Helpful Information For Measuring Samples; Sample Volume Requirements; Sample Measurement Accuracy And Reproducibility - Thermo Scientific NanoDrop Lite Plus User Manual

Helpful Information for Measuring Samples

Sample Volume Requirements

Sample Measurement Accuracy and Reproducibility

Thermo Scientific
Sample volume is important because it is essential that a proper liquid column is
formed and that the path between the upper and lower pedestals is bridged with
liquid sample which is held in place by surface tension.
The dominant factor determining the surface tension of a droplet is the hydrogen
bonding of the lattice of water molecules in solution. Generally, all solutes (including
protein, DNA, RNA, buffer salts and detergents) can reduce the surface tension by
interfering with the hydrogen bonding between water molecules. Although 1 μL volumes
are usually sufficient for most sample measurements, increasing the sample size to 2 μL
will ensure proper column formation for samples with reduced surface tension.
This is particularly true when working with protein samples which may contain
detergents. Such samples do not "bead" on the pedestal but tend to spread across
the measurement surface. To ensure that a liquid column forms in the gap between
the upper and lower pedestals, a 2 μL volume is indicated for such samples.
Protein samples, especially those containing detergents, are subject to bubble
formation. Extra care is required while pipetting onto the pedestal to ensure bubbles
are not present into the light path of the spectrophotometer.
Field experience indicates that the following volumes are sufficient to ensure
reproducibility: Aqueous solutions of nucleic acids: 1 μL
Aqueous solutions of purified protein: 2 μL
It is best to use a precision pipettor (0-2 μL) with precision tips to ensure that
sufficient sample (1-2 μL) is delivered. Lower precision pipettors (0-10 μL and larger)
are not as good at delivering 1 μL volumes to the lower pedestal. If the user is unsure
about the sample characteristics or pipettor accuracy, a 2 μL sample volume is
recommended.
Sample or aliquot heterogeneity and/or liquid column breakage may result in
erroneous or non-reproducible results. Follow the recommendations below to ensure
accurate and reproducible results:
• Ensure the pedestal surfaces are clean. A dirty pedestal (i.e., a pedestal with
sample dried on it) may cause erroneous absorbance readings (even negative
values) and signal saturation. It is good practice to clean the pedestal surfaces
with deionized water prior to starting a measurement.
Note Do not use a squirt or spray bottle to apply de-ionized water.
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NanoDrop Lite Plus User Guide
Helpful Information for Measuring Samples
NanoDrop Lite Plus User Guide 17