Water in HPLC

Under the Supervision of: PROF.DR. Sawsan Elmasry

Presented by: Reem Tawfeek

Points to be discussed

• Water used in HPLC.

• Lab water grades.

• Specifications of HPLC-grade water.

• how to determine water purity?

• Problems caused by poor quality water.

• Package and storage.

• Conclusion.

Water used in HPLC

• Water for HPLC may be prepared IN HOUSE (analytical labs may prepare its own HPLC water)

• (N.B. that what most frequently happens in Egyptian labs)

• Freshly prepared double distilled

water (within 24 hours) will be

convenient for HPLC applications

Water used in HPLC

• De-ionized water will be INCONVENIENT for HPLC application due to uncertainty of the absence of the microbial contamination

• OR labs could obtain water from HPLC-grade chemicals supplie

Lab Water GradesType 3 water r is the lowest grade, for

-glassware rinsing,

-heating baths and filling autoclaves,

-feed Type 1 lab water systems.

Type 2 water

- general laboratory applications eg.buffers

- as feed to Type 1 water systems,

- preparation of reagents for chemical analysis or synthesis

Type 1 water(reagent grade)) eg. milli-Q water

-HPLC mobile phase preparation

- blanks and sample dilution in GC, HPLC

- other advanced analytical techniques

the different water specifications based on the different water types

HPLC-water specifications

• According to B.P. 2010 water for chromatography isdeionized water with a resistivity of not less than 18 MΩ-cm

But is this enoughIt’s possible to have 18 MΩ-cm soup!

Can we use soup for HPLC?????

How to measure water purity?contaminants in water either

• Measured by resistivity( MΩ-cm at 25.00°C )

ionic

• Measured by TOC(ppb)organic

Sources of organic contaminants [Total organic carbon(TOC)]

• Leaching from purification media, tubing and containers,

• bacterial contamination

• absorption from the atmosphere

Poor quality water reduces chromatographic performance by

• affecting resolution and integration.

• introducing ghost peaks.

• altering stationary phase selectivity.

• and impacting baselines.

Ghost peaks

Isocratic baseline monitoring at 210 nm using 100% high TOC reagent water(10 Mᾩ·cm resistivity, 100 ppb TOC) (1 mL/min).

Using high-purity water(3 ppb TOC)

According to American Society for Testing and Materials ASTM Reagent grade water should contains

• Total organic carbon (TOC), max , 50 ppb

• Resistivity not less than 18 MΩ-cm

Packing and storage

• packaged in amber glass bottles

• sealed under a nitrogen with Teflon-lined fluorocarbon caps .

• Unless other instructions stated by SOP(standard operating procedures) Once the bottle is opened it should be used within 24 hours.

Conclusion

• We should switch from simple resistivity measurement to resistivity and TOC monitoring in order to be able to take reagent water quality for granted.

references

• W.M.A. Niessen, J. Chromatogr., A 856(1,2), 179–197 (1999).

• W. Byrne, Reverse Osmosis — A Practical Guide for Industrial Users. 2nd ed. (Tall Oaks Publishing,Littleton, Colorado, 2002).

• Standard Specification for Reagent Water American Society for Testing and Materials International 2001 Annual Book of Standards – Volume 11.01

• B. Srikanth, Ultrapure Water 15(3), 40–46(1998).

• K. Clark, M. Retzik, and D. Darbouret, Ultrapure Water 14(2), 21–24, (1997).

• C. Regnault, I. Kano, D. Darbouret, and S.Mabic, J. Chromatogr., A 1030(1–2), 289–295(2004).

• B. Stewart and B. Williamson, Am. Biotechnol. Lab., 16–18, December 2001.

• http://www.high-q.com/pdf/q&a_reagent_water_screen.pdf