A P P L I C AT I O N N O T E

Gas Chromatography/
Mass Spectrometry
Author:
Adam J. Patkin, Ph.D.

PerkinElmer, Inc.
Shelton, CT

Serial Dilution Workflow for

Automated Standard Preparation Introduction
Preparation of calibration standards for Gas
on the TurboMatrix MultiPrep+ Chromatography (GC) and Gas Chromatography/
Mass Spectrometry (GC/MS) analysis can be
expensive and time-consuming, especially when it needs to be done on a daily basis for routine analyses. This
expense and time is due to:
• Purchase of the required volumetric and transfer glassware
• Cleaning of the glassware to prevent carry-over
• Cost of purchase and disposal of high-purity solvents and analytical standards
• Skilled technician or scientist time to carefully prepare the standards
Automation of serial dilution standard preparation with the PerkinElmer TurboMatrix MultiPrep+
robotic autosampler:
• Eliminates the volumetric and transfer glassware, replacing it with syringes and disposable vials
• Eliminates cross contamination from reuse of volumetric and transfer glassware
• Can reduce the volume of solvent required from tens or hundreds of mL per calibration level to 1.6 mL or less
• Reduces the human time and skill requirements to filling vials with solvent and standards, and adding empty
vials to receive new dilution standards
In addition, the MultiPrep+ automation software can queue the preparation of a set of calibration standards to
occur immediately before they are injected along with analytical samples, or it can operate in a stand-alone mode.
 The Sample Manager software of the MultiPrep+ provides The workflow is to first use the 1000 µL syringe to add 900 µL
several methods of standards preparation. This Application of dilution solvent to all the vials. Next, the 100 µL syringe is
Note describes the “Serial Dilution” workflow. This dilution used to take 100 µL out of the neat stock sample in vial #54 and
workflow can prepare a series of standards in 2 mL vials from transfer it to vial #1. Vial #1 is now homogenized, optionally by
three to eight orders-of-magnitude dilution. This wide dynamic a high-speed vortex mixer, and if not, by the syringe sample
range can be very useful for the dilution of high concentration pumps with sample from vial #1 prior to the next step. Now,
commercial stock standards, preparing working standards, and 100 µL of the 10:1 dilution is transferred to vial #2 from vial #1.
for determining appropriate concentration working ranges for Vial #2 is now mixed. This continues for the specified number of
GC and GC/MS analysis. Once the working range has been dilutions. If two or three sets of dilution standards have been
determined, the “Calibration Dilution” workflow, described specified, they occur next.
elsewhere, may be useful because it gives more calibration
The four 10 mL vial Standard Wash station contains 10 mL of
points across a typical GC or GC/MS working range.
dilution solvent in each of vials #1, #2, and #3, for the dilution
solvent used to prepare replicate dilution sets 1, 2, and 3.
Workflow Overview
A user-specified Sample Manager method defines how many A GC injection sample list can be scheduled to occur
serial 10:1 dilutions are to be prepared and how many replicate immediately after the completion of standards preparation,
sets of dilutions. The method also defines all syringes, washing, for unattended operation.
mixing, temperatures, etc. Automated Serial Dilution is best for lower-volatility solvents
Calibration Dilution uses a 100 µL and a 1000 µL syringe to and analytes because of the needle holes put into the vial septa.
prepare up to three replicate sets of the specified number of For example, methanol or isooctane solvent with compounds
dilution steps. The final volume of each dilution level is 1 mL. The of higher boiling point may be quantitatively stable for several
vial positions are fixed in the tray, as shown in Table 1 and Figure 1. hours to days; long enough to prepare and inject. It is not
recommended for high-volatility solvents (e.g. diethyl ether,
Table 1. Vial Positions in Sample Tray. methylene chloride, carbon disulfide) or analytes which have
Vial (3 Replicates) Dilution Ratio boiling points at or below room temperature.
1, 10, 19 1 : 10 Dilution
Cost Savings for Solvents and Standards
2, 11, 20 1 : 100 Dilution
Table 2 shows the total quantity of diluent solvent and stock
3, 12, 21 1 : 1000 Dilution
standard solution required to prepare a single complete set of
4, 13, 22 1 : 10,000 Dilution
standards and three replicate standard sets. It assumes that all
5, 14, 23 1 : 100,000 Dilution
eight dilution levels are prepared, and that the diluent solvent is
6, 15, 24 1 : 1,000,000 Dilution the same as the syringe wash solvent. The total includes all syringe
7, 16, 25 1 : 10,000,000 Dilution diluent cleaning washes, and priming rinses with stock solution
8, 17, 26 1 : 100,000,000 Dilution and standards between dilution levels. Each replicate set starts
54 Neat Sample with a new diluent vial.

Table 2. Required Solvent and Standard Volumes.

# Sets of 8 Dilutions Solvent (mL) Standard (mL)
1 11.42 0.12
3 34.26 0.36

Each dilution level omitted saves 1.12 mL of solvent.

Smaller volumes of the stock solution can be optionally used
with the “bottom sensing” mode of syringe sampling, which
can take three 1 µL samples from as little as 5 µL of liquid in the
vial. (This mode requires a 23 Gauge liquid syringe and a conical
bottom vial, and is only possible for 2 mL sample vial trays.)

Figure 1. Sample Tray Layout.

2
Below are recommended MultiPrep+ configurations for Methods
Serial Dilution and a typical pre-run checklist. The TurboMatrix Sample Manager Calibration Dilution method is
shown in Table 3. The GC injection method is shown in Table 4.
Minimum Recommended MultiPrep+ Configuration Most parameters were used at their default values.
The Minimum Configuration Required to Perform Calibration
The PerkinElmer Clarus 690 GC used a 1 µL injection split 100:1
Dilution Is:
into a capillary split/splitless injector at 250 °C onto a PerkinElmer
• Robotic tool change syringe park station
Elite™ 5MS, 30 m x 0.25 mm ID x 0.25 μm column. Carrier gas
• Sample Tray holder
• 2 mL VT-54 rack was 1.5 mL/min helium (99.999 + % purity). The oven program
• Standard Wash station was 90 °C for 1.5 min, then ramp to 157 °C at 10 °C/min. The
• 1000 µL (57 mm needle, 23 Gauge) syringe and D8/57 tool (N6496004) split flow was reduced to 10:1 at 2 min to reduce gas
• 100 µL (57 mm needle, 23S Gauge) syringe and D7/57 tool (N6496002) consumption. The Clarus SQ 8T mass spectrometer operated
• 2 mL vials and caps under UltraTune conditions with a 250 °C transferline and ion
source, monitoring m/z 57 for the alkanes.
Suggested MultiPrep+ Configuration Enhancements
For homogeneous sample mixing and lower internal Table 3. Sample Manager Serial Dilution Method.
standard usage: Configuration
• Vortexer (strongly recommended) 100 μL Sample 1000 μL Solvent
P/N N6556086 P/N N6556089
• Magnetic metal crimp caps for 2 mL vials (required for Vortex mixing) Syringe Syringe
• L arge Wash station (N6496024), replacing Standard Wash for dilution Dilution Solvent
Dilution Vial Rack Rack 1 Standard Wash 1
solvent washing Station
• Solvent Module (N6496020), replacing Standard Wash for dilution solvent Wash Station Large Wash 1 Vortex Mixer Vortex Mixer 1
• 57 mm, 23 Gauge needles (required for bottom sensing vials) Dilute
• 1 0 µL (85 mm needle, 23S Gauge) syringe and D7/85 tool (N6496003) Dilution Steps 8 Repetitions 1
for GC injection
Sample Fill Rate Solvent Fill Rate
25 μL/s 50 μL/s
100 μL Syringe 1000 μL Syringe
Pre-Run Checklist Rinsing
1. Verify 1000 µL sample prep syringe is mounted. Stock Solution Dilution Sample
2 2
2. Verify 10 µL sample prep syringe is mounted. Rinse Cycles Rinse Cycles
3. (Optional) Verify 10 µL GC injection syringe is mounted. Wash 100 μL
Wash Cycles for
4. Place stock solution in Vial #54. Syringe After On 2
100 μL syringe
5. R eplace Standard Wash vials #1-#3 (depending on number of replicates) Each Sample
Wash Cycles for Rinse Volume for
with fresh 10 mL vials (to avoid carry-over) and dilution solvent. 2 10 μL
1000 μL Syringe 100 μL Syringe
6. R efill Large Wash station vials with syringe cleaning solvent.
Rinse Volume for
(#1 for internal standard, #2 for diluent). 20 μL
1000 μL Syringe
7. Put new, capped sample vials in tray positions being used (Figure 1).
Mixing
Mixing Speed 1200 rpm Mixing Time 6s
Stock Standards Preparation Advanced
Pesticide grade isooctane was used as the diluent and cleaning Height From
Bottom Sense
Off Bottom of 0.5 mm
solvent, and analytical standard grade n-decane, n-undecane, Stock Solution
Stock Solution
n-dodecane, n-tridecane, and n-tetradecane as the analytes.
Syringe Overfill 5% Wash Vial Depth 44 mm
The stock solution was prepared gravimetrically at 1.92, 2.34, Waste Port Sample Vial
1.73, 2.16, and 1.86 mg/mL, respectively. The naphthalene 12 mm 50 mm/s
Depth - Solvent Penetration Speed
internal standard was prepared at 1.01 mg/mL. Fast Expel for
50 μL Target Vial Depth 10 mm
1000 μL Syringe
Dilution Solvent Solvent Vial
44 mm 44 mm
Station Depth Depth
Stock Solution
30 mm Sample Pumps 5
Penetration Depth
Sample Vial
30 mm
Penetration Depth

3
 Table 4. Sample Manager Injection Method. Excellent linearity was observed over a wide linear dynamic
Configuration range. Examples are shown in Figure 2 and Figure 3.
Gas Chromatograph GC2 Syringe P/N N6556084
Pre-injection
Verify Barcodes Off Standard Wash 1
Wash Station
Post-injection Standard
Peltier Stack 1 none
Wash Station Wash 1
Peltier Stack 1
20 °C Peltier Stack 2 none
Temperature
Peltier Stack 2
20 °C
Temperature
General Syringe
Sample Vial Depth 30 mm Sample Pumps 6
Fill Volume 3 μL
Pre-injection Wash
Figure 2. n-Undecane Calibration Curve.
Wash Cycles 6 Wash Solvent 1 2
Wash Solvent 2 3 Wash Solvent 3 0
Wash Solvent 4 0 Syringe Fill Volume 3 μL
Syringe Flow Rate 2 μL/s
Sample Rinse
Sample Rinses 1 Rinse Volume 3 μL
Delay After Pull Up 2 s Delay After Aspirate 1s
Sampling
Sample Viscosity
2s Sample Fill Rate 2 μL/s
Delay
Prep Ahead Disabled
Inject Sample
Figure 3. n-Tridecane Calibration Curve.
Injection Mode Normal Injection Flow Rate 50 μL/s
Post-injection The mean R2 coefficient of determination was better than 0.999
0s
Dwell Time
over five orders-of-magnitude dilution, limited on the low end by
Post-injection Wash
trace analyte contaminants in the solvent.
Wash Cycles 6 Wash Solvent 1 2
Wash Solvent 2 3 Wash Solvent 3 0 Table 5. Coefficients of Determination for analyte linearity.

Wash Solvent 4 0 Syringe Fill Volume 3 μL Analyte R2 Decades

Syringe Flow Rate 5 μL/s n-Decane 0.9999 4
Advanced n-Undecane 0.9999 5
Pre-injection Dwell Height from Bottom n-Dodecane 0.9995 5
0s 0.5 mm
Time of Sample Vial
n-Tridecane 0.9997 5
Bottom Sense Sample Vial
Off 50 mm/s n-Tetradecane 0.9994 5
Sample Vial Penetration Speed
Waste Port Depth 10 mm Wash Vial Depth 40 mm
Sample Cleaning Sample Cleaning Conclusions
2 μL/s 2s
Fill Rate Viscosity Delay The Serial Dilution workflow of the PerkinElmer TurboMatrix
Air Gap 0 μL Injection Signal Mode PlungerDown MultiPrep+ robotic autosampler has been demonstrated to
Injector Penetration Injector Penetration
45 mm 100 mm/s produce dilutions across a wide dynamic range. This allows the
Depth Speed
preparation of fresh standards from stock solution using smaller
quantities of expensive high-purity analytes and solvents than
would be required by conventional manual volumetric methods.
It also reduces labor costs and opportunity for human error.

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