This Information Applies To: Agilent 6200 Series TOF and 6500 Series Q-TOF Systems
Issue
Instructions on how to correctly dilute the tune mix for 6200 Series TOF and 6500 Series Q-TOF systems depending on source type.
Background
If the Tune Report shows calibration abundances greater than 750,000 counts, or if MassHunter displays an error requesting dilution, then the tune mix will need to be carefully diluted to ensure accurate mass calibration. The instructions for dilution are listed in this article, specific to the source type installed on your TOF or Q-TOF system.
Items Required
Steps to Follow
To make dilutions, add components in the volumes shown in the following tables, depending on the source type installed or your system.
Dual AJS
Dual ESI
Multimode
Reference mix (if applies).
Dual AJS
| Component | Volume |
|---|---|
| ESI-L Low Concentration Tuning Mix | 10 mL |
| Ultrapure LC/MS Grade Acetonitrile | 88.5 mL |
| Ultrapure LC/MS Grade Water | 4.5 mL |
| 0.1 mM HP-0321 (included in the Biopolymer Reference Mass Kit) | 3 µL |
Table 1. Tuning mix dilution for Dual AJS source
Dual ESI
| Component | Volume (Positive Mode) | Volume (Negative Mode) |
|---|---|---|
| ESI-L Low Concentration Tuning Mix | 25 mL | 25 mL |
| Ultrapure LC/MS Grade Acetonitrile | 71.25 mL | 71.25 mL |
| Ultrapure LC/MS Grade Water | 3.75 mL | 3.75 mL |
Table 2. Tuning mix dilution for Dual ESI source
Multimode Source
| Component | Volume (Positive Mode) | Volume (Negative Mode) |
|---|---|---|
| MMI-L Low Concentration Tuning Mix | 25 mL | 25 mL |
| Ultrapure LC/MS Grade Acetonitrile | 71.25 mL | 71.25 mL |
| Ultrapure LC/MS Grade Water | 3.75 mL | 3.75 mL |
Table 3. Tuning mix dilution for multimode source
Reference Mix
6200 TOF and 6500 Series Q-TOF systems with Dual ESI or Dual AJS sources have a second nebulizer that is used to introduce reference mix into the source that corrects the mass axis for accurate mass data.
In 1 L of 95% Ultrapure LC/MS Grade Acetonitrile and 5% Ultrapure LC/MS Grade Water, use a pipettor to add the following individual calibrants mentioned in the following table depending on the source type installed on your system:
| Biopolymer Reference Mass Kit Component | Volume (Dual ESI) | Volume (Dual AJS) |
|---|---|---|
| TFANH4 | 0.5 mL | |
| Purine | 1.0 mL | 0.4 mL |
| HP-0921 | 0.45 mL | 1.0 mL |
Table 4. Tuning mix preparation for multimode source
There should be a minimum reference mix signal abundance of 10,000 counts for accurate reference mass corrections. (See Figure 1)
Figure 1. Spectrum scan showing the minimum abundance level of 10,000 counts (blue arrow) for reference mix masses.
