Advanced Instruments 3250 User manual

3255EN Rev. 22
The Advanced™ Osmometer
Model 3250
User’s Guide

2
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)

3
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
Table of Contents

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Advanced™Osmometer Model 3250 User’s Guide
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

5
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

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Advanced™Osmometer Model 3250 User’s Guide
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

7
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).

8
Advanced™Osmometer Model 3250 User’s Guide
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.

9
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.

10
Supplies, Parts and
Accessories
To order parts and accessories, contact the Advanced Instruments
Customer Service Department by using one of the following numbers.
• 800-225-4034 (toll-free within the USA and Canada)
• +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
ampule of Probe Bin Setting Fluid (Stainless Steel)
3D3700
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

11
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)

12
Calibrators and
Standards
To order calibrators and standards, contact the Advanced Instruments
Customer Service Department by using one of the following numbers.
• 800-225-4034 (toll-free within the USA and Canada)
• +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
mOsm (ten 5 mL ampules, two of each value)
3LA028
Protinol™3-Level Protein Control Kit (nine 3 mL bottles, three of
each level)
3MA028
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
500 mOsm/kg Calibration Standard (110 mL bottle) 3LA050
500 mOsm/kg Calibration Standard (ten 5 mL ampules) 3LA051
900 mOsm/kg Calibration Standard (110 mL bottle) 3LA090
900 mOsm/kg Calibration Standard (ten 5 mL ampules) 3LA091
1500 mOsm/kg Calibration Standard (110 mL bottle) 3LA150
1500 mOsm/kg Calibration Standard (ten 5 mL ampules) 3LA151
2000 mOsm/kg Calibration Standard (ten 5 mL ampules) 3LA201
3000 mOsm/kg Calibration Standard (ten 5 mL ampules) 3LA301

13
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.

14
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.

15
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 HO 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.

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Advanced™Osmometer Model 3250 User’s Guide
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 HO.
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.

17
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

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Advanced™Osmometer Model 3250 User’s Guide
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 HO.
One milliosmol (mOsm) is 10-3 osmols. Osmolality is defined as:
kg H O
2
Osmolality
= φ
nC
=
osmol
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.

19
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.

20
1Installation 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.