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.

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.

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 can be determined in the near UV at 280 nm due to absorption by tyrosine, tryptophan and phenylalanine amino acids; Abs 280 varies greatly for different proteins due to their amino acid content, and consequently the specific A280 factor for a particular protein must be determined (see also application sheet Protein Formulas).

The protein concentration can be calculated the following way:

• c prot. = (Abs. 280 * A280 factor) * lid factor * dilution factor
• With background correction:
• c prot. = (Abs. 280 – Abs. 320) * A280 factor * lid factor * dilution factor

This equation can be applied to other proteins if the corresponding factors are known (please note that the factor used by the NanoPhotometer™ is the reciprocal value of the extinction coefficient (l/g*cm) from a protein). The instrument can determine protein concentration at 280 nm and uses the above equation as default; the factors can be changed, and the use of background correction at 320 nm is optional.

The A280 Factor is based on the extinction coefficient of the protein [molecular weight/molar extinction coefficient (M-1*cm-1) or 1/extinction coefficient (l/g*cm)]

In the new NanoPhotometer™ software are the following protein A280 factors pre-programmed:
BSA (bovine serum albumin), serum albumin (mouse and human), lysozyme (chicken) and IgG (mouse).

There is also the possibility to enter custom factors. For correct calculation the following settings are needed, either the extinction coefficient (l/g*cm) or the molar extinction coefficient (M-1*cm-1) and the molecular weight (g/mol) of the protein.

Rapid measurements such as this at 280 nm are particularly useful after isolation of proteins and peptides from mixtures using spin and HiTrap columns by centrifuge and gravity, respectively.

The Protein Dye method determines proteins at 280 nm as well as dye incorporation. Common dye types are preprogrammed for convenience or a custom dye can be specified from 190-1100nm.

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/ml, 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 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 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 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 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.

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).

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.

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.

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.