Direct Detect User Guide - The University of Sydney
Contents
1. instrument module is convectively dried with a desiccant enclosed in a small removable multichambered pack If the desiccant is saturated and can no longer absorb humidity it must be replaced Both the hardware diagnostics icon Figure 23 and instrument status light on top of the instrument 5E in Figure 5 will turn yellow When you click on the hardware diagnostics icon Figure 24 the hardware diagnostics dialog appears and displays the message Direct Detect Spectrometer User Guide 12 2 Replacing the Desiccant continued Instrument internal humidity out of range Figure 24 M Millipore Direct Detect Fie Options Help Hardware diagnostics icon Last Sample Results Overall Status Message Instrument internal humidity out of range ua Jia Figure 24 Hardware Diagnostics interferometer warning Measuring Is still possible if the status light is yellow but the desiccant should be replaced as soon as possible Replace the desiccant as follows 1 2 Unplug the power plug The desiccant is accessible from the rear side of the spectrometer Figure 25 Remove the cover plate from the rear side by loosening the 4 Torx screws Figure 19 with the Torx screw driver supplied Take off the cover plate Direct Detect Spectrometer User Guide 27 12 2 Replacing the Desiccant continued Desiccant pack Figure 25 Replacing the d2. Direct Detect automated card holder Card insertion alignment arrow Status light Sample drying indicator light Card insertion light 7 1 2 The Direct Detect Automated Card Holder The automated card holder 5C accepts only the 4 sample Direct Detect Assay free cards The top of the card holder module contains two LED lights located at the bottom right corner 5F and 5G as viewed from the top The bottom LED light 5G is green whenever an Assay free card is inserted properly The top LED light 5F is yellow whenever the drying system heater and fan are active Periodically default setting is 15 minutes the software will initiate a reference measure ment which must be performed without a sample card inserted in the card holder If a card is found in the card holder the software will prompt you to remove it 8 Direct Detect Spectrometer User Guide 7 1 Direct Detect Spectrometer General Technical Features continued 7 1 3 Connecting Ports and LEDs The connecting ports e g power Ethernet and the system diagnostic LEDs are on the back of the instrument Figure 6 USB se TKD ARC ERR RES p EXT ACCESSORY TAP CFG RES POWER RA c 9 als ma of 4 sg rs Figure 6 Direct Detect Spectrometer rear side Connecting ports and LEDs Type Definition USB port Not available to user For instrument service use only 6C 6D 6E 6F 6G ETH LAN port The ETH LAN port
3. User Guide Direct Detect Spectrometer Notice The information in this document is subject to change without notice and should not be construed as a commitment by EMD Millipore Corporation Millipore or an affiliate Neither EMD Millipore Corporation nor any of its affiliates assumes responsibility for any errors that may appear in this document The M logo and Direct Detect are trademarks of Merck KGaA Darmstadt Germany Millipore and Amicon are registered trademarks of Merck KGaA Intel and Pentium are registered trademarks of Intel Corp Windows is a registered trademark of Microsoft Corp in the United States and or other countries Dell is a registered trademark of Dell Inc Torx is a registered trademark of Acument Intellectual Properties LLC Tween is a registered trademark of ICI Americas Inc 2012 EMD Millipore Corporation Billerica MA USA All rights reserved 00000833 Rev B 03 12 Contents Tove eten 1 Z Components SUD DIGG savannen 2 3 Symbols Used in this User Gr 3 4 Product LAG ING se sencecececeettentonteaeccecoecenuceonnteomeunthavaedcaneuesesaniaenndctdoesanstaussecteedeenseanseene 3 B STN Procul OMS ooren iea eE EEG ESEE NEEN 4 G INSTAL ATION ccssteeteeeeeeeeeeessesnnnnneeeeseeeeeeseseesssennnneeoooooeeeesssesessaasnnnesooeooeseesssssessneeneneags 4 NN 4 AEE 140015 1 EEE 5 Ooa ENN 6 6 4 Unpacking and Seting Up the Computer EE EN 6 65 Unpacking and Setting Up the Direct Detect Spectromete
4. 3 When the instrument validation dialog appears click on the Performance Qualification PQ Run Test button 1 Figure 28 The test takes about 5 minutes s M Millipore Direct Detect gt lt Fie Options Help User Name RHB Sample Name Volume pL z i a mes E Instrument validation aes re con and 0Q PQ Toei sm fe Fr Initiator Protein Peak 2 Instrument Yalidation x off Performance Qualification PQ This test compares the performance of the instrument on day to day basis Failure of this test can be due to degradation in the source or other component SSE Test expires in 0 days 71 Hours 43 Minutes fo CH Last Report gt Run Test Last Sample Results Card History off Operational Qualification 007 This test measures the operational performance of the instrument Failure of this test may indicate instrument misalignment or Failure Test expires in 365 days 23 Hours 59 Minutes Last Report gt Run Test 2 MOTE The instrument will not be able to perform sample measurement if either test has Failed Figure 28 Running OQ PO tests 4 To run the Operational Qualification OQ test click on the Operational Qualification Run Test Button 2 The test takes about a minute to run For information on interpreting test failures refer to section 11 4 12 6 Storage Store the Direct Detect spectrometer and Assay free cards in a clean dry environment
5. Carbohydrates Lipids Nucleic Acids Full Spectrum Last Sample Results Card History Spectrum Plot Curves Plot Statistics Figure 17 Direct Detect Software user interface Direct Detect Spectrometer User Guide 19 11 1 General Information continued Click on the hardware diagnostics icon and the hardware diagnostics dialog opens Figure 18 Hardware Diagnostics Overall Status Message End of lifetime is nearly reached ua JL a Figure 18 Hardware diagnostics dialog Instrument Status More information on hardware diagnostics Is available in section 5 1 of the Direct Detect Software User Guide If an issue cannot be resolved with the help of this diagnostics section and or the following troubleshooting tables refer to section 15 for information on how to contact Technical Service 11 2 Problems Indicated by Hardware Diagnostics Icon Symptom Cause Corrective Action Hardware diagnostics Spectrometer is still in the Wait until spectrometer initialization is completed icon is yellow initialization phase several minutes i i IR source has not yet The warm up takes about 7 minutes As soon as the a reached its operating IR source has reached its operating temperature the temperature hardware diagnostics icon will automatically turn green Wait until the warm up Is completed IR source nearing end of To confirm IR source issue click on the hardware operational lifetime diagnostics icon Figure 17 to o
6. OPUS DB Version OPUS 7 0 Build 7 0 122 DB 7 0 105 1106 for OPUS 7 0 Overall Test Result PASSED Test expires 12 25 2011 3 12 41 PM GMT 5 Test Date Time 12 15 2011 3 12 41 PM GMT 5 Test Spectra Path C Direct Detect V alidation Data 201 11215 151241 Date of last PQ Reference Measurement Comment ee Signaito Noise Test n Minimum Signal to Noise Limit area 1 1000 Signal to Noise measured 3458 KE Maximum 100 Line Deviation 2 0 Measured 100 Line Deviation 0 29 Wavenumber Accuracy Test Polystyrene 35 5 Deg C Peak is T corr Sample Material Polystyrene Specified Peak 1601 45 cm 1 Maximum Deviation 0 50 cm 1 Measured Peak 1601 13 cm 1 Corrected Peak 1601 25 cm 1 Measured Deviation 0 20 cm 1 Overall Test Result PASSED yf Figure 26 PQ test report 12 4 2 Operational Qualification Test OQ The Operational Qualification test checks instrument performance and compares this performance to the instrument specifications The OQ test is performed during final testing before an instrument is delivered to the customer It should also be performed after each major repair after replacement of optical components which may influence the instrument performance and after maintenance has taken place The factory default interval for the OQ test is 12 months Twelve months from the last OO test the software will alert you to run the instrument OQ test again You can use the instrument with an expired OQ
7. Spectrometer Remove all components from shipping boxes and lay out on work surface 6 5 1 Positioning and Connecting Components Figure 3 1 Place instrument on bench and put Netbook stand on top of instrument The front tab of the stand fits into the slot on top of the instrument 5B in Figure 5 2 Place Netbook computer on stand Figure 3 Rear side Connection ports 3 Turn the instrument computer assembly so that the rear of the instrument is facing you Figure 3 4 Connect one end of the ethernet cable to the instrument ETH LAN port 3A and the other end to the PC network LAN port 3B Direct Detect Spectrometer User Guide 6 5 Unpacking and Setting Up the Direct Detect Spectrometer cont 5 Connect the low voltage round end of the instrument power adapter to the power port of the instrument 3C Secure the connector with the clamp 6 Choose the instrument power cord with the correct plug configuration for local requirements and plug it into the power adapter 7 Plug both the PC and instrument power cords into a grounded power outlet A WARNING To avoid personal injuries and or property damage caused by electrical power connect the spectrometer and computer to a grounded power outlet Make sure that this power outlet complies with IEC International Electrotechnical Commission regulations 8 Turn on the PC NOTE The instrument is powered through the computer and does not have a
8. a measurement has been aborted the progress bar will display Measurement Aborted After a short period of time the instrument will return to Measure Card status 18 Direct Detect Spectrometer User Guide 11 Troubleshooting 11 1 General Information The status of the Direct Detect spectrometer is indicated by both the instrument status light located on the top right side of the instrument under the Netbook tray Figure 5 and the hardware diagnostics icon located in the upper right corner of the Direct Detect Software user interface Figure 17 The status light and hardware diagnostics icon colors are typically the same at any given time and indicate the following Green Instrument is in proper operating condition flashing green on the instrument indicates stand by mode Yellow Instrument is initializing or indicating a warning e g IR source has not yet reached its operating temperature the validity period of the instrument has expired the air humidity inside the spectrometer is too high Red Instrument has malfunctioned or has a defective component or an OQ PO test has expired or failed p h M Millipore Direct Detect x File Options Help User Name RHB ae ve Instrument validation icon Ne Gla and OQ PQ initiator Stend vm F Hardware diagnostics icon i ETTE n 11 16 18 AM en Average Conc 1 985 Std Deviation 0 05 CV 25 Protein
9. connects the spectrometer to the Netbook computer using the supplied data cable cross over 10Base T with RJ45 ports TKD LED The green TKD LED indicates that the interferometer mirror Is within the data acquisition range When data acquisition Is occurring the light is bright green The abbreviation TKD means Take Data ACC LED The yellow ACC LED indicates that the network is accessing the optics During transition of measuring data the LED is light yellow The abbreviation ACC means Access DIR ERR LED In normal operation mode the DIR ERR LED flashes pale red and indicates scanner movement If the LED lights permanently bright red there is an interferometer problem e g missing laser signal When this LED is permanently red no data acquisition is possible The abbreviation DIR means Direction and ERR means Error External Not available to user For instrument service use only accessory port TAP port Not available to user For instrument service use only Direct Detect Spectrometer User Guide 9 7 1 Direct Detect Spectrometer General Technical Features continued Type Definition 6H CFG switch CFG is a four fold DIP switch 1 ON and 2 OFF DHCP mode use last DHCP address 1 OFF and 2 ON use ON IP address 10 10 0 1 3 stay in boot loader do not load the application program 4 reserved The abbreviation CFG means Configuration SBY RES button Pressing this button for app
10. separate ON OFF switch It should be left on and powered up at all times 6 5 2 Initializing the Direct Detect Spectrometer 1 Refer to the Direct Detect Software User Guide for instructions on starting up the software and logging in section 1 1 2 During initialization the laser IR source and detector are switched on The IR source warms up the scanner moves forward and backward several times and tones once This phase takes several minutes and hardware diagnostics icon Figure 4 is yellow After the initialization has been completed successfully the hardware diagnostics icon turns to green but the instrument validation icon will be yellow This indicates that the PQ test has expired and needs to be run Refer to section 12 5 for instructions on running the PQ test Figure 4 Hardware diagnostics icon NOTE If the status indicator on either the instrument or the software is red or remains yellow after initialization is complete and the PQ test has been run refer to section 11 Troubleshooting Direct Detect Spectrometer User Guide 7 7 Direct Detect Spectrometer Parts and Functions 7 1 General Technical Features 7 1 1 The Instrument The Direct Detect spectrometer is a small compact FIIR spectrometer designed for measuring IR absorbance 5A 5B 5C 5D Figure 5 Direct Detect spectrometer Definition Instrument module with integrated electronics and optics Slot for Netbook stand tab
11. test but the software will query you each time a measurement Is initiated asking if you wish to proceed under the expired PQ condition The PQ test protocol consists of the following three tests amp Signal to noise test The signal to noise ratio determines the sensitivity of the spectrometer by calculating the average signal to noise ratio of ten 100 spectra A 100 spectrum is the ratio of two successively acquired single channel Spectra with no sample in the sample compartment The ratio of these two single channel spectra is used to generate a transmission spectrum 100 Line Test This test verifies that the 100 line does not shift over time lt measures the maximum deviation of a 100 line within a larger frequency range The average of ten measurements must not exceed the predefined limit Wavenumber Accuracy Test This test ensures that the frequency calibration of the instrument is within the defined limits using an internal polystyrene Standard sample Direct Detect Spectrometer User Guide 29 12 4 Description of Instrument Validation Tests continued Figure 26 shows a successful PQ test report OVP PQ Test Protocol Company MILLIPORE Operator Default Instrument Type Alpha Sample Compartment RT DLaTGS ZnSe Optics Configuration Fibre 1 with MIR KBr RT DLATGS Internal Pos 1 Accessory Millipore Direct Detect Instrument Serial Number M3 Instrument Firmw are Version 1 320 MDD 01 Oct 27 2011
12. the relevant safety precautions Failure to do so may cause property damage or personal injury Maintenance or repair procedures not described in this manual should be performed only by an EMD Millipore Corporation service engineer Refer to the Technical Assistance section for information on contacting Tech Service 12 1 Replacing the IR Source 12 1 1 IR Source Status Indicator The Direct Detect spectrometer Is equipped with a highly efficient low power air cooled infrared source with an average life span of 44 000 hours As the end of this life span Is approached the hardware diagnostics icon turns yellow When you click on the hardware diagnostics icon Figure 17 the hardware diagnostics dialog appears and displays the message End of lifetime is nearly reached Figure 18 Samples can still be measured but performance and reliability may be compromised and the IR source cat no DDSP00010 LA should be replaced as soon as possible If the source Is defective the hardware diagnostics icon turns red When you click on this icon the hardware diagnostics dialog appears and displays the message No source is connected Samples can no longer be measured and the IR source must be replaced 12 1 2 Replacing the IR Source AN WARNING During spectrometer operation the light source becomes very hot To avoid burns wait until the light source has cooled down before replacing it 1 Unplug the instrument power plug 2 On
13. Figure 11 Inserting sample card The arrow on the bottom right side of the card should be aligned with the arrow on the Direct Detect card holder module 5D in Figure 5 Gently release the card and the instrument will accept it for analysis When the card is inserted properly the instrument will respond by jogging the card holder up and down briefly The bottom green LED on Direct Detect card holder module will light and the system will produce a brief tone confirming correct placement of the Assay free card NOTE The software will not allow sample measurement if the card is not inserted properly Direct Detect Spectrometer User Guide 15 10 3 Measurement For detailed instructions on setting up a sample measurement refer to section 2 2 of the Direct Detect Software User Guide 10 3 1 General Measurement Procedure 1 Complete the sample measurement setup Figure 12 Check the Dry sample card box to allow the instrument to dry the sample User Name Card Name Samplel Method 654 Standards Therma 0 Cards Generated on 7 135 Curve Generated on 7 13 Sample Name vol L Cc me sample 1 Use last blank blank 1 Dry sample card Figure 12 Sample information NOTE The Direct Detect system comes with three preloaded calibration methods These pre existing methods may be used for all future measurements however we recommend preparation of fresh calibration method upon instrumen
14. Iris buffer concentration Add 450 uL of 1X PBS or exchange buffer a ty N 50 ul of ss 1 mg mL protein in PBS with 9 5 pin to pin to concentrate A gt r ey ee 1 mg mL protein in 500 pL of 500 uL of Vif A maim 50 mM Tris cn la mM Tris 50 mM Tris 50 mM PBS with 9 5 PBS with Tris 7 mM Tris 9 5 mM Tris Figure 8 Buffer exchange and concentration using an Amicon Ultra 0 5 device Direct Detect Spectrometer User Guide 13 10 How to Use the Direct Detect Spectrometer The instrument is controlled and operated by Direct Detect software installed on the Netbook computer Samples are introduced to the instrument module by inserting the Direct Detect Assay free card into the card holder Figure 11 An alignment notch on one corner of the card ensures correct card insertion into the card holder 10 1 Accessing Direct Detect Software The Direct Detect software can be accessed through the icon on the Notebook desktop Figure 9 a Direct Detect Figure 9 Direct Detect software desktop icon Each instrument user can be assigned a unique name and optional password Figure 10 The measurements performed by each user are stored and recalled based upon the user name M Direct Detect Login x UserName Administrator Administrator v Password TET Figure 10 Direct Detect login screen For detailed instructions on starting up the software refer to
15. an measure protein concentrations from 0 1 mg mL to around 30 mg mL the most accurate results are achieved within the recommended concentration range Samples to be analyzed are introduced to the system using the Assay free card which contains a novel IR transparent membrane The recommended sample volume is 2 uL per spot position 8 1 Buffer components The Direct Detect system is compatible with most commonly used buffers detergents and reducing agents however use of buffers which contain amides or amine functional groups may affect quantification Examples of buffers and respective concentrations which interfere with the spectral region used for protein quantification are listed in the table below Interfering Buffer Concentration mM Bicarbonate divalent EDTA Guanidine hydrochloride Guanidine isothyocyanate HEPES buffer pH 7 5 Sodium bicarbonate Sodium hydroxide Tris Urea Buffers containing more than 5 glycerol SDS and or Tween 20 surfactant should be avoided due to the excessive time required to achieve dryness Although the instrument comes with pre loaded calibration files fresh calibration with the buffer used for protein preparation is recommended 8 2 Materials Required but Not Supplied Protein standards Bovine Serum Albumin National Institute of Standards amp Technology SRM 927d optional amp Protein sample s Fixed volume precision pipette capable of precisely dispensing 2 ul a
16. at 15 30 C 32 Direct Detect Spectrometer User Guide 13 Specifications General Dimensions Weight approximate Power consumption Electronics Environmental conditions Safety regulations EMI regulations Performance Spectral range Resolution Wave number accuracy Wave number reproducibility Optics Design IR Source Computer Specifications Operating System Software Framework Screen Resolution Ethernet Cable Processor Random Access Memory RAM Graphics Card 22 x 30 x 25 cm 8 7 x 11 8 x 9 8 in width x depth x height 7 kg 15 4 lbs Maximal 100 240 VAC 50 60 Hz max 2 A Class 1 laser product contains 850 nm laser diode Operational temperature range 18 C 35 C 64 95 F Temperature variation max 1 C h and max 2 C day Humidity non condensing less than 80 relative humidity Installation site in a closed room max 2000 m above sea level Installation overvoltage category Pollution degree 2 Complies with EN IEC 61010 1 08 2002 Safety requirements for electrical equipment for measurement control and laboratory use Complies with EN IEC 61326 05 2004 Electrical equipment for measurement control and laboratory use EMC requirements 6000 500 cm Better than 2 cm Better than 0 05 cm Better than 0 01 cm 0 1 T Photometric accuracy Sealed optics housing convective desiccating Low voltage air cooled can be replaced lifetime 44 000 operatin
17. bchapter J Figure 2 Product label appears on both the back and the bottom of the instrument Direct Detect Spectrometer User Guide 3 5 Safety Precautions Review and understand the safety precautions below before installing and operating the Direct Detect spectrometer A WARNING Jo avoid danger of electric shock do not install the instrument in an area with a high humidity level Refer to Site Requirements in section 6 Do not touch the ethernet cable or universal power adapter plugs with wet hands lo avoid potential shock hazard choose the correct plug configuration and make sure that the power adapter is plugged securely into a properly grounded AC power outlet Make sure that the connection between the cable and the instrument Is secure Always ensure that the power supply input voltage matches the voltage available in your location Use only the power supplies that were supplied by EMD Millipore Corporation for use with the Direct Detect system Use of other power supplies may damage the system Minimize power draw from other instruments on the same power circuit Do not use with flammable or explosive liquids 6 Installation 6 1 Packaging Inspect the packaging for damage upon receipt If there are any signs of damage contact the responsible shipping company A WARNING Do not put a spectrometer showing signs of damage into operation as this may result in severe personal injuries and
18. ction and quantification of proteins Biomolecules are applied directly to a card based hydrophilic polytetrafluoroethylene PTFE membrane that is transparent in most of the infrared spectral region The Direct Detect system measures amide bonds in protein chains accurately quantifying an intrinsic component of every protein without relying on amino acid composition dye binding properties or redox potential Protein concentrations from 0 2 to 5 mg mL can accurately be determined from a minimal sample volume 2 uL without bio or immuno chemical staining Sample analysis takes only minutes and in most cases can be performed directly from the buffered or native solution The system is intended for research use only and is not for use in diagnostic procedures It has been validated with proteins and cell lysates representative of those currently in use in research laboratories Direct Detect Spectrometer User Guide 1 2 Components Supplied The Direct Detect system includes the following components Figure 1 Components supplied Direct Detect spectrometer Instrument power adapter with 4 power cord plug configurations EU UK US Japan Data cable crossover cable for 10Base T Ethernet standard with RJ45 connectors Dell Netbook computer with Direct Detect software installed Computer power adapter with world wide plug adapter Netbook stand Direct Detect Assay free cards package of 50 Direct Detect spotting
19. e information contact the office nearest you In the U S call 1 800 MILLIPORE 1 800 645 5476 Outside the U S go to our web site at www millipore com offices for up to date worldwide contact information You can also visit the tech service page on our web site at www millipore com techservice 16 EC Representative Millipore UK Ltd Fleming Road Kirkton Campus Livingston EH54 7BN UK 17 Standard Warranty The applicable warranty for the products listed in this publication may be found at www millipore com terms within the Terms and Conditions of Sale applicable to your purchase transaction Direct Detect Spectrometer User Guide 35
20. e of accuracy Figure 27 shows a successful OQ test report OVP OQ Test Protocol Company MILLIPORE Operator Default Instrument Type Alpha Sample Compartment RT DLaTGS ZnSe Optics Configuration Fibre 1 with MIR KBr RT DLATGS Internal Pos 1 Accessory Millipore Direct Detect Instrument Serial Number M3 Instrument Firmw are Version 1 320 MDD 01 Oct 27 2011 OPUS DB Version OPUS 7 0 Build 7 0 122 DB 7 0 105 1106 Overall Test Result PASSED Test expires 12 16 2012 9 32 23 AM GMT 5 Test Date Time 12 16 2011 9 32 23 AM GMT 5 Test Spectra Path C Direct Detect V alidation Data 201 11216 093223 Comment Resolution Test vw Water Vapor Band 1554 35 cm 1 Maximum Resolution 2 00 cm 1 Measured Resolution 1 63 cm 1 Sensitivity Test Y Measurement Region Start 2200 00 cm 1 Measurement Region End 2100 00 cm 1 Minimum S N 2000 Measured S N 3446 07 Energy Distribution Test Minimum Energy Value 5 00 Energy at 5000 00 cm 1 9 14 Minimum Energy Value 0 20 Energy at 500 00 cm 1 4 67 Energy at 7500 00 cm 1 0 01 Wavenumber Accuracy Test Water Vapor nf Expected Band 1576 130 cm 1 Measured Band 1576 129 cm 1 Maximum Deviation 0 050 cm 1 Measured Deviation 0 001 cm 1 Figure 27 OQ test report Direct Detect Spectrometer User Guide 31 12 5 Running the Instrument Validation OQ and PQ Tests 1 Make sure card holder is empty 2 Torun OO and or PQ tests click on the instrument validation icon
21. ectrometer status light does not light Corrective Action Check the power cord connection at the power outlet and the power port on the back of the spectrometer Figure 3 No voltage is applied Confirm proper voltage is at the power outlet Short circuit in the adapter of power cable Spectrometer is still in the initialization phase IR source has not yet reached its operating temperature Spectrometer status light is yellow Spectrometer status Instrument test has failed light is red Defective spectrometer component Direct Detect Spectrometer User Guide A short circuit is accompanied by a ticking sound in the power adapter Unplug the power cord immediately and contact Technical Service Wait until spectrometer initialization is completed several minutes The warm up takes about 7 minutes As soon as the IR source has reached its operating temperature the Status light will automatically turn green Wait until the warm up is completed To determine which instrument component is causing the test failure click on the hardware diagnostics icon Figure 17 to open the hardware diagnostics dialog Figure 18 If the laser interferometer electronics automation or detector is defective contact Technical Service If the IR source is defective refer to section 12 1 for instructions on how to replace it To find out which component is defective click on the hardware diagnostics icon Figure 17 t
22. esiccant 5 Pull out the used desiccant pack A pair of tweezers may be helpful 6 Remove the external packaging from the replacement desiccant pack 7 Insert the desiccant pack properly into the spectrometer as shown in Figure 25 A CAUTION Do not cut or tear the pack into single pieces or ingest the desiccant 8 Attach the cover plate and fasten the 4 Torx screws again Figure 19 9 Plug the power plug into the power outlet again After the new desiccant removes the moisture from the chamber approximately 30 minutes the instrument status light and hardware diagnostics icon will turn back to green 12 3 Cleaning The outer spectrometer surface can be cleaned with a damp or dry cloth Do not use detergents with organic solvents acids or bases A CAUTION Do not try to clean the interior of the spectrometer as this may lead to serious spectrometer damage 28 Direct Detect Spectrometer User Guide 12 4 Description of Instrument Validation Tests 12 4 1 Performance Qualification Test PQ The Performance Qualification test evaluates the spectroscopic performance and confirms proper function of the IR spectrometer To ensure that the instrument is working properly it is recommended that the PQ test be run on a weekly basis The factory default interval for the PO test is one week One week from the last PO test the software will alert you to run the instrument PQ test again You can use the instrument with an expired PQ
23. g hours Microsoft Windows 7 Microsoft NET Framework 2 0 installed automatically 1024 x 600 or greater RJ45 10BaseT 10 or 10 100 MBit Intel Pentium II 800 MHz or greater 2 GB XVGA 256 Mb RAM 32 bit color depth Open GL GDI Direct Detect Spectrometer User Guide 33 14 Ordering Information This section lists catalogue numbers for the Direct Detect spectrometer and replacement parts See Technical Assistance section for contact information You can purchase these products on line at www millipore com products Product Description Cat No Oty Pk Direct Detect Spectrometer Direct Detect Spectrometer includes instrument with power DDHW00010 WW 1 adapter Dell Netbook computer with power adapter Ether net cable Netbook computer stand 50 pack of Assay free cards spotting tray spare desiccant bag Torx screw driver Quick Start Guide Power adapters are configured for multiple countries Direct Detect Spectrometer Replacement Parts Accessories Direct Detect Assay free Cards Multiple colors DDAC00010 4P 50x4 Multiple colors DDAC00010 8P 50x8 Green DDAC00010 GR 50 Spotting Tray DDMISCO10 ST 5 Netbook Stand DDMISCO10 NS 1 Direct Detect IR Source DDSP00010 LA 1 Direct Detect Desiccant Bag DDSP00010 DE 1 Contact Technical Service to order see Technical Assistance section 34 Direct Detect Spectrometer User Guide 15 Technical Assistance For mor
24. ly collected spectra from a series of known protein concentrations To compile these files select Calibration mode no analysis from the Options menu and follow the instructions for collecting calibration spectra in section 4 2 of the Direct Detect Software User Guide Direct Detect Spectrometer User Guide 17 10 3 Measurement continued Section 4 2 of the Direct Detect Software User Guide details how to use the Calibration Method wizard to create or modify a calibration method file Figure 15 Calibration Method Generation Calibration Method Wizard Select Method Option Existing Method Files Method Info Create New Method Method Amide1 SURE Created 5 24 2011 11 08 44 AM O Modify Existing Method Modified 5 24 2011 11 08 44 AM User Name RHB Description Amide 1 calibration test Delete Method Region K region 1702 1602 cm 1 20 9873 20 5350 x Correlation Coefficient 1 0000 Path containing method files F Documents and Settings Roland My Documents Direct DetectyMethods EJ Figure 15 Calibration Method start page 10 4 Removing the Card After all 4 positions on the card have been read the instrument will sound a tone The card will rise to the initial insertion position and can be removed CAUTION Do not remove card during the reading To stop a reading in progress click on the Abort button Figure 16 Abort Figure 16 Abort button When
25. nd pipette tips NOTE To minimize pipetting error we recommend the use of a precision fixed volume pipette capable of dispensing 2 uL for applying samples to the Assay free card Adjustable pipettes are not recommended Direct Detect Spectrometer User Guide 11 8 2 Materials Required but Not Supplied continued Water PBS or other buffer see section 8 1 for a list of buffers that interfere with protein quantification amp Printer optional 9 Direct Detect Assay free Card and Sample Preparation 9 1 Direct Detect Assay free Card All concentration measurements are performed using the Assay free card Figure 7 CENTER NOTES als 1 lt INSERT L Figure 7 Direct Detect Assay free card The card contains four hydrophilic polytetrafluoroethylene PTFE membrane positions sized for easy sample application and surrounded by a hydrophobic ring to retain analyzed sample within the IR beam A 2 uL sample volume is recommended in order to assure proper sample application drying and measurement By design the positions on the card are numbered from 1 to 4 starting from the bottom Position 1 by software default is reserved for the blank measurement The blank is the buffer used to resuspend the protein but contains no protein One or more of the remain ing card positions are used for the protein samples The Assay free card can be inserted into the system and analyzed immediately after p
26. o open the hardware diagnostics dialog Figure 18 If the laser interferometer electronics automation or detector is defective contact Technical Service If the IR source is defective refer to section 12 1 for instructions on how to replace it 21 11 4 Problems Indicated by Instrument Validation Icon Instrument validation problems are indicated by the Instrument Validation Icon Figure 17 Symptom Instrument validation icon is yellow Instrument validation icon Is red 22 Cause An instrument validation test has expired An instrument validation test has failed Corrective Action To find out which instrument validation test has expired click on the instrument validation icon to open the instrument validation dialog Refer to section 5 2 of the Direct Detect Software User Guide for details on instrument validation To find out which instrument validation test has failed click on the instrument validation icon to open the instrument validation dialog Refer to section 5 2 of the Direct Detect Software User Guide for details on instrument validation Direct Detect Spectrometer User Guide 12 Maintenance The Direct Detect spectrometer is a low maintenance instrument and the operator can replace components with a limited service life Replacing the IR source and desiccant are described in this section A WARNING Perform only the maintenance procedures described in this manual and observe
27. or property damage Keep the original shipping boxes in case you need to return the spectrometer for servicing or repalr NOTE Instruments will not be accepted for repair if not in original packaging EMD Millipore Corporation will provide shipping boxes for a charge if original packaging has been discarded 4 Direct Detect Spectrometer User Guide 6 2 Site Requirements The user must provide an installation site that meets the following requirements Space requirement Stable and horizontal base which is able to bear the weight of at least 8 kilograms kg A clearance of at least 25 centimeters cm 10 inches in at the spectrometer rear side Environment Temperature range 18 35 C Humidity non condensing less than 80 relative humidity Temperature variations less than 1 C hour Do not install devices such as large electric motors heaters welding equipment radio transmitting equipment units emit ting pulsed NMRs or high powered lasers in close proximity to the Direct Detect spectrometer These devices can cause a Spectrometer malfunction Make sure that these types of devices are not connected to the same electrical circuit as the Direct Detect spectrometer Vibration Do not locate the Direct Detect spectrometer near vibration sources centrifuges vortexes ventilation hoods air condition ers etc or in rooms with intense floor vibrations Inductive electrical Do not install the Direct De
28. pen the hardware diagnostics dialog Figure 18 If IR source is indicated refer to section 12 1 for instructions on how to replace it Excessive humidity inside Wait 30 minutes to 2 hours for desiccant to remove instrument the humidity If after 2 hours the icon is still yellow refer to section 12 2 Replacing the Desiccant 20 Direct Detect Spectrometer User Guide 11 2 Problems Indicated by Hardware Diagnostics Icon continued Cause Instrument test has failed Symptom Hardware diagnostics icon is red Defective spectrometer component Corrective Action To determine which instrument component Is causing the test failure click on the hardware diagnostics icon Figure 17 to open the hardware diagnostics dialog Figure 18 If the laser interferometer electronics automation or detector is defective contact Technical Service If the IR source is defective refer to section 12 1 for instructions on how to replace it To find out which component is defective click on the hardware diagnostics icon Figure 17 to open the hardware diagnostics dialog Figure 18 If the laser interferometer electronics automation or detector is defective contact Technical Service If the IR source is defective refer to section 12 1 for instructions on how to replace it 11 3 Problems Indicated by Instrument Status Light top of instrument Cause Spectrometer is not connected properly to the power supply Symptom Sp
29. r sa ccczcetascccsccaccassncuctessnnckceeescecacesceaseecte 6 7 Direct Detect Spectrometer Parts and FUNCTIONS rnssvnvnnnnnnnnnnnnnnnnnnnevennnnnnnnnneennnnn 8 ENN 8 ER eg Computer DE EE EE 10 8 GE GU CNG EN SER EEr 11 Nr 11 82 Materials Required Dut Nor SN 11 9 Direct Detect Assay free Card and Sample Preparation rnnnnasvvrvrnnnnnnvnneennnnnnnerr 12 Oe Direct Detect NTN 12 S E 00 0 ENE EE eee eee 13 10 How to Use the Direct Detect Spectrometer sassrnnunnnnnnnvennnnnnnnnnnennnnnnnnnnennnnnnnenr 14 10 1 Accessing Direct EN 14 t0 2 Fe 15 OE S EEE NE 16 EGO TG 8 EEE NE EN 18 AV od LES 01 OIG EE EEE NM 19 VI FN NNN 19 11 2 Problems Indicated by Hardware Diagnostics ONA 20 11 3 Problems Indicated by Instrument Status Light top of instrument sssssrersrvesrersversserssersnen 21 11 4 Problems Indicated DY Instrument Validation ICON scnscnsssnscncsacavedsessvs disentiertsanavhasautiwncicsenepGiannionaueuts 22 Contents continued 12 13 14 15 16 17 VET du oe 1 EEE EE SEE 23 PENNER AN 23 NTN 26 gt EN 28 124 DET PIN SEN TN ES ran 29 125 free NNN 32 Vo SO 0 e E E E E eee eae 32 TONN 33 Ordering NT EIN de 34 Tecnnical AS Laser 35 ERT 35 Standard AE LE EEE NE 35 1 Introduction The Direct Detect infrared IR based quantification system is an innovative combination of software controlled instrumentation and EMD Millipore Corporation s advanced membrane technology optimized for the dete
30. rational Qualification instrument tests as follows r 3 11 Click on the instrument validation button RZ 12 When the instrument validation dialog appears click on the Performance Qualification Run Test button 1 Figure 23 The test takes about 5 minutes k Instrument alidation x off Performance Qualification PQ This test compares the performance of the instrument on day to day basis Failure of this test can be due to degradation in the source or other component Test expires in 0 days 71 Hours 43 Minutes Last Report gt Run Test C yf Operational Qualification 001 This best measures the operational performance of the instrument Failure of this test may indicate instrument misalignment or Failure Test expires in 365 days 23 Hours 59 Minutes Last Report gt Run Test 2 MOTE The instrument will not be able to perform sample measurement if either test has Failed Figure 23 Instrument validation dialog 13 After the test has finished run the Operational Qualification test by clicking on the Operational Qualification Run Test Button 2 14 If the instrument passes the test the hardware diagnostics icon turns green If the instrument fails refer to section 5 1 in the Direct Detect Software User Guide 12 2 Replacing the Desiccant Excessive humidity inside the spectrometer may cause H O bands in the spectrum To reduce H O bands to a minimum the air inside the Direct Detect
31. reparation The Direct Detect spectrometer has a built in drying system that will automatically dry the sample to the point where it will give an accurate reading The first sample position will typically take the longest period of time to dry with subsequent positions taking less time Including drying time the total measurement time for a card loaded with 2 ul samples does not exceed 5 minutes The Direct Detect Assay free card is intended for a one time protein spotting However already prepared cards can be stored and read again Direct Detect Spectrometer User Guide 9 2 Sample Preparation In general analysis of samples within the concentration range of 0 2 5 mg mL can be performed directly from the buffered solution Buffers containing more than 5 glycerol SDS and or Tween 20 surfactant should be avoided due to the excessive time required to achieve dryness For analysis of samples with protein content above 5 mg mL single step dilution is recommended Concentration determination of samples below 0 2 mg mL can be achieved after a simple spin concentration step using a centrifugal filter e g Amicon Ultra 0 5 device Use of centrifugal filters is also recommended for buffer exchange when the original buffer contains high concentration of components with amide or amine containing functional groups refer to section 8 Figure 8 is an example of a diafiltration protocol that concen trates the sample and reduces the
32. roximately 2 seconds activates deactivates the stand by mode Pressing this button for more than 6 seconds resets the spectrometer to the original state The abbreviation SBY means Standby and RES means Reset Do not use unless instructed to do so by service personnel 6J POWER port This port connects the spectrometer to the power supply via the Supplied power adapter and power cord The POWER port is for extra low voltage connectors Use only power cords supplies that have been supplied by EMD Millipore Corporation 6K Kensington lock The lock slot is an anti theft lock for mobile hardware Lock and Slot cable are not supplied 7 2 Direct Detect Computer and Software The Direct Detect spectrometer is designed to operate with the Direct Detect software that is pre installed on the Netbook computer For computer specifications refer to section 13 Direct Detect software may be updated periodically For information on the most up to date software go to www millipore com directdetect Register your instrument in order to receive notification about relevant software upgrades WARNING Do not install third party software screen savers etc as this may impact the performance of the Direct Detect system Direct Detect Spectrometer User Guide 8 General Guidelines The Direct Detect spectrometer is recommended for use with protein samples ranging from 0 2 to 5 mg mL Although the instrument c
33. section 1 1 of the Direct Detect Software User Guide NOTE A seven minute warm up cycle is required after initial power up and software activation The software will wait for seven minutes before allowing a sample measurement to begin When preparing to read samples we recommend that you power up the instrument and start the software prior to preparing the sample cards 14 Direct Detect Spectrometer User Guide 10 2 Loading and Installing the Card 1 Place the Assay free card s on the spotting tray or other clean surface Select an appropriate buffer blank preferably the same solution in which the protein has been prepared If that buffer is not fully characterized select the closest buffer possible Pipette 2 ul of the buffer solution to the default blank position on the card position 1 2 Pipette 2 uL of sample to sample positions 2 through 4 as needed Unused positions can be loaded and read at a later time as long as the buffer blank is appropriate for them Card positions can be labeled if desired NOTE The card can be allowed to dry at ambient conditions however the Direct Detect spectrometer has a built in drying system that can automatically dry the sample to the point where it will give an accurate reading 3 Insert the card vertically into the slot in the top of the card holder module with the card facing left and the alignment notch in the corner of the card oriented towards the center of the instrument
34. t installation refer to section 4 2 in the Direct Detect Software User Guide 2 Click on the Measure Card button Figure 13 The sample measurement takes about a minute and the measurement progress is shown in the lower status bar Measure Card W Figure 13 Measure sample 16 Direct Detect Spectrometer User Guide 10 3 Measurement continued The result from each measured sample position is displayed in the Last Sample Results tab Figure 14 of the main Direct Detect screen Concentration Sample1 mg ml 8 22 2011 11 16 18 AM 1 994 sample 3 i 2 041 sample2 1 919 sample 1 1 blank 1 Average Conc 1 985 Hold Ctrl key and select ee Std Deviation 0 05 Sample Number to Create remove from statistics Report CV 2 5 Yo Last Sample Results Card History Figure 14 Last sample results screen 3 For reporting options refer to sections 2 4 2 6 of the Direct Detect Software User Guide NOTE Periodically the software will initiate a reference measurement which must be performed without a sample card inserted in the card holder If a card is found in the card holder the software will prompt you to remove it 10 3 2 Calibration The Calibration Method Wizard in the Options menu is used to create or modify calibration method files that will be used to perform the analysis of the Assay free cards To create a calibration method you will need to have previous
35. tect spectrometer near any interference sources of potential inductive electrical interference e g pumps switching motors microwave ovens etc sources of high energy pulses or sources that might cause magnetic or radio frequency interference Power supply Voltage range 100 VAC to 240 VAC Frequency range 50 Hz to 60 Hz Protection class The power supply unit automatically adapts to most power sources commonly used Make sure that the power supply is easily accessible at all times Connect the Direct Detect spectrometer only to a grounded power outlet Take precautions to ensure uninterrupted power supply if there is the potential for power supply issues e g brownouts power surges frequent thunderstorms etc Direct Detect Spectrometer User Guide 5 6 3 6 4 6 5 Installation Configuration The Direct Detect spectrometer is configured to operate as a stand alone unit connected to the supplied Netbook computer PC using the factory set standard IP address 10 10 0 1 Unpacking and Setting up the Computer Unpack the Netbook computer power adapter and world wide plug adapter from the shipping box Choose the plug adapter with the correct configuration for local requirements and attach it to the plug Plug the low voltage round end of the power adapter into the power port of the PC but do not plug the computer into a power outlet at this time Unpacking and Setting Up the Direct Detect
36. test but the software will query you each time a measurement Is initiated asking if you would like to proceed under the expired OQ condition The OQ test protocol consists of the following four tests Wavenumber Resolution Test This test verifies the achievable spectrometer resolution by measuring a single channel single beam spectrum of the water vapor available inside the spectrometer using the maximum available resolution Direct Detect Spectrometer User Guide 12 4 Description of Instrument Validation Tests continued Signal Sensitivity Test This test is the same as the PQ signal to noise test except that the spectra are measured in a very small region 2200 2100 cm Energy Distribution Test This test verifies the achievable frequency range and the energy at the maximum position of the single channel spectrum Absolute values of energy are measured at different wavenumber positions These values are divided by the maximum value achieved and compared to acceptance values indicated in percent Optical components e g detector source and beam splitter as well as the card holder have an effect on the limits of the frequency range Wavenumber Accuracy Test he wavenumber accuracy test verifies that the frequency calibration of the spectrometer is within defined limits Water vapor Is used for this test because it has extremely narrow bands and the position of these bands can be measured with a very high degre
37. the back of the instrument loosen the 4 cover plate Torx screws with the Torx TX20 screw driver supplied Figure 19 Direct Detect Spectrometer User Guide 23 12 1 Replacing the IR Source continued Torx screws wow ETH LAN acr DIR SBY USB TKD ACC ERR EXT ACCESSORY CFG RES POWER Figure 19 Removing the cover plate 3 Take off the cover plate IR source IR source Torx screws Source plug Alignment groove Figure 20 Removing the IR source 4 Loosen the two mounting screws on the IR source Figure 20 with the Torx screw driver 5 Disconnect the green source plug Figure 20 6 Pull the source out from the spectrometer housing 24 Direct Detect Spectrometer User Guide 12 1 Replacing the IR Source cont Alignment groove Alignment pin Figure 21 IR source removed from instrument 7 Insert the new IR source into the spectrometer housing Make sure that the alignment pin fits into the alignment groove Figure 21 8 Secure the source by fastening the two Torx screws Figure 22 9 Plug the green source plug into the SRC female connector Figure 22 Replacing IR source Torx screws 10 Attach the cover plate and fasten the 4 Torx screws Figure 19 Plug the power plug to the power outlet again Direct Detect Spectrometer User Guide 25 12 1 Replacing the IR Source cont After replacing the IR source perform the Performance Qualification and Ope
38. tray Desiccant pack replacement 1 not shown Torx TX20 screw driver for replacing the IR source not shown Direct Detect Spectrometer Quick Start Card not shown 2 Direct Detect Spectrometer User Guide 3 Symbols Used in this User Guide The following symbols are used throughout this user guide and or on product labels and the user shall abide by indicated requirements Symbol Warning alerts you to actions that may cause personal injury or pose a physical threat rele Class I Laser product complies with 21 CFR Chapter 1 Subchapter J CE conformity marking Reference Declaration of Conformity for specific directives Do not discard with common solid waste at end of life Segregate with other waste electrical and electronic equipment WEEE and send to an appropriate facility for recycling For information on recycling EMD Millipore Corporation electrical and electronic products in the European Union please visit www millipore com weee Hot Surface 4 Product Labeling Direct Detect V Spectrometer IVI EMD Millipore Corporation S ml 290 Concord Rd Billerica MA 01821 Input 24V DC 2 0A 800 645 5476 REF DDHW00010 00 AN X SN 00000001 Made in Germany Manufactured 12 2011 For Research Use Only Not for use in diagnostic procedures C midd MA TKD ACC tel EXT ACCESSORY TAP CFG i POWER K Class 1 Laser Product Complies with 21 CFR Chapter 1 Su
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