Multidimensional Chromatography

Biomedical and Pharmaceutical Applications			303
Table 11.1 Biomedical applications of on-line heart-cut LC–LC
		Sample	First
Subsequent		preparationa	LC modeb
Analyte(s)	Sample matrix
LC mode(s)b	Detectionc	Reference
Drugs and Related Compounds
Ampicillin	human plasma	deproteination	C18
C18	post-Flu	28
	human urine	buffer dilution	C18
C18	post-Flu	28
-agonists	human/bovine urine	none	C18
C18	UV	22
	bovine urine	none	C18
C18	MS–MS	23
Basic drugs	human serum	liq – liq	ion-pair
C18	UV	37
		extraction	C18
Bupivacaine	human plasma	ultrafiltration	C8
CIEX	UV	24
Chloramphenicol	pig tissue	liq – liq	PRP
C18	UV	32
		extraction
Dexamethasone	bovine tissue	liq – liq	phenyl
silica; CN	UV	29
		extraction
Dopa and its	human/rat plasma	deproteination	CIEX
C18	EC	66
metabolite
Efletirizine	human plasma	SPE	C18
C18	UV	69
	human urine	dilution	C18
C18	UV	69
Glycyrrhizin and its	human plasma	SPE	C18
C18	UV	39
metabolite
Ibuprofen	human serum	liq – liq	ion-pair
C18; C18	UV	34
		extraction	C18
Manidipine and its	human serum	liq – liq	ion-pair
C18	UV	38
metabolite		extraction	C18
Mefenamic acid	human serum	liq – liq	C18

304		Multidimensional Chromatography
C18; C18	UV	34
		extraction
Melengestrol	bovine tissue	liq – liq	phenyl
silica; silica	UV	31
acetate	Table 11.1	extraction
		(continued)
		Sample	First
Subsequent		preparationa	LC modeb
Analyte(s)	Sample matrix
LC mode(s)b	Detectionc	Reference
Methandrostenolone	equine	liq – liq	phenyl
C18; C8	UV and	30
and its metabolites	plasma/urine	extraction
	MS–MS
Metyrapone	human	liq – liq	silica
Chiralcel OJ	UV	42
	plasma/urine	extraction
Phenyl-	human	liq – liq	ion-pair
C18	UV	36
propanololamine	plasma/urine	extraction	C18
Probenicid	rat plasma	deproteination	C18
ion-pair C18	UV	63
Propanolol	human plasma	liq – liq	ion-pair
C18	UV	35
		extraction	C18
Methotrexate	human urine	SPE	AIEX
C18	UV	26
Remoxipride	human plasma	liq – liq	CN
C18	EC	17
metabolite		extraction
Ro 23-7637	dog plasma	SPE	CIEX
C18	UV	27
Ro 24-0238	human plasma	SPE	CIEX
C8	UV	25
Ropivacaine	human plasma	ultrafiltration	C8
CIEX	UV	24
Sameridine	human plasma	ultrafiltration	C18
CIEX; CIEX	UV	16
TCV-116 and	human	liq – liq	C18
C18	Flu	64
its metabolites	serum/urine	extraction
Tipredane	rat urine	none	CN
C18	UV	18
metabolites

Biomedical and Pharmaceutical Applications			305
	human urine	none	C18
C1	UV	19
Vanillylmandelic	human urine	acidification,	ion-pair C18
AIEX	EC	65
acid		centrifugation
Various drugs	human	none	micel C8
C18	UV or Flu	33
	serum/plasma		or CN
Zidovudine- -D-	rat plasma	deproteination	C18
ion-pair C18	UV	63
glucuronide
Enantiomers of Drugs and Related Compounds
Amino acids	protein hydrolysates,	various	CIEX
Crownpak	post-Flu	43
	bacterial cultures,
CR( )
	food, urine
Artilide fumarate	human/anim.	SPE	C8
C18; Pirkle	pre-Flu	44
	plasma
Bupivacaine	human plasma	liq – liq	AGP
C18; C8	UV	54
		extraction
Chlortalidone	human whole	liq – liq	CN
-CD phenyl	UV	51
	blood	extraction
Dihydropyridine	dog plasma	liq –liq	AIEX
Ovomucoid	EC	49
calcium blocker		extraction
Ibutilide fumarate	human/animal	SPE	C8
C18; Pirkle	pre-Flu	44
	plasma
Ifosamide	human plasma	liq – liq	D,L-naphthyl-
Chiralcel OD	UV	55
		extraction	alanine
Ketoprofen	human plasma	deproteination	C18
C18; Ovomucoid	UV	47
Leucovorine	dog plasma	deproteination	BSA-silica
C18	UV	53
	human plasma	deproteination	phenyl
BSA-silica	UV	56
Manidipine	human serum	liq – liq	C18
C18	UV	45
		extraction

306		Multidimensional Chromatography
		Chiralcel OJ
Mefloquine	human plasma/whole	liq – liq	CN
silica;	UV	52
	blood	extraction
(S)-
naphthylurea
Metoprolol	human plasma	SPE	AGP
C18; C18	Flu	54
Metyrapol	human	liq – liq	silica
Chiralcel OJ	UV	42
	plasma/urine	extraction
	Table 11.1 (continued)
		Sample	First
Subsequent		preparationa	LC modeb
Analyte(s)	Sample matrix
LC mode(s)b	Detectionc	Reference
Oxazepam	human plasma	SPE	BSA-silica
C18; C18	UV	54
p-HPPHd	rat liver	liq – liq	C18
C18 with	UV	46
	microsomes	extraction
L-prolinamide
Pimobendan and its	human plasma	liq – liq extraction	silica
Chiralcel OD	UV	48
metabolite
Terbutaline	human plasma	SPE	phenyl
-CD C18	EC	51
	human plasma	SPE	AGP
C18; C18	EC	54
	human plasma	SPE	phenyl
-CD	EC or MS	57
Verapamil and	human plasma	liq – liq	C18
Ovomucoid	UV	50
its metabolites		extraction
Endogenous Compounds
Creatinine	human	buffer dilution	SEC
SEC; CIEX	UV	58
	serum/urine
CIEX;
Glutathione	cytosol of human	homogenization,	affinity
C8 or C18	UV	11
S-transferases	lung tissue	centrifugation
Hydroxy-	human urine	SPE	PRP
C18	EC	59
deoxyguanosine
Hydroxytryptamine	rabbit blood	centrifugation	CIEX

Biomedical and Pharmaceutical Applications			307
C18	EC	68
and its metabolite		on-line SPE
Immunoglobulin G	bovine serum	none	SEC
affinity	UV	60
and its multimers
Neopterin	human serum	centrifugation	C18
ion-pair C18	Flu	62
Proinsulin fusion	E. coli cells	sulfitolysis	SEC
AIEX	UV	61
protein
Riboflavin	human	deproteination	PRP
C18	Flu	67
	blood/plasma
Thiamineand	human	deproteination	PRP
ion-pair PRP	post-Flu	67
pyridoxal-phoshates	blood/plasma
Uric acid	human	buffer dilution	SEC
AIEX; SEC	UV	58
	serum/urine

aliq – liq, liquid – liquid; SPE, solid-phase extraction.

b affinity, affinity chromatography; AGP, immobilized 1-acid glycoprotein; AIEX, anion-exchange; - CD, -cyclodextrines; BSA, bovine serum albumin; C1, methylsilica; C8, octylsilica; C18, octadecylsilica; CIEX, cation-exchange; Crown pak CR ( ), octadecyl silica coated with chiral crown ether; CN, cyanopropylsilica; ion pair, ion-pair chromatography; micel, micellar chromatography; phenyl, phenylsilica; Pirkle, 3,5-dinitrobenzoyl-D-phenylglycine-bonded silica; PRP, polymeric reversed-phase; SEC, size-exclusion chromatography.

cEC, electrochemical detection; Flu, fluorescence detection; MS, mass spectrometric detection; pre-Flu, fluorescence detection after pre-column derivatization; post-Flu, fluorescence detection after post-column derivatization; UV, UV absorbance detection.

dp-HPPH, p-hydroxyphenyl phenylhydantoin.

Multidimensional Chromatography

Edited by Luigi Mondello, Alastair C. Lewis and Keith D. Bartle

Copyright © 2002 John Wiley & Sons Ltd

ISBNs: 0-471-98869-3 (Hardback); 0-470-84577-5 (Electronic)

12Multidimensional Chromatography: Industrial and Polymer Applications

Y. V. KAZAKEVICH and R. LOBRUTTO

Seton Hall University, South Orange, NJ, USA

12.1INTRODUCTION

In this present chapter, applications of multidimensional chromatography to industrial and polymer samples are described, together with general principles and details of the interfacing setups. The main focus is on complex analyte mixtures or samples that cannot be analyzed solely by using a single mode of chromatography. Multidimensional chromatography offers the ability to analyze certain components in a mixture, which is otherwise very difficult, when employing one type of analytical technique. The advantages of using multidimensional chromatography are increases in the selectivity and efficiency, plus identification of certain components in a multicomponent mixture.

The use of coupled column technology allows preseparation of complex samples of industrial chemicals and polymers and on-line transferring of selected fractions from a primary column to a secondary column for further separation. The resolution of individual components in a complex matrix and increased peak capacity may be obtained when using coupled column chromatography. Gas chromatography (GC) is a very efficient separation technique, but it is not always effective for the resolution of all components in complex mixtures due to either coelution or a lack of volatility. The preseparation of the complex sample may be carried out by using liquid chromatography (LC), supercritical fluid chromatogaphy (SFC), and even another GC column of different polarity. The first column may be used to isolate specific components and fractionate the chemicals by class or group before chromatographic analysis on the subsequent column. The optimal coupled column chromatography procedure includes the on-line sample pretreatment and cleanup prior to the final analytical technique, and results in faster analysis of the components. Coupled separation techniques could significantly minimize and often exclude off-line sample pretreatment and preseparation procedures, which usually include filtration through prepacked sample tubes, preparative thin layer chromatography, liquid–liquid partitioning, Soxhlet extraction, and supercritical fluid extraction. These off-line techniques could result in solute loss, contamination, long workup times and introduction of human error.

304	Multidimensional Chromatography

12.2 GENERAL

Multidimensional techniques have been applied for the analysis of polymer additives and polymer samples employed in polymer chemistry. Such additives, which include antioxidants (mainly sterically hindered phenols), are present in order to enhance the performance of the polymers and to ensure processing and long-term stability. Antioxidants, such as 2,6-di-tert-butyl-p-cresol and butylated hydroxytoluene (BHT), are added since many polymers are often subject to thermal and oxidative degradation. However, the additives themselves are also subject to oxidation, especially during processing or UV irradiation. The analysis of these additives and their transformation products has become very important for routine quality control, especially in the medical plastic and food packaging industries, where the identity and levels of potentially toxic substances must be accurately controlled and known (1). The use of multidimensional chromatography for the analysis of polymer additives in food products, including edible oils, are described in this contribution.

The usual means of identifying and quantifying the level of these additives in polymer samples is performed by dissolution of the polymer in a solvent, followed by precipitation of the material. The additives in turn remain in the supernatant liquid. The different solubilites of the additives, high reactivity, low stability, low concentrations and possible co-precipitation with the polymer may pose problems and lead to inconclusive results. Another sample pretreatment method is the use of Soxhlet extraction and reconcentration before analysis, although this method is very time consuming, and is still limited by solubility dependence. Other approaches include the use of supercritical fluids to extract the additives from the polymer and subsequent analysis of the extracts by microcolumn LC (2).

Multidimensional chromatography has also been applied for the analysis of industrial chemicals and related samples. Industrial samples which have been analyzed by multidimensional chromatography include coal tar, antiknock additives in gasoline (3), light hydrocarbons (4, 5), trihaloalkanes and trihaloalkenes in industrial solvents (6 – 8), soot and particulate extracts, and various industrial chemicals that might be present in gasoline and oil samples.

In this present chapter, the applications of multidimensional chromatography using various types of coupled techniques for the analysis of industrial and polymer samples, and polymer additives, are described in detail. The specific applications are organized by technique and a limited amount of detail is given for the various instrumental setups, since these are described elsewhere in other chapters of this volume.

12.3 LC–GC

High performance liquid chromatography (HPLC) is an excellent technique for sample preseparation prior to GC injection since the separation efficiency is high, analysis time is short, and method development is easy. An LC – GC system could be fully automated and the selectivity characteristics of both the mobile and stationary

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305

phases can be modified to effectively clean up unwanted components in sophisticated matrices. This, in turn, reduces the number of components actually present in the final analytical step (GC) by allowing heart-cut fractions of the selected peaks of interest. HPLC can effectively separate compounds based on chemical classes and can be used to enrich very dilute samples. The preseparation of components by HPLC prior to introduction into the GC system also increases the sensitivity. This can be achieved because of the efficient transfer of the whole HPLC fraction containing the compounds of interest and the efficient removal of interfering materials that may suppress the detection limits obtained by analysis during the GC step. The pretreatment of samples by HPLC offers three main advantages: preseparation into chemical classes by their different polarities, cleanup of the components of interest from a ‘dirty’ matrix, and sample enrichment (9).

Normally, most LC – GC applications use normal phase LC as the first separation method. Therefore, those low-boiling, non-polar solvents which are normally used in normal phase LC, plus small LC fraction volumes, are recommended. In addition, the volitization of the eluent which is being transferred is another important consideration. The volume of the transferred LC eluent may pose a problem due to column and detector overload and loss of resolution of the transferred fractions due to solvent overlap of the desired peaks. For these reasons, low eluent flow rates and relatively short LC retention times are normally used. The introduction of large volumes has an effect on the efficiency of the solute resolution and therefore the inlet temperature must be maintained so that it is close to the LC eluent boiling point. Ensuring the proper temperature avoids overload of the GC detector and column and thus prevents distorted, broad and even split peaks. These effects have been minimized by the introduction of retention gaps prior to the GC column of uncoated, deactivated capillary columns that allow the focusing and concentration of the solutes and have low retention capabilities. Solvent evaporation can be allowed in these retention gaps by two techniques, namely concurrent solvent evaporation (the GC initial temperature is higher than the boiling point of the eluent) and partial concurrent solvent evaporation (the GC initial temperature is lower than the boiling point of the eluent).

12.4LC–GC APPLICATIONS

12.4.1NORMAL PHASE LC – GC APPLICATIONS

The methods of analysis of polymer additives and chemicals, such as hydrocarbons, alcohols, etc., are not only restricted to the field of polymer chemistry but can also be applied for the analysis of such materials in the field of food chemistry. In addition, the analysis of polyaromatic hydrocarbons in edible oils has been of extreme importance. Polymeric packaging materials that are intended for food-contact use may contain certain additives that can migrate into the food products which are actually packaged in such products. The amounts of the additives that are permitted to migrate into food samples are controlled by government agencies in order to show

306	Multidimensional Chromatography

that the levels of migrated additives from polymeric materials are within the specified migration limits. The highest level of migration of these additives, on account of their lipopphilic nature, is usually seen in edible oils or fats. Many references to the analysis of oils by using HPLC – GC are given in the book by Grob (10). These food products are usually very difficult to analyze due to the complex nature of the oils and fats and usually many sample preparation steps are needed before chromatographic separation is applied. Therefore, the use of multidimensional chromatography has allowed for less sample handling, better quantitative results and efficient analysis of such samples. Usually, normal phase HPLC is used for sample preparation, including the separation of the additive from the oil and fats. Then, the eluent fraction containing the additive is transferred to either the GC system in the second dimension or to another LC column, followed by GC analysis.

Baner and Guggenberger (11) have reported the analysis of a Tinuvin 1577 poly- mer-additive-rectified olive oil, virgin olive oil, Miglyol 812 S, corn and sunflower oils by using on-line coupled normal phase HPLC – GC. These authors have reported the quantitative determination of Tinuvin 1577 from poly (ethylene terepthalate) (PET) and polycarbonate (PC) polymers in these oil samples. The HPLC column used for the analysis of virgin olive oil was a 125 mm 4.6 mm Spherisorb Si-5 m column with a guard column, with the eluent being 30% dichloromethane in hexane. A 50 L sample injection loop and a flow rate of 400 L min were used in this work. The LC GC interface was a 300 – 500 L transfer loop and was dependent upon the HPLC mobile phase flow rate and width of the Tinuvin 1577 HPLC peak. The GC column was a DB–5HT (15 m 0.25 mm, 0.1 m coating thickness) high-tempera- ture fused-silica capillary. The GC oven temperature program was 160 °C for 8 min (the sample transfer and solvent evaporation temperature), a 10 °C min ramp to 260°C, a 5 °C/min ramp to 320 °C, and finally a 10 °C/min ramp to 360 °C for 15 min. The GC conditions included a He flow rate of 0.9 ml/min at 160°C, nitrogen make-up gas and flame-ionization detection.

The Tinuvin 1577 (MW, 425.5) eluted on the silica column before the olive oil triglycerides. Then, the HPLC eluent fraction containing the Tinuvin 1577 was transferred into the GC unit by using a loop-type interface. The detection limit was found to be 0.19 0.07 mg L and the quantifiable limit was determined as being three times the limit of detection (LOD). Figure 12.1 shows the HPLC and GC chromatograms of the blank oil versus the detection limit concentration of Tinuvin 1577 in virgin olive oil. This method has also been applied to separate other higher- molecular-weight polymer additives such as Irganox 245 (MW, 586.8) and Irganox 1010 (MW, 1200), an antioxidant polymer additive.

12.5 SEC–GC APPLICATIONS

The premise of size exclusion chromatography (SEC) is that solute molecules are separated according to their effective molecular size in solution. SEC allows the separation of fractions according to their molecular weight and eliminates the

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Figure 12.1 Analysis of Tinuvin 1577 in 30% virgin olive oil (in hexane), showing (a) the gas chromatogram comparing the pure oil with a sample at the Tinuvin 1577 detection limit concentration, and (b) the corresponding liquid chromatogram. Reprinted from Journal of High Resolution Chromatography, 20, A. L. Baner and A. Guggenberger, ‘Analysis of Tinuvin 1577 polymer additive in edible oils using on-line coupled HPLC – GC’, pp. 669 – 673, 1997, with permission from Wiley-VCH.

high-molecular-weight material within the sample, thus preventing its transfer into the GC capillary column. The combination of SEC with capillary GC is very useful in the analysis of volatile compounds from complex mixtures.

A multidimensional system using capillary SEC– GC– MS was used for the rapid identification of various polymer additives, including antioxidants, plasticizers, lubricants, flame retardants, waxes and UV stabilizers (12). This technique could be used for additives having broad functionalities and wide volatility ranges. The determination of the additives in polymers was carried out without performing any extensive manual sample pretreatment. In the first step, microcolumn SEC excludes the polymer matrix from the smaller-molecular-size additives. There is a minimal introduction of the polymer into the capillary GC column. Optimization of the pore sizes of the SEC packings was used to enhance the resolution between the polymer and its additives, and smaller pore sizes could be used to exclude more of the polymer