Molecular Devices SpectraMax ABS User manual

5065390 C
January 2021
SpectraMax ABS
SpectraMax ABSPlus
Microplate Spectrophotometers
User Guide

SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
2 5065390 C
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©2021 Molecular Devices, LLC.
All rights reserved.

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Contents
Safety Information 4
Warnings, Cautions, Notes, and Tips 4
Chapter 1: Introduction 7
Computer Integration 8
Plate Controls 9
Cuvette Chamber 11
Temperature Regulation 12
Chapter 2: Setting Up the Instrument 13
Connecting Instrument Cables 14
Chapter 3: Getting Started 15
Chapter 4: Absorbance Read Mode 17
PathCheck Technology 18
Read Types 21
Chapter 5: Maintenance 23
Cleaning the Instrument 23
Replacing Fuses 24
Before You Move the Instrument 25
Chapter 6: Troubleshooting 27
Opening the Drawer Manually 31
Obtaining Support 32
Chapter A: Instrument Specifications 33
SpectraMax ABS Specifications 34
SpectraMax ABSPlus Specifications 38
Electromagnetic Compatibility 42
Appendix B: Accessories 43

SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
4 5065390 C
Safety Information
Information about the safe use of the instrument from Molecular Devices® includes an
understanding of the user-attention statements in this guide, the safety labels on the
instrument, precautions to follow before you operate the instrument, and precautions to follow
while you operate the instrument.
Make sure that everyone involved with the operation of the instrument has:
Received instruction in general safety practices for laboratories.
Received instruction in specific safety practices for the instrument.
Read and understood all Safety Data Sheets (SDS) for all materials being used.
Read and observe all warnings, cautions, and instructions. The most important key to safety is
to operate the instrument with care.
WARNING! If the instrument is used in a manner not specified by Molecular Devices,
the protection provided by the equipment might be impaired.
Warnings, Cautions, Notes, and Tips
All warning symbols are framed within a yellow triangle. An exclamation mark is used for most
warnings. Other symbols can warn of other types of hazards such as biohazard, electrical, or
laser safety warnings as are described in the text of the warning. Follow the related safety
information.
The following user attention statements might be displayed in the text of Molecular Devices
user documentation. Each statement implies the amount of observation or recommended
procedure.
WARNING! A warning indicates a situation or operation that could cause personal
injury if precautions are not followed.
CAUTION! A caution indicates a situation or operation that could cause damage to the
instrument or loss of data if correct procedures are not followed.
Note: A note calls attention to significant information.
Tip: A tip provides useful information or a shortcut, but is not essential to the completion
of a procedure.

Safety Information
5065390 C 5
Symbols on the Instrument
Each safety label found on the instrument contains an alert symbol that indicates the type of
potential safety hazard.
Symbol Indication
Consult the product documentation.
Potential biohazard.
Potential lifting hazard. To prevent injury, use a minimum of two people to lift the
instrument.
Potential heat hazard.
Required in accordance with the Waste Electrical and Electronic Equipment
(WEEE) Directive of the European Union. It indicates that you must not discard this
electrical or electronic product or its components in domestic household waste
or in the municipal waste collection system.
For products under the requirement of the WEEE directive, contact your dealer or
local Molecular Devices office for the procedures to facilitate the proper
collection, treatment, recovery, recycling, and safe disposal of the device.
- California proposition 65 requires businesses to provide
warnings to Californians about significant exposures to chemicals that cause cancer, birth
defects, or other reproductive harm.

SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
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Electrical Safety
To prevent electrical injuries and property damage, inspect all electrical equipment before
use and report all electrical deficiencies. Contact Molecular Devices technical support for
equipment service that requires the removal of covers or panels.
To prevent electrical shock, use the supplied power cord and connect to a properly grounded
wall outlet.
To ensure sufficient ventilation and provide access to disconnect power from the instrument,
maintain a 20cm to 30cm (7.9in. to 11.8in.) gap between the rear of the instrument and the
wall.
Power off the instrument when not in use.
Chemical and Biological Safety
Normal operation of the instrument can involve the use of materials that are toxic, flammable,
or otherwise biologically harmful. When you use such materials, observe the following
precautions:
Handle infectious samples based on good laboratory procedures and methods to prevent
the spread of disease.
Observe all cautionary information printed on the original containers of solutions before
their use.
Dispose of all waste solutions based on the waste disposal procedures of your facility.
Operate the instrument in accordance with the instructions outlined in this guide, and take
all the required precautions when using pathological, toxic, or radioactive materials.
Splashing of liquids can occur. Take applicable safety precautions, such as using safety
glasses and wearing protective clothing, when working with potentially hazardous liquids.
Observe the applicable cautionary procedures as defined by your safety officer when
using hazardous materials, flammable solvents, toxic, pathological, or radioactive materials
in or near a powered-up instrument.
WARNING! Never use the instrument in an environment where potentially damaging
liquids or gases are present.
Moving Parts Safety
The instrument contains moving parts that can cause injury. Under normal conditions, the
instrument is designed to protect you from these moving parts.
WARNING! If the instrument is used in a manner not specified by Molecular Devices,
the protection provided by the equipment might be impaired.
To prevent injury:
Never try to exchange labware, reagents, or tools while the instrument is operating.
Never try to physically restrict the moving components of the instrument.
Keep the instrument work area clear to prevent obstruction of the movement. Provide
clearance in front of the instrument of 18 cm (7.1 in.) for the plate drawer.
Note: Observe all warnings and cautions listed for all external devices attached to or in
use during the operation of the instrument. See the applicable user guide for the
operating and safety procedures of that device.

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Chapter 1: Introduction
The SpectraMax® ABS Microplate Spectrophotometer provides rapid and sensitive
measurements of a variety of analytes across a wide range of concentrations. The
SpectraMax® ABSPlus Microplate Spectrophotometer not only works on a wider range of (190
nm - 1000 nm) but it also adds the ability to read cuvettes. These instruments measure the
optical density (OD) of samples at selected wavelengths in a single read mode.
The high sensitivity and flexibility of the instrument make it useful for applications in the fields
of biochemistry, cell biology, immunology, molecular biology, and microbiology. Typical
applications include ELISA, nucleic acid, protein, enzymatic type homogeneous and
heterogeneous assays, microbial growth, endotoxin testing, and pipettor calibration.
The SpectraMax ABS supports the Visible Absorbance (ABS) read mode while the SpectraMax
ABSPlus supports the UV and Visible Absorbance (ABS) read mode with the following read
types:
Endpoint: At a single point in time.
Kinetic: Over a specified period of time.
Spectral Scan: Over a specified wavelength range.
The SpectraMax ABS reads 96-well plates.
The SpectraMax ABSPlus reads 96-well plates, 384-well plates, and cuvettes.
CAUTION! Although not recommended, you can also use strip well plates in both the
SpectraMax ABS and the SpectraMax ABSPlus.
You can integrate the SpectraMax ABS and the SpectraMax ABSPlus with the StakMax®
Microplate Handling System.
CAUTION! To prevent damage to the instrument, the height of the plate must not
exceed 17 mm, including the lid if the plate is lidded.
You use the Shake feature to mix the contents of the wells in a plate before each read cycle,
which makes it possible to perform kinetic analysis of solid-phase, enzyme-mediated
reactions. (Shake is not critical for liquid-phase reactions.)
Temperature controls allow the instrument to regulate the temperature of the plate chamber
from 5°C above ambient to 45°C.
CAUTION! Never touch the internal optic mirrors, lenses, filters, or cables. The optics are
extremely delicate, and critical to the function of the instrument.
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SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
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Computer Integration
Each Molecular Devices microplate reader is shipped with a license key for the SoftMax® Pro
Data Acquisition and Analysis Software that you install on the computer that you use to operate
the instrument. The SoftMax Pro Software provides integrated instrument control, data display,
and statistical data analysis.
You should install the SoftMax Pro Software on the computer before you set up the instrument.
Please be aware that some updates to the SoftMax Pro Software require a purchase. Contact
Molecular Devices before you update the software. To download the latest version of the
software, visit: https://www.moleculardevices.com/products/microplate-readers/acquisition-
and-analysis-software/softmax-pro-software#Order.
For information about the computer specifications that are required to run the software, the
software installation and licensing instructions, and the directions to create the software
connection between the computer and the instrument, see:
SoftMax Pro Data Acquisition and Analysis Software Standard Edition and MiniMax Imaging
Edition Installation Guide
SoftMax Pro Data Acquisition and Analysis Software - GxPEdition - Installation Guide for
the Multi Computer Setup
SoftMax Pro Data Acquisition and Analysis Software - GxPEdition - Installation Guide for
the Single Computer Setup
When you connect the microplate handler to a compatible microplate reader, the StakMax
Software runs with the SoftMax Pro Software that controls the microplate reader.
When you connect the microplate handler to the AquaMax Microplate Washer, the AquaMax
Software controls the StakMax Microplate Handler. See the AquaMax 2000/4000 Microplate
Washer User Guide .

Chapter 1: Introduction
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Plate Controls
The plate drawer is located on the front of the instrument and slides in and out of the plate
chamber. To open and close the plate drawer, use the button on the top front right of the
instrument or the SoftMax Pro Software. The arrow in the following image points to where well
A1 should be for reads.
The drawer closes for each read. Subsequent plate drawer operation is dependent on the
incubator setting:
When you open the drawer, if the incubator is off, then the drawer remains open.
When you open the drawer, if the incubator is on, then to maintain temperature control
within the plate chamber, the drawer closes after approximately 10 seconds.
Do not obstruct the movement of the drawer. If you must retrieve a plate after an error
condition or power outage and the drawer does not open, you can open the drawer manually.
See Opening the Drawer Manually on page 31.
The SpectraMax ABS can accommodate standard 96-well plates. The SpectraMax ABSPlus,
can accommodate standard 96-well plates and 384-well plates. When you do a read at
wavelengths below 340 nm, you must use special UV-transparent, disposable or quartz plates
to allow transmission of the deep UV spectra.
CAUTION! Although not recommended, you can also use strip well plates in both the
SpectraMax ABS and the SpectraMax ABSPlus.
CAUTION! To prevent damage to the instrument, the height of the plate must not
exceed 17mm, including the lid if the plate is lidded.
Not all manufacturers' plates are the same with regard to design, materials, or configuration.
Temperature uniformity within the plate may vary depending on the type of plate you use.

SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
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Note: Use high quality support frames or holders when you use strip-wells. You should
fill the frame or holder with empty strips to avoid warping.
Shake
To mix of the contents of the wells, you can define settings to shake the plate linearly along
the long axis at preset intervals.
Endpoint read type: Settings allow for the shaking of the plate for a user-defined number
of seconds and then reading at all selected wavelengths.
Kinetic read type: Settings allow for the shaking of the plate number for a user-defined
number of seconds before the initial read, and/or for a user-defined number of seconds
before each subsequent read.
Note: To enhance accuracy, the use of shake is recommended for ELISAs and other
solid-phase, enzyme-mediated reactions.

Chapter 1: Introduction
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Cuvette Chamber
The SpectraMax ABSPlus has a cuvette chamber on the top center of the instrument. Press
down on the cuvette chamber lid to open the lid and insert or remove a cuvette. The chamber
contains springs that position the cuvette in the proper alignment for a read. You must manually
close the cuvette door before you initiate a read.
Handle cuvettes on the frosted sides only. Place the cuvette into the chamber so that the
“reading” (clear) sides face the front and the rear of the instrument.
The guidelines for cuvette use in the instrument are the same as those that apply to any high-
quality spectrophotometer. You must ensure that the meniscus is comfortably above the light
beam in standard cuvettes and that the sample chamber in a microcuvette is aligned properly
with the beam. The light beam is 0.625 in (15.87 mm) above the cuvette bottom.
The instrument can accommodate standard-height (45 mm) and other cuvettes up to 56 mm in
height. The instrument does not accept short (25 mm high) microcuvettes. See Accessories on
page 43.
Not all manufacturers’ cuvettes are the same with regard to design, materials, or configuration.
Temperature uniformity within the cuvette can vary depending on the type of cuvette you use.

SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
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Temperature Regulation
When you power on the instrument the incubator is off. The temperature in the plate chamber
is ambient and isothermal.
The instrument is designed to regulate the temperature of the plate chamber from 5°C above
ambient to 45°C. The SoftMax Pro Software allows you to turn the incubator on and off to
adjust the plate chamber temperature. The temperature set point defaults to 37.0°C at start-up.
Accuracy of the temperature set point is guaranteed only if the set point is at least 5°C above
ambient. If the temperature set point is lower than the ambient temperature, the chamber
temperature remains at ambient. Temperature regulation is controlled by heaters only and,
therefore, cannot cool the temperature to a setting lower than ambient. Additionally, the
highest setting (45°C) can be achieved only if the ambient temperature is greater than 20°C.
Typically, the plate chamber reaches 37.0°C in less than 30 minutes. The plate chamber
temperature is maintained at the set point until you use the software to turn temperature
regulation off.
Should you turn the incubator back on after a momentary shutdown, allow about ten minutes
for the control algorithm to fully stabilize the plate chamber temperature.
Temperature regulation and control of the plate chamber is achieved through electric heaters,
efficient insulation, and temperature sensors. The heaters are located in the plate chamber,
which is insulated to maintain the temperature set point. The sensors are mounted inside the
chamber and measure the air temperature.
The temperature feedback closed-loop control algorithms measure the chamber air
temperature, compare it to the temperature set point, and use the difference to calculate the
regulation of the heating cycles. This technique results in accurate, precise control of the
chamber temperature with a temperature variation of the air inside the plate chamber of less
than 1.0°C. The temperature uniformity within the plate depends on its design and composition.
Note: The plate chamber temperature and cuvette chamber temperature display in the
SoftMax Pro Software.
The temperature sensors detect the temperature of the air inside the plate chamber, not the
temperature of the samples in the plate. If you use the instrument to warm the samples, use a
seal or lid on the plate to prevent evaporation of the sample. The seal or lid also helps to
maintain a uniform temperature. It can take an hour or more for a prepared sample to
equilibrate inside the plate chamber. You can speed up equilibration by pre-warming the
sample and the assay reagents to the desired temperature before you place the plate in the
chamber. Validate the data quality to determine whether the seal or lid can stay on the plate
for the read.

5065390 C 13
Chapter 2: Setting Up the Instrument
Before you unpack and set up the instrument, prepare a dry, flat work area that has sufficient
space for the instrument, host computer, and required cables. To provide access for
disconnecting power from the instrument, maintain a 20cm to 30cm (7.9in. to 11.8in.) gap
between the rear of the instrument and the wall. To ensure sufficient ventilation, do not block
the ventilation grids on the front and rear of the instrument.
The package contains the instrument and accessories to set up the instrument:
SoftMax Pro Software, product key, and installation guide
Instrument Installation Guide
USB computer connection cable
AC power adapter
For a complete list of the package contents, see the enclosed packing list.
Install the SoftMax Pro Software on the computer that operates the microplate reader. See the
SoftMax Pro Data Acquisition and Analysis Software Installation Guides .
The packaging is designed to protect the instrument during shipment. Tape is placed on the
cuvette door to protect the instrument from damage during shipment.
CAUTION! Do not touch or loosen screws or parts other than those specifically
designated in the instructions. Doing so could cause misalignment and possibly void the
warranty.
Note: Retain the shipping box and all packing materials for future transport needs.
CAUTION! When transporting the instrument, warranty claims are void if improper
packaging results in damage during transport.
CAUTION! Before unpacking the instrument, check the box for damage that occurred
during transportation. If any damage is noted, inform the supplier immediately and keep
the damaged packaging. Unless instructed otherwise by the supplier, do not continue
to unpack the instrument.
To unpack the instrument:
1. Open the top of the box.
2. Lift the accessories tool box and the instrument from the package, and then place the
instrument on a level surface.
3. Remove the packing material from both ends of the instrument, remove the instrument
from the plastic bag, and then set the instrument down carefully on a level surface.
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Connecting Instrument Cables
The power cord and USB cable connect to the ports on the rear of the instrument.
Illustration Part Number Description
5064799 USB computer connection cable, 3meter (9.8foot)
4400-0002 or 4400-0036 Power cord, 1meter (3.3foot)
Note: Before you connect or disconnect the power cord, make sure that the power
switch that is on the rear of the instrument is in the Off position.
1. Make sure that the power switch that is on the rear of the instrument is in the Offposition.
2. To use a computer to operate the instrument, connect the appropriate end of the supplied
USB cable to the USB port that is on the rear of the instrument, and then connect the other
end to a USB port on the computer.
3. Connect the supplied power adapter to the power port that is on the rear of the instrument,
and then connect the other end to a grounded electrical wall outlet.
4. Turn the instrument around so that the front of the instrument now faces you.
Note: Ensure no cables run beneath the instrument.
5. Remove the tape from the cuvette door on the SpectraMax ABSPlus.
6. Power on the instrument and wait for the plate drawer to open.

5065390 C 15
Chapter 3: Getting Started
Now that you have installed the SoftMax Pro Software on the computer, removed the tape
from the drawer and the cuvette port, and connected the cables, it is time to get started.
WARNING! Never use the instrument in an environment where potentially damaging
liquids or gases are present.
1. Turn on the instrument power switch located on the rear of the instrument. Wait for the
instrument to complete its diagnostic check and the plate drawer opens.
2. Power on the computer.
3. Start the SoftMax Pro Software and connect the software to the instrument. See the
SoftMax Pro Software user guide or application help.
4. Use the computer to define the protocol settings including plate type, read type, read
area, wavelength, detection speed, temperature, shake, and kinetic timing.
5. Use the computer to open the plate drawer or manually lift the cuvette chamber door. If
you adjust the chamber temperature, the drawer closes after approximately 10 seconds to
maintain temperature control.
6. Place the plate on the plate slide for a plate read (well A1 on the front right of the drawer)
or place the cuvette into the cuvette chamber for a cuvette read (springs hold the cuvette
in place) Handle cuvettes on the frosted sides only. Place the cuvette into the chamber so
that the “reading” (clear) sides face the front and the rear of the instrument.
7. Use the computer to close the plate drawer or manually close the cuvette door.
8. Use the computer to start the read.
9. The computer displays the read data and the plate drawer opens.
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5065390 C 17
Chapter 4: Absorbance Read Mode
The instrument uses the Absorbance (ABS) read mode to measure the Optical Density (OD) of
the sample solutions.
Absorbance is the quantity of light absorbed by a solution. To measure absorbance accurately,
it is necessary to eliminate light scatter. If there is no turbidity, then absorbance=optical
density.
A=log10(I0/I)=–log10(I/I0)
where I0 is intensity of the incident light before it enters the sample divided by the light after it
passes through the sample, and A is the measured absorbance.
The temperature-independent PathCheck technology normalizes absorbance values to a 1cm
path length based on the near-infrared absorbance of water.
The instrument allows you to choose whether to display absorbance data as Optical Density
(OD) or %Transmittance (%T).
Optical Density
Optical density (OD)is the quantity of light passing through a sample to a detector relative to
the total quantity of light available. Optical Density includes absorbance of the sample plus
light scatter from turbidity and background. You can compensate for background using blanks.
A blank well contains everything used with the sample wells except the chromophore and
sample-specific compounds. Do not use an empty well for a blank.
Some applications are designed for turbid samples, such as algae or other micro-organisms in
suspension. The reported OD values for turbid samples are likely to be different when read by
different instruments.
For optimal results, you should run replicates for all blanks, controls, and samples. In this case,
the blank value that will be subtracted is the average value of all blanks.
% Transmittance
%Transmittance is the ratio of transmitted light to the incident light for absorbance reads.
T=I/I0
%T=100T
where I is the intensity of light after it passes through the sample and I0 is incident light before it
enters the sample.
Optical Density and %Transmittance are related by the following formulas:
%T=102–OD
OD=2–log10(%T)
The factor of two comes from the fact that %T is expressed as a percent of the transmitted light
and log10(100)=2.
When in %Transmittance analysis mode, the instrument converts the raw OD values reported
by the instrument to %Transmittance using the above formula. All subsequent calculations are
done on the converted numbers.
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Applications of Absorbance
Absorbance-based detection is commonly used to evaluate changes in color or turbidity,
permitting widespread use including ELISAs, protein quantitation, endotoxin assays, and
cytotoxicity assays.
PathCheck Technology
The temperature-independent PathCheck technology normalizes absorbance values to a 1cm
path length based on the near-infrared absorbance of water.
The Beer–Lambert law states that absorbance is proportional to the distance that light travels
through the sample:
A = εcL
where A is the absorbance, εis the molar absorptivity of the sample, c is the concentration of
the sample, and L is the pathlength. The longer the pathlength, the higher the absorbance.
Microplate readers use a vertical light path so the distance of the light through the sample
depends on the volume. This variable pathlength makes it difficult to do extinction-based
assays and makes it confusing to compare results between microplate readers and
spectrophotometers.
The standard pathlength of a 1 cm cuvette is the conventional basis to quantify the unique
absorptivity properties of compounds in solution. Quantitative analysis can be done on the
basis of extinction coefficients, without standard curves (for example, NADH-based enzyme
assays). When you use a cuvette, the pathlength is known and is independent of sample
volume, so absorbance is directly proportional to concentration when there is no background
interference.
In a plate, pathlength is dependent on the liquid volume, so absorbance is proportional to both
the concentration and the pathlength of the sample. Standard curves are often used to
determine analyte concentrations in vertical-beam photometry of unknowns, yet errors can still
occur from pipetting the samples and standards. The PathCheck technology determines the
pathlength of aqueous samples in the plate and normalizes the absorbance in each well to a
pathlength of 1cm. This way of correcting the microwell absorbance values is accurate to
within ±4% of the values obtained directly in a 1cm cuvette.
Horizontal
light path
Vertical light path
Cuvette Microplate wells
PathCheck technology normalizes the data acquired from an absorbance read mode endpoint
read type to a 1cm pathlength, correcting the OD for each well to the value expected if the
sample were read in a 1cm cuvette. The instrument uses the factory installed water constant to
obtain the 1cm values. For the SpectraMax ABSPlus you can read a cuvette that contains
deionized water or buffer to use the Cuvette Reference correction method (typically not
necessary when you use aqueous solutions with minimal alcohol, salt, or organic solvent
content).

Chapter 4: Absorbance Read Mode
5065390 C 19
Note: You must select the PathCheck check box before a read because you cannot
apply the PathCheck technology after the read. After you read a plate with PathCheck
technology turned on, the software stores PathCheck information permanently within
the document.
Water Constant
The PathCheck technology is based on the absorbance of water in the near infrared spectral
region (between 900 nm and 1000 nm). If the sample is completely aqueous, has no turbidity
and has a low salt concentration (less than 0.5 M), the water constant correction method is
sufficient. The water constant is determined for each instrument during manufacture and is
stored in the instrument.
Note: The cuvette reference correction method that the software uses with the
PathCheck technology is different from the reference read of a cuvette that occurs
when you click the Ref button in the Cuvette Set section tool bar.
Eliminating the Pathlength Independent Component
Raw OD measurements of plate samples include both pathlength-dependent components
(sample and solvent) and a pathlength-independent component (OD of plate material). The
pathlength-independent component must be eliminated from the calculation to get valid
results that have been normalized by the PathCheck technology. You can do this using a plate
blank or using a plate background constant.
Using a Plate Blank
You can use this method if all samples in the plate are the same volume and the read does not
depend on the PathCheck technology to correct for variability in volumes.
To use a plate blank:
1. Designate a minimum of one well (preferably several) as Plate Blank.
2. Pipette buffer (for example, your sample matrix) into those wells and read along with the
samples. Do not use an empty well for a blank.
The instrument automatically subtracts the average of the blank wells from each of the
samples. The OD of the plate material is subtracted as part of the blank.
3. Select the Use Plate Blank check box in the Data Reduction dialog.

SpectraMax ABS and SpectraMax ABS Plus Microplate Spectrophotometers User Guide
20 5065390 C
Using a Plate Background OD
If your sample volumes are not identical or if you choose not to use a Plate Blank, then you
must use a Plate Background OD. Omitting a Plate Background OD results in artificially high
values after being normalized by the PathCheck technology.
To determine the Plate Background OD:
1. Fill a clean plate with water.
2. Read at the wavelengths you will use for the samples.
The average OD value is the Plate Background OD. If you intend to read your samples at more
than one wavelength, there should be a corresponding number of Plate Background OD
values for each wavelength.
Note: It is important that you put water in the wells and do not read a dry plate for the
Plate Background OD. A dry plate has a slightly higher OD value than a water filled plate
because of differences in refractive indices. Use of a dry plate results in PathCheck
technology normalized values that are lower than 1cm cuvette values.
Interfering Substances
Material that absorbs in the 900 nm to 1000 nm spectral region could interfere with PathCheck
technology measurements. Fortunately, there are few materials that do interfere at the
concentrations generally used.
Turbidity is the most common interference. If you can detect turbidity in your sample, you
should not use the PathCheck technology. Turbidity elevates the 900 nm measurement more
than the 1000nm measurement and causes an erroneously low estimate of pathlength. Use of
the Cuvette Reference does not reliably correct for turbidity.
Samples that are highly colored in the upper-visible spectrum might have absorbance that
extends into the near-infrared (NIR) spectrum and can interfere with the PathCheck technology.
Examples include Lowry assays, molybdate-based assays, and samples that contain
hemoglobins or porphyrins. In general, if the sample is distinctly red or purple, you should
check for interference before you use the PathCheck technology.
To determine possible color interference:
Measure the OD at 900 nm and 1000 nm (both measured with air reference).
Subtract the 900 nm value from the 1000 nm value.
Do the same for pure water.
If the delta OD for the sample differs significantly from the delta OD for water, then you should
not use the PathCheck technology.
Organic solvents could interfere with the PathCheck technology if the solvents have
absorbance in the region of the NIR water peak. Solvents such as ethanol and methanol do not
absorb in the NIR region, so the solvents do not interfere, except to cause a decrease in the
water absorbance to the extent of their presence in the solution. If the solvent absorbs
between 900nm and 1000nm, the interference would be similar to the interference of highly
colored samples. If you add an organic solvent other than ethanol or methanol, you should run
a Spectrum scan between 900 nm and 1000 nm to determine if the solvent would interfere
with the PathCheck technology.