Diode Array Detector Optimization

2

In This Section, We Will Discuss:

�The optical path for the diode array.

�How the diode array collects data.

�How to optimize the diode array.

DAD-SL

3

Tungsten lamp

Coupling

lens

Deuterium lamp

Source lens

(Achromat)

Holmium

oxide

filter

Support lens

Flow cell

Spectro

lens

Grating

Array

ProgrammableSlit

Optical Path – Agilent 1100/1200

4

Front View of the MWD or DAD

The inlet capillary can be attached directly to the column

Deuterium lamp Detector cellTungsten lamp

5

� Wavelength Range: 190 - 950 nm

� Lamps: Shine-through deuterium lamp (uv-range)

Tungsten lamp (vis-range)

� Slits: Programmable electromechanical; 1, 2, 4, 8 and 16 nm

� Noise: 0.8 x 10-5 AU at 254 and 750 nm

� 80 Hz, 8 signals, Data Never Lost, RFID tags, temperature

control for DAD-SL

� Flow Cells

Agilent 1100/1200 Diode Array Standard

Name Volume Path Length Max Pressure Part Number

Standard 13 µµµµL 10 mm 120 bar G1315-60022Semi-Micro 5 µµµµL 6 mm 120 bar G1315-60025Micro 2 µµµµL 3 mm 120 bar G1315-60024Semi-Nano 500 nL 10 mm 50 bar G1315-68724Nano 8 nL 6 mm 50 bar G1315-68716

Preparative -- 3 mm 120 bar G1315-60016Preparative -- 0.3 mm 20 bar G1315-60017Preparative -- 0.06 mm 20 bar G1315-60018

High Pressure 1.7 µµµµL 6 mm 400 bar G1315-60015

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DAD SL – Flow Cell Recommendations

13µl Standard Flow Cell:

� For highest sensitivity and linearity

� High-demanding quantitative work, e.g. analytical method development, QA/QC

� 4.6mm ID Columns (3mm)

5µl Semi-micro Flow Cell:

� Best compromise of sensitivity and selectivity

� For good quantitative and qualitative results, e.g. Screening, HT LC/MS/UV, Early Formulation Studies

� 3mm ID Columns (4.6 – 2.1mm)

2µl Micro Flow Cell:

� For highest selectivity

� Ultra-fast semi-quantitative work,

e.g. Screening Experiments, HT LC/MS/UV

� 2.1mm ID Columns

* Depends on analytical conditions and columndimension

+

++

+++

Signal /Noise Resolution*Flow Cell

Volume/Pathlengt

h

+++2 µl / 3mm

++5 µl / 6mm

+13 µl / 10mm

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Time

Wavelengthmsec

nm

LIGHT

SHIFT REGISTER

SWITCH

VIDEO LINE

READOUT CYCLE

PHOTODIODE

CAPACITOR

TRANSISTOR

Diode Array - Details

1024 Diodes

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Optimizing Step (Optical Diode Width)

Step defines the wavelength resolution

for spectral storage.

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Optimizing Slit

1 nm

2 nm

4 nm

8 nm

16 nm

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INFLUENCE OF RESPONSE TIME

0.1

mAU

Response

Time

2.3 sec

1.0 sec

0.3 sec

0.1 sec

Statistical noise of detector = 1

nn = number of data points

Peak Width

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min0.1 0.2 0.3 0.4 0.50

80Hz

PW=0.30sec

40Hz

PW = 0.33 sec

20 Hz

PW=0.42sec

10Hz

PW=0.67sec

5HzPW=1.24sec

• Sample: Phenone Test Mix

• Column: Zorbax SB-C18, 4.6x30, 1.8um

• Gradient: 50 -100% ACN in 0.3min

• Temperature: 50°, Flow Rate: 5ml

• Flow cell: 5ul

• Slit: 8 nm

• Signal: 245nm, Bandwith: 10nm

• Reference: 360nm, Bandwidth: 80nm

80Hz versus 20Hz

– 30% Peak Width

+ 30% Resolution

+ 40% Peak Capacity

+ 70% Apparent Column Efficiency

What’s the Benefit of 80Hz Data Acquisition RateDAD - SL

80Hz versus 10Hz

– 55% Peak Width

+ 90% Resolution

+ 120% Peak Capacity

+ 260% Apparent Column Efficiency

Do not use peak width smaller than necessary.

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Anisic

Acid

signalwavelength

bandwidth30 nm

reference bandwidth

referencewavelength350 nm

100 nm

Remember: This is just a guideline.

Use the Isoabsorbance Plot feature of Data

Analysis for optimization.

Sample Signal and Reference Optimization

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Bandwidth

(nm)

Signal

(10 AU)

Noise

(10 AU)

4

8

16

32

64

128

14.70

14.51

13.79

11.75

8.40

6.53

0.41

0.34

0.26

0.18

0.14

0.13

1.00

1.19

1.49

1.82

1.62

1.40

Relative

Signal/Noise

-4 -4

4 8 16 32 64 128

Signal/Noise

Signal

Noise

Instrument Bandwidth (nm)

Bandwidth

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0.1 maU

Bandwidth

32 nm

12 nm

4 nm

Influence of Sample Signal Bandwidth

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0.1 mAU

Reference wl

Reference wl

no reference

350 nm

550 nm

Time (min)

Influence of Reference Wavelength

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1 pmol each

wl 267 nm

ref 380 nm

wl 267 nm

no reference

PTH-A

SN

PTH-A

RG

PTH-A

LA

PTH-P

RO

PTH-P

HE

Grad.: 0.02 m KH PO / ACN, from 12% ACN to 45% ACN in 12 min2 4

Reference Wavelength and Gradient Elution

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Reference Selection

A

B

C

D

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Reference: Select Bandwidth

A

B

C

D

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A response at 280 nm indicates conjugated

double bonds and thus a bad oil quality

good quality bad quality

Multi-Signal Detection: Triglycerides in Olive

Oils

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WL1 (204 nm, Caffeine)

WL2 (222 nm, Hydrochlorothiazide)

WL1

WL2

WL4 (282 nm)

WL3

(260 nm)

WL3: Ref Wl to suppress Hydrochlorothiazide

WL4: Ref Wl to suppress Caffeine

g

Peak Suppression: Wavelength Selection

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Peak Suppression: Results

Hydrochlorothiazide

suppressed

Hydrochlorothiazide

+ Caffeine

Caffeine

suppressed

WL 222 nm

Ref 282 nm

WL 204 nm

Ref 260 nm

WL 222 nm

no reference

WL 204 nm

no reference

Hydrochlorothiazide

+ Caffeine

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Advantages of Twin Lamp Design

Twin Lamp1x10-5AU

Without Tungsten2x10-4 AU

min0 2 4 6 8

mAU

-0.2

0

0.2

0.4

0.6

700nm

Without Tungsten~1x10-5 AU

Twin Lamp 1x10-5 AU

min0 1 2 3 4 5 6 7 8 9

mAU

-0.2

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

254nm

ACN/Water 20/800.2 ml/min1 nm

Mobile phase:Flow rate:Slit:

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DAD SL Method Setup – Recommendations

Signals:

• Sample = WL of maximum absorbance

• BW = NBW of absorbance spectrum

• Reference = such that WL – 0.5 x Ref BW

lies outside Absorbance band

• Reference BW = 50-100nm

Slit

• For optimum spectral analysis choose slit

such that NBW/Slit > 10 for all compounds

• For maximum sensitivity slit can be

increased to 8 or 16nm

Peakwidth

• For optimum chromatographic resolution

choose Peakwidth equal or smaller than

FWHM of narrowest peak.

• For maximum sensitivity choose Peakwidth

twice as large as narrowest peak