606 Spectrophotometer Stasar Spectrophotometer-Stasar Operator’s Manual Part No. 25066x42 (Serial No. 20,000 and Above) Configuration A GILFORD a CORNING Laboratory Sciences Company. Spectrophotometer Stasar Operator’s Manual . Part No. 25066x42 . (Serial No. 20,000 and Above) Configuration A Change 1 PREFACE The Gilford Stasar Spectrophotometers are precision, reference spectrophotometers. This manual, prepared for you, the operator, tells how to get the most from your Stasar. Read it carefully, believe in what it tells you, and you can expect long and faithful service from your Stasar. Pay particular attention to the maintenance and operating instructions; poor ‘maintenance and operator error are primary causes of failures and malfunctions. This manual, Part Number 25066x42, Configuration A, was issued October 15, 1984, with Change | incorporated February 1, 1985, contains 52 pages of the issue indicated: Page Issue Preface ‘Change 1 ii and iv Original it and iii ‘Change 1 1-1 through 1-6 | 2-1 through 2-4 3-1 through 3-5 inal inal inal 4-1 through 4-7 inal , Sol Change 1 6-1 through 6-4, 6-6 through 6-12 Original : 6-5, 6-13 through 6-18 Change 1 7-1 through 7-8 Change 1 Writer/Editor: Tenley Orendorf Graphics: Ralph Focht, Dave Samiec Technical Assistance: Paul Fridenstine, Harry Debevec, George Ksenich ©1985, Ciba Coming Diagnostics Corp CHANGE |Title Page Section 1 - General Information. Introduction... Photometer.. SSSGSSSLLL Rapid Sampling System. Temperature Controlled Cuvette. Input/Output Provisions. = =f Section 2 - Installation Instructions. we Pel General... 21 Instrument Location 241 Dimensions. 21 Power Source.. 2-1 Unpacking. - 2-1 + 202 2-3 223 31 31 Principles of Absorption Spectroscopy.. 3-1 Spectrophotometer Components... 3.2 3-2 3-2 303 Round Cuvette Holder... 4 Photometer 4 Output Indicator. 4 £ Section 4 - Operating Instructions. General, Controls and Indicators.. Operating Procedures.. Stasar Il and I Preliminary Operation... Measuring Absorbance. Measuring Concentration. Temperature Verification... Setting Temperatur Round Cuvette Holder or Cuvette Accessory, Preliminary Operation... Measuring Absorbance. Measuring Concentration. EEEEEREESSEEIFTitle Section 5 - Hazard: Section 6 - Maintenance. General Maintenance Calibration. Care and Cleaning of Calibration Standard. Calibration with Model 1220 Calibration Standar: Wavelength Calibration Check.. Wavelength Calibration Adjustmen Absorbance Calibration Check.. Absorbance Calibration Adjustmen ‘Thermocuvette Maintenance, Rapid Sampling Cuvette As Required Maintenancs ‘Thermocuvette Inlet Tubing Replacemen Rapid Sampling Cuvette Inlet Tubing Replacement. Pinch Valve Maintenance... Pinch Valve Pinch-Point Adjustment.. Pinch Valve Tubing Replacement. Pinch Valve Replacement... Tungsten Lamp Replacement, Troubleshooting.. Section 7 - Parts List. Preventive Maintenance Parts. Illustrations Figure Title Ll Stasar Block Diagram. 1-2 Monochromator Optical Path.. 1-3 Rapid Sampling System. 1-4 Vacuum Pum 1-5 ii CHANGE |Figure Title el Stasar Back Panel... 1 22 Spectrophotometer Cover Retaining Screws. 22 1 23 Voltage Adjustment Switch Location. = 22 J 2-4 Vacuum Connections... 2-4 4 3-1 Stasar Block Diagram, 32 J 3-2 Monochromator Optical Path.. 3-3 = 33 Rapid Sampling System. 34 . Beh 1 : hel 4-2 . 43 Sample Actuator Bar Operating Positions.. . 4a Vacuum Adjustment. eon 45 Aspirating a Sample. 44 . 46 Round Cuvette Holder... 47 61 Cover Retaining Screws. 6-2 Stasar Cover in Resting Position 6-2 ‘ 6-3 1220 Calibration Standard. 6-2 5 6-4 Calibration Standard Compartment. 63 65 Stasar Controls. 6-4 . 6-6 1220 Standard in 64 6-7 Wavelength Adjustment Lever. 65 . 6-8 Cuvette Disassembly. 6-8 , 6-9 Cuvette Groove. 6-8 6-10 Cuvette Cleaning Too! 6-8 - ran Window Retainer Installation. 6-9 6-12 Rapid Sampling Cuvette. 611 . 6-13 Flaring Inlet Tubing 6-12 6-14 Pinch Valve. 6-13 6-15 Monochromator and Associated Parts.. 6-14 - 6-16 Lamp Assembly Mountingsevs 6-15 _ Tables 21 Voltage Range Switch Settings. ' 7aL O-Ring Compatibility Chart 7-2 (O-Ring Interchangeability Guide.. CHANGE 1Spectrophotometer - StasarINTRODUCTION The Gilford Stasar Spectrophotometers are dependable and highly accurate instruments operating in the visible portions of the spec- trum from 335 nm to 700 nm. Linear meas- urements of absorbance or concentration ate displayed on a bright, easy-to-read light emitting diode (LED) readout. A minimum of controls makes the Stasar series easy to operate and eliminates the need for exten- sive training time. Two versions of the Stasar Spectrophoto- meter are available to suit various needs, and are as follows: Stasar I Includes a rapid sampling system with a precision temper- ature cuvette. SAMPLE CONTAINER ‘THEAMOCUVETTE RAPID SAMPLING CUVETTE (OR ROUND. CUVETTE Section 1 General Information Stasar Il Includes the standard rapid sampling cuvette for applica- tions where temperature control is not necessary. This version can be updated to a Stasar IIL. StasarI Is the basic spectrophome- ter employing round cuvettes for sample handling. This version is no longer manufactured; however, existing instruments can be updated toa Stasar Il or Ill configuration. INSTRUMENT DESCRIPTION The Stasar Spectrophotometer, shown in block diagram in Figure 1-1, consists of a tungsten light source, diffraction grating PHOTOMULTIPLIER Tua iO TUNGSTEN ~ LIGHT HOt — sounce MONOCHROMATOR 340 SAMPLING SYSTEM Leo INDICATOR C=] PHOTOMETER ch ZERO CONTROL Figure 1-1, Stasar Block Diagram To Data Listen M4GENERAL INFORMATION monochromator, linear absorbance photome- ter, a four-place LED readout, a rapid sam- pling system, and a temperature controlled cuvette (Stasar Ill only). Provisions are included for thermal printers, auxiliary analog equipment, and inputs for remote control of sample and purge. LIGHT SOURCE. The Stasar light source is ‘@ single-filament tungsten lamp with long life and excellent stability. The lamp assem- bly is prefocused at the factory to simplify alignment and replacement in the field. MONOCHROMATOR. Monochromatic light is provided by a precision diffraction grating monochromator with narrow spectral band- width and minimal stray light characteris- tics. Wavelength is displayed on a digital DIFFRACTION | GRATING PHOTOMULTIPLIER, roe ReFLecring MIRROR, indicator and can be easily adjusted and calibrated. When the wavelength is adjusted below 400 nm, a blue stray light filter is automatically inserted in the light beam; when the wavelength is adjusted above 400 nm, a magenta attenuation filter is auto- matically inserted in the light beam. This, magenta filter also acts as a stray light filter above 600 nm. The monochromator light path is shown in Figure 1-2. PHOTOMETER. The Gilford Photometer Produces an output which is linear with absorbance. Conversion from transmittance to absorbance is made electronically. Photometric accuracy and linearity are high (Refer to Specifications). Another advan- tage includes direct linear conversion to concentration units. BAFFLE BAFFLE SPHERICAL, MIRROR exit REFLECTING cuverTE Sut MIRROR, Figure 1-2. Monochromator Optical Path 12u LED READOUT. Linear absorbance and con- centration measurements are displayed on a bright four digit LED indicator with a resol- ution of 0.001A. RAPID SAMPLING SYSTEM. (Stasar II and Iii) Vacuum draws samples into a rapid sam- pling cuvette which has a path-length of 10 mm. The sampling system provides a repro- ducible sample intake volume for similar samples, and vacuum and sampling time can be easily adjusted for samples of different viscosity or specific gravity. With an air purge between samples, the rapid sampling system minimizes carryover. Refer to Fig- ure 1-3. TEMPERATURE CONTROLLED CUVETTE. (Stasar Ill only) The temperature controlled ‘THEAMOCUVETTE SAMPLE = ACTUATOR BAR Sample _—! Time CONTROL GENERAL INFORMATION cuvette controls sample temperature direc- tly. There are three fixed temperature set- tings, 25°, 30°, and 37°C, and one setting which allows cuvette temperature to be ad- justed over the range of 25° to 37°C. Temperature may be displayed on the LED readout, by depressing the PUSH-TO-READ TEMPERATURE pushbutton. INPUT/OUTPUT PROVISIONS. Printers can be attached to provide a permanent printed record of absorbance, concentration, or ‘temperature readout. An analog signal from an external device can be connected to the analog input jack. Programmers, computers, or other auxiliary devices can be attached to provide external control of sampling and purging. VACUUM GAUGE To vacuum ‘SOURCE, vacuum receiver SAMPLE TIMING NETWORK Figure 1-3. Rapid Sampling System 13GENERAL INFORMATION GENERAL NOTES. Included as standard ‘equipment with Stasar II and Ill is the Model 3022 Vacuum Pump, Figure 1-#, and Model 3021 Vacuum Receiver, Figure 1-5. The vacuum receiver consists of a vacuum regu- VOLTAGE AND FREQUENCY LABEL POWER SWITCH Figure 1-4. Vacuurn Pump SPECIFICATIONS. SOLID STATE PHOTOMETER (OVERALL) ‘Absorbance Range « « Resolution...» Concentration Range Short Term Stability (Noise)*.. ++ +++ ++ Long Term Stability (Drift)* . . Photometric Accuracy «++ +++ Photometric Precision (Repeatability) . - Linearity Absorbance Calibration ‘Accessory Input - ++ OPTICAL SYSTEM Type of Monochromator . « slit Width . . Spectral Range... + Spectral Bandwidth « Wavelength Accuracy . « Wavelength Setting Precision . Light Source... ++ ++ Stray Light «+++ * After 30 minute warmup 4 sadard (Model 1220) « lator, gauge, and two bottles which serve as collector and reservoir. Also included with Stasar Il and Il is a dual-purpose calibration standard for calibration of wavelength and absorbance. COLLECTION CONTAINER VACUUM GAUGE vacuum RESERVOIR vacuum REGULATOR Figure 1-5. Vacuum Receivers + + 0,000 - 2.0008 + 0.001 «+ X 0.3 - 3.0 (0000 - 6000) continuously variable ceeeeeeee less than 0,001A (at 0A) less than 0,002A (at 2,000A) «+++ less than 0.005A/Hr (at 0A) 0,05%A, + 0,002A 0.005A maximum deviation Accuracy-1% stated value 0-7 volts Diffraction Grating Fixed at 0.4 nm 335 nm - 700 nm 340 nm - 650 nm when used with round cuvette holder 8 nm maximum at 360 nm 42m -.0.5.1m 2 Tungsten lamp with regulated variable voltage DC power supply less than 0.3% throughout the wavelength rangeGENERAL INFORMATION RAPID SAMPLING SYSTEM (Stasar II and III) “~~Sample Volume Vacuum Required . « Sample Timer Range «+ - + ++++Chamber Volume 44ul 6-15 inches Hg > + +Os1 ~ 1.0 seconds in 0.1 second steps + 10 mmp in standard cuvette Kel-F(Stasar 11) Aluminum with stainless steel liger (Stasar 1M) +++ Teflon! see ++ Quartz Glass Light Path... Cuvette Body Material . Inlet Tube... - 260+ Cuvette Window QUTPUT SYSTEM Measurement Display ...++.+++eeeee +++ 4-Place solid-state indicator DIGITAL OUTPUT Circuit « « «A/D Dual Slope Conversion Nominal 7 conversions per second 4 character output - 4 bits per character parallel BCD + » Positive 5V logic-Std TTL Positive 5V logic-Std TTL Type +++ Level Command Output (standard) Print Command Output (standard) TEMPERATURE CONTROLLER (Stasar Il!) Temperature Settings. -- 4 ++ 25°C, 30°C, 37°C, and variable from 25°C to 37°C 40.2°C - indicator reads To 0.1° of actual tempera- ture +0.05°C + « Tess than 20 seconds «+ less than 0.05°C/hour + Chamber Volume 4¢ul. Accuracy . . Regulation . Response . « Drift... : Thermocuvette Sample Volume . . PHYSICAL SIZE AND WEIGHT Height . « Width Depth . Weight . . cece eee ee eB inches (20,3 cm) verses 016.5 inches (41.9 cm) + 13.9 inches (35.2 cm) +33 pounds (14.9 kgm) + +++ 90-140 VAC, 50-60 Hz. Internal modifications to provide 180-280 VAC, 50- 60Hz operation. C.S.A. approved. POWER SOURCE 1-5GENERAL INFORMATION « Stasar Spectrophotometers should be mounted on a sturdy work bench in an area free from vibrations, drafts, and strong magnetic fields. ENVIRONMENTAL REQUIREMENTS ... . 15°C to 40°C OPERATING TEMPERATURE LIMITS .... STORAGE LIMITS .. ++ Temperature 0°C to 50°C Store the analyzer indoors; maximum humidity for prolonged storage is 0%-99% (non-condensing). Outdoor storage is recommended only in special reusable containers. To the best of our knowledge, routine storage inspection is not required, Return the 1220 calibration standard supplied with the analyzer, to the factory for recalibration once (1) a year with use, or once every three (3) years in storage. 16GENERAL These instructions contain all the in- formation necessary for unpacking and in- stalling your Stasar Spectrophotometer. INSTRUMENT LOCATION. The installation area should be free from drafts, vibrations, strong magnetic fields and sudden tempera- ture changes. Sufficient space should be available to allow for ample ventilation and access to wavelength and absorbance con- trol knobs. The system dimensions and power requirements are as follows: DIMENSIONS. Height .. . . «8 inches (20.3 cm) Width ..... 16.5 inches (41.9 cm) Depth ..... 13.9 inches (35.2 cm) Weight .... 33 pounds (14.9 kgm) POWER SOURCE. 115 volt operation (standard) Voltage: 90-140 VAC Frequency: 50/60 Hz Current: 0.7 Amp 230 volt operation (optional) Voltage: 180-280 VAC Frequency: 50/60 Hz Current: 0.35 Amp UNPACKING. The Stasar was carefully tested and inspected prior to shipment. Up- on receiving the instrument, check for any obvious damage which may have occurred during transit. Report any damage imme ately to the shipping agent. Check packing list to insure all components have been included. Carefully unpack the instrument and place on a sturdy work bench or table. Do not leave paper or other foreign material under the instrument, since air circulation could be restricted and result in erratic Section 2 Installation Instructions readings. Referring to the Maintenance Section of this manual, open the top cover and inspect the instrument for any internal damage or loose cables. If a disconnected cable or any damage is noted, contact your Gilford Service representative by dialing 1- 800-GILFORD. Referring to Figure 2-1, plug female line cord connector into receptacle on rear of Stasar labeled AC INPUT. Check the label next to the power switch to insure instrument is of the proper configuration for the supplied voltage and frequency. Refer to the Voltage Adjustment information in this section, Insure power switch is in the off Position. Connect the male end of the line cord to a grounded three pronged receptacle of proper voltage and frequency. Remove masking tape from cuvette chamber open- ing. For Stasar I installation, remove cuvet- te holder from shipping box and insert firmly into cuvette chamber. For Stasar II or Ill installation, remove cuvette from shipping box, moisten O-ring on tip with distilled water, and insert in cuvette chamber. If installing a Stasar Il, plug cable from cuvette into connector labeled J6 on rear of instrument. Refer to Figure 2-1. Twist connector lock to insure cable is tightly secured, RECEPTACLE Figure 2-1. Stasar Back PanelINSTALLATION INSTRUCTIONS. VOLTAGE ADJUSTMENT._ If it becomes necessary to operate this instrument on in- put power of voltage ratings other than ‘those specified on that data plate, make the following adjustments. 1, Turn off instrument power, then discon- nect the AC power cord from the receptacle ‘on the instrument back panel. 2. Disconnect vacuum tubing and all elec- trical cables from the spectrophotometer, then position the instrument on its back panel and loosen the two cover screws shown in Figure 2-2. NOTE Loosen the cover retaining screws approximately four full turns; however, do not complete- ly remove these screws. COVER RETAINING SCREWS Figure 2-2. Spectrophotometer Cover Retaining Screws (Bottom View) 3. Place the spectrophotometer in its nor~ mal, upright position and completely open the top cover. Locate the VOLTAGE CON- VERSION and VOLTAGE RANGE switches fon the bottom panel as shown in Figure 2-3. 22 VOLTAGE” RANGE VOLTAGE CONVERSION SWITCH SWITCH Figure 2-3. Voltage Adjustment Switch Location 4, Loosen but do not remove the two screws holding the VOLTAGE CONVERSION SWITCH plate. Remove the plate to gain access to the switch, 5. Use a small screwdriver to slide the switch to the alternate position so the volt- age value on the switch slide matches the input voltage being used. 6. Replace the VOLTAGE CONVERSION SWITCH plate and tighten the two mounting screws. 7. Replace the AC input fuse with one of proper voltage and current ratings. 8. The VOLTAGE RANGE SWITCH, shown in Figure 2-3, is normally set to either of the two NORM positions; however, it can be set for low, normal, or high prevailing line volt- age conditions. Select the appropriate switch settings as followst NOTE Exercise extreme care in the next step to avoid the position of severe or fatal electrical shock. Set a VOM or DVM to measure AC voltage, then connect the test leads to the AC receptacle being used for the spectro- photometer. Note the line voltage indicated on the meter, then set the voltage range switch according to Table 2-1.Table 2-1. Voltage Range Switch Settings LINE VOLTAGE SWITCH SETTINGS 90 VAC to 11I0VAC = LOW 105 VAC to 125 VAC | NORM* 120 VAC to 140 VAC HIGH 180 VAC to 220 VAC LOW 210 VAC to 250 VAC NORM* 240 VAC to 280 VAC HIGH ¥ Either of the two NORM settings may Be used. 9. To gain access to the VOLTAGE RANGE SWITCH, loosen but do not remove the two screws holding the VOLTAGE RANGE SWITCH plate, then remove the plate. 10. Use small screwdriver to place the switch slide to the appropriate position as indicated in Table 2-1. LL. Replace the VOLTAGE RANGE SWITCH plate and tighten the two mounting screws. 12, Close the instrument cover and tighten the two cover retaining screws. 13. Connect the instrument power cord to a grounded AC receptacle of proper voltage, frequency, and current ratings. VACUUM CONNECTIONS. (Stasar Il and Stasar Ill). The rapid sampling cuvette and ‘the thermocuvette require a vacuum source and waste receptacles. Stasar If and Ill are ESSURE vacuum VACUUM RECEIVER, vacuum PUMP INSTALLATION INSTRUCTIONS supplied with a vacuum pump and receiver bottles as part of the shipping kit. Always empty the first receiver bottle as soon as it is full, the second bottle is only a reservoir to protect the vacuum pump. Refer to Fig- ure 2-4, to perform the following steps. 1. Place vacuum receivers in a position to allow monitoring of the vacuum gauge dur- ing instrument operation. 2. Connect free end of neoprene tubing from vacuum receiver rubber stopper to teflon connector on rear of instrument labeled VAC. 3. Connect the free end of the neoprene tubing, from the teflon connector on the rear of the vacuum receiver, to the barbed fitting on the vacuum pump. 4. Check label on vacuum pump to insure proper configuration for the available line voltage and frequency. Connect vacuum pump to a grounded three pronged recepta- cle. POST INSTALLATION CHECK. Following installation of the Stasar perform the fol- lowing operational checks: 1. Turn power switch, located on lower left cabinet, to the ON position, 2, Allow approximately 15 minutes warm- up. 3. Turn vacuum pump power switch to the ON position. SPECTROPHOTOMETER Vacuum Connections 23INSTALLATION INSTRUCTIONS. 4, Adjust vacuum and sample time to aspir- ate a volume which yields the best repro- ducability. Refer to Operating Instructions in Section 4 of this manual. 5, Referring to Section 6, Maintenance, per- form Calibration with the Model 1220 Cali- 2H bration Standard, Or if a 1405x1 Standardi- zation Kit is available, perform the 340 and 550 Absorbance Calibration, Stray Light, Bandpass, and Linearity Calibration proce- dures as described in the Maintenance Sec- tion of this manual.GENERAL This section contains a description of the basic operating principles of the Stasar. This information is directed mainly toward the skilled laboratory technician and clinical bi- ‘ochemist who needs to understand how the instrument operates. The description of con- trols points out the location and function of each control on the instrument. PRINCIPLES OF ABSORPTION SPECTRO- SCOPY The ability to absorb radiation is a com- mon property of matter. The amount and wavelength of absorbed radiation depends on the molecular electron configuration of the absorbing species. Radiant energy of a specific wavelength can excite an electron from a ground state to a higher energy level. When this occurs energy is absorbed, and decreases in the total energy transmitted can be measured, If the path length or the concentration of the medium being measured is increased, the absorbance will also increase proportionately due to the availability of more absorbing molecules in the light path. The mathematical basis of absorption spectroscopy is based on the Beer-Lambert Law. Lambert observed that the fractional part of the light energy, absorbed in the thin layer of material, is dependent on both the absorbing species and the frequency of the incident radiation and is proportional to its thickness at constant temperature and pressure. (Absorbance = absorptivity x path length x concentration) Beer demonstrated that the absorption coefficient (A) introduced by Lambert was Section 3 Principles of Operation also directly proportional to the concentra~ tion of the absorbing molecules in a solution as shown in the following equations: T= folo@be A = abc = -loglo/I In these equations, Ip is the intensity of incident radiation upon the first surface, I is the intensity after transmission through the thickness b; ¢ is the concentration of the absorbing material; and a is the absorptivity which depends on the frequency of the radi- ation as well as on the nature of the sub- stance. The absorbance, A, is more directly related to the concentration and absorptivi- ty then is Tor the ratio of I to Io Absorb- ance is directly proportional to the sample concentration. Therfore, absorbance is com- monly used in quantitive measurements. The percentage of light transmitted through the sample, Ig is known as the transmittance, T. Spectrofhotometers differ in the method sed for determining Ilg,, the ratio of the intensity transmitted by°the sample to the intensity that would be transmitted if the sample were not present. The Stasar Series obtains an output in absorbance units by passing the amplified detector signal of transmittance (T) through a log converter. The Stasar Series makes this conversion using a constant voltage supply. Using this method, sensitivity and high voltage controls are not necessary and a high degree of linearity is obtained. Most measurements of the absorption of radiation must be made with a radiation source that emits a continuous range of frequencies (directly related to wavelength) This is characteristic of most bodies that emit radiation as a result of high tempera- 34PRINCIPLES OF OPERATION ture, and it becomes necessary to separate the ‘desired frequency specific for the chromophore being determined. SPECTROPHOTOMETER COMPONENTS ‘Stasar Spectrophotometers contain the following components for making measure- ments of absorbance or concentration: 1. A visible light source to provide radiant energy. 2. A monochromator to disperse radiation and provide monochromatic light. 3. A sampling system, which may be a thermocuvette, rapid sampling cuvette, or a round cuvette. 4. A photometer linear in absorbance to measure radiant energy in terms of absorb- ance. 5. An output indicator to provide the opera- tor with a visual indication of sample ab- sorbance, concentration or temperature. Refer to Figure 3-1 for a block diagram of ‘the components. SAMPLE CONTAINER, "THERMOCUVETTE RAPID SAMPLING CUVETTE ‘OR ROUND. CUVETTE LIGHT SOURCE. The light source is a Tungsten lamp supplied by a fully transistor ized, highly regulated power supply. Instead of the conventional method of changing measuring beam intensity by varying slit widths, Stasar uses a fixed slit and varies lamp intensity (measuring beam intensity) when the operator adjusts the zero control. The power supply then maintains a constant lamp voltage within close tolerances. In addition to excellent stability, lamp life is increased, since the light source is not al- ways at maximum intensity. The lamp is mounted on an adjustable plate, affixed to ‘the monochromator, to simplify alignment, MONOCHROMATOR. Monochromatic light is derived from a precision diffraction grat- ing monochromator providing wavelength accuracy to +2.0nm. Refer to Figure 3-2. Use of @ grating monochromator together with a fixed exit slit results in a nominal nm spectral bandwidth throughout the wavelength range. The fixed slit also elimin- ates the need for a complicated slit mech~ anism. With a fixed slit and a variable light source, measuring beam intensity is a func- tion only of light source intensity. runcsren LIGHT HOF a source MONOCHROMATOR < (Or- SAMPLING S¥STEM Leo PHOTOMETER INDICATOR oh ZERO CONTROL Figure 3-1. Stasar Block Diagram 32 TO DATA LISTERLane DIFFRACTION: ae GRATING PHOTOMULTIPLIER, a’ MIRROR ReFLecring PRINCIPLES OF OPERATION BARFLE BAFFLE SPHERICAL MIRROR exit REFLECTING cuverte SUIT MIRROR. sean Figure 3-2. Monochromator Optical Path The color of light passed through the fixed slit depends on the physical setting of the diffraction grating. The grating position {and thus the wavelength) is controlled by a micrometer screw adjustment mechanism directly ‘coupled to a digital counter. A positive stop mechanism at 311 nm and 726 Am prevents damage from out of range adjustments. The mechanism is designed to automatically insert a blue stray light filter in the light beam at wavelengths below 4Oonm. At wavelengths above 400nm, the mechanism automatically inserts 2 magenta beam attenuation filter which acts as a stray light filter above 600nm. SAMPLING SYSTEM. The rapid sampling system in the Stasar Il and II consists of the functional elements shown in Figure 3-3, plus a vacuum source. Vacuum is obtained from the vacuum pump and is applied through the vacuum receivers, the pinch valve, and the cuvette assembly. The pinch valve is electrically activated by a voltage from the sample timing network. — This voltage is applied to the valve only during the sample or purge cycle. The sample cycle is determined by the setting of the sample time control. The purge cycle continues as Jong as the sample actuator bar is held in ‘the purge position. Otherwise no voltage is applied to the pinch valve and it remains closed, keeping vacuum out of the rapid sampling cuvette. The Stasar IM rapid sampling thermocuvette contains Peltier effect thermoelectric devices to provide heating and cooling of the sample. The 33PRINCIPLES OF OPERATION ‘THERMOCUVETTE SAMPLE ACTUATOR BAR SAMPLE _—= TIME CONTROL SAMPLE TIMING NETWORK ‘To vacuum ‘SOURCE Ss PINCH VALVE Figure 3-3. Rapid Sampling System cuvette path length is 10 mm, and samples of 700 microliters are sufficient to obtain accurate readings of absorbance or concen- tration. Round Cuvette Holder. The round cuvette holder is designed to allow insertion of a 12 mm diameter by 75 mm long round cuvette. As shown in Figure 3-4, one round cuvette containing a sample can be inserted in the holder. The spring clip on the side of the cuvette holder retains the cuvette firmly in place. A cover fits over the cuvette holder to prevent room light from entering the sample during absorbance or concentration measurements. PHOTOMETER. The photometer features a Tog converter which compares the output current produced by the photomultiplier 34 tube to a reference current which is equal to zero absorbance (maximum light). The log converter then produces an output voltage directly proportional to absorbance and transmits this to the output indicator. Since the voltage supply is constant, no sensitivity or high voltage controls are required, and because the voltage output is linear to absorbance, linearity resolution is constant ‘throughout the 2.000A range of the Stasar. The feature of direct linearity in absorbance permits immediate and direct output readings in concentration units. OUTPUT INDICATOR. The output indicator is a four-place LED readout with a movable decimal point in concentration and acces- sory modes. The decimal point is fixed toBe aS aS eee eS coven ROUND CUVETTE SPRING: Figure 3-4. Round Cuvette Holder PRINCIPLES OF OPERATION the right of the most significant digit in the absorbance mode. The output indicator pro- vides a resolution of 0.001A throughout the 0.000A to 2.000A range of the instrument. NOTE The limit of concentration is from 1/3 to 3 times the actual absorbance of the sample; since 2,000A is the maximum linear absorbance, the upper useful limit of concentration is 6000 (decimal is movable). In addition to the LED readout, Stasar Provides a printer output for use with printers or computers. 35GENERAL, This section describes the controls and operating procedures for Stasar Il and Ml. Following Stasar Operation is a description of operation without a recorder. TEMP. SET ADJUSTMENT TEMPERATURE CALIBRATION, STANDARD ADJUST PUSH TO READ TEMPERATURE PUSHBUTTON WAVELENGTH CONTROL” WAVELENGTH, INDICATOR ON-OFF POWER: CONTROL SAMPLE, ACTUATOR BAR Section 4 Operating Instructions CONTROLS AND INDICATORS. Refer to Figure 41, for location of the following controls and indicators: The ON-OFF rocker switch, shown in Figure 4-2, control the main power to the spectrophotometer. SAMPLE TIMER CONTROL CON. CAL (CONCENTRATION CALIBRATION) os CONTROL CONTROL DECIMAL, CONTROL, DISPLAY PUSHBUTTON ABSORBANCE ABSORBANCE CONCENTRATION TEMPERATURE INDICATOR = ‘CONCENTRATION 21a INDICATOR vaLvE LAMP ACTUATION INDICATOR Figure 41. Stasar Controls odOPERATING INSTRUCTIONS POWER SWITCH Figure 4-2. Stasar Power Switch The WAVELENGTH control adjusts the wavelength within the useable range of 335 to 700nm. The WAVELENGTH indicator is mechanically coupled to the wavelength control and gives a direct readout of the operating wavelength. The ABS-CAL screwdriver adjustment permits absorbance calibration of the Stasar using the dual calibration standard or an alternative standard. This control is only used during instrument calibration. The PUSH-TO-READ TEMP button dis- plays the sample temperature on the instru- ment display. The DUAL CALIBRATION STANDARD can be used for both wavelength and absorb- ance calibration. ‘The TEMPERATURE control (Stasar IIl) prgvides, for g choice of four temperatures: 25°, 30°, 37°C and a variable TEMP SET position which allows cuvette temperature to be adjusted over a range of 25° to 37°C. The TEMP SET potentiometer on the control panel is accessible for use when the control panel is open. : NOTE Although the TEMP SET control settings allow cuvette tempera- ture adjustment over the entire range of 15°C to 50°C, the guar- anteed accuracy limits fall with in 25°C to 37°C. Temperature adjustment outside of the accur- acy limits is not recommended. a2 The SAMPLE TIMER control is a ten posi- tion switch which permits selection of sampling time. Sampling time together with sample viscosity, specific gravity, and a- mount of vacuum, determines aspirated sample volume. During sampling, vacuum is applied to the rapid sampling cuvette for the period of time selected by the SAMPLE TIME control. Sample time can be varied between approximately 0.1 second and 1.0 second, The CON. CAL. (concentration calibration) control permits adjustment of the instrument readout in the concentration mode to correspond with the concentration of the known samples. Subsequent measurements of unknown samples are displayed directly and accurately in concentration units. The CON-ABS-ACC control permits selec- tion of absorbance, concentration, or acces- sory modes of operation. In the ACC posi- tion, analog data applied to the J1 input can be processed by the analog-to-digital con- verter on the spectrophotometer and pre- sented on the LED readout. One of the two front panel lamps, marked ABS and CON, indicates whether the absorbance or concen- tration mode is activated. Both lamps ex- tinguish when ACC mode is selected, The DECIMAL POINT contro! permits manual placement of the decimal point in the digital indicator to the left of any of the four digits. This switch functions only when the CON-ABS-ACC control is placed to CON or ACC positions. The DISPLAY HOLD pushbutton retains the readout at its present value. ‘The ABSORBANCE/CONCENTRATION/- TEMPERATURE indicator consists of a four-place LED readout fixed decimal point in absorbance mode and movable deci- mal point in concentration and accessory modes. When the PUSH-TO-READ TEMP pushbutton is depressed, the temperature is displayed in the indicator window. The deci- mal point is fixed at two places from the left. The ZERO control permits adjustment of a reference blank to 0,000 before making measurements. The ABS indicator illuminates when the CON-ABS-ACC control is placed to ABS. The CON indicator illuminates when CON-ABS-ACC control is placed in the CON position. The valve actuation indicator illu- minates when the pinch valve is actuated and illuminates during sample or purge func- tion. The sample actuator bar, shown in Figure 4-3, has two operating actions; sample and purge. In the sample position, a timer is activated which opens the pinch valve for a precisely controlled period of time, drawing an accurately metered amount of sample into the cuvette. In the purge position, the timer is bypassed to keep the vacuum con- trol valve open continuously, allowing the system to be purged with air between samples. SAMPLE t FIRST STOP PURGE END STOP Figure 4-3. Sample Actuator Bar Operating Positions Initial movement of the sample actuator bar to the sample positon is physically limit- ed by a stop, to prevent unintentional ac| vation of the purge cycle. This stop, how- ever, is spring controlled so that additional operating force can activate the purge cycle. The valve actuation indicator on the front panel is useful in monitoring the oper- ation of the sample actuator bar; in the sample position, the indicator illuminates for a period of time determined by the setting of the sample time control; in the purge position, the valve actuation indicator illuminates until the sample actuator bar is released. The vacuum regulator on the vacuum receiver, shown in Figure 4-4, is shipped with Stasar II and III and permits adjustment of the amount of vacuum applied to the rapid sampling system and thus has a part in determining intake sample volume. OPERATING INSTRUCTIONS vacuUM GAUGE vacuum REGULATOR Figure 4-4. Vacuum Adjustment OPERATING PROCEDURES STASAR Tl_AND Ill. The following contains steps necessary for operation of the Stasar utilizing a rapid sampling cuvette to meas- ure absorbance and concentration. Also in- cluded is the procedure for measuring tem- perature in the thermocuvette. PRELIMINARY OPERATION. Perform the following procedure when initializing the instrument or when changing the viscosity of samples. When the instrument remains idle for a period of time, adjust the absorb- ance control for a reading of approximately 3.000A, this will reduce the voltage to the tungsten lamp, thus extending its life. 1, Turn instrument power on, allow approxi- mately 15 minutes warm-up. 2. Turn vacuum pump switch to the ON position. 3. Adjust the vacuum and sample time to aspirate a volume which yields the best reproducibility. When uncertain of the amount of vacuum and sample time, begin with a vacuum of 10 to 12 inches Hg and a sample time setting of 3 or 4. ‘ 3OPERATING INSTRUCTIONS, NOTE The sample timer, shown in Fig- ure 41, controls the amount of time the aspiration valve re- mains open. The vacuum control gauge on the reciever, shown in Figure 4-4, controls the speed at which the sample is pulled through the cuvette. When changing the sample volumes, adjust both the timing and vacu- um controls. If either adjustment is set too high the sample may purge completely through the system; if either adjustment is set too low sample may not be aspirated into the cuvette. The following are examples of vacuum and sample time adjustments. a. For aqueous samples, adjust the vacuum to 10-12 inches Hg, and set the sample time control for an intake of 0.7 ml from a graduated test tube. b. For highly viscous samples (such as PMS sugar, with 26 centipoises), adjust the vacuum to a higher level, up to 18 inches Hg. Set the sample time control for maximum sample intake. NOTE To obtain accurate measure- ments of absorbance and concen- tration when using sample test tubes containing as little as 500 ul (Stasar II) or 700 ul (Stasar 1D, sample inlet tubing on rapid sampling cuvette must be cut to 18 cm (7.1 inches) and 22 cm (8.6 inches) respectively. Insure that approximately 3 to & inches of inlet tubing extends downward past the cuvette handle, thus permitting test tubes up to 12x75 mm in size to be used. The rapid sampling cuvette is fur- nished with enough inlet tubing for initial installation and re- placement spares. When test tubes in excess of 12x75 mm are used, longer sample inlet tubing and slightly higher sample vol- umes may be required. MEASURING ABSORBANCE. After com- pleting preliminary operation, measure ab- sorbance as follows: 1. Position the CON-ABS-ACC control switch, shown in Figure 4-1, to ABS. 2. Select the desired wavelength by turning the wavelength contro! knob while monitor- ing the wavelength indicator. 3. Fully depress the sample actuator bar for at least 5 seconds to purge the system with air. 4, Aspirate a reference sample by depress- ing the sample actuator bar to the first position. Refer to Figure 4-5. Figure 4-5. Aspirating A Sample NOTE Insert the tip of the sample inlet tubing well below the surface of the liquid without touching the bottom of the container. 5. Adjust the zero control knob, shown in Figure 4-1, for a displayed reading of 0.000.OPERATING INSTRUCTIONS, NOTE The sample timer, shown in Fig- ure 41, controls the amount of time the aspiration valve re- mains open. The vacuum control gauge on the reciever, shown in Figure 4-4, controls the speed at which the sample is pulled through the cuvette. When changing the sample volumes, adjust both the timing and vacu- um controls. If either adjustment is set too high the sample may purge completely through the system; if either adjustment is set too low sample may not be aspirated into the cuvette. The following are examples of vacuum and sample time adjustments. a. For aqueous samples, adjust the vacuum to 10-12 inches Hg, and set the sample time control for an intake of 0.7 ml from a graduated test tube. b. For highly viscous samples (such as PMS sugar, with 26 centipoises), adjust the vacuum to a higher level, up to 18 inches Hg. Set the sample time control for maximum sample intake. NOTE, To obtain accurate measure- ments of absorbance and concen- tration when using sample test tubes containing as little as 500 ul (Stasar Ill) or 700 ul (Stasar 1D, sample inlet tubing on rapid sampling cuvette must be cut to 18 cm (7.1 inches) and 22 cm (8.6 inches) respectively. Insure that approximately 3 to 4 inches of inlet tubing extends downward past the cuvette handle, thus permitting test tubes up to 12x75 mm in size to be used. The rapid sampling cuvette is fur- nished with enough inlet tubing for initial installation and re- placement spares. When test tubes in excess of 12x75 mm are used, longer sample inlet tubing and ‘slightly higher sample vol- umes may be required. MEASURING ABSORBANCE. After com- pleting preliminary operation, measure ab- sorbance as follows: 1. Position the CON-ABS-ACC control switch, shown in Figure 4-1, to ABS. 2. Select the desired wavelength by turning the wavelength control knob while monitor- ing the wavelength indicator. 3. Fully depress the sample actuator bar for at least 5 seconds to purge the system with air. 4, Aspirate a reference sample by depress- ing the sample actuator bar to the first position. Refer to Figure 4-5. Figure 4-5. Aspirating A Sample NOTE Insert the tip of the sample inlet tubing well below the surface of the liquid without touching the bottom of the container. 5. Adjust the zero control knob, shown in Figure 4-1, for a displayed reading of 0.000. ee6. Fully depress the sample actuator bar for at least 5 seconds to purge the system with air. 7. Aspirate the sample and read the absorb- ance. Insure sample inlet tubing is well below the surface of the sample material without touching the bottom of the contain- er when aspirating. NOTE, Erratic readings may result from an insufficient sample intake. 8. Fully depress the sample actuator bar for at least 5 seconds to purge the system with ai 9. Repeat Steps 7 and 8 for subsequent measurements of like samples. Always in- troduce the reference blank and zero the instrument after changing wavelengths or reagents. 10. Perform the End-of-Run Cleaning pro- cedure described in Section 6 of this man- ual. MEASURING CONCENTRATION. After completing the Preliminary Operation pro- cedure, measure concentration as follows: 1. Position the CON-ABS-ACC control switch, shown in Figure 4-1, to CON. 2, Select the desired wavelength by turning the wavelength control knob, shown in Fig- lure 4-1, while monitoring the wavelength indicator. 3. Fully depress the sample actuator bar for at least 5 seconds to purge the system with air. 4, Aspirate a reference sample by depress- ing the sample actuator bar to the first position. Refer to Figure 4-5. NOTE Insert the tip of the sample inlet tubing well below the surface of the liquid without touching the bottom of the container. OPERATING INSTRUCTIONS 5. Adjust the zero control knob, shown in Figure 4-1, for a displayed reading of 0.000. 6. Fully depress the sample actuator bar for at least 5 seconds to purge the system with air. 7. Aspirate the sample with a known con- centration of the material of interest. In- sure sample inlet tubing is well below the surface of the sample material without touching the bottom of the container when aspirating. NOTE Erratic readings may result from an insufficient sample intake. 8. Adjust the CON, CAL. control, shown in Figure 4-1, for a digital readout correspond- ing to the known concentration. For exam- ple, if the concentration of a known sample is 20gm/100ml, adjust. the concentration calibration control for 2000 or 0200. 9. Position the decimal point control to indicate the desired units, for example, 20.00 or 020.0. 10. Fully depress the sample actuator bar for at least 5 seconds to purge the system with air. 11. Aspirate a sample and read the concen- tration. The concentration of the sample will be displayed directly in grams/100ml. NOTE When questioning whether the sample absorbance exceeds the linear 2.000A range of the in- strument, position the CON- ‘ABS-ACC control to the ABS po- sition. If the absorbance _ is greater than 2.000A, dilute the sample with the appropriate so- lution and apply the factor to the result. 12. Fully depress the sample actuator bar for at least 5 seconds to purge the system with air. +5OPERATING INSTRUCTIONS, 13. Repeat Steps 11 and 12 for subsequent measurements of like samples. Always in- troduce the reference blank and zero the instrument after changing wavelengths or reagents. 14, Perform the End-of-Run Cleaning proce- dure described in Section 6 of this manual. TEMPERATURE VERIFICATION (Stasar III a 1, Set the TEMPERATURE control switch, shown in Figure 4-1, to the desired tempera- ture setting. (25°C, 30°C, 37°C or TEMP SET for variable temperature settings) 2. Aspirate sample into the cuvette, refer- ring to Measuring Absorbance or Measuring ‘Concentration procedures. 3. Depress the PUSH-TO-READ TEMPERA- TURE pushbutton and monitor the display until the sample equilibrates at the selected temperature. Refer to Figure 41. 4, Read the temperature of the cuvette on the instrument display. SETTING TEMPERATURE (Stasar III only). To select a temperature other than the preset 25°, 30°, and 37°C, proceed as fol- lows: 1. Rotate the TEMPERATURE control switch to the extreme counterclockwise position. 2. Depress the PUSH-TO-READ_ TEMP switch while adjusting the TEMP SET con- trol, shown in Figure 4-1, with a small screwdriver until the digital readout indica- tes the desired cuvette temperature. Re- lease the switch. NOTE Although the TEMP SET control allows cuvette temperature ad- justment over the entire range of 15° to 50°C, the guarantees accuracy limits’ fall within 25 to 37°C. Temperature adjus ment outside the accuracy limits is not recommended. 3. Aspirate sample into the cuvette and allow approximately 20 seconds for the tem- perature to stabilize. Press the PUSH-TO- READ TEMP switch, then adjust the TEMP SET control for the desired cuvette temper- ature. Adjustment should be made in small increments to avoid temperature overshoot or undershoot. ROUND CUVETTE HOLDER OR CUVETTE ACCESSORY. This procedure describes the ‘Steps necessary to measure absorbance and concentration utilizing a round cuvette holder. When the instrument is to remain idle for a period of time, adjust the absorb- ance control knob for a digital readout of approximately 3.000A, this will reduce the voltage to the tungsten lamp, thus increas- ing its life. PRELIMINARY OPERATION. Turn instru- ment power on, allow approximately 15 min- utes warm-up. MEASURING ABSORBANCE. Following in- ‘Strument warm-up, refer to Figure 4-1, and measure absorbance as follows: 1. Position the CON-ABS-ACC control to ABS. 2, Select the desired wavelength by turning ‘the wavelength knob while monitoring the wavelength indicator. 3. Remove the cover from the round cuvette holder, see Figure 4-6, and insert a cuvette containing the reference blank. Replace the cover. 4, Adjust the zero control knob for a digital readout of 0.000. 5. Remove the cover from the cuvette hold- er and lift the cuvette containing the refer- ence from the holder. 6, Insert the cuvette containing the sample to be measured and replace the cover. Read the absorbance on the digital readout. 7. Remove the cover from the holder and remove the cuvette containing the sample.