqualitative analysis, 24quantitative analysis, 2526quinidine, 18, 19, 4647

RRaman, 14,17Raleigh, 17riboflavine, 4344

Sselectivity, 7, 18, 23, 25specifications, 14spectral libraries, 13

spectral mode, 67, 32spectral range, 21stray light, 1112

Raman, 11, 17Raleigh, 17

Tthree-dimensional plot, 12, 13, 17, 21, 34trichlorophenol, 31, 32

UUV/Visible absorption spectra, 6

Vvitamins, 4345

B2/B6, 4345

Wwarfarin, 48wavelength, 12

XXenon flash lamp, 1112

ZZorbax, 46, 48


54/54

Copyright 2000 Agilent Technologies

The information in this publication is

subject to change without notice.

All Rights Reserved. Reproduction,

adaptation or translation without prior

written permission is prohibited, except

as allowed under the copyright laws.

Printed in Germany 01/00

Publication Number 5968-9346E

www.agilent.com/chem

fluorescence detection in liquid chromatography (agilent technologies)

Documents