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Quantitative analysis methods for sugars

Suzanne de Goeij

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Suzanne de Goeij 2

MSc ChemistryAnalytical Sciences

Literature Thesis


by

Suzanne de Goeij

August 2013

Supervisor:dr. W.Th. Kok

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ABBR !"AT"#$S

AA anthranilic acidA!"" #$amino%enzoic ethyl esterA&' Atmos#heric &ressure 'onization

!G" !ackground "lectrolyte(A) (harged Aerosol )etection(TA! cetyltrimethylammonium %romide(*+," (a#illary *+one, "lectro#horesis)- )etection -imit) & 2/0$dimetho1y#henol"(- "lectro generated (hemilluminescence"-S) "va#orative -ight Scattering )etection"S' "lectros#ray 'onization

') lame 'onization )etector

G( Gas (hromatogra#hy3'-'( 3ydro#hilic 'nteraction -i4uid (hromatogra#hy3&A"( 3igh &erformance Anion$"1change (hromatogra#hy3&-( 3igh &erformance -i4uid (hromatogra#hy'(3 'nternational (onference on 3armonization-( -i4uid (hromatogra#hy-' -aser 'nduced luorescence

in inutes5 ulti#le 5eaction onitoringS ass S#ectrometry

6 & 7$*2$na#hthyl,$ $methyl#yrazolonen.r. 6ot 5e#orted&A) &ulsed Am#erometric )etection& & 7$#henyl$ $methyl$8$#yrazolone& T &hotomulti#lier tu%eQ- Quantitation -imit52 (orrelation coefficient5" 5eference5' 5efractive 'nde15S) 5elative Standard )eviation

S)S sodium dodecyl sulfateS' Selected 'on onitoringS96 Signal to noise ratioTem# Tem#eratureT S Tri ethyl Silyl:;$;'S :ltra ;iolet$;isi%le S#ectrosco#y

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"$% & A%%reviations ................................................................................................................................................

7 'ntroduction .................... ................ ................... ................... .............. .................... .................... ........... 8

2 -i4uid (hromatogra#hy *-(, ................... ................ .................... .................. .................. .................... ... 0

2.7 (olumns .................. ................... ............... .................... .................. ................... ................... ......... 0

2.2 )etectors in -i4uid (hromatogra#hy ............... ................... .................... .............. .................... .... =

2.2.7 "va#orating -ight Scattering )etection *"-S),........................ .................... .............. ............ =

2.2.2 5efractive 'nde1 )etector *5', .................. .................... ................... .............. .................... .... >

2.2. :ltra ;iolet )etection *:;,....................... .................... ................... .............. .................... .. 7?

2.2.< ass S#ectrometry * S, ................... ................. ................... .................. ................... ......... 77

2.2.8 luorescence )etection .................. ................. ................... ................... .................. ............ 7

2.2.0 (harged Aerosol )etection *(A), .................. .................... ................... .............. ................ 7<

2. )iscussion li4uid chromatogra#hy ................ ................... .................... .............. .................... ..... 78

3igh &erformance Anion$e1change (hromatogra#hy *3&A"(, .................... .................... .............. .... 27

.7 3&A"( @ith #ulsed am#erometric detection *&A), .................. .................... ............. ................. 27

.2 3&A"( @ith ass detection ................... ................. .................... ................ ................. ............... 22

. )iscussion high #erformance anion$e1change chromatogra#hy .................. .................... .......... 22

< Gas (hromatogra#hy *G(, .................. ................ .................... .................... .............. ................... ........ 28

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' "$TR#%(CT"#$

The se#aration and 4uantitative analysis of sugars is challenging for several reasons. To find a #ro#ertechni4ue/ column and detector for the se#aration and 4uantitative detection of sugars is difficult.-i4uid chromatogra#hy *-(, is most @idely used to se#arate sugars due to the availa%ility. 3o@ever

traditional reversed #hase columns cannot %e used for underivatized sugars/ as the stationary #hase @illnot #rovide the re4uired retention and s#ecialized columns are necessary. )etection of sugars facestrou%les as their structure contains no chromo#hores. )etection %y :;$;'S/ as commonly used in 3&-(/is not #ossi%le in sugar analysis. )etection techni4ues such as eva#orate light scattering *"-S),/refractive inde1 *5',/ ass s#ectrometry * S, can %e used %ut every detector has its o@n limitations. Allli4uid chromatogra#hy methods in com%ination @ith several detectors @ill %e discussed in cha#ter 2.Another ty#e of li4uid chromatogra#hy that can %e used for sugar analysis is high$#erformance anion$e1change chromatogra#hy @ith #ulsed am#erometric detection *3&A"(B&A),. Anionic se#aration and&A) detection im#roves the sensitivity and s#ecificity com#ared to other -( methods. 3&A"($&A) @ill %ediscussed in cha#ter .

)ue to their high #olarity/ hydro#hilicity and lo@ volatility/ saccharides have to %e converted into volatileand sta%le derivatives/ i.e./ trimethylsilyl or acetate derivatives/ %efore G( analysis. 'n general G(methods @ith ') or S detection #rovide a good se#aration of sugars and a good sensitivity/ %utre4uire #rior ste#s of reduction and derivation/ @hich are very time consuming and not very #ractical inroutine analysis. The use of G( for sugar se#aration @ill %e discussed in cha#ter

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) L"Q("% C*R#MAT#GRA+*, -LC.

Se#aration and detection of sugars can %e done @ith li4uid chromatogra#hy using different column ty#esand detectors. 'n li4uid chromatogra#hy there are various @ays of analysing sugars/ a distinguish can %emade %et@een columns and detection techni4ues. )ifferent columns @ill result in other se#aration of

sugars and results in different validation #arameters like detection limits.

)/' Columns

The stationary #hases that can %e used for the -( se#aration of native *underivatized, sugars are cyano$or amino silica$%onded columns/ cationic e1change resins *see cha#ter , or hydro#hilic stationary#hases.

A column #acked @ith hydrogen sulfonated divinyl %enzene$styrene co#olymer #articles can %e used forthe se#aration of sugars %y ion$e1change chromatogra#hy. 7$2 The addition of acetonitrile *0C, to themo%ile #hase *?.?

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After derivatization of the sugars for :; or fluorescence detection/ se#aration can %e #erformed on anormal silica %ased ( or (7 column/ and numerous e1am#les are re#orted in the literature. =$ $>

Ag%levor et al. re#orted the analysis of %iomass sugars on a &revail column @ith #olymeric %eads asstationary #hase and found the analysis of the standard sugars on the &revail had a strong de#endence

on the com#osition of the mo%ile #hase. or %aseline se#aration an isocratic run at 78C @ater and 8Cacetonitrile @as used. 7? A chromatogram of the develo#ed method %y Ag%levor et al. is #resented infigure 2.

igure 2F se#aration of sugars in %iomass %y a #olymeric %ased column and "-S) detection/ re#roducedfrom reference 7?.

)/) %etectors in Li0uid Chromatogra1hy

(ommonly used detectors in li4uid chromatogra#hy are :;/ "-S)/ 5'/ (A)/ fluorescence and S. "verydetector has its advantages and disadvantages in the detection of sugars. 'n the #aragra#h %elo@ alldetectors @ill %e discussed for the detection of sugars and in ta%le 7 B 8 details of the method aredescri%ed *#age 70$2?,.

)/)/' va1orating Light Scattering %etection - LS%.

The "va#orating -ight Scattering )etector *"-S), is an universal/ nons#ecific detector in @hich the signalintensity is related to the concentration of the analyte in the effluent/ %ut not to its o#ticalcharacteristics. An "-S) detects all non$volatile solutes after eva#oration of the solvent of the column

effluent %y light dis#ersion on the solid analyte #articles formed. The detector is useful in the4uantitative determination of non$chromo#horic com#ounds and can %e used in com%ination @ithgradients/ although attention should %e given to the fact that non$volatile salts cannot %e used as %ufferin the eluent.A disadvantage of the "-S) is the lo@ re#roduci%ility/ a slightly lo@ sensitivity to lo@ molecular @eightcom#onents and the non$linearity of the detector. Quantification can %e done %y using a #olynomicorder or %i$logarithmic cali%ration line.

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The detection of non$chromo#horic and non$volatile com#ounds makes "-S) a suita%le techni4ue fordetection of sugars.

-iu et al. develo#ed a method for :; a%sor%ing com#ounds and sugars in one. )etection @as done %y :;and "-S) in #arallel. )etection limits re#orted for the sugars %y "-S)/ @ere com#ara%le to the results

@ith a :; detector for :;$a%sor%ing com#onents in the same sam#les.7

They also validated the recoveryand #recision and found good accuracy @ith recovery in the range of >>.> C. The intra$ and inter$day re#eata%ility of the retention time and the #eak area @ere satisfactory/ @ith C5S) values less than7.72 C. 7 A chromatogram of the se#aration of the sugars %y -iu et al. is #resented in figure .Sharma et al. also validated an -($"-S) method according to the '(3 guidelines and found com#ara%lerecoveries in the range of >>.> C. The #recision results sho@ed the lo@ values of intra$ and inter$day C 5S) of the retention times and #eak areas E 7C. < Also !handari et al. validated the method andfound recoveries of > .7 B > .0 C and sho@ed #recision results of E 2C of intra$ and inter$day C 5S) ofretention times and #eak areas. 8 The re#orted 5S) values of !handari et al. *E 2 C, are slightly higher

com#ared to the 5S) values of Sharma et al. *E 7 C, and -iu et al. *E 7.72 C,. !handari et al. used ane1traction ste# for the sugars and took this e1traction into the validation data @hich can cause a higherC 5S).

igure F (hromatogram of a !iorad 3& $ =3 column @ith "-S) detection/ se#aration of sugars in%iomass/ re#roduced from reference 7.

The lo@est detection limits @ith the detection of "-S) @ere re#orted %y Ag%levor et al. The se#aration@as #erformed on a &revail car%ohydrate "S column. The re#orted detection limits are a factor 7? lo@ercom#ared to the other references. 7?

The com%ination of -( and "-S) is re#orted in several other articles. 'n ta%le 7 *#age 70, thesea##lications are re#orted @ith their s#ecific #erformance #arameters.

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)/)/) Refractive "nde2 %etector -R".

A 5' detector detects com#onents %ased on the refraction of light in solution. When com#onents areeluting from the column/ the com#osition change in the analytical and reference cell is recorded/ @hichchanges its #hotorefractive level. As a result/ the amount of light received %y the light$receiving section

changes/ sho@ing a #eak @hich can %e detected. Any com#onent in the eluent can %e detected @hichmake 5' suita%le for sugar detection.The res#onse of 5' detection results in linear cali%ration curves. Dne of the disadvantages of 5' detectionis the tem#erature sensitivity/ small fluctuations in tem#erature results in high variations. 5' detectioncannot %e used in com%ination @ith gradients @hich makes method develo#ment limited.

(havez$Servin et al./ (astellari et al. and !arreira et al. all re#orted validated methods for sugarse#aration @ith the use of 5' detection. The detection limits are in the same order *see Ta%le 2,/although different columns @ere used for the se#aration. 2$0$77

(astellari et al. validated the method and found a #recision of E7C and a recovery of >>. C for fructose

and >>.0C for fructose. They also validated the difference %et@een direct injection and injection aftersam#le clean u#. 6o significant difference @as o%served. 2 !arreira et al. determined the #recision of themethod %y re#eata%ility *intra$day, and intermediate #recision *inter$day,. The intra$day #recision @asfound to %e E7.>. C. 0 (havez$Servin et al. found re#eata%ility results of lessthan 7C and re#roduci%ility of less than =.?C. 5ecoveries in all sugars @ere %et@een > and 77 C. 77

-o#es et al. descri%ed a 3&-($5' method @ith the use of a chiral column. 72 The method allo@s thecar%ohydrate identification and determination of the a%solute configuration *d or l, and simultaneouslyalso determine the configuration of the anomeric centre *H or I, of the monosaccharide.

They also studied the effect of the column tem#erature on the se#aration.72

The elution #rofiles of thesugars are de#ended on column tem#erature. The change @as not uniform for all monosaccharidesF fore1am#le/ @hen the tem#erature @as increased/ the elution #rofile of ly1ose %ecame @orse/ @hile forglucose the %est elution #rofile @as achieved at

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)/)/3 (ltra !iolet %etection -(!.

'n li4uid chromatogra#hy :;9:;$;'S detectors are fre4uently used to measure com#onents sho@ing ana%sor#tion s#ectrum in the ultraviolet or visi%le region. An :; detector is e4ui##ed @ith a deuteriumlam# *)2 lam#, as a light source and has a light ranging from 7>? to ? nm.

As a lot of com#onents e1hi%it a chromo#horic grou#/ so a %road range of su%stances can %e detected.Sensitivity of the detection de#ends on the com#onent. 3o@ever the lack of chromo#hores in thestructure #revents the use of :; detection for native sugars. Therefore/ the derivatization of sugars isnecessary to a##ly :; detection. The reagents 7$#henyl$ $methyl$8$#yrazolone *& &, and #$amino%enzoic ethyl ester are the most #o#ular la%els that react @ith reducing car%ohydrates under mildconditions. =$

igure ? B 72? C.

-v et al. #erformed a full validation of the method as descri%ed %y the '(3 guidelines. They re#orted 5S)C values for intra$ and inter$day #recision of less than 2.< C for the retention time and less than

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Suzanne de Goeij 72

3ammad et al. used a tri#le 4uadru#ole S in 5 *multi#le reaction monitoring, modes. Theyanalysed the sugars as their alditol acetate anion adducts M N(3 (D 2O

$ using electro s#ray ionization innegative ion mode. Although very lo@ detection limits are re#orted *#g range, the #recision is around 8Cand the average recovery is around 0?C for all sugars. This is e1#ected since it might %e that the sugarsare not com#lete re$acetylated @ith acetic anhydride/ or undergo side reaction. 70 A chromatogram of the

se#aration develo#ed %y 3ammad et al. is #resented in figure 8.

igure 0F ast se#aration of sugars %y -($ S/ re#roduced from reference 70. *1ylose *a,/ galactose *%,/mannose *c,/ glucose *d,/ fucose *e,/6$acetylgalactosamine *f,/ 6$acetylglucosamine *g, *h and ' are nonsugar com#ounds,,

atias et al. used S @ith "S' in full scan modes for identification and S' *selected ion monitoring, for4uantification. Sugars @ere detected in #ositive ion mode as their sodium adducts M N6aO N. 5ecoveriesof the target sugars ranged from >8.< B >=.= C and the re#roduci%ility of the injection @as re#orted asless than .< C. 7=

ass s#ectrometry in com%ination @ith li4uid chromatogra#hy and the se#aration of sugars is re#orted

in literature. A fe@ a##lications and validation #arameters are #resented in ta%le < *#age 7>,. oremethods @ere found ho@ever @ithout validation #arameters. 7=$7

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Suzanne de Goeij 7

)/)/5 6luorescence %etection

luorescence detection is also re#orted in com%ination @ith a##lications for the se#aration anddetections of sugars.'n fluorescence su%stances a%sor%s light at one @avelength and emit light at another @avelength.

Su%stances have s#ecific @avelengths of light that it a%sor%s *e1citation @avelengths, and emits*emission @avelengths,.

luorescence detection is suita%le for trace analysis due to generally having high sensitivity andselectivity *not detecting im#urities,. There are not many com#onents that originally emit fluorescence*natural fluorescence,. 3o@ever/ after reaction @ith a fluorescence reagent *derivatization, a lot ofcom#ounds can %e detected as fluorescent su%stances. This method makes it #ossi%le to measurevarious com#onents @ith high sensitivity.

Since sugars contain no fluoro#hores/ derivatization of the sugars is re4uired. (ommon fluorescent tagsused for la%elling of the monosaccharides #rior to 3&-( analysis areF anthranilic acid *AA,/ 2$

amino%enzamide/ 2$amino#yridine/ #henyl isothiocyanate/ >$fluorenylmetho1ycar%onylhydrazine/ =$amino$ Kakita used %enzamidide asderivatization reagent and re#orted high sensitivity/ good linearity and good re#roduci%ility. 7> 5acaityteet al. used anthranilic acid *AA, as derivatization reagent. >

5acaityte et al. develo#ed 2 different methods for the se#aration of sugars/ 3&-($fluorescence and (+"$-' *laser induced fluorescence,/ %ut only validated the 3&-( method. !oth methods are suita%le for these#aration and detection of sugars/ each @ith their advantages and disadvantages. 5ecoveries %et@een>0.7 and 7??.2C @ere determined. The re#eata%ility @as found as less than .

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Suzanne de Goeij 7<

Kakita et al. validated the method and found the 5S) for the retention time varied %et@een ?.?8 B ?. 0Cand for the fluorescence intensity %et@een 7.7< B 7.02C. 7>

Sensitive methods @ith fluorescence detection are availa%le for 4uantification of sugars and aresummarized in the ta%le %elo@/ ta%le 8 *#age 2?,. Although lo@ detection limits can %e reached/

derivatization is al@ays re4uired/ @hich makes the method not suita%le for routine analysis.

)/)/7 Charged Aerosol %etection -CA%.

(harged Aerosol )etection *(A), is a recent develo#ed detector %ased on the detection of scattered#articles/ like in "-S). The eluent coming from the -( is ne%ulized using a flo@ of nitrogen and theresulting aerosol is trans#orted through a drift tu%e @here the volatile com#onents and solvents areeva#orated. The dried #article stream is charged @ith a corona discharge needle/ resulting in anelectrical charge measured %y an electrometer. -ike "-S)/ the (A) is mass$de#endent and is calleduniversal. 2? Aerosol detectors are @ell$suited to detect all non$volatile analytes/ regardless of @hether these analytes

contain a chromo#hore. These #ro#erties make aerosol detectors suita%le for the detection of sugars. 27

A #erformance evaluation %et@een "-S) and (A) is re#orted %y ;ervoort et al. itting of thee1#onential factor in the cali%ration e4uation sho@ed that for the chosen concentration interval thelinearity of the (A) is much %etter than that of the "-S). 'n general the (A) is more sensitive than "-S)/the #eak sha#e is little @orse in (A) *higher tailing factor, and the re#eata%ility of %oth detectors is stillaround a factor 2 @orse com#ared to :; detection. 2?

A##lications of sugars se#arations are re#orted/ ho@ever @ithout validation #arameters. 27$22

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Suzanne de Goeij 78

)/3 %iscussion li0uid chromatogra1hy

)ifferent ty#es of li4uid chromatogra#hy columns are availa%le for the se#aration of sugars. 'n all casesse#aration of sugars is challenging and method develo#ment is re4uired to o#timize se#aration inreasona%le retention times.After se#aration/ detection is also difficult as sugars contain no chromo#hores to analyse them @ith :;/@hat mostly is done in -(.

The use of "-S) in com%ination @ith the se#aration of sugars has %een re#orted %y different authors.With the use of different columns for the se#aration/ detection limits in the same order @ere found *in

the order of ?.7 B 7 µ g #er injection,. A recovery of > B >>.> C @as re#orted and an intra$ and inter$day#recision as C 5S) of the retention time and #eak are E 2 C.

A recent develo#ed detector for the analysis of sugars is the (A) detector ho@ever the #erformance iscom#ara%le to the "-S) detector.

)etection of sugars %y 5' @as re#orted in the first a##lications of the analysis of sugars. )etection limitsare in the same range as re#orted in "-S) a##lications. -o#es et al. did some method develo#ment %yvarying the column tem#erature on a chiral column and 5' detection. 72 (astellari et al. found e1cellentrecoveries *P>>. C, and #recision results E7C. 2 3o@ever other authors re#orted lo@er recoveries*%et@een > B 77 C, and higher #recision values.

ass s#ectrometry is also an o#tion for the detection of sugars. Several a##lications are re#orted @heresugars @ere analysed as their sodium adduct/ acetate adduct or chloride adduct. (om#ared to thedetection limits of other detectors/ the lo@est limits @ere re#orted %y 3ammad et al. using a 4uadru#ole

S/ detection limits in #g range #er injection are re#orted. 70 Although very lo@ detection limits arere#orted *#g range, the #recision is around 8C and the average recovery is around 0?C for all sugars.

(om#ara%le #recision values are found in other a##lications.

or detection %y :; a derivatization reagent is re4uired. A##lications are re#orted @here 7$&henyl$ $methyl$8$#yrazolone = and #$amino%enzoic ethyl ester is used as derivatization reagent/ @ith recoveries%et@een >? B 72? C. The re#orted detection limits of %oth references are in the nanogram range/ @hichis significant lo@er com#ared to the re#orted detection limits %y "-S) and 5'. The re#orted values for#recision are E2. and %enzamidide 7> as derivatization reagent. The detectionlimits of sugar se#aration @ith the use of a fluorescence detector are significant lo@er com#ared to the"-S) and 5' detection/ ho@ever a derivatization ste# is al@ays re4uired.

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Ta%le 7 F Dvervie@ of -($"-S) methods for the analysis of sugars.(olumn ethod details Sugars *retention

time min a,-inear range* g9ml,

52

!iorad 3& $ =328? mm 1

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Ta%le 2F Dvervie@ of -($5' methods for the analysis of sugars

(olumn ethod details Sugars *retentiontime min a,

-inear range* g9ml,

52

Tracer car%ohydratescolumn 28? mm 1 >?.>>>

"uros#her 7??$8632 column

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Ta%le F Dvervie@ of -($:; methods for the analysis of sugars


-inear range* g9ml,

52 5S)

5&$(7 column

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Ta%le 8F Dvervie@ of -($ luorescence methods for the analysis of sugars.


-inear range* g9ml,

52

TSKgel Amide$ ?column #acked @ithchemically %ondedcar%amoylBsilica gel

"luentF Gradient =.8 min linear gradient fromA acetonitrileB@ater/ * ?F2?, to ! acetonitrileB@ater/ *0?F

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Suzanne de Goeij 27

3 *"G* + R6#RMA$C A$"#$8 &C*A$G C*R#MAT#GRA+*, -*+A C.

Another ty#e of li4uid chromatogra#hy is 3igh &erformance Anion$"1change (hromatogra#hy *3&A"(,@ith #ulsed am#erometric detection *&A),. 3&A"($&A) is often used for the se#aration of sugars. 'tre4uires a dedicated system and s#ecial detector. All references make use of a &A ty#e *&A$79&A$7?9&A$

2?9&A$7??9&A$2??, of column.2 $2

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Suzanne de Goeij 22

5aessler et al. tested the sta%ility of the retention times after su%se4uent injections *at the same day,and found 5S) E7C. 2<

3/) *+A C 9ith Mass detection

!eside &A) detection/ S detection can %e used in com%ination @ith 3&A"( although this is a

technological challenge. Ty#ical alkali acetate and hydro1ide eluents are not com#ati%le @ithatmos#heric #ressure ionization *A&', due to their non$volatility and high conductance. !ruggink et al.created a system @ith a desalting device installed %et@een the column and the S. The desalterconverts the alkali hydro1ide and acetate into @ater and acetic acid continuously e1changing the alkalications %y hydronium ions using a selective cation e1change mem%rane and a regenerant. 28 !ruggink etal. created a 3&A")$ S system @here 4uantification @as done in S' mode. 'n this case S @as onlyused for structure elucidation. 'n the validation of %oth detectors it a##eared that the detection limits of

S @ere 7? times higher com#ared to &A) detection. 28 The sugars se#arated %y &A) and S detectionare #resented in figure >/ the differences in sensitivity of the detectors is clearly visi%le.

igure >F Se#aration of glucose *7,/ fructose *2, and sucrose * , %y 3&A") chromatogra#hy @ith &A) andS detection/ re#roduced from reference 28.

3/3 %iscussion high 1erformance anion8e2change chromatogra1hy

3igh #erformance anion$e1change chromatogra#hy @ith &A) detection is a sufficient @ay of se#aratingand detecting sugars. )ifferent ty#e of columns can %e used to o#timize the se#aration. The correlationcoefficient of the cali%ration curves is in all re#orted references P?.>>.

)etection limits re#orted @ith 3&A")$&A) are in the nanogram range and are com#ara%le to -( @ith :;or fluorescence detection after derivatization or -( @ith S detection.!ruggink et al. 28 develo#ed a system to use S as detection in com%ination @ith 3&A"(. They alsore#orted im#ressive detection limits @ith &A) detection ho@ever they are the %uilder and o@ner of the3&A"( system.

'n ta%le 0 3&A"( methods and validation #arameters are #resented. ore 3&A"( methods are re#ortedin literature/ @ithout validation #arameters. 2=$2

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Ta%le 0F Dvervie@ of 3&A"($&A) * S, methods for the analysis of sugars


-inear range* g9ml,

52 5S) C

(ar%o&ac &A$7?28? 1

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Suzanne de Goeij 20

edeiros et al. determined the re#roduci%ility of the analytical #rocedure through the relative standarddeviation of re#licate measurement. The 5S) values ranged from 8.8 B >. C. ?

ernandez$Artigas et al. re#orted a 5S) range of . B 8.0C for the #recision study. 2 uzfai et al.re#orted an average 5S) of . C. 8

A chromatogram of the se#aration of different trimethylsilylated sugars in gum is #resented in figure 7?.

igure 7?F (hromatogram of trimethylsilylated sugars in gum ara%ic analysed %y G($ S/ re#roducedfrom reference .

4/3 %iscussion gas chromatogra1hy

The main advantage of using gas chromatogra#hy is the high resolution in relative short retention times/although derivatization of the sugars is al@ays re4uired. The re#orted detection limits are the nanogramrange/ com#ara%le to 3&A")$&A) or -( @ith derivatized sugars for :; or fluorescence.'n G( different ty#es of derivatization reagent are availa%le/ methylation/ acetates and silyllation. 'n allre#orted references the trimethylsilyl ether is used/ the good volatility and sta%ility characteristics of thederivatives formed make trimethylsilyl *T S, ethers the most #o#ular derivatives a##lied to G( analysisof saccharides. ost re#orted methods used S as detection/ due to the e1tra information o%tained@ith S. 6ot many a##lications could %e found for 4uantitative sugars se#aration and detection %y G($

S or G($ '). This could %e due to the lo@ recovery values re#orted *0 and 77 C,/ relative high#recision results *around 8C, and the e1tra @ork of the derivatization. The use of G($ S to identifysugars is mainly used in the re#orted references

'n ta%le = a##lications of analysis of sugars using G( are summarised. ore a##lications can %e foundho@ever @ithout validation #arameters. $

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Ta%le =F Dvervie@ of G( methods for the analysis of sugars

(olumn ethod details Sugars *retention time min a,*H/ I if a##lica%le,

-inear range* g9ml,

52

)!8$ S ca#illarycolumn ? m 1?.28mm '.). andfilm thickness of?.28 m

(arrier gasF 3e(onstant flo@ 7. m-9min.

S source tem#F 2 ? ('njectionF S#lit less mode/ s#lit less time

? s.'njector tem#F 2 ?J('njection volumeF 7 lTem# #rogramF (onsisted of injection at08 ( and hold for 2 min/ tem#eratureincrease of 0 (9min to ?? (/ follo@ed%y an isothermal hold at ?? ( for 78minTotal analysis timeF ?7.2 B 72?

5ecovery inrange 0 $77 C

?.> 8?.>>>?.>>=?.>>>

actor our fused$silica ca#illarycolumn; $8 ms/ ? m 1?.28 mm/ ?.28 m

(arrier gasF 3e(onstant flo@ 7. m-9min)etectionF S'njectionF s#lit9s#litless mode'njector tem#F 2 ? J('njection volumeF 7 lTem# #rogramF initial tem#erature 08J( *held for 2 min,/ increased at 0 J(9minto ?? J( *held for ? min,Total analysis timeF 8? min

Ara%inose *7>.0>/ 7>.=8,ylose *27.80/ 22.8?,

Glucose *2/ 27.7>,

ylitol *27. >,annitol *28.27,

All range ?.7 B78?

?.>>=?.>>?.>>=?.>>=?.>>=?.>>8?.>>??.>><?.>>=?.>>0

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(olumn ethod details Sugars *retention time min a,*H/ I if a##lica%le,

-inear range* g9ml,

52

The fused silicaca#illary column($ 2 28Q(29SG-$7 28 m 1 ?.28 mm'.)./ ?.28 mm filmthickness coated@ith 7??Cdimethyl#olysilo1ane

(arrier gasF 62(onstant flo@ 7.8 m-9min)etectionF ')'njection s#lit ratioF 7F2?'njection tem#F 2>?J()etector tem#F ??J(Tem# #rogramF 'nitial tem#erature @as7 ? J(/ follo@ed %y a heating rate of

J(9min to 2 ?J(. The finaltem#erature @as maintained for 2 minTotal analysis timeF not re#orted

ructoseGlucoseSucrose

altose5affinose

n.r. ?.>>>?.>>?.>>?.>>?.>>=

?m 1 ?.28 mm '.). fused$silicacolumn coated@ith a ?.28 - filmof S"$8<

(arrier gasF 3e)etectionF S'nlet #ressure of 77? k&aS#lit$s#litless injector'njection tem#F ??J(Tem# #rogramF from 708J( u# to2 8J(at2J(9minTotal analysis timeF 8 min

ylose *7=.=>,Ara%inose*7 .>2,5hamnose*27.

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5 CA+"LLAR, L CTR#+*#R S"S -C .

!esides li4uid and gas chromatogra#hy/ a num%er of analytical methods are descri%ed using ca#illaryelectro#horesis as se#aration techni4ue for the se#aration of sugars. (" utilizes an o#en tu%ularca#illary/ @hich can %e ra#idly flushed @ith fresh %uffer directly after detection of interesting #eaks and

%e #re#ared for the ne1t injection. 't is a #o@erful techni4ue concerning the sim#licity/ short analysistime/ efficiency and lo@ sam#le consum#tion.

)ue to the pKa of monosaccharideLs %eing more than 77/ monosaccharideLs are negatively charged instrong %asic running %uffer and can %e further se#arated under a fi1ed electric field. or im#roving these#aration efficiency/ some surface$active agents/ such as S)S/ tetrahydrofuran *T3 ,/he1adecyltrimethylammonium %romide *(TA!,/ etc. can %e added into the running %uffer. 0

5/' %etection in Ca1illary lectro1horesis

As @ell as -( and G( chromatogra#hic methods/ detection of sugars in (" is challenging %ecause of thea%sence of chromo#horic grou#s. 'n this revie@ the (" se#aration of sugars is descri%ed and com#ared@ith three ty#es of detectionF :; after derivatization =$ $ >/ indirect :; detection

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Suzanne de Goeij ?

igure 77F )erivatization scheme of 7$*2$na#hthyl,$ $methyl$8$#yrazolone *6 &, for sugars/ re#roducedfrom reference .

Wang et al. tested an increasing concentration of methanol added to the %ackground electrolyte

solution/ @hich resulted in increasing resolution of the sugars to %e se#arated. The %est se#aration @asachieved at

Uou et al. validated the method and found good re#roduci%ility @ith 5S)C of the migration time and#eak area/ res#ectively/ from ?.

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Suzanne de Goeij 7

5/'/) "ndirect (!

As discussed #reviously/ direct :; detection of sugars is im#ossi%le. 3o@ever adding an :; activereagent to the %ackground electrolyte makes indirect :; detection #ossi%le. A highly alkaline #3condition @as used in order to charge car%ohydrates negatively and to #romote migration to@ards the

anode. "lectroosmotic flo@ @as reversed to the direction of the anode %y addingcetyltrimethylammonium %romide (TA!, to the electrolyte. All references use (TA! in com%ination @ithan :; active com#ound in the !G". 5izelio et al. selected sor%ate as the co$ion %ecause it is a strongchromo#hore @ith mo%ility similar to that of fructose/ glucose and sucrose.

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Suzanne de Goeij 2

5/'/3 Am1erometric: lectrochemical %etection

Another o#tion in (" detection is electrochemical detection/ es#ecially am#erometric detection.

This detection techni4ue is suita%le for analysis of sugars %ecause the electro active hydro1yl grou#s incar%ohydrates can %e catalytically o1idized to #roduce significant current res#onses on the surface ofmetal electrodes such as co##er and nickel disk electrodes. This is re#orted in several #a#ers. < $

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3u et al. and Uang et al. @ere a%le to se#arate their interested sugars @ithin ? minutes @ere (oa et al.managed the se#aration of the desired sugars @ithin 78 minutes.

3u et al. validated the method on #recision and found the #recisions of migration time and #eak current*in terms of relative standard deviation*5S),, @ere ?.7= $ ?.2

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Ta%le F Dvervie@ of (" methods using :; detection for the analysis of sugars

(a#illary ethod details Sugars *migrationtime min a,

-inear range* g9ml,

52 5S) C

fused$silicaca#illaryFTotal length < .8cm/ effectivelength

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Ta%le >F Dvervie@ of (" methods using indirect :; detection for the analysis of sugars.


-inear range* g9ml,

52

&olyamide$coatedfused silicaca#illary778cm length *7?8cm to detector,8? m '.)./ =8

m D.).

!G"F 2/0$dimetho1y#henol *) &, m /cetyltrimethylammonium %romide *(TA!, 7.8 m#3 72.?Tem#F Am%ient;oltageF Z7 k;'njectionF hydrodynamic #ressure at 8? m%ar for 7 sDn$column :; detection at 22> 8?.>>><?.>>><?.>>>7?.>> >?.>>

:ncoated fused$silica0?.? cm totallength/ .8 cmeffective length/8? m '.). and

=8 m D.).

!G"F 2? mmol9- sor%ic acid/ ?.2 mmol9- (TA!/

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Ta%le 7?F Dvervie@ of (" methods using am#erometric9electrochemical detection for the analysis of sugars.


-inear range* g9ml,

52

&olyimide$coatedfused silica (a#illary>?.>>?.>>

!are fused$silicaca#illary 0? cm long@ith 28 m innerdiameter and 0?

m outer diameter

!G"F 72? mmol9- 6aD3 solution;oltageF 72 k;'njectionF "lectro$kinetically at 72 k; for 7? s)etection #otential ?.08 ;Total analysis timeF ? min

ucoseGalactoseGlucose5hamnoseAra%inose

ructoseylose

All sugars 7 B7??

?.>>?.>>?.>=?.>>?.>>?.>>?.>>

A 0? cm length of 28m '.). and 0? m

D.). fused silica(a#illary

!G"F 8? mmol9l 6aD3 solution;oltageF 70 k;'njectionF 70 k;90 sWorking #otential is ?.08 ;Total analysis timeF 2? min

Sucrosealtose

Glucoseructose

7 B 8?? ?.>>?.>>?.>>?.>>

A 28$ m i.d. >?$cmlong uncoated fused$silica ca#illary

!G"F #3 . ? mmol9- %orate containing 2?C*v9v, A(6 7?? mmol9- %orate detection %uffercontaining 8.? mmol9- 5u*%#y, 2N

;oltageF 78 k;'njectionF 7? k;9 sWorking #otential 7.2 ; *vs. Ag9Ag(l,Total analysis timeF 2? min

ylose5hamnoseGlucose

?.?7 B

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%The electrochemical cell consisted of a #latinum au1iliary electrode/ a co##er disk @orking electrode and an Ag9Ag(l * mol9electrodec A three electrode system/ @hich consisted of a (u disk electrode or diameter ?? m as @orking electrode/ a saturated calomeas reference electrode/ and a #latinum @ire as counter electrode/ @as used in %oth electrochemistry and detection e1#erimentsd A co##er$disk electrode @ith 7

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7 C#$CL(S"#$

The se#aration and 4uantitative analysis of sugars is challenging for several reasons. To find a #ro#ertechni4ue/ column and detector makes se#aration and detection of 4uantitative sugars analysis achallenge.

-i4uid chromatogra#hy is the most #o#ular techni4ue to detect sugars although there are severalchallenges to overcome. S#ecial columns are on the market for sugars se#aration/ %ut in all casesmethod develo#ment is still re4uired and not al@ays o#timum %aseline se#aration is achieved. Ty#icalanalysis times are %et@een ? B 8? minutes.

)etection of sugars in -( gives several #ossi%ilities/ "-S)/ 5'/ :;/ S/ luorescence and (A)/ all @ith theiro@n advantages and disadvantages.

A disadvantage of the "-S) is the lo@ re#roduci%ility/ slightly lo@ sensitivity to lo@ molecular @eightcom#onents and the non$linearity of the detector. An advantage is relative sim#le use of the detector.

The use of "-S) in com%ination @ith the se#aration of sugars has %een re#orted %y different authors.With the use of different columns for the se#aration/ detection limits in the same order @ere found *inthe order of ?.7 B 7 µ g #er injection,. A recovery of > B >>.> C @as re#orted and an intra$ and inter$day#recision as C 5S) of the retention time and #eak are E 2 C.

The res#onse of 5' detection results in linear cali%ration curves. A disadvantage in 5' detection is thetem#erature sensitivity/ small fluctuations in tem#erature results in high variations. 5' detection cannot%e used in com%ination @ith gradients @hich makes method develo#ment limited. )etection limits of 5'are in the same range as "-S) a##lications. (astellari et al. found e1cellent recoveries *P>>. C, and#recision results E7C. 2 3o@ever other authors re#orted lo@er recoveries *%et@een > B 77 C, andhigher #recision values/ re#eata%ility E7.8C and re#roduci%ility E=.?C.

ass s#ectrometry is also an o#tion for the detection of sugars. ass s#ectrometry for the detection ofsugars is difficult due to the lo@ efficiency of ionization related to the lo@ acidic character of sugars.Several a##lications are re#orted @ere sugars @ere analysed as their sodium adduct/ acetate adduct orchloride adduct. (om#ared to the detection limits of other detectors/ the lo@est limits @ere re#orted %y3ammad et al. using a 4uadru#ole S/ detection limits in #g range #er injection are re#orted. 70 Althoughvery lo@ detection limits are re#orted *#g range, the #recision is around 8C and the average recovery isaround 0?C for all sugars. (om#ara%le #recision values are found in other a##lications.!eside 4uantification/ the use of S can generate information a%out chemical structures @hich can %every useful develo#ment #hases.

or detection %y :; a derivatization reagent is re4uired. A##lications are re#orted @here 7$&henyl$ $methyl$8$#yrazolone = and #$amino%enzoic ethyl ester is used as derivatization reagent/ @ith recoveries%et@een >? B 72? C. The re#orted detection limits are in the nanogram range/ @hich is significant lo@ercom#ared to the re#orted detection limits %y "-S) and 5'. The re#orted values for #recision are E2.

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Suzanne de Goeij >

)ifferent derivatization reagents are a##lica%le for detection %y fluorescence. -o@ detection limits arere#orted @ith the use of anthranilic acid > and %enzamidide 7> as derivatization reagent. The detectionlimits of sugar se#aration @ith the use of a fluorescence detector are significant lo@er com#ared to the"-S) and 5' detection/ and com#ara%le to the detection limits found %y :; after derivatization/ ho@evera derivatization ste# is al@ays re4uired.

Another ty#e of li4uid chromatogra#hy is high$#erformance anion$e1change chromatogra#hy @ith &A)detection. )ifferent ty#es of columns can %e used to o#timize the se#aration. The correlation coefficientof the cali%ration curves is in all re#orted references P?.>>. )etection limits re#orted @ith 3&A")$&A)are in the nanogram range and are com#ara%le to -( @ith :; or fluorescence detection afterderivatization or -( @ith S detection. !ruggink et al 2 develo#ed a system to use S as detection. Theyalso re#orted im#ressive detection limits @ith &A) detection ho@ever they are the %uilder and o@ner ofthe 3&A"( system.

!esides -(/ gas chromatogra#hy is an o#tion for the se#aration and detection of sugars. The mainadvantage of using gas chromatogra#hy is the high resolution in relative short retention times. Adisadvantage is that derivatization of the sugars al@ays is re4uired. 'n all re#orted references thetrimethylsilyl ether is used/ the good volatility and sta%ility characteristics of the derivatives formedmake trimethylsilyl *T S, ethers the most #o#ular derivatives a##lied to G( analysis of saccharides. 6otmany a##lications could %e found for 4uantitative sugars se#aration and detection %y G($ S or G($ ').This could %e due to the lo@ recovery values re#orted *0 and 77 C,/ relative high #recision results*around 8C, and the e1tra @ork of the derivatization. ost re#orted methods used S as detection/ dueto the e1tra information o%tained @ith S.

(" is a good alternative for the se#aration of sugars. 't is a #o@erful techni4ue concerning the sim#licity/short analysis time/ efficiency and lo@ sam#le consum#tion. 'n relative short retention times *@ithin Y ?minutes, se#aration of different sugars can %e achieved. or the o#timization of the re4uired se#arationof the sugars different ca#illaries can %e used. )ue to the pKa of monosaccharideLs %eing more than 77/monosaccharideLs are negatively charged in strong %asic running %uffer/ like %orate or 6aD3 %uffer.&laying around @ith the concentration of the running %uffer can im#rove the se#aration.After se#aration a fe@ #ossi%le detection methods are availa%le :; after derivatization/ indirect :; andelectrochemical detection. :; after derivatization al@ays re4uires e1tra @ork @here the recovery needsto %e taken into account. 5e#orted recoveries are P>8C. 'ndirect :; results in significant higherdetection limits com#ared to :; @ith derivatization and electrochemical detection. An advantage ofindirect :; is that limited e1tra @ork is re4uired. All detectors re#orted re#eata%ility results of themigration time and #eak are E8C/ mostly E7C for migration time. 5e#roduci%ility results re#orted areE0C.

)e#ending on the se#aration needed/ amount of sam#les to %e analysed and the re4uired detectionlimits different methods can %e a##lied. or traces of sugars in routine analysis in the food industry/ a3&A")$&A) can %e used. or %ulk analysis for research #ur#oses @hen having lots of sam#les a -($"-S)method is sufficient. When traces of sugars are not analyses routinely/ -($:; or ("$:; can %e a##lied. 'fthe sam#le contains lot of different sugars and high resolution is needed/ a G( method is the %est o#tion.

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Suzanne de Goeij *2?77,782>$78 0M70O -.A. 3ammad/ . . Saleh/ .;. 6ovotny and U. echref/ Multiple% eaction Monitoring #i-uid

$!ro(atograp!y Mass Spectro(etry for Monosacc!arides $o(positional Analysis of lycoproteins, [.Am. Soc. ass S#ectrom/ 2? *2??>, 722>$2?0M22O 5.W. )i1on and G. !altzell/ 'eter(ination of levoglucosan in at(osp!eric aerosols using !ig! perfor(ance li-uid c!ro(atograp!y )it! aerosol c!arge detection, [ournal of (hromatogra#hy A/ 77?>*2??0, 27

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