SmartSpec™ Plus Spectrophotometer Instruction Manual Catalog Number 170-2525 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
Table of Contents Page Section 1 General Safety Information .....................................................................1 1.1 1.2 Caution Symbol......................................................................................................1 Precautions.............................................................................................................1 Section 2 Packing List ..............................................................................................
Warranty ! The SmartSpec Plus Spectrophotometer and accessories are warranted against defects in materials and workmanship for one year. If any defects occur in the instruments or accessories during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts at its discretion without charge. The following defects, however, are specifically excluded: ! 1. Defects caused by improper operation. 2.
Section 1 General Safety Information This instrument is intended for laboratory use only. This product conforms to the "Class A" standards for electromagnetic emissions intended for laboratory equipment applications. It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or in the same circuit as those applications. The user should be aware of this potential and take appropriate measures to avoid interference. 1.
Section 3 Installation 3.1 Environmental Requirements To ensure correct operation and stable performance over an extended period of time, install the SmartSpec Plus in a location which meets the following conditions: • Room temperature between 15 and 35°C. Not recommended for cold room use (4°C). • Not exposed to direct sun light. • Not subject to direct or continuous vibration. • Not subject to intense magnetic or electromagnetic fields. • Relative humidity between 0–95%.
Section 4 Introduction 4.1 Identification of System Components 5 2 4 1 3 Fig. 1. View of the SmartSpec Plus Spectrophotometer. Instrument 1. Keypad interface 2. Display 3. Sample compartment door 4. Integrated thermal printer 5. Printer paper compartment door Change menu and select options by pressing these buttons. Press a single button to perform action described on the menu display. 2 x 24 alphanumeric backlit LCD display. Displays various menus and options available.
11 10 9 12 Fig. 3. View of the SmartSpec Plus sample compartment. 9. Cuvette chamber Insert cuvette into the cuvette chamber. Ensure that the cuvette is placed in the proper orientation with respect to the light path. 10. Cuvette storage compartment Storage space for cuvette. 11. Cuvette spring Aligns cuvette. Accommodates various cuvette configurations. 12. Light path Light path axis. 14 15 13 Fig. 4. View of the SmartSpec Plus integrated thermal printer paper with door removed. 13.
UV/Visible Spectroscopy is based on the absorption of light as a function of wavelength. All spectrophotometers have a light source that generates light of specific wavelengths. The SmartSpec Plus uses a xenon flash lamp as its light source. Xenon flash lamps have several advantages over the conventional light sources in that they reduce warm up time, do not heat up the instrument and have a lamp life of 10 to 15 years.
The Lambert-Beer’s law identifies the relation between sample concentration and the intensity of transmitted light. Absorbances and % transmittances are defined by the following equations: % Transmittance (%T) %T = It/I0 * 100 = 10-εcl * 100 Absorbance or optical density (A or O.D.
4.2 Setup When the SmartSpec Plus is not carrying out an assay, you can enter the Setup routine by pressing Setup. To proceed through Setup without changing the current setting, press Enter. Most of the current settings can be changed by pressing Select until you see your desired choice; sometimes you have to use the number pad to change an entry. In either case, press Enter after the selection is made. When you have gone all the way through Setup, SmartSpec Plus will automatically return to Home screen.
3. Background compensation. When using the DNA/RNA assay, you can choose to have SmartSpec Plus automatically subtract background absorbance readings collected at 320 nm from the A260 and A280 measurements. If the background subtraction option is invoked, then the A320 is subtracted from the A260 and A280 readings before these measurements are displayed and before they are used in the calculation of nucleic acid concentration. The default condition is to not subtract the A320 background.
7. New Operator. When SmartSpec Plus prints a report, it includes the name of the operator (if specified). You can add a new operators name to the list of operators in Setup. Want to add the name of a new operator? No Press Select to toggle from No to Yes and the screen will change to Enter new operator's name: (8 char): The keyboard will automatically switch to the alpha mode and you can use the keys to enter a name with as many as eight characters. To enter an A in the first space, press the 2 key once.
12. Time convention. You may choose to print time in 12-hour or 24-hour mode. Toggle between the choices by pressing Select. and then press Enter. Select time display mode 13:12 24-hr e.g. Press Select for Select time display mode 12-hr e.g. 1:12 13. Date convention. You can choose to print the date in either MM/DD/YY or DD-MMYY mode. Toggle between the choices by pressing Select and then press Enter. Select date display mode e.g.
The serial port parameters are: 9600 baud rate, 8 data bits, no parity, 1 stop, and XON/XOFF flow control. These parameters are pre-set in the unit. When using a serial printer set the printer to the serial port parameters specified above. Please note that the data are sent from the SmartSpec Plus to a computer or serial printer as hex characters. If your computer or printer is not set to accept these characters please refer to the instrument’s instruction manual for correct settings.
4. When the SmartSpec Plus sends data, either when requested from Scan, or Kinetics, or when the user activates the data echo toggle in Setup, the data streams to the terminal window. 5. Capturing data in HyperTerminal and saving the data in a file: In the main HyperTerminal window menu, first click Transfer and then click Capture Text. Transfer Capture Text . . . [give file name and path] e.g. c:\program files\accessories\hyperterminal\capture.
4.5 Keypad Descriptions Print Abs Conc A260:A280 Dilution Factor Setup Assay Buttons DNA/RNA SmartSpec Plus will print data on the current sample, a user-selectable range of samples or all samples in the current assay. SmartSpec Plus will automatically display the absorbance reading of the current sample and the wavelength being read. If more than one wavelength is being read, press Abs to toggle among the different wavelengths.
Protein Scan Kinetics OD600 λ Read Blank Read Sample Select Enter SmartSpec Plus will automatically read one or more wavelengths depending on the type of assay specified (Bradford, Lowry, BCA or UV Protein) or the user can specify a wavelength. SmartSpec Plus will convert absorbance data to concentration by use of a standard curve, except for the case of UV Protein in which the absorbance readings are multiplied by user-selectable constants.
Cancel Cancels the last entry or causes an exit from Setup. When editing sequence information, press Cancel to delete a base. When Options are displayed, press Cancel to return to the assay. Used to access other menus or screens. Also used to move the cursor. Feeds paper through the printer. Used when removing or installing a paper roll. The Alpha key allows the user to toggle between alpha and numeric input for naming stored standard curves or naming operators.
ii. Choose standard curve option. a. Create a new standard curve. b. Recall a standard curve from memory. c. No standard curve. SmartSpec Plus will not be able to convert absorbance to concentration. C. Scan i. Set upper and lower limits of scan. (200 nm to 800 nm) ii. Choose whether to subtract background and, if so, specify background wavelength. iii. Choose fast or slow scan. iv. For the fast scan, choose number of successive scans. D. Kinetic i. Choose wavelength to read. ii.
Section 6 Operating Instructions Note: It is Good Laboratory Practice (GLP) to use the SmartSpec Plus spectrophotometer to measure absorbances in the range of 0.100 to 1.500 AU. 1. Choose an assay. SmartSpec Plus has six different assay keys in the second row of buttons. When you press one of them, it signals SmartSpec Plus to clear its memory of absorbance readings from the previous assay, to reset the sample number to 1, and to reset the dilution factor to 1.
The SmartSpec Plus Bradford, Lowry, BCA and Other Protein assays use standard curves created by measuring the absorbances of solutions of known concentration to determine the concentration of unknown samples. If you do not want to construct a new standard curve, you may recall one from the on-board memory. SmartSpec Plus has the capability of storing up to ten standard curves in memory. The stored curves are numbered automatically from 0 to 9, or you can overwrite the number.
Limitations of the methods. The linear regression, when fit properly, generally produces reliable results at the extremes of the range of the standard curve. Results calculated from mean absorbance data outside the range of the standard curve may be unreliable. (For the purposes of this discussion, the range of the standard curve is defined as the absorbance range between the highest and lowest mean absorbances of the standards used to form the standard curve.
2. Choose sample replicates. Samples may be read in replicate and you do not need to have the same number of replicates for each different sample. For each sample replicate group, SmartSpec Plus will calculate a mean absorbance and standard deviation. When the full report is printed, each individual absorbance is reported along with the mean and standard deviation of the replicate group.
• Press the right arrow key to change the number of sample replicates. Enter number of sample replicates: _____ B. Sample replicates. • Place the first replicate of your first sample in the cuvette chamber and press Read Sample. Before it collects the absorbance data, SmartSpec Plus will ask for the number of replicates for the first sample. Sample #1 Enter number of replicates: 3 >=Option • Use the number keys to input the number of replicates and press Enter.
• Press A260:A280 to see the ratio displayed A260= x.xxx A260:A280 = • For DNA oligos and RNA oligos, press Conc to see the concentration displayed in units of pmoles/µl. A260= Conc= • A280= x.xxx x.xxxx x.xxx xxx.xxx Samp #1 pmoles/µl To read the next sample, insert the cuvette in the chamber and press Read Sample. From any of the above screens you can press Enter to return to the Ready screen. Ready to Read absorbance <=Exit Assay >=Options B. Protein.
• Now press Select and the negative sign will be displayed in front of 0.500. Min AU scale: +/- -0.500 Max AU scale: • Press Enter again and now define the upper limit. The upper limit must be a positive number. After the scan data are printed, or if you choose not to print the scan data, you are next offered the opportunity to download the data via the serial port to a computer. Press when ready to accept serial data.
• If you want to download the data, make sure the connections are made to the computer and then press Enter. If you do not want to download the data, press Cancel. Ready to Read Absorbance <=Exit Assay >=Options After the data are downloaded SmartSpec Plus will reset for the next kinetic data collection. From the Ready screen, you can change any of the parameters for the kinetic data collection. E. OD600.
8. Exit the Assay. You can exit the assay by pressing the left arrow key when the Ready screen is displayed. Ready to read absorbance <=Exit Assay >=Options SmartSpec Plus will prompt you to save a newly created standard curve (for protein assays) and then prompt you to print a complete report of absorbances, concentration and A260:A280 ratios. The report will also show means and standard deviations for each standard or unknown sample replicate group.
• After the default conversion factor is accepted or changed, SmartSpec Plus is ready to measure nucleic acid absorbance and convert that absorbance to concentration. Ready to read absorbance <=Exit Assay >=Options B. DNA oligonucleotides and RNA oligonucleotides. If you choose DNA oligo or RNA oligo, you must provide the molecular weight of the oligo and either the molar extinction coefficient or the mass/absorbance conversion factor. If you don’t know them, then SmartSpec Plus can estimate them for you.
b. If you do not know the molecular weight. Enter oligo composition: #A:___#C:___#G:___#T:___ • Use the number keys to input the number of A’s, then press Enter to move the cursor to the next field and enter the number of C’s. After entering the number of T’s (or U’s for RNA oligos), press Enter. SmartSpec Plus will calculate the molecular weight and display it along with the molar extinction coefficient. Mol Ext Coef=xxxxxxx MW=xxxxx >=Continue • ii. Press the right arrow key to continue the assay.
• Press > to continue the assay, and the screen will display Ready to read absorbance <=Exit Assay >=Options • SmartSpec Plus is now ready to collect absorbance data. b. If you do not know the molecular weight. Enter oligo composition: #A:___#C:___#G:___#T:___ • Use the number keys to input the number of A’s, then press Enter to move the cursor to the next field and enter the number of C’s. After entering the number of T’s (or U’s for RNA oligos), press Enter.
• Use the number keys to input the length of the oligo and press Enter. You can enter a length of up to 99 nucleotides. After you press Enter, SmartSpec Plus will calculate and display the extinction coefficient and molecular weight. Mol ext coef= MW= __________ >=Continue The molar extinction coefficient is displayed with units of liter mole-1 cm-1 • Press the right arrow to continue the assay.
• Press the right arrow to continue the assay. SmartSpec Plus will now be ready to measure the absorbance of your oligonucleotide and to convert those absorbance data into concentrations of µg/ml and pmol/µl. Mol ext coef= __________ MW= _________ >=Continue Nucleic Acid Calculations Molecular Weight (MW): • Length (L): MW ≈ L * 310 - 61 • Sequence or Composition: DNA: MW = (#A * 312.2) + (#C * 288.2) + (#G * 328.2) + (#T * 303.2) - 61 RNA: MW = (#A * 312.2) + (#C * 288.2) + (#G * 328.2) + (#U * 289.
7.2 Protein Assay 1. Choosing the type of assay. Press Protein and the display becomes Select assay: Bradford As you press Select, other options are displayed: Select assay: Lowry Select assay: UV Protein Select assay: BCA Select assay: Other Press Enter when the correct type of assay is listed. SmartSpec Plus knows what wavelength to read for the first four assays, but if you select Other, then you must enter the wavelength to be read.
3. Standard Curves. Once you have selected the type of assay, SmartSpec Plus will ask if you want to make a new standard curve. You can decline to make a standard curve, in which case it will not be possible for SmartSpec Plus to convert absorbance readings to concentration; you can recall a saved standard curve from memory, or you can create a new standard curve. Do you want to make a new standard curve? Yes If you want to make a new standard curve, press Enter and skip ahead to part B below.
• In order to see the information about the standard curve, press Select to toggle from No to Yes and press Enter. r2=x.xxx CONC=ABS*M +I 0=Show more >=Continue r2 refers to the square of the correlation coefficient and is a value between 0 and 1, with 1 representing a perfectly straight line (see discussion above). The general equation of the curve fit is presented in the top right part of the display Concentration = Absorbance*Slope + Intercept • Press 0 to see the slope.
• Each time you place a new blank into SmartSpec Plus, press Read Blank. After the absorbance reading is collected, the instrument is zeroed and the display will show Blanking Spectrophotometer You can check the absorbance reading at any time by pressing Abs. A750 = 0.000 >=Continue • Press the right arrow key to return to the assay.
i. If there are no standard replicates. SmartSpec Plus will direct you to enter the concentration of each standard and then to measure the absorbance of each standard. Enter concentration of standard #1: ________ • Use the number keys to input the concentration of standard #1 and press Enter. You may enter concentrations with as many as eight significant digits, but all results are reported with four significant digits because SmartSpec Plus absorbance data are measured to four significant digits.
• If you have varying numbers of replicates for the different standards, press Select to toggle from Yes to No and press Enter. Skip ahead to b. If you have the same number of replicates for each standard, press Enter. a. If there are the same number of replicates for each standard. You can have as many a nine replicates for each standard Enter the number of standard replicates: ___ • Use the number keys to input the number of standard replicates and press Enter.
After all replicates of standard #1 are measured, SmartSpec Plus asks for the concentration of standard #2 and directs you to put the replicates into SmartSpec Plus one at a time until all are read. Enter concentration of Standard #2: __________ Insert Rep #1 of Std #2 and PRESS Read Sample • • • Insert Rep #9 of Std #1 and PRESS Read Sample This process continues until all replicates of all standards have been read.
b. If there are varying numbers of standard replicates. Each time SmartSpec Plus prepares to read a standard, you will be prompted for the number of replicates for that standard and then for the concentration of the standard. After all replicates of the first standard are read, then SmartSpec Plus will prompt for the number of replicates of the second standard and then for the concentration of the second standard. This will continue until the last replicate of the last standard is read.
After all replicates of standard #1 are measured, SmartSpec Plus asks for the number of replicates of standard #2 and then the concentration of standard #2 before prompting you to insert each of the replicates of standard #2.
The display will show either A750 = X.XXX Std #1 Rep #2 >= Continue • or A750 = X.XXX = - X.XXX Std #1 Mean >= Continue Press the right arrow key to return to the assay. After the last replicate of the last standard is measured, SmartSpec Plus will construct the standard curve. C. Editing standard curve data. After the absorbance of the last replicate of the last standard has been read, SmartSpec Plus will offer you a chance to edit the data set before constructing the standard curve.
• You can choose to blank the instrument again before you re-read your sample by pressing Enter at this screen. If you do not want to re-zero SmartSpec Plus, press Select to toggle from Yes to No and press Enter. SmartSpec Plus will now direct you to insert blank (if you chose that option above) and the to insert the specified standard. When the absorbance data have been collected, SmartSpec Plus will offer you an opportunity to continue re-reading other samples.
• Press 0 to see the intercept. Press the right arrow to return to the assay or press the left arrow key to see the slope displayed again. Intercept (I)=xx.xx e xx <=Back 0=More >=Continue If the slope or intercept has a value greater than 9999 or less than 0.0001, it will be shown in exponential notation. In all cases with SmartSpec Plus, e represents the base 10, not the base of the natural logarithm. For example, 100,000 would be represented as 1.000 e 05 and 0.00001 would be represented as 1.000 e-05.
If you want to rescale the Y axis, press Enter. If you want to rescale the X axis, press Select to toggle the display and press Enter. Rescale standard curve? Absorbance (X-axis) When you rescale the X axis, you must input the new maximum X value and press Enter. A new maximum Y value is automatically calculated and the graph is plotted. Max conc=150.0000 Enter new max: When you rescale the Y axis, you must input the new maximum Y value and press Enter.
7.3 Spectral Scanning SmartSpec Plus can collect data over a range of wavelengths and then plot those absorbances. There are both Fast and Slow scans: the fast scan generally takes a minute or less to accomplish while the slow will take several minutes, but results in more accurate absorbance data and a smoother curve. The scanning time is dependent on the range scanned and the absorbance of the sample. You can improve the accuracy of fast scans by specifying more than one repetition of the scan.
• Use the number keys to input the number of successive scans and press Enter. SmartSpec Plus will now display the Ready screen. If you want to invoke the smoothing option or the subregion option, choose Options from the Ready screen. Ready to read absorbance <=Exit Assay >=Options 1. Repetitions 2. Range 3. Background >=More The first three options allow you to change the conditions already specified above. Press the right arrow key to see the other options. 4. Subregions 5.
7.4 Kinetics When you press Kinetic, you begin by specifying the reading wavelength. Enter reading wavelength nm for kinetic assay: • Use the number keys to input the wavelength to be read and press Enter. Enter duration of timed seconds assay : • Use the number keys to input the duration of the assay in seconds and press Enter. You may collect data for as long as 9999 seconds.
7.5 OD600 This assay is useful for monitoring growth of bacterial cultures. When you press OD600, SmartSpec Plus will display the current conversion factor and offer you the choice of accepting or modifying it. A600 1.0=xx.xexx cell/ml Is this factor OK? Yes • If the displayed conversion factor is acceptable, press Enter. Otherwise, press Select to toggle from Yes to No and press Enter. Enter new conversion factor; xx.
7.6 Lambda λ This assay allows you to collect absorbance data from up to three different wavelengths simultaneously. No concentrations are calculated. When you press λ, SmartSpec Plus will prompt you first for the number of wavelengths to read. Enter number (1–3) of wavelengths to read: 3 • Use the number keys to input the number of wavelengths to be read and press Enter. SmartSpec Plus will then ask for each individual wavelength.
Section 8 Troubleshooting Troubleshooting Table Problem Unit does not power up Cause Solution Power switch in off position. 1. Check power switch on back of unit. No power from outlet. 2. Check power source. Bad power cord. 3. Replace. LCD display difficult to read Back lighting burned out. 1. Call Bio-Rad service. Contrast setting out of adjustment. Printer jam Thermal paper caught between print head and thermal plate when tearing off paper. 1.
Problem RS232 serial port/no response Cuvette does not fit in cuvette chamber Negative values/no absorbancy Fluctuations Cause Incomplete system initialization during power up. PC setup incorrect/wrong instruct mode. Cable's not fully connected/ wrong adaptor. Serial Port not selected in Setup Solution 1. Power down unit/Restart unit. 2. Check spreadsheet program setup. 3. Check all connections. Cuvette spring in wrong location. Cuvette spring missing. 4.
4. Dampen cloth with water. Use it to clean the instrument from the outside, including the display and the keyboard. 9.2 Cleaning Quartz Cuvettes It is important to maintain the cleanliness of quartz cuvettes so that residues from prior analyses do not contribute errors to future measurements. For most biological samples, the following cleaning procedure will help keep these cuvettes working optimally for your spectrophotometery system. 1. Wash cuvette with warm water. 2.
• • • • • Trim leading edge of paper roll with scissors if uneven or torn. Feed paper into slot opening until it stops. Press the "Paper Feed" key located on the keypad and guide the paper into the printer until it grabs and self feeds. Continue to press "Paper Feed" button until paper comes out of printer outlet located on top of case as shown. Wind any excess paper back on roll and drop new paper roll with shaft into grooves in paper storage area as shown. Re-install printer door cover.
Section 11 System, Accessories, and Reagents for the SmartSpec Plus 11.1 Product Information Catalog Number Product Description SmartSpec Plus Spectrophotometer and Accessories 170-2525 SmartSpec Plus Spectrophotometer 170-2502 Standard 1–3.5 ml Cuvette, quartz 170-2503 Semi-microvolume 1.4 ml Cuvette, quartz 170-2504 Microvolume 700 µl Cuvette, quartz 170-2505 Sub-microvolume 100 µl Cuvette, quartz 170-2415 Standard Cuvette, 1–3.5 ml, polystyrene disposable cuvettes, 100 223-9950 Standard 3.
11.3 Cuvette Selection Guide Catalog Number Minimum Volume Maximum Volume Cuvette Type 170-2502 1,000 µl 3,500 µl Standard cuvette, quartz 10 mm 170-2503 500 µl 1,400 µl Semi-microvolume cuvette, quartz 10 mm 170-2504 200 µl 700 µl Microvolume cuvette, quartz 10 mm 170-2505 80 µl 100 µl Sub-microvolume cuvette, quartz 10 mm Pathlength 11.4 Selecting the Appropriate Cuvette The SmartSpec Plus has 1 mm, 2 mm, 5 mm and 10 mm pathlengths.
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