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New Mexico State University
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 Waters HPLC/MS

Screen MassLynxGeneral Operating Conditions and Paramaters

Background -- The instrument is really two intrumental modules combined to make one very powerful analytical tool, managed by software called, MassLynx. The opening screen for MassLynx software used for instrument control and data analysis is shown at the left. There are several modules.

The first module is a high-performance liquid chromatograph (HPLC). Waters Alliance 2690 Separations Module has the following capabilities: HPLC

Bullet Four-solvent mixing in gradient or isocratic mode
Bullet Continuous seal wash system
Bullet In-line vacuum degassing
Bullet Refrigerated autosampler compartment
Bullet Heated column compartment

The second module is a mass spectrometer (MS). The Waters-Micro
MSmass ZQ2000 Mass Spectrometer has the following capabilities:
Bullet electrospray ionization probe (atmospheric pressure)
Bullet Z-spray extraction system (dual orthoganol sample cones)
Bullet cone gas to minimize cone fouling
Bullet hexapole for ion focusing
Bullet single quadropole system
Bullet photomultiplier system to prevent electron mulitplier fouling

The MS continuously monitors the HPLC solvent flow. When a compound of interest (analyte) elutes from the separations module, the MS first vaporizes the HPLC solvent to remove it, and then it ionizes the compound. The MS then electronically propels the ion through an extraction cone, then into a molecular-mass sorter and finally, into a particle counter. A computer monitors the particle count for a range of masses and ion charges. The values generated provides a mass spectrum that shows information about an analyte's identity as well as its concentration in the sample.

HPLC is a chemistry based tool for quantifying and analyzing mixtures of chemical compounds. It's used to find the amount of a chemical compound within a mixture of other chemicals. An example would be to find out how much caffeine there is in the cup of coffee (or tea, or cola). First dissolve the sample in a solvent (like water or alcohol), thus the term LIQUID chromatography and then a detector measures response changes between the solvent itself, and the solvent plus the sample when passing through. The electrical response is digitized and sent to a data system.

A solvent delivery System - pushes the solvent stream through the instrument at constant flow rate
into a column - a stainless steel tube packed with silicon beads that HPLC Systemseparates an analyte (the caffeine) from other compounds (like sugar), a detector - or an optical sensor that detects changes in the characteristics of the solvent stream, plus a data system which controls the system components followed by storing, processing, and displaying data. In a liquid chromatographic process a liquid permeates through a porous solid stationary phase and elutes the solutes into a flow-through detector.

The stationary phase is usually in the form of small-diameter (5-10 mm) uniform particles, packed into a cylindrical column. The typical columnHPLC Columns is constructed from a rigid material (such as stainless steel or plastic) and is generally 5-30 cm long and the internal diameter is in the range of 1-9 mm. Column - A tube which contains the stationary phase. The stationary phase differentially interacts with the sample's constituent compounds as they are carried along in the mobile phase. The most efficient columns produce the sharpest peaks, which gives better separation by minimizing band spreading. A tight narrow sample "band" is produced when a columns' stationary phase is uniformly packed (mechanical). The packing material adds "chemical" band spreading (tailing, pore depth etc.).

An other PADDetector (in series or parallel to the MS) is a UV/VIS: Waters 996 photodiode array detector. Detector - Instrument in the chromatographic system which senses the presence of a compound passing through, and provides an electronic signal to a recorder or computer data station. Output is usually peaks, that is, the chromatogram. The detector consists of:
o UV – Ultraviolet light
o Lamp
o Grating/Lens - Wave length
o FlowCell
o PhotoDiode


Conventional HPLC detection relies solely on peak retention time for LC-MSidentification, in order to identify unknown peaks after a separation, greater specificity is requred from the detector. A specific, non-universal HPLC detector, may optimize detection of known compounds, without prior identification of the samples componets.

The Waters integrated system allows identification of peaks in HPLC separations. It separates complex mixtures and identifies peaks of interest. The integrated photodiode array (PDA) detection and mass spec detection is the heart of the unit. The combination PDA and MS data from a single HPLC injection for productes positive compound identification. The PDA detector also helps to ascertain peak homogeneity. Each chromatographic peak generates a positive Electron Ionization (EI) mass spectra. These spectra are characteristic "fingerprints" for compounds, which, unlike ambiguous molecular weight information, can identify compounds. Automated library search for compounds to match both mass spec and PDA spectra against commercially available and/or user libraries for sample identification of analysis.

The MASSLYNX.INI file contains current settings for all MassLynx windows andLogo dialog boxes. When a new user logs on a new Username.INI file is created. Each time this user uses MassLynx any changes to the current settings are saved to this file. It is possible under some conditions that one of the settings in Username.INI may become corrupted causing problems with the operation of MassLynx. A default INI file is present in the C:\MASSLYNX directory saved under the name MASSLYNX.SAV. In cases where the INI file has become corrupted this backup file can be copied to Username.INI to restore a set of uncorrupted default parameters

To restore MASSLYNX.SAV using Windows Explorer
1. Save any parameter files needed in MassLynx e.g. tuning parameter files.
2. Close down MassLynx.
3. Press Start button, choose Windows NT Explorer from the Programs option.
4. Select the MassLynx directory
5. Highlight the MASSLYNX.SAV file and select Copy from the Edit menu.
6. Choose Paste from the Edit menu. This will give a file called Copy of MassLynx.sav.
7. Delete the USERNAME.INI file , e.g. Administrator.ini, and select the copy of Masslynx.sav. Click on the name of the file until a rectangular box appears around the name, then type in USERNAME.INI, or choose rename from the Edit menu.
8. Restart MassLynx.
9. This will set the MassLynx system, for this user, back to a default state, for example the current raw data file will be V50 etc.

A nitrogen generator is used to produce a constant supply of high purity gas to the instrument. The nitrogen generator is an alternative to traditional gas supply (cylinders, or Dewars of liquid nitrogen).

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