detection qualification and types of detectors in hplc.pdf
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Detection Qualification and Typesof Detectors in HPLC
Dr. Shulamit LevinMedtechnica
http://www.forumsci.co.il/HPLC
Detection in HPLC
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The most common HPLC detectors:UV/Vis
Fixed wavelengthVariable wavelengthDiode array
Refractive indexFluorescenceElectrochemical
Less common:ConductivityMass-spectrometric (LC/MS)Evaporative light scattering
DetectorsBeer's LawAbsorbance = Extinction Coefficient x
Pathlength x Concentration
Reduce Pathlength Reduce Concentration
Only for monochromatic light
Beer's Law
Absorbance = Extinction Coefficient x Pathlength x Concentration
ABExtinction Coefficient
Concentration Concentration
A b s o r b a n c e
A U
T r a n s m
i t t a n c e
%AU %T T0 100 1.0001.0 10 0.1002.0 1 0.0103.0 0.1 0.001
T=solvent/sample A=log(solvent/sample)
A B
*
*
*
*
**
*
*
*
*
Single Wavelength UV Detector
Optical Light Path
Mercury, Zincor Cadmium
Source Lamps
Dual
Photodiode
Sample side
Reference side
Wavelength Aperture Plate Wavelength
Filter
Flow Cell
Detection in HPLC
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UV-VIS Detector Optical Bench
Deuterium Arc Lamp
RotatingDiffraction
Grating190 to 600nm
Taper-CellFlowCell
Beam Splitter Mirrors
DualPhotodiode
ApertureSlit
IlluminationLens
Beam-Defining Apparatus
Optical Light Path
Sample side
Reference side
UV Detection of AccQ-Tag AminoAcid Derivatives
SampleName: Cult Std Vial: 1 Inj: 1 Ch: 486 Type: Standard
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00
AU
Minutes
A s p a r t
i c A c i
d
G l u t a m
i c A c i
d
H y d r o x y p r o l
i n e
S e r
i n e
A s p a r a g
i n e
A M Q
G l y c i n e
G l u t a m
i n e
H i s t i d i n e
N H 3
T h r e o n i n e
A r g
i n i n e
A l a n i n e
P r o
l i n e
A l p h a - a m
i n o b u t y r
i c a c
i d
T y r o s i n e
C y s
t e i c A c i
d
V a
i n e
M t e h i o n
i n e
O r n
i t h i n e
L e u c
i n e L y
s i n e
P h e n y
l a l a n i n e
T r y p t o p
h a n I s
o l e u c i n e
Liquid fromcolumn
Extraction of 3D Data
Wavelength
A b s o r b a n c e Spectrum
Time
A
b s o r b a n c e
Chromatogram
1
2
Detection in HPLC
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Minutes
nm
4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
260.00 260.00280.00 280.00300.00 300.00
320.00 320.00
340.00 340.00360.00 360.00
380.00 380.00400.00 400.00420.00 420.00
440.00 440.00
nm
0.00000.0000
0.00020.0002
0.00040.0004
0.00060.0006
AUAU
Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm
PDA Spectrum Index Plot DNPH Derivatives 0.25 ng Each Peak
Maximum Impurity Detection
260.00 260.00
280.00 280.00300.00 300.00320.00 320.00340.00 340.00360.00 360.00380.00 380.00
400.00 400.00420.00 420.00
440.00 440.00
nm
Minutes
nm
-0.00010 0.00010
0.00000 0.00000
0.00010 0.00010
0.00020 0.00020
0.00030 0.00030
18.40 18.60 18.80 19.00 19.20
AU AU
Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng 360nm 996PDA 360.0 nm
Hexaldehyde 2,5-Dimethylbenzaldehyde
Coelution of DNPHHexaldehyde and2,5-Dimethylbenzaldehyde
PDA and fluorescent DetectorComparisons for Aflatoxin Analysis
SampleName: Aflatoxin Mix Vial: 2 Inj: 1 Ch: SATIN Typ e: Standard
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
mV
Minutes
AflatoxinG2
AflatoxinG1
AflatoxinB2
AflatoxinB1
UV at 360 nm 31 point smoothedUV at 360 nmFluorescence 365 ex 455 em
Photo-diode arrayChromatographic and Spectral Sensitivity
1.6 2.4 3.2 A b s o r b a n c e
Minutes
0.07 mAU0.15 ng Ethylparaben
210.0 250.0 290.0nm
0.07mAU0.530 AU
Detection in HPLC
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The most common HPLC detectors:UV/Vis
Fixed wavelengthVariable wavelengthDiode array
Refractive indexFluorescenceElectrochemical
Less common:ConductivityMass-spectrometric (LC/MS)Evaporative light scattering
Detectors
LAMP
SLED or Incandescent
R
R
S
To Amplifier
No sample = n
With sample = n+
X = Const x n
Differential Refractive Index Detector
Refractive Index Detection withDifferential RI - Sugars
-160.00
-140.00
-120.00
-100.00
-80.00
-60.00
-40.00
-20.000.00
20.00
40.00
60.00
80.00
100.00
120.00
5.00 6.00 7.00 8.00 9.00 10.00 11.00
mV
Minutes
Fructose
Dextrose Sucrose
Maltose Lactose
SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard
Bagel Extract
Refractive Index Detection withDifferential RI - Sugars
SampleName: Sugar Stds -500 ng each
0.0000002
5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00
Minutes
Sucrose
DextroseFructose
00000000
0.0000004
del
RIU
Detection in HPLC
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Refractive Index Detection withDifferential RI - Polymers
0.0050.00
100.00
150.00200.00250.00300.00
350.00400.00450.00
500.00550.00600.00650.00
700.00750.00800.00
18.00 20.00 22.00 24.00 26.00 28.00 30.00
MV
Minutes
1 2 6 0 0 0 0
9 6 4 0 0
5 5 7 0
SampleName: GPC STDS
Dow 1683
2 8 9 0 0 0 0
1 9 0 0 0 0
1 0 3 0 0
192300
SensitivityRefractive Index Detector
D e
l R I U
5.0 6.0 7.0 8.0Minutes
1 2
250 ng on column1=Tristearin2=Myristic acid
Styragel HR 0.5,4.6 x 300 mm,35C, 0.35 mL/min
dRI sensitivity =32X, 32C
The most common HPLC detectors:UV/Vis
Fixed wavelengthVariable wavelengthDiode array
Refractive index
FluorescenceElectrochemical
Less common:ConductivityMass-spectrometric (LC/MS)Evaporative light scattering
DetectorsFluorescence Detectors
Excitation filter
LAMP
Cell
Emission filter
Photomultiplier
Short pass - transmits all wavelengths below a specified cutoff Long pass - transmits all wavelengths above a specified cutoff Band pass - blocks all wavelengths outside a specified band
Detection in HPLC
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Fluorescence Detector Optical Bench
LAMP
Photomultipliertube
EmissionGrating
ExcitationGrating
Mirror
Torroidal Mirror
Beam Splittter
FlowCell
Mirror
Torroidal Mirror
Photodiode
ExcitationSlit
Emission
Slit
SensitivityFluorescence Detector
0.1 pg Anthracene
Excitation = 251 nmEmission = 406 nm
Minutes
0.0 1.0 2.0 3.0 4.0 5.0
5 mV
mV
Fluorescence vs. UV Detection
Minutes
AMQ
20.00 40.00 60.00
R e s p
o n s e
AccQ-Tag amino acidanalysis
FluorescenceExcitation=250 nm
Emission=395 nm
UV 254 nm
The most common HPLC detectors:UV/Vis
Fixed wavelengthVariable wavelengthDiode array
Refractive index
FluorescenceElectrochemical
Less common:ConductivityMass-spectrometric (LC/MS)Evaporative light scattering
Detectors
Detection in HPLC
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Electrochemical Detector
Reference Electrode
Working Electrode
Electrolyte (mobile phase)
Auxiliary Electrode
Analyte is oxidized or reduced
+ -
As compounds are oxidized or reduced, a current proportional to concentration is produced.
Electrochemical Detection ofCatecholamines & Related Compounds
1 . N or ep in eph er in e 1 50 pp b2. Epinepherine 200 ppb3. Normetanepherine 50 ppb4. Dopamine 200 ppb5. Metanepherine 2 00 ppb6. 3-Methoxytyramine 75 ppb
7. 4-Methoxytyramine 500 ppb
2.00 4.00 6.00 8.00 10.00 12.00
Minutes
0.00
nAmps
1 2
3
45
6
7
Pulsed Amperometric Detectionof Monosaccharides
1. Fucose2. Galactosamine3. Glucosamine4. Galactose5. Glucose6. Mannose
20.00Minutes5.00
mV
0.00
300
12
34
5
6
The most common HPLC detectors:UV/Vis
Fixed wavelengthVariable wavelengthDiode array
Refractive index
FluorescenceElectrochemical
Less common:ConductivityMass-spectrometric (LC/MS)Evaporative light scattering
Detectors
Detection in HPLC
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Conductivity Detector
Mobile phase
Mobile phase plus sample
Conductivity Detection of SevenAnion Standard
0.70
1.05
1.40
S
0.00 5.00 10.00 15.00 20.00 25.00Minutes
1
2
3
45
6 7
1. Fluoride2. Chloride3. Nitrite4. Bromide5. Nitrate6. Phosphate7. Sulfate
1 ppm2 ppm4 ppm4 ppm4 ppm6 ppm4 ppm
Column:Eluent:Flow rate:Injection vol.:Detection:
Waters IC-Pak Anion HCBorate/Gluconate2.0 mL/min100 LDirect Conductivity
Conductivity and UV Detectors inSeries
S
0.00
0.40
0.80
1.20
1.60 1 2 3
45
6
7
0.00 4.00 8.00 12.00 16.00 20.00 24.00Minutes
0.00
0.01
0.02
0.03
0.04
0.05
AU
3
4
5 Column:Eluent:
Flow rate:Injection vol.:
Waters IC-Pak Anion HR1.2 mM Sodium Carbonate/1.2 mM Sodium Bicarbonate1.0 mL/min50L
Detection: Direct Conductivity afterSuppression
Detection: UV (PDA) at 214 nm
1. Fluoride2. Chloride3. Nitrite4. Bromide5. Nitrate6. Phosphate7. Sulfate
1 ppm2 ppm4 ppm4 ppm4 ppm6 ppm4 ppm
Applications
Sensitivities for compounds such as phenol, catecholamines,nitrosamines, andorganic acids arein thepicomole (nanogram)range.
The mobile phase must be made electrically conductive,usuallyby the addition of a suitable salt:
Ion Exchange
Reversed Phase and Ion-Pair RP
No normal phase separations
Detection in HPLC
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The most common HPLC detectors:UV/Vis
Fixed wavelengthVariable wavelengthDiode array
Refractive indexFluorescenceElectrochemical
Less common:ConductivityMass-spectrometric (LC/MS)Evaporative light scattering
Detectors
The scattered light is detected by asilicone photodiode located at a
90 angle from the laser. Thephotodiode produces a signalwhich is sent to the analogoutputs for collection. A light trapis located 180 from the laser tocollect any light not scattered byparticles in the aerosol stream.
EVAPORATIVE LIGHT SCATTERING
Radioactive Detector Primarily used for the measurement of 3H, 14C, and 32P,beta-emitters and many soft gamma and positron emittersencountered in bio-medical researchand pharmaceuticalqualitycontrol.
How LC-MS Works
MassSpectrum
HPLC
Source Analyzer IonDetector
DataSystem
LC/MSInterface
Separation
Desolvation
Ionization Sorting of Ions Detection DateProcessing
x
Detection in HPLC
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Mixture
2.00 4.00 6.00 8.00 10.00Time0
Int.1: MassChromatogram
5.054.65
3.82
0.74
8.62
5.65
8.02
10.62
Mixture
2.00 4.00 6.00 8.00 10.00Time0
Int.1: MassChromatogram
Mixture
2.00 4.00 6.00 8.00 10.00Time0
Int.1: MassChromatogram
5.054.65
3.82
0.74
8.62
5.65
8.02
10.62
T otal- I on-Current Chromatogram
with poor resolution
Mix
60 80 100 120 140 160 180 2 00 2 20 240 2 60 280 300 3 20 340m/z0
100
%
(10.696) 1: ScanES+4.34e5262.87
59.99
213.90
195.98120.8068.92 98.85
76.87 128.82 170.92
222.87
235.87
240.88
263.87
264.85
267.91287.01 309.02333.84
Mix
60 80 100 120 140 160 180 2 00 2 20 240 2 60 280 300 3 20 340m/z0
100
%
(10.696) 1: ScanES+4.34e5
Mix
60 80 100 120 140 160 180 2 00 2 20 240 2 60 280 300 3 20 340m/z0
100
%
(10.696) 1: ScanES+4.34e5262.87
59.99
213.90
195.98120.8068.92 98.85
76.87 128.82 170.92
222.87
235.87
240.88
263.87
264.85
267.91287.01 309.02333.84
LC-MS
190
Electron Multiplier
I n l e t
RingElectrode,Rf
EndCapElectrode
AxialModulation
IonIon TrapsTraps
+ + +++ + ++
Types of Mass
SpectrometersAnalyzers
Types of Mass
SpectrometersAnalyzers
Ti m e O f F l i g h t M a s s A n a l y z e r sTi m e O f F l i g h t M a s s A n a l y z e r s
SOURCE
DETECTORREFLECTRON MODE
R E F L E C T R O N O ND E T E C T O RL I N E A R M O D E
DRIFT TUBE
SOURCE
DETECTORREFLECTRON MODE
R E F L E C T R O N O F F DETECTORLINEAR MODEDRIFT TUBE
Ti m e O f F l i g h t M a s s A n a l y z e r sTi m e O f F l i g h t M a s s A n a l y z e r s
SOURCE
DETECTORREFLECTRON MODE
R E F L E C T R O N O NDETECTORLINEAR MODEDRIFT TUBE
SOURCE
DETECTORREFLECTRON MODE
R E F L E C T R O N O F F D E T E C T O RL I N E A R M O D EDRIFT TUBE
2 2 0
IonSource
Slit
Magnetic sector
Electrostatic Sector
( E S A )
Detector
SlitNier-Johnson-Geometry (EB)
SectorSector Mass SpectrometersMass S pectrometers
199
FTFT -- ICR ICR --SpectrometerSpectrometer
DCDC
DC
Source
Filament
Transferoptic
Trapping Plates
TransmitterPlates
Receiver Plates
Sender
Elektroden Electrodes
Y
ZX
Magnetic Fielt B
77
IonSource
Detector
nonresonant Ion
resonantIon
dcandRfVoltages
TheThe Quadrupole AnalysatorQuadrupole Analysator
Time (min)
1.00 2.00 3.00 4.00 5.000
100
0
100
0
100
0
100
0
100 MW=2958.11e4
2.56
MW=2802.21e5
1.57
MW=2641.26e5
2.16
MW=2601.53e51.29
TIC3.50e5
1.571.29 2.16
2.56
Ding Time (min)
1.00 2.00 3.00 4.00 5.000
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
1008.11e4
2.56
2.21e51.57
1.26e52.16
1.53e51.29
TIC3.50e5
1.571.29 2.16
2.56
Ding
10 L injection of 200 ng/mL sample (in 40%MeOH),1=Propranolol, 2=Doxepin, 3=Nortriptyline,4=Trimipramine, 65/35 0.1 % Formic Acid / MeCN0.2 mL/min
1 00 1 25 1 50 1 75 2 00 2 25 2 50 2 75 3 00Mass/Charge (m/z)
0
100
0
100
100
0
1004.59e4295
296
1.13e5280
281
7.67e4264
233 265
9.25e4260
261
0O
N
N
N
NH
O NH
OH (1)
(2)
(3)
(4)
10 L injection of 200 ng/mL sample (in 40%MeOH),1=Propranolol, 2=Doxepin, 3=Nortriptyline,4=Trimipramine, 65/35 0.1 % Formic Acid / MeCN0.2 mL/min
1 00 1 25 1 50 1 75 2 00 2 25 2 50 2 75 3 00Mass/Charge (m/z)
0
100
0
100
100
0
1004.59e4295
296
1.13e5280
281
7.67e4264
233 265
9.25e4260
261
0O
N
O
N
N
N
NHNH
O NH
OH
O NH
OH (1)
(2)
(3)
(4)
Fast LC-MS Analysis2.1 x 50 mm ( 5 m) HARDWARE - ES/APCI Ion SourceHARDWARE - ES/APCI Ion Source
ESI and APCI are easily interchangeable in seconds withoutventing the system
ESI and APCI use a unique counter electrode to optimizesampling from the liquid spray and to aid sample desolvation
Automatic Probe recognition
Detection in HPLC
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ESI-MS Ion FormationESI-MS Ion Formation
n = 20
n = 22
n = 18
n = 16, m/z = 1060
n = 23, m/z = 738
n = 21
n = 19
n = 17
Horse Heart Myoglobin
n = 20
n = 22
n = 18
n = 16, m/z = 1060
n = 23, m/z = 738
n = 21
n = 19
n = 17
Horse Heart Myoglobin
Mass RangeMass RangeMultiply Charged MoleculesMultiply Charged Molecules
Calculated MassAcquired Mass rangeAcquired Mass range
APCI Probe Equipped With aAPCI Probe Equipped With aHeated Nebulizer Heated Nebulizer
From LCColumn
NebulizerGas
Heater Block
Makeup Gas
Corona Discharge Needle
S M S S MSS S S M MS
MSH+SH+
SH+
SH+
SH +M
+ MH +S
+ ToMass Analyzer
760 torr vacuum
Generates molecular weight and structural information APCI flow rate: 0.2 to 2mL/minute Option: Crossflow interface
Liquid Chromatography- Mass Spectrometery(LC-MS)
Detection in HPLC
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Triple Quadrupoles MS-MS
Modes of Operation
Triple Quadrupoles MS-MS
Modes of Operation
Multiple Reaction Monitoring
Typically used in Quantitative Work ofTriple Quadrupoles
Multiple Reaction Monitoring
Typically used in Quantitative Work ofTriple Quadrupoles
MS 1 CollisionCell
MS2
StaticStatic
Multiple Reaction Monitoring
Typically used in Quantitative Work ofTriple Quadrupoles
Multiple Reaction Monitoring
Typically used in Quantitative Work ofTriple Quadrupoles
MS1 CollisionCell
MS2
StaticStatic
D a u g h t e r ( P r o d u c t ) I o n S p e c t r aD a u g h t e r ( P r o d u c t ) I o n S p e c t r a
M S 1 C o l l i s i o nC e l l
M S 2
S c a n n i n gS t a t i c
D a u g h t e r ( P r o d u c t ) I o n S p e c t r aD a u g h t e r ( P r o d u c t ) I o n S p e c t r a
M S 1 C o l l i s i o nC e l l
MS 2
S c a n n i n gS t a t i c
Parent (Precursor) Ion Spect raParent (Precursor) Ion Spect ra
M S1 Col li sionCell
MS 2
Sta t i cScann ing
Parent (Precursor) Ion Spect raParent (Precursor) Ion Spect ra
M S1 Col li sionC e l l
MS 2
StaticScanning
C o n s t a n t N e u t r a l L o s s S p e c t r aC o n s t a n t N e u t r a l L o s s S p e c t r a
M S 1 Col li s i onCell
MS 2
S c a n n i n gS c a n n i n g
C o n s t a n t N e u t r a l L o s s S p e c t r aC o n s t a n t N e u t r a l L o s s S p e c t r a
M S1 Co l li s i onC e l l
MS 2
S c a n n i n gScann ing
BASIC DETECTOR REQUIREMENTS
Low drift and noise level (trace analysis).High sensitivity.
Fast response for high performance systems.Wide linear dynamic range (quantitation).Low dead volume (minimal peak broadening & remixing of theseparated bands).Insensitivity to changes in type of solvent, flow rate, andtemperature.Operational simplicity and reliability.Tuneable, so that detection can be optimized for differentcompounds.Preferably non-destructive.
An ideal LC detector should have the following properties:
Detector Criteria
SelectivitySensitivity and detection limitStabilityLinear range
Dynamic RangeReproducibilityEffect on peak shapeMaintenance
PROPERTIES OF DETECTORS
SELECTIVITY
UNIVERSALSPECIFIC
A selective detector allows one to seeonly components of interest despite oftheir co-elution with any others.
Detection in HPLC
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PROPERTIES OF DETECTORS
CONCENTRATION
RE
SPONSE
Sensitivity of a detector is not the minimumamount that can be detected.
SENSITIVITY PROPERTIES OF DETECTORS
h noise
h signal
h signal
= 2 x
h noise
DETECTION LIMIT
Limit of detectionLowest concentration that can be detectedSignal-to-noise ratio of 2:1 or 3:1
Limit of quantitationLowest concentration that can be determinedwith acceptable precisionSignal-to-noise ratio of 10:1
Detector Sensitivity Chromatographic SensitivitySignal-to-Noise Ratio
Noapparentnoise
Minutes
2.8 3.0 3.2 3.4
0.2 AU0.001AU
Minutes
2.00 3.00 4.00
Noise
Detection in HPLC
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Increase Signal-to-Noise Ratio
Signal-to-noise (S/N)is peak height to
noise
Increase S/N byincreasing peakheight
Increase S/N bydecreasing noise
3:1
6:1
8:1
Factors Increasing UV SignalIncrease sample concentrationIncrease injection volumeChoice of wavelength (s)Low volume flow cell
Flow cell pathlength
Optics bench designLamp energyWavelengthsMobile phase compositionPump pulsationElectronics
Factors Affecting Noise in UV Detectors
Chromatographic SensitivitySingle Wavelength vs Maxplo t
-0.002
0.000
0.002
0.004
0.006
0.008
0.010
A U
0.0 2.0 4.0 6.0
Minutes
220 nm
0.0 2.0 4.0 6.0Minutes
Maxplot
PROPERTIES OF DETECTORS
BASELINE STABILITY
SHORT RANGE
LONG RANGE
DRIFT
NOISE
Detection in HPLC
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PROPERTIES OF DETECTORS
TEMPERATURE, FLOW RATE, EL ECTRONICS
REPEATABILITY OF RESPONSE
PROPERTIES OF DETECTORS
MAINTENANCE AND COST
EASY HANDLING OF FLOW-CELL
EASY A/D CONVERSION
SAFETY
Detection in HPLC
Dr. Shulamit Levin, Medtechnica
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