The NanoPhotometer™ UV/Vis spectrophotometer Complete solution for ultra low and standard volume applications The NanoPhotometer™ is designed to provide the utmost flexibility in photometry. The outstanding technology combines ultra low and standard volume applications for improved concentration measurement and estimation of purity in one UV/Vis spectrophotometer. The fully developed software enables more user comfort with the established methods and assists with the predefined Functions self-programming to meet future needs. Various photometric functions, e.g. single and multi wavelength measurement combined with kinetic methods, opens up a wide range applications. The full wavelength scan (190 nm - 1100 nm) allows curve interpretation for attainment of more detailed scientific knowledge over whole spectrum. Get your result in only 20 seconds Analysis without cuvettes The sample (0.7 to 10 µl) is pipetted directly onto the active measurement window Closing of the lid. Taking the measurement. After the measurement the sample can be retrieved and stored for later use. Cleaning of the measurement window is easily performed with a fluff free tip or Kimwipe – no cross contamination, no dilution errors, no carry over effects Removing of sample residues from the lid mirror with a fluff free tip or Kimwipe. The photometer is ready for the next sample. Advanced Technology The NanoPhotometer™ spectrophotometer is a simple-to-use UV/Visible instrument. It is the perfect composition in highest quality to cover all photometric applications in molecular biology, biochemistry, and microbiology. The CCD array detector (1024 pixels) and the pulsed xenon lamp guarantee most accurate measurements. The spectrophotometer has no moving parts, which is the basis of the rapid scanning operating system within the range from 190 nm and 1100 nm. The high performance instrument operates with wavelength reproducibility (± 1 nm) and accuracy (± 2 nm) at a spectral bandwidth of 5 nm. Within the photometric range of - 0.3 to 2.5Absorbance, respectively 0 to 199%T, the NanoPhotometerTM performs with highest linearity and reproducibility. The back-lit liquid crystal display is very easy to navigate with the alphanumeric entry and navigation arrow keys on the hard wearing, spill proof membrane keypad After switching on the instrument will perform a series of self-diagnostic checks for wavelength calibrations. Planview of the NanoPhotometer Function The sample is pipetted onto the centre of the measuring window. Due to the integrated beam deflexion and the use of fibre-optic light conductors the sample can be measured directly on the measurement window. Using one of the four available lids of the microliter cell (lid 10, lid 50 and optional lid 5 or lid 100) different liquid columns of defined pathlength are created. Virtual dilutions of up to 1/100 are generated in comparison to a standard 1 cm cuvette measurement, without a manual dilution of the sample. Dilution errors are avoided and the sample can be retrieved after the measurement.   Sample analysis made easy LabelGuard Applications for ultra low sample volume measurements Cuevette Applications for standard sample volume measurements Functions for self-programming methods User Methods for storage ofself-configured methods Utilities Instrument set up   Applications Nucleic acids: dsDNA, ssDNA, RNA, and Oligonucleotides Labeling: efficiency dye incorporation rate for microarray experiments Proteins: Bradford, Lowry, BCA, Biuret, and UV Protein (A280) Cell density: for microbiology and cell culture For more user comfort, only three steps are necessary to enter method of choice.   The NanoPhotometer™ is equipped with pre-programmed Applications for highest user comfort. Nucleic acids dsDNA, ssDNA, RNA, and Oligonucleotides, Labeling efficiency Proteins Bradford, Lowry, BCA, Biuret, and UV Protein (A280) Cell density for bacterial cell cultures Nucleic Acids Application Nucleic Acids Quantification It is well established that for determination nucleic acid concentration in solution the absorbance at wavelength 260 nm (A260) is used. The function describing the concentration to absorbance relation is the Lambert-Beer Law: A = e * c * d. The absorbance (A) is the product of the substance specific extinction coefficient (e), the concentration of the absorbing sample (c), and the optical pathlength in cm (d). A solution of dsDNA in a 10 mm pathlength cell with an optical density of 1.0 has a concentration of 50 µg/ml. The NanoPhotometer™ uses the factors 50, 40, 37 and 33 as defaults for dsDNA, ssDNA, RNA and oligonucleotides, respectively, and compensates factors for dilution and varying pathlength . Nucleic Acids Estimation of Purity Depending on the extraction/purification or synthesis/purification method of the nucleic acids different impurities can be expected (TRIzol, humic acids, carbohydrates, Guanidine thiocyanate, nucleotides, peptides, EDTA, phenol and protein). It is recommended to include OD ratio measurement (A260/A280 and A260/A230) for purity estimation. Ratio A260/A280 In solution, pure DNA and RNA typically have A260/A280 ratios of 1.8 and 2.0. If the absorbance ratio is significantly less, the nucleic acid is probably contaminated with protein. Accurate quantification of nucleic acid is not reliable without prior purification, and the efficacy of this can be judged by the A260 /A280 ratio. Ratio A260/A230 For RNA samples the ratio values <2.0 point out genomic DNA contamination. Successful DNase I treatment displays in ratio values > 2.0. Ratio values <1.5 indicate impurities of extraction chemicals or incompletely removed constituents of cells. Note: Both ratio values can also be perturbed easily by pH, even if the nucleic acid samples are clean. Use buffers around 7.5 for your measurements. Use of Background Correction Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280nm, respectively, is sometimes used to compensate for the effects of background absorbance. The wavelength used is 320 nm and it can allow for the effects of turbidity, high absorbance buffer solution and the use of reduced aperture cells. The NanoPhotometer™ offers the option of background correction at 320 nm for nucleic acid determination. Dye incorporation rate To determine the dye incorporation rate, the absorbance reading at the wavelength reported for maximum absorbance of the fluorescence dye is used. The corresponding extinction coefficient of the dye is used in the Lambert-Beer Law to determine the dye concentration (c = A / (e * d)). Comparing these values with the DNA concentration gives a dye incorporation rate. FOI = C(dye) / C(nucleic acid) Example: Frequency of Incorporation (FOI) of Cy3 per 1000 bases: FOI(Cy3) = 58.5 * A550/A260 The user can switch between two options of result presentation: a data table or a scan plot. Use of Background Correction The absorbance reading of dye labelled nucleic acids at 260 nm is affected by the dye contribution. To obtain accurate concentration values, the contribution has to be eliminated using correction factors. The NanoPhotometer™ offers the option to select the background correction for dye contribution of absorbance reading at 260 nm for nucleic acid determination.   Protein Applications 1. Protein UV The Protein UV method determines Proteins at 280nm. The effect of nucleic acid in the protein solution due to strong nucleotide absorbance at 280 nm can be compensated by measuring Abs 260, and following the equation of Christian Warburg: Protein (mg/ml) = 1.55*A280 - 0.76* A260 The use of background correction at 320 nm is optional. 2. BCA The BCA method depends on reaction between Cupric ions and peptide bonds, but in addition combines this reaction with the detection of Cuprous ions using bicinchoninic acid (BCA), giving an absorbance maximum at 562 nm. The BCA process is less sensitive to the presence of detergents used to break down cell walls. Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad. The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on). The user can switch between two options of result presentation: a data table or a scan plot. 3. Bradford The Bradford method depends on quantitating the binding of a dye, Coomassie Brilliant Blue, to an unknown protein and comparing this binding to that of different, known concentrations of a standard protein at 595 nm (default setting); this is usually BSA, bovine serum albumin. Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on).     The user can switch between two options of result presentation: a data table or a scan plot. 4. Lowry The Lowry method depends on quantifying the colour obtained from the reaction of Folin-Ciocalteu phenol reagent with the tylsryl residues of an unknown protein and comparing with those derived from a standard curve of a standard protein at 750nm; this is usually BSA, bovine serum albumin Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on).   The user can switch between two options of result presentation: a data table or a scan plot. 5. Biuret The Biuret method measures the reaction between Cupric ions and peptide bonds in an alkali solution, resulting in the formation of a complex absorbing at 546 nm. Select units of measurement: µg/µl, pmol/µl, µg/ml, mg/dl, µg/l, mg/l, g/l, mmol/l, µmol/l, U/l, %, ppm, ppb, conc, none. Calibration mode: Either measurements of prepared standards or manually enter data via keypad The Calibration Screen shows the calibration values and allows standards to be measured. A graph will display the results and the fitted curve as measurements are made. Poor reading can be repeated (with replicates on).   The user can switch between two options of result presentation: a data table or a scan plot. OD 600 Cell Density Applications The method OD 600 is used to determinate the absorbance at 600 nm. Bacterial cell cultures are routinely grown until the absorbance at 600 nm (known as OD600; default setting) reaches approximately 0.4 prior to induction or harvesting. A linear relationship exists between cell number (density) and OD 600 up to approx. 0.6. Select units of measurement: OD, cells/ml. With units OD selected the results output are OD values. If cells/ml was selected two further parameters are displayed (Factor and Multiplier). The measured absorbance in these turbid samples is due to light scattering, and not the result of molecular absorption. A calibration curve can be determined by comparing measured OD 600 to expected OD 600 determined by using an alternative technique (e.g. microscope slide method). Open for future development For special photometric applications or future development, a selection of pre-defined features for general photometric functions enables to create own methods. The configured methods can be saved in nine empty folders (nine methods per folder) under User Methods. The method folder names are editable to allow self-organisation. Complete solution for ultra low and standard volume applications The NanoPhotometerTM is equipped with a selection of pre-defined general photometric features to provide the utmost flexibility in photometry. Scientists are enabled to configure own user methods easily. 1. Single Wavelength The feature Single Wavelength allows simple absorbance (Abs) or transmission (%T) measurements at a single user defined wavelength. The result windows shows the amount of light passed through a sample relative to a reference. 2. Concentration The feature Concentration determines the concentration of samples from a single wavelength reading as a Colorimetric assay. The amount of light that has passed through a sample relative to a reference is multiplied by a factor to obtain the concentration. The factor may be a known figure, or may be calculated by the instrument by measuring a standard of known concentration. The units to present the results are selectable (µg/ml, µg/µl, pmol/µl, mg/dl, mmol/l, µmol/l, g/l, mg/l, µg/l, U/l, %, ppm, ppb, conc or none). 3. Wavescan The feature Wavescan records an absorption (Abs) or transmission (%T) spectrum between two user defined wavelengths, maximum range 190 nm to 1100nm. The spectral plot allows simple identification of peak height and position. Benefit The curve interpretation of the entire spectrum provides most detailed information for a sample. 4. Kinetics The feature Kinetics performs a colorimetric assay which follows the change in absorbance as a function of time at fixed wavelength. Kinetic studies are routinely used for the enzymatic determination of compounds in food, beverage and clinical laboratories by measuring NAD/NADH conversion at 340nm. The change of absorbance per minute (A/min), concentration (A/min x factor) and correlation coefficient (calculated from a best fit of the data points) are displayed. 5. Standard Curve The feature Standard Curve is a colorimetric assay at a single wavelength based on a user programmed curve. The preparation of a multi-point calibration curve from standards of known concentration to quantify unknown samples is a fundamental use of a spectrophotometer; this instrument has the advantage of being able to store this curve as a method, using up to 9 standards. Besides, the data value used for the Standard Curve Calibration can be entered manually. The units to present the results are selectable: µg/ml, µg/µl, pmol/µl, mg/dl, mmol/l, µmol/l, g/l, mg/l, µg/l, U/l, %, ppm, ppb, conc or none. 6. Multi Wavelength The feature Multi Wavelength determines absorbance (Abs) or transmission readings at up to 5 user defined wavelengths relative to a reference. The results are presented in a scan plot covering the range of wavelengths selected and highlighted the relevant wavelength with cursors and a table of values. 7. Absorbance Ratio The feature Absorbance Ratio calculates the ratio of absorbance readings from two user defined wavelength. The amount of light that has passed through a sample relative to a reference at two wavelengths. Volume of the sample and dilution factor can be entered. The unit to present results are selectable: µg/ml, ng/µl, µg/µl. The NanoPhotometer™ offers the possibility for user-configured methods. Technical Specifications Photometric specifcations Photometric mode Absorbance, %Transmission, concentration, scan, ratio, multi and single wavelength, kinetics in ?Abs*factor/min Kinetics mode Start time, end time, interval, numbers of points TBA. Analysis mode LSQ, rate, factor, curve Scan range Full wavelength scan 200nm – 950nm Wavelenght range 190nm - 1100nm Slew rate Measurement time for full spectrum acquired simultaneously in less then <5 sec Photometric range -0.3 – 2.499 Abs0-199% T Photometric Reproducibility ±0.003Abs (0 to 0.5Abs)±0.007Abs (0.5-1.0Abs) @ 260nm Photometric Accuracy ±0.005A or ±1% of the reading, whichever is the greater Wavelength reproducibility < ±0.2nm Wavelength accuracy ± 2 nm Zero stability ±0.003Abs/hour after 20min warm up @ 340nm Noise 0.002A rms at 0A @ 260nm0.005A (pk to pk) at 0A @ 260nm Bandwidth 5 nm Stray Light <0.5% at 220nm and 340nm NaNO2 Optical arrangements Optical arrangements Dual channel Czerny Turner with flat grating, 1024 pixel CCD array Lamp Xenon flash lamp Lifetime 109 flashes Warranty 1 year Performance Verification Auto diagnostics when switched on Methods Programming Method storage Up to 90 user-defined methods Built-in methods for nucleic acids (dsDNA, ssDNA, RNA, Oligos), labelling efficiency (dye incorporation for microarrays), proteins (Bradford, Lowry, BCA, Biuret, UV), bacterial cell density (microbiology) Predefined functions for Self-programming methods: single and multiple wavelength, concentration, wavelength scan, kinetics, standard curve, and ratio calculation Standard Cuvettes Cuvettes cells 10mm cuvette cells with outside dimension 12.5mm x 12.5mm. Quartz SUPRASIL cells are recommended. Cuvette compartment 10mm cell (outside dimension 12.5mm x 12.5mm) Cuvette storage 8 x 10mm cells Electrical Power External autoranging power supply 225 VA Operating voltages 90 - 250 V, (50 Hz / 60 Hz), Max 30VA Operating environment Temperature 10 - 35 °C Cooling Natural air, no internal fan Heated cell holder Not necessary Input / Output Ports USB USB is standard for connection to a PVC for direct data download for spreadsheet calculations, printout and data storage Built-In Printer Internal Serial interface to optional external printer. Graphics to be printed out on thermal high stability paper, 56mm wide, and 40 columns BlueTooth Internal Serial interface to optional Blue Tooth wireless link for connection to a PC Safety The instruments is designed to conform with: EN-61010-1:2001 and EN 6010-2-101: 2002 Certification To Article 5 of the Low Voltage Directive (73/23/EEC), the EMC Directive (89/336/EEC) and the IVD Directive The standards used shall be EN 61010-1: 2001 safety requirements for electrical equipment for measurement, control and laboratory use EN 61326-2.3: 1998 electromagnetic compatibility - generic emission standard Electrical equipment for measurement, control and laboratory use EN 61000-4-6: 1992 electromagnetic compatibility - generic immunity standard part 1. Residential, commercial and light industry Specifications are subject to change without notice.