Spectrophotometry is a fundamental technique in molecular biology used to quickly determine the concentration of nucleic acids, such as DNA and RNA, in a laboratory sample. While nucleic acids absorb maximally at 260 nm, the purity of the sample must also be assessed using specific absorbance ratios. The 260/280 ratio primarily indicates protein contamination, but the 260/230 ratio specifically signals the carryover of chemical compounds from the purification process.
Interpreting the 260/230 Ratio and Ideal Values
The 260/230 ratio serves as a sensitive indicator of residual chemical contamination in a purified nucleic acid sample. This ratio is calculated by dividing the absorbance reading at 260 nm (nucleic acid signal) by the absorbance reading at 230 nm (contaminant signal). A low ratio means the 230 nm reading is disproportionately high compared to the 260 nm signal, indicating significant impurities.
For highly purified DNA or RNA, the expected 260/230 ratio commonly falls within the range of 2.0 to 2.2. A value substantially below 2.0 suggests contamination, and readings below 1.5 often signal severe impurity that may negatively affect downstream applications.
A low 260/230 ratio also carries the risk of concentration overestimation, as some contaminants may cause a slight crossover in absorbance at 260 nm. This suggests that the calculated concentration of the nucleic acid might be inaccurately high. Accurate interpretation of this ratio is a routine quality control step before proceeding with further molecular work.
Specific Contaminants That Absorb at 230 nm
The primary cause of a low 260/230 ratio is the incomplete removal of chaotropic salts, such as guanidinium thiocyanate or guanidine hydrochloride. These salts are potent absorbers around 230 nm and are used in column-based purification kits to lyse cells and bind nucleic acids. Their carryover into the final sample drastically lowers the ratio.
Residual organic solvents, such as phenol or Trizol reagent, are another common source of 230 nm absorption. These compounds are used in traditional extraction methods and exhibit strong absorbance near 230 nm. Incomplete phase separation during extraction allows traces of these chemicals to remain in the final aqueous phase.
Common 230 nm Absorbing Contaminants
Several other chemicals used during purification can also contribute to a reduced ratio:
- Chaotropic salts (e.g., guanidinium salts)
- Organic solvents (e.g., phenol, Trizol)
- Chelating agents (e.g., EDTA)
- Buffer salts
- Non-ionic detergents (e.g., Triton X-100, Tween 20)
- Residual carbohydrates or polysaccharides (especially from plant samples)
- Glycogen, often added as a carrier during precipitation
Practical Impact on Downstream Molecular Assays
The presence of 230 nm absorbing contaminants is problematic because these chemicals often function as potent inhibitors of enzymatic reactions. Chaotropic salts, for example, interfere with the structure and function of various enzymes fundamental to downstream molecular assays. Even relatively small amounts of these residual chemicals can significantly compromise experimental success.
In polymerase chain reaction (PCR) and quantitative PCR (qPCR), contaminating salts and organic solvents directly inhibit the DNA polymerase enzyme. This inhibition leads to poor amplification efficiency, inaccurate quantification, or complete reaction failure, often requiring costly repetition. Similarly, reverse transcriptase, which synthesizes complementary DNA (cDNA) from RNA, is highly sensitive to these inhibitors, resulting in low yield or poor quality template.
For next-generation sequencing (NGS) library preparation, the consequences of a low 260/230 ratio are particularly severe. The NGS workflow relies on precise enzymatic steps, such as adapter ligation and amplification, all of which are easily inhibited by residual contaminants. A low ratio indicates the sample is unsuitable for sensitive, enzyme-based molecular applications, potentially causing low library yields or sequencing run failure.
Strategies for Improving Nucleic Acid Purity
When a low 260/230 ratio is observed, the primary corrective action is optimizing the wash steps in the purification protocol to ensure complete removal of residual chemicals. Performing an additional wash with the kit’s ethanol-based wash buffer can significantly reduce chaotropic salts. Allowing the wash buffer to sit on the column membrane for an extended period, such as one minute, before centrifugation facilitates more thorough contaminant removal.
Incomplete drying of the silica membrane is a common error, as residual ethanol contributes to a low ratio. After the final wash, the column must be centrifuged multiple times to remove all traces of ethanol before elution. For severe contamination, the sample should be purified again using a secondary clean-up kit or ethanol precipitation followed by multiple washes with 70% ethanol.
Using a pre-warmed elution buffer can improve the nucleic acid yield. This helps raise the ratio by increasing the signal at 260 nm relative to the contaminant signal at 230 nm.