Sample dilution variable volume pipettes

Once the compound of interest has been isolated from the sample matrix, it is often necessary to dilute the sample in order to analyse it within the validated assay range (see Chapter 8). A test injection may have to be performed in order to assess the sample concentration in the first instance. Once it has been established that the sample does require dilution, this can easily be performed using calibrated glass (larger volumes) or variable volume pipettes (smaller volumes). 

You have been asked to dilute a liver extract 10 times using mobile phase as the solvent. Following an extraction procedure, you have a volume of 200 pL (solution A). Using a variable volume automatic pipette, draw up 100 pL of solution A and dispense this into a suitable glass or plastic vial. Using a second variable volume automatic pipette, transfer 900 pL of mobile phase into the same vial. Stopper the vial and shake or invert to mix. The sample solution has been diluted 10 times. This is often referred to as a 1 in 10 or 1 to 10 dilution. 

When preparing a dilution series, it is important to maintain aseptic conditions (Section 13.8). For example, dilution blanks must be flamed when opened to transfer sample (Fig. 13.2). Sterile 1.0 mL pipettes (0.1 mL graduations) or variable volume pipettors equipped with sterile plastic tips must be used to aseptically transfer liquid between dilution blanks. Sterile pipettes or pipette tips should be used only once, and then a new pipette or tip is used. Pipettors can be sterilized by wiping down their surfaces with 70% v/v ethanol and should be calibrated yearly. 

Some techniques are known to provide higher variability than others. The choice of an appropriate method at the outset can improve precision. For example, a volume of less than 20 mL can be measured more accurately and precisely with a syringe than with a pipette. Large volumes are amenable to precise handling but result in dilution that lowers sensitivity. The goal should be to choose a combination of sample preparation and analytical instrumentation that reduces both the number of sample preparative steps and the RSD. Automated techniques with less manual handling tend to have higher precision. 

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