1a hplc instruments

7/29/2019 1a HPLC Instruments

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Chapter 2

HPLC Instruments


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Content

z Composition of the HPLC system

z Pumps

z Injectors

z Detectors


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The Chromatographic System

Solvent

Pump

AutoSampler

HPLC Column

Detector

Waste

Data System


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Criteria for HPLC Pumps (Analytical)

z Constructed of materials that are chemically

resistant to the mobile phase

z Capable of delivering flow at min. 4000 p.s.i.

z Pulse-free

z Flow delivery 0.01 - 5mL/min or greaterz Flow reproducibility


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Types of HPLC Pumps

z Reciprocating pumps

Single piston

Dual piston, parallel

Dual piston, serialz Positive displacement pumps

Syringe type

z Pneumatic


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Dual Piston, Parallel Pump


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Dual Piston, Parallel Pump

Plunger Movement


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Pump Head

Outlet check valve

Plunger seal

Ball and seat

Plunger

Inlet check valve

Filter cup


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Inlet/Outlet Check Valves

Inlet check valve (510/515)


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Dual Piston, Serial Pump

Flow SchematicsCritical Functions

1. Optional gradient proportioning valve(GPV)

2. Check valves

3. Primary head plunger4. Primary pressure transducer

5. Accumulator head plunger

6. System pressure transducer

7. Independent piston drive motors

AB

CD

Eluents1

2

2

PrimaryPrimary

AccumulatorAccumulator

5

4

6To System

3

7

7


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Injector Types

z Manual injectors

Fixed loop

Variable volume

z Autosamplers/autoinjectorsFixed loop

Variable volume


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Criteria for Injectors

z Allow multiple samples to be injected using an automatedor semi-automated process

z

High injection precisionz Must have a needle washing routine

Virtually eliminates cross-contamination between samples

z Minimal wasted sample

z Should be useable for analytical to semipreparative LCz Least possible change to the solvent flow path

z Make-before-Break (MBB) switching

No interruption of flow when the injector valve moves betweenLoad and Inject positions

Prolongs the life of certain columns


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Fixed loop Injection Valve

Manual/Automatic (Rheodyne)

Initial fluid element Fluid element after flow

Tube wallSample loop

Laminar flow

pump

column

Position 1 (load) Position 2 (inject)


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Schematic of a Variable Volume

Autosampler(Waters 717/2695)


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Normal Configuration


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Sample Withdrawal


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Sample Injection

S f


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Schematics of a Fixed Loop Injector(Waters 2795)


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Autosamplers,

Sample Formats, Vials

z Crimp cap

Cheapest solution Needs a crimping tool

z Snap cap

Easy to use - no tools Traditional or New LectraBondTM versions

z Screw cap The universal version

Traditional or New LectraBondTM versions

z Limited volume vials Inserts

With internal taper

Needle opening


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HPLC Detectors

z Operates by registering an output in response to sample

detection.z A linear relationship is expected between response of the

detector and concentration of the sample, and calibration

techniques are designed to promote this relationship.

z Not all detectors are linear and effects from other

components of the HPLC system may cause the detector

to deviate from a linear relationship between response

and concentration.


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HPLC Detectors

z HPLC detectors can be classified as:

Solute property detectors -respond to physical or

chemical properties of the solute that are

generally not exhibited by the mobile phase.

Bulk property detectors -respond to an overall

change in the physical property of mobile phase

with and without solute.

There is no single detector that can be employed for all

HPLC separations the magic box!


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Criteria for HPLC Detectors

z High sensitivity

z Negligible baseline noise

z Large linear dynamic range

z Response independent of variations in operating

parameters (pressures, temperature, flow-rateetc.)z Response independent of mobile phase

z Low dead volume

z Non-destructive

z Stable over long periods of operations.

z Selective


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UV/Visible Detectors

z Most frequently used detector in HPLC

analysisz Compounds must contain a UV absorbing

chromophore

z Must work in the linear range of Beers Law

A = b c

Molar extinction coefficient cell path length (cm)

Sample concentration (moles/l)Absorbance


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Schematics of a Variable

Wavelength UV/Vis Detector


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The Photodiode Array

(PDA) Detectorz Advantages

Can provide UV spectra across peaks

Can be used to assess peak purity/ spectral

homogeneity

Can be used to track peaks via their UV spectra(library search)


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Optical Resolution

Influence on Linearity

1 nm10 nm #2

10 nm #1

AU

Concentration

Narrow spectral

peak #1 with awide bandpass (10nm) is non-linear

230 240 250 260Wavelength, nm

Absorbanc

e

1 nm

10 nm

1

2


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Example of UV Spectra

Spectrum index plotSpectrum index plot


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Example of Library Search

U V Cutoffs for Some


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U.V. Cutoffs for Some

Common Solvents

SolventSolvent UV CutoffUV Cutoff SolventSolvent UV CutofUV Cutof

WaterWater 180180 N-HeptaneN-Heptane 197197MethanolMethanol 205205 CyclohexaneCyclohexane 200200N-PropanolN-Propanol 205205 Carbon tetrachlorideCarbon tetrachloride 265265

AcetonitrileAcetonitrile 190190 ChloroformChloroform 245245THFTHF 225225 BenzeneBenzene 280280

AcetoneAcetone 330330 TolueneToluene 285285

Methyl acetateMethyl acetate 260260 Methylene chlorideMethylene chloride 232232Ethyl AcetateEthyl Acetate 260260 TetrachloroethyleneTetrachloroethylene 280280NitromethaneNitromethane 380380 1,2-Dichloroethane1,2-Dichloroethane 225225

All wavelengths reported in nm.

Remember that Solvents chosen can affect detection!!

UV cutoff = Wavelength at which solvent absorb 1 AU


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Fundamentals of Fluorescence

Some Applicationsz Environmental

Polyaromatic Hydrocarbons

Phenols, carbamates

z Food and Beverage

Alfatoxins in Food Products

Dyes

z Biotech and Pharmaceuticals Derivatized amino acids

(AccQ-Tag or OPA)

Conjugated and aromatic systems are most likely to exhibit fluorescence


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Refractive Index Detectors

z First HPLC detector developed

z

Typically referred to as Universal detectors Detects all dissolved solutes- non-specific

RI response depends on the difference in RI between mobile

phase and solute(s).

Sensitivity reaches maximum when RI differences are greatest.

z Utilizes Snells Law Principles

g sensitivity but only for isocratic runs

Commonly used for:

Sugars, Polymers and Fatty Acids


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Schematic of an RI Detector

LAMP

LED orIncandescent R

S

To Amplifier

No sample

With sample

R

S


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Conductivity Detectors

z Generally used for ion Chromatography

z Detects the ability of analyte to carry a charge

solutes are ionic (that is, acid and bases)

Inorganic anions and cations

z Sensitivity is at the low ppb (ng/L) level

z Typically used with isocratic systems but canutilize isoconductive gradients


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Schematics of Conductivity Detector

Mobile phase

Mobile phase andsolute(s)


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Electrochemical Detector (ECD)z Destructive-detection mode

z

Sample is electrochemically modified in the cell with theconcomitant generation of current detected as response.

z High sensitivity with picogram (10-12 grams) to high

femtogram (10-15 grams) range.z Proper selection of buffer, electrode and voltage is critical

to success as well as conditioning the mobile phase.


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Some Compound Types


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Some Compound Types

Sensed by ECDOxidation Reduction

Phenolic Ketones

Oximes Aldehydes

Dihydroxy Oximes

Mercaptans Conjugated acids

Peroxides Conjugated esters

Hydroperoxides Conjugated unsaturation

Aromatic Amines, diamines Activated halogens

Purines Aromatic halogens

Heterocyclic Rings Nitro compounds

Heterocyclic rings


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Benefits of MS Detection

z Additional information Molecular weight

Structure

z Better Selectivity Possible Quantification of UV co-eluted compounds

Shorter analysis

z Sensitivity In Sir mode, MS detection can be much more sensitive than

UV detection (100 times)

z Cross check UV and MS library search Improved compounds characterization and differentiation

z Speed up method development Improve impurities identification

Improve impurities characterization


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Additionnal Information

277 278 279 280 281 2820

2,000

4,000

6,000

8,000

10,000

12,000

Parent Ion Region

MW 278

100 150 200 250 3000

2,000

4,000

6,000

8,000

10,000

12,000

m/z

Intensity

[M+H]+

m/z

Positive ion mode,ESI, 2 ul/min, 50%MeOH:49%H2O:1% HOAc

0.3 mg/ml

100 150 200 250 3000

2,000

4,000

6,000

8,000

10,000

m/z

Intensity

[M+H]+

Fragment Ion

Region

183 184 185 186 187 188 1890

2,000

4,000

6,000

8,000

10,000

m/z

C H N O S6 8 3 2

m/z = 186

NH

N

N

CH3

CH3

NH2 S

O

O

Sulfamethazine

92

156

186

At High Cone Voltage

(fragmentation occurs)

At Low Cone Voltage

(typically a Protonated Molecule is

observed)


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z Need for Selectivity.

Matrix interference

very similar structures, impossible separation

Better Selectivity


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z The molecules are difficult to detect.

Zero or poor UV absorbance or Fluorescence

Matrix interference

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00Time9

100

%

-149

100

%

3: Diode ArrayTIC

3.45e6

TIC8.96e5

Sensitivity

UV

MS


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z Need for better Qualitative Information

Component Confirmation Mass Spectrum (next to UV-Spectrum)

Library

Valuable information on Unknowns

UV and MS Libraries

Choosing a Detector


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Choosing a Detector

for HPLC RI UV/VIS Fluor. ECD Cond. MS

Response Universal Selective(Chromaphor) Selective(Fluorophor) Selective(Redox) Selective(Ions) Selective(Ionizable)

Sensitivity gram nanogram picogram picogram picogram picogram

LinearRange

104

105

103

106

105

103

Flow

Sensitive

Yes No No Yes Yes Yes

Temp.Sensitive

Yes No No Yes Yes No


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