Section 15- Troubleshooting
Analyze a fresh replicate of the blanking solution as though it were a sample by selecting ‘Measure’ (F1). The result should be
a spectrum that varies no more than 0.050 A (10mm absorbance equivalent).
Wipe the blank from both measurement pedestal surfaces with a laboratory wipe and repeat the process until the spectrum is
within 0.005 A (1mm path).
Confirm that reference (blank) solution and solvent are the same material
Buffers often absorb in the UV range and therefore it is critical to blank the instrument with exactly the same material that the sample is
suspended in.
Confirm that your sample is not too dilute
Measuring samples at or near the detection limit will result in measurements that can vary a significant amount. Refer to the
‘Measurement Concentration Range’ of the application module that you are using for the applicable measurement range.
Confirm instrument accuracy and reproducibility with CF-1
This is a potassium dichromate calibration standard available from NanoDrop Technologies and its distributors. It is a good practice to
check the instrument’s performance every six months with a fresh vial of CF-1.
260/280 Ratio
Many researchers encounter a consistent 260/280 ratio change when switching from a standard cuvette spectrophotometer to the
NanoDrop
®
ND-1000 spectrophotometer. The three main causes for this are listed below:
Change in sample acidity
Small changes in solution pH will cause the 260:280 to vary
**
. Acidic solutions will under-represent the 260:280 ratio by 0.2-0.3, while a
basic solution will over-represent the ratio by 0.2-0.3. If comparing the NanoDrop
®
ND-1000 Spectrophotometer to other
spectrophotometers, it is important to ensure that the pH of an undiluted sample measured on the ND-1000 is at the same pH as the
diluted sample measured on the second spectrophotometer.
**
William W. Wilfinger, Karol Mackey, and Piotr Chomczynski,
Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid
Purity:
BioTechniques 22:474-481 (March 1997)
Wavelength accuracy of the spectrophotometers
Although the absorbance of a nucleic acid at 260nm is generally on a plateau, the absorbance curve at 280nm is quite steeply sloped.
A slight shift in wavelength accuracy will have a large effect on 260:280 ratios. For example, a +/- 1 nm shift in wavelength accuracy
will result in a +/- 0.2 change in the 260:280 ratio. Since many spectrophotometers claim a 1 nm accuracy specification, it is possible to
see as much as a 0.4 difference in the 260:280 ratio when measuring the same nucleic acid sample on two spectrophotometers that are
both within wavelength accuracy specification.
Nucleotide mix in your sample
The five nucleotides that comprise DNA and RNA exhibit widely varying 260:280 ratios
**
. The following represent the 260:280 ratios
estimated for each nucleotide if measured independently:
Guanine: 1.15
Adenine:
4.50
Cytosine: 1.51
Uracil:
4.00
Thymine: 1.47
The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260:280 ratios
for the four nucleotides present. It is important to note that the generally accepted ratios of 1.8 and 2.0 for DNA and RNA are “rules of
thumb”. The actual ratio will depend on the composition of the nucleic acid. Note: RNA will typically have a higher 260:280 ratio due
to the higher ratio of Uracil compared to that of Thymine.
***
Leninger, A. L.
Biochemistry
, 2
nd
ed., Worth Publishers, New York, 1975
Unusual Spectrum
A sample that exhibits jagged “cuts” out of the spectrum, but an otherwise normal shape, may be the result of detector saturation. This
can be caused by the software selecting too high of an integration time due to a dirty sample pedestal upon startup. Try cleaning both
sample pedestals thoroughly and restarting the software. For reference, examples of spectra generated with a saturated detector are
shown below.
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