A spectrophotometer blank has to contain everything the sample contains except the analyte. When a solution has been prepared or diluted with bacteriostatic water, the blank must therefore be bacteriostatic water too, and preferably from the same vial. The reason is the preservative: 0.9% benzyl alcohol is 83 mmol/L of an aromatic compound, and aromatic rings absorb ultraviolet light.
Where benzyl alcohol absorbs
Benzyl alcohol has an absorption band centered near 257 nm with a molar absorptivity of roughly 200 L·mol−1·cm−1. At 83 mmol/L in a 1 cm cell that corresponds to an absorbance in the tens, far beyond the range of any instrument, so undiluted bacteriostatic water is effectively opaque from about 240 to 275 nm. The band falls away steeply above 270 nm, but at 280 nm the residual absorbance is still measurable, typically somewhere between a few hundredths and a few tenths of an absorbance unit depending on the instrument bandwidth, and that is enough to matter for low-concentration measurements at 280 nm. Above about 300 nm, and everywhere in the visible range, benzyl alcohol is transparent and bacteriostatic water behaves as water.
Practical consequences
- Measurements below about 290 nm: bacteriostatic water is the wrong diluent. Any analyte band in the 240–275 nm region is buried, and even a matched blank cannot recover a signal from a region where the blank itself is opaque. Use plain sterile water or a buffer for the dilution and for the blank.
- Measurements at 280 nm: possible, provided the blank is bacteriostatic water at exactly the same concentration as in the sample and the analyte absorbance is well above the residual. Matching matters because the blank subtraction is subtracting a value that may be comparable to the signal.
- Measurements above 300 nm and in the visible range: the preservative does not interfere; bacteriostatic water is an acceptable blank.
Why the blank should come from the same vial
Benzyl alcohol oxidizes slowly to benzaldehyde in an opened vial, and benzaldehyde absorbs about sixty times more strongly (its maximum near 249 nm has a molar absorptivity of roughly 12,000). Converting just one part in a thousand of the preservative to benzaldehyde adds about one absorbance unit at 249 nm in a 1 cm cell and a measurable amount at 280 nm. Two vials with different histories can therefore have different UV baselines even though both are within specification. Prepare the sample and the blank from the same vial, opened on the same day, and record the lot number. See benzyl alcohol oxidation.
Matching the preservative concentration
If the stock solution was prepared in bacteriostatic water and diluted with bacteriostatic water, every dilution contains 0.9% benzyl alcohol and a bacteriostatic water blank matches all of them. If the stock was prepared in plain water and diluted with bacteriostatic water, the preservative concentration in each dilution depends on the dilution factor (a 1:10 dilution contains 0.81%), and each dilution needs a blank made with the same proportion of bacteriostatic water and plain water. See preparing a dilution series.
Checklist
- Quartz cuvettes for any measurement below 340 nm.
- Blank and sample from the same vial and lot; note the date the vial was opened.
- Below 290 nm, switch to a preservative-free diluent for both sample and blank.
- Record the diluent lot in the method record. See labeling and record-keeping.
Related
Bacteriostatic water in analytical chemistry: when the preservative interferes · Benzyl alcohol: chemical and physical properties · Physical properties of bacteriostatic water
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