Acetonitrile is the most commonly used organic mobile phase component due to its unique properties that set it apart from the other LC solvents (midrange solvent strength, excellent solvent performance, generation of well-defined chromatographic peaks, and lower viscosity and lower UV cutoff compared to alcohols and ethers).1 Acetonitrile is typically a by-product of the large-scale production of acrylonitrile (from ammonia and propylene) and may contain a wide range of very low-level impurities, including acrylonitrile, α(β)-methacrylonitrile, cis/trans butenenitrile, acetaldehyde, acetone, methanol, ethyl cyanide, acrolein, allyl alcohol, propenoic acid, oxazol, and acetic acid.2–4 Trace amounts of the above impurities may remain in HPLC acetonitrile even after a sophisticated purification process. Some of these trace impurities not only can lead to high baseline and ghost peaks, interfering with the qualification and quantification of analytes, but can also contaminate the column and block the system, resulting in malfunction of the instrument.
As an authority for the standards of test methods for analytical reagents in the U.S., the ACS Committee on Analytical Reagents clearly defines the specifications for HPLC-grade acetonitrile in Reagent Chemicals (9th ed.). The specifications for HPLC acetonitrile should include specifications for general use and HPLC suitability. The general-use specifications include GC purity, water, titratable acid or base, and residue upon evaporation. The HPLC applicability specifications include UV absorbance and HPLC blank gradient elution baseline.5
In this paper, the two HPLC suitability specifications for acetonitrile and their importance to HPLC applications are explained in detail, and operational considerations and factors influencing test results are discussed through detailed experimental data.
Two different HPLC systems were used in this work, and specific conditions are described in the text. Figures 1 and 2 were obtained using an Agilent 1100 HPLC system comprised of a degasser (G1379A), quaternary pump (G1311A), automatic sampler (G1313A), and DAD detector (G1315B) (Agilent Technologies, Wilmington, DE). The rest of the chromatograms were generated using an Alliance® 2695 HPLC system and 2996 PDA detector from Waters (Milford, MA). A ZORBAX C18 column was supplied by Agilent, and the B&J C8 column was acquired from Honeywell Burdick & Jackson.
The UV spectra were obtained using a Cary 4000 UV-VIS spectrophotometer from Varian (Palo Alto, CA). The reference cell was 1 cm quartz, and the sample cell was 1 cm or 5 cm quartz.
Chemicals and reagents
HPLC-grade acetonitrile was from Honeywell Burdick & Jackson and other HPLC solvent suppliers. High-purity water was supplied by a Milli-Q ultrapure water system (Millipore, Bedford, MA) and Honeywell Burdick & Jackson, and acetic acid (AR) was supplied by Sigma (St. Louis, MO).
Results and discussion
Significance of UV absorbance specification for HPLC acetonitrile
UV absorbance background is critical for HPLC acetonitrile for two reasons. First, most organic impurities contribute to UV absorption. Minimal UV absorbance of acetonitrile indicates minimal organic impurities in acetonitrile. Second, the most commonly used detection mode in HPLC instruments is the UV detector, which means the lower the UV absorbance of acetonitrile, the lower the chromatographic baseline background, and hence the higher the sensitivity and the lower the detection limit.
Figure 3 - UV absorbance vs wavelength curves for HPLC acetonitrile from different manufacturers using a 1-cm cell (high-purity water as reference).
Figure 3 shows that UV spectra of HPLC-grade acetonitrile from various suppliers are different, especially in short wavelength range (300–190 nm). This may be due to difficult-to-remove impurities and levels in acetonitrile resulting from the different purification techniques of each solvent supplier. Since most analytes are monitored below 300 nm using a UV detector in HPLC analysis, most solvent suppliers set up specific UV absorbance specifications in the above wavelength range.
Factors influencing the measured value of UV absorbance
Figure 4 - UV absorbance vs wavelength curves for HPLC acetonitrile from different manufacturers using a 5-cm cell (high-purity water as reference).
During the test experiment, many factors influence the measured value of UV absorbance:
1. Optical distance (cell length). In the range 0.2–0.8 AU, the measured value of absorbance (A) has minimal error; otherwise, the error of absorbance measured is relatively large. If using a 1-cm cell, the absorbance of acetonitrile is still far below 0.2 AU, even at a short wavelength range of 250~210 nm; thus there is a risk of significant measurement error. According to Beer’s law, the absorbance of solvent is proportional to the optical distance of sample; hence, by increasing the sample cell length, the absorbance of solvent can be increased, allowing it to fall into the range of 0.2–0.8 AU to reduce the measurement error. Figure 4 shows UV spectra of different acetonitrile samples scanned from 400 nm to 190 nm using a 5-cm cell. It can be seen from the curves that, using the 5-cm cell, most UV absorbance of acetonitrile at low wavelengths falls into the range 0.2–0.8 AU, which indicates that the data are more accurate. In addition, it is easier to see the UV absorbance difference of diverse acetonitrile samples compared to a 1-cm cell. In view of this, some manufacturers such as Honeywell Burdick & Jackson set up the UV absorption specifications of acetonitrile using both 1-cm and 5-cm cells.
2. Instrumental parameters. Since the absorbance of acetonitrile is extremely low, the instrumental parameters should be set up in such a way as to gain optimized sensitivity and accuracy. The double light beam mode overcomes the error resulting from light source energy variation with time under a single light beam mode, and makes it more convenient to scan in the full UV light wavelength range. Use of the baseline correction function of the spectrophotometer removes the interference from instrument variations in the light source, detector, and sample cell.6 Because the noise of the instrument increases with scanning speed, about one-half the maximal scanning speed is recommended.
Figure 5 - UV absorption profile of HPLC-grade acetonitrile using air and water as reference (1-cm cell).
3. Reference substance. Regarding the reference substance chosen for the UV absorbance measurement of solvent, there are two possibilities. Reagent Chemicals (9th ed.) uses water as a reference substance, while China Pharmacopeia employs air as a reference substance to measure the UV absorption of solvents. When using air as a reference substance, the measured UV absorbance of acetonitrile is lower than that using water as a reference, and is typically negative at the wavelengths ranging from 400 to 250 nm. This is because air has higher UV absorption than water, which is contributed by oxygen and carbon dioxide in the air. Figure 5 shows that the UV absorbance difference caused by the reference difference is significant—up to 0.03 AU. It is necessary to learn whether air or water is used as a reference when we read the UV absorbance data on the Certificate of Analysis report provided by the solvent supplier.