NanoDrop A260/A230 Ratio: Purity Assessment Explained
By Dr. Zubair Khalid, DVM, MS, PhD ·
Introduction to NanoDrop and the A260/A230 Ratio
What is a NanoDrop?
The NanoDrop is a microvolume ultraviolet-visible (UV-Vis) spectrophotometer manufactured by Thermo Fisher Scientific that measures the absorbance of light by biomolecules in solution. Unlike conventional spectrophotometers that require cuvettes holding 50–100 µL of sample, the NanoDrop uses surface tension to hold a 1–2 µL droplet between two optical fibers, eliminating the need for cuvettes and enabling measurements of highly concentrated samples without dilution. This design has made the NanoDrop the standard instrument in molecular biology laboratories for quantifying nucleic acids (DNA and RNA) and proteins.
The instrument works by generating light from a xenon flash lamp, which passes through the sample droplet. A spectrometer then measures the intensity of light that passes through, comparing it to a reference measurement taken during the blanking step. The instrument reports absorbance values at multiple wavelengths, most importantly 260 nm, 280 nm, and 230 nm, along with calculated ratios between these values. The two most commonly reported ratios are A260/A280, which primarily indicates protein contamination, and A260/A230, which indicates contamination by a broader range of substances including chaotropic salts, carbohydrates, and organic solvents. For a detailed discussion of the A260/A280 ratio, see our article on Nanodrop A260/a280.
The A260/A230 Ratio Defined
The A260/A230 ratio is the quotient of absorbance measured at 260 nm divided by absorbance measured at 230 nm. It serves as an indicator of nucleic acid purity, specifically detecting contamination by substances that absorb light at 230 nm. These contaminants include phenol, guanidine hydrochloride, guanidine thiocyanate, EDTA, carbohydrates, and some proteins. A pure nucleic acid sample typically exhibits an A260/A230 ratio in the range of 2.0–2.2. Values significantly below this range indicate the presence of contaminants that may interfere with downstream applications such as restriction enzyme digestion, PCR, sequencing, or transfection.
The ratio is particularly important because it is more sensitive than the A260/A280 ratio to certain contaminants. For example, guanidine salts used in silica-column-based RNA purification kits absorb strongly at 230 nm but have minimal absorbance at 280 nm, meaning a sample could have an acceptable A260/A280 ratio of 1.8–2.0 while still being heavily contaminated with guanidine. The A260/A230 ratio catches this contamination that the A260/A280 ratio would miss.
Principle of UV Spectrophotometry in Nucleic Acid Quantification
Absorbance at 260 nm
Nucleic acids absorb ultraviolet light maximally at 260 nm due to the aromatic ring structures of the purine and pyrimidine bases. Adenine, guanine, cytosine, thymine, and uracil all contain conjugated double-bond systems that absorb light in the 250–270 nm range, with a peak absorbance at approximately 260 nm. The Beer-Lambert law governs this relationship: A = εcl, where A is absorbance, ε is the molar extinction coefficient, c is the concentration, and l is the path length.
The NanoDrop uses a path length of 1 mm (0.1 cm) for standard measurements, which is one-tenth the path length of a conventional cuvette. This short path length allows measurement of samples with concentrations up to 15,000 ng/µL for double-stranded DNA without dilution. The instrument automatically converts the measured absorbance to a 1 cm path length equivalent for reporting purposes.
For double-stranded DNA, an absorbance of 1.0 at 260 nm corresponds to approximately 50 ng/µL. For single-stranded RNA, the conversion factor is approximately 40 ng/µL, and for single-stranded oligonucleotides, it is approximately 33 ng/µL. These conversion factors are built into the NanoDrop software and are used to calculate nucleic acid concentration from the measured absorbance.
Absorbance at 230 nm
The 230 nm wavelength is in the far-UV region of the electromagnetic spectrum. Many organic compounds absorb light at this wavelength because they contain peptide bonds, aromatic amino acids, or other functional groups with electronic transitions in this range. The absorbance at 230 nm is not specific to any single class of molecules; rather, it captures a broad spectrum of potential contaminants.
Key contaminants that absorb at 230 nm include:
- Phenol: Used in traditional phenol-chloroform extraction methods, phenol has significant absorbance at 230 nm with a peak around 270 nm.
- Guanidine salts: Guanidine hydrochloride and guanidine thiocyanate are chaotropic agents used in many commercial nucleic acid purification kits. They absorb strongly at 230 nm.
- EDTA: Ethylenediaminetetraacetic acid, commonly used to chelate divalent cations in buffers, absorbs at 230 nm.
- Carbohydrates: Polysaccharides and other sugars absorb at 230 nm and can co-purify with nucleic acids, particularly from plant tissues.
- Some proteins: While proteins absorb primarily at 280 nm due to tryptophan and tyrosine residues, the peptide bond itself absorbs at 210–230 nm.
The A260/A230 ratio thus provides a broad measure of contamination by organic compounds that absorb in the far-UV range.
How the Ratio is Calculated
The NanoDrop measures absorbance at multiple wavelengths simultaneously using a diode array or charge-coupled device (CCD) detector. The instrument records the absorbance at 260 nm and at 230 nm, then calculates the ratio as:
A260/A230 = Absorbance at 260 nm ÷ Absorbance at 230 nm
Both absorbance values are corrected for the blank measurement, which is taken using the buffer or solution in which the nucleic acid is dissolved. The blanking step is critical because buffers themselves absorb UV light, and failure to blank properly will skew both the absolute absorbance values and the calculated ratios.
The NanoDrop software also applies a baseline correction by subtracting the absorbance at 340 nm, where neither nucleic acids nor common contaminants absorb significantly. This correction accounts for any light scattering caused by particulate matter in the sample, which would otherwise inflate absorbance readings across all wavelengths.
What Does the A260/A230 Ratio Indicate?
Expected Values for Pure Samples
For pure nucleic acid samples, the expected A260/A230 ratio is 2.0–2.2. This range applies to both double-stranded DNA and RNA. The theoretical maximum for a perfectly pure nucleic acid sample is approximately 2.2, though values up to 2.4 are occasionally observed in practice.
The ratio is slightly higher for RNA than for DNA in some cases, but the difference is small and not diagnostically significant. What matters is whether the ratio falls within the acceptable range. Values between 1.8 and 2.0 are generally considered acceptable for most downstream applications, though they may indicate minor contamination. Values below 1.8 warrant investigation and possible purification.
It is important to note that the A260/A230 ratio is not a fixed constant; it depends on the buffer composition, pH, and ionic strength of the solution. For example, nucleic acids dissolved in Tris-EDTA (TE) buffer at pH 8.0 will have a different A260/A230 ratio than the same nucleic acids dissolved in water at pH 7.0. This is because the absorbance of nucleic acids at 230 nm is pH-dependent, with higher pH values generally increasing absorbance at this wavelength.
Interpreting Low Ratios
An A260/A230 ratio below 1.8 indicates contamination by substances that absorb at 230 nm. The lower the ratio, the more severe the contamination. A ratio of 1.5–1.8 suggests moderate contamination that may still allow successful downstream applications, while a ratio below 1.5 indicates significant contamination that will likely interfere with enzymatic reactions.
Low ratios most commonly arise from:
- Residual guanidine salts from column-based purification kits
- Residual phenol from organic extraction
- EDTA carried over from elution buffers
- Carbohydrates co-purified from plant or fungal sources
- High concentrations of certain buffer components
The interpretation of a low ratio depends on the downstream application. PCR and restriction enzyme digestion are relatively tolerant of moderate contamination, while sensitive applications such as next-generation sequencing library preparation, transfection, and in vitro transcription are more susceptible to inhibition.
Interpreting High Ratios
A260/A230 ratios above 2.2 are less common but can occur. Values up to approximately 2.4 are generally not a cause for concern and may simply reflect the specific buffer conditions or the absence of any UV-absorbing contaminants. However, ratios significantly above 2.4 should be interpreted with caution, as they may indicate:
- An error in blanking, where the blank solution absorbed more at 230 nm than the sample solution
- The presence of compounds that absorb at 260 nm but not at 230 nm, inflating the ratio
- Very low nucleic acid concentration, where the absorbance values are near the detection limit and the ratio becomes noisy
In practice, a high A260/A230 ratio is rarely a problem. The more common and more concerning scenario is a low ratio indicating contamination.
Common Contaminants Affecting the A260/A230 Ratio
Phenol and Guanidine
Phenol is used in traditional liquid-liquid extraction methods for nucleic acid purification. In this procedure, a sample is mixed with phenol and chloroform, and after centrifugation, the aqueous phase containing nucleic acids is separated from the organic phase containing proteins and lipids. Residual phenol in the aqueous phase absorbs strongly at 230 nm and can significantly lower the A260/A230 ratio.
Guanidine salts, specifically guanidine hydrochloride and guanidine thiocyanate, are chaotropic agents that denature proteins and are used in many commercial nucleic acid purification kits. These kits typically use silica membranes or magnetic beads that bind nucleic acids in the presence of high concentrations of guanidine salts. If the washing steps are insufficient, residual guanidine remains in the eluted sample and absorbs at 230 nm.
The presence of guanidine contamination is particularly insidious because it does not significantly affect the A260/A280 ratio. A sample contaminated with guanidine may have an A260/A280 ratio of 1.9–2.0, which appears acceptable, while the A260/A230 ratio reveals the problem. This is why the A260/A230 ratio is an essential complement to the A260/A280 ratio for assessing nucleic acid purity. For more information on the A260/A280 ratio, see our article on Nanodrop 260/280.
Carbohydrates and Polysaccharides
Carbohydrates and polysaccharides are common contaminants when isolating nucleic acids from plant tissues, fungal samples, or certain bacterial species. These molecules have hydroxyl groups and glycosidic bonds that absorb UV light in the 230 nm range. Plant tissues, in particular, contain high levels of polysaccharides that can co-precipitate with nucleic acids during alcohol precipitation.
The challenge with carbohydrate contamination is that it is more difficult to remove than guanidine or phenol contamination. Standard ethanol precipitation may not effectively separate nucleic acids from polysaccharides, and additional purification steps such as size-exclusion chromatography or specialized extraction buffers may be required. Carbohydrate contamination is often suspected when the A260/A230 ratio is low but the A260/A280 ratio is normal, and when the sample has a viscous or mucilaginous appearance.
EDTA and Other Salts
EDTA is a chelating agent commonly included in DNA storage buffers such as TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). EDTA absorbs UV light at 230 nm, and if a sample is eluted in a buffer containing EDTA, the A260/A230 ratio will be lower than if the same sample were eluted in water or a buffer without EDTA.
This is an important consideration when interpreting A260/A230 values. A DNA sample eluted in TE buffer will typically have an A260/A230 ratio of approximately 1.8–2.0, while the same DNA eluted in water will have a ratio of 2.0–2.2. The difference is due to the absorbance of EDTA at 230 nm, not to any difference in the DNA itself.
Other salts, including sodium acetate, sodium chloride, and ammonium acetate, can also affect the A260/A230 ratio, though their absorbance at 230 nm is generally weaker than that of EDTA. High concentrations of these salts can also affect the refractive index of the solution, which can influence the measurement.
How to Measure the A260/A230 Ratio with a NanoDrop
Sample Preparation
Proper sample preparation is essential for obtaining reliable A260/A230 measurements. The nucleic acid sample should be thoroughly mixed before measurement to ensure homogeneity. If the sample was stored frozen, it should be thawed completely and mixed by pipetting or gentle vortexing before measurement.
The sample should be free of particulate matter, as particles can scatter light and inflate absorbance readings. If the sample appears turbid or contains visible particulates, it should be centrifuged briefly (e.g., 10,000 × g for 1 minute) and the supernatant used for measurement.
The choice of blank solution is critical. The blank should be the same buffer in which the nucleic acid is dissolved. For example, if the DNA is eluted in TE buffer, the blank should be TE buffer. If the DNA is in water, the blank should be water. Using a different buffer for the blank than the sample buffer will introduce systematic errors in both the absorbance values and the calculated ratios.
Measurement Procedure
The measurement procedure for a NanoDrop instrument follows a standardized protocol:
- Initialize the instrument: Open the NanoDrop software and select the appropriate application (e.g., "Nucleic Acid" for DNA or RNA quantification).
- Blank the instrument: Pipette 1–2 µL of the blank solution onto the lower optical pedestal. Lower the upper arm to make contact with the droplet, and select "Blank" in the software. The instrument measures the absorbance spectrum of the blank and uses it as the reference for subsequent measurements.
- Measure the sample: Wipe the pedestals with a lint-free laboratory wipe. Pipette 1–2 µL of the nucleic acid sample onto the lower pedestal, lower the upper arm, and select "Measure" in the software.
- Record the results: The software displays the absorbance spectrum, the absorbance at 260 nm, 280 nm, and 230 nm, the calculated concentration, and the A260/A280 and A260/A230 ratios.
- Repeat for additional samples: Wipe the pedestals between samples to prevent cross-contamination. The blank should be re-measured periodically, especially if the instrument has been idle for more than a few minutes or if a new batch of samples is being measured.
- Clean the instrument: After all measurements are complete, clean the pedestals with a lint-free wipe and a small amount of distilled water or 70% ethanol.
Interpreting the Readout
The NanoDrop software reports the A260/A230 ratio alongside the concentration and the A260/A280 ratio. The readout also displays the full absorbance spectrum from 220 nm to 350 nm, which can be useful for diagnosing contamination. A pure nucleic acid sample will show a smooth absorbance curve with a peak at 260 nm and a relatively flat baseline. Contamination is often visible as an elevated baseline or additional peaks in the spectrum.
When interpreting the readout, it is important to consider the concentration of the sample. The A260/A230 ratio becomes unreliable at very low concentrations (below approximately 10 ng/µL) because the absorbance values are near the detection limit of the instrument, and the ratio of two small numbers is subject to high variability. For accurate ratio measurements, the nucleic acid concentration should be above 20 ng/µL.
Factors That Can Skew the A260/A230 Ratio
pH and Buffer Composition
The absorbance of nucleic acids at 230 nm is pH-dependent. At pH values below 6.0, the absorbance of nucleic acids at 230 nm decreases, which can artificially elevate the A260/A230 ratio. Conversely, at pH values above 8.5, the absorbance at 230 nm increases, which can lower the ratio.
This pH dependence means that the same DNA sample will give different A260/A230 values depending on the buffer in which it is dissolved. For example, DNA in 10 mM Tris-HCl (pH 8.0) will have a lower A260/A230 ratio than the same DNA in water (pH 7.0). This is not a measurement error; it reflects the genuine pH-dependent absorbance properties of the nucleic acids.
The buffer composition also matters beyond pH. As discussed above, EDTA absorbs at 230 nm and will lower the A260/A230 ratio. Other buffer components, such as Tris itself, also have some absorbance in the far-UV range. For this reason, it is best practice to elute nucleic acids in water or a low-ionic-strength buffer (e.g., 5 mM Tris, pH 8.0) when accurate A260/A230 measurements are needed.
Sample Concentration
The A260/A230 ratio is concentration-dependent at both extremes of the measurable range. At very low concentrations (below 10 ng/µL), the absorbance values are small, and the signal-to-noise ratio is poor. The ratio of two small, noisy numbers can vary substantially between replicate measurements.
At very high concentrations (above 3,000 ng/µL for the standard 1 mm path length), the absorbance at 260 nm may exceed the linear range of the detector, leading to inaccurate absorbance readings. The NanoDrop automatically adjusts the path length for concentrated samples, but this adjustment introduces its own uncertainties.
For reliable A260/A230 measurements, the sample concentration should be in the range of 20–2,000 ng/µL. If the sample is more concentrated, it should be diluted with the same buffer used for the blank. If the sample is less concentrated, the measurement should be interpreted with caution, and replicate measurements should be taken to assess variability.
Instrument Calibration and Maintenance
The NanoDrop requires regular calibration and maintenance to provide accurate measurements. The xenon lamp has a finite lifespan and its output decreases over time, which can affect absorbance readings across all wavelengths. The optical fibers and pedestals must be kept clean, as residue from previous samples can absorb UV light and contaminate subsequent measurements.
Most NanoDrop instruments include a self-calibration function that uses a reference standard (typically a dye solution with known absorbance properties) to verify the accuracy of the wavelength and absorbance measurements. This calibration should be performed regularly according to the manufacturer's recommendations. For detailed instructions on instrument operation and maintenance, refer to the Nanodrop Eight User Manual or the Nanodrop Ultra User Manual.
If the instrument has not been calibrated recently, or if the lamp has exceeded its recommended lifespan, the A260/A230 values may be unreliable. In such cases, the absolute absorbance values at 260 nm and 230 nm should be examined for consistency with expected values for the sample type.
Troubleshooting Low A260/A230 Ratios
Re-precipitation with Ethanol
The most straightforward method for removing contaminants that lower the A260/A230 ratio is ethanol precipitation. This procedure exploits the differential solubility of nucleic acids versus most contaminants in ethanol solutions.
To re-precipitate nucleic acids:
- Add salt: Add sodium acetate (pH 5.2) to a final concentration of 0.3 M, or ammonium acetate to a final concentration of 2.5 M. The salt neutralizes the negative charge on the phosphate backbone, allowing the nucleic acids to precipitate.
- Add ethanol: Add 2.5 volumes of ice-cold 100% ethanol for sodium acetate precipitation, or 2 volumes for ammonium acetate precipitation. Mix thoroughly by inverting the tube.
- Incubate: Incubate at -20°C for at least 30 minutes, or at -80°C for 15 minutes. Longer incubation times improve precipitation efficiency, especially for small nucleic acids.
- Centrifuge: Centrifuge at 12,000–15,000 × g for 15–30 minutes at 4°C. The nucleic acid pellet should be visible at the bottom of the tube.
- Wash: Carefully remove the supernatant and wash the pellet with 70% ethanol. This step removes residual salt and contaminants that remain in the supernatant. Centrifuge again at 12,000 × g for 5 minutes and remove the supernatant.
- Dry and resuspend: Air-dry the pellet for 5–10 minutes to remove residual ethanol. Resuspend in water or the desired buffer.
Ethanol precipitation is effective at removing guanidine salts, phenol, and many other organic contaminants. However, it is less effective at removing carbohydrates and polysaccharides, which can co-precipitate with nucleic acids.
Column Purification
If ethanol precipitation does not adequately improve the A260/A230 ratio, or if the contaminant is a carbohydrate or other molecule that co-precipitates with nucleic acids, column-based purification may be necessary. Commercial silica-column kits are available for both DNA and RNA purification and are effective at removing a wide range of contaminants.
The principle of column purification is that nucleic acids bind to a silica membrane in the presence of high concentrations of chaotropic salts, while contaminants pass through. After washing with an ethanol-containing buffer to remove residual salts, the nucleic acids are eluted in water or a low-salt buffer.
When using column purification to improve the A260/A230 ratio, it is important to follow the manufacturer's protocol carefully, paying particular attention to the washing steps. Incomplete washing is a common cause of residual guanidine contamination, which will keep the A260/A230 ratio low.
When to Proceed Despite Low Ratio
There are situations where proceeding with a low A260/A230 ratio is acceptable. If the downstream application is relatively tolerant of contaminants, such as standard PCR or restriction enzyme digestion, moderate contamination (A260/A230 of 1.5–1.8) may not affect the results. In such cases, the time and sample loss associated with additional purification may not be justified.
However, for sensitive applications such as next-generation sequencing, quantitative PCR, in vitro transcription, or transfection, a low A260/A230 ratio should be addressed before proceeding. Contaminants can inhibit enzymes, interfere with nucleic acid modification reactions, or reduce transfection efficiency.
It is also worth noting that the A260/A230 ratio is just one indicator of nucleic acid quality. The integrity of the nucleic acid, as assessed by agarose gel electrophoresis or microfluidic analysis, is equally important. A sample with a low A260/A230 ratio but intact, high-molecular-weight nucleic acid may perform better in downstream applications than a sample with a perfect A260/A230 ratio but degraded nucleic acid.
Common Pitfalls and Best Practices
Pitfall: Ignoring the Blank
One of the most common errors in NanoDrop measurements is failing to blank the instrument correctly. The blank must be the exact same buffer in which the nucleic acid is dissolved. If the blank is water but the sample is in TE buffer, the absorbance of the TE buffer will be subtracted from the sample measurement, leading to an artificially low absorbance at 230 nm and an artificially high A260/A230 ratio.
Conversely, if the blank is TE buffer but the sample is in water, the absorbance of the TE buffer will be added to the sample measurement, leading to an artificially high absorbance at 230 nm and an artificially low A260/A230 ratio.
The blank should also be re-measured periodically, especially if the instrument has been idle or if the buffer composition has changed. A fresh blank should be measured for each new batch of samples.
Pitfall: Measuring Too Concentrated Samples
The NanoDrop can measure samples up to 15,000 ng/µL for double-stranded DNA, but the accuracy of the measurement decreases at very high concentrations. The instrument adjusts the path length automatically, but this adjustment introduces uncertainty, particularly for the A260/A230 ratio.
For accurate A260/A230 measurements, the sample concentration should be below 2,000 ng/µL. If the sample is more concentrated, it should be diluted with the same buffer used for the blank. The dilution factor should be recorded so that the concentration can be calculated correctly.
Best Practice: Consistent Pipetting
The NanoDrop measurement is sensitive to the volume and placement of the sample droplet. A volume that is too small (less than 1 µL) may not form a complete bridge between the two optical fibers, leading to an inaccurate measurement. A volume that is too large (more than 2 µL) may overflow the pedestal, also causing inaccuracies.
Consistent pipetting technique is essential for reproducible measurements. The pipette should be calibrated regularly, and the same pipette should be used for both the blank and the sample. The droplet should be placed in the center of the lower pedestal, and the upper arm should be lowered gently to avoid introducing air bubbles.
For best results, each sample should be measured in duplicate or triplicate, and the average of the measurements should be used. This is particularly important for samples with low concentrations, where the measurement variability is higher.
Pitfall: Cross-Contamination Between Samples
The pedestals of the NanoDrop must be wiped clean between measurements to prevent cross-contamination between samples. Residual nucleic acid from a previous sample will contribute to the absorbance of the next sample, skewing both the concentration and the ratios.
The pedestals should be wiped with a lint-free laboratory wipe between each measurement. If the previous sample was highly concentrated, the pedestals should be cleaned with a small amount of distilled water or 70% ethanol before the next measurement.
Best Practice: Examining the Full Spectrum
The NanoDrop software displays the full absorbance spectrum from 220 nm to 350 nm. This spectrum contains valuable diagnostic information that is not captured by the A260/A230 ratio alone. A pure nucleic acid sample shows a smooth curve with a peak at 260 nm and a relatively flat baseline. An elevated baseline or additional peaks in the spectrum indicate contamination.
For example, a peak at 280 nm suggests protein contamination, while an elevated baseline across the entire spectrum suggests particulate matter or turbidity. Examining the spectrum can help identify the source of contamination and guide the appropriate purification strategy.
Summary and Key Takeaways
The A260/A230 ratio is a critical parameter for assessing nucleic acid purity, complementing the more commonly discussed A260/A280 ratio. While the A260/A280 ratio primarily detects protein contamination, the A260/A230 ratio detects a broader range of contaminants, including guanidine salts, phenol, EDTA, and carbohydrates. These contaminants absorb light at 230 nm and can interfere with downstream applications even when the A260/A280 ratio appears acceptable.
For pure nucleic acids, the expected A260/A230 ratio is 2.0–2.2. Values below 1.8 indicate contamination that may warrant further purification, while values above 2.4 are unusual and may indicate measurement artifacts. The ratio is influenced by buffer composition, pH, and sample concentration, and these factors must be considered when interpreting results.
The NanoDrop provides a rapid, low-volume method for measuring the A260/A230 ratio, but accurate measurements require proper blanking, consistent pipetting, and attention to sample concentration. When the ratio is low, ethanol precipitation or column purification can remove contaminants and improve nucleic acid quality.
For a comprehensive understanding of nucleic acid purity assessment, it is essential to consider both the A260/A230 and A260/A280 ratios together, along with the full absorbance spectrum and the integrity of the nucleic acid as assessed by gel electrophoresis. For more information on related topics, see our articles on RNA Nanodrop 260/230, Nanodrop A280 Protein Concentration, and Nano-300 Nanodrop.
Frequently Asked Questions
What is a good A260/A230 ratio for DNA?
A good A260/A230 ratio for DNA is 2.0–2.2. Values between 1.8 and 2.0 are generally acceptable for most downstream applications, though they may indicate minor contamination. Values below 1.8 warrant investigation and possible purification. The ratio can be slightly lower (approximately 1.8–2.0) if the DNA is eluted in a buffer containing EDTA, which absorbs at 230 nm.
Why is my A260/A230 ratio low?
A low A260/A230 ratio (below 1.8) indicates contamination by substances that absorb light at 230 nm. Common culprits include residual guanidine salts from column-based purification kits, phenol from organic extraction, EDTA from storage buffers, and carbohydrates from plant or fungal sources. The specific contaminant can often be identified by examining the full absorbance spectrum and considering the purification method used.
Does the A260/A230 ratio indicate protein contamination?
The A260/A230 ratio is not the primary indicator of protein contamination. Proteins absorb primarily at 280 nm due to tryptophan and tyrosine residues, so protein contamination is better assessed by the A260/A280 ratio. However, the peptide bonds in proteins do absorb at 210–230 nm, so severe protein contamination can lower the A260/A230 ratio. For a detailed discussion of protein contamination assessment, see our article on Nanodrop A260/a280.
Can I use the A260/A230 ratio to assess RNA purity?
Yes, the A260/A230 ratio is applicable to RNA purity assessment. Pure RNA should have an A260/A230 ratio of 2.0–2.2, similar to DNA. Guanidine thiocyanate, which is commonly used in RNA purification kits, absorbs strongly at 230 nm, so a low A260/A230 ratio in RNA samples often indicates residual guanidine contamination. For more information specific to RNA, see our article on RNA Nanodrop 260/230.
What does an A260/A230 ratio above 2.2 mean?
An A260/A230 ratio above 2.2 is generally not a cause for concern, provided the value is not excessively high (above 2.4). Slightly elevated ratios may reflect the specific buffer conditions or the absence of any UV-absorbing contaminants. However, very high ratios may indicate an error in blanking, where the blank solution absorbed more at 230 nm than the sample solution, or very low nucleic acid concentration, where the ratio becomes noisy.
How can I improve my A260/A230 ratio?
To improve a low A260/A230 ratio, the contaminants must be removed. Ethanol precipitation with sodium acetate or ammonium acetate is effective for removing guanidine salts, phenol, and many organic contaminants. Column-based purification using silica membranes is more effective for removing carbohydrates and other contaminants that co-precipitate with nucleic acids. Ensuring complete removal of the wash buffer during column purification is critical for eliminating residual guanidine contamination.
Is the A260/A230 ratio affected by pH?
Yes, the A260/A230 ratio is pH-dependent. The absorbance of nucleic acids at 230 nm decreases at pH values below 6.0 and increases at pH values above 8.5. This means that the same nucleic acid sample will give different A260/A230 values depending on the pH of the buffer. For consistent results, nucleic acids should be measured in a buffer with a defined pH, ideally between 7.0 and 8.5.
Key Takeaways
- The A260/A230 ratio measures nucleic acid purity by comparing absorbance at 260 nm (nucleic acids) to absorbance at 230 nm (contaminants).
- Pure DNA and RNA samples have A260/A230 ratios of 2.0–2.2; values below 1.8 indicate contamination.
- The A260/A230 ratio detects contaminants that the A260/A280 ratio misses, particularly guanidine salts, phenol, EDTA, and carbohydrates.
- The ratio is pH-dependent and buffer-dependent; always blank with the exact buffer in which the sample is dissolved.
- Low ratios can be improved by ethanol precipitation or column purification, but the appropriate method depends on the contaminant.
- The A260/A230 ratio is unreliable at very low concentrations (below 10 ng/µL) and should be interpreted with caution for such samples.
- Always examine the full absorbance spectrum and consider both the A260/A230 and A260/A280 ratios together for a complete assessment of nucleic acid purity.