Sponsored Content by DeNovix Inc.Reviewed by Olivia FrostSep 13 2024
This article is based on a poster originally authored by DeNovix.
Absorbance and fluorescence are distinct yet complementary approaches for determining the concentration and quality of biological materials. Choosing the right quantification techniques for biological samples is critical in any quality control procedure.
Absorbance
Absorbance methods measure the amount of light absorbed by a sample at a specific wavelength. The absorbance is directly proportional to the sample's concentration, enabling accurate quantification.
Microvolume
- 1 µL sample volume
- No sample dilution is required
Image Credit: DeNovix Inc.
Cuvette
- Larger sample volume
- Higher sensitivity compared to Microvolume
Image Credit: DeNovix Inc.
Fluorescence
Fluorophores bind to the sample of interest and are excited by a specific wavelength of light, with emission measured at a higher wavelength. The sample fluorescence is then compared to known standards for quantification.
0.5 mL PCR tubes
- 200 µL of assay volume
- 1-20 µL of sample
- Select from a variety of fluorophores and assay kits.
Image Credit: DeNovix Inc.
Applications
Absorbance applications
- dsDNA
- RNA
- ssDNA
- Protein A280
- Peptides
- Kinetics
- Labeled DNA/Protein
- OD600
- Colorimetrics
- UV/Vis
Fluorescence applications
- dsDNA
- RNA
- ssDNA
- Protein
- Basic Fluorometer
- Custom Assays
DeNovix DS-11 FX+ Spectrophotometer / Fluorometer feature comparison
Absorbance
- Sensitivity: 0.75 ng/µL
- Specificity: Not analyte-specific
- Dynamic Range: 37500 ng/µL
- Sample Volume: 0.5 to 1 µL
- Detect Contamination: Detects co-extracted contaminants
- Assay Cost: Zero cost/sample
- Speed: Load and measure in seconds
Fluorescence
- Sensitivity: 0.0005 ng/µL
- Specificity: Highly specific
- Dynamic Range: 4000 ng/µL
- Sample Volume: 1 to 20 µL
- Detect Contamination: No contamination information
- Assay Cost: Range of assays
- Speed: Assay setup required
The verdict
Absorbance
A quick and simple method for quantifying a wide range of samples with no assay costs or setup time. Microvolume measurements are possible throughout a large dynamic range, including all routinely used sample concentrations. Contaminants can be recognized, providing extra quality control.
Fluorescence
Fluorescence methods offer enhanced sensitivity, enabling quantification down to sub-picogram per microliter concentrations. These assays are highly specific to the analyte, ensuring accurate quantification without interference from contaminants. Assay kits and standards of known concentration are required for these measurements.
Combining measurements
Image Credit: DeNovix Inc.
The DS-11 Series Spectrophotometer / Fluorometer allows for both absorbance and fluorescence measurements in a single instrument. Combining these two methods increases confidence and accuracy in sample measurements, making it essential for protocols in applications like Next Generation Sequencing.
Download this infographic from DeNovix Inc., which compares key aspects of absorbance and fluorescence quantification and explains when to use each method or combine the results for optimal outcomes.
Download the full poster to learn more
About DeNovix, Inc.
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