Table of Contents - Applied Biosystems

Table of Contents - Applied Biosystems

Q Trap LC/MS/MS TurboIonSpray Ion Source Manual Optimizing the TurboIonSpray

Optimizing the TurboIonSpray

The following section outlines the practical considerations that must be made when optimizing the TurboIonSpray performance. It is intended to provide the information necessary to aid the operator in quantifying the separate operating parameters.

TurboIonSpray performance depends on the following factors:

• Sprayer position

• TurboIonSpray (IS) voltage

• Nebulizer gas (GS1)

• Curtain gas (CUR)

• Heater gas and temperature (GS2 and TEM)

• Declustering potential (DP)

• Solvent composition

Optimum performance on standard compounds is relatively easy to achieve and little adjustment or tuning is required once the source is optimized.

With experience, the operator will develop a personal optimization method that works best. In general, once the sprayer is set up and optimum factors have been determined, little or no readjustment of the values is required on a day-to-day basis.

For tuning purposes, a compound with a known molecular ion should be introduced either by continuous infusion or flow injection. Infusion is preferable because it provides a continuous flow of sample. The tuning compound should have characteristics similar to the sample to be analyzed during normal operation and should be introduced at the same liquid flow rate.

CAUTION! When optimizing the sprayer position, make certain not to spray directly down the orifice. Spraying down the orifice may contaminate the vacuum interface and vacuum chamber ion optics and could impact instrument performance.

CAUTION! To avoid possible damage to the instrument, always view the sprayer tip through the side port when adjusting its position.

CAUTION! If the source is to be left unattended while in operation, ensure that an

LC shutoff is in use to prevent flooding of the plenum chamber.

TurboIonSpray Probe Position

The position of the TurboIonSpray Probe relative to the orifice and to the heater probe is an important factor in optimizing TurboIonSpray performance. The probe should point between 5 and 10 mm off axis with respect to the center of the orifice. The distance of the heater probe from the orifice plane is fixed, but the TurboIonSpray can be adjusted using the scale on the side of the sample inlet arm. Changing from low solvent flow rates

(40 µL/min) to high solvent flow rates (1 mL/min) requires that the TurboIonSpray be

11

Optimizing the TurboIonSpray Q Trap LC/MS/MS TurboIonSpray Ion Source Manual repositioned further away from the orifice to prevent solvent penetration through the orifice into the mass spectrometer.

Optimal

TurboIonspray

Target

Curtain Plate

Orifice

40 µL/min

200 µL/min

1 mL/min

TurboIonSpray positioning across the orifice

Also, as the aqueous composition of the carrier solvent increases at high flow rates

(1 mL/min), the more visible the spray becomes and the farther away from the orifice it should be directed. See the preceding figure where the areas indicated in the diagram for the different flow rates are the optimum target areas for the TurboIonSpray liquid spray.

The circle immediately around the orifice (for example, the part of the orifice plate that is visible when viewing the front of the interface) should remain clear of solvent or solvent drops at all times.

The best position is usually a few millimeters off axis to the bottom of the curtain plate aperture. Multiply charged proteins and peptides introduced at a few microliters per minute usually require the sprayer to be as close as possible to the curtain plate.

TurboIonSpray Voltage (IS)

In positive mode, compounds usually require a high probe voltage of between 4000 and

5500 V. In negative mode, compounds usually require a lower voltage, between

–3000 and –4500 V.

Note: If the TurboIonSpray voltage is set too high a blue glow can be seen at the tip of the

TurboIonSpray indicating a corona discharge. This will result in decreased sensitivity and stability of the ion signal.

Nebulizer Gas (GS1)

Optimize for signal stability and sensitivity. Typically a value of 10 to 45 psi is used as applied by the applications computer.

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Q Trap LC/MS/MS TurboIonSpray Ion Source Manual Optimizing the TurboIonSpray

Curtain Gas (CUR)

The curtain gas ensures a stable, clean environment for the sample ions entering the mass spectrometer. The gas curtain prevents air or solvent from entering the analyzer region of the instrument while permitting the sample ions to be drawn into the vacuum chamber by the electrical fields generated between the vacuum interface and the TurboIonSpray needle. The presence of the solvent vapor or moisture in the analyzer region of the mass spectrometer contaminates the Q0 rod set causing a reduction in resolution, stability, and sensitivity, and an increase in chemical background noise.

In order to prevent instrument contamination, the curtain gas should be optimized at the

highest possible setting, (never below 11 psi) that does not result in a significant reduction in signal intensity.

Heater Gas (GS2)

The heater gas (Gas 2) aids in the evaporation of solvent, which aids in increasing the ionization of the sample. The higher the liquid flow or the higher the aqueous composition of the solvent, the higher the heater gas temperature and gas flow required. However, too high a temperature can cause premature vaporization of the solvent, and result in a high chemical background noise, while too high a heater gas flow can produce a noisy or unstable signal.

The following table provides recommended operating conditions for the TurboIonSpray at three different flow rates. For each flow rate, the curtain gas (from setting 11 to 45 at the applications computer) should be as high as possible. The solvent composition used for optimization was 1:1 water, acetonitrile. These conditions represent a starting point from which to optimize TurboIonSpray. By an iterative process, the various settings can be optimized using flow injection analysis to obtain maximum signal-to-noise for the compound of interest.

TurboIonSpray Suggested Operating Settings

Flow

Rate

(µL/min)

TurboProbe

Temperature

(°C)

Heater Gas (GS2) Nebulizer Gas

(GS 1) Position

40 300

(200–500)

200 400

(300–500)

L/min psi

6

(4–8)

65

(40–90)

7

(4–8)

75

(40–90) psi

20

(10–30)

30

(15–35)

V

2

3

H

0

5

1000 475

(450–500)

8

(4–8)

90

(40–90)

50

(20–55)

6 10

V = vertical position

H = horizontal position

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Optimizing the TurboIonSpray Q Trap LC/MS/MS TurboIonSpray Ion Source Manual

Turbo Temperature (TEM)

The quantity and type of sample affect the optimal TurboIonSpray temperature. At higher flow rates, the optimal temperature increases. A more significant factor is the composition of the solvent. As the organic content of the solvent increases, the optimal probe temperature should decrease. With solvents consisting of 100 percent methanol or acetonitrile, the probe performance may optimize as low as 300 °C. Aqueous solvents consisting of 100 percent water at flows approximately 1 mL/min require a minimum probe temperature of 425 °C. Normal optimization is usually performed in increments of

25 °C.

The TurboIonSpray is normally used with sample flow rates of 40 µL/min to

1000 µL/min. The heat is used to increase the rate of evaporation and this improves ionization efficiency resulting in increased sensitivity. For recommended temperatures,

see the TurboIonSpray Suggested Operating Settings table on page 13.

CAUTION! Do not operate the TurboIonSpray with probe temperatures greater than

500 °C.

Declustering Potential (DP)

Optimal declustering potential operating conditions with the TurboIonSpray source should be set high enough to reduce the chemical noise, but low enough to avoid fragmentation.

Start with the declustering potential (DP) at 20 V.

Note: The fragmentation energy of a compound is a function of its structure and molecular weight. Generally, lower molecular weight compounds require less energy — lower declustering potential — to induce fragmentation. For some labile compounds, DP values of between 5 and 10 may be required with the TurboIonSpray.

In general terms, the higher the declustering potential, the greater the energy imparted to the ions entering the analyzing region of the mass spectrometer. The energy helps to decluster the ions and to reduce the chemical noise in the spectrum, resulting in an increase in sensitivity. Increasing the voltage beyond optimal conditions can induce fragmentation before the ions enter the mass filters, resulting in a decrease in sensitivity.

In some instances, fragmentation is a valuable tool that provides additional structural information.

Solvent Composition

Commonly used solvents and modifiers are acetonitrile, methanol, propanol, water, acetic acid, formic acid, ammonium formate, and ammonium acetate. The modifiers such as triethyl amine (TEA), sodium phosphate, trifluoroacetic acid (TFA), and sodium dodecyl sulfate are not commonly used because they complicate the spectrum with their ion mixtures and cluster combinations. They may also suppress the strength of the target compound ion signal. The standard concentration of ammonium formate or ammonium acetate is from 2 to 10 millimole per liter for positive ions and 2 to 50 millimole per liter for negative ions. The concentration of the organic acids is 0.01% to 0.5% by volume.

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Q Trap LC/MS/MS TurboIonSpray Ion Source Manual Optimizing the TurboIonSpray

Source Exhaust Pump

WARNING! The TurboIonSpray source exhaust is a safety measure and must be kept operational to ensure continued safe operation.

The source exhaust system is required for TurboIonSpray operation. The exhaust pump draws the solvent vapors from the enclosed source chamber and delivers them to a trap at the rear of the instrument chassis where they can be collected. The source exhaust system is interlocked to the system electronics, such that if the source exhaust pump is not operating to specification, the instrument electronics are disabled.

The exhaust system lowers the pressure in the source slightly below atmospheric pressure.

If the pressure in the source rises beyond a preset trip point, the instrument high voltage power supply is disabled.

WARNING! The source exhaust pump must be vented to either an external fume hood, or external exhaust source.

WARNING! Standard laboratory rules should apply when using or handling flammable compounds with APCI source.

Since this source includes a heating element with an operating temperature above the flammability point of some solvents, it is important to maintain and verify the API instrument before each use of the APCI source.

The pressure switch for the exhaust line must be tested before each use by shutting off the source exhaust gas supply. If the hose is connected to a forced ventilation system, disconnect the hose from the drain bottle. Fault messages will be displayed on the monitor indicating the source exhaust gas is off, which verifies that the pressure switch is working.

If the fault messages are not displayed the pressure switch is defective and the APCI source must not be used. A service call is mandatory. If the above procedure is not followed, the ion source pressure sensor may inadvertently allow the system to operate when the ion source is not being properly exhausted. When the source is not properly exhausted, vapor can escape through the heated nebulizer probe and condense within the probe's electrical wiring. This could cause a short circuit and the possibility of a fire if flammable solvents are used.

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Optimizing the TurboIonSpray Q Trap LC/MS/MS TurboIonSpray Ion Source Manual

16

Exhaust supply connection points

Operating Tips

1. When running the TurboIonSpray, with or without the heater probe on, run the curtain gas at as high a flow rate as possible without decreasing the signal. This will help to: a) Prevent penetration of the curtain gas, which can produce a noisy signal.

b) Prevent contamination of the orifice.

c) Increase the overall signal to noise ratio.

2. The liquid spray from the TurboIonSpray should be directed away from the orifice in order to: a) Prevent contamination of the orifice.

b) Prevent piercing of the curtain gas, which can create an unstable signal.

c) Prevent electrical shorting due to the presence of the liquid.

3. The higher the liquid flow, or the higher the aqueous composition of the solvent, the higher the temperature and heater gas (Gas 2) flow required. However, too high a temperature can cause thermal degradation of the compound, and result in a high chemical background noise, while too high a heater gas flow can produce a noisy, or unstable signal.

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