This Information Applies To: Agilent Isocratic LC Systems, Agilent Refractive Index Detectors (RID)
Mobile phase preparation method impacts retention time reproducibility between injections on the same instrument, by other operator, or similar instruments. It impacts systems that use premixed mobile phases including isocratic pumps or the refractive index detector (RID).
Usually, a contraction or expansion of the volume occurs when mixing different solvents, so the method used to mix the solvents will result in different actual compositions. An example is the water:methanol mixture, which is widely used in liquid chromatography. This will result in volume expansion due to exothermic like condition. A mixture of acetonitrile and water on the other hand will result in solvent contraction.
A 50:50 water:methanol solution can be prepared using the following techniques:
A. Inline mixing: The solvents are mixed by the pump 50% each channel. This method is not recommended when using a RID.
B. Vol/Vol: Measure separately 250 mL water and 250 mL methanol and then mix them. The mobile phase is pumped 100% in one channel of the pump. Premixing volumes that have been measured separately should provide the similar results from different users. The slight variations will be due to user's ability to accurately measure the solvent and whether a volumetric flask or graduated cylinder is used.
C. Mix-to-mark: Transfer 250 mL of methanol to a volumetric flask and fill to 500 mL with water. The mobile phase is pumped 100% in one of the channels. The result also will be different if water is added first, then fill to volume with methanol.
D. Weight/weight: 200 g of methanol are mixed with 200 g of water. These weights are measured separately before combining. The resulting mixture is pumped using just one channel of the pump. This is not a typical method, but it is more accurate, because weight is independent of temperature and removes user's ability to accurately measure volume. To obtain more similar results to the Vol/Vol method, the weight must be calculated based on the solvent's density.
|Warning: Always wear Personal Protective Equipment when handling solvents to prepare mobile phases|
In the following example, the same sample is injected using 50:50 water:methanol as mobile phase, prepared in different ways as mentioned before. The results show significant changes in the elution patterns (see Figure 1).
- LC System: Agilent 1100 series with quaternary pump
- Column: ZORBAX Eclipse XDB-C8 Rapid-Resolution (3.5 µm), 4.6 x 50 mm (p/n 935967-906)
- Detection: UV 254 nm
- Flow: 1 mL/min.
- Temperature: ambient
Figure 1. Effect of different mobile phase preparations of 50:50 water:methanol.
A. Inline mixing B. Vol/Vol C. Mix-to-mark D. Weight/weight
1. Uracil 2. Butylparaben 3. Naphthalene 4. Dipropylpthalate 5.Acenaphthene
Tips: The vol/vol method is the most like the inline mixing produced by a binary or quaternary pump
While mobile phases can be prepared by any of the preceding preparation methods, it is important that it is properly documented, and understood by all the operators in the lab.
See more information in the following application note: Effect of Mobile Phase Preparation on Chromatography
Learn how to effectively operate your Agilent LC System:
HPLC-0GEN-1021r - Handling HPLC Solvents e-learning course on Agilent University