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Approaches to conserve

solvent in HPLC analysis

Daniela Daniel, PhD


Application Chemist – Shimadzu do Brasil
Why reduce solvent consumption ?

1) Price: low cost/analysis

Shortage of acetonitrile increase the price


(October 2008)

2) Eco friendly analytical methods


How to reduce solvent consumption ?

• Methodologies review

• Efficient use of LC systems

• Solvent recycle

• Alternative solvents

• Ultra-fast HPLC systems


Approaches to save solvent

Can you change analytical No Daily conservation


conditions? Recycle of mobile phase
Utilization of gradient system
Yes

Is use of acetonitrile critical for No


separation? Use of methanol

Yes

Can you modify your instrument


No
or purchase a new instrument? Change the column

Yes

Transfer to semi-micro conditions


Transfer to ultra-fast HPLC system
Approaches to save solvent

Can you change analytical No Daily conservation


conditions? Recycle of mobile phase
Utilization of gradient system
Yes

Is use of acetonitrile critical for No


separation? Use of methanol

Yes

Can you modify your instrument


No
or purchase a new instrument? Change the column

Yes

Transfer to semi-micro conditions


Transfer to ultra-fast HPLC system
Daily Conservation

Use of automation tool

Automatic start-up and shut-down of HPLC system

Use of methanol for column wash

Methanol can be utilized for column wash in most cases


Recycle of Mobile Phase

Principle

Eluate from column is re-used as a mobile phase by returning back to


reservoir bottle automatically

Only “clean” eluate is re-used.

Advantages

Modification of analytical conditions is not necessary

It is easy and effective for simple samples

Application

Isocratic elution only


Recycle of Mobile Phase

Solvent recycle valve kit (option)

For UV-VIS detectors


SPD-20A/20AV

SPD-10Avp/10AVvp
Solvent recycle valve kit
Operation
for SPD-20A/20AV
Parameter: threshold and delay time
(can be set from LCsolution software or detector front panel)
Recycle of Mobile Phase

Possibility of contamination of mobile phase


There is a possibility that contaminants are recycled if the contaminants can’t
be detected at measuring wavelength

Countermeasure
Risk reduction of contaminants by dual
wavelength monitor
Use of Gradient System

Utilization of gradient system for isocratic analysis

Reduction of waste of unused mobile phase


Approaches to save solvent

Can you change analytical No Daily conservation


conditions? Recycle of mobile phase
Utilization of gradient system
Yes

Is use of acetonitrile critical for No


separation? Use of methanol

Yes

Can you modify your instrument


No
or purchase a new instrument? Change the column

Yes

Transfer to semi-micro conditions


Transfer to ultra-fast HPLC system
Use of Methanol

• Different separation selectivity

• Different elution performance

• Absorption at shorter wavelength

• High viscosity (column pressure)


Approaches to save solvent

Can you change analytical No Daily conservation


conditions? Recycle of mobile phase
Utilization of gradient system
Yes

Is use of acetonitrile critical for No


separation? Use of methanol

Yes

Can you modify your instrument


No
or purchase a new instrument? Change the column

Yes

Transfer to semi-micro conditions


Transfer to ultra-fast HPLC system
Change the Column

Short column

 tR is reduced

 N is reduced
Column with small internal diameter

 Easy optimization of analysis method

 Small adaptation in the HPLC system


Column with reduced particle size

 tR is reduced

 Pressure is increased
Change the Column

Shima-pack VP-ODS (150 x 4.6mm, 4.6 µm)

Peaks:

1)Acetophenone

2)Propiophenone

3)Butilphenone

4)Valenophenone

5)Hexaphenone

6)Heptaphenone

7)Octanophenone

Shim-pack XR-ODS (75 x 3.0 mm, 2.2 µm)


Approaches to save solvent

Can you change analytical No Daily conservation


conditions? Recycle of mobile phase
Utilization of gradient system
Yes

Is use of acetonitrile critical for No


separation? Use of methanol

Yes

Can you modify your instrument


No
or purchase a new instrument? Change the column

Yes

Transfer to semi-micro conditions


Transfer to ultra-fast HPLC system
Transfer to Semi-micro conditions

• Solvent saving with maintaining separation

• Column with small internal diameter

• Modification of analytical conditions

(Flow rate, injected volume, etc.)

• Modification of HPLC instruments

Narrow pipe to minimize dispersion, Detector cell and gradient mixer


Approaches to save solvent

Can you change analytical No Daily conservation


conditions? Recycle of mobile phase
Utilization of gradient system
Yes

Is use of acetonitrile critical for No


separation? Use of methanol

Yes

Can you modify your instrument


No
or purchase a new instrument? Change the column

Yes

Transfer to semi-micro conditions


Transfer to ultra-fast HPLC system
Ultra Fast system

Ultra
Fast
Liquid
Chromatography
Effective reduction of mobile phase consumption (1/5 – 1/10)
High throughput analysis
Two approaches: Particle size and temperature
Particle size and separation

Separation with resolution using small particle size

Flow rate: 0.2 mL/min Flow rate: 0.7 mL/min

Column Plate number Column Plate number


Column A 3,590 Column A 2,320 (down)
Column B 5,080 Column B 4,500 (even)
Column C 6,810 Column C 8,010 (up)
XR-ODS Columns

Substrate Totally porous spherical,


high purity silica gel
Particle Size 2.2 um

Stationary Phase C18, C8, Phenyl, SIL

Surface Treatment Endcapping

Carbon Load 18.4%

Maximum Pressure 35MPa*

Shim-pack XR-ODS (2,2 µm)


Temperature effect

Best column efficiency: Increase N and decrease the pressure

Mobile phase: H2O/ACN (3/7, v/v)

Detection: 245 nm.

Peaks: 1: acetophenone

2: propilphenone

3: butilphenone

4: pentilphenone

5: hexanophenone

6: heptanophenone

7: octanophenone
UFLC - Prominence

Solvent consumption: 10.5 mL

Peaks
1:actophenone,
2:propiophenone,
3:butyrophenone,
4:valenophenone,
5:hexanophenone,
6:heptanophenone,
7:octanophenone.

Solvent consumption: 2.3 mL

Mobile phase: water/acetonitrile (3/7, v/v)


Temperature: 40℃
UFLC - Prominence
Solvent consumption: 12.8 mL

Peaks
1:cefadroxil,
2:cephaprin,
3:cefaclor,
4:cefalexin,
5:cephradine,
6:cefotaxime.
7:cefazolin,
8 :cefuroxime,
9 :cefoperazone,
10 :cefloxitin, 1
1 :cefamandole A,
12 :cephalothin, 1
3: cefamandole B

Solvent consumption: 1.7 mL

Mobile phase : A: 0.1% formic acid-water, B: acetonitrile, Gradient elution


Temperature : 40℃
Flow rate : 1.0mL/min
Method tranfer programm

Method Transfer Program HPLC method: 50 minutes


Parameter Input Sheet
This program supports the parameter setup operations required when transferring from a conventional LC to Prominence UFLC.
Enter the parameters from the conventional LC into the blank boxes below and click [Optimize] to display the estimated optimal
chromatographic conditions and instrument environment for an ultrafast LC using a Shim-pack XR Series column.
The suitability of the chromatographic conditions produced by this program is not guaranteed. The estimated parameters may be
incorrect due to differences in the column characteristics. Evaluate the suitability of the displayed chromatographic conditions before using
them.

A value must be entered in each box with a thick border. When these values are entered, the
button at the right changes to [Optimize]. Click this button to display the parameters. Ready

must be filled in.

Chromatographic Conditions

Column : Shim-pack VP-ODS 4.6 mm i .d . 150 mm long 4.6 µ m


Mobile phase A: 20 mmol/L (Sodium) Phosphate <pH 2.5> Init. Conc.; 80 %
B: Acetonitrile Init. Conc.; 20 %
Gradient program
No. Time A Conc. B Conc.
1: min % %
2: min % %
3: min % %
4: min % %
5: min % %
6: min % %
7: min % %
8: min % %
9: min % %
Flow rate : 1 mL/min
Temperature : 40 °C
Detection Wavelength: 254 nm
Response: 1000 ms 20 50 100 500 1000 1500

Sample volume : 10 µ L

Instrument Environment Peak Information

Mixer volume : 1.7 mL Minimum elution time : 5 min


0.1 0.5 1.7 2.6

Cell volume : Conventional Analysis time : min


Conventional Semi-micro

2.0 3.0 4.6

1: Maintain separation 30 50 75 100

Transfer Conditions : 2

2: Reduce analysis time Resolution tolerance ; %


3

3: Designate column dimensions and flow rate


mm i .d . mm long mL/min

UFLC method: 9 minutes


Minimized Analysis Cycle time

Prominence UFLC employs a fast autosampler (10sec /10uL injection)

Mobile phase: H2O/ ACN (4/6 to 2/8 in 0.4 min, gradient), Flow rate: 3 mL/min, Temperature: 80C, Detection: 245 nm, Sampe volume: 4L
Ultra-fast LC-MS system

LCMS-2020
Ultra-fast LCMS-2020

UF switching technology

High-speed polarity switching

UF scanning technology

Fast scanning speed

UF sensitivity

Superior sensitivity in ultra high speed analysis


UF Scanning Technology

Scanning speed of 15,000 u/sec is achieved without sacrificing


sensitivity or resolution, even during high-speed scans
UF Switching Technology

Polarity switching between positive/negative in 15 msec enables


analysis of the fastest LC peaks in both modes
UF Sensitivity

Reserpine analysis (1pg) S/N = 350


UF Detection of Catechins
Ultra-fast detection of catechins in Positive/Negative modes by LCMS-2020
(x100,000)
2.25 Analysis of catechins in tea

1 1: (-)-gallocatechin,
2
2.00 2: (-)-epigallocatechin,
3 4
3: (+)-catechin,
1.75
4: (-)-epicatechin,

5 6 5: (-)-epigallocatechin gallate,
1.50
6: (-)-gallocatechin gallate,
1.25 m/z 307 7: (-)-epicatechin gallate,
7 8
m/z 291 8: (-)-catechin gallate
Positive
1.00 m/z 459
9, 10: methylated catechins
1
m/z 443
9 OH
0.75 2 10
m/z 473 OH
3 4
m/z 305 HO O
0.50 5 6
m/z 289 negative
7 8 OH
0.25 m/z 457
OH
m/z 441
9 10 (-)-epicatechin
0.00 m/z 471

0.0 0.5 1.0 1.5 2.0 2.5 3.0 min


Approaches to save solvent

Can you change analytical No Daily conservation


conditions? Recycle of mobile phase
Utilization of gradient system
Yes

Is use of acetonitrile critical for No


separation? Use of methanol

Yes

Can you modify your instrument


No
or purchase a new instrument? Change the column

Yes

Transfer to semi-micro conditions


Transfer to ultra-fast HPLC system
The trends in HPLC…
The impasse…

High Resolution x Fast Analysis


Prominence UFLCXR

High Speed & High Resolution


Increased pressure tolerance
up to 66MPa (9,570psi)

Optimized flow path for less dispersion


Tubing, needle seal, autosampler valve

Wider range application


Use of longer column for high resolution analysis

Ultra fast analysis by MeOH mobile phase

Higher resolution column, Shim-pack XR-ODS II


2.2um particle with 8nm pore
Current HPLC…
Picos:

1: Acetofenona

HPLC 2: Propiofenona
3: Butilfenona
4: Valerofenona
5: Hexanofenona
6: Heptanofenona
7: Octanofenona

Speed

UFLC

Resolutio
n
UFLCXR
New ultrafast HPLC system
NEXERA

•Highest resolution

•Highest speed

•Ultra-high pressure (up to 130 MPa)


NEXERA with LCMS-2020
www.shimadzu.com.br
www.empesa.net

Muchas Gracias !