3250

The Advanced™ Osmometer
Model 3250
User’s Guide
3255EN Rev. 22
Advanced™ Osmometer Model 3250 User’s Guide
Copyright
This user’s guide is copyrighted by Advanced Instruments with
all rights reserved. Under copyright laws, this guide may not be
reproduced in any form, in whole or part, without the prior written
consent of Advanced Instruments.
© 2019 Advanced Instruments.
Advanced Instruments has reviewed this guide thoroughly. All
material contained within is believed reliable, but the accuracy and
completeness are not guaranteed or warranted, and are not intended
to be representations or warranties concerning the product described.
Microsoft® and Windows® are registered trademarks of Microsoft
Corporation in the United States and other countries. Intel® is a
registered trademark of Intel Corporation in the United States and
other countries. All other trademarks are the property of Advanced
Instruments.
Hot-Line™ Service
If you have any questions or problems regarding the proper operation
of your instrument, please contact our Hot-Line Service department by
calling one of the following numbers.
• 800-225-4034 (toll-free within the USA and Canada)
• +US 781-320-9000 (elsewhere)
• 781-320-0811 (fax)
2
Table of Contents
Copyright.............................................................................................................. 2
Hot-Line™ Service.............................................................................................. 2
Safe Use............................................................................ 7
Symbol Conventions......................................................................................... 7
General Cautions................................................................................................ 8
FCC Requirements............................................................................................9
Supplies, Parts and Accessories............................... 10

Calibrators and Standards...........................................12
Foreword......................................................................... 14
Intended Use..................................................................................................... 14
Principles of Freezing-Point Osmometry................................................15
Instrumentation................................................................................................ 16
Freezing-Point Thermodynamics.............................................................. 16
Definitions............................................................................................................18
Installation and Setup................................................ 20

Step 1 — Find a location for the instrument........................................ 20
Step 2 — Obtain additional items ........................................................... 20
Step 3 — Unpack the instrument...............................................................21
Step 4 — Install the heat transfer fluid...................................................22
Step 5 — Load printer paper (Figure 1)..................................................23
Step 6 — Power up the instrument..........................................................25
Step 7 — Prime the heat transfer fluid pump......................................25
Step 8 — Run a diagnostic test..................................................................27
Step 9 — Set date and time........................................................................27
Step 10 — Set language preference.........................................................27
Step 11 — Check initial factory calibration.............................................28
Step 12 — Proceed to Chapter 2................................................................28
3
Instrument Operation..................................................29
Hazardous material cautions...................................................................... 29
Function of major components ............................................................... 30
Operating head (Figure 3, Item 1)........................................................30
Freezing chamber (Figure 3, Item 8)..................................................30
Measurement and control circuits (inside) ....................................... 31
Electronic circuits (inside)........................................................................ 31
Display panel (Figure 3, Item 4)............................................................ 31
Keypad (Figure 3, Item 4) ...................................................................... 32
Printer (Figure 3, Item 9)......................................................................... 33
Supervisor/Operator keyswitch (Figure 5) ...................................... 33
Power module (Figure 5).........................................................................34
RS-232 port (Figure 5)..............................................................................34
Barcode port (Figure 5)...........................................................................34
Sample preparation........................................................................................35
Sample handling..............................................................................................35
Repeatability Tips........................................................................................... 36
Verifying calibration daily.............................................................................37
Sample test procedure................................................................................. 38
Sample test errors........................................................................................... 41
Changing operating settings..................................................................... 42
0. Select Setup Item...................................................................................43
1. Set Block Bin #.........................................................................................44
2. Set Sample Bin #....................................................................................44
3. Dis/Enable I.D. #.....................................................................................45
4. Set Stir Amplitude.................................................................................45
5. Set Date/Time..........................................................................................46
6. Dis/Enable Beeper.................................................................................46
7. Set Serial Rate.......................................................................................... 47
8. Dis/Enable Xon/Xoff............................................................................. 47
9. Serial Number.......................................................................................... 47
10. Select Language...................................................................................48
11. Product/Test............................................................................................48
12. Data Capture...........................................................................................48
13. Assistance................................................................................................48
4

14. Set "Buzz" Amplitude.........................................................................48

15. Set "Buzz" Point....................................................................................49
16. Select Range...........................................................................................50
17. Zero Calibration...................................................................................... 51
Using the RS-232 port....................................................................................51
Standards and Quality Control.................................52

Repeatability and accuracy.........................................................................52
Standards and controls.................................................................................52
Maintenance of standards............................................................................53
Quality control implementation.................................................................53
Calibration......................................................................55
Calibration procedure....................................................................................55
Calibration notes..............................................................................................57
Troubleshooting and Service.....................................58

Service and maintenance cautions.......................................................... 58
Obtaining service............................................................................................ 60
To return an instrument for repair or replacement:....................... 61
Routine maintenance.................................................................................... 62
Shutdown and storage................................................................................. 62
Troubleshooting checks............................................................................... 63
Internal diagnostic tests............................................................................... 64
1. Statistics...................................................................................................... 65
2. Recall results............................................................................................66
3. A/D Tests...................................................................................................66
4. Stir/Freeze Test.......................................................................................68
5. Probe Bin Test..........................................................................................68
7. Display/Print Test....................................................................................70
8. Beeper Test...............................................................................................70
9. Keypad Test..............................................................................................70
10. Head Up/Down Test ............................................................................ 71
11. Event Record............................................................................................ 71
Fuse replacement.............................................................................................71
Sample probe checks and replacement.................................................73
5
Mandrel, probe, and stir/freeze wire alignment .................................77

Alignment using the alignment tool.................................................... 77
Alternate method—using a sample tube containing 0.2 mL of
water................................................................................................................. 79
Stir/freeze amplitude adjustment............................................................ 80
Troubleshooting Table.................................................83
Product Specifications............................................... 90
Regulatory Notices.......................................................93
Warranty and Warranty Duties.................................97
Supplemental RS-232 Information..........................101
Symbol Definitions.....................................................104
Product Disposal and Recycling............................. 107
Service Log..................................................................109
6
Safe Use
To reduce the risk of bodily injury, electric shock, fire, and damage to
your instrument, please read and observe the precautions in this User’s
Guide.
• If the product is used in a manner not in accordance with the
equipment design, operating instructions or manufacturer’s
recommendations, the operation of the product may be impaired to
the extent that a safety hazard is created.
• Do not attempt to perform electrical work if you are not fully
qualified. This manual is not a substitute for electrical training.
Symbol Conventions
The exclamation point within an equilateral triangle is intended

to alert the user to the presence of important operating
and maintenance (servicing) instructions in the literature
accompanying this product.
The lightning flash with arrowhead symbol within an equilateral
triangle is intended to alert the user to the presence of
uninsulated dangerous voltage within the product’s enclosure
that may be of sufficient magnitude to constitute risk of
electric shock to persons.
The static symbol within an equilateral triangle is intended
to alert the user to the presence of internal components that
could be damaged by static electricity.
This static symbol is intended to alert the user to the presence
of a specific component that could be damaged by static
electricity.
This symbol indicates the presence of alternating current (AC).
7
This symbol indicates the presence of a fuse
This symbol indicates the presence of protective earth ground.
This symbol indicates the power is ON.
This symbol indicates the power is OFF.
Note Additional symbol definitions are available in Symbol Definitions.
General Cautions
• This product should be operated only with the type of power source
indicated on the product’s electrical ratings label. Refer to the
installation instructions included with the product.
• If the power cord provided is replaced for any reason or if an
alternate cord is used, the cord must be approved for use in the
local country. The power cord must be approved for the product’s
listed operating voltage and be rated at least 20% greater than the
ampere ratings marked on the product’s electrical ratings label. The
cord end that connects to the product must have an IEC 60320
connector.
• Plug the product into an approved grounded electrical outlet.
• Do not disable the power cord’s grounding pin.
• If an extension cord or power strip is used, make sure that the cord
or strip is rated for the product, and that the total ampere ratings of
all products plugged into the extension cord or strip do not exceed
80% of the cord’s or strip’s rating limit.
• Route power cords so that they will not be walked on, tripped on,
or pinched by items placed upon or against them. Pay particular
attention to the plug, electrical outlet, and the point where the cord
exits the product.
8
Safe Use
• Do not pull on cords and cables. When unplugging cords or cables,

grasp the corresponding connector.
• Do not install or use this product in any area subject to extreme
short-term temperature variations, or locations that exceed the
specified operating environment temperatures.
• Never use this product in a wet area.
• To avoid injury or fire hazard, do not operate this product in an
explosive atmosphere.
• Do not install or use the product on an unstable, non-level work
surface.
• Do not operate this product with the covers removed or unsecured.
FCC Requirements
WARNING Changes or modifications to this unit not expressly
approved by Advanced Instruments could void the user’s
authority to operate the equipment.
• This equipment has been tested and found to comply with the limits
for a Class B digital device, pursuant to Part 15 of the FCC Rules.
These limits are designed to provide reasonable protection against
harmful interference in a residential installation. This equipment
generates, uses, and can radiate radio frequency energy and, if not
installed and used in accordance with the instructions, may cause
harmful interference to radio communications. However, there is no
guarantee that interference will not occur in a particular installation.
If this equipment does cause harmful interference to radio or
television reception, which can be determined by turning the
equipment off and on, the user is encouraged to try to correct the
interference by one or more of the following measures:
–– Reorient or relocate the receiving antenna.
–– Increase the separation between the equipment and receiver.
–– Connect the equipment into an outlet on a circuit different
from that to which the receiver is connected.
–– Consult the dealer or an experienced radio TV technician for
help.
9
Supplies, Parts and
Accessories
To order parts and accessories, contact the Advanced Instruments
Customer Service Department by using one of the following numbers.
• +US 781-320-9000 (elsewhere)
• 781-320-3669 (fax)
Part description Part no

2-Amp Time Delay (T) Fuse for 100-130V 70022R
Clapper 3C2241
Sample Tubes, Disposable 0.2 or 0.25 mL 3LA825
Heat Transfer Fluid 3DA811
Heat Transfer Fluid Filter 4D3710R
Air Filters (package of 6) 3D2340
Printer Paper (5 rolls) FLA835
Operator/Supervisor Keys 3D3185
Probe/Stir Wire Alignment Tool Kit 3LA700
Replacement Sample Probe with Mandrel and one 10-mL 3D3700
ampule of Probe Bin Setting Fluid (Stainless Steel)
Mandrel 3LH500
One-way Check Valve 4D3705R
Sample Tube Rack 3LA846
RS-232 Cable, 3 meters RS232-CABLE
Service Manual 3255SM
Stir/Freeze Coil 3D2404R
Stir/Freeze Wire (pack of 2) 3LH243
10
Supplies, Parts and Accessories
Part description Part no

Quick Instruction Card (English/French) 3250-7
Quick Instruction Card (German/Spanish) 3250-70
Quick Instruction Card (Italian/Swedish) 3250-71
User’s Guide 3255
Yoke 3LH230
Power Cord (specify voltage and country)
11
Calibrators and
Standards
To order calibrators and standards, contact the Advanced Instruments
Customer Service Department by using one of the following numbers.
• +US 781-320-9000 (elsewhere)
• 781-320-3669 (fax)
Description Part No.

Clinitrol™ 290 mOsm/kg Reference Solution (ten 5 mL ampules) 3LA029
Five-Level Osmolality Linearity Set: 100, 500, 900, 1500 and 2000 3LA028
mOsm (ten 5 mL ampules, two of each value)
Protinol™ 3-Level Protein Control Kit (nine 3 mL bottles, three of 3MA028
each level)
Renol™ Urine Osmolality Controls (eight 3 mL bottles, two levels) 3LA085
0 mOsm/kg Calibration Standard (ten 5 mL ampules) 3MA000
100 mOsm/kg Calibration Standard (110 mL bottle) 3LA010
100 mOsm/kg Calibration Standard (ten 5 mL ampules) 3LA011
12
Calibrators and Standards
Note Advanced Instruments cannot guarantee the stated instrument

performance specifications and accuracy of test results unless
Advanced Instruments brand consumables are used with the
instrument. Use of consumables from manufacturers other than
Advanced Instruments is not recommended and may adversely
affect system calibration, performance, operation, and accuracy
of test results. For information on obtaining these standards
and controls, contact Advanced Instruments or an authorized
representative.
13
Foreword
Intended Use
Advanced™ Osmometers use the technique of freezing-point depression
to measure osmolality. Osmolality is the total solute concentration of an
aqueous solution. Osmometers measure the number of solute particles
irrespective of molecular weight or ionic charge. This information is
useful to the following disciplines:
• Clinical, emergency and sports medicine
• Medical research
• Biotechnology and pharmaceutical research and manufacturing
• Food and beverage manufacturing
• Environmental research and monitoring
• Academic research
• Industrial applications
When used by a trained operator in clinical applications, the
osmometer provides results that assist in establishing the proper
diagnoses and treatments for patients with disorders involving
water and electrolyte imbalances. Osmometers will test virtually any
biological fluid including, but not limited to, whole blood, plasma, urine,
feces, sweat, and tissue homogenate. Operation of the instrument is
deemed to be moderate complexity under CLIA and FDA guidelines.
14
Foreword
Principles of Freezing-Point Osmometry

When a solute is dissolved in a pure solvent, the following changes in
the solution’s properties occur:
• the freezing point is depressed,
• boiling point is raised,
• osmotic pressure is increased, and
• vapor pressure is lowered.
These are the so-called colligative or concentrative properties of the
solution which, within reasonable limits, change in direct proportion
to the solute concentration; in other words, the number of particles in
solution.
Of the colligative properties, measurement of the freezing point allows
the concentration of an aqueous solution to be easily determined with
great precision.
The freezing point of pure H₂O is precisely +0.010 °C. One mole of a
non-dissociating solute such as glucose (where the solute does not
dissociate into ionic species, but remains intact), when dissolved in
1 kilogram (kg) of water will depress the freezing point by 1.858 °C.
This change is known as the freezing point depression constant for
water. The freezing point depression also depends upon the degree
of dissociation of the solute. If the solute is ionic, the freezing point is
depressed by 1.858 °C for each ionic species. For example, if one mole
of sodium chloride were to completely dissociate into two ionic species
(Na+ and Cl-) in 1 kg of water, the freezing point would be depressed
by 3.716 °C. However, dissociation is never complete. Interference
between solute molecules reduces dissociation by a factor called the
osmotic coefficient.
In a simple solution such as glucose or sodium chloride in water, the
freezing point can be measured and the unit concentration easily
determined from an equation or a reference table. However, the
equation is unique for each solute. In a more complex solution, all
ionized and non-dissociated species contribute to the freezing-point
depression and the concentration of each solute cannot be easily
determined.
15
Each of the colligative properties has a similar problem, and though

each of the colligative properties changes in direct proportion to
the solute concentration, each requires a different mode and unit
of measurement. Osmolality is a common unit of concentration
measurement that can be used to relate all the colligative properties to
each other, and to other concentration units. Because of its universality,
most osmometry applications regularly use osmolality, expressed
as mOsm/kg H₂O, as the common unit of concentration rather than
applying further conversion factors.
Instrumentation
Advanced™ Osmometers are devices for the determination of the
concentration of solutions by means of freezing-point measurement.
Advanced Osmometers utilize high-precision thermistors to sense the
sample temperature, to control the degree of supercooling and freeze
induction, and to measure the freezing point of the sample. They can
routinely determine differences of ±1 mOsm/kg H₂O.
Freezing-Point Thermodynamics
The quickest and most precise way to measure the freezing point of a
solution is to supercool it several degrees below its freezing point. It is
unstable in this state, and a mechanical agitation induces crystallization.
The heat of fusion suddenly liberated causes the sample temperature
to rise toward a plateau temperature, where a liquid/solid equilibrium
occurs. The equilibrium temperature is, by definition, the freezing
point of the solution. Managing the plateau temperature for precise
measurement is the basis for several patents issued to Augustus Fiske.
The time over which liquid/solid equilibrium develops and is maintained,
is a function of the speed with which the heat-of-fusion is liberated
vs. the speed it is transferred away, or absorbed, by the surrounding
environment. This ratio can be slowed and the equilibrium time
stretched, to give a distinct plateau height measurable to 0.001 °C.
16
Foreword
Sensitive thermistor probes monitor the sample temperature

and control the thermoelectric cooling element. Microprocessor
control and automated operation minimize imprecision due to
operator technique.
The following Standard Freezing Curve illustrates the
temperature of a sample as it progresses through the freezing
cycle and shows the action of the instrument at each stage of
the cycle.
Standard Freezing Curve
17
Definitions
Solution: A homogeneous mixture of solute and solvent in which the
solvent is usually the major component, and the solute is the minor
component.
Concentration: The ratio of solute to a given amount of solvent (molal),
or ratio of solute to solution (molar).
The amount of solute is usually expressed in terms of moles (i.e., gram
molecular weights). One mole = 6.028 x 1023 molecules (Avogadro’s
number). One mole of glucose (180.2 g) and one mole of sodium
chloride (58.4 g) each contain Avogadro’s number of molecules.
Common units of concentration are:
• Molality: Moles of solute per kilogram of pure solvent.
• Osmolality: Osmols of solute particles per kilogram of pure solvent.
As noted above, most ionic solutes do not completely dissociate.
Osmolality is a unit of concentration that takes into account the
dissociative effect. Osmolality is usually expressed in mOsm/kg H₂O.
One milliosmol (mOsm) is 10-3 osmols. Osmolality is defined as:
osmol
Osmolality = φnC =
kg H 2O
where:
ø = osmotic coefficient, which accounts for the degree of molecular
dissociation.
n = number of particles into which a molecule can dissociate.
C = molal concentration of the solution.
• Molarity: Moles of solute per liter of solution.
18
Foreword
• Osmolarity: Osmols of solute particles per liter of solution.

Although molarity and osmolarity may be common units of
measurement in other branches of chemistry, they are not
used in osmometry because the ratio of solute to solution is
not linear. Molality and osmolality are linear, independent of
the effect of temperature and volume displaced by solute. A
calculated conversion between units of molality and molarity
is complex and generally unnecessary when the terms are
properly understood.
Freezing Point/Melting Point: The temperature at which the
liquid and solid phases of a substance will remain together in
equilibrium.
Freezing-Point Depression: When a solute is added to a
solvent, the freezing point of the solvent is lowered. In aqueous
solutions, one mOsm of solute per kilogram of water depresses
the freezing point by 1.858 millidegrees Celsius (m °C).
Supercooling: The tendency of a substance to remain in the
liquid state when cooled below its freezing point.
Crystallization Temperature: Aqueous solutions can be induced
to freeze (i.e., crystallize) most reliably when supercooled.
When supercooled, agitating the solution (freeze pulse)
induces crystal formation. The crystallization temperature is
the temperature at which crystallization is induced. During
crystallization, the heat of fusion raises the temperature of the
sample to an ice/water freezing-point plateau.
Heat of Fusion: The heat released when the mobile molecules of
a liquid are frozen into rigid ice crystals.
Freezing-Point Plateau: The constant temperature maintained
during the time that ice and liquid exist in isothermal
equilibrium after crystallization occurs.
19
1 Installation and Setup
In order to set up your instrument properly, it is important

that you read and follow the steps in this section. Please
follow these steps carefully and be sure to read Instrument
Operation before attempting to run tests on your instrument.
Step 1 — Find a location for the instrument

When choosing a location for your new osmometer, be sure to meet
the following criteria.
• Adequate space. The dimensions of the instrument are 12.5 ×17.5 ×
11.5 inches (32 × 44 × 29 cm). Be sure to keep your workplace free
of debris and allow 6 inches (15 cm) of clear space on each side of
the instrument for fan-driven air circulation.
• Electric outlet availability. Your instrument will need to operate
within five feet of a properly grounded, three-prong electrical
outlet capable of continuously supplying 2 amperes at 100-240V.
If the instrument is not grounded properly, its operation may be
impaired and a safety hazard may exist. Therefore, be sure to test
the outlet and record the results before operating your instrument.
Step 2 — Obtain additional items

To operate your instrument, you will need to obtain the following
items:
• Soft, no-lint, non-ionic paper tissues for wiping the sample probe.
• Clean, dry 0.2-mL or 0.25-mL pipette.
20
Installation and Setup
Step 3 — Unpack the instrument

To unpack your osmometer, take the following steps.
1. Carefully unpack your osmometer, accessories and supplies and
inspect them for shipping damage. Use the enclosed packing list to
verify that all items have been received.
2. Save your osmometer’s shipping boxes and packaging material in
case future transport of the instrument becomes necessary.
3. If any item on the packing list appears to be missing from your
shipment, please search carefully through and under all packing
materials. If the item is not found, notify your receiving department
immediately. Advanced Instruments can only be responsible for
items reported missing within 10 days of a shipment’s arrival.
4. If you receive any damaged items, save the cartons and packing
material those items came in for inspection by the insurer. The
carrier, dealer, and Advanced Instruments must be notified within
24 hours in order for your warranty and insurance to apply. Have
the transportation company inspect items, fill out a Report of
Concealed Damage, and file your claim. Then, notify Advanced
Instruments immediately for repair or replacement.
5. Complete the online warranty card to register your product.
Quantity Part No. Description
1 3250 The Advanced™ Model 3250 Osmometer
1 3D3185 Operator/Supervisor Keys (set of 2)
1 FL0408 Paper Roll Holder
1 Power Cord (as specified)
1 3LA700 Probe/Stir Alignment Tool Kit
1 3LA846 Sample Tube Rack
1 3250-7 Quick Instruction Card (English/French)
1 3250-70 Quick Instruction Card (German/Spanish)
1 3250-71 Quick Instruction Card (Italian/Swedish)
1 3255 User’s Guide
1 135007PM Warranty Card
Table 1: Model 3250 Osmometer Packing List
21
Step 4 — Install the heat transfer fluid

1. Pull forward the top of the heat transfer compartment door to
release the magnetic latch, then pull the door all the way open (see
Figure 4).
2. Locate the bagged plastic tubes protruding through the back wall
of the compartment. Remove and discard the plastic bag from the
ends of the tubes.
3. A heat transfer fluid filter should already be mounted on the smaller
of the two plastic tubes. If replacement is necessary, force the
tubular end of the heat transfer fluid filter at least ¼" or 6 mm into
the free end of the smaller of the two tubes.
Caution The heat transfer liquid contains hazardous chemicals. Consult
the material safety data sheet (MSDS) and use appropriate
personal protective equipment.
4. Open a bottle of heat transfer fluid.
5. Insert the plastic tube with the heat transfer fluid filter into the
bottle of heat transfer fluid so as to locate the filter at the bottom
of the bottle.
6. Insert the free end of the larger of the two plastic tubes into the
neck of the heat transfer fluid container to return the heat transfer
fluid to the container for recirculation.
7. Stand the container of heat transfer fluid in the plastic tray in the
heat transfer fluid compartment, making sure the filter remains low
in the heat transfer fluid. Press the bottle into the retainer clip.
8. Close the compartment door.
Note When the heat transfer fluid level reaches the REPLACE line on
the bottle label, discard remaining solution and replace with a
new bottle of heat transfer fluid (Part No. 3DA811). Continued
use when fluid is below this level will change the
thermodynamics of the test.
Note It is not recommended to top off the existing bottle of heat
transfer fluid, due to moisture and growth that exist. Always
discard the old bottle and fluid and replace with a new bottle.
22
Note Should it become necessary to move the instrument to another

location after the heat transfer fluid has been installed, take
appropriate precautions to prevent fluid spillage.
Step 5 — Load printer paper (Figure 1)

1. Fully depress the printer cover release button located immediately
in front of the printer cover, then lift up on the printer cover to
open. Release the printer cover release button.
2. Unroll about 6" (15 cm) of paper from the paper roll.
3. Insert the roller into the paper roll.
4. Place the paper roll and roller into the V-shaped roller supports, as
shown in the upper-right photo of Figure 1. Make sure the paper
roll is oriented as shown, feeding from the bottom of the roll. When
both ends of the roller are in proper position, the paper roll should
be level. If the paper roll is not level, carefully reseat the roller
between the roller guides.
5. Hold the paper down onto the surface of the osmometer and center
the paper on the printer tear bar. When the paper is centered,
gently close the printer cover. Press on the top of the printer cover
to make sure it is firmly latched with the printer mechanism.
6. The printer paper is loaded and ready for operation.
Caution Do not attempt to pull the paper through the printer, as this
may damage the mechanism. Either release the printer cover
by fully depressing the printer cover release button and lifting
up on the cover, or use the Feed button on the keypad to
advance the paper.
23
Figure 1: Load Printer Paper
24
Step 6 — Power up the instrument

Power up the 3250 in the following manner:
1. Connect the power cord to the 3250 and the power outlet.
2. If the 3250 has been installed as instructed in the previous steps,
turn the POWER switch on. The display will begin to scroll, and the
printer will begin printing.
3. Record the displayed software revision and the block and sample
probe bin numbers reported by your instrument in the service log
at the end of this user’s guide. When all of the instrument data has
been displayed, the operating head will rise and Press START to
Continue will begin to scroll across the display.
Caution If a power interruption occurs, turn the instrument off at once.
Leave it turned off for at least 5 seconds after power has been
restored (even if power restoration is immediate).
Step 7 — Prime the heat transfer fluid pump

The first time the instrument is being started, you will need to prime
your heat transfer fluid pump using the TEST menu as follows:
1. Insert sample tube in well and press the TEST key. The display will
change to Select Test Item.
2. Press the < key to step the display backward to Head Up/Down
Test.
3. Press the START key. The display will change to [START] Test
[STOP].
4. Press the START key.
25
Primer Pump
Fluid Return
Tube
Figure 2: Prime the Heat Transfer Fluid Pump
5. Open the heat transfer fluid door to access the small primer pump
(Figure 2). Squeeze the primer pump while the operating head is
cycling, making sure to squeeze at the same time that the operating
head reaches the bottom of its cycle. Observe the fluid return
line from the cooling well back to the bottle. Once fluid begins
to appear, continue priming for five more cycles to remove any
trapped air. Stop using the primer pump and watch the return line
to make sure that a small amount of fluid continues to flow through
the return line with each head up/down cycle. Then press the STOP
key to raise the operating head and exit to the test menu.
6. Press the STOP key a second time to exit from the test menu to
Press START to Continue.
26
Step 8 — Run a diagnostic test

With a sample tube containing fluid such as a calibration standard or
deionized water in the freezing chamber, press the START key. The
microprocessor will respond with the display, Running Diagnostics,
while it lowers the operating head, cools the freezing chamber and
completes a series of internal diagnostic checks. If the diagnostic
checks are not successfully completed or the freezing chamber does
not cool properly, a diagnostic message will be presented.
Note Any error reported prior to or during diagnostics will require the
operator to press STOP before any of the keyboard commands
can be used.
If the instrument has just been turned on, it takes a few minutes to
cool the freezing chamber. If the heat transfer fluid pump has not been
primed as described in step 7, the time will be longer.
When the freezing chamber has been cooled and the internal
diagnostic checks have been completed, the operating head rises,
Osmometer Ready is displayed, and an audible tone is sounded.
Step 9 — Set date and time

If you wish, you may now change your date and time settings by using
the instructions found in Chapter 2 of this user’s guide.
Step 10 — Set language preference

The 3250 can be operated in multiple languages. Your instrument will
initially be set for English. If you would prefer to operate the instrument
on one of the other languages, you should change the language setting
as described in 10. Select Language.
27
Step 11 — Check initial factory calibration

Your instrument has been carefully calibrated (including the optional
Zero calibrator) by the manufacturer, but to verify that this calibration
is accurate within your operating environment, run tests using low and/
or high range calibration standards before testing samples. The number
and type of tests that you run should be determined by your own
laboratory’s standard protocol. Use the operating technique described
in Chapter 2 when running these materials.
If you determine that the initial calibration is incorrect, recalibrate as
described in Chapter 4.
Note If the 3250 has just been moved from a different location, it
should be allowed to warm up for 20 to 30 minutes before
running calibration verification tests.
Step 12 — Proceed to Chapter 2

If you have followed the steps outlined in this chapter, your instrument
is ready for use. To learn how to operate your instrument, read the
next chapter, Instrument Operation. We strongly recommend that you
read the entire second chapter before attempting to operate your
osmometer.
28
2 Instrument Operation
In order to run your instrument properly, it is important that you
read and adhere to the instructions in this section. For
information on calibration, see Calibration.
Hazardous material cautions

• WARNING: Handle all biohazardous materials according to
established good laboratory practices and follow your institution’s
exposure control plan. Persons handling human blood and body
fluid samples must be trained in blood-borne hazards and observe
universal precautions. Universal precautions is an approach to
infection control, where all human blood and body fluids are
treated as if known to be infectious. Use personal protective
equipment such as gloves, gowns, etc., to prevent exposure. Store
biohazardous materials in regulated waste containers and dispose
of these materials in a safe and acceptable manner that is in
compliance with all country, state and local requirements.
CAUTION The heat transfer fluid contains hazardous chemicals.
Consult the material safety data sheet (MSDS) and use
appropriate personal protective equipment.
• Heat transfer fluid can become contaminated with sample material
which may be considered a biohazard. Use appropriate cautions
during removal and disposal of sample tubes after testing to
prevent heat transfer fluid remaining on the tube exterior from
contacting personnel or the instrument surface.
• If a biohazardous material is spilled on or inside the equipment,
decontaminate the equipment using a 1% bleach solution, or as
outlined by those policies and procedures established within your
institution.
• To avoid injury or fire hazard, do not operate this product in an
explosive atmosphere.
29
Function of major components

Operation of the instrument will be quicker and easier if you become
familiar with the locations and functions of the components, systems
and controls described below before proceeding further.
Operating head (Figure 3, Item 1)

The operating head contains a stir/freeze wire with electrical means to
vibrate it; an ultra-stable, ultra-precise thermistor sample probe; and
devices to automatically locate the probe and sample in the freezing
chamber.
Supervisor/Operator
Operating Head (1)
Keyswitch, Power Switch,
Fuse Holder, Barcode,
and RS-232 Connector on
Back Panel (see Figure 5) Stir/Freeze Wire (2)
Sample Probe (3)
Printer (9)
Freezing
Chamber (8) Display Panel
and Keypad (4)
Heat Transfer
Fluid Filter (5)
Air Filter (7)

Replace Heat
Transfer Fluid
Filter Line (6)
Figure 3: Model 3250 Components and Controls
Freezing chamber (Figure 3, Item 8)

Sample cooling is performed by a thermoelectric cold stage. The
chamber contains a small amount of heat transfer fluid for optimum
cooling capacity.
30
Instrument Operation
Measurement and control circuits (inside)

The microprocessor-controlled circuits automatically measure and
control the dynamic temperature of the sample freezing chamber.
Process calibration data and sample temperature information. They also
present calibrated test results on the digital display.
Electronic circuits (inside)

The main circuitry is contained on two printed circuit boards in a
motherboard/daughter board configuration. More in depth technical
details are available in the unit service manual sold separately.
Processor Board: The processor board is the smaller of the two printed
circuit boards and contains the Intel 80C186EB central processor, two
flash EPROMs, RAM, Realtime clock, watchdog circuit, and glue logic.
Application Board: The application board contains the circuits for
controlling and interfacing with the other subsystems such as the
keypad, display, head motor, cooling assembly, RS-232, printer and
barcode ports, etc. The application board also contains two 20-bit A/D
converters for reading the sample and block probe thermistors.
Display panel (Figure 3, Item 4)

The display panel displays:
• Test status and results.
• Status messages when the instrument is turned on or when
functions are selected.
• Pertinent error messages when fault conditions occur. (Messages
longer than 20 characters scroll across display.)
31
Keypad (Figure 3, Item 4)

The keypad allows operator input to the microprocessor. The layout and
functions of the keypad switches are explained below.
Figure 4: Keypad Layout and Functions
FEED Advances printer paper

SETUP Activates the setup menu
TEST Activates diagnostic test menu
CALIB Initiates calibration procedure
1 through 0 Allows numeric input, as required

< and > Steps through menu items
CLEAR Clears some data in setup mode and when using
sample identification
ENTER Confirms sample identification and/or setup menu
items
START Starts tests and setup procedures
STOP Cancels the procedure in process.
Refer to Symbol Definitions for more information on keypad symbols.
32
Printer (Figure 3, Item 9)

The printer records on paper the displayed test results, sample
identification, and other necessary information.
Supervisor/Operator keyswitch (Figure 5)

The Operator position of the Supervisor/Operator keyswitch provides
a means of locking out the setup and calibration functions of the
instrument to help prevent unauthorized changes.
The Operator keyswitch position allows access to the sample and test
function only; the Supervisor keyswitch position allows access to the
setup, test and calibration functions, as well as the sample and test
functions.
Barcode Port
Supervisor/Operator
Keyswitch
Power Module
RS-232 Port
Figure 5: Back Panel
33
If the SETUP or CALIB key is pressed while the Supervisor/ Operator

keyswitch is in the Operator position, a Supervisor Key Needed; Press
STOP to Continue message is continually scrolled across the display
until STOP is pressed. When STOP is pressed, the message will change
back to Ready, re-enabling the sample test function.
Power module (Figure 5)

The power module on the rear of the instrument contains the following
components.
• Power switch
The rocker-style power switch controls the power to the instrument.
The power switch may be left on continuously; the 3250 enters
standby mode automatically if idle for more than 5 minutes.
• Power cord connector
The power cord connector accommodates a power cord suitable for
the power available.
• Fuse holder
The fuse holder contains the instrument’s necessary fuses. For
instructions on replacing fuses, see Chapter 5.
RS-232 port (Figure 5)
The RS-232 port allows you to output your instrument’s data/
messages to an external device, such as a computer. For more detailed
information dealing with the RS-232 port, please read the section titled
Using the RS-232 port, which is found later in this chapter.
Barcode port (Figure 5)

A D-type, 15-pin barcode port is provided in the back of the 3250 for
connecting and providing power to such a device. For proper operation,
the barcode port requires a 1200 bps, RS232-level signal providing
asynchronous serial data containing 1 start bit, 8 data bits, 1 stop bit
and no parity.
34
Signal Pin Direction

+5V DC 1 to reader
receive data 10 from reader
gnd/earth 9 common
Table 2: Barcode Port Connections
A suitable barcode scanner is available from Advanced Instruments. To

interface with the 3250, the barcode scanner must be programmed as
follows, referring to the scanner users guide.
• 1200 bps
• CR suffix
• disable beep after good decode
• triggerless trigger mode (optional)
Sample preparation
No special sample preparation is required. Body fluids such as serum or
plasma may be used directly.
Note Additives such as anticoagulants from collection tubes may
contribute significantly to the measured osmolality.
Particulate matter and certain bacteria can also cause premature
crystallization; in blood, it is normally eliminated by centrifugation.
Particulate matter in urine may be removed by centrifugation or
filtration through an inert filter.
Sample handling
Different sample tube sizes and styles should not be intermixed
because each may require a different instrument adjustment and/
or calibration. Sample tubes should be as uniform as possible in
composition, shape and size. For optimum results, you should always
use 3LA825 sample tubes.
35
Repeatability Tips
• Sample tube cleanliness is extremely important for repeatability.
Glass tubes should be washed in hot water containing a nonionic
detergent, rinsed in distilled water, and thoroughly dried before
each use.
• Always treat all samples uniformly before the test. Treat
standards and reference solutions the same as the unknowns.
Do not intermix different tube styles.
• To avoid contamination and evaporation, cover all samples not
immediately being tested.
• Use the same sample size consistently (i.e., during calibration,
testing, etc.).
• For repeat runs, use another sample from the same source or
thaw the original sample before repeating. If you must rerun the
same sample or use it for another procedure, remove it from
the freezing chamber as soon as possible and keep it covered
between tests.
• Always use the same operating procedure as proper, consistent
procedure is the key to repeatable results.
• The first reading in any given period of operation may be
slightly off because of temperature conditioning or residual
contamination of the probe. Subsequent readings on aliquots
of the same sample should group within the specifications as in
Appendix B, if you practice good operating techniques.
• If an occasional sample produces irregular results, discard
obviously discrepant readings as long as the instrument has
been producing accurate readings repeatedly. Repeat the
reading in question.
• Do not reuse plastic sample tubes or cleaning materials. Discard
after each use.
36
Glass tubes generally are not as uniform in shape and size as plastic
tubes, nor do they thermally isolate test samples as well. The 3250
operating parameters are optimized for Advanced Instruments plastic
sample tubes (Part No. 3LA825); glass sample tubes may not provide
the same repeatability. If you need further information concerning
sample tube requirements, please contact Advanced Instruments as
recommended in Chapter 5.
Samples should always be pipetted or measured. 0.25‑mL or 0.2‑mL
samples may be used in Advanced Instruments sample tubes. To
achieve the specified performance, we recommend that you use 0.25-
mL samples; 0.2-mL samples require somewhat more critical probe and
stir/freeze wire adjustments for similar precision in results. The 3250
should be calibrated with the same sample size used for testing.
Verifying calibration daily

Like most measuring instruments, osmometers need calibration against
standards. Your 3250 was calibrated at the factory using the optional
Zero calibrator. The probe and calibration parameters are stored in
parameter RAM which is powered by an internal battery when the
instrument power is off or disconnected. Thus, when Osmometer Ready
is displayed, your 3250 osmometer is calibrated and ready to run.
However, the calibration should always be verified during operation to
be sure that none of the parameters affecting calibration have changed.
(If recalibration is required, see Chapter 4).
Note To verify the precision of your instrument, test products with
known values within your reportable range (i.e., Clinitrol™ 290
mOsm/kg, Protinol™ Protein-based Controls, Renol™ Urine
Osmolality Controls).
Following is the recommended procedure for instrument operation:
37
Sample test procedure

1. Remove the sample tube from the freezing chamber (see Figure 3,
Item 8).
2. Gently wipe the probe, stir/freeze wire, mandrel and the top of the
freezing chamber (in that order) with a soft, lint-free, non-ionic
paper tissue dampened with distilled water to remove anything that
might contaminate the sample to be tested. Be careful not to bend
the probe or stir/freeze wire.
3. Select a reference solution or NaCl standard with a freezing point
close to that expected for your unknown (Advanced™ Clinitrol™ 290
Reference Solution is recommended for serum). Swirl and carefully
open an ampule of the reference solution (or NaCl standard)
selected.
4. If the expected freezing point is in the 1500-4000 mOsm range, the
high range should be selected. If the freezing point is not within the
range selected, an error message will be displayed, requesting the
range to be changed (see Setup Menu, later in this chapter).
5. Select a clean sample tube (see Repeatability Tips).
6. Pipette or measure a sample of the selected reference solution or
NaCl standard into the clean sample tube and place the tube in the
freezing chamber.
7. Press the START key.
I.D. #: If I.D. # has been enabled via the setup menu (see
3. Dis/Enable I.D. #), the instrument operator will be
prompted for a sample or user identification number of up
to 13 digits at this time from either the keypad or optional
barcode scanner.
a. At the I.D. # prompt, the present setting may be retained by
pressing ENTER.
b. The present setting may be erased entirely by pressing CLEAR or
erased one digit at a time from the right by repeatedly pressing <.
38
c. A new number may be entered one digit at a time from the

left via the numeric keypad, unless the number was input from
a barcode scan in which case pressing < will erase the whole
number.
d. Press ENTER to store the new setting and continue the test. Press
START to Continue will begin to scroll across the display.
e. Press START (Pressing STOP would cancel the test).
The rest of the test is completely automatic. The operating head
will lower the sample probe into the sample tube in the freezing
chamber. The fan will come up to full speed and the freezing
chamber will cool.
The sample temperature will follow the freezing curve shown in the
Foreword, and will be displayed as soon as it falls below 0 °C.
The 3250 will freeze the sample at a fixed crystallization point,
follow the plateau development, determine the osmolality, and lock
the result on the display.
Note If the STOP key is pressed while a test is in progress, the
test will be discontinued and the 3250 will reset itself for
the next test.
Note Sample ID# information will be printed on the printout and
presented via the RS-232 port at the end of the test.
8. When the display reads Osmolality XXX mOsm, the operating head
will automatically return to the start position, and the printer will
print out the results.
Note If the wrong osmolality range has been selected, an error
message will replace the osmolality report. Use the select
range procedure to select the required range; then test
another aliquot.
39
Note Results are displayed with the (-) negative temperature sign
removed, per industry practice. However, if testing a sample
with a near 0 mOsm value, it may be possible for a positive
temperature value to be displayed with the negative sign
preceding the mOsm value.
9. Remove the sample tube from the cooling well.
CAUTION The heat transfer fluid contains hazardous chemicals.
CAUTION Heat transfer fluid can become contaminated with sample
material which may be considered a biohazard. Use
appropriate cautions during removal and disposal of sample
tubes after testing to prevent heat transfer fluid remaining
on the tube exterior from contacting personnel or the
instrument surface.
10. Gently wipe the probe, stir/freeze wire, mandrel and the top of the
freezing chamber (in that order) with a soft, clean and dry, lint-free,
single-use paper tissue after each test, to avoid contaminating the
next sample. Be careful not to bend the probe or stir/freeze wire.
11. Repeating steps 1-9, test two to four more aliquots of the same
reference solution or standard to check repeatability and accuracy
before running tests on unknown samples. Follow the techniques
recommended in Repeatability Tips. When checking calibration and
repeatability with standards or reference solutions, it is usually best
to average the readings from three to five aliquots of each sample
to avoid error.
40
If you are running the instrument for the first time, it is

recommended that you follow the Clinitrol™ Reference Solution
samples with bracketing calibration standards to check the
instrument accuracy and linearity over the range of interest.
–– If the instrument accuracy on the reference solution (and/or
calibration standards) is not satisfactory, refer to Calibration.
–– If the instrument repeatability on the reference solution (and/or
calibration standards) is not satisfactory, refer to Standards and
Quality Control.
–– If the accuracy and repeatability on the reference solution (and/
or calibration standards) are satisfactory, you may begin testing
unknown samples, using exactly the same test procedure as for
the reference solution and standards.
12. Always wipe the probe, stir/freeze wire, mandrel and the top of
the freezing chamber with a soft, clean and dry, lint-free, single-
use paper tissue after the last test, to avoid having contaminating
material dry on the probe. Be careful not to bend the probe or stir/
freeze wire.
13. Leave an empty sample tube in the freezing chamber to prevent
debris from entering the well.
The 3250 may be left on continuously. When idle for five minutes or
more, it automatically enters standby mode: the fan slows, the freezing
chamber warms slightly and the display changes to the date and time.
The 3250 remains in the standby mode until another test is needed. It
automatically exits from standby mode when any keypad is pressed.
Sample test errors

Occasionally a test will not run to completion and your instrument will
display an error message. Refer to the Troubleshooting Table at the
end of this user’s guide for an explanation of a particular message.
41
Changing operating settings

Your 3250 has been individually set up at the factory for normal
operation, but SETUP may be required for changing the date and time
or other parameters for your individual needs.
Note NOTE The Supervisor/Operator keyswitch must be in the
Supervisor position to enable use of the SETUP menu. If the
SETUP key is pressed while the Supervisor/Operator keyswitch is
in the Operator position, a Supervisor Key Needed; Press Stop to
Continue message will be displayed.
Specific details for each setup menu item follow.
Setup Menu Items

0. Select Setup Item
1. Set Block Bin #
2. Set Sample Bin #
3. Dis/Enable I.D. #
4. Set Stir Amplitude
5. Set Date/Time
6. Dis/Enable Beeper
7. Set Serial Rate
8. Dis/Enable Xon/Xoff
9. Serial Number
10. Select Language
11. Product/Test
12. Data Capture
13. Assistance
14. Set "Buzz" Amplitude
15. Set "Buzz" Point
16. Select Range
17. Zero Calibration
Table 3: Setup Menu Items
42
0. Select Setup Item

This menu item is displayed first when SETUP is pressed and allows
either direct selection or sequential selection of a menu item.
• Direct Selection: For direct selection, press START at Select Setup
Item. The display will change to Setup Menu #: 0. At Setup Menu
#: 0, enter the number of the desired setup item (from the Setup
Menu, Table 3) on the keypad and press ENTER. The display will
change to the selected menu item.
• Sequential Selection: For sequential selection, repeatedly press the
> switchpad to step the display forward or < to step the display
backward through the setup menu list. The displayed parameter
list is cyclic; pressing > at the last item cycles the display to item 0.
Continue pressing > or < until the desired menu item is displayed.
Note that while direct, numerical selection is only available at Select
Setup Item, > or < stepping is available from any menu position.
When the desired menu item is displayed, press the START switchpad
to select the item, display the current setting and enable changing the
setting.
The current setting is indicated either by the symbol * or a numeric
value and may be changed by pressing > or < (or using the item-
specific instructions below).
Press ENTER to store a new setting (or STOP to restore the original
setting). The display will revert to the title of the parameter selected.
Then one may press: START to display the new setting, > or < to step to
the next SETUP item, STOP to exit the SETUP menu, or TEST to change
to the TEST menu.
43
1. Set Block Bin #

This menu item displays the current block probe bin setting and allows
the setting to be changed.
The block probe bin setting should only need to be changed when
a new block probe of a different bin number is installed in your
instrument.
Upon entering Set Block Bin #, the current setting will be displayed. To
change the setting, enter your new block probe bin number by means
of the numeric keypad. Press ENTER to store the new setting or STOP
to restore the original setting. You may also use the > < keys to step
through the available choices. Once you press ENTER, you will be asked
to confirm the change. Press ENTER to confirm.
Changing the block probe bin number forces recalibration of the
instrument.
2. Set Sample Bin #

This menu item displays the current sample probe bin number setting
and allows the setting to be changed.
The sample probe bin number should only need to be changed when
a new sample probe of a different bin number is installed in your
instrument. Use the sample probe bin test procedure in the diagnostic
tests section (Chapter 5) of this user’s guide to determine the bin
number of a replacement probe.
Upon entering Set Sample Bin #, the current setting will be displayed.
To change the setting, enter your new sample probe bin number by
means of the numeric keypad. Press ENTER to store the new setting or
STOP to restore the original setting. You may also use the > < keys to
step through the available choices. Once you press ENTER, you will be
asked to confirm the change. Press ENTER to confirm.
Changing the sample probe bin number forces recalibration of the
instrument.
44
3. Dis/Enable I.D. #
This menu item enables a means of entering an identification number
for each sample test result. When enabled, each test prompts for an
identification number to be entered via the numeric keypad or optional
barcode scanner. The identification numbers entered are displayed
and sent to the printer port and the RS-232 port with the sample test
results.
ID numbers may not exceed 13 characters.
1. At Dis/Enable I.D. #, press START to display [on] < I.D. # * [off]. The
current setting is indicated by the symbol, *.
2. At [on] < I.D. # * [off], press < or > to toggle the I.D. # option on or
off.
3. Press ENTER to store the new setting (or STOP to restore the
original setting).
4. Set Stir Amplitude
This menu item displays the current sample stir amplitude setting and
allows the setting to be changed.
1. At Set Stir Amplitude, press START to display Amplitude = xx.
2. At Amplitude = xx, look across the tip of the stir/freeze wire from
the side to view and estimate the sample stir vibration amplitude.
If properly adjusted, the stir/freeze wire should vibrate principally
fore and aft and should never strike the probe. The recommended
stir amplitude adjustment procedure may be found in Chapter
5, Troubleshooting and Service. Pressing > increases the stir
amplitude; pressing < decreases the stir amplitude.
original setting).
45
5. Set Date/Time
This menu item enables changing the date/time format and resetting
the date and time.
At Set Date/Time, press START to display the current date and time.
Any change must be made in this order:
1. Use < to toggle to the date format you require (mm/dd/yy or
dd/mm/yy).
2. Use > to toggle to 24hr format, am or pm, as required.
3. Press CLEAR to clear the entire setting.
4. Enter the current date and time (including zeros), via the numeric
keypad, one digit at a time from left to right. Time must be entered
in 24-hour format.
5. Use > to toggle 24hr format to am or pm, as required.
6. Use < to toggle to the date format you require (mm/dd/yy or
dd/mm/yy).
original setting). If an invalid setting is attempted, the instrument
will reject the setting and beep three times when ENTER is pressed.
6. Dis/Enable Beeper
The 3250 keypad in itself has no audible indication that a key has been
adequately pressed. The Dis/Enable Beeper menu item enables an
audible indication.
1. At Dis/Enable Beeper, press START to display [on ] < beeper * [off].
The current setting is indicated by the symbol, *.
2. At [on] < beeper * [off], press < or > to toggle the keypad beeper
on or off. Neither selection will affect the invalid-selection beeper
except that, when the keypad beeper is on, an invalid selection will
invoke two beeps, when off, only one.
original setting).
46
7. Set Serial Rate

This menu item enables changing the serial rate of the data sent to the
RS-232 port.
1. At Set Serial Rate, press START to display Serial Rate = xxxx.
2. At Serial Rate = xxxx, the serial-port baud rate may be reset by
means of the < or > key to either 1200, 2400, 4800, 9600, or 19200
baud.
original setting).
8. Dis/Enable Xon/Xoff
This menu item enables or disables the Xon/Xoff handshaking protocol
for RS-232 communication.
1. At Dis/Enable Xon/Xoff, press START to display [on] < Xon/Xof *
[off]. The current setting is indicated by the symbol, *.
2. At [on] < Xon/Xof * [off], press < or > to enable or disable serial-
port Xon/Xoff handshaking.
original setting).
Note Sending a CTRL-S from the connected PC will suspend RS-232
output until you send a CTRL-Q. While suspended, data will
accumulate in the UART output buffer. However, output buffer
memory is limited, and data will be lost if the output remains
suspended for too long.
9. Serial Number
1. At Serial Number, press START to display the instrument serial
number (Serial #: 123).
2. Press STOP to return to the setup menu. This number is set by the
factory and cannot be changed by the operator.
47
10. Select Language

This menu item enables selecting one of the alternate languages.
1. At Select Language, press START to display the current setting.
2. Press < or > to select one of the available languages.
original setting).
11. Product/Test
Product/Test is for factory use only and has no field function.
12. Data Capture

Pressing START at this SETUP menu item will bring you into an ON/
OFF selection menu.
When set to ON, the freezing curve output data will be presented to
the serial port independent of the system’s one-way hold feature. This
data is updated every 100 ms after crossing 0 °C and may be captured
with any RS-232 compatible computer program for use in plotting
freezing curves.
13. Assistance
Pressing START at this SETUP menu item will present you with
information on how to contact Advanced Instruments for supplies and
service. Important settings information which may be needed by our
service personnel will also be presented.
14. Set "Buzz" Amplitude

Note The amplitude of the freeze pulse has been optimized by the
factory. Adjustment of this setting is recommended only under
the supervision of an authorized service technician.
This menu item displays the current "buzz" amplitude setting and
allows the setting to be changed. The number displayed has no unit of
measure and may vary from assembly to assembly.
48
1. At Set Buzz Amplitude, press START to display Amplitude = xxx

and activate a one-second freeze pulse.
2. Each press of the START keypad will produce an additional one-
second freeze pulse.
3. Pressing the > key will increase the setting up to a maximum of 255.
Pressing the < key will decrease the setting to a minimum of 150.
original setting).
15. Set "Buzz" Point
This menu item enables setting the temperature at which crystallization
is induced by the freeze pulse (buzz) in each range.
Most freezing point osmometers supercool all samples in the
0-2000 mOsm range to approximately 3000 on the mOsm scale. The
3250 buzz points default to 3000 for the lower range and 4800 for the
upper range. Because the test results may be affected somewhat by the
amount of supercooling, these default buzz points are recommended
for general use and for the most reliable comparison of data.
Certain fluids may not freeze reliably at the default buzz point,
however, or may give more repeatable results with either more or
less supercooling than the default buzz point provides. The 3250 is
designed to accommodate these, as well as the more ordinary types of
samples.
1. At Set “Buzz” Point, press START to display the current
crystallization setting, displayed as “Buzz” Point = xxxx. To change
the crystallization point:
2. At “Buzz” Point = xxxx, the present setting must be cleared before
the buzz point can be changed. Press CLEAR to clear the setting.
Alternatively, the < > keys may be used to increase or decrease the
value, one digit at a time.
3. Enter the desired buzz point (including zeros), via the numeric key-
pad, one digit at a time from left to right. The 3250 buzz point can
be set between 2000 and 5400 on the mOsm scale in either low or
high range.
49
Note The buzz point should be at least 1200 mOsm higher than
the expected test value.
original setting).
Note Each time the buzz point is changed, the instrument
displays the message Check Calibration, a reminder that
the instrument calibration is affected by the amount of
supercooling.
16. Select Range

This menu item enables optimizing the 3250 test parameters for a
wider osmolality range than can be accommodated by a single set of
test parameters. Two ranges are provided:
• Low range sets the 3250 to operate between 0 and 2000 mOsm.
• High range sets the 3250 to operate between 1500 and 4000 mOsm.
Independent calibration parameters are maintained for each range (see
Chapter 4 for calibration instructions). Generally, each range requires a
different crystallization temperature ("buzz" point). See item 15, above.
To change the test range:
1. At Select Range, press START to display [low] range >[high].
2. At [low] range >[high], press < or > to toggle between the low and
high ranges.
original setting).
Note Each time the range is changed, the instrument displays the
buzz point for the range selected.
50
17. Zero Calibration

This menu item allows the user to activate or deactivate the zero
calibration (for low range only). Enabling this will prompt the user to
test three (3) replicates of the zero (0 mOsm) calibrator (3MA000) as
part of the low range calibration.
To set Zero Point Calibration on/off:
1. At Zero Calibration, press START to display [ON]< Zero Cal? *[OFF].
2. At [ON]< Zero Cal? *[OFF], press < or > to toggle between Zero
Calibration ON/OFF.
3. Once set press ENTER to confirm.
Using the RS-232 port

The RS-232 port (see Figure 5) allows you to output to an external
device, such as a computer. Almost every item of information displayed
by your instrument is also transmitted over the RS-232 port, including
test results, all error messages, and most display data from the
diagnostic menu.
The default serial data rate for communications is 9600 bps (bits per
second), though you may alternatively select 1200, 2400, 4800, and
19200 bps.
The DB-9 RS-232 port on your instrument conforms to the DTE EIA-
232C standard and can reliably communicate over shielded cable up to
10 meters in length, depending on the baudrate you use.
Note that your instrument is only designed to support unidirectional
communication with an external device. At this time, there is no
protocol for bidirectional communication.
For a sample RS-232 Port Setup, please see the Supplemental RS-232
Information at the end of this user’s guide.
Note This instrument requires the use of a null modem RS-232C cable.
There are several variations on null modem cables. Advanced
Instruments recommends that you purchase a RS-232C cable
direct from our factory.
51
3 Standards and Quality
Control
Repeatability and accuracy

Two important measures of quality control are repeatability and
accuracy. Repeatability is sometimes called reproducibility, sometimes
precision. However described, repeatability is essential in nearly all
forms of measurement, and is fundamental to accuracy. Repeatability
is not accuracy; repeatability underlies accuracy. Repeatability is a
measure of the ability of a method to obtain the same result time after
time on the same sample.
Repeatability or precision is usually quantified as the standard
deviation (S.D.) of a set of measurements normally distributed about a
mean: ±1 S.D. about the mean denotes the range of values within which
68% of the measurements will fall; ±2 S.D., 95%.
By contrast, accuracy is a measure of how close to the true value a
method or measuring device has come. One can repeat without being
accurate, but one cannot be accurate without repeating well.
Standards and controls

To be accurate, all measurements ultimately must be referred to highly
reliable independent standards. Advanced™ Osmometers are calibrated
with standards consisting of stable sodium chloride solutions of known
freezing points which bracket those of the expected unknowns. These
standards are related to the most fundamental work on freezing
points of dilute solutions published and are traceable to the National
Institute of Standards and Technology (formerly the National Bureau
of Standards). We also use and provide a sodium chloride reference
solution for checking osmometer accuracy at a point close to the
osmolality of normal blood serum.
52
Standards and Quality Control
Osmometers used for testing biological solutions should be checked

additionally by making determinations on actual biological solutions or
controls formulated to simulate biological solutions. Several biological
controls are commercially available and may be recommended
or specified in your area. Contact your Advanced Instruments
representative to obtain Protinol® Protein-based Controls or Renol™
Urine Osmolality Controls. Please check with your professional
associations and regulatory bodies for their guidelines and/or
requirements, and refer to our recommendations contained herein.
Maintenance of standards
Doubts about the accuracy of standards destroy confidence in
the accuracy of the test. Advanced™ osmometer standards are
specified to be within very close tolerances. Several of the sodium
chloride-based reference and calibration solutions designed for the
Advanced™ Osmometers are sealed in glass ampules to insure against
any possibility of alteration during transit or storage. However, we
recommend that you test samples from each newly-received lot and
maintain records of your test results on osmometer standards for
reference.
Quality control implementation

The following recommendations outline the minimum requirements for
quality control:
• Read, understand and follow the instructions and recommendations
in the user’s guide as closely as practical.
• The instrument repeatability and accuracy should be checked
each time it is used, as recommended in step 9 of the sample
test procedure section. Run at least two levels of standards or
controls daily. Prepare, maintain and follow additional verification
procedures specifically adapted to your own laboratory
requirements as necessary.
• Repeatability tips are specified in the previous chapter. It depends
to a certain extent upon operator technique and can usually be
improved by carefully following the suggestions in the repeatability
tips section.
53
• Instrument accuracy is determined by comparing your results on

precisely-known standards and/or controls with their specified
values. The instrument accuracy is adjustable; if it is unsatisfactory,
it should be corrected by recalibrating, as instructed in Chapter 4.
However, it has been shown that frequent unnecessary recalibration
will introduce inaccuracy. The calibration of a freezing point
osmometer in good condition and properly operated will not shift
or drift.
• Follow quality control requirements of your associations and
regulatory bodies.
• Advanced Instruments recommends the following usage schedule
of quality control materials to ensure proper instrument operation,
result reporting, and Good Laboratory Practice:
–– Clinitrol™ 290 Reference Solution should be used daily to ensure
that the instrument is performing properly and has not shifted.
–– If your laboratory is required to run matrix-based control
solutions, Protinol™ Serum Protein Control and Renol™ Urine
Control should be run daily to monitor precision and create an
audit trail that can be tied to patient results.
–– Linearity should be verified using the five-level linearity set as
required by your laboratory.
54
4 Calibration
This chapter describes the procedure for calibrating your
instrument. If you have questions or problems regarding the
calibration procedure, please see Troubleshooting and Service.
Calibration procedure
Aside from requiring manual sample introduction, calibration of the
3250 is automatic. Following the procedure suggested below, you
simply select CALIB and initiate freezing-point tests on each of two
calibration standards required for the selected range.
The calibration standards required are:
• Low range: 100 and 1500 mOsm/kg H₂O
• High range: 1500 and 3000 mOsm/kg H₂O
The 3250 also offers an optional Zero calibration (0 mOsm/Kg H₂O) in
the low range only (see 17. Zero Calibration)
Internally, the 3250 accomplishes calibration in two steps for each
range. First, it records the raw means of the results of freezing point
tests on standards at each of the two calibration levels. Then it
performs the necessary conversion calculations and calibrates the
display. Each range is independently calibrated.
Check the calibration frequently, as recommended in Chapter 2, but do
not recalibrate when the calibration is satisfactory. It has been shown
that unnecessary recalibration will introduce inaccuracy. The calibration
of a freezing point osmometer in good condition and properly
operated will not shift or drift.
Both the High and Low ranges were initially calibrated at the factory.
However, each time the buzz point is changed, the calibration should
be checked and recalibrated if necessary. Also, anytime the sample or
block probe bin numbers are changed, recalibration will be required.
55
The calibration procedure is as follows:

1. At Osmometer Ready (or the date and time standby display), press
CALIB.
The CALIB key will light and the display will quickly change to
Calibration, then prompt you for the first calibration standard.
2. If the sample probe is replaced, the sample probe bin # must be
determined and set before the 3250 can be recalibrated.
3. Load the standard sample prompted by the display into the
freezing chamber and press START.
The display will change to Cooling Sample, the operating head will
lower, a calibration test will be run and the results will be displayed
and recorded internally.
Continue loading and testing standard samples at the first
calibration level until prompted by the display for a sample at the
second calibration level.
Three samples are required at the first calibration level of the Low
range. Do not load samples at the second calibration level until
prompted by the display; to do so may cause a Calibration Out of
Range; Repeat Calib error message.
At least six samples are required at the second calibration level of
the Low range, and at each calibration level of the High range.
When calibration tests have been acceptably completed at the first
calibration level, the instrument will calculate and store the mean
of the raw values of the closest tests, then prompt the operator
for samples at the second calibration level. The calibration will not
actually be changed until all calibration tests have been acceptably
completed at both calibration levels.
When six calibration tests have been acceptably completed at the
second calibration level, the instrument will calculate and store the
mean of the raw values of the closest tests, recalibrate the readout
and display Calibration Complete.
4. At Calibration Complete press STOP to return to Osmometer Ready,
CALIB to restart the calibration sequence, or START to lower the
operating head and start a regular freezing point test.
56
Calibration
Calibration notes
• The 3250 will retain its previous calibration data until it completes a
recalibration and the display reads Calibration Complete.
• Although it is possible for the 3250 to detect certain gross
calibration errors such as reversal of the standards, it remains the
responsibility of the operator to be sure to introduce accurate
standards of the specified freezing point values.
• Pressing STOP during recalibration will cancel the current
calibration test but allow calibration to be resumed with another
sample. Pressing STOP a second time will cancel recalibration and
retain the previous calibration.
• Good operating technique is as important during calibration as
during normal operation (see Repeatability Tips). If the calibration
test results are not acceptably repeated throughout the six tests
at each calibration level, the instrument will report Calibration out
of Range and the entire calibration procedure must be redone. If
the repeatability problem recurs despite good operating technique,
please call or write immediately for
Hot-Line service.
Note Pressing ENTER in Set Block Bin # or Set Sample Bin # will
erase calibration and force a full recalibration of the instrument.
Note If the instrument has lost all previous calibration data, or you
have reset the block or sample bin numbers, you will see the
message Recalibration Needed. Pay close attention to the
displayed prompts. You will be requested to run a slightly
different sequence than described above.
57
5 Troubleshooting and
Service
This chapter contains very basic information to help you solve
problems that might arise with your osmometer. Please read all
instructions very carefully, and if a solution cannot be found in
this guide, contact Advanced Instruments for Hot-Line Service.
Service and maintenance cautions

• Do not perform any service or maintenance yourself, except as
detailed in the User’s Guide.
• Unplug the power cord prior to opening or removing covers, or else
you may be exposed to electric shock, excessive temperatures, or
mechanical hazards.
• Performing service or maintenance not detailed in the User’s
Guide, with or without a Service Manual, should only be done by a
qualified service technician.
• Never restrict airflow into or out of the product. Occasionally, check
the air vents for blockage.
• Wipe the exterior of the product with a soft, damp cloth as needed.
Using cleaning products other than those specified, may discolor or
damage the finish.
• If the product requires service for any of the following reasons,
unplug the product from the electrical outlet and refer service to a
qualified service technician.
–– The power cord, extension cord, power strip, or power input
module is damaged.
–– Liquid has been spilled into the interior of the product.
–– A foreign object has fallen into the product.
–– The product has been dropped or damaged by a falling object.
58
Troubleshooting and Service
–– There are noticeable signs of overheating or a burning odor.

–– The product does not operate normally when you follow the
operating procedures.
–– The main supply fuse(s) or any internal fuse(s) continually fail.
• A discharge of static electricity from contact with the human body
or other conductor may damage system boards or static sensitive
devices. Never perform internal maintenance without following
recommended static protection procedures.
• The product is equipped with operator accessible fuses. If a fuse
blows, it may be due to a power surge or failure of a component.
Replace the fuse only once. If the fuse blows a second time, it is
probably caused by failure of a component part. If this occurs, refer
service to qualified service personnel. Always replace the fuse with
one of the same rating, voltage, and type. Never replace the fuse
with one of a higher current rating.
• When servicing the product, use only factory-specified parts.
• The product contains an integral lithium battery that is not user-
serviceable.
WARNING When returning this product for service, or shipping this
product to a second location, remove all heat transfer fluid
and hazardous specimens and decontaminate the product
before packaging for shipment. If the product cannot be
decontaminated, consult with your shipping agent on
appropriate packaging and marking.
59
Obtaining service
Before contacting Advanced Instruments for Hot-Line Service,
be sure to read through this user’s guide for instructions on
routine adjustments, instrument care and troubleshooting. If this
information does not solve your problem, call the appropriate
number below.
• +US 781-320-9000 (elsewhere)
• 781-320-0811 (fax)
If you purchased your instrument outside of the U.S. or Canada,
please contact your Advanced Instruments authorized dealer
for service or repair.
When contacting our service personnel, please have ready
the model and serial numbers from the label on the back of
your instrument, your user’s guide or service manual, and
the symptoms of your problem. You should use a telephone
as close to your instrument as possible to facilitate making
recommended diagnostic checks. If you need to order parts or
service, a purchase order from your purchasing agent will be
necessary.
A service technician may assist in making minor repairs over
the phone, providing you with recommended parts (or part
numbers), or may issue an authorization to ship the instrument
for factory repair.
60
To return an instrument for repair or replacement:

1. Notify our service department to obtain an RMA (Returned Material
Authorization).
CAUTION The heat transfer liquid contains hazardous chemicals.
2. Open the heat transfer fluid compartment in the front of the
instrument. Remove the smaller plastic tube from the heat transfer
fluid container; temporarily leave the larger plastic tube in the heat
transfer fluid container.
3. Place an empty sample tube in the freezing chamber. Run the Head
Up/Down Test for a few minutes to pump the heat transfer fluid
remaining in the system out into the heat transfer fluid container.
Remove, cap and retain the heat transfer fluid container.
4. Tape or tie a small plastic bag over the ends of the heat transfer
fluid plastic tubes and fluid filter to retain any leakage. Lower the
operating head into the empty sample tube in the freezing chamber
to protect the probe during shipment.
5. Carefully pack and send everything except supply items.
Be sure to prepay for any shipment to the factory. Advanced
Instruments cannot accept collect shipments without prior factory
approval. Please insure the shipment, or accept the damage risk.
61
Routine maintenance
Daily. Checks you should perform to keep your instrument in peak
condition are:
• Calibration Repeatability: Readings each day on separate aliquots of
the same standards should not deviate beyond the repeatability and
drift limits indicated in Appendix B.
• Heat-transfer fluid: Heat-transfer fluid will evaporate from the well
and droplets will cling to the sample tubes as they are removed.
Check the contents of the heat transfer fluid reservoir occasionally;
replace the fluid when the level drops below the REPLACE line on
the bottle.
CAUTION The heat transfer liquid contains hazardous chemicals.
Monthly. Maintenance you should perform:
Clean or replace the air filter (Figure 3, item 7) monthly; replace as
necessary. A dirty filter affects the ability of the fan to cool internal
components; a rise in internal temperature may adversely affect
reliability.
Shutdown and storage

Overnight or weekend:
1. Rinse the probe clean with distilled water and wipe it dry.
2. Turn off the instrument, if desired.
3. Do not cover the instrument unless it is turned off.
One week or longer:
1. Rinse the probe clean with distilled water and wipe it dry.
2. Turn off the instrument.
3. Unplug the power cord from the wall outlet.
4. Cover the instrument.
62
Troubleshooting checks
Check operational requirements. If you are experiencing difficulties
with your instrument, first carefully review the operational requirements
listed in the product specifications and the recommended setup and
operating procedures. Check the heat transfer fluid level. Make sure
that the heat transfer fluid pump is fully primed.
Check fuses. You will find the power switch and fuse holder beside the
power cord connector on the back panel of the instrument. Switch the
power switch to the off position and disconnect the power cord. Use a
small flat-bladed screwdriver or similar tool to pry out the fuse holder.
Visually check for a blown fuse. If there is any doubt, test the fuses
with a continuity checker or ohmmeter or simply replace them with
new fuses.
Check probe and stir wire. Run the A/D Test and the Probe Bin Test
to evaluate the probe operation. Refer to the Mandrel, Probe, and Stir/
Freeze Wire Alignment section for proper alignment of the sample
probe and the stir wire. Refer to the Stir/Freeze Amplitude Adjustment
section to properly adjust the stir and freeze amplitudes.
Check error messages. The software of your instrument is designed in
such a way that any incomplete task will be associated with an error
message, many self-explanatory, that will help you discover the source
of your problem. You can find all error messages and descriptions of
what they mean in Appendix A.
63
Internal diagnostic tests

The diagnostic menu allows any of a series of tests to be run
to check one functional subsystem of the instrument, or to
perform some necessary adjustment or setup.
An item on the TEST menu may be selected in either of the
following two ways:
• At Select Test Item, repeatedly press the > key to step the
display forward or < to step the display backward through
the list. The list is cyclic; pressing > at the last item cycles
the display to item 0. Continue pressing > or < until the
desired test is displayed. While the desired test is displayed,
press the START key.
• Or, at Select Test Item, press the START key. The display
will change to Test Menu #: 0. Type the number of the
desired test on the numeric keypad. The 0 on the display
will change to the number typed, if valid. Then press ENTER.
The display will show the selected test. Next, press the
START key.
• At Select Test Item, the following tests are available:
0. Select Test Item
1. Statistics
2. Recall Results
3. A/D Tests
4. Stir/Freeze Test
5. Probe Bin Test
6. Barcode Test
7. Display/Print Test
8. Beeper Test
9. Keypad Test
10. Head Up/Down Test
11. Event Record
64
Descriptions of the test menu items and instructions for their use
follow:
1. Statistics
The statistics option allows you to recall the stored results from a
selected last number of tests and calculate their average, standard
deviation and coefficient of variance. To use this option, use the
following procedure.
1. At Osmometer Ready, press the TEST key and then the > key,
respectively.
2. Press START to enter the statistics mode. Your instrument will
request the number of tests to be considered when computing
statistics. Enter a number from 2 to 30.
3. Press the left ENTER key to display the average of the selected
number of last results.
4. Press the left < key to display the standard deviation of the
selected number of last results.
5. Press the left < key a second time to display the coefficient of
variance of the selected number of last results.
6. To return to the diagnostic menu, press the STOP switchpad.
7. To return to Osmometer Ready, again press the STOP key.
Note The instrument will output all three values to the printer and
serial port at step 3. For example:
Based on Last 5 Samples
AVG = 901.40 mOsm
Std Dev = 1.14 mOsm
CV = 0.13%
65
2. Recall results
The recall results feature of the Model 3250 allows you to display and
print the results of your last 30 tests. If you need to see any or all of
these results, take the following steps.
1. At Osmometer Ready, press the TEST key to enter the diagnostic
test menu.
2. Use the > or < keys to cycle through your choices until you see
Recall Results.
3. Press START to enter the recall results mode. Your instrument will
display and print the data of the most recent test result.
4. Use the > or < keys to see the results stored in the recall buffer.
5. Continue in this manner until you have recalled the necessary
results. For example:
#30 09/06/2005 01:04:29 PM
ID NONE
Result = 903 mOsm
6. To return to the diagnostic menu, press the STOP key.

7. To return to Osmometer Ready, press the STOP key again.
3. A/D Tests
This set of tests may be used to test the block probe, the cooling
assembly and the sample probe. The A/D tests may also be used
to check the accuracy of the A/D conversion channel or to monitor
the A/D channel for drift over time or temperature (by temporarily
replacing the sample probe with a precision resistor).
On entry, the A/D tests will display [Start] Ready?.
1. At [Start] Ready?, pipette or measure a sample of 1500 or 3000
m °C standard into a sample tube, place the tube in the freezing
chamber, and press the START key.
On entry, the A/D tests display the target cooling block
temperature, the channel being read, and the current channel
reading or duty cycle.
66
The display is in the form, off Blk NNNN.NN ohm, where off
indicates that a target temperature has not yet been selected, Blk
indicates that the block probe channel is being tested, NNNN.NN is
a numeric read-out of the probe, and ohm indicates the units of the
readout. These readings are updated continuously.
2. Pressing the < key sequentially changes the target cooling well
block temperature from off to +1, to -8, in steps of -1 °C to off, etc.
3. Pressing the > key sequentially changes the channel and readout
units from block probe resistance in ohms, to sample probe
resistance in ohms, to block temperature in tenths of a degree
Celsius, to sample temperature in tenths of a degree Celsius, to
cooling block duty cycle (NN % on), to block resistance in ohms,
etc.
CAUTION The temperature values displayed are based on the block
probe resistance which the instrument is configured for.
Thus, if the block probe bin number has been incorrectly
set, both the displayed temperatures and resistances will
be incorrect, as well as the actual temperature of the
sample cooling well.
While the target temperature is off, the block probe is warm and its
resistance should be less than 4,990 ohms. As the temperature is
set colder, the resistance will increase.
4. Press STOP to raise the operating head and exit to the test menu.
67
4. Stir/Freeze Test
This menu item enables visually and aurally checking the stir and
freeze vibration amplitudes. Please refer to the Stir/freeze amplitude
adjustment section shown later on in this chapter for instructions.
5. Probe Bin Test

This test is used to determine the resistance and bin number of the
sample probe at a specific temperature (-0.093 °C). It is essentially the
same as any other sample test except that the sample probe resistance
and correct bin number are determined and displayed, in place of the
sample freezing point.
On entry, the probe bin test will display [Start] Ready?.
1. At [Start] Ready?, pipette or measure a sample of probe bin-
setting fluid into a clean sample tube, place the tube in the freezing
chamber and press the START key. The 3250 will run a special
freezing-point test to determine the sample probe resistance and
bin number.
Note In an emergency, H₂O may be used in place of probe bin-setting
fluid for this test. The bin number thus determined will be
acceptable for bin setting though the probe resistance displayed
will be that at the freezing point of the H₂O sample used and will
be different from that at a freezing point of -0.093 °C.
The display will flash Cooling Sample (as in a normal test) until the
sample probe resistance reaches 4990 ohms. Then the test proceeds
normally except that the display is in ohms rather than mOsm. At
the end of each test, the final display will be the test result in ohms
and the sample probe bin number (e.g., 12345.67 Ohms Bin 10).
2. A test in progress can be stopped by pressing the STOP key.
3. Another test may be run by replacing the sample and pressing
START again.
68
At a specific freezing point, each probe has a unique resistance

which should vary very little from test to test. Therefore, the results
of this test may be used as a troubleshooting aid, in conjunction
with the sample-probe checks described in section Sample probe
checks and replacement, shown later in this chapter.
4. Record the sample probe resistance and bin number in the service
log at the end of this user’s guide (and/or other convenient
location); then press the STOP key.
The display will revert to Probe Bin Test.
5. Press SETUP and follow the procedure 2. Set Sample Bin # to reset
the sample probe bin setting as necessary.
Note Recalibration is required each time the sample probe is
replaced and/or the sample probe bin number is reset.
If running this test produces the message, Reset Probe
Configuration instead of the sample probe resistance and bin
number, the sample probe bin number is not available from the
instrument memory and must be reset.
6. Press SETUP and follow the procedure 2. Set Sample Bin #
described in Chapter 2 to preset a random sample probe bin
number, such as 5. Then press TEST and rerun the probe bin test.
Finally, as necessary, press SETUP and use the 2. Set Sample Bin
# procedure to correct the sample probe bin setting. If the Reset
Probe Configuration message persists, please refer to Chapter 5
Obtaining service to get help.
69
6. Barcode Test
This test performs a continuous check of the barcode port.
1. Press the START key to enter the test. At [START] Test [STOP] you
may press START once the barcode scanner is connected, or press
STOP to return to the main menu.
After you press START you may begin scanning barcodes. If your
code will not scan, check the manufacturers user’s guide to verify
correct setup of the scanner for your symbologies.
2. Press STOP to exit to the test menu.
Note This test will only work after power on diagnostics have been
completed.
7. Display/Print Test
This test is a simple check of the display and printer. On entry, a series
of characters should scroll across the display (and a single line of
the same characters should print out on the printer). The characters
displayed have been chosen to illuminate every dot in the character
matrix. This makes it possible to distinguish any dots that no longer
work, on the printer as well as the display.
Press STOP to exit to the test menu.
8. Beeper Test
This test exercises the beeper.
1. On entry, [on] beeper [off] is displayed. Press the < key to cause
the beeper to beep repeatedly; press the > key to stop the beeper.
2. Press STOP to exit to the test menu.
9. Keypad Test
This test enables checking each key on the keypad, as well as the
lamps of each illuminated key. As each key is pressed, its name should
be displayed and the lamp of each illuminated key should light.
Press the STOP key twice to exit to the test.
70
10. Head Up/Down Test

This test enables selectively lowering and raising the operating head. It
may be used to check the operation of the head motor drive, to prime
and drain the heat transfer fluid pump, and to check for obstructions in
the fluid system.
CAUTION To protect the sample probe, place a sample tube in the
freezing chamber before running this test.
1. On entry, [START] Test [STOP] is displayed. Press START to

continuously cycle the operating head down and up.
2. Press STOP to raise the operating head and exit to the test menu
3. Press STOP to exit from the test menu to Osmometer Ready (if the
initial diagnostics had not been completed when the test menu was
entered, the 3250 will complete them before Osmometer Ready is
displayed).
11. Event Record
This menu item displays the last 200 recordable events of the
instrument, and is used when servicing the instrument.
Fuse replacement
If you determine that your instrument is not functioning because of
blown fuses, you will need to replace the fuses using the following
procedure:
1. Switch the power switch to the off position and disconnect the
power cord.
2. Use a small flat-bladed screwdriver or similar tool to remove the
fuse holder door. Remove the fuse holder.
3. Double-check the values marked on the fuses. The Model 3250 will
automatically adjust for voltages between 100VAC and 240VAC,
but appropriately rated fuses (2) must be installed. Use 5 x 20 mm,
250V, time delay (Type T): 2-Amp.
4. Re-install the fuse holder into the back of the instrument.
71
5. Reconnect the power cord and switch the power switch to the on
position. The instrument should start up as normal.
Power Switch
Fuse Holder
Figure 6: Fuse Replacement
72
Yoke (1)
Sample Probe
Connector (10) Clapper (3)
Stir/Freeze Wire
Coil (2)
Setscrews (4)
Head
Mandrel
Setscrews (9)
Mandrel (5)
Sample Probe
Setscrew (6)
Stir Wire Slot in
Mandrel
Sample Probe (8)
Stir/Freeze Wire (7)
Figure 7: Operating Head
Sample probe checks and replacement

Glass-tipped probes are very sensitive and quick to respond but are
somewhat susceptible to pushing, pulling and bumping damage. Such
damage often causes varying electrical leakage to ground through
the sample and may usually be detected via close visual inspection or
disconnecting the connector and checking the probe resistance. This
can be done in place without disturbing the physical adjustment of the
probe.
73
The following procedure is recommended for testing and/or replacing

the sample probe:
1. Place an empty sample tube in the freezing chamber
(Figure 3, Item 8) to catch any extraneous material that might fall
in.
2. Unplug the instrument; it is not sufficient to just turn it off.
3. Remove the head cover by removing the two top screws and pulling
the cover straight up.
4. Unplug the sample probe connector (Figure 7, Item 10). The probe
leads are electrically accessible through the side slots in the
connector. Use care not to touch or bend the connector pins. Soil
or salt from your fingers can increase the electrical resistance of the
connection.
5. With the probe at room temperature (20 °C - 25 °C), the resistance
between the probe leads should be in a range of 2000 - 2800
ohms. Immerse the tip of the probe into a container of ice and
water (~0 °C). The resistance should rapidly increase to a range of
5500 - 6100 ohms.
With the tip of the probe immersed in a concentrated NaCl solution
(do not wet the probe leads or connector with the salt water), the
resistance between either probe lead and the solution should be
greater than 20 megohms. Carefully rinse the probe clean following
this test.
Probe damage is indicated by any deviation from the resistance
parameters given above.
If sample probe replacement is indicated:
6. Loosen the two setscrews (Figure 7, Item 9) holding the mandrel in
the head chassis. Lower the probe and mandrel down over the stir
wire.
7. Fit a new sample probe by pushing the probe connector and wires
up through the hole in the head chassis before seating the mandrel.
74
8. Then raise the mandrel up around the stir wire. Align the mandrel
so the stir-wire channel is perpendicular to the front edge of the
chassis and so the flange is flush, with no gaps between the flange
and the underside of the chassis. Retighten the two setscrews to
hold the mandrel in place. Do not overtighten—overtightening will
damage the mandrel.
9. The probe length is factory adjusted to the mandrel, and should
not require further adjustment. Proper positioning of the probe
and stir wire can be quickly confirmed with the probe and stir-
wire alignment tool provided with the instrument, or the method
described in the Mandrel, probe, and stir/freeze wire alignment
section shown later on in this chapter.
10. Reconnect the probe connector on the head chassis, taking care
once again not to touch or bend the connecting pins.
11. Arrange the leads so that they will not touch the clapper (Figure 7,
Item 3) or be pinched when the head cover is replaced.
12. Replace the head cover and its two attaching screws.
13. Reconnect the power cord and turn the instrument on.
14. Make sure the Supervisor/Operator keyswitch is in the Supervisor
position while using the SETUP and CALIB menus (see Figure 5).
15. When the operating head rises and Press START to Continue begins
to scroll across the display, press the START key.
16. When the display changes to Osmometer Ready, press the TEST
key. The display will change to Select Test Item.
17. At Select Test Item, repeatedly press the > key until the display
reads Probe Bin Test.
18. At Probe Bin Test, press START. The display will change to [Start]
Ready?.
19. At [Start] Ready?, remove the empty sample tube from the freezing
chamber, pipette or measure a sample of probe bin setting fluid
into a clean sample tube, place the tube in the freezing chamber
and press the START key. The 3250 will run a special freezing point
test to determine the sample probe resistance and bin number.
75
Note In emergency, H₂O may be used in place of bin-setting fluid

for this test. The bin number thus determined will be
acceptable for bin-setting though the probe resistance
displayed is that at the freezing point of the H₂O sample
used, and will be different from that for bin-setting fluid.
20. Record the sample probe resistance and bin number in the Service
Log at the end of this user’s manual; then press the STOP key. The
display will change to Probe Bin Test.
21. Press SETUP to enter the SETUP menu, then repeatedly press the
key until the display reads Set Sample Bin #.
22. At Set Sample Bin #, press the START key. The display will report
the current sample bin number setting.
23. If the current sample bin number setting is not the same as that just
determined, enter the correct bin number by means of the numeric
keypad; then press the ENTER key. The display will change to Set
Sample Bin #; then press the STOP key to return to Osmometer
Ready. If the current sample probe bin setting is the same as that
just determined, simply press the STOP key twice to return to
Osmometer Ready.
24. Press the CALIB key and calibrate the instrument as recommended
in Chapter 4. Recalibration is required each time the sample probe
is replaced.
76
Mandrel, probe, and stir/freeze wire alignment

Alignment using the alignment tool
Proper alignment and adjustment of the mandrel, probe and stir/freeze
wire are of utmost importance to assure accuracy, precision and proper
operation of the instrument.
Note After making any probe and/or stir/freeze wire adjustments, the
instrument calibration must be checked and adjusted as
necessary.
To facilitate positioning of the probe and stir/freeze wire, a probe
alignment tool is included with the instrument and with each
replacement sample probe. Replacement probes for the 3250 are
pre‑mounted in a mandrel at the proper vertical location. To realign the
probe and/or stir/freeze wire, use the following procedure:
Mandrel
Make sure the stir wire

does not rub on the sides
of the slot in the mandrel.
Raised V
Centerline
This portion of the stir wire
should
be parallel to sample probe,
Alignment Tool
and the space should be
about .04 inches (1 mm).
Stir Wire
Sample Probe
Bottom of End of stir wire should

sample probe rest on this small step.
should rest on
this level.
Figure 8: Probe and Stir/Freeze Wire Alignment
77
1. Slide the tool squarely up onto the mandrel, round end first, with
the stir/freeze wire located as shown in Figure 8.
2. Bend the metal probe stem gently by hand, as necessary, to align
the probe along the RAISED V CENTERLINE of the probe alignment
tool.
3. With the alignment tool squarely up on the mandrel, the bottom
of the probe should rest on the main level, as shown in Figure 8.
If vertical adjustment is necessary, first loosen the probe setscrew.
Then raise or lower the probe within its mandrel as necessary, and
then retighten the probe setscrew gently (overtightening will crack
or distort the thin plastic wall of the mandrel). Recheck the probe
position.
4. The bottom of the stir/freeze wire should be even with the small
step, as shown in Figure . As necessary, remove the head cover,
loosen the stir/freeze wire setscrew(s) and reset the stir/freeze wire.
Retighten the stir/freeze wire setscrew(s). Then bend the wire, if
necessary, so that the lower portion is parallel to the sample probe
and spaced about .04 inches (1 mm) away from the sample probe.
The stir/freeze wire should be
about the width of the stir/
freeze wire away from the
The flange of the mandrel must mandrel where it exits from
be flush with the head chassis. the mandrel.
The tip of the stir/freeze wire

should be about the width of
the stir/freeze wire away from
the probe. Bend the wire if
necessary. The tip of the stir/
freeze wire should be even with
The black bead in the probe tip must
the top of the black thermistor
be in the center of a 0.2-mL sample
bead inside the glass probe
in the sample tube, at equal distances
tip. Loosen the stir/freeze wire
from all nearby surfaces. It must be
setscrews to enable vertical
the same distance from the bottom of
adjustment. Re-tighten the
the tube as it is from the side walls.
setscrews gently to avoid
cracking the thin side wall of
the mandrel.
Figure 9: Mandrel, Probe and Stir/Freeze Wire Adjustment
5. After any probe or stir/freeze wire position adjustment, check and

adjust the stir/freeze amplitude, as necessary, as recommended in
Stir/freeze amplitude adjustment.
6. After making any probe and/or stir/freeze wire adjustments, check
the instrument calibration and recalibrate as necessary.
78
Alternate method—using a sample tube containing 0.2 mL of

water
Proper alignment and adjustment of the mandrel, probe and stir/freeze
wire are of utmost importance to assure accuracy, precision and proper
operation of the instrument.
Note After making any probe and/or stir/freeze wire adjustments, the
instrument calibration must be checked and adjusted as
necessary.
1. Pipette a 0.2-mL sample of H₂O or any of the standards into a
sample tube. Then hold the tube up on the mandrel in the position
shown in Figure 9. Note in which direction the tip of the probe
must be moved (if any) to perfectly center its black sensor bead in
the sample volume. Remove the sample tube.
2. If vertical adjustment of the probe tip is necessary to center
the black bead in the 0.2-mL sample volume, loosen the probe
setscrew (Figure 7, Item 6), then manually raise or lower the probe
within the mandrel. Retighten the setscrew gently (overtightening
will crack or distort the thin plastic wall of the mandrel). Recheck
as described in step 1 above.
3. If horizontal adjustment is necessary to center the black bead in the
sample volume, bend the metal probe stem gently in the required
direction by hand. Recheck as described in step 1, above.
4. Check and adjust the stir/freeze wire position according to the
recommendations in Figure 9.
5. After any stir/freeze wire position adjustment, check and adjust the
stir/freeze amplitude, as necessary, as recommended in the next
section, Stir/freeze amplitude adjustment.
79
Stir/freeze amplitude adjustment

The stir and freeze vibration amplitudes are affected by the probe, stir/
freeze wire and mandrel positions. If the one-second freeze pulse is not
strong enough to freeze samples reliably, visually check the freeze and
stir vibration amplitudes as follows:
1. Make sure the Supervisor/Operator keyswitch is in the Supervisor
position while using the TEST and SETUP menus (see Figure 5).
2. Press the TEST key. The display will change to Select Test Item.
3. At Select Test Item, press the START key. The display will change to
Test Menu #: 0.
4. At Select Menu #: 0, press the 3 key; then press ENTER. The
display will change to Stir/Freeze Test.
5. At Stir/Freeze Test, press the START key. The display will change to
[stir] [freeze].
6. At [stir] [freeze], a freeze pulse will occur each time the > key
is pressed. Watch the tip of the stir/freeze wire and press >. If
properly adjusted, as recommended in Chapter 2, 14. Set "Buzz"
Amplitude, the stir/freeze wire should produce a loud "buzz,"
vibrate principally fore and aft, and never strike the probe.
Look from the side of the instrument as you press > again; the
freeze-pulse vibration blur at the very tip of the stir/freeze wire
should span at least ½" or 13 mm for one second, then cease.
If the freeze vibration is not enough, first remove the head cover by
removing the top screws and pulling straight up and make sure that
there are absolutely no metal chips between the clapper and the
coil armature. Even a single iron chip will greatly reduce the freeze
amplitude and must be carefully wiped out of the magnetic gap.
Then recheck the stir/freeze amplitude.
If the freeze vibration is still not enough, use either or both of the
following adjustments:
80
1. Adjust the freeze "buzz" amplitude (default = 223; range = 150-255)

as described in 14. Set "Buzz" Amplitude. In general, increasing
this setting will increase the freeze vibration. However, caution
should be used since too high a value may cause the clapper to
become trapped by the electromagnetic coil.
2. Adjust the clapper spacing. Refer to Figure 7. Bend the yoke (Item
1) to locate the clapper (Item 3) closer to or further away from the
coil (Item 2), whichever is found to increase the stir/freeze wire tip
vibration.
3. Changing the coil-to-clapper spacing probably will adversely affect
the stir/freeze wire tip location; after bending the yoke, realign the
stir/freeze wire according to the recommendations in the Mandrel,
probe, and stir/freeze wire alignment section.
Check the freeze vibration amplitudes as instructed in steps 1 through
6 of this section. Optimum spacing between the coil and clapper
should provide at least ½" or 13 mm of tip vibration.
Reinstall the operating head cover. Do one last freeze test with the
operating head cover reinstalled; the freeze pulse characteristics are
slightly different with it in place.
If the freeze vibration is still not enough, do not proceed further
without obtaining Hot-Line® service as recommended in Obtaining
service.
If you succeed in obtaining enough freeze vibration, the stir vibration
amplitude must be readjusted as follows:
1. Press the SETUP key. If the Supervisor/operator keyswitch is in the
Supervisor position, the display will change to Select Setup Item.
2. At Select Setup Item, press the START key. The display will change
to Setup Menu #: 0.
3. At Setup Menu #: 0, press the 4 key, then press ENTER. The
display will change to Set Stir Amplitude.
4. At Set Stir Amplitude, press the START key. The display will change
to Amplitude = xxx, with xxx being some two- or three-digit
number around 98.
81
Looking from the side of the instrument, you should be able to see
a stir vibration blur at the tip of the stir/freeze wire. For 0.2 mL to
0.25 mL osmometer operation, the stir vibration blur at the very tip
of the stir/freeze wire should be between 3/32" and ⅛" or 2 and 3 mm.
Stirring is necessary to minimize temperature gradients in viscous
samples but more than ⅛" or 3 mm can cause poor repeatability
or frothing which may make it impossible to reliably determine the
freezing point.
If the stir vibration amplitude is not correct, press > to increase the stir
amplitude and the xx value or press < to decrease the stir amplitude
and the xx value. Continue to increase or decrease the xx value as
necessary, until the stir vibration blur at the very tip of the stir/freeze
wire is between 1/32" and ⅛" or between 2 and 3 mm.
As this adjustment is completed, you may wish to hold a sample tube
containing a 0.2-mL sample of either an NaCl standard or H₂O up in
position on the probe and stir/freeze wire to make sure that froth is not
produced.
When the stir amplitude is correct, you may wish to record the stir-
amplitude xx value in the Service Log for reference. Press ENTER to
store the new setting. The display will change to Set Stir Amplitude.
At Set Stir Amplitude, press the STOP key to exit the SETUP program
(or > or < to step to another SETUP item).
When STOP is pressed, the display will change to Osmometer Ready
and the 3250 is ready for freezing point tests.
IMPORTANT After completing any probe and/or stir/freeze wire

adjustments, check the instrument calibration and
recalibrate as necessary.
82
Appendix A
Troubleshooting Table
Make sure that the instrument is unplugged before removing the
instrument cover!
Problem/Message Explanation
Abrupt loss of power If you lose power to your instrument, we
recommend that you check that your outlet is
providing the correct amount of power. Check
that your cord is firmly plugged into both the
instrument and the outlet. Visually inspect the
fuses, and change as necessary.
A/D Init Failure Error is self-correcting. If the error persists, contact
Advanced Instruments for Hot-Line Service.
A/D High Filter Error
A/D Low Filter Error
A/D Cal Mode Error

Baudrate Error Reset baudrate. If the error persists, contact
Block Probe Open? If you see this message, check the probe wiring and
then try restarting your instrument. If the problem
persists, contact Advanced Instruments for Hot-
Line Service.
Block Probe Failure If you see this message, first try restarting your
instrument. Make sure the block probe is plugged
into the main board. If the problem persists,
contact Advanced Instruments for Hot-Line Service.
Buzz Point Default Set If you see this message, the value stored in memory
for the range at which the instrument is set was
invalid. The default value has been restored. Verify
your desired setting and check your calibration.
If the problem persists, contact Advanced
Instruments for Hot-Line Service.
83
Calibration out of If you see this message, verify that your operator
Range; Repeat technique is correct. Then, recalibrate. If the
problem persists, contact Advanced Instruments for
Hot-Line Service.
Cooling System Error If you receive this message, check to make sure
that the thermoelectric is plugged in, check the
thermoelectric LED on the application PCB, then
try restarting your instrument. Second, check that
your instrument’s cooling chamber contains enough
heat transfer fluid. If the problem persists, contact
Count Error: Parameter Turn unit off and on. Custom settings and probe bin
RAM numbers may need to be reset. If the error persists,
EPROM Failure This message indicates a memory error with your
FLASH memory. Try restarting your instrument, and
ignore if it does not reoccur.
Error Reading Barcode Check to see if barcode scanner is damaged.
Make sure that the barcode is no longer than 13
characters. Cancel barcode and repeat reading.
ESC TRAP Turn unit off and on. If the error message does not
persist and no other error message is displayed,
ignore this message. If the error persists, contact
Event Record Lost This message indicates that the event record stored
in memory has been corrupted. Try restarting your
instrument. If this does not solve your problem,
Fan System Failure Check to make sure that the fan is plugged in, and
that the fan isn’t blocked. Check the fan driver
LED on the application board. Try restarting your
84
FPE_INTOFLOW Turn unit off and on. If the error message does not
persist and no other error message is displayed,
FPE_INTDIV0 ignore this message. If the error persists, contact
FPE_INVALID Advanced Instruments for Hot-Line Service
FPE_ZERODIVIDE
FPE_OVERFLOW
FPE_UNDERFLOW
FPE_INEXACT
FPE_STACKFAULT
FPE_EXPLICITGEN
FPE_UNDEFINED
Head Not Down This error indicates that the sensor at the head
down position was not tripped. Clear any blockage
and try restarting your instrument. If this does not
solve your problem, contact Advanced Instruments
for Hot-Line Service.
Head Not Up This error indicates that the sensor at the head up
position was not tripped. Clear any blockage and
try restarting your instrument. If this does not solve
your problem, contact Advanced Instruments for
Hot-Line Service.
Insufficient Data This message indicates that not enough valid
test results exist in memory for calculating the
requested statistics.
Low Battery The lithium battery contained inside the NVRAM
memory is too low to maintain stored information,
calibration, and system settings. The instrument
will function normally as long as the main power
remains on. Contact Advanced Instruments for Hot-
Line Service.
85
Low Fluid Level in The heat transfer fluid pump may be
Freezing Chamber malfunctioning, or there may be a leak or
obstruction in the fluid system. Run the Head Up/
Down Test while observing the priming of the
heat transfer fluid pump and the flow of fluid into
and out of the freezing chamber. If leakage or
an obstruction is suspected, contact Advanced
Instruments for Hot-Line Service.
Memory Allocation Error Turn unit off and on. If the error message does
not persist and no other message is displayed,
ignore this message. If the error persists, contact
No Parameters in RAM Error is self-correcting. This will restore default
settings. Custom settings and probe bin numbers
will need to be reset. If the error persists, contact
No Plateau, Repeat Test Your instrument was unable to detect a freezing
plateau, and was therefore unable to give a result.
Check your technique and try again. If the message
persists, obtain Hot-Line Service.
No response when the Try restarting your instrument. If this does not solve
START or TEST key is your problem, Contact Advanced Instruments for
pressed Hot-Line Service.
Out of Range This message will appear if the plateau value
exceeds the upper limit of 4000 mOsm. It will
also appear if the buzz point setting limits are
exceeded.
Parameter RAM Failed This message tells you that a new software
or New Software version has been installed since you last powered
Version the instrument or that the information stored
in parameter RAM has been corrupted. The
instrument will reload factory defaults.
Printer fails to print Make sure there is paper in the printer. If there is
not, install paper per the instructions in Step 5
— Load printer paper (Figure 1). If there is paper
in the printer, make sure that the printer cover is
completely and securely closed.
86
Printer advances paper Check to make sure the paper roll is properly
and appears to be installed per the instructions in Step 5 — Load
printing, but the paper printer paper (Figure 1). The paper must be feeding
is blank. from the bottom of the roll, as shown in Figure 1.
RAM Failure This message indicates a memory error with the
system RAM. Try restarting your instrument and
ignore if it does not reoccur. Your instrument may
load factory defaults.
Recalibration Needed This message indicates that you need to re-
calibrate your instrument. Recalibrate closely
following the instructions in Calibration. If the error
message repeats, obtain Hot-Line Service.
Reset Probe Config Most likely, this message indicates that you should
reset your block and sample probe bin numbers as
described in Calibration. If this does not solve the
problem, contact Advanced Instruments for Hot-
Line Service.
Result Over Range, Try This message indicates the final result is too high
Higher Setting for the low range setting. Select high range and try
again.
Result Under Range, Try This message indicates the final result is too low
Lower Setting for the high range setting. Select low range and try
again.
Results not repeatable If your results are not repeatable, first check that
(too scattered) your operator technique is sound. Second, use
the A/D test to ensure that your sample probe is
working correctly. If you have questions or if this
does not isolate the problem, please call Hot-Line
Service for assistance.
Sample Did Not Freeze... Test another aliquot of that sample in a clean
sample tube. Try testing samples known to be
within the range of the instrument.
87
Sample Freeze Error... This message can be displayed for a number of
reasons. Check your technique and the condition of
your sampler. If neither of these is the source of the
problem, you should check your sample and block
probe numbers. If this does not solve your problem,
Sample Pre-freeze... Test another aliquot of that sample in a clean
sample tube. Check/correct the probe bin
numbers. Correct the stir/freeze wire alignment
and adjustment as necessary. Try to minimize the
effects of materials that tend to cause premature
crystallization. Make sure samples contain no
bubbles or froth before testing. Decrease the
sample stir amplitude, if necessary, to avoid
generating bubbles before the freeze buzz occurs.
If this does not solve your problem, you should
check the probe bin number and obtain service if
necessary.
Sample Probe Failure Switch the instrument off, then on. Check the
sample probe by running the A/D tests. If the error
message does not persist and other error messages
are not displayed, ignore this message. Otherwise,
Sample Probe Open? Switch the instrument off, then on. Make sure the
sample probe is plugged into the main board.
Check the sample probe by running the A/D tests.
If the error message does not persist and other
error messages are not displayed, ignore this
message. Otherwise, contact Advanced Instruments
for Hot-Line Service
Standards Reversed? This message will appear during the calibration
Repeat Calibration procedure if the instrument detects that the low
and high calibration standards may have been
mixed up and were entered in the wrong order.
Retry the calibration, being sure that the standards
are correct.
88
Stir System Failure This error message indicates a problem with
your stir/freeze wire system. Try restarting your
Supervisor Key This feature or action has been locked out by use
Needed... of the supervisor/operator keyswitch.
System Error: This error message indicates a system error with
Communications the communications ports. Try restarting your
Test Time-out Error This message indicates that your instrument was
unable to complete the test in the allotted time.
Be sure that your operator technique is sound and
retry the test. If the problem persists, check your
block probe number. If you need more assistance,
Thermoelectrics System This error message indicates a problem with
Failure your thermoelectrics. Check the thermoelectric
LED on your application PCB. Try restarting your
Unknown Error This error message indicates a software event has
occurred internally, which has no definition. Try
restarting your instrument and ignore if it does not
reoccur.
89
Appendix B
Product Specifications
Electrical
Power Requirement: 100 to 240 VAC (50/60 Hz)
Fuses (2): 250V time delay (Type T): 2-Amp

Power Consumption: 95 Watts
Memory Backup: Integral lithium cell; 10-year life (typical);
(not user-replaceable)
Sample Volume: 0.20 to 0.25 mL
Sample Capacity: Single sample
Readout: 20-character vacuum fluorescent display
Units: mOsm/kg H₂O
Ranges:
Low: 0 to 2000 mOsm/kg H₂O
High: 1400 to 4000 mOsm/kg H₂O
Resolution: 1 mOsm/kg H₂O

Communications: Onboard printer, DTE EIA-232/V.24 (RS-232) serial
port and optional barcode scanner
Performance at
Reference Conditions1
Linearity: Less than 0.5% from a straight line
Repeatibility:
0 to 400 mOsm: Std. Deviation < 2 mOsm/kg H₂O
400 to 4000 mOsm: Std. Deviation < 0.5% of value mOsm/kg H₂O
Drift: Less than 1 mOsm/kg H₂O per month
1 Reference Conditions: 20 °C to 25 °C (68 °F to 77 °F); 40% to 60% Relative Humidity; tolerances of reference or calibration
solutions excluded.
90
Performance Over
Operating Conditions
Temperature effects: Less than 1 mOsm/kg H₂O for every 5 °C (9 °F)
ambient temperature change.
Operating Conditions
Temperature: 18 °C to 35 °C (64 °F to 95 °F)
Humidity: 5 to 80% relative humidity (non-condensing)
Storage Temp.: -40 °C to +45 °C (-40 °F to +113 °F)
Start-up Time: Immediately from stand-by; under 5 minutes from
power-on
Test Time: Approximately 2 minutes per sample in low range;
approximately 3 minutes in high range
Sound Level: Measured at operator’s position < 45 dB(A) Typ.
≈ 86 dB(A) max. (2 sec.)
Dimensions inches centimeters
Width: 13.0 33.0
Depth: 18.0 45.7

Height: 12.0 30.5
With head up: 16.0 40.6
Weight pounds kilograms
Net: 23.0 10.4
Shipping: 34.0 15.4
Warranty: One-year limited warranty on workmanship and all
parts except glass, plastic, and parts warranted by
their makers.
91
Product Specifications
Certification:
Refer to Regulatory Notices for applicable

standards.
Installation Class: I
Over-Voltage Category: II
Pollution Degree: 2
Moisture protection: IPX0 (ordinary)
92
Appendix C
Regulatory Notices
• This product has been designed and manufactured in
accordance with U.S., Canadian, and European regulatory
requirements as outlined below. Modifications made to this
product that are not expressly approved in writing by the
manufacturer will void the user’s authority to operate this
product, previously issued factory approvals, and the user’s
rights under the warranty.
• The distributor or dealer may have applied additional
local, national, or international approvals to this product.
Consult the distributor or dealer for more information and
documentation.
• Connections to this product must be made with shielded
cables. Use of non-shielded cables may violate RFI/EMI
limits.
93
Regulatory Notices
Regulatory approval type Description

This product has been listed by ETL testing
laboratories as being in compliance with
the requirements of UL 61010-1, “Electrical
U.S. Safety
Equipment for Laboratory Use; Part 1: General
Requirements”. The “US” in the lower right of the
ETL mark demonstrates this listing.
This product has been listed by ETL testing
laboratories as being in compliance with the
requirements of CAN/CSA C22.2 No. 61010-1,
“Safety Requirements for Electrical Equipment
Canadian Safety
for Measurement, Control and Laboratory Use
- Part 1: General Requirements”. The “C” in the
lower left of the ETL mark demonstrates this
listing.
This product meets the intent of Directive
2014/30/EU Conformity - EMC for
Electromagnetic Compatibility. Compliance was
demonstrated using the following standards,
as listed in the Official Journal of the European
CE Declaration of Communities: Consult the Declaration of
Conformity - EMC Conformance certificate shipped with the
product for the latest update.
• EN 61326-1, & EN 55011, CISPR 11, Group 1, Class
B, “Electrical Equipment for Measurement,
Control, and Laboratory Use”.
94

2014/35/EU, the Low Voltage Directive.
Compliance was demonstrated using the
following standards as listed in the Official
Journal of the European Communities: Consult
the Declaration of Conformance certificate
shipped with the product (if required) for the
latest update.
CE Declaration of
Conformity - Low • EN 61010-1, “Safety Requirements for
Voltage Electrical Equipment for Measurement,Control
and Laboratory Use - Part 1: General
Requirements”.
• EN 61010-2-101, “Safety Requirements for
Electrical Equipment for Measurement, Control
and Laboratory Use - Part 2-101: Particular
Requirements for In Vitro Diagnostic (IVD)
Medical Equipment”.
A CB report and certificate have been issued
CB Report
for this product. A copy of this certificate is
CB Certificate
available upon request.
The osmometer, along with the calibrators and
controls manufactured by Advanced Instruments,
are listed with a U.S. Department of Health and
Human Services, Food and Drug Administration,
as:
U.S. FDA Listing
Osmometer Class 1
Calibrators Class 1
Controls Class 1
The osmometer, along with the calibrators and
controls manufactured by Advanced Instruments,
are licensed with Health Canada, Therapeutic
Products Directorate, Medical Devices Bureau,
as:
Health Canada License
Osmometer Class 2
Calibrators Class 2
Controls Class 2
95
Regulatory Notices

98/79/EC for In Vitro Diagnostic Medical
EC Declaration of
Devices. Consult the Declaration of Conformance
Conformity - IVD
certificate shipped with the product (if required)
for the latest update.
This device complies with Part 15 of the FCC
Rules. Operation is subject to the following two
FCC - Part 15, Subpart B, conditions: (1) this device may not cause harmful
Class B interference, and (2) this device must accept any
interference received, including interference that
may cause undesired operation.
This Class B digital apparatus complies with
Canadian ICES-003.
Canadian ICES-003
Cet appareil numérique de la lasse B est
conforme à la norme NMB-003 du Canada.
This Class B digital apparatus complies with
Japan VCCI
VCCI technical requirement V-3.
2012/19/EU for Waste Electrical and Electronic
EC Declaration of
Equipment (WEEE). Consult the Declaration
Conformity - WEEE
of Conformance certificate shipped with the
product (if required) for the latest update.
EC Declaration of 2011/65/EU for “Restriction of the Use of
Conformity - RoHS Certain Hazardous Substances in Electrical and
Electronic Equipment.”
96
Appendix D
Warranty and Warranty
Duties
By accepting and operating ADVANCED INSTRUMENTS

this instrument, the user and WARRANTS THAT IT:
Advanced Instruments agree
to the following responsibilities 1. Has produced equipment
which constitute contractual equal to or exceeding that
warranties and conditions of any competitive product
between the seller and the user in the same price range in
for the maximum benefit and standards of design, material
usefulness of the instrument. and workmanship.
97
Warranty and Warranty Duties
2. Knows of no defects in design 3. The instrument will not be

or materials which may cause altered without written
bodily injury. approval from Advanced
3. Will endeavor to advise Instruments.
the user of changes or 4. Advanced Instruments will be
improvements in the notified immediately if any
instrument as they are injury occurs in any association
developed, so that the user with the instrument and will be
may take steps to improve the allowed prompt and thorough
safety and performance of examination of the instrument
his equipment throughout its in question.
useful life. 5. Advanced Instruments will
4. Will replace or repair be held harmless in cases of
equipment according to the injury arising (see definitions
guarantee on the attached below):
warranty. a. Beyond the useful life of the
5. Will cooperate closely in equipment.
common defense of any b. From unreasonable use.
accident involving this
equipment, or third-party c. When Advanced Instruments
suit against the user or is not immediately notified
operating personnel, if advised of said injury.
immediately by the user of the d. From interpretation of
occurrence of any accident. results.
THE USER WARRANTS THAT:
1. The instrument will be used
DEFINITIONS
reasonably. 1. "Useful life" is:
2. The instrument will be a. The same as the
regularly maintained according depreciation life in the
to this manual, including a log Internal Revenue Service
of all service, tests and repairs guidelines, whether or not
performed on the equipment, the user actually depreciates
and records will be kept of all the instrument, but not to
requests for repair made to exceed 10 years from date of
Advanced Instruments where delivery to the user.
such repairs were beyond
the ability of local service
personnel.
98
b. Only during the time 3. "Immediate notification" is:

when the user has not a. Recognition that time is
loaned, leased or resold of the essence when any
the equipment to any third accident, malpractice or
party. product liability arises
2. "Reasonable use" is use: which involves Advanced
a. According to the instructions Instruments equipment
supplied by Advanced b. Notification to Advanced
Instruments (assuming Instruments immediately
English-reading personnel (the same day, if possible)
or supervision). If neither in the event of injury to any
the supervisor nor the person in circumstances
operator reads English, involving Advanced
the user agrees to obtain Instruments equipment in
accurate translations of which Advanced Instruments
the instrument labels, might be named as a
instructions, user’s guides defendant in any form of
and/or manuals provided. litigation.
b. Under direct, on-the-job c. Allowing Advanced
supervision of the supervisor Instruments or its
or other professional in representatives, immediate,
charge. full, and thorough
c. In which there are no known examination of Advanced
defects or uncorrected Instruments equipment, and
repairs. all records pertaining to
such equipment.
d. Only for the purpose stated
in the instructions provided
with the instrument.
e. In which the equipment has
been maintained according
to the instructions provided.
99
Warranty and Warranty Duties
d. Only during the time e. Only during the time when

the equipment has been the user has not altered
maintained on a regular the equipment in any way
basis as prescribed by without written approval
Advanced Instruments. If the from Advanced Instruments.
user is in an area which has
no local service, Advanced
Instruments may require
a local service person
(understood to mean the
person actually performing
the “hands-on” service of
the equipment) to attend
and pass a reasonable
maintenance and repair
course.
100
Appendix E
Supplemental RS-232
Information
The DB-9 RS-232 port on your instrument conforms to the DTE
EIA-232 standard and can reliably communicate over shielded
cable up to 10 meters in length, depending on the baud rate
you use. Almost every item of information displayed by your
instrument is also transmitted over the RS-232 port, including
test results, all error messages, and most display data from the
diagnostic menu.
Data is transmitted asynchronously as 1 start bit, 8 data bits
and 1 stop bit, with no parity. Each message transmitted
from the communication port is terminated by the sequence,
Carriage Return (0D Hex), Line Feed (0A Hex). Note that your
instrument is only capable of outputting information. At this
time, there is no protocol for talking to the instrument.
This product’s serial port output is based on ASCII code
protocols. No special codes are used to identify errors, test
results, or normal operational messages. Connection to any
data collection or LIMS system will require the user or a
third party to write custom interface software in order to
recognize and parse the text strings of interest. Advanced
Instruments does not maintain a central list of text strings
used in the various versions of software created, nor have we
adopted any standard protocol with respect to the format of
these messages. To determine the format and type(s) of text
strings contained in this instrument, users should follow the
instructions in this section and save the output for evaluation
by the interface programmer.
101
Supplemental RS-232 Information
At this time Advanced Instruments has not investigated any

third-party software for use or recommendation for the purpose
of importing data into PC applications, such as Microsoft® Excel
or a customer’s LIMS system.
Sample RS-232 Setup

As a typical example of a communications program setup, the
following instructions will describe the process necessary for
using your instrument in conjunction with Hyperterminal (or
similar applications) and a null modem cable, available from
Advanced Instruments, as shown in the diagram below. You can
use this procedure to be sure that your instrument and cable
are operating correctly.
1. Create a Hyperterminal connection using the Hyperterminal
program.
2. While working within that connection, choose the Properties
selection from the File menu.
3. Select the Configure button.
4. Choose the following settings for the Configure menu and
click on the OK button.
Bits per second See user’s guide.
Data bits 8
Parity None
Stop bits 1
Flow control None
5. Select the Settings menu.
6. Chose TTY for the Emulation option.
7. Select ASCII setup.
8. Set the Sending option to Echo Typed Characters Locally.
9. Set the Receiving option to Wrap Lines that Exceed Terminal
Width.
You should at this point see all instrument output in the
Hyperterminal window of your computer screen.
102
Null Modem Cable
Shield connects
to metalized
connector housing
at both ends.
103
Appendix F
Symbol Definitions
On-Off Functional Arrow
Feed Printer
Interrupt Enter
Test RS232
Start Bar Code
Stop Attention
Record Review Caution Hot Surface
Setup Dangerous Voltage
Calibration Lifting Hazard
Cancel; Delete Calibrator
Content Do Not Open Top
104
Control Handle With Care
Negative Control Toxic
Positive Control Use Blade To Open
Flammable Do Not Re-Use
Fragile For In Vitro Diagnostic Use
Irritant European Conformity
Keep Dry Temperature Limit
Date Manufactured See Instructions
Sterile Lot Number
Non-Sterile Use By; Expiration Date
Serial Number Authorized Representative
Solution Part Number
Sufficient for [x]

Latex-Free
Tests
Open Here Diluent
105
Symbol Definitions
See Instructions for

Low Fluid Level
Temperature Guidelines
Keep Hands Clear Potential Puncture Hazard
Electronic Equipment -
Manufacturer
Dispose of Properly
Biohazard Laser Hazard
Cut Hazard Static Discharge
106
Appendix G
Product Disposal and

Recycling
International concern about environmental pollution resulting from
improper disposal of products and materials at the end of their useful
life has resulted in an increase in legislation to control the methods and
procedures used to handle waste electrical and electronic equipment.
While the regulatory status in some regions of the world has
progressed to the point where formal legislation is already in effect,
many other regions are in the process of creating similar legislation
or adopting that already in existence in other areas. The result in the
years ahead will be more stringent control over disposal of products
and recycling of their components once they are withdrawn from use.
Since regulations governing the disposal of your instrument and
accessories may vary depending upon your geographic location,
the following guidelines are provided to assist you in identifying the
options available to you once the decision has been made to replace or
dispose of this product:
• Contact the supplier who sold you the product. Whether this was
Advanced Instruments itself, or one of its authorized dealers, this
supplier should be knowledgeable about the national and local
regulations governing disposal and recycling of products in your
area. In some cases, this supplier may be legally obligated to
accept the product from you and arrange for proper disposal or
recycling with no further involvement on your part. Alternately, the
supplier can provide you with specific instructions for actions that
you can take to dispose of the product properly.
107
Product Disposal and Recycling
• Contact your local government agency responsible for waste

collection and disposal. They can identify procedures and
restrictions in effect to ensure proper disposal, and available
locations where products can be sent.
• Contact Advanced Instruments Hot-Line Service:
–– 800 225-4034 (toll-free within the USA and Canada)
–– +US 781-320-9000 (elsewhere)
–– 781-320-0811 (fax)
Service personnel will provide you with contact information for
local disposal, or instructions for returning the product directly
to Advanced Instruments.
108
Appendix H
Service Log
3250
Mode: _____________________________________________________
Serial Number:_______________________________________________
Software Revision:____________________________________________
Sample Probe Number:________________________________________
Block Probe Number:__________________________________________
Date Problem/Symptom Action
109
Service Log
Date Problem/Symptom Action
110

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The Advanced™ Osmometer

Supplies, Parts and Accessories............................... 10

Installation and Setup................................................ 20

14. Set "Buzz" Amplitude.........................................................................48

Standards and Quality Control.................................52

Troubleshooting and Service.....................................58

Mandrel, probe, and stir/freeze wire alignment .................................77

The exclamation point within an equilateral triangle is intended

This symbol indicates the presence of alternating current (AC).

This symbol indicates the presence of a fuse

This symbol indicates the presence of protective earth ground.

This symbol indicates the power is ON.

This symbol indicates the power is OFF.

Note Additional symbol definitions are available in Symbol Definitions.

• Do not pull on cords and cables. When unplugging cords or cables,

Part description Part no

Part description Part no

Description Part No.

Note Advanced Instruments cannot guarantee the stated instrument

Principles of Freezing-Point Osmometry

Each of the colligative properties has a similar problem, and though

Sensitive thermistor probes monitor the sample temperature

Standard Freezing Curve

• Osmolarity: Osmols of solute particles per liter of solution.

In order to set up your instrument properly, it is important

Step 1 — Find a location for the instrument

Step 2 — Obtain additional items

Step 3 — Unpack the instrument

Table 1: Model 3250 Osmometer Packing List

Step 4 — Install the heat transfer fluid

Note Should it become necessary to move the instrument to another

Step 5 — Load printer paper (Figure 1)

Figure 1: Load Printer Paper

Step 6 — Power up the instrument

Step 7 — Prime the heat transfer fluid pump

Figure 2: Prime the Heat Transfer Fluid Pump

Step 8 — Run a diagnostic test

Step 9 — Set date and time

Step 10 — Set language preference

Step 11 — Check initial factory calibration

Step 12 — Proceed to Chapter 2

Hazardous material cautions

Function of major components

Operating head (Figure 3, Item 1)

Sample Probe (3)

Air Filter (7)

Figure 3: Model 3250 Components and Controls

Freezing chamber (Figure 3, Item 8)

Measurement and control circuits (inside)

Electronic circuits (inside)

Display panel (Figure 3, Item 4)

Keypad (Figure 3, Item 4)

Figure 4: Keypad Layout and Functions

FEED Advances printer paper

1 through 0 Allows numeric input, as required

Printer (Figure 3, Item 9)

Supervisor/Operator keyswitch (Figure 5)

Figure 5: Back Panel