Quantifying Additives in Polymers using Multiple Heart-Cutting 2D-LCStephan Buckenmaier1 and Matthias Pursch2

1 Agilent Technologies, Research and Development, Hewlett-Packard-Str. 8, 76337 Waldbronn, Germany2 Dow Deutschland Anlagengesellschaft mbH, Analytical Technology Center (ATC), Industriestr. 1, 77836 Rheinmuenster, Germany

Introduction

Conclusions

The growing need to quantify low compound levels in complex matrices

requires analytical tools with advanced separation capability and sensitivity.

Industrial applications include analysis of process intermediates, pesticides

derived from natural products, or additives in food or polymer matrices.

Hexabromocyclododecane (HBCD) has been used as flame retardant additive

in polystyrene (PS). Such complex matrix poses a significant analytical

challenge. Insufficient separation can spoil accuracy and precision (Fig. 3) and

cause suppression of the ionisation in MS (e.g. Poster PSB-MULTI-22).

2D-LC has been demonstrated to markedly enhance separation performance

compared to conventional 1D-LC.

Multiple heart-cutting (MHC) 2D-LC allows for storage of aliquots from 1D in

parallel to 2D-analysis. This disconnects 1D and 2D time scales so that both

dimensions can operate under more optimal conditions.

This work presents an automated MHC 2D-LC approach for the quantitative

analysis of target compounds in complex matrices illustrated on the example of

HBCD in PS.

Experimental

Results and Discussion

Samples: 0.5 g PS was dissolved in 3 mL dichloromethane. Part of polymer

was crashed using 10 mL ACN. Spiking experiments used known

concentrations of HBCD in ACN during the precipitation step to yield HBCD

amounts in polymer of 0.02, 0.04, 0.10, 0.20, and 1.00 wt-%. The resulting

slurry was filtered with a 0.45 μm polypropylene membrane.

2D LC Setup: Data acquisition used an Agilent 1290 Infinity 2D-LC system with

multiple heart-cutting operated with OpenLAB CDS Edition C.01.07. Fig. 1:

Schematic of the system.

.

Results and Discussion

Excerpt of method development

Fig. 2: Overlay of 1D-LC separations of HBCD standard and real PS foam

samples (one without HBCD and one spiked to give 0.1% HBCD in PS)

acquired using a C18 column.

• Presence of multitude of individual components in this polymer sample

presents a significant challenge for a 1D-LC separation (more than 50 peaks

observed).

• HBCD isomers (α, β, γ) well separated from each other but overlap with

peaks of the polymer matrix (see inset).

• Use of longer columns and/or different gradients did not solve this peak

overlap issue.

• Accurate quantitation impossible, especially at low levels of HBCD in PS.

Results and Discussion

Fig. 3: Overlay of 1D-LC separations acquired using a phenyl column. HBCDstandard, PS sample spiked with HBCD, and real sample.

First dimension (1D, blue path):

• 1290 Infinity: Quaternary pump (0.35 mL/min), autosampler (inj.-V 6 µL),

TCC (30°C), DAD with 10 mm flow cell (λ = 220 nm, 5 Hz).

• Column: Zorbax SB-Phenyl 150 × 2.1 mm, 1.8 μm.

• Mobile phases: A = water, B = MeOH, and C = THF

• Gradient:

Second dimension (2D, red path):

• 1290 Infinity: Binary pump (2.00 mL/min), TCC (50°C), DAD with 60 mm flow

cell (λ = 220 nm, 80 Hz).

• Column: Zorbax SB C18, 50 × 3.0 mm, 1.8 μm.

• Mobile phases: A = water, B = ACN.

• Gradients from 60 to 85%B in 1.50 min; 2D cycle time = 2.00 min.

Multiple heart-cutting interface:

• 2-pos/4-port duo valve connected to two selector valves (parking decks) with

in total twelve sampling loops (40 µL).

• Switch of the duo valve places decks in sampling or 2D-analysis position.

• Switch of selector valves provides access to loop positions.

Fig. 2

Fig. 1

Fig. 3

Fig. 5

• MHC 2D-LC has solved the separation problem with all HBCD isomers

being baseline separated from the polymer matrix.

Fig. 6

• (A): A heart-cut #1 was made at 5.70 min (blank, not shown) followed by

three consecutive cuts across region-1 (start at 7.40 min) and four cuts

across region-2 (at 8.15 min).

• (B) On basis of the MHC algorithm, Deck-B, connected to 1D (blue path),

has parked cuts #2−4 in loops-1 to -3 and remains in an idle position (loop-4)

until the 2D-cycle of cut #1 (red path) has finished.

• (C): The Duo Valve switch has submitted Deck-B to 2D-processing and

Deck-A has parked cuts #5−8.

Fig. 5 (A): Representative 2D-separations of cut #4 that contains HBCD isomer

α, and Panel (B) those of cut #7 that contains β and γ.

• The phenyl stationary phase in combination with methanol as the organic

modifier shifted the HBCD peaks to higher retention times relative to the PS

matrix compared to the C18 separation in Fig. 2.

• At first glance, replacing C18 by phenyl and corresponding eluent

chemistries have a negative effect on the separation in that they caused co-

elution of HBCD β- and γ-isomers with large PS peaks.

• However, such difference in selectivity is beneficial in the context of 2D-LC

separations.

2D-LC experiment

Fig. 4 (A): Expanded view of Fig.3 illustrating the cutting scheme. Panels (B),

(C) show snapshots of the 2D-LC monitor (part of the 2D-LC software), which

traces the actual state of the interface during the 2D-LC experiment. The decks

are displayed schematically, each with six slots representing the sampling

loops.

Fig. 4

2D-LC Repeatability

Fig. 7: Superimposed 2D-results for cuts #4 and #7 from ten consecutive

injections of real HBCD polymer sample (0.16 wt-%) .

2D-LC Quantification

Fig. 6 (A): 2D-results of cuts #5−8 obtained at the lowest level measured. Panel

(B): Plots of peak area versus HBCD level obtained from cuts #4, #8, and #7,

respectively, for isomers α, β, and γ. Panel (C): Plots of peak area versus

HBCD level obtained when peak area of each isomer across the multiple heart-

cuts was summed to give the total area attributed to a particular compound.

• Isomer-γ exemplifies “walking through the 1D peak” pattern. Abundance in

2D increases from cut #6 to #7 and then decreases in cut #8.

• Reasonable quantitation results obtained from single cut quantitation with

R2-values > 0.99.

• Quantitiation results significantly improved with R2-values ≥ 0.9999 when

total 2D-peak area was used.

• The limit of quantitation (LOQ, S/N = 10) was estimated at 0.01 wt-% HBCD

in PS for the β-isomer peaks, which were of low intensity.

• Good repeatability obtained in 2D for retention as well as peak area.

• Acceptable precision for quantification purposes calculated for isomer-α in

cut #3, isomer-β in cut #8, and isomer-γ in cut #7 with 9.0%, 1.3%, and

5.2%, respectively.

• RSD-values significantly improved when using the total 2D-peak area for

calculation with 0.3%, 3.0%, and 1.5% for isomers α, β, and γ.

• For all HBCD isomers, a combined peak area RSD of 0.7% was obtained.

Table: % RSD data calculated for peak areas of each single cut and those for

total peak areas.

Fig. 7

MHC 2D-LC is a powerful technique for targeted analyses in complex matrices.

This was demonstrated for the determination of HBCD in polystyrene (PS) using

the combination of phenyl column in 1D and C18 column in 2D operated with

MeOH and ACN gradients, respectively.

By the means of the MHC technology that provides automated fractionation for

specified areas from the 1D chromatogram, 2D-LC successfully separated

HBCD and PS, which was not achieved with conventional 1D-LC.

Grouping of 2D-peak areas of a compound obtained from multiple cuts across a

1D-peak cluster “forgives” potential peak shifts in 1D, whether they are due to

changes in operating conditions, sample composition, or column chemistry.

Quantitation performance was demonstrated down to a level of 0.01 wt-%

HBCD in PS with regression coefficients close to unity (R2 ≥ 0.9999) and peak

area RSD-values ≤ 3%.

HPLC 2015 - PSB-MULTI-21

This work has meanwhile been published:

Anal. Chem. 2015, 87, 5310−5317