CHROMA M260
Colorimeter
Operator Manual
Copyright © 2010, Sherwood Scientific Ltd., All rights reserved.
260 91 000 Issue 1 26th May 2010 ECN 613
G:\DOCUMENT CONTROL\Manuals\Chroma\26091000 Operator Manual M260 Issue 1 #20864 on.doc
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Contents
Contents ………………………………………………………………………2
Intended Use ………………………………………………………………………4
Chapter One Introduction .................................................................................. 5
1.1 Contacting Sherwood Scientific Ltd .......................................................... 5
1.1.1 If your Model 260 Colorimeter was supplied by a third party ....... 5
1.1.2 If the Instrument was obtained from Sherwood Scientific Ltd ...... 5
1.2 Using this Manual ..................................................................................... 6
1.3 Safety ........................................................................................................ 6
1.4 Reagents .................................................................................................... 6
1.5 Sample Preparation .................................................................................... 6
1.6 Warranty ................................................................................................... 6
Chapter Two Installation .................................................................................... 7
2.1 Location .................................................................................................... 7
2.2 Services Required ...................................................................................... 7
2.3 Unpacking ................................................................................................. 7
2.4 Assembly .................................................................................................. 7
2.5 Connecting Peripheral Devices .................................................................. 7
2.5.1 Printer .......................................................................................... 7
2.5.2 Thermostatted Cuvette Holder (267 86 001) ................................. 8
2.6 Instrument Setup ....................................................................................... 8
Chapter Three Scientific Background .................................................................. 9
3.1 Historical ................................................................................................... 9
3.2 Principles of Colorimetry........................................................................... 9
3.3 Clinical Chemistry ..................................................................................... 9
Chapter Four Specifications ...............................................................................10
4.1 Sample Requirements ...............................................................................10
4.1.1 Total Volume Required: ..............................................................10
4.2 Measurement Range .................................................................................10
4.2.1 Operating Range .........................................................................10
4.2.2 Resolution...................................................................................10
4.2.3 Warm Up Time ...........................................................................10
4.2.4 Wavelength Range ......................................................................10
4.2.5 Alternative Filters .......................................................................10
4.2.6 Thermostatted Cuvette Holder (267 86 001) ................................10
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Chapter Five Product Description .....................................................................11
5.1 Controls and Indicators .............................................................................11
5.1.1 Typical Front Panel Layout .........................................................11
5.1.2 The LCD Display: .......................................................................11
5.1.3 Buttons .......................................................................................11
5.1.4 Modifying Numbers etc. .............................................................12
5.2 Sample Heating and Incubation Times ......................................................12
5.3 Operating Modes ......................................................................................13
5.3.1 Warm Up ....................................................................................13
5.3.2 Main Menu .................................................................................13
5.3.2.1 Setup ..........................................................................................13
5.3.2.2 Simple ........................................................................................13
5.3.2.3 Last .............................................................................................13
5.3.2.4 Method .......................................................................................13
5.4 Menu Structure .........................................................................................14
Chapter Six Operation .....................................................................................15
6.1 Start Up ....................................................................................................15
6.2 Zeroing and Calibration ............................................................................15
6.3 Simple Method .........................................................................................16
6.4 Concentration Methods .............................................................................17
6.5 Time Based Measurements .......................................................................17
6.5.1 End-Point Methods .....................................................................17
6.5.2 Kinetic Measurements.................................................................17
6.5.3 Incubation and Measurement Times ............................................18
6.6 Automated Measurements ........................................................................18
6.7 Sample Numbering ...................................................................................18
Chapter Seven Method Setup ...............................................................................19
7.1 Factor Method - Setup Procedure ..............................................................19
7.2 Concentration Method - Setup Procedure ..................................................20
7.3 End Point Method with Factor - Setup Procedure......................................21
7.4 End Point Method with Calibrator - Setup Procedure ................................22
7.5 Kinetic Method with Factor Method - Setup Procedure.............................23
7.6 Kinetic Method with Calibrator – Setup Procedure ...................................24
Chapter Eight Method Run Details .....................................................................25
8.1 Factor Method - Run Procedure ................................................................25
8.2 Concentration Method - Run Procedure ....................................................26
8.3 End Point Method with Factor - Run Procedure ........................................27
8.4 End Point Method with Calibrator - Run Procedure ..................................28
8.5 Kinetic Method with Factor – Run Procedure ...........................................30
8.6 Kinetic Method with Calibrator– Run Procedure .....................................32
Appendix A Warranty .....................................................................................34
Appendix B Gelatin and Interference Filters ..................................................36
Appendix D Time Based Methods for Clinical Analysis .................................37
Appendix E Simple Factor Procedure .............................................................40
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Intended Use
This CHROMA Model 260 Colorimeter is intended to be used by professional
chemists/biochemists. Programming the unit is easier with detailed understanding of
the mechanisms of both the test kits to be used in conjunction with the instruments
and the underlying principles of colorimetry.
Please note that the drop–in Gelatin filters, although sealed, are subject to
deterioration when exposed to excessive heat and humidity. Storage in a cool area and
in a plastic bag with silica gel moisture absorbent pack is recommended.
The unit incorporates a “Warm-up” time period which can be over-ridden. If this is
done then care needs to be taken over the validity of the results.
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Chapter One Introduction
1.1 Contacting Sherwood Scientific Ltd
1.1.1 If your Model 260 Colorimeter was supplied by a third party
i.e. a Distributor in UK or Overseas, the first contact should be that Distributor
for all issues of supply and operation. Please note here the details of the
Company where you purchased this unit:
Company……………………………………………
Tel Number…………………………………………
Contact Name……………………………………….
Email………………………………………………..
1.1.2 If the Instrument was obtained from Sherwood Scientific Ltd
Contact for all issues of supply and operation should be sent to:
info@sherwood-scientific.com
Tel +44 (0)1223 243444
Fax +44 (0)1223 243300
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Introduction (continued)
1.2 Using this Manual
Reading the Operator Manual will allow the user to operate the Colorimeter.
The user will be able to enter new methods, edit existing methods and run
methods.
The performance to be expected is listed in Chapter 4 of this Manual.
Your CHROMA Programmable Colorimeter incorporates an Interactive
Interface, designed to be used in conjunction with third party water-testing and
clinical diagnostic kits. The Programming Instructions are shown in Chapter 7
of this manual and the Methods of Operation for programmed tests are in
Chapter 8.
1.3 Safety
Your Model 260 Colorimeter operates at low voltage (12 Volts DC), powered
from a universal self adjusting power supply.
CARE should be taken to use the correct adapter for the user’s local supply.
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1.4 Reagents
The CHROMA range of Colorimeters is designed to be used with
commercially available diagnostic and water testing kits. The programming
options should accommodate the vast majority of applications. Please contact
your kit supplier for the details of the sample preparation, measurement
procedure, standard concentrations, factors and calculations. These can then be
programmed into the colorimeter.
1.5 Sample Preparation
Samples should be prepared according to the protocols of the reagent kit
manufacturers. The total volume of reagent and sample required for the
Colorimeter to operate satisfactorily is dependent on the profile of the cuvette
used. These are listed in Chapter 4 (see 4.1.1) of this manual.
1.6 Warranty
The Sherwood Scientific warranty statement is included at Appendix A.
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Chapter Two Installation
2.1 Location
The Colorimeter should be placed on a stable bench out of direct sunlight and
with sufficient space to accommodate a separate heating block if necessary.
2.2 Services Required
The Model 260 Colorimeter is supplied with a universal power supply.
The Colorimeter can also be used with a car battery or any 12V battery giving
1.25A for field use when fitted with the appropriate battery lead (471 88 200).
Visit www.sherwood-scientific.com for a complete list of accessories,
consumables and spares.
2.3 Unpacking
The Model 260 Colorimeter is packed in a custom designed box, which will
protect the instrument for both air-freight and sea-freight transport. The box
contains:
Description Part No.
M260 Colorimeter 26000009
Box 100 Cuvettes as per packing list
Spare Bulb 001 44 137
Universal PSU with Multi-plug Adapters 001 53 313
Cuvette Holder 252 11 002
Operator Manual 260 91 001
Lead, instrument to printer 926 09 052
2.4 Assembly
Remove the Colorimeter from the packaging.
Insert the Cuvette Holder into the Instrument.
2.5 Connecting Peripheral Devices
2.5.1 Printer
Connect the printer lead to the RS232 output at the rear of the Colorimeter.
Ensure that the printer is set up to the following condition:
9600 baud
8 bit
Parity None
Stop bit 1
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Connecting Peripheral Devices (continued)
2.5.2 Thermostatted Cuvette Holder (267 86 001)
The thermostatted cuvette holder (if ordered) should be inserted with the
power lead taken through the drain hole in the instrument before inserting the
holder into the instrument. This device will, when connected to a power
source, automatically settle to 37º C with the indicator light showing green.
NB the thermostatted cuvette holder is not connected electronically to the CHROMA
Colorimeter and its set point cannot be altered from 37ºC. It will only accommodate
the micro cuvette 001 26 120.
2.6 Instrument Set-up
The CHROMA Model 260 is based on the very reliable and stable Sherwood
design but now incorporates full programmability.
However it is still possible to operate the “Simple” protocol which enables the
Absorbance and % Transmission of a sample to be measured, without
involving programming, after Auto-zeroing the instrument.
In addition, simple Factor and Concentration experiments may also be carried
out, without need to create Methods or include Sample Naming, if such an
approach is better suited for your requirements.
The Model 260 is supplied without wavelength filters which must be ordered
separately. This model is thus suitable to be used as a colorimeter dedicated to
applications where only a few different wavelength filters are required.
A full list of available Gelatin and Interference Filters is given in Appendix B.
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I
Chapter Three Scientific Background
3.1 Historical
We can trace the heritage of the Sherwood CHROMA range of colorimeters
back to the very first Model 2 Colorimeter developed for a Dr Rose at
Hammersmith Hospital in London by Evans Electro-Selenium Ltd. (EEL) in
the 1940s. Prior to the Model 2 the concentrations of analytes by reference to
their colour was accomplished by the optical matching by eye; the Tintometer
approach. The Model 2 was superseded by the original Model 252 colorimeter
and then in the 1980s by the CHROMA models 254 and 257. Sherwood now
has brought the line into the 21st century and included programmability with
the models 260, 264 and 267.
3.2 Principles of Colorimetry
The two most basic laws which describe the foundations of a quantitative
analysis by photometry are attributed to Lambert and Beer. Lambert’s Law
states that the intensity of transmitted light decreases logarithmically as the
path length increases arithmetically. Beer’s Law states that increasing the
concentration of analyte had the same effect as a proportional increase in the
path length. Both laws use a constant related to the amount of light absorbed
per analyte molecule, i.e. specific absorption. (This property has been given
many names in the literature, such as molar absorptivity and extinction
coefficient. These names are simply trying to introduce the concept that
different analyte molecules are able to absorb a different amount of light per
molecule, which relates to the energy states of their electrons).
The combined laws are now simply referred to as Beer’s Law and are
expressed as follows: -
log
10
where a is an absorptivity constant, b is related to path length and C is analyte
concentration
or - A = abC
Since in most analytical work, the path-length is kept constant using a 1 cm
cell, the calibration graph generated for a particular analyte is linear with a
measurement given in absorbance units over a wide range.
Once the linearity of a calibration curve has been established, an analyst has
the convenient option of entering a factor, f on the 260 Colorimeter and
reading out in Concentration units directly, shown as follows: -
C = f A
where the factor, f, combines the previous path length and absorptivity factors,
a and b.
3.3 Clinical Chemistry
For a detailed description of time based and kinetic enzyme measurements as
it applies to Clinical Chemistry see Chapter Six, Seven and Eight.
For an overview of time based methods with calculations see Appendix D.
I0
= abC
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Chapter Four Specifications
4.1 Sample Requirements
When used with test kits, the manufacturer of the kit will specify the
individual sample parameters depending on the particular assay.
4.1.1 Total Volume Required:
Standard cuvettes: 1.4ml Part No: 471 88 300
Semi-micro cuvette: 600µl Part No: 471 88 400
Micro cuvette 400µl Part No: 001 26 120
4.2 Measurement Range
When used with test kits, the manufacturer of the kit will specify the
individual measurement ranges depending on the particular assay.
4.2.1 Operating Range
0 to 2.999 Abs
100.0 to 0 %T
0 to 9999 Conc
4.2.2 Resolution
0.001 Abs
0.1 %T
0.001 Conc
4.2.3 Warm Up Time
15 minutes. (Instrument monitors this).
4.2.4 Wavelength Range
400 to 750nm.
4.2.5 Alternative Filters
• Interference Filters wavelengths 405-660 nm band pass ± 5 nm
• Hi Spec filter at 430 nm ± 1 nm for EBC beer colour method
4.2.6 Thermostatted Cuvette Holder (267 86 001)
Controls to 37° C ±0.1°C
Only for use with micro-cuvette (001 26 120)
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Chapter Five Product Description
5.1 Controls and Indicators
5.1.1 Typical Front Panel Layout
5.1.2 The LCD Display:
Has three lines with a maximum of 16 characters per line. When results are
displayed the digits are shown over two rows in double size.
The buttons were designed to be universally understood.
5.1.3 Buttons
The Select buttons
These allow various choices to be displayed in the Menu sequence.
The Enter button
Allows the user to select the choice displayed in the Menu. It also
accepts a reading or result and operates the Print function.
The Back Space button
Allows the user to go back one or more steps in the protocol.
Pressing these two buttons simultaneously will take the user straight
back to the start up screen.
Traditional Zero/Blank Button.
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Product Description (continued)
5.1.4 Modifying Numbers etc.
Where there are numbers to change i.e. concentration factor values or letters to
change, i.e. to label tests and to define sample numbers or names, then the
Enter button changes the decade or letter position and the select buttons are
used to change the value of the number or letter.
For example to enter a concentration factor of 100 the user would do the
following when prompted:
Press to leave the 0 in the first unit column
Press to get 1 in the second unit column then press to accept
then press to leave 0 in the 3d and 4th unit column.
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5.2 Sample Heating and Incubation Times
Our protocols only cover the functions of the Colorimeter. They do not control
the use of a heating block or water bath. We indicate when a particular
incubation or measurement should start and an alarm sounds before the end of
the incubation/measurement. This allows the user to transfer the cuvette back
to the colorimeter for the measurement. Provision is made to enter the
temperature of a particular test in the programmed methods, but this is for
completeness of method recording and reporting purposes only.
Note: For any test requiring samples to be incubated at a specified
temperature (especially time based i.e. kinetic and end-point tests) it is critical
that the specified temperature is actually attained by reagents and samples
alike. Failure to ensure that is the case will give rise to results which are
incorrect. As an example, low results are often caused by poor temperature
transfer to the sample; often exacerbated by the use of micro cuvettes in an inappropriate heater block or a heater block which is controlling at the “set”
temperature but is actually lower than stated. The user is advised to establish
the correct settings for their sample preparation “system” before embarking on
the analysis of critical samples. A difference of just one degree Celsius from
the required setting can yield results as much as 10% in error.
.
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Product Description (continued)
5.3 Operating Modes
5.3.1 Warm Up
There is a 15 minute warm up period built into the start up routine. This can be
over-ridden by pressing the Enter button
5.3.2 Main Menu
5.3.2.1 Set-up
While the Model 260 Colorimeter is warmed, you can enter Setup to:
• set time and date format
• set the actual time and date
• select (Uart) the RS232 settings for printer
• set the LCD screen contrast.
(for a full list of setup options see the Model 260 Menu structure on the next
page).
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5.3.2.2 Simple
To use the colorimeter for a simple Absorbance measurement, (or %
Transmission), Simple mode is used. This only requires instrument with
appropriate filter selected to be zeroed against a blank solution (See 6.3)
prior to measurement of samples. Straightforward Factor and
Concentration experiments may also be performed in Simple Mode; they
just require the entering of a factor or measurement of a ‘known’ standard
in addition to blanking before measurement of samples.
NB In the “Simple” mode there is no sample labelling or operator identifier, or
storage of the methodology used.
5.3.2.3 Last
Takes the user straight back to the last used method.
5.3.2.4 Method
Entering the Method screen gives two options.
1. Recall
Allows an existing Method to be selected, which can then be:Run, Edited, Deleted or Printed.
2. New
Allows a New Method to be entered and stored.
See Chapter 7 for detailed method entry procedures.
The Model 260 Colorimeter Menu Structure is displayed on the next page.
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5.4 Menu Structure
Main Menu
Simple
Last
Method
Setup
Absorbance
% Transmission
Recall
New
Time Format
Date Format
Set Clock
Language
Uart
AutoPrint
Contrast
Run
Edit
Delete
Concentration
Factor
Time Based
Print
Endpoint
Kinetic
Update Firmware
Factor
Concentration
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NB. Uart is for RS232 parameter setup
If you enter “Update Firmware” the only
way out, is to switch the power off to the
unit.
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Chapter Six Operation
6.1 Start Up
Switch on the Colorimeter and, if needed, a heater block or water bath for sample
and standard preparation. Allow the heater block, or water bath to get to the
control temperature.
NB The nominal temperature setting point of a Heating Block should be
checked with an accurate thermometer in a liquid sample in a cuvette (of the
same profile you intend to use with your samples) in the block to ensure that the
desired temperature is achieved.
6.2 Zeroing and Calibration
Once the correct temperature has been reached and the Colorimeter has ended its
warm up phase and shows “Ready” then the correct filter should be inserted.
When the appropriate filter has been put in the filter slot, the user should press the
Enter key to move on to the next step in the procedure/method.
It is important that the cuvette holder and filter are in their correct positions
and that nothing is in the cuvette holder at this point.
The detector will saturate and the unit will “freeze” if return is pressed
before the filter is in place. If that happens, switch the power off and then on
again to recover normal operation.
The filter required and the actual procedures for zeroing and calibration are
prompted by the menu system for each analytical procedure required.
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Operation (continued)
6.3 Simple Method
The simple method allows measurement of Absorbance or % Transmission of a
sample. Here there is no calibration procedure. The instrument is zeroed against a
blank solution before a reading is taken of the sample. Put the blank cuvette in the
cuvette holder and press the zero button when prompted. Then put the sample
cuvette in the cuvette holder and the absorbance or % Transmission reading will
be displayed. To print the result, if a printer is connected, press the enter button.
When all the sample readings have been taken, press the back space button.
Many applications for the Colorimeter involve measurement of the concentration
of an analyte either by:-
1. Using a factor supplied by the test kit manufacturer.
2. Comparison with a calibrator (or ‘standard’) of known concentration.
In the Model 260 Colorimeter’s ‘Simple’ Menu, these two facilities are
respectively denoted as:-
1. Factor.
2. Concentration.
Within the Simple Menu the Model 260 allows Factor or Concentration
experiments to be performed very simply if the user does not require to save and
store a method protocol or to enter sample identifiers (mane or numbers). The
instrument, with appropriate filter selected, is zeroed against a blank solution after
which the user is prompted to enter a factor (or a standard of known concentration
and that concentration value) before the instrument is then ready to take a reading
and calculate a result for an unknown sample or samples.
For detailed operational flow charts of the ‘Simple’ Factor or Concentration
procedures, please refer to Appendix E.
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Operation (continued)
6.4 Concentration Methods
As stated, the majority of applications for the Colorimeter will be the
measurement of the concentration of an analyte either by:-
1. Using a factor supplied by the test kit manufacturer.
2. Comparison with a calibrator (or “standard”) of known concentration.
In the 260 series colorimeters, these two methods are respectively denoted as:-
1. Factor Method
2. Concentration Method
The ‘Method’ section within the Model 260’s Menu allows for methods to be
constructed, saved, recalled and operated for assistance with GLP and SOP
procedures. In addition, these ‘Methods’ allow the operator to enter sample
identifiers (names or numbers) for subsequent sample/result correlation.
Detailed illustration of the ‘Setup’ and “Running” of these method types is given
in Chapter 7 and 8 respectively of the Manual.
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6.5 Time Based Measurements
Some measurements, particularly of clinical samples, require time to develop an
absorbing reactant, which then allows the presence of the analyte of interest to be
determined and quantified. These have two categories:-
6.5.1 End-Point Methods
Used when the colour needs time to develop but is then stable for, usually, at least
an hour.
6.5.2 Kinetic Measurements
This Procedure is used, for example, when the analyte is an enzyme which
catalyses a particular reaction. Higher concentrations of the analyte result in faster
rates of reaction. The rate of change of adsorption is calculated having measured
the adsorption at two different times in the constant rate period of the reaction
(pre-determined by the test kit manufacturer).
Both time based measurement types can be made using a known factor or in
comparison to a calibrator (or “Standard”), both supplied by the test kit
manufacturer.
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Operation (continued)
Consequently, in the Model 260 Colorimeter, four method types can be entered,
edited and/or run to accommodate the methodologies outlined above. They are
respectively denoted as:-
1. End Point Method with Factor
2. End Point Method with Calibrator
3. Kinetic Method with Factor
4. Kinetic Method with Calibrator
In many cases the sample itself may contain interfering coloured substances. The
protocols provided in the Model 260 Colorimeter require a sample blank to be
measured and then the sample. The blank result is subtracted from the absorbance
of the reacted sample (this is done automatically by the colorimeter software).
Detailed Illustration of the Setup and “Running” of these method types is given in
Section 7 and 8 respectively of the Manual.
6.5.3 Incubation and Measurement Times
Many kinetic measurements procedures require an initial incubation time before a
second reagent is added. This time is sometimes referred to as the “delay” time.
Sherwood differentiates between the incubation and the “Measurement” time
during which the reaction takes place and the change in absorbance is measured.
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6.6 Automated Measurements
The main feature of the New CHROMA Colorimeter is its programmability. Once
the correct methodology is selected and e.g. the correct calibration concentration
value and wavelength is programmed, each measurement operation is prompted
by the instrument. Once named and saved the method can be recalled from a list
of methods by name.
6.7 Sample Numbering
The Model 260 Colorimeter allows both numerical and alpha-numerical sample
numbering to be used so that a result can be printed against the original sample
number. This is important where Good Laboratory Practice is employed.
It is possible to accept a sequential number as the default option.
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Chapter Seven Method Set-up
The following flowcharts give detailed illustration of entering a new Method of a type
accommodated by the Model 260 Colorimeter software (outlined in Section 6.4 and 6.5).
7.1 Factor Method – Set-up Procedure
Select Method from Main Menu using >> then press ↵↵↵↵
Select New from Method Menu using >> then press ↵↵↵↵
Select Factor from Mode Menu using >> then press ↵↵↵↵
Select wavelength from list* using >> then press ↵↵↵↵
Set Factor value. Use >> and ↵↵↵↵ for each number column
Note sample temperature if required using >> then ↵↵↵↵
(This facility only allows a record of the test temperature used to be printed
for traceability. The colorimeter does not control sample temperature).
Set concentration units for the test kit using >> then ↵↵↵↵
Enter a method name using >> and then ↵↵↵↵ for each letter
Select sample labelling format using >> then press ↵↵↵↵
You can print the method and check it before you save it
You can edit, save or cancel your new method
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7.2 Concentration Method – Set-up Procedure
Select Method from Main Menu using >> then press ↵↵↵↵
Select New from Method Menu using >> then press ↵↵↵↵
Select Concentration from Mode Menu using >> then press ↵↵↵↵
Select wavelength from list* using >> then press ↵↵↵↵
Set Standard concentration value as outlined in the test kit. Use
>> then press ↵↵↵↵ for each number column
Set concentration units using >> then ↵↵↵↵
Note sample temperature if required using >> then ↵↵↵↵
(This facility only allows a record of the test temperature used to be printed
for traceability. The colorimeter does not control sample temperature).
Enter a method name using >> and then ↵↵↵↵ for each letter
Select sample labelling format
You can print the method and check it before you save it
You can choose to edit, save or cancel your new method
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7.3 End Point Method with Factor – Set-up Procedure
Select Method from Main Menu using >> then press ↵↵↵↵
Select New from Method Menu using >> then press ↵↵↵↵
Select Time Based from Mode Menu using >> then press ↵↵↵↵
Select Endpoint from Mode Menu using >> then press ↵↵↵↵
Select wavelength from list*using >> then press ↵↵↵↵
Select No on Use Calibrator screen using >> then press ↵↵↵↵
Set Factor value using >> then ↵↵↵↵ for each number column
Set concentration units using >> then ↵↵↵↵
Set Incubation time using >> then ↵↵↵↵ for each number column
Note sample temperature if required using >> then ↵↵↵↵
(This facility only allows a record of the test temperature used to be printed
for traceability. The colorimeter does not control sample temperature).
Enter a method name using >> and then ↵↵↵↵ for each letter
Select sample labelling format
You can print the method and check it before you save it
You can choose to edit, save or cancel your new method
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7.4 End Point Method with Calibrator – Set-up Procedure
Select Method from Main Menu using >> then press ↵↵↵↵
Select New from Method Menu using >> then press ↵↵↵↵
Select Time Based from Mode Menu using >> then press ↵↵↵↵
Select Endpoint from Mode Menu using >> then press ↵↵↵↵
Select wavelength from list* using >> then press ↵↵↵↵
Select Yes on Use Calibrator Screen using >> then press ↵↵↵↵
Set Calibrator value. Use >> then ↵↵↵↵ for each number column
Select required units using >> then ↵↵↵↵ for each unit’s column
Set Incubation time. Use >> then ↵↵↵↵ for each number column
Note sample temperature if required using >> then ↵↵↵↵
(This facility only allows a record of the test temperature used to be printed
for traceability. The colorimeter does not control sample temperature).
Enter a method name using >> and then ↵↵↵↵ for each letter
Select sample labelling format
You can print the method and check it before you save it
You can choose to edit, save or cancel your new method
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7.5 Kinetic Method with Factor Method – Set-up Procedure
Select Method from Main Menu using >> then ↵↵↵↵
Select New from Method Menu using >> then ↵↵↵↵
Select Time Based from Mode Menu using >> then ↵↵↵↵
Select Kinetic from Mode Menu using >> then ↵↵↵↵
Select wavelength from list* using >> then ↵↵↵↵
Select No on Use Standard? Screen using >> then ↵↵↵↵
Set Factor value. Use >> then ↵↵↵↵ for each number column
Select required units using>> then ↵↵↵↵ for each unit’s column
Set Incubation time. Use >> then ↵↵↵↵ for each number column
Set measuring period. Use >> then ↵↵↵↵ for each number column
Note sample temperature if required using >> then ↵↵↵↵
(This facility only allows a record of the test temperature used to be printed
for traceability. The colorimeter does not control sample temperature).
Enter a method name using >> and then ↵↵↵↵ for each letter
Select sample labelling format
You can print the method and check it before you save it
You can choose to edit, save or cancel your new method
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7.6 Kinetic Method with Calibrator – Set-up Procedure
Select Method from Main Menu using >> then ↵↵↵↵
Select New from Method Menu using >> then ↵↵↵↵
Select Time Based from Mode Menu using >> then ↵↵↵↵
Select Kinetic from Mode Menu using >> then ↵↵↵↵
Select wavelength from list* using >> then ↵↵↵↵
Select Yes on Use Standard? Screen using >> then ↵↵↵↵
Set Standard value using >> then ↵↵↵↵ for each number column
Select required units using>> then ↵↵↵↵ for each unit’s column
Set Incubation time. Use >> then ↵↵↵↵ for each number column
Set measuring period. Use >> then ↵↵↵↵ for each number column
Note sample temperature if required using >> then ↵↵↵↵
(This facility only allows a record of the test temperature used to be printed
for traceability. The colorimeter does not control sample temperature).
Enter a method name using >> then ↵↵↵↵ for each letter
Select sample labelling format
You can print the method and check it before you save it
You can choose to edit, save or cancel your new method
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Chapter Eight Method Run Details
The following flow charts give detailed illustration of running a Method of a type
accommodated by the Model 260 Colorimeter, as outlined in Sections 6.4 and 6.5.
8.1 Factor Method - Run Procedure
Select Method from main menu using >> then ↵↵↵↵
Select Recall from Method Menu using >> then ↵
Select Run from Recall Menu using >> then ↵
Select required Method using >> then ↵
Method details will move up and down in display.
↵
↵↵
↵
↵↵
↵
↵↵
Check details are correct and press ↵↵↵↵.
Insert the filter as prompted then ↵↵↵↵
The cuvette holder must be empty when you press ↵↵↵↵
Insert the blank in the cuvette holder and then press 0
The instrument will be set to Zero. Press ↵
↵
↵↵
Enter sample number using >> then ↵
Put the sample in the cuvette holder when prompted. Press ↵
The sample OD will be shown on the display. Press ↵
The concentration of the sample will be displayed. Press↵
Choose Yes or No to run another sample then press ↵
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↵ for each number option
↵↵
↵
↵↵
↵
↵↵
↵
↵↵
↵
↵↵
25
8.2 Concentration Method - Run Procedure
Select Method from Main Menu using >> then ↵↵↵↵
Select Recall from Method Menu using >> then ↵↵↵↵
Select run from Recall Menu using >> then ↵↵↵↵
Select required Method using >> then ↵↵↵↵
Method details will move up and down in display.
Check details are correct and press ↵↵↵↵.
Insert the filter as prompted then ↵↵↵↵
The cuvette holder must be empty when you press ↵↵↵↵
Sherwood Scientific Limited
Insert the blank in the cuvette holder and then press 0
The instrument will be set to zero. Press ↵↵↵↵
Put the standard into the cuvette holder and press ↵↵↵↵
The OD of the standard will be displayed. Press ↵↵↵↵
Enter sample number using >> then ↵↵↵↵ for each number option
Put the sample in the cuvette holder and press ↵↵↵↵
The sample OD will be displayed. Press ↵↵↵↵
The sample concentration will be display. Press ↵↵↵↵
Select yes to run another sample then press ↵↵↵↵
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8.3 End Point Method with Factor - Run Procedure
Select Method from Main Menu using >> then ↵↵↵↵
Select Recall from Method Menu using >> then ↵↵↵↵
Select Run from Recall Menu using >> then ↵↵↵↵
Select the named method you want to run using >> then ↵↵↵↵
Method details will move up and down in display.
Check details are correct and press ↵↵↵↵.
Insert the filter as prompted then ↵↵↵↵
The cuvette holder must be empty when you press ↵↵↵↵
Put your sample and sample blanks in your pre-heated bath or
heater block and press ↵↵↵↵ to start the colorimeter timer
Just before the incubation time ends a beep will alert the user
Enter sample number using >> then ↵↵↵↵ for each number option.
Put the blank in the cuvette holder and press 0
The absorbance will be set to 0.000. Press ↵↵↵↵
Put the sample in the cuvette holder and press ↵↵↵↵
The sample absorbance will be shown and stored. Press ↵↵↵↵
The sample concentration will be shown. Press ↵↵↵↵
Test Another. Select Yes or NO using >> and press ↵↵↵↵
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8.4 End Point Method with Calibrator - Run Procedure
Select Method from Main Menu using >> then ↵↵↵↵
Select Recall from Method Menu using >> then ↵↵↵↵
Select Run from Recall menu using >> then ↵↵↵↵
Select the named method you want to run using >> then ↵↵↵↵
Method details will move up and down in display.
Check details are correct and press ↵↵↵↵.
Insert the filter as prompted then ↵↵↵↵
The cuvette holder must be empty when you press ↵↵↵↵
Put the calibrator blank in a pre-heated water bath or heater
Just before the incubation time ends a beep will alert the user
Put the blank in the cuvette holder and press the 0
The absorbance will be set to 0.000. Press ↵↵↵↵
Put the calibrator in the cuvette holder and press ↵↵↵↵
The calibrator absorbance will be shown. Press ↵↵↵↵
continued on next page
block and press ↵↵↵↵ to start the colorimeter timer
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End Point Method with Calibrator - Run Procedure (continued)
Enter the sample number using >> then ↵↵↵↵ for each position
When prompted, put the sample blank and press the 0
The absorbance will be set to 0.000. Press enter↵↵↵↵
Put the sample in the cuvette holder and press ↵↵↵↵
The sample absorbance will be displayed and stored. Press ↵↵↵↵
The sample concentration will be shown. Press ↵↵↵↵
Test Another. Select Yes or NO and press ↵↵↵↵
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8.5 Kinetic Method with Factor – Run Procedure
Select Method from Main menu using >> then ↵↵↵↵
Select Recall from Method Menu using >> then ↵↵↵↵
Select Run from Recall menu using >> then ↵↵↵↵
Select required Method using >> then ↵↵↵↵
Method details will move up and down in display.
Check the details are correct and press ↵↵↵↵.
Insert the filter as prompted then ↵↵↵↵
The cuvette holder must be empty when you press ↵↵↵↵
Enter sample number using the >> then ↵↵↵↵ for each number
Put the Sample in the preheated water bath or heater block
and press ↵↵↵↵ to start the colorimeter timer
Just before the incubation time ends a beep will alert the user
Insert the sample in the cell holder and press ↵↵↵↵
The optical density of the sample will be displayed. Press ↵↵↵↵
Return the sample to the incubator and press ↵↵↵↵ on the
colorimeter to start the timer again
Just before the incubation time ends a beep will alert the user
Put the sample in the cuvette holder again and press ↵↵↵↵
Continued on next page
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Kinetic Method with Factor – Run Procedure (continued)
The new optical density of the sample will be displayed. Press ↵↵↵↵
The change in Sample OD per minute will be displayed. Press ↵↵↵↵
The concentration of the sample will be displayed. Press ↵↵↵↵
Select Yes or No and press ↵↵↵↵
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8.6 Kinetic Method with Calibrator– Run Procedure
Select Method from Main Menu using >> then ↵↵↵↵
Select Recall from Method Menu using >> then ↵↵↵↵
Select Run from Recall Menu using >> then ↵↵↵↵
Select required method using >> then ↵↵↵↵
Method details will move up and down in display.
Check the details are correct and press ↵↵↵↵.
Insert the filter as prompted then ↵↵↵↵.
The cuvette holder must be empty when you press ↵↵↵↵
Put the Standard in the pre-heated water bath or heater block
and press ↵↵↵↵ to start the colorimeter timer
Just before the incubation time ends a beep will alert the user
Insert the standard in the cuvette holder and press ↵↵↵↵
The optical density of the Standard will be displayed. Press ↵↵↵↵
Return the standard to the incubator and press ↵↵↵↵ on the
colorimeter to start the timer again
Just before the incubation time ends a beep will alert the user
Insert the standard in the cuvette holder again and press ↵↵↵↵
Continued on next page
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Kinetic Method with Calibrator– Run Procedure (continued)
The new optical density of the Standard is shown. Press ↵↵↵↵
The change in Optical density of the Standard is shown. Press ↵↵↵↵
Enter the sample number using >> then ↵↵↵↵ for each number
Put the sample in the pre-heated water bath or heater block
and press ↵↵↵↵ to start the colorimeter timer
Just before the incubation time ends a beep will alert the user
Put the sample in the cuvette holder and press ↵↵↵↵
The optical density of the Sample will be shown. Press ↵↵↵↵
Return the sample to the incubator and press ↵↵↵↵ on the
colorimeter to start the timer again
Just before the incubation time ends a beep will alert the user
Insert the sample in the cuvette holder again and press ↵↵↵↵
The new optical density of the Sample will be shown. Press ↵↵↵↵
Change in Optical density will be shown. Press ↵↵↵↵
The sample concentration will be displayed. Press ↵↵↵↵
Choose Yes or No in answer to test another using >> then ↵↵↵↵
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Appendix A Warranty
Sherwood Scientific Limited
Product Warranty Statement
_________________________________________________________________________________________
Warranty Term: 12 Months
Sherwood Scientific Ltd (Sherwood) warrants, subject to the conditions itemised within
this document, through either Sherwood personnel or personnel of its authorised
distributors, to repair or replace free of all charges, including labour, any part of this
product which fails within the warranty time specified above, appertaining to this
particular product. Such failure must have occurred because of a defect in material or
workmanship and not have occurred as a result of operation of the product other than in
accordance with procedures described in the instructions furnished with this product.
Conditions and specific exceptions that apply to the above statement are as follows:
1. End-user warranty time commences on the date of the delivery of product to enduser premises.
2. Free of all charges’ statement applies only in areas recognised by Sherwood as
being serviced either directly by its own personnel, or indirectly through
personnel of an authorised distributor. Products purchased outside these areas
requiring service during the warranty period will incur charges relative to the
travel/transit costs involved. However, products purchased in such areas will be
serviced during the warranty period free of all charges providing they are
returned, carriage paid, to either Sherwood or by pre-arrangement to an authorised
Sherwood distributor.
3. All maintenance (other than operator maintenance as described in the
instructions), repairs or modifications have been made by Sherwood or Sherwood
authorised personnel.
4. This product has where applicable been operated using Sherwood specified
supplies and reagents.
5. Sherwood reserves the right to make any changes in the design or construction of
future products of this type at any time, without incurring any obligation to make
any changes whatsoever to this particular product.
6. Reagents, supplies, consumables, accessories and user maintenance items are not
included in this warranty.
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Product Warranty Statement (continued)
7. Repairs or replacement of any part failing due to abnormal conditions
including the following, are excluded from this warranty:
a. Flood, lightning, earthquake, tornado, hurricane, or any other natural or
man-made disaster.
b. Fire, bombing, armed conflict, malicious mischief or sprinkler damage.
c. Physical abuse, misuse, sabotage or electrical surge.
d. Damage incurred in moving the product to another location.
8. User agrees to permit Sherwood personnel or personnel of its authorised
distributor to make changes in the product which do not affect results
obtained, but do improve product reliability.
Representations and warranties purporting to be on behalf of Sherwood made by any
person, including distributors and representatives of Sherwood, which are inconsistent
or in conflict with the terms of this warranty (including but not limited to the
limitations of the liability of Sherwood as set forth above), shall not be binding upon
Sherwood unless reduced to writing and approved by an officer of Sherwood Scientific
Ltd.
Except for the obligations specifically set forth in this warranty statement, in no event
shall Sherwood be liable for any direct, indirect, special, incidental, or consequential
damages, whether based on contract, tort or any other legal theory and whether
advised of the possibility of such damages.
Neither Sherwood nor any of its third party suppliers makes any other warranty of any
kind, whether expressed or implied, with respect to Sherwood Products.
Sherwood Scientific Ltd.,
1 The Paddocks,
Cherry Hinton Road,
Cambridge,
CB1 8DH,
England
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Appendix B Gelatin and Interference Filters
252 15 001
252 16 001
252 17 001
252 18 001
252 19 001
252 20 001
252 21 001
252 22 001
252 26 001
252 31 001
471 79 700
471 79 800
471 88 900
471 89 000
471 89 100
FILTER, GELATIN, 430NM
FILTER, GELATIN, 470NM
FILTER, GELATIN, 490NM
FILTER, GELATIN, 520NM
FILTER, GELATIN, 540NM
FILTER, GELATIN, 580NM
FILTER, GELATIN, 600NM
FILTER, GELATIN, 710NM
FILTERS, GELATIN, SET OF 8
FILTER, GELATIN, 410NM
FILTER, INTERFERENCE, 430NM
FILTER, HIGH SPEC, 430NM
FILTER, INTERFERENCE, 405NM
FILTER, INTERFERENCE, 510NM
FILTER, INTERFERENCE, 546NM
471 89 150
471 89 152
471 89 155
471 89 200
471 89 300
471 89 400
FILTER, INTERFERENCE, 600NM
FILTER INTERFERENCE, 620NM
FILTER, INTERFERENCE, 636NM
FILTER, INTERFERENCE, 660NM
FILTER, INTERFERENCE, 680NM
FILTER, INTERFERENCE, 725NM
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Appendix D Time Based Methods for Clinical
Analysis
The laboratory analysis of enzymes in blood plasma is useful in the diagnosis and
monitoring of various diseases especially where there is damage to tissues and organs
of the body e.g. the heart, the liver and bone. Much of the raised enzyme activity,
compared to the normal reference range, is due to enzymes within the cells of the
tissues or organs being released into the bloodstream as a result of damage or rupture
of the cell walls.
The more clinically important enzyme test requests made to laboratories are:-
• The enzymes AST (aspartate aminotransferase) and ALT(analine
aminotransferase) known as the aminotransferases are often requested together :-
o Increased levels of AST in blood plasma may be found in cases of acute
hepatitis, necrosis of the liver, in severe crush injuries and after heart
attacks.
o Increased levels of ALT are found in the same liver diseases as is AST.
• High plasma levels of the enzyme Gamma GT are found in alcohol-associated
liver disease e.g. cirrhosis, in hepatitis and may be moderately raised after certain
drug therapy.
• Increased levels of LDH are found after acute damage to the liver or the main
muscles, in cases of tumours known as lymphoma and in certain types of anaemia.
A form of this enzyme termed HBDH is raised in plasma after heart attacks
(myocardial infarction).
• Increased plasma levels of ALP are found in various diseases of the liver and
bone.
• Raised levels of creatine kinase (CK) are found after heart attack and in trauma to
muscle.
The role of these enzymes in the cells of the body is to act as a catalyst where they
convert a substrate to another molecule and by-product. The enzyme actually
accelerates the reaction. By careful analysis of the enzyme reaction on the substrate,
where parameters such as temperature and pH are optimised, the activity of the
enzyme in plasma can be easily determined.
There are two types of method in which enzymes or metabolic substrates can be
measured in clinical chemistry:
1. End Point method
2. Reaction Rate method
Each method follows similar principles.
• Enzymes are simply used as reagents in excess to turn the blood
substrate under investigation into a product that can be measured e.g.
glucose by end point method.
• The enzyme of clinical significance can be measured by observing the
rate at which the reaction proceeds and hence the rate that the substrate
or products increase of decrease over a period of time e.g. LDH at 340
nm by the rate of reaction.
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Appendix D (continued)
1. End Point Reaction
If the assay is intended to measure the concentration of the substrate then excess
enzyme is present and the reaction goes to completion e.g. glucose by the enzyme
glucose oxidase.
i) The substrate (Sample) mixture is placed in the cuvette. This is brought to
temperature (externally in a heating device).
ii) The cuvette is then placed in the colorimeter, the enzyme is added and the
instrument is immediately zeroed.
iii) The cuvette is placed in the heating device for a period which is usually
between 10-30 minutes (this is called the incubation period).
iv) At the end of the measurement time the final absorbance (OD) is measured.
A standard sample of known concentration of substrate and a blank sample containing
no substrate are both treated in exactly the same way.
Calculation
After subtracting the blank absorbance reading from both the Standard and the
Sample, the ratio of the Sample Absorbance and the Standard absorbance is multiplied
by the standard concentration to obtain the concentration of the unknown. (OD refers
to absorbance in the calculation below).
(OD sample-OD blank) x concentration of Standard = Concentration of Sample
(OD standard –OD blank).
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Appendix D (continued)
2. Reaction Rate
This method of analysis is used where an enzyme itself is to be measured and the
substrate is added in excess to the reaction cuvette.
The calculation measures the rate of change of either the initial substrate or a
particular product of the reaction. This is then either compared with the rate of change
of an enzyme concentration of a fixed standard or simply multiplied by a given factor.
Due to complex reactions taking place a delay time or incubation as well as the
measuring time may be used.
A. For immediate Reaction Rate (i.e. no incubation time)
i) The substrate is brought to the appropriate temperature externally.
ii) Place cuvette in light path and sample (or standard) is added.
iii) Initial absorbance is recorded and Start initiated.
iv) Sample removed and placed into heater for Measure Time.
v) 20 seconds before end of Measure Time buzzer sounds to remind user
to prepare to replace cuvette into colorimeter.
vi) At exact end of Measure Time cuvette is in light path and final
absorbance noted.
Calculation
∆ OD sample x factor = sample concentration
or ( ∆ OD sample ) x Standard concentration = Concentration of Sample
∆ OD Standard
B. For Delayed Reaction Rate (i.e. with Incubation time)
Substrate is brought to temperature externally
i) Sample (or standard) added.
ii) Incubation Time started.
iii) 20 seconds before end of Incubation time buzzer sounds reminder.
iv) Cuvette placed in colorimeter and at end of incubation Time
absorbance value is noted and Start of Measurement time is pressed.
v) Sample removed and placed into heater for Measure Time.
vii) 20 seconds before end of Measure Time buzzer sounds to remind user
to prepare to replace cuvette into colorimeter.
viii) At exact end of Measure Time cuvette is in light path and final
absorbance noted.
Calculation
∆ OD sample X factor = sample concentration
or (∆ OD sample ) x concentration of standard = concentration of sample
(∆ OD Standard)
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Appendix E “Simple” Factor Procedure
Select Simple from Main Menu using >> then ↵↵↵↵.
Select Factor from Main Menu using >> then ↵↵↵↵.
Insert the filter as prompted, then ↵↵↵↵.
The cuvette holder must be empty when you press ↵↵↵↵
Insert the blank in the cuvette holder and then press 0
The instrument will be set to Zero and immediately prompt
the user to enter the Factor given for the test kit in use.
Enter the test kit factor using >> then ↵↵↵↵ for each number
option.
(In this example we have entered a factor of 15).
Once the factor has been entered the display changes to
present the Absorbance measured and the calculated
Concentration.
Insert a sample in the cuvette holder and the display
shows Absorbance, measured and calculated
Concentration. Remove the sample cuvette, replace it
with another and the Absorbance and calculated
Concentration will be displayed immediately. It is
recommend you wait at least 10 seconds for the sample to
settle before taking a reading. Once you have finished
measuring all your samples press
“Select Mode” Screen.
to return to the
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Appendix E (continued)
“Simple” Concentration Procedure
Select Simple from Main Menu using >> then ↵↵↵↵.
Select Concentration from Main Menu using >> then ↵↵↵↵.
Insert the filter as prompted then ↵↵↵↵.
The cuvette holder must be empty when you press ↵↵↵↵.
Insert the blank in the cuvette holder and then press 0.
The instrument will be set to Zero and immediately
prompt the user to enter the concentration of the
standard to be measured.
Enter the standard concentration using >> then ↵↵↵↵ for
each number option.
(In this example we have entered a concentration of 28).
Insert the standard in the cuvette holder, wait 10
seconds and then press ↵↵↵↵.
The instrument will display Absorbance measured and
the Concentration of the standard.
Insert a sample in the cuvette holder and the display
shows Absorbance, measured and calculated
Concentration. Remove the sample cuvette, replace it
with another and the Absorbance and calculated
Concentration will be displayed immediately. It is
recommend you wait at least 10 seconds for the sample
to settle before taking a reading. Once you have finished
measuring all your samples press
“Select Mode” Screen.
Sherwood Scientific Limited
to return to the
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