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CARRYOVER IMPROVEMENT ACHIEVED THROUGH INSTRUMENT DESIGN CHANGES AND NEEDLE WASH OPTIMIZATION FOR HPLC SYSTEMS
Amanda B. Dlugasch, Jennifer Simeone, and Patricia R. McConville Waters Corporation, Milford, Massachusetts, USA
INTRODUCTION Sample carryover is a common problem for analytical labs and
may adversely affect chromatographic methods. Sample carryover
occurs when material from an injection is present in subsequent
injections. There are several factors that can influence carryover
including the chemistry of the sample analyte as well as the
injector design of the High Performance Liquid Chromatographic
(HPLC) system. The injector design is important not only to
minimize the potential for carryover, but also to properly clean the
components of the needle that are vulnerable to contamination. A
HPLC system based upon a flow through-needle design is capable
of providing improved carryover performance due to the interior of
the needle being washed by the mobile phase during the analysis
and the exterior of the needle being washed with an appropriate
wash solvent. Modifying the injector design provides a significant
reduction of the sample carryover with optimal washing
procedures of the needle. The needle wash, therefore, is an
important factor in which depends on the sample. Optimally, the
wash will be able to easily solubilize the analyte(s) of interest. In
this study, we will examine the improved carryover obtained after
design optimization of the HPLC system injector and the
optimization of the needle wash solvent over a wide range of
sample types.
METHODS LC Systems: Alliance: Alliance e2695 Separations Module with 100
μL syringe 2998 PDA Detector and CH-30 equipped
with passive column preheater. Firmware 3.03
2018 Alliance: Alliance e2695 Separations Module
with 100 μL syringe 2998 PDA Detector, CH-30
equipped with passive column preheater and the
e2695 Enhancement1 Kit (p/n: 700011805)
Firmware 3.04
Caffeine:
Column: XBridge C18 4.6 x 50 mm, 3.5 µm
(p/n: 186003031)
Needle Wash: Methanol
Isocratic: 70:30 Water:Methanol
Chlorhexidine:
Column: CORTECS C18 Column, 2.7 µm, 3 mm x 100 mm
(p/n: 186007372)
Needle Wash: 50:50 Water:Acetonitrile
Isocratic: 67:33 0.1% TFA in Water: 0.1% TFA in
Acetonitrile
Coumarin:
Column: CORTECS C18 Column, 2.7 µm, 3 mm x 100 mm
(p/n: 186007372)
Needle Wash: 90:10 Water:Acetonitrile
Isocratic: 90:10 Water:Acetonitrile
Quetiapine: Quetiapine Fumarate Assay USP 40 NF35 S11
Column: XBridge BEH C8 Column, 5 µm, 4.6 mm x 250
mm (p/n: 186003018)
Needle Wash: 90:10 Water:Acetonitrile
70:30 Methanol:Water
Isocratic: 54:7:39 Methanol:Acetonitrile:Buffer
Buffer: 2.6 g/L of dibasic ammonium phosphate
adjusted to pH 6.5 with phosphoric acid
References
1. Quick Reference Guide: 2018 Alliance Enhancements. Waters User Manual 720006332EN. 2018 June.
2. Jenkins, Tanya, Michael Waite. Low Sample Carryover with Key Performance Indicators on the Alliance HPLC System. Waters Technology Brief 720004534EN. 2012 Dec.
3. Official Monographs, Quetiapine Fumarate USP 40 NF35 S1, United States Pharmacopeia and National Formulary (USP 40-NF35 S1) Baltimore, MD: United Book Press, Inc.; 2017. p. 5939.
4. Dolan, John. Autosampler Carryover. LCGC Europe. Volume 19, Issue 10, pg 522-529. http://
www.chromatographyonline.com/autosampler-carryover-3?id=&pageID=1&sk=&date=
RESULTS
CONCLUSION The 2018 Alliance HPLC System incorporates a newly designed
seal pack1 that improves needle wash flow over the exterior of the
injector needle.
The newly designed seal pack results in a significant reduction in
carryover for a wide range of chemical compounds.
The 2018 Alliance HPLC System can reduce the amount of carryover present regardless of the specific compounds being
analyzed.
The optimal needle wash setting and needle wash solvent composition is essential to decreasing sample carryover, therefore
application specific.
When combined, the needle wash mode and the needle wash composition significantly reduce carryover on the 2018 Alliance
HPLC System.
Figure 1. Structures of caffeine, chlorhexidine, coumarin and quetiapine fumarate.
Table 1. Carryover results for caffeine, chlorhexidine, coumarin and quetiapine fumarate on the Alliance HPLC System and the 2018 Alli-ance HPLC System using the default ‘Normal’ wash mode setting. *The carryover instrument specification is based upon the compound caffeine under specific method conditions.
Compound Alliance HPLC
System
2018 Alliance HPLC System
Carryover Improvement
Caffeine 0.0011% 0.00016% 6.9x
Chlorhexidine 0.010% 0.0005% 20x
Coumarin 0.0002% 0.00006% 3.3x
Quetiapine fumarate
0.028% 0.019% 1.5x
System Specification*
< 0.01% < 0.0025% NA
Figure 3. The observed impact of the advanced features for the needle wash modes for coumarin and quetiapine fumarate on the 2018 Alliance HPLC System.
Figure 4. Carryover results for the two different needle wash compositions on the 2018 Alliance HPLC System.
Figure 2. The chromatographic results for the Alliance HPLC Sys-tem (red) and the 2018 Alliance HPLC System (blue) for the post-challenge blank are represented in the figure above for each of the compounds, caffeine, chlorhexidine, coumarin, and quetiapine fu-marate. The black chromatogram represents the challenge or stan-dard solution.
Caffeine
Chlorhexidine
Coumarin
Quetiapine Fumarate
Caffeine Challenge
Solution
Alliance
Carryover
2018 Alliance
Carryover
Chlorhexidine
Standard Solution
Alliance
Carryover
2018 Alliance
Carryover
Coumarin Challenge
SolutionAlliance
Carryover
2018 Alliance
Carryover
Quetiapine
Standard Solution
Alliance
Carryover
2018 Alliance
Carryover
