Exam 3

Covers powerpoints 15-20

Ionization energy

Ionization energy

amount of energy required to expel and electron from its outermost shell

Quadrupoles

• 2 sets

  • 2 vertical are positive

  • 2 horizontal are negative

• Use electrostatic attractions and momentum to get ions through the quarupoles

Time of Flight MS

• all ions leave ion source at the same time WITH same kinetic energy

• larger the ion, the slower the velocity, the longer it takes to traverse through the field-free region

Key components of tuning and calibration GC/MS

• mass accuracy

  • properly assigns masses for calibrant

  • important for database searching

• Mass resolution

  • can distinguish between adjacent masses

  • important for isotope id

• Appropriate sensitivity

  • dynamic system must be adjusted as the source gets dirty

  • need for consistency

• No air leaks

  • need to minimize secondary interactions

  • secondary fragements

  • loss of focus to the detector

Two types of high vacuum pumps

1. Turbomolecular pump

   1. has fixed blades that spin like jet engine

   2. most common

2. Diffusion pump

   1. high molecular fluid is heated

Repeller, IonFocus, EntLens, EntOffs

optimized voltages determined from autotune

Emission and EIEnergy

energies associated with the filament necessary to ionize and create fragments

Filament

indicates which of the two is in use

Stepsize

value between scans

Samples

number of readings taken for the profile data

Averages

number of averages done to the sampled scans

HEDEnab

• high energy dynode

• reduces background signal

EMVolts

• voltage of the tube electron multiplier tube

• over time higher voltages are required to maintain consistent abundances

Centroid data

• typical mass spectra

  • has bars for the ions

• smaller file size

• some information loss

profile data

• looks like a typical chromatogram

  • has peaks that resolve to baseline

• large file size

• no information loss

Manual tune

• can be used to diagnose issues

  • leaks

  • pump oil

  • column bleed

• can manually input settings if there are issues in tune or with data

Ion source differences

• High pressure

  • ion source is located IN high vacuum

• Atmospheric pressure

  • ion source is located OUT of high vacuum

Where is the mass analyzer located?

in the high vacuum

Electrospray ionization

• sample is dissolved in polar solvent

• pumped through stainless steel capillary with voltage between 2000-4000 volts

• liquid is aerosolized as it exits the capillary

  • solvent is evaporated by drying gas and removed by vacuum pumps

• additional fragmentation can be done in this area

  • fragmentor

  • cone voltage

• 2 modes

  • positive

  • negative

positive mode

• best suited to basic drugs that form a stable HCl salt

  • [M+H]+ is the primary ion formed

  • [M+nH]ⁿ⁺ and [M+Na⁺]⁺ can also be formed

• lose electrons

negative mode

• best suited to acidic drugs that form stable Na salts

  • [M-H]^-, [M-nH]^n-, and [M+I^-]^-

• lose hydrogen

Cone voltage

• extracts ions from atmospheric pressure region of the ion source into the high vacuum region of the mass analyzer

• to induce in-source fragmentation (CID) for structural determination

• for declustering heavily hydrated ion sin order to reduce their mass

• Typical cone voltage is 10-60 V

Homolytic cleavage

• breaking of a covalent bond in such a way that each fragment gets one of the shared electrons

• produces free radicals

Heterolytic cleavage

• breaking of a covalent bond in such a way that one atom gets both of the shared electrons

• occurs in polar bonds

• electrons will move to the most electronegative atom

Alpha cleavage

• happens at the alpha position on the molecule

• lose the electron from oxygen radical + bond adjacent

• loss from other side of the molecule

Beta cleavage

• occurs for both homolytic and heterolytic cleavages

• breaks bond in the beta position

Alcohol preferred fragmentation

• cleavage of C-C bond next to oxygen

• loss of water may occur

• possible small molecular ion

Amine preferred fragmentation

• small molecular ion is an odd number if odd number of N atoms

• Base peak forms from cleavage adjacent to the C-N bond

Deconvolution Reporting Software

• noise is defined and compensation factored in

• scan skew is compensated for

• true peak max is found

  • to the nearest 1/10th scan

• Full spectrum deconvolution= all ions used

  • resulting spectra called components

• Components matched against Target Library

  • full spectrum used

GC/MS interpretation procedure

• Identify molecular ion if present

• Evaluate any isotopic observations

• use isotopes to calculate probable carbons numbers for molecule and/or fragments

• Review all losses observed to determine substructures

• review major fragments

• hypothesize a molecular structure consistent with above observations

• CONFIRM Hypothesis with additional data

  • chemical ionization MS

  • High res MS

  • IR

  • NMR

Steps to generate a “good” spectrum

• scan for masses that are high enough

• ensure that there are enough points across the peak

  • scan speed

• subtract the background

  • column bleed

  • co-eluting peak

  • dirty chromatogram

• Scan averaging

  • scan skew

• look for signal saturation

• nitrogen rule

• Rings and double bonds

Nitrogen rule

If molecular mass of an unknown compound to the nearest integer value is an odd number, then the compound contains an odd number of nitrogen atoms

What does HPLC stand for?

High Performance Liquid Chromatography

Basic Components of liquid chromatography

• Mobile phase

• degasser

• high pressure pump

• mixing (proportioning) valve

• Pulse-dampener

• pre-column/guard column

• column

• injector system

• oven

• detector system

HPLC mobile phase

• Typically a binary system

  • Mobile phase A and mobile phase B

• quaternary system (4 solvents)

• polar solvent (aqueous)

• non-polar solvent (organic)

• Modifieres

• Filters/frits for containers

Modifiers

additives to maintain pH (buffers, ion-pairing agents, inorganic salts, organic amines) and other agents that can affect performance

Degassers

remove any dissolved gases in liquid

• sonication

• vacuum filtration

Why a degasser?

• all liquids have dissolved gasses from the air

• dissolved gases reduce pump flow stability, detector baseline, and stability, and increase detector noise

• influence analytical results

Types of pumps

• Reciprocating pump

  • majority of commercial HPLC use this design

• Displacement pumps

  • produce flow that is independent of viscosity and back pressure

    • syringe pump

    • aka metering pump

• Pneumatic pump

  • mobile phase in container is pressurized to force the liquid out at a pre-defined rate

Requirements of HPLC pumping system

• generation of pressures up to 6000 psi

• pulse free output

• flow rates ranging fom 0.1-10 mL/min

• flow control and flow reproducibility of 0.5% or better

• Corrosion-resistant components

Mixing chamber

mobile phases brought together and mixed depending upon the requested proportions

Isocratic= static proportions

Gradient=dynamic proportions

Pulse damper

tubing stretches when pressure rises so extra flow flow from the pump at this moment is accommodated by extra volume of the damper

Acts as a shock absorber to smooth out pulsating flow

Pre-column

small disposable cartridges packed with a frit and some column material

designed to catch any particulate material so that the column does not become clogged

Column

• Dimensions

  • 4.6mm x100 mm (id x length)

• Particle size

  • 3.5 um for HPLC

  • 2 um for UPLC

• Pore size

  • 300 angstrom

• Stationary phase

  • C18, C8, CN, etc.)

Reverse Phase HPLC

• Stationary phase is NON-POLAR

• Mobile phase is POLAR

• most commonly used

• base on partitioning of analyte between aqueous mobile phase and analytical column

• Polar analytes less retained

Normal phase HPLC

• Stationary phase is POLAR

• Mobile phase is NON-POLAR

• non-polar analytes less retained

UPLC

accommodates sub 2 um particles and very high operating pressure

significant improvements in resolution over HPLC and/or faster runtimes

Isocratic mode

constant proportion of mobile phase A and mobile phase B employed. Good for target compound analysis

Gradient mode

employs a constantly varying proportion of mobile phase A to mobile phase B starting highly aqueous (A) and changes to highly organic (B). Allows for multiple analyte detection in a single run

Ion-paring mode

An ion pair reagent is added to the mobile phase to bond to the charged species to shield and cancel apparent changes to allow for retention on the stationary phase

Factor affecting liquid chromatography

• Flow rate

• temperature

• mobile phase composition

  • solvent strength

  • mode

    • isocratic vs gradient

• Amount injected

• pH stationary phase

• column attributes/dimesions

LC detectors

• Uv-Vis

  • Uses absorbance of UV radiation at specific wavelengths

• Diode array

  • entire spectrum is monitored instead of 1 wavelength. Range is 210-300 nm.

• Fluorescence

  • energy of a specific (excitation) wavelength is absorbed. Compound gives off energy at another (emission) wavelength

• Electrochemical

  • based on the measurement of electrical output of a chemical being oxidized or reduced

• MS and tandem MS

LC problems

• plugged frit

• column contamination

• plugged packing

• leak

• flow incorrect

• split peaks from injection solvent effects

• fronting peak

• peak splitting

• Column overload

• Phase collapse

  • irresesevible

  • silica in column gets tangled and does not stick straight out to catch particles like it should

Why HPLC?

• low temperature analysis

• polar compounds do not require derivatization

• numerous detectors

• can be interfaced directly to MS

• numerous column phases

• can inject aqueous samples directly

• more variables to work with

  • pH

  • mobile phase composition

  • many column phases

  • derivatization to enhance performance

  • enatiometic separation

Other HPLC techniques

• Size Exclusion chromatography

• Gel-permeation chromatography

Ion chromatography

• mode of HPLC

  • strong ion exchange columns

  • strong ionic strength mobile phase

• useful for determination of anions

LC applications

• pharmaceuticals

• vitamins

• chiral separations

• proteins

• peptides

• amino acids

• drugs

HPLC vs GC

• HPLC

  • non-volatile samples

  • thermally unstable compounds

  • resolution not as good

  • macromolecules

  • inorganic and ionic samples

  • more complex interface to MS

• GC

  • volatile and thermally stable

  • rapid analysis

  • simple instrumentation

  • good resolution

  • easily interfaces to MS

  • limited in injection solvents

Analyte Polarity

can predict the retention time of analytes relative to each other based on functional groups and how they interact with the mobile and stationary phases.

Mobile phase polarity

• Needs to mix with water

• cannot be 100% aqueous or 100% organic

• Water is most polar and hexane is most non-polar

Stationary phase polarity

• What kind of molecules do we want to stick to the column?

• Silica is most polar and ODS (octodecyl-silica) is most non-polar

Properties of good HPLC detectors

• Selectivity-can look at specific types of molecules

• Sensitivity- should match needs of analysis. If looking in ng/mL, then need super sensitive detector like MS

• Minimal background- ignores it to focus on analytes

• Linear response- needs a large dynamic range to allow for quantitation

• Non-destructive

• HPLC UV-vis is like GC FID

Detector sensitivity

• Refractive index- LOW (1-5 ug)

• Conductivity- LOW (10-50 ng)

• UV-Vis- MEDIUM (0.5-1.0 ng)

• Electrochemical- HIGH (50-500 pg)

• Fluorescence- HIGH (10-100 pg)

• Mass Spectrometer (10-100 fg)

As the components elute from the column they pass into a detector where some physicochemical property of the analyte is measured (the response).

True

Use the checkboxes to indicate which three of the following statements are TRUE

• Solubility in the mobile phase may preclude the HPLC analysis of very large molecules

• In HPLC mobile phase components are selected to ensure sample solubility

• GC is suitable to analyze volatile components

Pick the correct answer

Mass spectrometer produce spectra that can assist with the identification of sample components for both GC and LC

Reversed phase HPLC uses a system in which the mobile phase is more polar than the stationary phase

True

Select the option that best completes the sentence.  In HPLC analytes with strong affinity for the stationary phase

Elute later than those with lower affinity for the stationary phase

Use the checkboxes to indicate which two of the following statements are TRUE (analyte)

1. HPLC analysis has no volatility issues; however, the analyte must be soluble in the mobile phase

2. HPLC can analyze samples over a wide polarity range and is able to analyze ionic samples

Use the checkboxes to indicate which two of the following statements are TRUE (mobile phase)

1. HPLC analysis has no volatility issues; however, the analyte must be soluble in the mobile phase

2. HPLC uses a liquid mobile phase to transport the analytes (sample) through the column

HPLC is able to analyze samples which are both polar and non-polar.

True

The majority of  HPLC detectors are destructive.

False

Use the checkboxes to indicate which three of the following statements are TRUE (detectors)

1. Some HPLC detectors are non-destructive

2. Mass Spectrometers can be used as detection systems for HPLC or GC to assist with analyte identification

3. HPLC should be selected instead of GC when dealing with high molecular weight analytes

Use the checkboxes to indicate which two of the following statements are TRUE (protein)

1. Large proteins and peptides can be analyzed by HPLC

2. HPLC is usually carried out at or around room temp

Use the checkboxes to indicate which two of the following statements are TRUE  (chromatogram)

1. High efficiency (narrow) chromatographic peaks have a better chance of being resolved from one another

2. Good chromatographic peaks are symmetrical

Identify the various components of the UV flow cell and detector:

• A=inlet capillary

• B=outlet capillary

• C=collimated light

• D=photodiode

• E=flow cell window

Which of the following properties is essential for mobile phase components to be compatible with charged aerosol detection?

Volatile

The recommended minimum signal to noise ratio for the limit of detection (LOD) of a detector for HPLC is:

3:1

Identify the monochromator type from the schematic diagram of the fluorescence detector blow

• A=excitation monochromator

• B=emission monochromator

Identify the components of the diode array detector:

• A=Achromatic lens system

• B=flow cell

• C=slit

• D=grating

• E=photodiode

Match the chromatographic output with the detector response time used for the data acquisition:

• A=0.1 second

• B=2.3 seconds

Which of the processes shown represents fluorescence

B

Which of the chromatograms below was acquired using a reference wavelength?

B

Match the output shown below with the DAD slit width setting used for the data acquisition:

• A= 16 nm slit width

• B= 1 nm slit width

Which of the following buffers would not be suitable for evaporative light scattering detectors?

Sodium phosphate

From the analyte UV spectrum below select an appropriate reference wavelength setting for diode array data acquisition:

350 nm

A variable wavelength detector can be used to achieve which of the following

Measure absorbance at a single wavelength

What is the parameter X marked on the detector response schematic below?

Noise

The linear range of an analytical method represents

The concentration range over which the signal obtained is directly proportional to the analyte amount

Match the spectrum obtained with the DAD bandwidth setting used to acquire the data:

• A= 8nm bandwith

• B=30 nm bandwith

Identify the component in the variable wavelength detector shown that is used to select the desired wavelength for the absorbance measurement

E

To increase charged aerosol detector response for low volatility analytes which of the detector parameters should you adjust?

Increase evaporation temperature

Identify the excitation and emission maxima from the method development experiment below:

• A= excitation wavelength

• B=emission wavelength

Which 3 of the following could cause excessive baseline noise in evaporative light scattering detectors?

1. non-volatile mobile phase components

2. column bleed

3. nebulizer temperature set too high

In reversed phase HPLC the term sacrificial base refers to

A base that is sued to minimize unwanted secondary silanol retention

The chromatograms shown were obtained with four different mobile phase systems (same sample, reversed phase conditions): 30%THF, 30% acetonitrile, 30% methanol, and 40% methanol. Which chromatogram was obtained with 30% methanol?

Chromatogram 1

Rank the molecules shown in order of retention . Place the LEAST retained molecule in the first place and the MOST retained molecule in the fourth place

• C is 1st

• B is 2nd

• A is third

• D is fourth

When increasing mobile phase water content in reversed phase HPLC the retention time for the presented analyte will:

Increase

Match the chromatogram with the mobile phase composition that was used for the separation

• A=30% organic

• B=50% organic

• C= 70% organic

• D=15% organic

Sodium acetate has a pK_a of 4.8. Over which pH range would you recommend it as a suitable buffer system?

3.8-5.8