m co c. in
pHOpticaTM micro
www.wpiinc.com
Fiber Optic pH System
for pH microsensors
INSTRUCTION MANUAL
PC-controlled one-channel fiber optic pH system for pH
microsensors; excitation wavelength of 470nm; quartzquartz glass-fibers of 140 μm outer diameter connected
by ST-fiber connectors;
Table of Contents
Table of Contents
1
Preface.............................................................................................................................1
2
Safety Guidelines ...........................................................................................................2
3
Description of the pHOptica micro Device...................................................................4
4
Required Basic Equipment............................................................................................7
5
pH-Sensitive Microsensors: Sensors and Housings ..................................................8
5.1
Housings of pH-Sensitive Microsensors............................................................11
5.1.1
Needle-type - pH Micro-Sensor (NTH)............................................................12
5.1.2
Implantable pH-Micro-Sensor .........................................................................14
Description of pHOptica micro Software ...................................................................17
6
6.1
Software Installation and Starting the Instrument.............................................17
6.2
Function and Description of the pHOptica micro Program ..............................18
6.2.1
Menu Bar ........................................................................................................19
6.2.2
Control Bar......................................................................................................24
6.2.3
Graphical Window...........................................................................................30
6.2.4
Status Bar .......................................................................................................31
6.3
Subsequent Data Handling ..................................................................................31
7
Calibration and Measurement .....................................................................................33
7.1
Buffers for Calibration..........................................................................................33
7.2
Mounting the Implantable Microsensors............................................................33
7.3
Mounting the Needle-Type Microsensors ..........................................................34
7.4
pHSolver-v07.exe used for calculation of the calibration values.....................37
7.5
Calibration/Measurement of a pH Microsensors ...............................................39
7.6
Some Advice for Correct Measurement .............................................................40
7.6.1
Signal drift due to photo-decomposition .........................................................40
7.6.2
Performance proof ..........................................................................................41
General Instructions: ...................................................................................................42
8
8.1
Warm-Up Time ......................................................................................................42
8.2
Maintenance ..........................................................................................................42
9
Technical Data ..............................................................................................................43
9.1
General Data..........................................................................................................43
9.2
Analog Output and External Trigger...................................................................45
9.3
Technical Notes ....................................................................................................46
9.4
Operation Notes....................................................................................................46
10
Appendix .......................................................................................................................47
10.1
Basics in Optical Sensing....................................................................................47
10.1.1
10.1.2
10.1.3
10.1.4
10.1.5
Major Components of Fiber-Optic Microsensors ............................................47
Luminescence Decay Time ............................................................................47
Advantages of Optical Lifetime Based Sensors..............................................48
Dynamic Quenching of Luminescence ...........................................................49
Dual Lifetime Referenced (DLR) Optical Sensors ..........................................50
Table of Contents
10.1.6
10.1.7
Fluorescence (Förster) Energy Transfer (FET) ..............................................52
Literature.........................................................................................................53
Preface
1
1
Preface
Congratulations!
You have chosen a new innovative technology to read out chemo-optical sensors!
The pHOptica micro is a compact, portable, PC-controlled fiber-optic phase detection device
to read out pH sensors. The data evaluation is PC supported.
The pHOptica micro was specially developed for very small fiber-optic microsensors. It is
based on a novel technology, which creates very stable, internal referenced measured
values. The pHOptica micro is useful to read out sensors with average luminescence decay
times in the range of 1 µs to 0.6 ms.
Optical sensors (also called optodes) based on decay time measurements have important
advantages over intensity based optodes:
•
•
•
•
•
They are small
They are independent on fluctuations of the light source
Their signal does not depend on the flow rate of the sample
They are less sensitive to photo bleaching and dye leaching
They are not influenced by intensity fluctuations due to fiber bending
Therefore, they are ideally suited for the examination of small sample volumes, long term
measurements in difficult samples, and for biotechnological applications.
A set of different microsensors, flow through cells and integrated sensor systems is available
to make sure you have the sensor which is ideally suited to your application.
Please feel free to contact our service team to find the best solution for your application.
Safety Guidelines
2
2
Safety Guidelines
PLEASE READ THESE INSTRUCTIONS CAREFULLY BEFORE WORKING WITH THIS
INSTRUMENT!
This device has left our works after careful testing of all functions and safety requirements.
The perfect functioning and operational safety of the instrument can only be ensured if the
user observes the usual safety precautions as well as the specific safety guidelines stated in
these operating guidelines.
-
Before connecting the device to the electrical supply network, please ensure that the
operating voltage stated on the power supply corresponds to the mains voltage
-
The perfect functioning and operational safety of the instrument can only be
maintained under the climatic conditions specified in Chapter 9 "Technical Data" in
this operating manual.
-
If the instrument is moved from cold to warm surroundings, condense may form and
interfere with the functioning of the instrument. In this event, wait until the
temperature of the instrument reaches room temperature before putting the
instrument back into operation.
-
Balancing, maintenance and repair work must only be carried out by a suitable
qualified technician, trained by us.
-
Especially in the case of any damage to current-carrying parts, such as the power
supply cable or the power supply itself, the device must be taken out of operation and
protected against being put back into operation.
-
If there is any reason to assume that the instrument can no longer be employed
without a risk, it must be set aside and appropriately marked to prevent further use.
-
The safety of the user may be endangered, e. g., if the instrument
•
•
•
•
is visibly damaged;
no longer operates as specified;
has been stored under adverse conditions for a lengthy period of time;
has been damaged in transport
-
If you are in doubt, the instrument should be sent back to the manufacturer WPI for
repair and maintenance.
-
The operator of this measuring instrument must ensure that the following laws and
guidelines are observed when using dangerous substances:
•
•
•
•
EEC directives for protective labor legislation;
National protective labor legislation;
Safety regulations for accident prevention;
Safety data-sheets of the chemical manufacturer
The pHOptica micro is not protected against water spray;
The pHOptica micro is not water proof;
The pHOptica micro must not be used under environmental conditions which cause watercondensation in the housing;
The pHOptica micro is sealed;
The pHOptica micro must not be opened;
We explicitly draw your attention to the fact that any damage of the manufactural seal will
render of all guarantee warranties invalid.
Safety Guidelines
3
Any internal operations on the unit must be carried out by personal explicitly authorized by
WPI and under antistatic conditions.
Needle-type sensors are housed in extremely sharp syringe needles. Avoid injury by
handling the needle carefully. Please pay attention to all safety guidelines for safe handling
of sharp needles and syringes. Beware of injuring with the needle as well as with the sensor
tip. The glass fiber can break if pricked into the skin and can cause inflammation.
The pHOptica micro may only be operated by qualified personal.
This measuring instrument was developed for use in the laboratory. Thus, we must assume
that, as a result of their professional training and experience, the operators will know the
necessary safety precautions to take when handling chemicals.
Keep the pHOptica micro and the equipment such as PT1000 temperature sensor, power
supply and sensors out of the reach of children!
As the manufacturer of the pHOptica micro, we only consider ourselves responsible for
safety and performance of the device if
• the device is strictly used according to the instruction manual and the safety guidelines
• the electrical installation of the respective room corresponds to the DIN IEC/VDE
standards.
The pHOptica micro and the sensors must not be used in vivo examinations on humans!
The pHOptica micro and the sensors must not be used for human-diagnostic or
therapeutically purposes!
Description of the pHOptica micro Device
3
Description of the pHOptica micro Device
The pHOptica micro is a precise single channel
Fiber Optic pH System for pH microsensors based
on quartz-quartz glass-fibers of 140 μm. The small
outer dimensions, low power consumption and a
robust box make it ready for portable and field use.
For operation, a PC/Notebook is required. The
pHOptica micro is controlled using a comfortable
software, which also saves and visualizes the
measured values.
The pHOptica micro has 2 analog outputs (0-5 V)
and one trigger input (TTL) to be connected to a
data logger. Analog connectors are BNC
connectors.
The analog outputs are programmable to deliver
pH, temperature, or the raw values (phase shift or
amplitude), and the data are called via computer
and RS232 (digital) or using the external trigger
input (analog).
pHOptica micro instruments features:
•
•
•
•
high precision
portable (battery power optional)
analog/digital data output
external temperature measurement
Extension to a multi-channel
multi-analyte system
Using a computer port extender providing multiple
RS232 ports, up to eight single devices (OXY MICRO,
OXY MINI, pHOptica micro, …) can be connected to
one single computer. This multi-instrument set-up offers
a highly flexible method to create multi-channel, multianalyte measuring systems including additional
temperature-compensation of each channel.
4
Description of the pHOptica micro Device
Front Panel
ELEMENT
DESCRIPTION
POWER
ON/OFF switch
SENSOR
ST fiber connector
L1
Control
LED
Temp
Connector for PT 1000
temperature sensor
FUNCTION
Switches the device ON and OFF
Connect the fiber-optic sensor here.
red:
instrument off;
green: instrument on;
orange: stand by;
Connect the PT 1000 temperature sensor for
temperature compensated measurements here.
5
Description of the pHOptica micro Device
6
Rear Panel of the pHOptica micro device
Two standard BNC connectors are added for analog output channels 1 and 2, another one
for external trigger input.
The electrical specifications of all rear panel connectors are given in technical specification
sheet. Please read also the technical notes to avoid mistakes.
ELEMENT
DESCRPTION
FUNCTION
12 VCD
Line adapter for power
supply
RS 232
RS232 interface
(male)
Connect the device with a RS232 data cable to your
PC/Notebook here.
CH 1
Analog out
(channel 1)
Connect the device with external devices, e.g. a data
logger
CH 2
Analog out
(channel 2)
Connect the device with external devices, e.g. a data
logger
EXT TRIG
External trigger input
Connector for 9 - 36 V DC power supply.
Connect the device with external devices, e.g. data
logger with a trigger output, pulse generator
Required Basic Equipment
4
Required Basic Equipment
• pH meter pHOptica micro*
• Software pHOpt_v1.exe for pHOptica micro*
• PC / Notebook
(System requirements:
Windows 95/98/2000/Millenium/NT 4.0/XP; Pentium processor, at least 133 MHz, 16 MB
RAM)
• RS 232 Cable *
• Line adapter (110 - 220 V AC, 12 V DC) *
• Temperature sensor PT 1000*
• Analyte-sensitive microsensor
The sensors are mounted into different types of housings
• Vessels for calibration solutions
We recommend Schott laboratory bottles with a thread which can be obtained by VWR International
(ordering number: 215L1515)
• Laboratory support with clamp, sensor-manipulator
*: scope of supply
7
8
Fiber-Optic Microsensors: Sensors and Housings
5
pH-Sensitive Microsensors: Sensors and Housings
Optical pH Sensors designed for pHOptica micro device are based on the new Dual
Luminophor Reference method (see also appendix). They consist of an inert long decay time
reference dye and a short decay time indicator dye, which changes its fluorescence intensity
due to the pH-value. The measured value, the average decay time, represents the ratio of
the two fluorescence intensities. Therefore, the signal is internal referenced and insensitive
to fluctuations of the light source.
Dynamic range
Optical pH sensors respond according to the mass acting law, hence, they do not show a
linear behavior like potentiometric pH electrodes. This limits their use to a dynamic range of
approximately 3 to 4 pH units. On the other hand, the resolution can be very high in the
optimal range. Figure 5.1 shows a typical response curve of a pH sensor. Increasing the pH,
the signal - in our case the phase angle Φ - decreases. The phase angle Φ can be related to
the pH as shown in Figure 5.2. The theoretical aspects are explained more detailed in the
appendix.
10
60
9
55
8
50
45
phase
7
pH
Imax
Imin
dpH
pH0
6
5
40
: 18.77
: 56.33
: 00.64
: 06.51
35
30
4
25
3
0
1
2
3
time, min
4
20
15
4
5
6
7
8
9
pH
Figure 5.1 Response of pH micro-sensor pH-HP-5.
Figure 5.2 Effect of the phase angle of a pH
microsensor (pH-HP-5) on different pH values.
The characteristic of a typical sensor are listed below:
Dynamic range: pH 5.5 to 8.5
Resolution: 0.01 pH
Drift due to bleaching: 0.03 per 1000 measuring points
Response time: less then 30 sec
Please note that the given values may differ from your special device and sensoric
application.
9
Fiber-Optic Microsensors: Sensors and Housings
Resolution
7.00
Continous mode (measurement each second)
6.95
pH
6.90
6.85
6.80
0.00
2.00
4.00
6.00
8.00
10.00
time, min
Figure 5.3. Resolution of the pH sensor can be up to 0.01 pH
Cross sensitivities
While pH electrodes are influenced by sulfide, electromagnetic fields or flow velocity the
optical pH measurement is interfered by ionic strength. This problem can be overcome by a
calibration with buffers of similar ionic strength than the sample.
55
50
45
is50
is100
is200
phase
40
35
30
25
20
15
4
5
6
7
8
9
pH
Figure 5.4 Cross-sensitivity to ionic strength – we recommend to calibrate the sensor in pH-adjusted
media!
10
Fiber-Optic Microsensors: Sensors and Housings
Sensor drift
Like all optical chemo sensors, optical pH-sensors are suspect to photobleaching. Due to this
process the measured intensity (amplitude) is decreasing while the phase of the sensor is
increasing. This process is quite slow and depends on the used light intensity and the
duration of illumination. If you are in doubt whether a measured increase in phase is due to
photobleaching or decreasing pH of the sample, please lower the light intensity. If the drift is
not getting smaller, your signal drift is due to pH changes in your sample.
8
7.8
7.6
7.4
7.2
pH 7
6.8
6.6
6.4
6.2
6
0
200
400
600
800
Measuring points
1000
1200
1400
Figure 5.5 Drift due to bleaching: 0.03 per 1000 measuring points
Typical bleaching rates are listed below.
1000 Measuring points
1 hour in sec. Mode
1 d in 1 min Mode
Sensor Drift due to Bleaching
0.03 pH
0.1 pH
0.05 pH
Please notice, that bleaching rates also depend on the adjusted light intensity of the LED and the
individual instrument and sensor.
Temperature dependency
pH Micro sensors display, as any other pH sensors, a distinct temperature sensitivity. Some
typical calibration curves at different temperatures are shown below:
11
Fiber-Optic Microsensors: Sensors and Housings
55
50
5 °C
15 °C
20 °C
30 °C
35 °C
40 °C
45
Phase
40
35
30
25
20
15
4
5
6
7
8
9
10
pH
To obtain most reliable results we recommend to calibrate at the same temperature as used
for measurement. Higher temperature mimics lower pH. At pH 7 a deviation of about 0.08 ph
/ 5° C occurs.
Limitations
The offered pH sensors for pHOptica micro were specially designed for physiological
samples and media. Samples with extremely low ionic strength and low buffer content may
be not measured correct. To test whether the sample can be measured correct, try to
perform the calibration procedure with a buffer system which is as similar to the sample as
possible. If the calibration procedure does not result in a sigmoidal shaped calibration plot,
the system is not suited for the sensor. Please contact our service team for special sensors.
The measurement can also be influenced by small, highly fluorescent molecules like
fluorescein or rhodamin in the sample.
The sensors do not stand pH above 9 for prolonged time and organic solvents.
Response time
The response time (t90) of the pH sensor is dependent from the diffusion rate of protons
through the sensor layer, hence, the response time is dependent from the thickness of the
sensor layer and the stirring rate. The typical response (t90) time of a pH-micro sensor is
below 30 sec.
5.1
Housings of pH-Sensitive Microsensors
WPI fiber-optic pH microsensors are based on 140 µm silica optical fibers. To protect the
small glass fiber tip against breaking, suitable housings and tubings around it, depending on
the respective application, were designed.
WPI offers the following standard designs:
Fiber-Optic Microsensors: Sensors and Housings
5.1.1
Needle-type - pH Micro-Sensor (NTH)
Needle-type pH micro-sensors are miniaturized
fiber optic chemical sensors designed for all
research applications were a small tip size of ca.
140 µm are necessary. Needle-type pH microsensors are ideal for pH profiling in sediment
and biofilms with a high spatial resolution. The
pH-sensitive tip of the optical fiber is protected
inside a stainless steel needle. Such a design is
the best for an easy penetration through a
septum rubber or any other harsh material as
well as secure for transportation. For
measurement purposes the sensor must be
pushed out from the needle.
12
Fiber-Optic Microsensors: Sensors and Housings
Schematic drawing of the NTH (Needle-type) micro-sensor
male fiber-optic plug
spring
safety nut
fiber cable
~
syringe plunger
~
needle plastic base
syringe housing
syringe needle
transport block
needle plastic base
glass fiber
protective plastic cap
sensor tip
syringe needle
FEATURES
•
•
•
•
•
•
•
•
High spatial resolution (ca. 140 µm)
Penetration probe for insertion into semisolids like sediments or biofilms
Penetration through septa
Easy to handle
Different needle size available
No reference electrode needed
High temporal resolution
Insensible towards electrical interferences and magnetic fields
SPECIFICATION
Tip size
140 µm
Measuring range
5 – 9 pH
Response time
30 sec
Resolution
Up to 0.01 pH
13
Fiber-Optic Microsensors: Sensors and Housings
Drift
(per 1000 Measuring points)
0.03 pH
Temperature Range
from 5 °C to + 50 °C
Cross-Sensitivity
Optical pH sensors display slight cross sensitivity to
ionic strength (salinity) and towards small
fluorescent molecules like dissolved indicator dyes.
14
ORDER
The NTH (Needle-type) pH sensors are offered with options to be specified in the purchase order
form. The order code key shown below (see also the example) defines the parameters. Please,
choose the parameters that best meet Your requirements.
NTH
pH
HP5
L
NS
Code of pHNeedle-Type
sensor coating
Housing Analyte pH
Length of
Glass Fiber
- 2.5 m
-5m
- 10 m
Stainless Needle
Length [mm] / diameter [mm]
- 20 / 0.4
- 40 / 0.4
- 40 / 1.2
- 120 / 0.8
EXAMPLE
NTH
pH
HP5
L
5
NS 40 0.4
With this code you will order a microsensor mounted in a needle-type housing (NTH), with the pH
sensitive coating HP5 with a glass fiber length of 5 m (L5) mounted in a stainless needle of 40 mm
length and 0.4 mm diameter (NS 40/0.4) .
5.1.2
Implantable pH-Micro-Sensor
Implantable pH micro-sensors are the
miniaturized fiber optic chemical sensor
designed for all research applications were a
small tip size of 140 µm are necessary.
Implantable pH micro-sensors are ideal for
costumer-specific set up. They were applied
for implantation in blood circuits and for
profiling with a high spatial resolution
Fiber-Optic Microsensors: Sensors and Housings
Schematic drawing of the Implantable micro-sensor
FEATURES
•
High spatial resolution (ca. 140 µm)
•
Easy to handle
•
Most flexible for a wide range of applications
•
High temporal resolution
•
Insensible towards electrical interferences and magnetic fields
SPECIFICATION
Tip size
140 µm
Measuring range
5 – 9 pH
Response time
30 sec
Resolution
Up to 0.01 pH
Drift
(per 1000
points)
Measuring 0.03 pH
Temperature Range
from 5 °C to + 50 °C
Cross-Sensitivity
Optical pH sensors display
slight cross sensitivity to ionic
strength
(salinity)
and
towards small fluorescent
molecules
like dissolved
indicator dyes.
15
16
Fiber-Optic Microsensors: Sensors and Housings
ORDER
The Implantable pH sensors are offered with options to be specified in the purchase order
form. The order code key shown below (see also the example) defines the parameters.
Please, choose the parameters that best meet Your requirements.
IMP
Implantable
Housing
pH
HP5
900/
600/
140/
inner plastic cable
Code of pHdiameter
[µm] / length [cm]
sensor coating
600
µm
/
0
to
customer request
Analyte pH
outer plastic cable
length of bare glass fiber
Diameter [µm] / length [m]
Diameter [µm] / length [mm]
900 µm / 0 10
140 µm / 1 to customer request
EXAMPLE
IMP
pH
HP5
900/ 5
600/ 1
140/ 2
This is the order code for the implantable micro-sensor (IMP) with the pH sensitive coating
HP5, with a fiber cable length of 5 m (900/5), a cable plastic jacket length of 1 cm (600/1), a
bare optical fiber length of 2 mm (140/2)
Description of pHOptica micro Software
6
17
Description of pHOptica micro Software
This software is compatible with Windows 95/98/2000/Millenium/NT4.0/XP.
6.1
1.
2.
3.
4.
5.
Software Installation and Starting the Instrument
Insert the supplied disc/CD into the respective drive. Copy the file pHOpt_v1.exe onto
your hard disk. (for example, create C:\pHOptica_micro\ pHOpt_v1.exe). Additionally,
you may create a link (Icon) on your desktop.
Connect the pHOptica micro via the supplied serial cable to a serial port of your
computer. Tighten the cable with the screws on your computer and on the pHOptica
micro.
Connect the power supply.
Please close all other applications as they may interfere with the software. Start the
program pHOpt_v1.exe with a double click. The following information window appears:
If the right com port is adjusted this information window disappears within a few
seconds. If the wrong com port is adjusted you are asked to set the right com port:
With a right mouse click onto ‘com port’ you are able to set the right com port. Please
confirm your selection by clicking the ‘OK’ button. The information window disappears if
the right com port is adjusted.
Description of pHOptica micro Software
6.2
Function and Description of the pHOptica micro Program
The window shown below is displayed after starting the software pHOpt_v1.exe:
The program has 4 main sections:
1. Menu bar
2. Graphical window
3. Status bar
4. Control bar, divided into numerical display, control buttons and warning lights
menu bar
control buttons
warning lights
numerical display
graphical window
18
Description of pHOptica micro Software
6.2.1
Menu Bar
File
Æ Exit
Charts
Æ pH
Display
Æ Zoom
Æ AutoScaleY1
Æ Undo Zoom
Æ Temperature
Æ Amplitude
Æ Phase
Print
Æ Charts
Æ Clear Charts
Æ Dimensions
Settings
Æ Com Port
Æ Instrument Info
Æ analog settings
Æ LED Intensity
Æ Frequency
File
Exit
Closes the program.
Charts
The respective charts of the measurement can be displayed (√) or hidden
pH:
The measured pH value
Temperature:
The measured temperature
Amplitude:
The magnitude of the sensor signal
Phase:
Phase angle, the raw data
Display
Zoom:
AutoScaleY1 is the default setting. AutoScaleY1 means that the y-axis is scaled
automatically.
Undo Zoom: The original display is recovered; see also graphical display
Clear Charts: The graphs shown on the display is cleared.
19
Description of pHOptica micro Software
20
Dimensions:
You can adjust the number of
measurements points on the x-axis
shown in the display (maximum
number of points are 5000)
Furthermore, you can adjust the
micromum and the maximum of the yaxis.
The AutoScaleY1 function is switched
off.
Print
Charts: The charts shown in the display can be printed
Settings
ComPort
The serial comport (com1 – com20) for the serial interface (RS 232) can be chosen in this
window. COM 1 is the default setting. If you choose the wrong Com port, the information
window ‘Connect the instrument to the PC and choose the right com-port’ does not
disappear.
Instrument Info:
Here you can find the version of the software and some important settings of the instrument.
If you have a problem with the pHOptica micro device, please contact our service team and
have the software and instrument information ready.
To change back to the graphical window click the ‘Measure Chart’ button.
Description of pHOptica micro Software
Instrument Info
Software Info
21
Description of pHOptica micro Software
22
LED-Intensity
With the current of the LED you can adjust the amount of light illuminating the sensor.
You can choose between an ‘Auto Adjust’ of the LED where the pHOptica micro adjusts the
optimal LED current itself, or you can select ‘Advanced’ where you can adjust the LED
current yourself.
If you increase the LED current, the signal amplitude increases, since a higher light density
illuminates the sensor. Higher amplitudes lead to more stable values and therefore higher
resolution, but also to increased photobleaching.
Auto Adjust:
To make the adjustment of the LED intensity automatically, just click the button ‘Start Auto
Adjust’. Please check that the Microsensors has been connected to the instrument.
The automatically adjustment of the LED intensity is finished when in the status window the
message ‘Auto adjustment finished’ appears. Click the ‘Close’ button to confirm the
settings.
Description of pHOptica micro Software
23
Advanced:
Click the ‘Advanced’ button to change the LED current manually. Values between 10 and
100 % are possible. After clicking the ‘confirm’ button you can see the change of the
amplitude in the window below.
Please note:
By increasing the light intensity you increase the amplitude of the sensor. This leads to
smoother phase signals. However, increasing the light intensity can increase photobleaching,
which decreases the shelf-life of your sensor.
Frequency - This option is only for advanced users with special adapted sensors.
Please refer to the special advice of our service team.
With the menu frequency you can adjust the optimal modulation frequency of your analytesensitive indicator dye. Please chose 49: 44.86 kHz for the pH sensor pH-HP5.
If you change the settings you have to restart the software.
Description of pHOptica micro Software
24
Analog output
Here you can choose which data are exported via the analog output. The pHOptica micro
device has two analog outputs and one trigger input. The desired data sources (temperature,
amplitude, phase) can be chosen via the dialog box.
Equivalence coefficient
temperature
amplitude
phase
6.2.2
1 : 0.1 (e.g. 208mV = 20.8°C)
1 : 20 (e.g. 1110mV = 22200 relative units)
1 : 0.025 (e.g. 1100mV = 27.50°)
Control Bar
Numerical display
The measured pH is shown in the display window.
Description of pHOptica micro Software
25
Temperature measurement:
The actual temperature value of the sample (in the case of temperature compensated
measurements) is displayed in the temperature window.
If measurement is performed without temperature compensation, the manual inserted
temperature is displayed with the hint that temperature measurement is off–line.
Control buttons:
The way to start a measurement is
(1) Calibration of the sensor by input of calibration values
(2) Start Measurement with Assistant
(3) Log Data
Calibration:
Type in the calibration values. Calibration values are obtained using pHSolver-v07.exe.
Please see 7.1 pHSolver-v07.exe used for calculation of the calibration values for
details
Description of pHOptica micro Software
26
Measurement:
The measurement assistant is opened (default setting).
Quick Start:
The measurement is started. The measurement settings are continuous mode which means
that each second a new measurement data is recorded. The measurement is temperature
compensated i.e. a temperature sensor has to be connected. If no temperature sensor is
connected the following warning window appears.
Click the ‘Close’ button if you want to continue the measurement without temperature
compensation. The temperature is set to 20 °C by the software. Connect the temperature
sensor if you want to perform a temperature compensated measurement.
If you want to change the measurement settings click the ‘Advanced start’ button.
Please note:
The measurement values are not stored. Click the ‘Log Data’ button to store the
measurement data.
Advanced Start:
In the ‘Advanced Start’ mode it is possible to adjust user-defined measurement settings.
In the ‘Sampling Rate’ window you can select the desired measurement mode with a dropdown menu.
Description of pHOptica micro Software
27
By clicking the drop down menu you can choose from ‘fast sampling’ (update rate each 250
– 450 ms) to the ‘60 min’ mode where each hour a measuring point is recorded.
The speed of recording a measurement point in the ‘fast sampling’ mode is about 250 ms
when no temperature sensor is connected and the analog output settings are switched off
and decreases to about 450 ms when connecting a temperature sensor or activating the
analog output channels.
Please note:
The sensor shelf life can be increased using a slower measuring mode since the effect of
photo-bleaching is reduced. The illumination light is switched off between sampling. A further
advantage using a high measuring mode is that huge amounts of data for long-time
measurement can be avoided.
Dynamic averaging
The ’dynamic average’ defines number of averaged
measured values. The higher the running average, the
longer the time (sampling time) used for averaging. The
higher the running average is set, the smoother the
measurement signal (maximum 25 samples); The
default setting is 4.
Additional temperature measurements
In the ‘temperature compensation’ window you can decide whether you want to measure
with or without temperature measurement.
If you want to measure with the additional temperature sensor Pt1000, click the ’on’ button.
Please ensure that the temperature sensor Pt1000 is connected to the pHOptica micro,
before you click the ‘Start’ button to continue. The window where you can enter the
temperature manually is disabled.
Description of pHOptica micro Software
28
If you want to measure without temperature compensation, choose the ’off’ button. You will
now be requested to enter the temperature of the sample manually. Click the ’Start’ button to
start the measurement.
Log Data:
To store the data of your measurement click the ‘Log Data’ item. Next to the ‘Log Data’ item
an information window displays whether the actual measurement is stored to a file (logging)
or not (no logging);
The measurement description which you can enter in the text field ‘Enter a description to
the header of the file’ is stored in the Ascii File.
By clicking the button ‘Choose File’, you can select the location where you want to store the
data. Choose as file extension *.txt. Click the ’speichern’ button to confirm your settings.
Description of pHOptica micro Software
29
By clicking the ‘Stop Log Data’ item you stop data logging which is displayed by the blinking
‘no logging’ in the information window next to it.
Stop Measurement
The measurement is ended by a left click on the ’stop’ button in the control bar.
Description of pHOptica micro Software
30
Warning Lights:
At the right bottom of the window you can find the amplitude, phase angle and three warning
lights. The warning lights are explained below:
amplitude:
red:
green:
Amplitude is too low, the sensor tip may be damaged or sensor
cable may not be connected
Amplitude is critically low, replacement of the sensor is
recommended
amplitude is correct
red:
green:
phase angle is out of limits
phase angle is in normal range
yellow:
phase:
ambient light: red:
green:
background light (e.g. direct sunlight, lamp) is too high. Decrease
of false light is recommended
ratio of sensor signal to false light is acceptable
By clicking the ‘Display Raw Values’ button, the raw data of
phase angle and amplitude are displayed next to the warning
lights.
6.2.3
Graphical Window
The respective sensor signal is displayed according to the selection of the 4 control buttons
pH, phase, amplitude and temperature (menu chart). The raw values (the phase angle in
degrees and the sensor amplitude in mV) can also be displayed by clicking the button
‘Display Raw values’. The temperature is given in [°C]
Zoom Function:
1. Press the left mouse button and drag from left to right to enlarge a certain area of the
graphical window. The graphical window displays the selected data points and is not
actualized with new data.
2. Press the left mouse button and drag from right to left to recover the original display, or
click the ‘Undo Zoom’ button in the display menu under zoom.
31
Description of pHOptica micro Software
6.2.4
Status Bar
sw
sw
sw
sw
sw1: Displays the serial port which is used for communication of the pHOptica micro device
with the PC
sw2: Displays the file name in which the measurement data are stored. „No storage file
selected“ is displayed if no file was selected (no data storage).
sw3: Displays the start time of the measurement
sw4: Displays the actual time
6.3
Subsequent Data Handling
In the head of the ASCII file, you find the description of your measurement which you have
entered by storing the file.
Below you find the ‘instrument info’ containing all important settings of the instrument and
firmware and the calibration values and the date of the calibration
.
The ‘software info’ below contains the version number of the pHOptica micro software, date
and time of the performed measurement. If there is a problem with the pHOptica micro
device, please contact our service team and have the software and instrument information
ready.
Below, you find the ‘measure mode settings’ containing the dynamic averaging, and the
measuring mode.
The following rows, separated by semicolons, list the measuring data. The first two rows
contain the date and time, the third the log-time in minutes. The pH values are stored in the
fourth row. The raw data - phase angle in [°] and the amplitude in [mV] - are stored in the
fifth and sixth row, respectively. The seventh row contains the temperature in °C measured
by PT1000 temperature sensor. Raw data can be used for user defined recalculations
according to the formulas and tables of the delivered Excel sheet
The eigthth raw of the Ascii file displays possible error messages.
32
Description of pHOptica micro Software
***** DESCRIPTION ***********
pHOptica micro sample#1
***** INSTRUMENT INFO *******
IDENTIFICATION
PHIboard number : 20030007
PM number
: 20030110
Serial number : pH-1-micro-AOT-03-007
MUX channel
: OFF - 00
PARAMETERS
Signal LED current: 050
Ref LED current : 058
Ref LED amplitude : 88397
Frequency
: 049
Sending interval : 0001
Averaging
:5
Internal temp : 26.8 C
SYSTEM SETTINGS
APL function
: OFF
Temp compensation : ON - ch a
Analog out
: chA t chB t
RS232 echo
: ON
CALIBRATION
Sensor type
:1
Imax
: 22.00
Imin
: 54.00
dpH
: 00.66
pH0
: 07.00
Date (ddmmyy) : 241003
Pressure (mBar) : 0020
FIRMWARE
Code v1.088PH IAP: 07/31/03, 10:45:01
Xilinx built
: 20/08/02 (MM/DD/YY)
Reset condition : SLEEP
***** SOFTWARE INFO *********
pHOpt_v1 - 01/2004
24.10.2005
10:43:16
******MEASURE MODE SETTINGS**
Dynamic Averaging
4
measure mode
1 sec
start time
10:37:44
date(DD/MM/YY)
time/hh:mm:ss logtime/min pH
24.10.2005 10:43:16
0
24.10.2005 10:43:17
0.017
24.10.2005 10:43:19
0.034
24.10.2005 10:43:20
0.051
24.10.2005 10:43:21
0.068
24.10.2005 10:43:22
0.086
24.10.2005 10:43:23
0.103
24.10.2005 10:43:24
0.12
24.10.2005 10:43:25
0.137
24.10.2005 10:43:26
0.154
24.10.2005 10:43:27
0.171
24.10.2005 10:43:28
0.188
24.10.2005 10:43:29
0.205
phase/°
5.09
5.075
5.068
5.08
5.08
5.07
5.105
5.105
5.085
5.098
5.093
5.128
5.12
amp
52.3
52.34
52.35
52.32
52.32
52.35
52.27
52.26
52.31
52.28
52.29
52.2
52.22
temp/°C
3464
3475
3487
3461
3477
3494
3479
3468
3454
3464
3468
3481
3465
ErrorMessage
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
21.5 E0
Measurement
7
33
Calibration and Measurement
Calibration of the sensors is recommended before each measurement. Each calibration is
only valid for the corresponding sensor and should be repeated before beginning a new
measurement. Especially, after longer measurements (more than 1000 measuring points) the
sensor should be re-calibrated.
7.1
Buffers for Calibration
For the calibration of pH Micro-sensors at least 4 different buffers are needed. To obtain best
results a similar composition as the measured sample is recommended. For example, for
measurement in cell culture media calibration in buffers with an ionic strength of 140 mM and
a phenol red concentration of 15 mg/l is ideal. Please notice, that the pH range of the
calibration should exceed the pH range of the measurement – e.g. it is not favorable to
calibrate with buffers of pH 4.0, 5.0, 5.5, and 6.0 and to measure about pH 7. Calibration is
only correct for interpolation, not for extrapolation.
7.2
Mounting the Implantable Microsensors
1.
Remove the microsensor carefully from the protective cover. The microsensor is
protected with a glass housing during the transport.
2.
Fix the glass housing microsensor with a clip to a laboratory support or a similar stable
construction.
We strongly advise you not to handle with microsensors without the support especially when the sensor tip is extended.
34
Measurement
3.
Remove the protective cap from the male fiber plug and connect it to the ST-plug of the
pHOptica micro device. The female fiber-plug of the pHOptica micro has a groove in
which the spring of the male fiber-plug of the microsensor has to be inserted. The safety
nut must be carefully attached while turning and is locked by turning slightly clockwise.
Be careful not to snap off the fiber cable.
ST-connector
male fiber plug
7.3
Mounting the Needle-Type Microsensors
1.
Remove the microsensor carefully from the protective cover. The needle-type
microsensor is housed in 0.4 x 40 mm syringe needle mounted to a 1 mL plastic syringe
housing with integrated PUSH & PULL - IN & OUT mechanism. The syringe needle is
protected with a protective plastic cap (A).
2.
Carefully remove the protective plastic cap (A) covering the syringe needle.
When doing so, grip the plastic base of the needle tightly. The syringe needle must not
be removed from the syringe housing. Work carefully!
35
Measurement
tightly grip
the needle base
3.
Fix the microsensor with a clip to a laboratory support or a similar stable construction.
We expressly warn you not to handle with microsensors without the support especially when the sensor tip is extended.
4.
Remove the protective cap from the male fiber plug and connect it to the ST-plug of the
pHOptica micro device. The female fiber-plug of the pHOptica micro has a groove in
which the spring of the male fiber-plug of the microsensor has to be inserted. The safety
36
Measurement
nut must be carefully attached while turning and is locked by turning slightly clockwise.
Be careful not to snap off the fiber cable.
ST-connector
male fiber plug
5.
The glass fiber with its sensing tip is prevented from slipping using a transport block (B).
Remove the transport block from the hole in the syringe housing. Now it is possible to
retract or extend the glass fiber with its sensor tip by pushing or pulling the plunger.
Before pushing out the sensor tip, make sure that you have removed the protective
plastic cap and have some space in front of the syringe needle.
sensor tip
Measurement
37
WHEN GLASS-FIBER WITH ITS SENSOR TIP IS PUSHED OUT, HANDLE WITH CARE.
THE GLASS FIBER IS UNPROTECTED AND MIGHT BREAK
7.4
pHSolver-v07.exe used for calculation of the calibration values
To receive pH values it is necessary to determine calibration values of the sensor. This can
be done by the use of pHSolver-v07.exe. Other fitting programs (e.g. Origin or MathLab)
which can handle the Boltzman equation can be used too.
The delivered File pHSolver-v07.exe does all the calculations. It calculates a Boltzman
curve fit for the measured values.
Ensure that dot instead of comma is used as decimal separation (US-Standard)
1)
Copy the files libraryfiles.exe and pHSolver-v07.exe to a folder named pH-solver.
2)
Start libraryfiles.exe by doubleclick and follow the instructions. If the software asks you
if an existing file should be overwritten, please click on NO.
3)
Start the software pHsolver-v07 by double click.
4)
Type in the calibration values of the sensor data sheet into the respective "inital value"
fields.
Measurement
5)
Type in the pH-phase value pairs of your calibration (first pH value of the buffer, then
space, then phase, enter, next pair.) Use at least 5 values.
Type in calibration values
Or use the paste data function (for example copy a set of data of a excel sheet).
Or open a data file containing the data in the format:
38
Measurement
6)
7.5
39
Click on "least square fit" and you will get the set of calibration data in the calibration
result area. You can store the data in a file by pressing “transfer data”. If the "initial
guess" is far from the real data, the software may ask you to try again - please just
click on yes, the software will find a new guess on its own.
Calibration/Measurement of a pH Microsensors
1.
Connect the pHOptica micro via the RS232 cable to your computer.
2.
Switch on the pHOptica micro and connect the sensor as shown in Chapters 7.2 to 7.3
depending on the sensor in use.
3.
Start the pHOptica micro software on your computer and start the measurement (see
chapter 6.2.2).
4.
Calibrate the pH sensor, pH-sensitive foil with the calibration solutions (e.g. buffer
solutions of pH 5, pH 6, pH 7, and pH 8). To minimize the response time, slightly stir the
buffer solution. Please ensure that the sensor is dipped in the buffer solution.
5.
Wait about 3 minutes until the phase angle belonging the respective buffer is constant
(the variation of the phase angle should be smaller than ± 0.1°)
6.
Wash the sensor tip with distilled water to clean it from respective buffer solution before
taking the next calibration value.
7.
Open the file pHSolver-v07.exe and enter the pH-values of the used buffer (i.e. 4.0, 5.0,
6.0, 7.0, 8.0 and 9.0) and the corresponding phase values in the calibration value
window (see above).
8.
Press the "least square fit” button.
9.
The calibration results phasemax, phasemin, dpH, pH0 are shown and the calibration curve
is displayed.
Measurement
40
10. Wash the sensor with distilled water to clean it from buffer components.
7.6
7.6.1
Some Advice for Correct Measurement
Signal drift due to photo-decomposition
pH – sensor-sensors are suspect to photobleaching which results in a drift of the sensor to
lower pH. Photobleaching is higher at basic pH. Photobleaching depends at the amount of
light. Therefore you should reduce the amount of light. The pHOptica micro device offers two
ways of reducing the amount of light.
First, you can lower the amount of measuring points in the case of long term measurements
by choosing the second or minute mode in the measuring assistant (see chapter 6.2.2.
Control Bar, Advanced Measurement)
Second, you can lower the LED current:
Choose LED-Intensity in the Settings menu of the menu bar.
With the current of the LED you can adjust the amount of light illuminating the sensor spot.
By increasing the light intensity you increase the amplitude of the sensor. This leads to
smoother phase signals. However, increasing the light intensity can increase photobleaching,
which decreases the shelf-life of your sensor.
Bleaching always mimic lower pH. For long term measurements the drift can be estimated by
the amount of measurement points and LED intensity. The sensor should be recalibrated
after the measurement and the calibration values compared to the initial values to obtain
maximum accuracy.
Please notice, that the noise (and therefore the resolution) of the signal depends on the
amplitude of the sensor. Lowering the LED current will lead to lower photobleaching but
increased noise of the sensor. The best resolution is achieved if the amplitude of the sensor
is more then 5000. An amplitude of less then 2000 will result in a high noise of up to 0.1 pH.
41
Measurement
7.00
Continous mode (measurement each second)
6.95
pH
6.90
phase/° Led100
phase/° LED 15
6.85
Linear ( phase/° Led100)
6.80
0.00
Linear ( phase/° LED 15)
2.00
4.00
6.00
8.00
10.00
time, min
Figure 7.3 Phase resolution illumination the sensor tip with 15% and with 100 % LED current,
respectively.
7.6.2
Performance proof
If you want to prove the performance during the past measurement, please check the
calibration buffers by inserting the sensor tip in the buffer solution pH 6 and pH 7 when you
have finished your measurement. If the device displays the correct pH values, the sensor
worked perfectly during the whole measurement.
If you are in doubt whether the sensor has to be changed, please follow this procedure:
1) Expose the sensor to pH 4 – Buffer solution (e.g. Certipur Buffersolution pH 4, Merck,
109435) and adjust intensity to a value of more then 2000 (ideally more then 10000)
by adjusting the LED current (see page 20). If this is not possible and the fiber cable
and connection to the sensor is OK, change the sensor. The measured phase must
be between 60 and 48 ° - otherwise check frequency (must be 049) or change
sensor.
2) Expose the sensor to pH 9 – Buffer solution (e.g. Certipur Buffersolution pH 9,
Merck, 109461). The measured phase must be between 35 and 10° - otherwise
change sensor.
General Instructions
8
General Instructions:
8.1
Warm-Up Time
42
The warm up time of the electronic and opto-electronic components of the pHOptica micro is
5 min. Afterwards stable measuring values are obtained.
8.2
Maintenance
The instrument is maintenance-free.
The housing should be cleaned only with a moist cloth. Avoid any moisture entering the
housing! Never use benzine, acetone, alcohol or other organic solvents.
The ST-fiber connector of the sensor can be cleaned only with lint-free cloth. The sensor tip
may be rinsed only with distilled water. Please ensure, that no sample residues are inside the
syringe needle. If necessary, rinse the glass-fiber with distilled water.
43
Technical Data
9
Technical Data
9.1
General Data
MODES
pH
range: 4.5 – 8.5
resolution: 0.01 pH units
Electrical temperature sensor
range: 0 - 50 °C
(Pt 1000)
resolution: ± 0.5 °C
accuracy: ± 2° C
OPTICAL OUTPUT / INPUT
Optical connector
ST compatible, Core/Center 100/140
Channels
1
Wavelength
470 nm
TEMPERATURE SENSOR INPUT
Lemo Connector Size 00
DC INPUT
Connector for Pt-1000 temperature sensor
1
4
2
3
DC-Range :
12 V/1250mA up to 18V/900mA
Technical Data
44
DIGITAL OUTPUT
communication protocoll
serial interface RS232
19200 Baud, Databits 8, Stoppbits 1, Parity none, Handshake
none
instrument output:
on RJ11 4/4 plug
Interface cable to PC:
RJ11 4/4 to DSub9:
ENVIRONMENTAL CONDITIONS
Operating temperature
Storage temperature
0 to +50ºC
-10 to +65ºC
Relative humidity:
up to 95%
OPERATION CONTROL
LED at the front panel:
red:
instrument off
green: instrument on
orange: stand by
DIMENSIONS
length: 185 mm;
width: 110 mm;
height: 45 mm;
weight: 630 g;
Technical Data
9.2
45
Analog Output and External Trigger
The device is supplied with a dual programmable 12bit analog output with galvanic isolation
and an external trigger input.
•
ANALOG OUTPUT
GENERAL SPECIFICATION - ANALOG OUTPUT
Channels
Connector
Resolution
Output range
Galvanic isolation
Shortcut protection
2
BNC
12 bit
0 to 4095mV (±2mV max. error)
500V rms
Yes
Programmable to
temperature, amplitude, phase by software
Equivalence coefficients :
temperature
1 :: 0.1
amplitude
1 :: 10
phase
1 :: 0.025
(i.e. : 208 mV = 20.8°C)
(i.e. : 2220 mV = 22200 relative units)
(i.e. : 1100 mV = 27.50°)
Update rate:
The update rate is dependent on the sampling rate of the software.
If an external trigger is used, the update rate is equivalent to the trigger pulse rate.
DC SPECIFICATION - ANALOG OUTPUT
Resolution
temperature
amplitude
phase
± 2mV Î ± 0.2°C
± 2mV Î ± 20 relative units
± 2mV Î ± 0.05°)
Accuracy error
± 10mV
Technical Data
•
46
EXTERNAL TRIGGER INPUT
GENERAL SPECIFICATION Channels
Connector
Input voltage range
Trigger mode
Normal state
Isolation
EXTERNAL TRIGGER INPUT
1
BNC
TTL-compatible / up to 24V
Low-High-Low
(Input must be kept Low for at least 50µs)
no current
500V rms
Timing Specifications:
Min rise &fall time for trigger
Max rise &fall time for trigger
Min pulse length
Min pause length
Min periode length
9.3
15ns (see TTL-specification)
2 ms
3 ms
10 ms
13 ms
Technical Notes
Power Adapter
pHOptica micro should always be used with the original power adapter (110220VAC/12VDC). As an alternative power source a battery can be used that meets the DC
input voltage given in technical specification. The battery adapter cable is available as an
additional accessory.
Analog Outputs
WARNING: The analog outputs are not protected against any input voltage! Any voltage
applied to the analog outputs can cause irreversible damage of the circuit.
RS232 Interface
The unit uses special interface cable. Another cable can cause the unit’s malfunction.
Optical Output (ST)
The ST connector is a high precision optical component. Please keep it clean and dry.
Always use the rubber cap to close the output when not in use.
9.4
Operation Notes
Measurement
To achieve the highest accuracy pHOptica micro should be warmed-up for 5min before
starting the measurement. Please see the details of measurement process described in
pHOptica micro manual.
Temperature Compensation
No other than supplied temperature sensor could be used with the unit. The use of any other
temperature sensor can damage the device.
47
10 Appendix
10.1 Basics in Optical Sensing
10.1.1
Major Components of Fiber-Optic Microsensors
In optical chemical sensors, the analyte interacts with an indicator and changes its optical
properties. The result is either a change in the color (absorbance or spectral distribution) or
the luminescence properties (intensity, lifetime, polarisation). Light acts as the carrier of the
information.
The major components of a typical fiber optical sensing system are
•
•
•
•
a light source to illuminate the sensor (laser, light emitting diode, lamps)
an optical fiber as signal transducer (plastic or glass fiber)
a photodetector (photodiode, photomultiplier tube, CCD-array)
the optical sensor (indicator immobilised in a solid matrix)
glass-fiber
with its sensor tip
OF
ST
syringe needle
LEDsig
silica fiber coupler
sensor housing
PMT
ST
microoptode
LEDref
Figure 11.1 Schematic drawing of the optical setup of
a measuring system with Microsensors
(LED: light emitting diodes, PMT: photomultiplier, OF:
optical filters, ST: fiber connector)
10.1.2
Figure 11.2 Scheme of a sensor
Luminescence Decay Time
The pHOptica micro measures the luminescence decay time of the immobilised luminophore
as the analyte dependent parameter.
τ = f([O2])
(1)
The pHOptica micro uses the phase-modulation technique to evaluate the luminescence
decay time of the indicators. If the luminophore is exited with a sinusoidally intensity
modulated light, its decay time causes a time delay in the emitted light signal. In technical
terms, this delay is the phase angle between the exiting and emitted signal. This phase angle
is shifted as a function of the analyte concentration. The relation between decay time τ and
the phase angle Φ is shown by the following equation:
tan Φ
2π ⋅ f mod
(2a)
tan Φ = 2π ⋅ f mod ⋅ τ
(2b)
τ ≡ tan Φ ≡ Φ ≡ f([O2])
(2c)
τ=
τ: luminescence decay time; Φ: phase angle; fmod: modulation frequency
48
I/Imax
I/Imax
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
reference signal
Φ1
τ0
τ1
Φ0
-5
0
5
10 15
time [µs]
20
25
Figure 11.3 Schematic of the single
exponential decay (t0 > t1).
10.1.3
30
-5
0
5
measuring
signal
10 15 20
time [µs]
25
30
Figure 11.4 The luminophore is excited with sinusoidally
modulated light. Emission is delayed in phase expressed by the
phase angle F relative to the excitation signal, caused by the
decay time of the excited state
Advantages of Optical Lifetime Based Sensors
•
•
•
•
the signal is independent of changes in flow velocity;
they are insensible towards electrical interferences and magnetic fields;
long-term stability and low drift;
using silica fibers, it is possible to measure in samples while physically separate from the
light source and detectors;
• light conducting fibers are able to transport more information than power currents
(information can be simultaneously transferred, e.g., intensity of light, spectral distribution,
polarisation, information such as decay time or delayed fluorescence);
The measurement of the luminescence decay time, an intrinsically referenced parameter,
has the following advantages compared to the conventional intensity measurement.
• The decay time does not depend on fluctuations in the intensity of the light source and the
sensitivity of the detector;
• The decay time is not influenced by signal loss caused by fiber bending or by intensity
changes caused by changes in the geometry of the sensor;
• The decay time is, to a great extent, independent of the concentration of the indicator in
the sensitive layer Î Photobleaching and leaching of the indicator dye has no influence
on the measuring signal;
• The decay time is not influenced by variations in the optical properties of the sample
including turbidity, refractive index and coloration.
49
The measurement of the luminescence decay time, an intrinsically referenced parameter,
has the following advantages compared to the conventional intensity measurement.
• The decay time does not depend on fluctuations in the intensity of the light source and the
sensitivity of the detector;
• The decay time is not influenced by signal loss caused by fiber bending or by intensity
changes caused by changes in the geometry of the sensor;
• The decay time is, to a great extent, independent of the concentration of the indicator in
the sensitive layer Î Photobleaching and leaching of the indicator dye has less influence
on the measuring signal;
The decay time is not influenced by variations in the optical properties of the sample
including turbidity, refractive index and coloration.
10.1.4
Dynamic Quenching of Luminescence
The principle of measurement is based on the effect of dynamic luminescence quenching by
molecular oxygen. The following scheme explains the principle of dynamic luminescence
quenching by oxygen.
emission of
light
1
absorption of light
excited
state
.
energy transfer
by collision
Î no emission of light
2
Figure 11.5 Principle of dynamic quenching of luminescence by molecular oxygen
(1) Luminescence process in absence of oxygen
(2) Deactivation of the luminescent indicator molecule by molecular oxygen
The collision between the luminophore in its excited state and the quencher (oxygen) results
in radiationless deactivation and is called collisional or dynamic quenching. After collision,
energy transfer takes place from the excited indicator molecule to oxygen which
consequently is transferred from its ground state (triplet state) to its excited singlet state. As
a result, the indicator molecule does not emit luminescence and the measurable
luminescence signal decreases.
A relation exists between the oxygen concentration in the sample and the luminescence
intensity as well as the luminescence lifetime which is described in the Stern-Volmerequation (1). Here, τ0 and τ are the luminescence decay times in absence and presence of
oxygen (I0 and I are the respective luminescence intensities), [O2] the oxygen concentration
and KSV the overall quenching constant
50
I0 τ 0
=
= 1 + K SV ⋅ [O 2 ]
I
τ
I = f([O 2 ])
(3)
τ = f([O 2 ])
I:
I0:
τ:
τ 0:
KSV:
Luminescence intensity in presence of oxygen
Luminescence intensity in absence of oxygen
Luminescence decay time in presence of oxygen
Luminescence decay time in absence of oxygen
Stern-Volmer constant (quantifies the quenching efficiency and therefore the sensitivity
of the sensor)
[O2]: oxygen content
1.0
(B)
(A)
5
0.8
4
0.6
3
I0/I or τ0/τ
I/I0 or τ/τ0
6
0.4
2
0.2
0
20
40
60
80
1
100
oxygen content [%]
Figure. 11.6 (A) Luminescence decrease in the presence of oxygen. (B) Stern-Volmer plot.
Indicator dyes quenched by oxygen are, for example polycyclic aromatic hydrocarbons,
transition metal complexes of Ru(II), Os(II) and Rh(II), and phosphorescent porphyrins
containing Pt(II) or Pd(II) as the central atom.
10.1.5
Dual Lifetime Referenced (DLR) Optical Sensors
The measurement of intensity is simple in terms of instrumentation but its accuracy is often
compromised by adverse effects such as drifts of the opto-electronic system and variations in
the optical properties of the sample including fluorophore concentration, turbidity, coloration
and refractive index. Therefore, efficient referencing methods are required for quantification
of intensity signals. Among those, ratiometry, i.e., the measurement of the fluorescence
intensity at two or more wavelengths of a single indicator fluorophore or an indicator
fluorophore plus an inert fluorescence standard, is common to reference fluorescence
intensity. However, this method requires two separate optical channels thus complicating the
optical setup. For example, the drift in the sensitivity of both channels can be different, as
51
can be the intensities at two excitation wavelengths. Light scatter and signal loss caused by
fiber bending (e.g. in fiber optic sensors or certain sensortiterplate readers) further contribute
to effects not compensated by two-wavelength referencing.
Alternatively, the measurement of the fluorescence decay time, an intrinsically
referenced parameter, is hardly affected by fluctuations of the overall fluorescence intensity.
The decay time of most pH-sensitive indicator dyes, however, is in the nanosecond time
scale requiring a sophisticated and expensive instrumentation which limits the use in sensor
application.
WPI uses new and general logic to reference fluorescence intensity signals by decay
time measurement. In contrast to the most common ratiometric method, where luminescence
excitation or emission is measured at two wavelengths, this scheme uses a couple of
luminophores with different decay times and similar excitation spectra. An analyte-insensitive
µs-lifetime luminophore is combined with an analyte-sensitive ns-lifetime fluorophore, and a
method is presented how to convert fluorescence intensity into a phase shift. Preferably, the
reference dyes display decay times in the sensorsecond or millisecond time domain to
simplify the opto-electronic system.
The phase-modulation (frequency-domain) method is a well-established technique for the
measurement of luminescence decay times and was described in chapter 12.1.4. The phase
angle Φ measured at a single modulation frequency (fmod) reflects the luminescence decay
time (τ) provided that the decay is single-exponential (equation 4):
τ=
tan Φ
2πf mod
(4)
In this scheme, two luminophores are used. The first, referred to as the indicator, has a short
decay time (τind), the second acting as the reference standard has a decay time in the µs
range (τref). Ideally, the two luminophores have overlapping excitation and emission spectra
so that they can be excited at the same wavelength and their fluorescence can be detected
using the same emission window and photodetector. The phase shift Φm of the overall
luminescence obtained at a single frequency depends on the ratio of intensities of the
reference luminophore and the indicator dye. Φm can be represented as the superposition of
the single sine wave signals of the indicator and the reference luminophore (see Figure
11.7).
The reference luminophore gives a constant background signal (ref) while the
fluorescence signal of the indicator (ind) depends on the analyte concentration. The average
phase shift Φm directly reflects the intensity of the indicator dye and, consequently, the
analyte concentration. The modulation frequency is adjusted to the decay time of the
reference dye.
2
2
overall signal
reference
(A)
0
amplitude
amplitude
1
indicator
(=LED frequency)
-1
(B)
overall signal
reference
1
0
indicator (=LED)
-1
Φind Φm Φ
ref
Φind Φm Φref
-2
-2
0
45
90
135
180
Φm [°]
225
270
315
360
0
45
90
135
180
Φm [°]
225
270
315
360
Figure 11.7 Phase shift of the overall luminescence (Φm), the reference (Φref) and the indicator (Φind).
Fluorescence of the indicator in (A) absence and (B) presence of the analyte.
Equations 5 and 6 show the superposition of the phase signals of the reference dye,
which possess a constant decay time and luminescence intensity, and the indicator:
52
A m ⋅ cosΦ m = A ref ⋅ cosΦ ref + A ind ⋅ cosΦ ind
A m ⋅ sinΦ m = A ref ⋅ sinΦ ref + A ind ⋅ sinΦ ind
(5)
(6)
where A is the amplitude of either overall signal (m), luminophore (ref), or indicator (ind), and
Φ is the phase angle of either the overall signal (m), the luminophore (ref), or the indicator
(ind), respectively. In case the modulation frequency (fmod) is optimal, tan Φref is described by
equation 7
tanΦ ref = 2π ⋅ f mod ⋅ τref = 1
(7)
and Φind can be written as
tan Φ ind = 2π ⋅ f mod ⋅ τ ind =
2π ⋅ τ ind τ ind
=
2π ⋅ τ ref τ ref
(8)
The reference luminophore has a decay time that is orders of magnitude longer than that of
the indicator. Consequently, Φind can be set equal to zero in equation 9, since at low
modulation frequencies (in the kHz range) there is no phase shift.
tan Φ ind =
τ ind τind << τref
⎯⎯ ⎯
⎯→ 0 ⇒ Φ ind → 0
τ ref
(9)
The decay time of the reference luminophore is not affected by the analyte, hence:
Φref = constant Î tan Φref = constant ÎΦref = constant
(10)
Therefore, equations 5 and 6 can be simplified to give 11 and 12, respectively:
A m ⋅ cos Φ m = A ref ⋅ cos Φ ref + A ind
A m ⋅ sin Φ m = A ref ⋅ sin Φ ref
(11)
(12)
Dividing equation 11 by 12 results in a correlation of the phase angle (Φm) and the intensity
ratio of the indicator dye (Aind) and reference luminophore (Aref) :
A
A ⋅ cos Φ ref + A ind
A m ⋅ cos Φ m
1
= cot Φ m = ref
= cot Φ ref +
⋅ ind
sin Φ ref A ref
A ref ⋅ sin Φ ref
A m ⋅ sin Φ m
(13)
In this equation, cotΦm reflects the referenced intensity of the fluorescence indicator. A linear
relation is obtained between cot (Φm) and the ratio of Aind/Aref, because the phase angle of
Φref of the reference luminophore was assumed to be constant. This method is referred to as
Dual Lifetime Referencing (DLR).
10.1.6
Fluorescence (Förster) Energy Transfer (FET)
The term fluorescence energy transfer refers to a non-radiative transfer of excited state
energy from a donor (D) to an acceptor (A) and results from a dipole-dipole interaction
between donor and acceptor. Non-radiative energy transfer does not involve the emission
and reabsorption of photons. A transfer, where the acceptor dye reabsorbs photons emitted
by the donor is called radiative transfer or inner filter effect. The rate of non-radiative energy
transfer (kT) depends on the fluorescence quantum yield of the donor, the overlap of the
emission spectrum of the donor with the absorption spectrum of the acceptor, and their
relative orientation and distance. The theory was derived by Förster, who gave a quantitative
expression of kT between a donor and acceptor pair at a fixed separation
distance r (equation 14).
53
8.71 ⋅ 10 23 ⋅ κ 2 ⋅ Φ d
1 ⎛R ⎞
kT =
⋅J = ⎜ 0 ⎟
6
4
τd ⎝ r ⎠
r ⋅ n ⋅ τd
6
(14)
with
∞
J = ∫ FD (λ) ⋅ ε A (λ) ⋅ λ4 ⋅ dλ
(15)
0
where Φd and τd are the quantum yield and lifetime of the donor in absence of the acceptor,
n is the refractive index of the medium, r is the distance between donor and acceptor and κ2
is a factor describing the relative orientation in space of the transition dipoles of the donor
and acceptor. The overlap integral J, represented in equation 15, expresses the degree of
spectral overlap between donor emission and the acceptor absorption. Fd(λ) is the corrected
fluorescence intensity of the donor in the wavelength range λ to λ+dλ with the total intensity
normalized to unity and εA(λ) is the extinction coefficient of the acceptor at λ. The Förster
distance R0 is the donor-acceptor critical transfer distance at which radiative decay and nonradiative energy transfer are equally probable. Remarkably, the efficiency depends on the 6th
power of r.
Many optical sensors exploit the principle of energy transfer. The potential of
ET-based sensors relies on the fact that well-investigated absorbance based indicators can
be applied by adding an analyte-insensitive fluorescent donor with a sufficiently large
spectral overlap. Sensors for pH, carbon dioxide and ammonia have been realized by energy
transfer from a pH-insensitive donor to a pH-sensitive acceptor. Additionally, luminescence
energy transfer is a convenient way to overcome the lack of suitable lifetime based indicators
since the color change of an absorber can be converted into a decay time information.
Energy transfer measurements require a constant separation distance between the
D-A pair which does not vary during the excited state lifetime of the donor. This can be
achieved by covalently linking donor and acceptor via spacer groups. An alternative way to
fix the separation distance is the formation of donor-acceptor ion-pairs.
10.1.7
Literature
If you want to find out more about this subject, we recommend the following publications.
• Wolfbeis O.S. (Ed.), Fiber Optic Chemical Sensors and Biosensors, Vol. 1&2, CRC,
Boca Raton (1991).
• Klimant I., Wolfbeis O.S., Oxygen-Sensitive Luminescent Materials Based on
Silicone-Soluble Ruthenium Diimine Complexes, Anal. Chem., 67, 3160-3166 (1995).
• Klimant I., Kühl M., Glud R.N., Holst G., Optical measurement of oxygen and
temperature in sensorscale: strategies and biological applications, Sensors and
Actuators B, 38-39, 29-37 (1997).
• Holst G., Glud R.N., Kühl M., Klimant I., A sensoroptode array for fine-scale
measurement of oxygen distribution, Sensors and Actuators B, 38-39, 122-129 (1997).
• Klimant I., Meyer V., Kühl M., Fiber-optic oxygen Microsensors, a new tool in aquatic
biology, Limnol. Oceanogr., 40, 1159-1165 (1995).
• Klimant I., Ruckruh F., Liebsch G., Stangelmayer A., Wolfbeis O.S., Fast Response
Oxygen Microsensors Based on Novel Soluble Ormosil Glasses, Mikrochim. Acta,
131, 35-46 (1999).
• Klimant I., Huber Ch., Liebsch G., Neurauter G., Stangelmayer A., Wolfbeis O.S., Dual
Lifetime Referencing (DLR) - a New Scheme for Converting Fluorescence Intensity
into a Frequency-Domain or Time-Domain Information, in New Trends in
54
Fluorescence Spectroscopy: Application to Chemical and Life Sciences, Valeur B. &
Brochon J.C. (eds.) Springer Verlag, Berlin (2001). chap. 13, 257-275.
• Klimant, I., Verfahren und Vorrichtung zur Referenzierung von Fluoreszenzintensitätssignalen, Ger Pat Appl DE 198.29.657 (1997)
• Huber Ch., Klimant I., Krause Ch., Werner T., Mayr T., Wolfbeis O.S., Optical Sensor for
Seawater Salinity, Fresenius J. Anal. Chem., 368, 196-202 (2000).
• Huber Ch., Klimant I., Krause Ch., Wolfbeis O.S., Dual Lifetime Referencing as Applied
to an Optical Chloride Sensor, Anal. Chem., 73, 2097-2103 (2001).
• Huber Ch., Klimant I., Werner T., Krause Ch., Wolfbeis O.S., Nitrate-Selective Optical
Sensors Applying a Lipophilic Fluorescent Polarity-Sensitive Dye, Anal. Chim. Acta,
449, 81-93 (2001).
• Huber Ch., Werner T., Krause Ch., Klimant I., Wolfbeis O.S., Energy Transfer Based
Lifetime Sensing of Chloride Using a Luminescent Transition Metal Complex, Anal.
Chim. Acta, 364, 143-151 (1998).
• Preininger C., Mohr G.J., Fluorosensors for Ammonia Using Rhodamines
Immobilized in Plasticized Poly(vinyl chloride) and in Sol-Gel; A Comparison Study,
Anal. Chim. Acta, 342, 207-213 (1997).
• Kosch U., Klimant I., Werner T., Wolfbeis O.S., Strategies to Design pH Optodes with
Luminescence Decay Times in the Sensorsecond Time Regime, Anal. Chem., 362,
73-78 (1998).
• Krause Ch., Werner T., Huber Ch., Klimant I., Wolfbeis O.S., Luminescence Decay
Time-Based Determination of Potassium Ions, Anal. Chem., 70, 3983-3985 (1998).
• Neurauter G., Klimant I., Wolfbeis O.S., Sensorsecond Lifetime-Based Optical Carbon
Dioxide Sensor Using Luminescence Energy Transfer, Anal. Chim. Acta, 382, 67-75
(1999).
55
pHOpticaTM
fiber-optic phase detection device
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components and parts, shall be free from detects in material and workmanship for a period of one year*
from the date of receipt. WPl´s obligation under this warranty shall be limited to repair or replacement, at
WPI´s option, of the equipment or defective components or parts upon receipt thereof f.o.b. WPI, Sarasota,
Florida,
U.S.A. Return of a repaired instrument shall be f.o.b. Sarasota.
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