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Microbore high-performance liquid chromatography mass spectrometry
Znternationai Journal of Mass Spectrometry and Ion Physics, 46 (1983) 189-192 Elsevier Scientific
Publishing
Company,
Amsterdam
-
189
Printed in The Netherlands
MICROBORE HIGH-PERFORMANCE LIQUID CHRCMATOGRAPHYMASS SPECTRCVlETRY M.S.Lant’,
D.E.Gamesl, S.A_Westwoodl and B.J.Woodhall' 1 Department of Chemistry, University College, PO Box 78, Cardiff, CFl 1XL. 2ICI Pharmaceuticals Division, Hurdsfield Industrial Estate, Macclesfield,
Cheshire, SK10 2NA, England.
ABSTRACT The performance of PTFE and glass lined stainless steelmicrobore liquid chromatography columns are compared for liquid chromatography mass spectrometry (LC/?IS) using a moving belt system. The latter columns give better chromatographic efficiencies and are more durable- Advantages which accrue from this type of LC/MS include improved sensitivity and ability to handle high percentage aqueous phases. A system for microbore gradient LCjMS is described.
INTRODUCTION One of the major problems in combining a liquid chromatograph with a mass spectrometer is handling the high gas volumes generated by the flow rates of mobile phase used for conventional liquid chromatography (LC). The various types of interface for combined liquid chromatographymass spectrometry (LC/MS) have been recently reviewed (ref. l), and most experience problems in handling all the eluent from a conventional liquid chromatograph. An attractive way of overcoming the problem is to use packed (ref. 2) or open tubular capillary (ref. 3) or microbore (ref. 4) columns for LC, since the mobile phase flow rates are considerably reduced. Direct coupling of open tubular capillary (ref. 3,s) and ultra microbore (ref. 6) LC columns with a mass spectruneter has been demonstrated. However the easiest approach to the generation of low flow rates (5 - SO& min -I) is the use of packed microbore coluTIns,since the technical problems encountered are easier to overcome. Microbore LC/MS has been carried out with interfaces of the direct liquid introduction (ref. 7-9) jet (ref. lo), vacuum nebulizing (ref. 11) and moving belt types (ref. 9, 12, 13). Enhanced sensitivity was obtained in most of these studies, however until recently (ref. 8,9) little attention was paid to chromatographic efficiencies and routine performances equivalent to those obtainable with conventional columns have been difficult to obtain. We report here on our studies of microbore LC/MS using a moving belt interface, with two systems, a JASCO microbore liquid chromatograph which uses OOZO-7381/83/0000-0000/$03.00
Q
1983
Elsevier Scientific
Pubiishing Company
190
flexible PTFE collnnnsand a Waters LC pump modified forlow flow rates, used with Whabnan glass lined stainless steel microbore columns. (Xlrobjectives in this study have been, to develop microbore LC/MS capable of operating at chromatographic efficiencies comparable with those obtained from conventional colLannsover long periods, to improve the capability of moving belt systems in handling of high percentage aqueous mobile phases, to improve the sensitivity of the system and reduce the necessity of using the infrared heater to remove solvent, since this can cause sample decomposition. EXPERMENTAL The conditions and column packing methods used for our studies with the JASCO microbore liquid chrcmatograph have reported previously (ref. 9)_
For
the other studies reported in this paper a Waters 6ooOA liquid chromatography -1 ~_w"pmodified by the manufacturers for flow rates down to lO$ min , a Valco valve loop injector fitted with a O.Zr_Linternal loop and Whafman @aidstone, Kent, U-K_) 250 x lmm glass lined stainless steel columns packed with Partisil 10 ODS-3 or Partisil 10 C& were used.
LC/MS was performed on moving belt
interfaces, fitted with Finnigan 4COO or VG-70H mass spectrometers. Mass spectral data was acquired and processed using Incas data systems. Kapton belts were used and the LC column was positioned so that it was almost in contact with the moving belt of the interface. RESULTS AND DISCUSSION Our initial studies in this area utilized a JASCOmicrobore liquid chromatograph, Early studies proved disappointing in that colunn efficiencies only of the order of loo0 theoretical plates were obtained. this can cause in LC/MS investigations is
The problems that
illustrated by our studies of an
extract of timbo powder which contains two pairs of isomeric rotenoids, each pair have very similar mass spectra and hence cannot be resolved by use of the data system (ref, 13). Considerable improvements in chromatographic efficiencies were obtained by packing the PTFE columns at higher pressure and in excess of 7ooO theoretical plates were obtained by X/MS for standard mixtures of naphthalene and biphenyl with reasonable retention times. The technique was used with a wide variety of sample types and gave improved sensitivities, since background from solvent impurities was reduced and the requirements for a splitting with aquems mobile phases avoided. In addition by feeding ethanol onto the belt behind the LC column aqueous mobile phases containing high percentages (80%) of water are readily handled. (ref. 9). One of the problems with microbore LC is that of column overload. Injection in excess of 10~ of sample leads to degradation of chromatographic
191
performance.
This becomes a problem when small amounts of one compound are
being sought in the presence of .a large amount of another conbpound. We hav-e developed colon
switching techniques
In spite of the considerable JASCO system, chromatographic
to alleviate this problem
improvements we were able to effect with the
efficiencies did not match those obtainable with
conventional columns and the performance rapidly.
(ref. 9).
of the columns deteriorated
fairly
We next decided to investigate the use of glass lined stainless steel
colrnnns of 1 mm i-d. with a conventional pumping system modified rates and capable of use
at
for low flow-
much higher pressures than the JASCO system.
initial studies were once again
tir
disappointing, but whilst we were ywrsuing
them we were loaned some commercial columns of this type and investigated their performance.
EI LC/YS of a mixture of naphthalene and biphenyl, using a glass
lined stainless steel column (250 x 1 mm) packed with Partisil 10 CDS-3 and a -1 mobile phase of methanol + water (80:20) at a flow rate of 2Od min , gave approximately biphenyl
11,500 theoretical plates for naphthalene
(K' = 2.4) 11,800 theoretical plates.
(K' = 1.8) and for
This is comparable with standard
columns and we have utilized the system for a wide range of compound types without noticable deterioration Use of these colons sensitivities, measured gibberellins
in column performance
has resulted in considerable
(ref. 14). improvements in IX/%
in terms of sample injected on column.
are illustrative of this fact.
Our studies of
For gibberellin A2 using convent-
ional LC it is necessary to inject between 3 - 5+g of sample onto the LC column to obtain useful EI spectra.
Although the EI spectrum does not provide
relative molecular mass data this is obtainable from positive and negative chemical ionization
(CI) spectra.
IJsing a glass lined stainless steel microbore
column a similar quality EI spectrum was obtained
from a 20 ng injection of
gibberellin A3. In order to study complex mixtures of varying polarity the use of gradient sys&ns
is desirable.
Using two Waters pumps modified for low flow rates and a
Waters solvent programmer we have been able to devise a microbore system.
TO minimize
gradient LC
dead volume from the point of mixing to the column inlet,
the high pressure noise filter in one of the LC pumps was by-passed, the solvents from the two pumps were mixed in a mixing tee and fed directly to the microbore column.
The system has been used successfully for LC/NS studies of
extracts of samples from test wellson land fill sites and gradient systems consisting of e.g. acetonitrile
+ water (2O:SO) (A) and acetonitrile
going from 0 to 100% B were used to resolve these complex mixtures
(B)
(ref. 1.5).
CONCLUSIONS The use of microbore
LC/MS with interfaces of the moving belt type enables,
better on-column LC/?.% sensitivities
to be obtained, and high percentage aqueous
192 mobile
phases to bc used without probIcms.
used with
thcsc systems
Gratlicnt clution can he effectively
and one advantage over I,C/VS systems 01~ the direct
introduction type is that flow progrann1ing and rapid analysis by USC of higher flow rates can be used without Comparative
studies of PTFE and
the necessity of splitting the solvent. glass
that the latter are more cffcctive lifetimes and their use for-IX/%
1
inctlstainless steel
columns indicate
in terms of collnllncl-ficiencies and column is strongly advocated.
hCKNOwt,EtX;I,vINrs 1 , We thank Whatman Ltd for the loan of glass lined stainless steel colrnrns. Two of us thank the SRC and ICI c M.S.I,.) and the ARC (S.A.W.) for financial support.
We are indebted to the SRC and Royal Society for assistance in the
purchase of mass sI,ectrometric and liquid chromatographic
equipment.
REFEREACES 1. P.J.Arpino and G.Cuichon, Anal.Chem., 51(1979) 682R-701A; W.II.McFadden, .J.Chromatogr.Sci., 17(1979) 2-16; 18(1980) 79-115; D.E.Games, Hiomed. Mass Spectrom., X(1981) 454-462; in ll.R.Morris (Ed.), Soft Ionization Biological ?lass Spectromctry, Ileydcn, London, 1981, pp.54-68. 2. M.Novotny, J.Chromatogr.Sci., 18(1980), 473-478. 3. D.Tshii and T.Takeuchi, .J.Chromatogr.Sci., 18(1980) 462-472. 4. R.P.W.Scott, J.Chrorn;~togr-.Sci. , 1#[1!180) 49-54. 5. R.Tijssen, J.P.A.Blcumer, A.I,.C.Smit and M.E.Van Kreveld, .J. Chromatogr. , 218(1981) 137-165. T.Takeuchi, D.Ishii, A.Saito and T.Ohki, ,J.lIighResoln.Chrom~~togr., 6. Chromatogr. Commun., 5 (1982) 91-92. 57-63; .J..J.Brophy,D.Nelson and J.D.IIenion, J.Chromatogr.Sci., l!l(l9Sl) 7. M.K.Withers, Int. J.Mass Spectrom. Ton Phys., 36 (1980) 205-212; K.II.Schafer and K.Levsen, J.Chromatogr., 206(1981) 245-252; E.Yamauchi, T.Mizuno and K.AZUIIl~,Shibak Shitsuryo Runseki, 28(1980) 227-234: 8. P.Krien, G.Devant and M.Ilardy, .J.Chromutogr., 251 (1982) 129-139. D.E.Games, M.S.Lant, S.A.Westwood, M..J.Cocksedge, N.Evans, J.Williamson 9. and R.J.Woodhall, Biomed.Mass Spectrom., 9(1982) 215-224. 10. T.Takeuchi, Y.IIirata and Y.Ukumara, Anal.Chem., 50(1978) 659-660. 11. S.Tsuge, Yu. Yoshida, T.Takeuchi, K.Mochizuki, N.Kokubun and K.Hibi, Chem.Biomed.Environ. Instrum., lO(1980) 405-418. 12. W.H.McFadden, H.L.Schwartz and S.Evans, J.Chromatogr., 122(1976) 389-396. 13. D.E.Games, P.Hirter, W.Kuhnz, E.Lewis, N.C.A.Weerasinghe and.S.A.Westwood, J.Chromatogr., 203(19X1) 131-138. 14. N.J.Alcock, L.Corbelli, D.E.Games, M.S.I,ant and S.A.Westwood, Kiomed. Mass Spectrom. submitted. 15. M.G.Foster, O.Meresz, D.E.Games and S.A.Westwood, unpublished work.
Publishing
Company,
Amsterdam
-
189
Printed in The Netherlands
MICROBORE HIGH-PERFORMANCE LIQUID CHRCMATOGRAPHYMASS SPECTRCVlETRY M.S.Lant’,
D.E.Gamesl, S.A_Westwoodl and B.J.Woodhall' 1 Department of Chemistry, University College, PO Box 78, Cardiff, CFl 1XL. 2ICI Pharmaceuticals Division, Hurdsfield Industrial Estate, Macclesfield,
Cheshire, SK10 2NA, England.
ABSTRACT The performance of PTFE and glass lined stainless steelmicrobore liquid chromatography columns are compared for liquid chromatography mass spectrometry (LC/?IS) using a moving belt system. The latter columns give better chromatographic efficiencies and are more durable- Advantages which accrue from this type of LC/MS include improved sensitivity and ability to handle high percentage aqueous phases. A system for microbore gradient LCjMS is described.
INTRODUCTION One of the major problems in combining a liquid chromatograph with a mass spectrometer is handling the high gas volumes generated by the flow rates of mobile phase used for conventional liquid chromatography (LC). The various types of interface for combined liquid chromatographymass spectrometry (LC/MS) have been recently reviewed (ref. l), and most experience problems in handling all the eluent from a conventional liquid chromatograph. An attractive way of overcoming the problem is to use packed (ref. 2) or open tubular capillary (ref. 3) or microbore (ref. 4) columns for LC, since the mobile phase flow rates are considerably reduced. Direct coupling of open tubular capillary (ref. 3,s) and ultra microbore (ref. 6) LC columns with a mass spectruneter has been demonstrated. However the easiest approach to the generation of low flow rates (5 - SO& min -I) is the use of packed microbore coluTIns,since the technical problems encountered are easier to overcome. Microbore LC/MS has been carried out with interfaces of the direct liquid introduction (ref. 7-9) jet (ref. lo), vacuum nebulizing (ref. 11) and moving belt types (ref. 9, 12, 13). Enhanced sensitivity was obtained in most of these studies, however until recently (ref. 8,9) little attention was paid to chromatographic efficiencies and routine performances equivalent to those obtainable with conventional columns have been difficult to obtain. We report here on our studies of microbore LC/MS using a moving belt interface, with two systems, a JASCO microbore liquid chromatograph which uses OOZO-7381/83/0000-0000/$03.00
Q
1983
Elsevier Scientific
Pubiishing Company
190
flexible PTFE collnnnsand a Waters LC pump modified forlow flow rates, used with Whabnan glass lined stainless steel microbore columns. (Xlrobjectives in this study have been, to develop microbore LC/MS capable of operating at chromatographic efficiencies comparable with those obtained from conventional colLannsover long periods, to improve the capability of moving belt systems in handling of high percentage aqueous mobile phases, to improve the sensitivity of the system and reduce the necessity of using the infrared heater to remove solvent, since this can cause sample decomposition. EXPERMENTAL The conditions and column packing methods used for our studies with the JASCO microbore liquid chrcmatograph have reported previously (ref. 9)_
For
the other studies reported in this paper a Waters 6ooOA liquid chromatography -1 ~_w"pmodified by the manufacturers for flow rates down to lO$ min , a Valco valve loop injector fitted with a O.Zr_Linternal loop and Whafman @aidstone, Kent, U-K_) 250 x lmm glass lined stainless steel columns packed with Partisil 10 ODS-3 or Partisil 10 C& were used.
LC/MS was performed on moving belt
interfaces, fitted with Finnigan 4COO or VG-70H mass spectrometers. Mass spectral data was acquired and processed using Incas data systems. Kapton belts were used and the LC column was positioned so that it was almost in contact with the moving belt of the interface. RESULTS AND DISCUSSION Our initial studies in this area utilized a JASCOmicrobore liquid chromatograph, Early studies proved disappointing in that colunn efficiencies only of the order of loo0 theoretical plates were obtained. this can cause in LC/MS investigations is
The problems that
illustrated by our studies of an
extract of timbo powder which contains two pairs of isomeric rotenoids, each pair have very similar mass spectra and hence cannot be resolved by use of the data system (ref, 13). Considerable improvements in chromatographic efficiencies were obtained by packing the PTFE columns at higher pressure and in excess of 7ooO theoretical plates were obtained by X/MS for standard mixtures of naphthalene and biphenyl with reasonable retention times. The technique was used with a wide variety of sample types and gave improved sensitivities, since background from solvent impurities was reduced and the requirements for a splitting with aquems mobile phases avoided. In addition by feeding ethanol onto the belt behind the LC column aqueous mobile phases containing high percentages (80%) of water are readily handled. (ref. 9). One of the problems with microbore LC is that of column overload. Injection in excess of 10~ of sample leads to degradation of chromatographic
191
performance.
This becomes a problem when small amounts of one compound are
being sought in the presence of .a large amount of another conbpound. We hav-e developed colon
switching techniques
In spite of the considerable JASCO system, chromatographic
to alleviate this problem
improvements we were able to effect with the
efficiencies did not match those obtainable with
conventional columns and the performance rapidly.
(ref. 9).
of the columns deteriorated
fairly
We next decided to investigate the use of glass lined stainless steel
colrnnns of 1 mm i-d. with a conventional pumping system modified rates and capable of use
at
for low flow-
much higher pressures than the JASCO system.
initial studies were once again
tir
disappointing, but whilst we were ywrsuing
them we were loaned some commercial columns of this type and investigated their performance.
EI LC/YS of a mixture of naphthalene and biphenyl, using a glass
lined stainless steel column (250 x 1 mm) packed with Partisil 10 CDS-3 and a -1 mobile phase of methanol + water (80:20) at a flow rate of 2Od min , gave approximately biphenyl
11,500 theoretical plates for naphthalene
(K' = 2.4) 11,800 theoretical plates.
(K' = 1.8) and for
This is comparable with standard
columns and we have utilized the system for a wide range of compound types without noticable deterioration Use of these colons sensitivities, measured gibberellins
in column performance
has resulted in considerable
(ref. 14). improvements in IX/%
in terms of sample injected on column.
are illustrative of this fact.
Our studies of
For gibberellin A2 using convent-
ional LC it is necessary to inject between 3 - 5+g of sample onto the LC column to obtain useful EI spectra.
Although the EI spectrum does not provide
relative molecular mass data this is obtainable from positive and negative chemical ionization
(CI) spectra.
IJsing a glass lined stainless steel microbore
column a similar quality EI spectrum was obtained
from a 20 ng injection of
gibberellin A3. In order to study complex mixtures of varying polarity the use of gradient sys&ns
is desirable.
Using two Waters pumps modified for low flow rates and a
Waters solvent programmer we have been able to devise a microbore system.
TO minimize
gradient LC
dead volume from the point of mixing to the column inlet,
the high pressure noise filter in one of the LC pumps was by-passed, the solvents from the two pumps were mixed in a mixing tee and fed directly to the microbore column.
The system has been used successfully for LC/NS studies of
extracts of samples from test wellson land fill sites and gradient systems consisting of e.g. acetonitrile
+ water (2O:SO) (A) and acetonitrile
going from 0 to 100% B were used to resolve these complex mixtures
(B)
(ref. 1.5).
CONCLUSIONS The use of microbore
LC/MS with interfaces of the moving belt type enables,
better on-column LC/?.% sensitivities
to be obtained, and high percentage aqueous
192 mobile
phases to bc used without probIcms.
used with
thcsc systems
Gratlicnt clution can he effectively
and one advantage over I,C/VS systems 01~ the direct
introduction type is that flow progrann1ing and rapid analysis by USC of higher flow rates can be used without Comparative
studies of PTFE and
the necessity of splitting the solvent. glass
that the latter are more cffcctive lifetimes and their use for-IX/%
1
inctlstainless steel
columns indicate
in terms of collnllncl-ficiencies and column is strongly advocated.
hCKNOwt,EtX;I,vINrs 1 , We thank Whatman Ltd for the loan of glass lined stainless steel colrnrns. Two of us thank the SRC and ICI c M.S.I,.) and the ARC (S.A.W.) for financial support.
We are indebted to the SRC and Royal Society for assistance in the
purchase of mass sI,ectrometric and liquid chromatographic
equipment.
REFEREACES 1. P.J.Arpino and G.Cuichon, Anal.Chem., 51(1979) 682R-701A; W.II.McFadden, .J.Chromatogr.Sci., 17(1979) 2-16; 18(1980) 79-115; D.E.Games, Hiomed. Mass Spectrom., X(1981) 454-462; in ll.R.Morris (Ed.), Soft Ionization Biological ?lass Spectromctry, Ileydcn, London, 1981, pp.54-68. 2. M.Novotny, J.Chromatogr.Sci., 18(1980), 473-478. 3. D.Tshii and T.Takeuchi, .J.Chromatogr.Sci., 18(1980) 462-472. 4. R.P.W.Scott, J.Chrorn;~togr-.Sci. , 1#[1!180) 49-54. 5. R.Tijssen, J.P.A.Blcumer, A.I,.C.Smit and M.E.Van Kreveld, .J. Chromatogr. , 218(1981) 137-165. T.Takeuchi, D.Ishii, A.Saito and T.Ohki, ,J.lIighResoln.Chrom~~togr., 6. Chromatogr. Commun., 5 (1982) 91-92. 57-63; .J..J.Brophy,D.Nelson and J.D.IIenion, J.Chromatogr.Sci., l!l(l9Sl) 7. M.K.Withers, Int. J.Mass Spectrom. Ton Phys., 36 (1980) 205-212; K.II.Schafer and K.Levsen, J.Chromatogr., 206(1981) 245-252; E.Yamauchi, T.Mizuno and K.AZUIIl~,Shibak Shitsuryo Runseki, 28(1980) 227-234: 8. P.Krien, G.Devant and M.Ilardy, .J.Chromutogr., 251 (1982) 129-139. D.E.Games, M.S.Lant, S.A.Westwood, M..J.Cocksedge, N.Evans, J.Williamson 9. and R.J.Woodhall, Biomed.Mass Spectrom., 9(1982) 215-224. 10. T.Takeuchi, Y.IIirata and Y.Ukumara, Anal.Chem., 50(1978) 659-660. 11. S.Tsuge, Yu. Yoshida, T.Takeuchi, K.Mochizuki, N.Kokubun and K.Hibi, Chem.Biomed.Environ. Instrum., lO(1980) 405-418. 12. W.H.McFadden, H.L.Schwartz and S.Evans, J.Chromatogr., 122(1976) 389-396. 13. D.E.Games, P.Hirter, W.Kuhnz, E.Lewis, N.C.A.Weerasinghe and.S.A.Westwood, J.Chromatogr., 203(19X1) 131-138. 14. N.J.Alcock, L.Corbelli, D.E.Games, M.S.I,ant and S.A.Westwood, Kiomed. Mass Spectrom. submitted. 15. M.G.Foster, O.Meresz, D.E.Games and S.A.Westwood, unpublished work.