Chromatography focus on
Automation of a Complex, Multi-Step Sample Preparation Using the Standalone Agilent 7696A WorkBench
James D. McCurry, PhD,Senior Scientist, Agilent Technologies
In analytical chemistry, sample preparation can be as simple as adding a solvent or as complex as performing chemical reactions to improve the instrumental measurements that follow. While sample preparation is a critical component to any chemical measurement, chemists rarely look forward to performing this job, especially if it is complex, boring and involves handling unpleasant chemicals. As a result, manual sample preparation can be the source of many errors and poor precision. To help reduce errors and improve precision, many manual sample preparations are done using with large amounts of chemicals and expensive volumetric glassware to make handling, dispensing, and measuring easier.
A good example of a difficult manual preparation is ASTM method D6584. This method measures the free and total glycerin content in B100 biodiesel to assure good product quality [1]. Since the various glycerins found in biodiesel are not volatile, they cannot be measured using gas chromatography (GC). Method D6584 describes a sample preparation protocol to derivatise these compounds with a trimethylsilation reagent so they can be analysed with GC. The steps for this sample preparation are complex, time consuming, and use pyridine; a toxic solvent with a distinctly unpleasant odour. This assures the unpopularity of this procedure.
The Agilent 7696A Sample Prep WorkBench is a standalone instrument specifically designed to perform automated sample preparation [1,2]. It uses two 7693A injection towers to volumetrically transfer liquids between 2-mL vials. The vials containing various chemical resources, standards and samples are housed in three 50-positions trays. The sample tray compartment houses a robotic arm to move vials, a vortex mixing station and a sample heating station.
Designing the 7896A WorkBench Procedure
The ASTM D6584 preparation procedure can be completely described in six individual steps as shown in Table 1. When done manually, this prep consumes large amounts of standards, reagents, solvents and disposable glassware. Since the Agilent WorkBench uses smaller 2-mL vials, this procedure can be scaled down by a factor of 10. The WorkBench also uses two pipetting syringes to transfer liquids, thus eliminating the expense of disposable glassware. Table 1 also shows how each step was scaled to accommodate the 2-mL vials used by the WorkBench.
Table 1. ASTM method D6584 uses a six step derivatisation of glycerins in biodiesel to prepare the samples for analysis by high temperature GC. Since the Agilent 7696A Sample Prep WorkBench uses 2-mL vials, the manual sample must be scaled down 10:1.
Steps Manual Sample Prep in 15-mL Vials
1 Add 100 mg B100 to a 15 mL vial with Teflon screw cap 2 Add 100 uL ISTD1 solution (butanetriol) to the vial 3 Add 100 uL ISTD2 solution (tricaprin) to vial
4 Add 100 uL derivatization reagent (MSTFA) to vial and mix 5 React at room temperature for 15 minutes 6 Add 8 mL n-heptane to vial and mix
10:1 Scaling --> WorkBench Sample Prep using 2-mL Vials
Add 10 mg B100 to a 2 mL vial with Teflon screw cap Add 10 uL ISTD1 solution (butanetriol) to the vial Add 10 uL ISTD2 solution (tricaprin) to vial
Add 10 uL derivatization reagent (MSTFA) to vial and mix React at room temperature for 15 minutes Add 800 uL n-heptane to vial and mix
Before building a WorkBench sample prep, we first defined the chemical resources needed to prepare the biodiesel samples and where those resources were positioned in the WorkBench trays. Table 2 shows each resource, their tray positions and the pipetting syringe parameters used to dispense each resource. The WorkBench software also provides a graphic, overhead view of the resources in the sample trays as shown in Figure 1. In this example, we show ten samples in tray positions 1 to 10 and ten n-heptane resource vials that will be used for each sample. The n-heptane vials are stored in tray positions 101 to 110.
Table 2. Four chemical resources are needed to completely derivatise glycerins in biodiesel. The resources, tray positions and syringe parameters are set in the WorkBench software. The syringe draw speeds are used to load each resource into the syringe. The syringe dispense speeds are used to transfer the resource into the 2-mL sample vials.
Tray
Chemical Resource ISTD1 (1000 ug/ml
butanetriol in pyridine)
ISTD2 (8000 ug/mL tricaprin in pyridine
MSTFA derivatization reagent
n-Heptane
Position 51
52 53
Syringe Size
100 uL 100 uL 100 uL 101 - 110 250 uL
Syringe Draw Speed
250 uL/min 250 uL/min 250 uL/min 500 uL/min
Syringe Dispense Speed
500 uL/min 500 uL/min 500 uL/min 2000 uL/min
Figure 1. The WorkBench software provides an overhead view of each chemical resource in the sample trays. For this example, in addition to the chemical resources, ten samples were placed in tray positions 1 to 10.
Sample weighing cannot be performed using the WorkBench because there is no analytical balance. Since weighing 10 mg of biodiesel can be very challenging, an Eppendorf Reference Adjustable-Volume Pipettor (10-100 uL) was used to transfer the sample. Weighing 10mg of biodiesel was done by manually pipetting 11.4uL of biodiesel into tared 2-mL vials and recording the weight to the nearest 0.1mg.
To mimic the manual sample prep workflow, individual WorkBench methods were created for each step outlined in Table 2. For instance, we created a method called ADD_ISTD1.M to add the first internal standard solution (ISTD1) to every sample before adding the second internal standard (ISTD2) using method ADD_ISTD2.M. With this approach, we only needed to wash the syringe with solvent after switching to a different resource. This greatly reduces the amount of wash solvent needed and allows more samples to be prepared before refilling the wash solvent reservoirs. The final ‘script’ for the WorkBench sample prep, including the syringe wash steps, is shown in Table 3. To run the complete sample prep, each method is run by the WorkBench sequence queue as shown in Figure 2.
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56
